Durban University of Technology - Abstracts: Poster Presentations · 2016. 1. 18. · University of the Free State Intracellular gas bubbles in yeasts P52 Ms Thobeka Mhlongwe Stellenbosch

Abstracts: Poster Presentations

Poster Presenter Affiliation TitleofResearchPaper

P1 MsAngeliqueduPreez

UniversityofPretoria

Pantoeaananatisstrainsfromdifferentniches:aflagellinglycosylationislandcomparison

P2 MrAnthonyOtigbu

UniversityofFortHare Assessmentofantibacterialactivityofanindigenoushoney

P3 MsLesley-AnneCaine

UniversityofFortHare

AntimicrobialresistanceandphylogeneticprofilingofEscherichiacoliisolatedfromrawcow'smilkintowcommercialdairyfarmsoftheEasternCapeprovince,SouthAfrica

P4 MrCharlesHunter

UniversityofKwaZulu-Natal

MALDI-TOFMS:arobustscreeningmethodforprofilingandgroupingaerobicendospore-formingbacteriaascandidatebiocontrolagents

P5 MrLethukuthulaNgobese

UniversityofKwa-ZuluNatal

ConstitutiveFLO1overexpressioninS.cerevisiaestrainsbearingdeletionsincellwallbiogenesisrelatedgenes

P6 MrsDikonketsoMatjuda

AgriculturalResearchCouncil

Analysesoftheimpactsofbacteriologicalseepageemanatingfrompigfarmingonthenaturalenvironment

P7 MrsKeletsoMashile

NorthWestUniversity

Serotypediversityandreassortmentbetweenhuman/animalrotavirusstrainsintheNorthWestProvince,SouthAfrica

P8 MsSarishaSinghUniversityofKwaZulu-Natal

Pyrolysis/GC-MSprofilingtoidentifycarbonandnitrogeninfluenceonpolyhydroxyalkanoateproductionbyBacillusthuringiensis

P9 MsSarishaSingh UniversityofKwaZulu-Natal

Screeningforpolyhydroxyalkanoateproductionbybacteriaisolatedfromsludgeandeffluentobtainedfromapapermill

P10 MsMunangiwaTshivhase

UniversityofLimpopo

EvolutionaryengineeringofXylosefermentingyeastsisolatedintheKrugerNationalPark,LimpopoProvince

P11 MrsKousarHoorzook

UniversityofJohannesburg

Comparisonofoptimisedin-houseDNAextractionmethodtoavailablecommercialwatertestingkitsusingquantitativereal-timePCR



Factorsaffectingabsolutequantitativereal-timePCRfromwatersamples



Singlestep11geneM-PCRforthedetectionofdiarrhoeagenicE.coliinclinicalandenvironmentalwatersources

P14 MrOlufemiAkanbi


Isolationandidentificationofselectedpathogenicbacteriafromfarmedandwildduskykob(Agyrosomusjaponicus)harvestedfromEasternCapeProvinceofSouthAfrica

P15 MsKaminiGovender


Over-expressionofFLO11encodedmannoproteininSaccharomycescerevisiae

P16 MrArchiePhiri UniversityofLimpopo MicrobialandchemicaldynamicsduringMarulafermentation

P17 MsWendyLangenhoven

ARCInfruitec-Nietvoorbij

DetectionoftumourigenicAgrobacteriuminsoilandplantmaterialinWesternCapevineyards

P18 MsSonamBaliram

AgriculturalResearchCouncil-ISCW

Evaluationofphosphatesolubilisingmicroorganismsisolatedfromgardensoilfortheirabilitytosolubiliserockphosphate

P19 MrTshimangadzoNdiitwani


MOTIFsearch,asequence-basedapproachtoidentifyingtertiaryalcoholesterhydrolasesfrombacterialgenomes

P20 MsNkatekoMasingi

UniversityofLimpopo

Anoxybacillussp.,athermophilicβ-glucosidaseproducingbacteriumisolatedfromahotspringinZimbabwe

P21 DrSarinaClaassens

North-WestUniversity

Phospholipidfattyacidvsmetabolomicsanalysisforprofilingofmicrobialcommunities

P22 MrMashuduMukhuba


ComparisonofcommerciallyavailableDNAextractionkitsfortheisolationofbacterialandarchaealDNAfromvariousstagesintheanaerobicdigestionprocess

P23 MsSannieKatlegoMashigo

AgriculturalResearchCouncil-ISCW

StockpiledsoilsfromcoalminesinMpumalangaregion:impactsonbiologicalcharacteristicsofthesoil

P24 MsNondumoMakhanya

UniversityofKwaZulu-Natal CloningandexpressionofaputativeM.tuberculosissecretoryprotein

P25 MrCalloteDube UniversityofFortHare

InvitrostudyonH.pylori-ureaseinhibitionbyactivesolventextractsofSouthAfricanhoney

P26 MsNompumeleloNyembe


TwitchingmotilityanddevelopmentofabiofilmassayforXylophilusampelinus

P27 MrKingsleyEbomah


AssessmentofthequalityindicesoftheNahoonandEasternbeachesinEasternCapeProvince,SouthAfrica

P28 MrDonMvududu UniversityoftheWitwatersrand

ScreeningofCMM6andAMM2transgeniccassavalinesforresistancetocassavamosaicdisease

P29 DrLubanzaNgoma


ScreeningofEndophyticBacteriatowardstheDevelopmentofCottageIndustry:AninVitroStudy

P30 MsKeitiretseMolefe


DeterminationoffactorsthatinfluencereproductiveconditionsincowsintheruralfarmsoftheNgakaModiriMolemadistrictoftheNorthWestProvince

P31 MsMellisaNaidoo

DurbanUniversityofTechnology

Profilingofunknownbacterialculturesisolatedfromtissuecultureplantingsusingphenotypicmicroarray

P32 MrSipheleleSibisi UniversityofKwaZulu-Natal

Investigatingbiosurfactantproductionbysponge-associatedbacteriaandassessingtheirpotentialanti-biofilmactivity

P33 MrsNaziaShaikh UniversityofKwaZulu-Natal

Identificationofeffluxpumpactivityinfish-associatedflavobacteriumsppisolates

P34 MrsLucretiaGovender


LipophilicextractiveprofilesofeucalyptusspeciesusedindissolvingpulpproductioninSouthAfrica:effectsofstorage

P35 MrAyodejiOsunla


Physicochemicalandmicrobiologicalqualitiesofwaterandsoilsamplesfromgroundnutoilproducingindustry

P36 MsNikitaNankoo UniversityoftheWitwatersrand

Structuralcharacterizationofthecell-to-cellmovementandreplicationproteinsofSouthAfricancassavamosaicvirus

P37 MsMorongwaMathipa

UniversityofLimpopo

Assessingtheinfluenceofminingactivitiesonthebio-physicochemicalqualityofsurfaceandgroundwaterforhumanuseintheTubatseMunicipality

P38 DrCharlesEmekaObiukwu

ImostateUniversity,Nigeria

Modelsonthekineticsofphenolutilizationinammoniumphosphatesupplementedphenolladenrefineryeffluent



PhysiologicalResponseofPeriophthalmuspapillioasBiomarkerofAquaticEcosystemPollution



In-vitroantimicrobialstudiesoflocalNigerianspicesagainstentericpathogens

P41 MrsToyinAdelowotan


ComparisonoftheMLSTTypingandCCM-PCRAssayWorkflowsforthedeterminationoftheSequenceTypesofStaphylococcusaureusisolates

P42 MrAyodejiIdowu UniversityofFortHare

TheeffectoftemperatureanddistanceonpoliovirustiterfromclinicalspecimensofacuteflaccidparalysiscasesinNigeria

P43 MrMaropengCharlesMonyama UNISA GroupBstreptococcuscolonizationinpregnantwomenatDr.George

MukhariHospital,SouthAfrica

P44 MrOluwatayoE.Abioye


Thesynergisticeffectsofn-hexanefractionofParkiabiglobosa(Jacq.)barkextractandselectedstandardantibioticsonbacterialisolates

P45 MrTaiwoFadare UniversityofFortHare

MicrobiologicalassessmentofairinsomeselectedsawmillandfurniturefactoryenvironmentsinOsunState,SouthWesternNigeria

P46 DrIdayatGbadamosi

UniversityofIbadan,Nigeria

EssentialoilconstituentsandinvitroantimicrobialactivityoftherootsofMondiawhitei(hook.F.)skeels

P47 MsPhilippaFranzini

UniversityofPretoria TheroleofdesertinsectmicrobialsymbiontsontheCarbonCycle

P48 MsLeticiaMosina UniversityofPretoria

Cloning,PurificationandCrystallisationofabi-functionalPaenibacillusmucilaginosusexoglucanase

P49 MrsTovhowaniRamulongo


ImmunedampeningandrefocusingofaSAT2foot-and-mouthdiseasevaccinestrain

P50 MrsThandoMrwetyana

UniversityofFortHare Sharkliver,apotentialsourceofantimicrobialagents

P51 MrKhumishoDithebe

UniversityoftheFreeState Intracellulargasbubblesinyeasts

P52 MsThobekaMhlongwe

StellenboschUniversity

EntomopathogenicfungiassociatedwithinvasivewaspsintheWesternCaperegion

P53 MrJuliusHellmuth

UniversityoftheFreeState

EvaluatingERIC-PCRasamoleculartypingmethodofAvibacteriumparagallinarum

P54 DrOsuolaleYinkaTitilawo


FingerprintsandprevalenceofmultidrugresistantEscherichiacolipathovarsinselectedsurfacewatersinSouthwestNigeria

P55 DrOsuolaleYinkaTitilawo


EssentialoilconstituentsandinvitroantimicrobialactivityoftherootofMondiawhitei(hook.F.)Skeels

P56 MrCasperBrink StellenboschUniversity

ThedevelopmentofamicrobialgrowthpromotingmixtureforcommercialAspalathuslinearis(rooibos)plants

P57 DrNwabisaMehlomakulu


Aerobicfermentationasanalternativetoreducingethanollevelsinwinemaking

P58MrLukasMarthinusDuPlooy

UniversityoftheFreeState GasbubbleformationinthegenusSaccharomyces.

P59 MrWicoSander UniversityoftheFreeState Investigationintotheeffectoffattyacidsontheyieldofrotavirusinfection

P60 MsKaylaLawson StellenboschUniversity

Themicrobialcommunitiesassociatedwithahoneybee(Apismellifera)hive

P61 MsJeanetteVanNiekerk

UniversityoftheFreeState DiagnosticsofBeakandfeatherdiseasevirus

P62 MsTersiaConradie

StellenboschUniversity

Sunnysideup:usinganantioxidantproducingbacteriumtoenhancepigmentationineggyolksoflayinghens

P63 MsMapulaMaropola

UniversityoftheWesternCape

Screeningformagnetotacticbacteriainmarine,freshwateranddesertsedimentsofNamibiaandSouthAfrica

P64 MsBiancaPieterse


Theroleofcytochromep450intheproductionofprostaglandinE2bySaccharomycescerevisiae

P65 MrRodneyHart AgriculturalResearchCouncil

CharacterisationandevaluationofnovelSaccharomycescerevisiaehybridsfortheproductionofaromaticSauvignonBlancwineproduction

P66 MsNicoleKennedy


DeterminationofQACresistantStaphylococcusaureusstrainsfrommastitissamples

P67 MrAndriesvanderWalt

UniversityofPretoria MicrobialcommunityecologyoftwoNamibDesertfairycirclebiotopes

P68 MsNtombifuthiShezi RhodesUniversity Bio-prospectingasoilmetagenomelibraryforcarbohydrateactive

esterases

P69 MsCorneliaLianiMeyburgh


Novelyeast-basedexpressionsystemfortheproductionofsubunitvaccines

P70 MrOluwasegunKuloyo


Substratecharacterizationandheterologousexpressionoftheself-sufficientCYP505E3fromAspergillusterreus

P71 MsElke Coetsee

UniversityoftheFreeState LongrangePCRtodetectHP2andMU-likephages

P72 MsJi-YunLee UniversityoftheFreeState

BacteriophagelambdaendolysinexpressionforavianpathogenicEscherichiacolitreatment

P73 MsNthabisengZeldaMokoena


TheinfluenceofCandidaalbicansphenotypicplasticityonexpressionofvirulencefactors

P74 MsPreciousLetebele


DifferentialmetabolomicsandtranscriptomeanalysisofKluyveromycesmarxianusonxyloseandglucoseascarbonsubstrates

P75 MsShaniceAdams


HeterologousexpressionofasiderophorefromaPseudoalteromonassppassociatedwithamarineinvertebrate

P76 MrGodfredTanih UniversityofFortHare

Incidences,speciesdistributionandantimicrobialresistanceofEnterococcussppisolatedfromfaecalandwatersamplesfromthreecommercialfarmsinAmatoledistrictsEasternCape,SouthAfrica

P77 MsVictoriaOntong

UniversityoftheWesternCape Actinomycetes:keepingrooibosplantshealthynaturally

P78 MrRaimondovanVuuren

UniversityoftheWesternCape CharacterisinganovelDNApolymerasefromNamibianmetaviromes

P79 MrRelebohileMatobole


ElicitationofsecondarymetaboliteexpressionfrommarinespongeisolatesfromAlgoaBay,SouthAfrica

P80 MrWoutervanderWesthuizen


DevelopmentofanISS-basedavianpathogenicEscherichiacolivaccineinEscherichiacoliandYarrowialipolytica

P81 DrThabisoMotaung


Thepheromoneexporter,HST6p,controlsstressresponsesandmorphogenesisinC.albicans

P82 MsLondiweKhumalo


Assessmentofpigmentedhalophilcbacteriafortheimprovementofrejectbrineevaporationrates

P83 MsNontobekoMvubu


Canonicalpathways,networksandtranscriptionalfactorregulationbyclinicalstrainsofM.tuberculosisinpulmonaryepithelialcells

P84 MsNontandoHadebe


Isolationandcharacterizationofprebioticoligosaccharidesfromalgalextractsandtheireffectongutmicroflora

P85MsLangutaniKhambani


Isolationandcharacterizationofrhizobacteriafortheproductionofsiderophoresandtheirin-vitroantagonisticactivityonselectedphytopathogens

P86 MsElizabethFamewo


PhylogeneticanalysisofbacterialpopulationsofPernapernal.inAlgoabay,EasternCapeProvince,SouthAfrica:implicationsforpublichealth

P87 MrGoitsemangMakete


IsolationandidentificationofpotentialprobioticbacteriafromSouthAfricanSaanengoatsmilk

P88 MsChristianaMojisolaOwoseni


ChlorineInactivationofE.coliIsolatesfromSelectedWastewaterTreatmentPlantsintheEasternCapeProvince

P89 MsZiyandaDlamini


Efficacyofprobioticbacteriainmanagementofpostweaningdiarrhealsyndromeonweanedpiglets

P90 MrJustinHoff

ARCInfruitec-Nietvoorbij

Theuseofchromogenicmedia(candida)forthescreeningofwinerelatedyeastspecies

P91 MrJustinHoff ARCInfruitec-Nietvoorbij

IdentificationofaceticacidbacteriainspontaneousSauvignonBlanc,PinotageandMerlotfermentations

P92 MrJustineFri UniversityofFortHare

CulturedandWildDuskyKob(Argyrosomusjaponicus),areservoirofhumanpathogenicvibriospecies

P93MsMariaCatharinaGrobbelaar

UniversityoftheWesternCape BioactiveactinobacteriaassociatedwithAspalathuslinearis

P94MsKhumbuzileNokwandaBophela


CharacterizationandpathogenicityofEnterobacterspeciesassociatedwithbacterialblightanddie-backofEucalyptusseedlingsandcuttings

P95 MrMatthewvanWyngaard

UniversityofKwaZuluNatal

Controlofbacterialcontaminationinprawnhatcheriesusingaphage-basedsolution

P96 MsZiyandaMmango


Isolationandscreeningofcellulolyticmicroorganismsfromdecayinglignocellulosicbiomass

P97 MsMotlagomangKhantsi


MolecularcomparisonofintraspecificvariationinthebacterialstrainsresidentintherhizoplaneandrhizosphereofBambaranut(Voandzeiasubterraneal.Thouars)

P98 MsAgnesMohapi NorthWestUniversity

EpidemiologicalsurveyofsalmonellacontaminationonbeefcarcassesinabattoirsandbeefproductsintheNorthWestProvince,RepublicofSouthAfrica

P99 MsKgomotsoSetsetse

NorthWestUniversity

Determinationofmicrobialcontaminantsofinfectiousdog-to-dogbitewoundspresentedatDaleBeighleVeterinaryHospital

P100 MsBBagheri StellenboschUniversity

Employingcontrolledmulti-starterfermentation,anewperspectiveinyeastdynamics

P101 MrKoosMmutle NorthWestUniversity

Effectofdosevariationsofrabiesvaccineoncanineantibodytitreresponse

P102 MsTintswaloMgiba

NorthWestUniversity

CryptosporidiuminfectioninsheepandgoatsaroundMafikeng,SouthAfrica

P103 MrMotlapeleMutle

NorthWestUniversity

Determinationofquarterlysubclinicalmastitisusingdifferentscreeningmethodsindairycattle

P104 MrIsaacMosimane

NorthWestUniversity

SerologicalprevalencestudyofbovineviraldiarrhoeaincommunalfarmsaroundMafikeng

P105 MrMolatlhegiSethaiso

NorthWestUniversity

IdentificationofgeneralcoliformbacteriafrommilkcollectedfromlactatingcowsamongstselectedfarmsteadofMafikengvillages

P106 DrMxolisiMasango


Characterizationofcelldeathcausedbydiplodiatoxinanddipmatol,mycotoxinsofStenocarpellamaydis

P107 MrNerveZhou LundUniversity AdaptationthroughchromosomalrearrangementsinadaptivelyevolvedLachanceakluyver

P108 MrNjomHenryAkum


PriorinfectionofwheatwithrustinducesresistancetoRussianwheataphid

P109 MsAsisiphoNkohla


IsolationandscreeningofcellulolyticandxylanolyticbacteriafromlignocellulosicbiomassfromAliceTown

P110 MsRobynVisserCapePeninsulaUniversityofTechnology

Marineactinomycetesasasourceofnovelantimicrobialagents

P111 MsShandreWeels

CapePeninsulaUniversityofTechnology

Determiningantimicrobialpropertiesofphenolicsdetectedinpeatsamples

P112 DrRobynRoth CSIRBiosciences Co-expressionofsulfhydryloxidaseanddisulphideisomeraseresultintheproductionofsolubleCRM197inEscherichiacoli

P113 MsPFMhlanga UniversityofKwa-ZuluNatal

AntimicrobialandantioxidantpropertiesoffungalendophytesfromKigeliaafricana

P114 MsSelishaNaidoo UniversityofKwaZulu-Natal

Revivalandcharacterizationofendospore-formingbacteriafromanancientsedimentcoreobtainedfromtheMfabenipeatland,SouthAfrica

P115 MrsShimaAbdulgader

UniversityofCapeTown

LongitudinalprevalenceandantibioticsusceptibilityofnasopharyngealcarriageofStaphylococcusaureusinhealthyinfantsandtheirmothersinabirthcohortintheWesternCape

P116 MsShivaniGoolab CSIR Theuseofbacterialcellsurfacedisplayasabrucellosisantigendelivery

system

P117 DrAtheeshaSingh UniversityofJohannesburg

Survivingtheacidbarrier:responsesofVibriocholeraeO1andO139tosimulatedgastricfluid

P118 MsTanzelleOberholser

UniversityofPretoria Rootassociatedmicroorganismsincropproductivity

P119 DrThabisoEricMotaung

UniversityoftheFree-State

PhenotypicswitchinginCandidaalbicansinfluencesprostaglandinE2production

P120MsYeshonaSewsunker


Doesthevolumematter?Aninsightintomodellingandoptimizationofbiohydrogenproductionacrossscales

P121 MsAshiraChanderman


Production,PurificationandCharacterizationofphytasefromEnterobacterspp.ACSS

P122 MrJohnsonZininga

DurbanUniversityofTechnology ScreeningandProductionofathermoandacidstablePhytaseproducer

P123 MrMelvinMakolomakwa


Batchandfed-batchproductionofphytasefromThermomyceslanuginosus

P124 MsAlveeraSingh DurbanUniversityofTechnology

AntimycobacterialandmoleculardockingstudiesofpentacyclictriterpenesisolatedfromleavesofBuddlejasaligna

P125 MrArumugamNanthakumar


ExploringlignocellulosicbiomassofSouthAfricancropsforxylooligosaccharideproduction

P126 MrDiimiseniNekhumbe


ComparativestudyofcyanatehydrataseproductionbydifferentstrainsofthermophilicfungusThermomyceslanuginosus

P127 MsLeeanthaNaicker

DurbanUniversityofTechnology Antimicrobialandantioxidantactivitiesofpiperidinederivatives

P128 MrAbeenChetram

DurbanUniversityofTechnology Invitropharmacologicalscreeningofnovel1,2,4triazolederivatives

P129 MsVuyokaziBelewa

NelsonMandelaMetropolitanUniversity

ModeofinhibitionofTulbaghiaviolaceaonAspergillusflavus

P130 MrNjabuloNene UniversityofKwa-ZuluNatal

Developmentofsemi-definedmolassesasastandardisedlaboratoryyeastculturemedium

P131 MrSydwellLanga

AgriculturalResearchCouncil Administrationofprobioticsinpigsandtheireffectonweightgain

P132 Ms.EvashneeRampersad


CreationofaxylanasedeficientstrainofThermomyceslanuginosusbyrecombinantDNAtechnology.

P133 MrMondeThabiCapePeninsulaUniversityofTechnology

Oxidationofphenoliccompoundsfromrooibos(Aspalathuslinearis):improvingantioxidantpotential

P1

PantoeaananatisSTRAINSFROMDIFFERENTNICHES:AFLAGELLIN

GLYCOSYLATIONISLANDCOMPARISON

DuPreez,A.1,DeMaayer,P.2andCoutinho,T.A.1

1DepartmentofMicrobiologyandPlantPathology,ForestryandAgriculturalBiotechnology Institute

(FABI), University of Pretoria, Pretoria 0002, South Africa. 2Centre for Microbial Ecology and

Genomics,UniversityofPretoria,Pretoria0002,SouthAfrica

Pantoeaananatis isaplantpathogenicenterobacteriumthat infectsabroadrange

ofagronomiccrops.Thepathogenicitymechanismsofthisbacteriumremainlargely

unknown. However, flagella have recently been shown to play a vital role in the

infectionprocess.Asthemainstructuralproteinoftheflagellum,flagellin,ishighly

immunogenic and capable of host defense avoidance. Recent studies identified a

genomic island in theplant pathogenPseudomonas syringaewhichplays a role in

glycosylation of flagellin, masking it from host detection. A comparative genomic

analysisofseventeenP.ananatisstrainsforwhichgenomesarecurrentlyavailable

revealedthepresenceofaflagellinglycosylationisland(FGI)locatedadjacenttothe

flagellin(fliC)genes.ThisFGIisbetween9and22.7kbinsizeandcontainsbetween

7 and22protein coding sequences (CDSs). LocalizedBlastP comparisonof the FGI

CDS sets indicated extensive variability in the CDS content. The flagellins are

glycosylated with distinct sugars, and distinct acetyl, methyl and formyl side

branches. The seventeen sequencedP. ananatis strains could be divided into four

groupson thebasis of their distinct FGIs. Thepresenceof a FGI could explain the

broadrangeofhoststhatP.ananatiscaninfectwithouthostdetection.

P2

ASSESSMENTOFANTIBACTERIALACTIVITYOFANINDIGENOUSHONEY

EXTRACTSONHelicobacterpyloriUREASE

OtigbuA.C1,ClarkeA.C1,AkanbiO1,FriJ1.

1Department of Biochemistry&Microbiology, University of Fort Hare, P.MB. X1314, 5700, Alice, South

Africa.

Honey is one of such medically important natural products. The Eastern Cape pure

honeyprocessedfromraw,pure,strainednectarsource(Scutiamyrtina) inPortAlfred

was used for the study to evaluate the antibacterial potential of anti-H. pylori

compounds obtained from an indigenous honey variety in controlling H. pylori

infection.Twosolvents(DichloromethaneandPetroleumspirit)wereusedforextraction

of antibacterial compounds from the crude Honey using the liquid-liquid method of

solventextraction.369Cclinical isolatesofH.pyloriwasusedfortheexperimentwhile

ATCC43526strainsservedaspositivecontrolstrain.Minimuminhibitoryconcentrations

(MICs)of theextractsweredeterminedusing theagardilutionmethodandcompared

with a standard antibiotic, clarithromycin (CLA) used for H. pylori treatment.

DichloromethaneextractofPureHoneyshowedhighinhibitionforpurifiedformsofthe

enzymeswith27% inhibition forH.pylori369Cureaseand20% inhibition forH.pylori

ATCC urease but however displayedweak inhibition against the control enzyme, Jack

Beanureaseshowing5%inhibitionwhilethePetroleumspiritextractdemonstratedvery

weak inhibitionforall formsoftheenzymesshowing10%inhibitionforH.pylori369C

urease,and5% inhibition forH.pyloriATCCureaseand traceof inhibitionagainst the

controlenzyme,JackBeanurease

P3

P4

MALDI-TOFMS:AROBUSTSCREENINGMETHODFORPROFILINGANDGROUPING

AEROBICENDOSPORE-FORMINGBACTERIAASCANDIDATEBIOCONTROLAGENTS

Hunter,C.H.1,Laing,M.D.2,Wallis,F.M.1andSchmidt,S.1

1DisciplineofMicrobiology,SchoolofLifeSciences,UniversityofKwaZulu-Natal,PrivateBagX01,

Scottsville,3209.

2DisciplineofPlantPathology,SchoolofAgricultural,EarthandEnvironmentalSciences,Universityof

KwaZulu-Natal,PrivateBagX01,Scottsville,3209.

Matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass

spectrometry(MS)isanon-destructiveionizationmethodthatprovidesafast,sensitive

and accurate means of analyzing and characterizing biomolecules over a broadm/z

range. MALDI-TOF MS was assessed as a means of identifying and grouping aerobic

endospore-forming bacteria as potential biocontrol agents and screening them for

lipopeptide production. Twenty-seven isolates, selected for their antagonism of

Rhizoctonia.solaniinvitrowereevaluated.Massspectrawerecollectedinthem/zrange

2000to20000foridentificationpurposes,andinthem/z750to2500rangeforprofiling

secondarymetaboliteproduction.Initialattemptstoidentifyisolatestothespecieslevel

usingBiotyper-basedidentificationsoftwaremetwithlimitedsuccess.Extensionofthe

databaseusing“in-house”massspectraprofilesofisolatesidentifiedonthebasisof16S

rRNA and gyrA gene sequence analysis significantly improved the ranking of isolate

identification matches achieved. Cluster analysis of mass spectra allowed for the

relationshipsbetweenisolatestobeassessedandprovidedameansofgroupingisolates

fordereplicationpurposes.MALDI-TOFalsoprovidedaconvenientmeansofdetecting

bioactive lipopeptides (viz., surfactin, iturins, fengycin) directly from whole cell

preparations,cellextractsandevencrudeculturefiltrates.Overall,MALDI-TOFMSwas

found to meet the requirements for a practical yet robust technique suitable for

processinglargenumbersofbacterialisolatesforbiocontrolscreeninganddereplication

purposes.

P5

CONSTITUTIVEFLO1OVEREXPRESSIONINS.CEREVISIAESTRAINSBEARINGDELETIONS

INCELLWALLBIOGENESISRELATEDGENES

NgobeseLM,GuptharASandGovenderP

SchoolofLifeSciences,Biochemistry,UniversityofKwaZuluNatal,SouthAfrica

Todate, ithasbeenreportedthatthefivedominantFLOgenesinS.cerevisiaeencode

forafamilyofglycosylphosphatidylinositol(GPI)linkedglycoproteinsthatarecommonly

referred to as flocculins. The adhesionphenotypes that are associatedwith individual

FLOgeneshavebeenextensivelyresearchedandwellcharacterized.However,farlessis

understoodaboutthecellularmetabolicroutesthatleadtotheirbiochemicalsynthesis

and incorporation into the yeast cell wall. Additionally our fairly limited current

understandingofthefinemoleculararchitectureofthesemannoproteinspredominantly

reliesondatageneratedfrominsilicopredictiveresearchstudies.Inthisresearchstudy

ageneticengineeringapproachwillbeemployedtoconstitutivelyoverexpresstheFLO1-

encodedmannoprotein in thewild typeBY4741haploid laboratoryS. cerevisiae strain

andinmutantBY4741strainsbearingadisruptivedeletionineithertheirKNR4orGPI7

cell wall biogenesis related genes. As such the effect of the gene deletions on the

intensityoftheflocculationphenotypewillshedlightonthecontributionofthedeleted

gene products in biochemical processing of FLO1 mannoproteins. Additionally it is

envisioned that the transgenic yeast strains overexpressing FLO1mannoproteins will

provide a viable alternative for the large scale isolation and purification of the intact

mannoprotein especially if it were to be released into the growth medium by FLO1

overexpressingdeletiontransgenicstrains.Thisglycoproteinreservoircanbeutilisedin

the structural analysis of FLO1mannoproteins. In relation to the transgenicwild type

BY4741-F1Pstraindecreasedflocculation intensitywasobserved intheBY4741ΔKNR4-

F1P strain. BY4741ΔGPI7-F1P displayed a flocculation intensity thatwas similar to the

BY4741-F1P strain. Interestingly, 10% higher protein concentrationswere observed in

thespentgrowthmediumofthetransgenicBY4741ΔKNR4-F1Pstraintherebyseemingly

suggestingthepossibilityoftheFLO1mannoproteinbeingreleasedextracellularly.

P6

ANALYSESOFTHEIMPACTSOFBACTERIOLOGICALSEEPAGEEMANATINGFROMPIG

FARMINGONTHENATURALENVIRONMENT

D.S.Matjuda1,RAdeleke2&O.AAiyegoro1

1Gastro intestinal Microbiology and Biotechnology, Agricultural Research Council- Animal Production

Institute,Irene,SouthAfrica.2SoilHealthUnit,AgriculturalResearchCouncil-InstituteofSoilClimateandwater,Arcadia,SouthAfrica.

Modern pig farming production may cause exposure of bacterial pathogens and

introductionofresistancegeneduetopig’sdroppingsandlackofseepagemanagement.

Thisresearchwascarriedouttoinvestigatetheeffectsofseepageemanatingfromapig

farmon its receiving environment and to detect the presence of antibiotic resistance

gene.Soilandwatersampleswerecollectedmonthlyforaperiodofsixmonths(March-

August2013). Sampleswere collected8different siteswithin thepig farm.Procedure

followed for analysing samples includes viable cell counts of 101 to 108 dilutions,

physicochemicalanalyses,antibioticsusceptibilitytest,APIidentificationofisolates,and

Moleculardetectionofresistantgene.Theviablecells insoilsamplesfrom0cfu/mlto

2.44 x 1010cfu/ml, and ranged from 5.00 x 101 to 5.05 x 109 in water sample.

Physicochemicalparametersofwaterandsoilsamplesshowedunacceptablehighlevels

thanthemaximumpermissible limitssetbyDepartmentofWaterAffairsandForestry

(DWAF). Salmonella spp, Proteus spp, E.coli1 etc. were isolated from soil and water

samples from the pig farm. Isolates were highly resistant to 8 out of 17 antibiotics

tested,Themostresistancegenesdetectedinmostisolateswereaa(6’)-le-aph(2”)-la,

VanA,VanB,andOtrB.Theresultshowedthatpigfarmseepageisimpactingnegatively

on the natural environment in the vicinity of pig farm due to observed high bacterial

contaminationwithpresenceofpathogenicbacteriawithantibioticresistancegene,and

increasedlevelsofphysicochemicalparameterssoilandwater

P7

P8

PYROLYSIS/GC-MSPROFILINGTOIDENTIFYCARBONANDNITROGENINFLUENCEON

POLYHYDROXYALKANOATEPRODUCTIONBYBacillusthuringiensis

Singh,S.1,Permaul,K.2,Govinden,R.1,Lekha,P.3,Sithole,B.3,

1UniversityofKwaZulu-Natal(Westvillecampus),MicrobiologyDepartment,UniversityRoad,Westville,

PrivateBagX54001,Durban,4000.2DurbanUniversityofTechnology,BiotechnologyandFoodTechnology,P.O.Box1334,Durban4000.3

Forestry&ForestProductsResearchCentre,POBox17001,Congella,4013,SouthAfrica

Thereisstronginterestinresearchofbiologically-derivedpolymersfortheproductionof

bioplastics. This is because microorganisms can produce plastic building components

such as polyhydroxyalkanoate (PHA) grown in environments rich in excess carbonbut

limitedinnitrogen,phosphorus,oxygenormagnesiumcontents.Thecurrentstudyused

pyrolysis/GC-MS (Py/GC-MS) to investigate and compare PHA production by Bacillus

thuringiensiswhengrownina30:1ratioofcarbon:nitrogensource.Thirty-onedifferent

nutrient combinations were used, each consisting of glucose, sucrose, cellulose,

fructose,carboxymethylcellulose(CMC),starch,lactoseorglycerolascarbonsourceand

NH4Cl,(NH4)2SO4,yeastextractortryptoneasnitrogensources.PHAmonomerswere

notdetectedwhenB.thuringiensiswasgrownin(NH4)2SO4incombinationwithanyof

thecarbonsources.GrowthinNH4Clincombinationwithcelluloseorfructoseresulted

in only oleic acid and 2-butenoic acid 4,4-dimethoxy being synthesized, respectively.

Growthwithyeastextractortryptonewithglucose,sucrose, fructose,CMC, lactoseor

glycerol,resultedintheproductionofoleic,palmitoleic,crotonicandcis-vaccenicacids.

The combinations of fructose with yeast extract and tryptone with glucose were the

most successful as 6 and5different PHAmonomersweredetected, respectively, and

eachmonomer product consisted of 2, 16, 18 or 19 carbon atoms. Organic nitrogen

sources yielded more complex monomers, varying from 4 to 19 carbons in length.

Py/GC-MSwaseffectivefortherapidcharacterisationofPHA-productionafteralteration

ofcarbonandnitrogensources.

P9

SCREENINGFORPOLYHYDROXYALKANOATE-PRODUCTIONBYBACTERIAISOLATEDFROM

SLUDGEANDEFFLUENTOBTAINEDFROMAPAPERMILL

Singh,S.1,Permaul,K.2,Lekha,P.3,Sithole,B.3,Govinden,R.1

1UniversityofKwaZulu-Natal (Westville campus),MicrobiologyDepartment,UniversityRoad,Westville,

PrivateBagX54001,Durban,4000.2DurbanUniversityofTechnology,BiotechnologyandFoodTechnology,P.O.Box1334,Durban4000.3Forestry&ForestProductsResearchCentre,CSIR,POBox17001,Congella,4013,SouthAfrica

Polyhydroxyalkanoates (PHAs) are naturally-occurring polymers utilised in bioplastics.

Theyare synthesisedbyGram-positiveandGram-negativebacteriaasaby-productof

their cellular processes. Interest now lies in the isolation of new strains capable of

utilizing inexpensive carbon sources for PHA-production. The present study involved

screening forand isolatingPHA-producingbacteria, fromeffluentandsludgeobtained

fromthreesitesatalocalpapermill.ScreeningforPHA-andpolyhydroxybutyrate(PHB)-

producing bacteria involved using Nile Blue A agar and Sudan black staining assays,

respectively. Thereafter, their colonymorphology,Gram-reaction and cellmorphology

wereassessed.Intotal,69PHA-producingbacteriawereisolated.Variablefluorescence

responseswereobserved:36.2%fluorescedyellow,44.9%fluorescedorangeand18.8%

fluoresced yellow-orange. Of the isolates, 92.8%were positive for PHB-production by

appearing blue-black. A 26 day time-course study on isolate E19 indicated that PHB

granule accumulation increases over time. Varieties of colony and cell morphologies

werenoted,whichdifferedamongstthesampledsites.Slimy,creamcolouredcolonies

wereexhibitedby26.1%oftheisolateswhereas11.6%ofthecoloniesexhibiteddistinct

spreadingedges.TwoisolatesobtainedfromabypassdrainsamplewereGram-positive

cocci.Ofthe48isolatesisolatedfromtheeffluentafterclarification,52.1%wereGram-

positiveand45.8%wererod-shapedbacteria.Of the19 isolatesobtained fromsludge

onawall,84.2%wereGram-negativeand52.6%werecoccibacteria.Thusfar,itcanbe

concluded that the isolates were capable of PHA- and/or PHB-production. It appears

these isolates could potentially produce PHAswhereby effluent and/or sludge can be

utilisedasacheapcarbonsource.

P10

EVOLUTIONARYENGINEERINGOFXYLOSEFERMENTINGYEASTSISOLATEDINTHE

KRUGERNATIONALPARK,LIMPOPOPROVINCE

M.Tshivhase;E.L.JansenvanRensburg;D.C.LaGrange;I.Ncube

DepartmentofBiochemistry,MicrobiologyandBiotechnology.UniversityofLimpopo.PrivateBagX1106,

Sovenga,0727.

Energy demand and environmental concerns has resulted in the use of renewable

energy sources such as plant materials. Evolutionary engineering is an effective

approach to improve efficiency of ethanol production. The aim of this study was to

improve locally isolated yeasts for growth on high concentrations of xylose, high

temperatures and high concentrations of acetic acid, through adaptation. The best

adapted yeast strain was then compared to the parental strain and Pichia stipitis

(NRL72Y) in a bioreactor at optimal conditions. Seven yeastswere used in this study.

Pichia kudriavzeii (kp34ey) adapted well to extreme conditions. It produced 4.2 g/l

ethanolcomparedto0.8g/lfortheparentalstrainand0g/lforP.stipitisonxyloseas

solecarbonsourceinthepresenceofaceticacid(3g/l)at37°C.Boththeadaptedand

parentalP.kudriavzeiikp34eyyeastsproducedmoreethanolfermentingat30°Cinthe

presenceofaceticacidwiththeadaptedstrainproducing6.1g/lethanol,theparental

strain 3.2 g/l andP. stipitis 3.2 g/l. The enhanced adaptation process for the yeastP.

kudriavzeii kp34ey proved to be successful with a five-fold improvement of ethanol

productioncomparedtotheparentalstrainat37°C.

P11

COMPARISONOFOPTIMISEDIN-HOUSEDNAEXTRACTIONMETHODTOAVAILABLE

COMMERCIALWATERTESTINGKITSUSINGQUANTITATIVEREAL-TIMEPCR

HoorzookK.B.andBarnardT.G.

WaterandHealthResearchCentre,FacultyofHealthSciences,UniversityofJohannesburg,POBox17011,

Doornfontein2028,Johannesburg,SouthAfrica

Molecular methods have traditionally been performed on single isolates and from

enrichments.Enrichmentsprovidehigherbacterialcounts,onlyindicateviablebacteria,

cannotestimatecountsandonlygetanideaofpresenceandabsence.Viablebutnon-

culturable bacteria cannot be isolated by standard culture – based methods, the

simplestwaytoovercomethiswouldbetoisolateDNAfrombacterialcellsconcentrated

directlyfromthewatersamples,thuscircumventingtheneedforculturability.Theaim

wastodevelopamethodtoconcentratethebacterialcellsdirectlyfromwatersamples

followedbyDNAextractionfromthecells.Thereafter,comparethe inhousemodified

DNA extraction method with commercially available water testing kits. The DNA

extraction by membrane filtration was tested with four types of membranes namely

Polycarbonate (Poly), Nitrocellulose (NC), Nitrocellulose-acetate (NA) and Polyether-

sulphone(PES).PolywasusedinthemodifiedinhouseDNAextractionmethodandwas

comparedtofouravailablecommercialwatertestingkits,namelyWaterMasterTMDNA

purification kit (Separations), Ultra CleanWater DNA isolation kit (Optima Scientific),

AquadienTM kit (Biorad) and Metagenomic DNA isolation kit for water (Separations)

usingoptimisedgadquantitativereal-timePCR.TheinhouseDNAextractionmethodR2

andslopewere(0.99and0.99)(-3.48and-3.65)respectivelyfor2repeats.Incontrastto

R2andslopetoWaterMasterTMDNApurificationkit(R2=0.34andR2=0.73)(-5.73and-

4.45); Ultra Clean Water DNA isolation kit (R2=0.97 and R2=0.28) (-3.89 and -8.84);

AquadienTMkit(R2=0.98andR2=0.77)(-3.59and-5.94);MetagenomicDNAisolationkit

(R2=0.65andR2=0.77)(-3.83and-4.89).Thein-houseDNAextractionprotocolforwater

testinghas thepotential for goodDNA recovery, repeatability and reproducibility and

qualityforPCRanalysis,itisalsosuitableandcosteffectivealternativetotheavailable

commercialDNAextractionwatertestingkits.

P12

FACTORSAFFECTINGABSOLUTEQUANTITATIVEREAL-TIMEPCRFROMWATER

SAMPLES


WaterandHealthResearchCentre,FacultyofHealthSciences,UniversityofJohannesburg,POBox17011,


Quantitativereal-timePCR(qPCR)combinestheadvantageoftheconventionalPCRwitha real time detection of the amplification products, and can be adapted to highthroughputanalysis.qPCRanalysisisaffectedbytheeffectiveconcentrationofbacterialcellsfromwater;theDNAextractionefficiency;theinfluenceofinhibitorysubstancesonthe qPCR; the standard curve preparation and the influence of background bacteriaduringtheDNAextractionprocess.StandardcurveisacrucialstepforqPCR,thereareno standard procedure followed for the construction of these curves. The aimof thisstudywastoprovideinformationonthefactorsaffectingquantitativeanalysiswhichwillbeasteppingstoneindeterminingtheE.colipathogenvirulencegenestohousekeepinggeneratiosforeachE.colipathogenicgroup.E.coliwasusedasamodelorganismsinceit is used as an indicator organism but can also cause infection. Standard curves forabsolutequantificationfortheeaeAgene(EPECandEHEC)andgadgene(commensalE.coli)was successfully optimised. The following considerations need to be taken intoaccount before quantification: a) the effect of backgroundDNA, b) the effect of DNAextractiononeachdilution.Therefore,threeadditionalstandardcurveswereincluded.Standard curve 1 was constructed using tenfold diluted extracted DNA in water;Standardcurve2wasconstructedbydilutingEHECcells tenfold inpreviouslyenrichedbroth and extracting DNA from each individual diluted tube. Standard curve 3 wasconstructed by using tenfold diluted extracted DNA in extracted total coliforms (TC)brothDNA. Standard curve 4was constructed by diluting EHEC cells tenfold in steriledistilledwaterandextractingDNAfromthedilutedcells. Instandardcurve1,which isthe norm in creating a standard curve has a goodmean of 0.99 and the SDwas low(0.0081). Compared to standard curve 2 (mean 0.98 and SD 0.005), standard curve 3(mean 0.98 and SD 0.019) and Standard curve 4 (mean 0.95 and SD 0.028) the SDincreases for standard curve 3,4 and the mean decreases for standard curve 2.Variabilityalso is shown inupperand lowercopynumberdetectionbetween the fourstandard curves. It is very important to consider the factors such as influence ofbackground DNA as well as extracting each dilution to include variability of the DNAextractionwhencreatingastandardcurveforabsolutequantification.

P13

SINGLESTEP11GENEM-PCRFORTHEDETECTIONOFDIARRHOEAGENICE.coliIN

CLINICALANDENVIRONMENTALWATERSOURCES


WaterandHealthResearchUnit,FacultyofHealthSciences,Universityof Johannesburg,POBox17011,


Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC)groups,ofwhichtheDECgroupsareclassifiedinto5groups.TherearemediaavailablefortheisolationofComECandselectedDECtypes.Conformationalstepsareperformedafterculturing if it is requiredto test for thepresenceofDEC, increasing thecostandtime required for obtaining the results. Molecular biology methods such as thepolymerasechainreaction(PCR)havebeenusedtoidentifymicro-organismsaccordingto their specific genetic makeup. The aim of this study was to develop a single-stepmultiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genesassociatedwithDECandComEC,withtheinclusionofcontrolstomonitorinhibition.Atotal of 701 samples, taken from clinical and environmental water sources in SouthAfrica,wereanalysedwiththeoptimisedm-PCRwhichtargetedtheeaeA,stx1,stx2,lt,st, ial,eagg,astAandbfpvirulencegenes.Themdhandgapdhgeneswereincludedasaninternalandexternalcontrol,respectively.Theexternalcontrolgene(humangapdhgene)wasdetected in all the sample indicating thatnoPCR inhibitionwereobtained.The internal control gene (mdh)wasdetected in100%of theenvironmental and85%(202/239)oftheclinicalisolates,confirmingtheisolatesasE.coliPCRpositivesamples.AlltheDECtypesweredetectedtovaryingdegrees inthemdhpositiveenvironmentaland clinical isolates. The E. coli asta toxin was detected in 35% of themdh positiveenvironmentalisolatesand17%ofthemdhpositiveclinicalisolates.Interestingly25%ofthetoxinpositiveenvironmentalisolatesand17%ofthetoxinpositiveclinicalisolatesdidnotcontainanyotherofthevirulencegenestestedfor.Thisresultisveryimportantsince in 1996 during an outbreak of gastrointestinal illness was caused by E. coli0166:H15 which possessed no entero-pathogenicity-associated genes other than theasta gene. In conclusion, the optimised 11 gene multiplex PCR reaction could besuccessfullyused for theconfirmationofE.coli isolatesaswellas the identificationofpathogenic E. coli types from the isolates. TheMPCR was also successful in showingmonitoring for PCR inhibition to ensure the correct reporting of results. The use ofmultiplex PCR techniques still offers researchers the opportunity to screen for awiderangeofpathogensinasingletest.

P14

ISOLATIONANDIDENTIFICATIONOFSELECTEDPATHOGENICBACTERIAFROMFARMED

ANDWILDDUSKYKOB(Agyrosomusjaponicus)HARVESTEDFROMEASTERNCAPE

PROVINCEOFSOUTHAFRICA

AKANBI,O.E.1,Paterson,A.2andClarke,A.M.1

1DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,PrivateBagX1314,Alice,5700.2SouthAfricanInstituteofAquaticBiodiversity,SAIAB,SouthAfrica.

Formanycenturies,fishhavebeenoneofthemainsourcesoffoodandisstillregarded

asanimportantfoodbecauseofitsrichnessinprotein,vitaminsandminerals.InAfrica,

it is one of the cheapest sources of protein and has an advantage over other foods

because of its palatability, deliciousness and quick digestion. However, this important

foodcanalsobea sourceof foodbornepathogens. This studyaimedat isolatingand

identifyingShigelladysenteriae,SalmonellatyphiandVibriospeciesinskin,gillandgut

offarmedandwildfish,duskykob,harvestedfromtheEasternCapeProvinceofSouth

Africa.Tissuesamples(skin,gill,gut)ofthefishandwaterwereculturedonappropriate

media and confirmed using API 20E. Pseudomonas flourescens, Serratia marcescens,

Enterobacter cloacae, Aeromonas hydrophila, Chryseobacterium meningosepticum,

Photobacteriumdamselae,PseudomonasaeruginosaandPseudomonasluteolawereall

isolated fromdusky kob in thewild. In the fish farm, Serratia liquefaciens Salmonella

spp.,Shigellaspp.,Enterobactercloacae,Vibrioalginolyticus,Vibriofluvialis,Aeromonas

salmonicida, Enterobacter aerogenes, Shiwanella putrefaciens, Burkholderia cepacia,

Raoultella ornithinolytica and E.coli were all identified. Physico-chemical parameters

performedon fish farmwater conformed toSouthAfricanGuidelines foraquaculture.

Although there was a low concentration of highly human pathogenic agents such as

Vibriocholera,SalmonellatyphiandShigelladysenterae,duskykobactsasareservoirof

opportunistic pathogenswhich are potential health hazard to human consumerswho

areimmune-compromised.

P15

OVER-EXPRESSIONOFFLO11ENCODEDMANNOPROTEININSaccharomycescerevisiae

Govender,K.1,Pillay,K.2andGovender,P.2

1DepartmentofBiochemistry,SchoolofLifeSciences,UniversityofKwaZulu-Natal,PrivateBagX54001,

Durban,4000,SouthAfrica.

Yeast cell wall mannoproteins that are released by Saccharomyces cerevisiae during

fermentation has captivated researchers world-wide because these mannoproteins

exhibit many advantageous properties. These adhesins are associated with many

adhesionphenotypessuchasinvasivegrowth,pseudohyphalformation,flocculationand

biofilmformation.AminoacidsequencesofFloproteinshavebeendiscoveredbasedon

theanalysisof thegenomic sequenceofyeast. In this study itwasenvisaged that the

over-expression of a dominant FLO11 gene will generate the desired adhesin in

sufficient quantities. A novel strategy was utilized which incorporated genetic

manipulation of S. cerevisiae laboratory strains to over-express and release cell wall

mannoproteins into the growth medium. As such, the FLO11-encoded cell wall

mannoprotein was over-expressed in S. cerevisiae strains containing a gene deletion

related to cellwall biosynthesiswhichwas previously shown to promote extracellular

hyper-secretion. This study utilized a polymerase chain reaction (PCR)-based cloning

strategyinordertoproducetransgenicstrainsofS.cerevisiaeinwhichthenativeFLO11

open reading frame (ORF) was placed under the transcriptional control of the

constitutive TEF1 promoter. The research data revealed that the strategy employed

resulted in theconstitutiveexpressionof theFLO11ORF in the transgenicstrains.The

effectoftheadhesioncharacteristicsattributedtoFLO11gene-basedtransformantswas

evaluated. The flocculation potential and invasive growth tendencies of FLO11 gene-

basedtransformantsandwildtypestrainswereestablished.

P16

MICROBIALANDCHEMICALDYNAMICSDURINGMARULAFERMENTATION

A.Phiri,PfareloShandukani,DanieLagrange,MathabathaSetati,KgaboMoganedi

UniversityofLimpopo,DepartmentofBiochemistry,MicrobiologyandBiotechnology,PrivateBagx1106,

Sovenga,0727

Inmanylocalcommunitiesthemarulafruitisfrequentlypreparedintonon-alcoholicand

alcoholicbeverages fordomesticor commercialpurpose in thisprovidesa reasonable

income to the families who have turned marula beer into an enterprise during the

marula season. Fermentation of marula beer is mainly mediated by the natural

microorganismassociatedwiththemarula fruit,hencethebeerhasashortshelf-life (

only2-4daysdependingontheambienttemperature)duetothismixedfermentation

process.Thedynamicsduringmarulabeerfermentationwerestudiedthroughprofiling

of the microbial and chemical changes throughout the fermentation process, using

marulabrewsproducedindifferentcommunities.Chemicalprofilingoftheunfermented

marula juice was achieved using both GC and HPLC assays. Preliminary screening

showed a complex mixture of volatile fatty acids (VFA) and alcoholic compounds.

ComparableamountsofVFAssuchasbutyricacid,valericacid,caproicacid,heptanoic

acidwereobservedinallmarulafruitjuicesfromdifferentlocalities.Formationofacetic

acid, propionic acid and various other yet unidentified smaller molecular weight

compoundsstarttoemergefromday2offermentationandtheconcentrationincreases

proportionatelywithfermentationtime.ThemicrobiotawasidentifiedwithMALDI-TOF

Bio-typer and ARISA assays. The following generawere identified frommarula brews

sampledsequentiallyduringthefermentationprocessforprofiling:Bacillusmegaterium,

Erwiniapapaya,Klebsiellapneumoniae,Bacillusamyloliquefaciens,Seratiamarcescens.

Lactobacillus species emerged as fermentation proceeded. Though not all compounds

and microorganisms have been identified yet, analysis based on these preliminary

resultsshowspresenceofvariedenvironmentalmicrobesassociatedwithmarulafruits.

This is the first studywhichwill elucidateon the chemical andmicrobial changes that

occur throughout the fermentation process and will assist in identifying spoilage

microorganismsassociatedwithunpalatablemarulabrews

P17

DETECTIONOFTUMOURIGENICAGROBACTERIUMINSOILANDPLANTMATERIALIN

WESTERNCAPEVINEYARDS

Langenhoven,W.E.andPetersenY.

ARCInfruitec-Nietvoorbij,CultivarDevelopment,PrivateBagX5026,Stellenbosch,7599,SouthAfrica.

Agrobacterium vitis, the predominant causal agent of grapevine crown gall, results in

severeeconomic losses ingrapevinecultivationworldwide.Visualsymptomexpression

ofthediseaseappearsasgallsonrootsorthebaseofthevine,whereassymptomson

nurserymaterialareusuallyobservedatthegraftunionsandatnodeswherebudshave

been removed. The resultant tumours inhibit plantphysiological functions resulting in

stuntedgrowthandsubsequentyieldloss.During2013,anumberofgrapevinenurseries

intheWesternCapeProvincereportedtheoccurrenceofsymptomsassociatedwithA.

vitis. This necessitated the need to develop a protocol to accurately determine the

presenceof thepathogen for screeningofpropagationmaterial andplanting sites. To

addressthisneed,primersspecificforA.vitis,Agrobacteriumspecies,aswellasprimers

targeting tumourigenic strains were assessed for their suitability for a standard PCR

diagnostictest.TheseprimershavebeentestedagainstDNAisolatedfromfieldsamples

of soil and root material collected from symptomatic grapevines. Preliminary results

haveindicatedthepotentialtodetecttumourigenicAgrobacteriumspp.frombothplant

andsoilsamples.Onceoptimised,thedetectionprotocolwillhavepositiveimplications

for disease management strategies in nurseries, and new as well as in established

vineyards.

P18

EVALUATIONOFPHOSPHATESOLUBILISINGMICROORGANISMSISOLATEDFROM

GARDENSOILFORTHEIRABILITYTOSOLUBILISEROCKPHOSPHATE

Baliram,S.1andAdeleke,R.A.1,2

1AgriculturalResearchCouncil–InstituteofSoil,ClimateandWater(ARC-ISCW),600BelvedereStreet,

Arcadia,Pretoria0001,SouthAfrica.2UnitforEnvironmentalScienceandManagement,North-WestUniversity(PotchefstroomCampus),

Potchefstroom2520,SouthAfrica.

Phosphate is one of the nutrients in soil that occurs in low concentrations. Due to it

being a limiting nutrient to plants, it is often applied to soil in the formof fertilizers.

Theseappliedfertilizerscontainingphosphatesorthephosphatesfoundinsoilarenot

utilized to their full capacity by plants. This is attributed to the fact that part of the

phosphatesbecome insoluble and immobilisedwithin the soil. Bacteria and Fungi can

reverse this effect by breaking down insoluble phosphates to amore soluble form, a

form inwhichplants canuse.Themechanismsused for the solubilisationof insoluble

phosphate include acidification, chelation and exchange reactions. Thesemechanisms

are performed by certain microorganisms known as Phosphate Solubilising

Microorganisms (PSM). They can be found in almost all soils. This research aimed at

isolating and identifying PSM from ordinary garden soil. Soil samples were collected

wherebacteriaandfungiwereisolatedusingtheNationalBotanicalResearchInstitute's

phosphate growth medium (NBRIP) with tri-calcium phosphate as a source of

phosphate. The isolated PSM will be screened for their ability to solubilize rock

phosphate. Rock phosphate is a more insoluble, natural mineral or lower grade ore

whichismorecostefficientcomparedtotri-calciumphosphate.

P19

MOTIFSEARCH,ASEQUENCE-BASEDAPPROACHTOIDENTIFYINGTERTIARYALCOHOL

ESTERHYDROLASESFROMBACTERIALGENOMES

Ndiitwani,T.1,Photolo,M.1,Tlou,M.G.1

1DepartmentofBiochemistry,FacultyofScience,UniversityofJohannesburg,KingswayCampus,Auckland

Park,2006

Tertiary alcohols (TAs) are chiral compounds that show versatile biological activity in

natureandasaresult,havebecome interestingtargetsasbuildingblocks forvaluable

pharmaceutical compounds. The enantiomers thatmake up these compounds display

differentbiologicalactivitiesandthisthereforenecessitatestheirresolutioninorderto

isolate the desired activity. Esterase catalyzed enantioselective hydrolysis of tertiary

alcoholesters(TAEs)isoneapproachthatiscurrentlybeingexplored,duetothemany

advantagesassociatedwithusingbiocatalysts.However, this is limitedbythefact that,

onlya fewesterasesof theGGG(A)Xmotif family/TAEhydrolaseshavebeen identified

and characterized in this regard. These limitations indicate that there is a need to

expand the biocatalysis "toolbox" through studies aimed at identifying new TAE

hydrolases with improved properties. In this study, we report on the MOTIF search-

based screening of the genomes of several bacterial species for the GGG(A)X family

esterases.ThesearchresultedintheidentificationofputativeTAEhydrolasesfromthe

genomesofE.coli,P.syringae,P.aureginosaandM.tuberculosis.Todate,theesterase

from P. syringae(1000 bp) has been amplified, subcloned into pET-20b(+) and

functionally expressed on tributyrate agar in E. coli BL21 (DE3). Screening for TAE

hydrolaseactivityusing the linalyl acetateplate assay yieldeda false-positive reaction

andasa result, theassay is currentlybeingoptimised.However, successful functional

expressionof the gene indicates thatMOTIF search is an effective tool for identifying

theseenzymesfrombacterialgenomes.

P20

ANOXYBACILLUSSP.,ATHERMOPHILICβ-GLUCOSIDASEPRODUCINGBACTERIUM

ISOLATEDFROMAHOTSPRINGINZIMBABWE

Masingi,N.N.andNcube,I.

UniversityofLimpopo,DepartmentofBiochemistry,MicrobiologyandBiotechnology.PrivatebagX1106,

Sovenga0727.

Athermophilicβ-glucosidaseproducingstrainofAnoxybacillusflavitharmuswasisolated

from a hot spring in Zimbabwe. The A. flavithermus strain is a gram positive spore

formingmotilebacteriacapableofgrowthonglucose,fructose,sucrosemannose,avicel

and cellobiose as sole carbon sources. Growth was observed between 30 and 70 0C

(optimum 60) and at pH 7.5-9 (optimum 8). The microorganism hydrolysed starch,

gelatinand tributyrinandcouldgrow in thepresenceofup to5%NaCl.Phylogenetic

analysisbasedonthe16SrDNAgenesequenceindicatedthatthestrainbelongedtothe

genus Anoxybacillus. The Anoxybacillus strain showed a 98 % sequence similarity to

Anoxybacillus flavithermusAK1,Anoxybacillus flavithermus KW,Anoxybacillus sp JS40

and 97 % similarity to Anoxybacillus pushchinoensis. However, the physiological and

biochemical characteristics of the isolate suggest that the Anoxybacillus flavithermus

isolated may be a novel strain. The bacterial strain produces a thermophilic β-

glucosidasewithanoptimaltemperatureof600CandpHoptimaof7-7.5.Underoptimal

conditionswithpNPGasasubstratetheenzymehasahydrolyticactivityof15.1U/ml.

Theβ-glucosidasegenewassequencedandthededucedproteinsequenceshowsthat,

the enzyme belongs to glycosyl hydrolase family 1 (GH1), according to sequence

similaritywithotherGH1β-glucosidases.TheA.flavithermusstrainshowspotentialasa

producerofthermostableβ-glucosidaseenzymethatisofparticularimportanceforthe

saccharification step in bioethanol production. The saccharification efficiency of the

enzymestillneedstobeinvestigated.

P21

PHOSPHOLIPIDFATTYACIDVSMETABOLOMICSANALYSISFORPROFILINGOF

MICROBIALCOMMUNITIES

Willers,C.1,1JansenvanRensburg,P.J.2,andClaassens,S.

1UnitforEnvironmentalSciencesandManagement,North-WestUniversity,PrivateBagX6001,

Potchefstroom,2531.2FocusAreaHumanMetabolomics,North-WestUniversity,PrivateBagX6001,

Potchefstroom,2531.

Phospholipid fatty acid (PLFA) analysis has been a staple in determining microbial

communitystructureformanyyears.However,thetechniqueistimeconsuming,labour

intensiveandfraughtwithpotentialpitfallsduetothemanydifferentstepsinvolved.To

add to the problem, there are various extraction techniques and modifications to

existing methods that have been published over a number of years. Different

researchersfollowdifferentmethodsandnosinglestandardisedmethodforextraction,

fractionationandanalysishasbeenagreedupon.Inaddition,resultsfromPLFAanalysis

may be interpreted in different ways. All of these factors constrain comparability

betweenstudies.TheaimofthisstudywastocomparetraditionalapproachesforPLFA

analysis with a metabolomics based approach for characterisation of microbial

community structure. The different approaches were evaluated based on analyses of

pure bacterial cultures, homogenised soil samples and soil samples treated with

chemical fumigants and biofumigants. Results obtained showed that a metabolomics

based approach holds potential for further development as a technique to profile

microbial communities. However, the PLFAmethodwith fractionation is still relevant

and may be refined by optimising certain steps in the extraction and fractionation

procedures.AnintegrativeapproachusingelementsfrombothPLFAandmetabolomics

basedmethodscanofferenhancedprofilingofmicrobialcommunities.

P22

COMPARISONOFCOMMERCIALLYAVAILABLEDNAEXTRACTIONKITSFORTHE

ISOLATIONOFBACTERIALANDARCHAELDNAFROMVARIOUSSTAGESINTHE

ANAEROBICDIGESTIONPROCESS

MukhubaM1,2,RoopnarainA1,AdelekeR1andMyerM2

1ARC-InstituteforSoil,ClimateandWater,AgriculturalResearchCouncil,600,BelvedereStreet,Arcadia,

Pretoria0001,SouthAfrica.2UniversityofSouthAfrica, CnrChristiaandeWet&PioneerAvenue,Floridacampus,1709

TraditionaltechniquesfortheisolationofbacterialandarchaealDNAfromstoolsamples

especiallycowdungaretimeconsumingand laborious.TheuseofDNAextractionkits

forDNAextractionisconsideredtobeanattractivealternative.Inthecurrentstudy,6

commercially available DNA extraction kits were tested. Genomic DNAwas extracted

from samples obtained from a running biodigester (samples from feed, digester and

slurry). DNA was extracted in triplicate using both manual and automated DNA

extraction kits and the quantity andquality of the extractwas analysedusing a qubit

fluorimeter.Universalbacterial(341F/907R)andarchaealprimers(344F/915R)equipped

with GC-clamp was used for PCR. The respective PCR products was separated using

Denaturing Gradient Gel Electrophoresis (DGGE). The DGGE analysis enables the

assessmentof the impactof theextractionkiton the rDNA fingerprints. Thisenables

theselectionoftheidealkitwhichaccuratelyportraysthecompletemicrobialdiversity

oftherespectivesamples.Computersoftware(GelComparsoftwarepackage)wasused

foradvancedgelanalysis toobtainrefinedresults.Diversity indices (Shannon-Weaver)

werecalculated.TheleastfrequentDGGEbandswereexcisedandtheDNAwaseluted

andsequenced.Acostanalysisonthevariousextractionkitswasalsodone.

P23

STOCKPILEDSOILSFROMCOALMINESINMPUMALANGAREGION:IMPACTSON

BIOLOGICALCHARACTERISTICSOFTHESOIL

Mashigo,SK.1,2,Rasheed,A.1,2andBezuidenhout,C.2

1MicrobiologyandEnvironmentalBiotechnologyResearchGroupAgriculturalResearchCouncil–

InstituteforSoil,Climate&Water600,BelvedereStreet,Arcadia,0001,Pretoria,SouthAfrica2UnitforEnvironmentalScienceandManagement,NorthWestUniversity-Potchefstroomcampus,Private

BagX6001,Potchefstroom,2520,SouthAfrica

During opencast mining, the physical, chemical and microbiological properties of soil

change as a result of soil stripping and storage. Soil microbiota is a key soil quality

indices which can be affected by stockpiling. The aim of this study was to use the

functionalpropertiesofmicroorganisms fromstockpiledasbiological indicatorsof soil

quality.Agricultural(unmined)soilservedasacontrol.β-glucosidaseandureaseactivity

were determined from soil samples. Pure bacterial isolateswere further screened for

phosphate solubilisation, nitrogen fixation and indole acetic acid (IAA). Isolates from

stockpiled soil did not produce IAA, but could fix nitrogen and solubilise insoluble

phosphate.However,isolatesfromunminedsoilshowedpotentialtosolubiliseinsoluble

phosphate, fix nitrogen and produce IAA. There was a higher concentration of β-

glucosidase and urease from stockpiled soils in comparison to unmined soils. Higher

concentrations of β-glucosidase in the soil suggests deficiency of carbon related

compounds in the soil.Urease are important enzymes for nitrogen cycling in the soil.

Increasedureaseactivityinthesoilisanindicationofadeficiencyinnitrogenlevels.To

copewithlowproportionofnitrogen,microorganismsmaysecreteureasetometabolise

ureatoyieldammoniaandcarbamate.Theprocessofstockpilinghaspotentialnegative

effectsonsoilquality.

P24

CLONINGANDEXPRESSIONOFAPUTATIVEM.tuberculosisSECRETORYPROTEIN

MakhanyaN,ChilizaTE,PillayB

Discipline of Microbiology, School of Life Sciences, University of KwaZulu-Natal Private Bag X54001,

Durban4000,SouthAfrica

M. tuberculosis (Mtb) secretory proteins are involved in host-pathogen interactions,

facilitatinghost cell invasionandareassociatedwith virulence. Secretoryproteins are

idealcandidatesfordevelopmentofarapidTBantigendetectiontestbecausetheycan

be easily detected in the blood and other bodily fluidswithoutmuch invasiveness. In

order to select suitable candidate antigens, 18Mtb-specific proteins were analysed,

using bioinformatics tools, to identify secretory proteins. Only one of the 18 proteins

was predicted, with SignalP 4.0, to encode a signal peptide sequence required for

secretion, the Rv0397A protein. NCBI Blast of the 82 amino acid protein sequence

confirmedthisproteinasspecifictoMtbandM.bovisspecies.The246bpRv0397Agene

was amplied with specific PCR primers and successfully cloned into pGEX-6P-1 and

transformedintoE.coliBL21.ProteinexpressionwasinducedwithIPTGandanalysedby

SDS-PAGEandwesternblot.Usinganti-GSTmonoclonalantibody,a35kDaproteinwas

detectedbywesternblot,confirmingfusionofthe9kDaRv0397Atothe26kDaGSTtag.

Rv0397A isaputativeproteinofunknownfunction.However,mostMtbproteinswith

unknown function have been shown to be associated with virulence. The successful

cloningandexpressionofRv0397Awillallowfurthercharacterizationanddetermination

ofthefunctionofthisprotein.TheuniquenessofRv0397AinMycobacteriummakesita

goodcandidateforuseindevelopmentofMtbantigendetectionassayfordiagnosisof

activeTB.

P25

InvitroSTUDYONH.pylori-UREASEINHIBITIONBYACTIVESOLVENTEXTRACTSOF

SOUTHAFRICANHONEY

Dube,C.

MicrobialPathogenicityandMolecularEpidemiologyResearchGroup,DepartmentofBiochemistryand

Microbiology,FacultyofScienceandAgriculture,UniversityofFortHare,PrivateBagX1314,Alice5700,

SouthAfrica

Honey is one of the natural products that have demonstrated a broad-spectrum of

antibacterialactivityagainstpathogenicmicro-organisms ((Jedderetal.,1985;Ndipet

al., 2007 andManyi-Loh et al., 2010). It exhibits a complex mixture of sugars, small

amounts of organic acids, phenolic acids, flavonoids, proteins, minerals, vitamins,

enzymes and other phytochemicals (Aliet al., 2009; Silveret al., 2013). Research has

shown that the geographical location plays an important role in determining the

bactericidalpropertiesofdifferenthoneytypes(Alzahranietal.,2012).Theobjectiveof

this study is to search, analyze and compare anti-urease active compounds extracted

fromSouthAfricanhoneydiversitieswithManukahoney fromNewZealand,which is

respectedfor itsbroadspectrumantibacterialactivity.Ureasewillbeextractedfroma

standard strain of H. pylori and from biopsy cultures of samples obtained from

symptomatic patients who visited Livingstone Hospital, Port Elizabeth, South Africa

betweenMayandDecember2008.Fiveorganicsolventsstartingwiththenon-polarto

polar(Petroleumspirit,nHexane,Dichloromethane,Ethylacetateandmethanol)have

beenusedforcompoundextraction fromhoney.H.pyloriurease inhibitionactivityby

honeyextractswillbedoneusingureaseassaykitandthecheckerboardmethodwillbe

employed to assess extracts for synergy. Lineweaver-Burk plots will be used to

determine Michaelis-Menten constants. Currently we are working on the urease

inhibitionbyhoneyextractsaswellas theLiquidChromatographyMassSpectrometry

(LCMS) analysis of the South African honey extracts. On completion, this study will

revealcompoundsinhoneywithanti-H.pyloriureaseeffect.

P26

TWITCHINGMOTILITYANDDEVELOPMENTOFABIOFILMASSAYFORXylophilus

ampelinus

Nyembe,N.P.P1,2.,Petersen,Y1.,Ludidi,N.2andNdimba,B.K1,2.

1ARCInfruitec-Nietvoorbij,PrivateBagx5026,Stellenbosch,75992DepartmentofBiotechnology,UniversityoftheWesternCape,PrivateBagX17,Bellville,7535

Xylophilusampelinus,thecausalagentofbacterialblightandcankerofgrapevines,has

long been a threat in theWestern Cape table grape industry. The bacterium causes

massivedestruction in infected vineyards, causingmajor commercial problemsdue to

yield loss and reduced life expectancy of the infected vines. This study serves to

investigate the role played by bacterial motility during biofilm formation by X.

ampelinus. Bacteria can move using a number of mechanisms, two of which are

twitching and swarming motility. Twitching motility is facilitated by type IV pili (Tfp)

whereasswarmingmotilityisflagella-dependent.Transmissionelectronmicroscopywas

usedtoconfirmthepresenceofpiliandtheflagellum.X.ampelinusTfpmutantswere

constructedandused toconfirmtwitchingmotilityaswellas study the importanceof

Tfp genes biofilm formation. Subsurface twitching on agarwas observed for thewild

typecellswhereasTfpmutants twitchingmotility varied.Thegrowthconditions for in

vitrobiofilm formationweredetermined.Celladherence toglass tubesandglassslide

coverslipswerevisualisedusingcrystalvioletstaining.Biofilmformationwasmonitored

for30daysandinitialattachmentfirstobservedaftertendays,afterwhichthebiofilm

increased in density as it matured. Biofilm formation is known to play a vital role in

pathogenicity of bacteria, therefore these preliminary results will be applied to the

functional characterisation of biofilm formation in X. ampelinus. Insights on these

pathogenicity factors may provide improved strategies for plant protection and

eventuallycontrolmeasures.

P27

AssessmentofthequalityindicesoftheNahoonandEasternbeachesinEasternCape

Province,SouthAfrica

1,2EbomahKE,1,2ManiSand1,2OkohAI

1SAMRCMicrobialWaterQualityMonitoringCentre,UniversityofFortHare,Alice5700,SouthAfrica.2AppliedandEnvironmentalMicrobiologyResearchGroup(AEMREG),DepartmentofBiochemistryand

Microbiology,UniversityofFortHare,Alice5700,SouthAfrica.

Weevaluated thequality indicesof thebeachwaterofNahoonandEasternbeaches.

Elevensamplingsites,alongthebeacheswereselectedincludingsixforNahoonbeach

anditsandfiveforEasternbeach.Beachwatersampleswerecollectedbi-monthly,over

a period of twelve months (September 2014 to August 2015) and analysed using

standard procedure. The physicochemical qualities of theNahoon beachwaterswere

obtained as follows: turbidity (4 – 31 NTU); temperature (18 – 25 °C), pH (7 - 8),

electricalconductivity (10.1–70.1µS/cm),andtotaldissolvedsolids(6.4–46.1mg/l);

whereasEasternbeachsitehad:turbidity(2–30NTU);temperature(18–25°C),pH(7-

8), electrical conductivity (10.1 – 67.1 µS/cm), and total dissolved solids (7.4 – 49.5

mg/l).MicrobiologicalqualitiesforNahoonbeachfollowtheorder:faecalcoliform(101–

104CFU/100ml),Escherichiacoli(101–104CFU/100ml),Enterococcus(101–102CFU/100

ml);whilemicrobiologicalqualitiesforEasternbeach:faecalcoliform(101–104CFU/100

ml), Escherichia coli (100 –103CFU/100ml), Enterococcus (101 –103 CFU/100 ml). Our

findingssuggeststhatthequalitiesoftheNahoonandEasternbeachwatersfellshortof

thesetguidelinesforrecreationalwaterqualityandthusconstitutepublichealthriskto

beachusersandswimmers.

P28

SCREENINGOFCMM6ANDAMM2TRANSGENICCASSAVALINESFORRESISTANCETO

CASSAVAMOSAICDISEASE

1ReyM.E.C.,1MvududuD.and1MoraloM.

1SchoolofMolecularandCellBiology,Universityof theWitwatersrand, Johannesburg,

SouthAfrica

With growth in population and climate change, cassava can provide one solution for

foodsecurityandasourceofstarchforindustrialusesandbiofuelsinSouthAfrica,and

othercountriesintheSADCregion.Oneofthesevereconstraintsoncassavaproduction

is cassava infecting begomoviruses (CBV), including African cassava mosaic virus

(ACMV).CBVisresponsibleforsignificantyieldlossofthestarchytubers.Theaimofthis

research is todevelop virus resistance through genetic engineering (GE) of cassava to

express hairpin RNA (hpRNA) silencing constructs against CBV. Our laboratory has

developed CMM6 and AMM2 transgenic lines transformed with a stacked ACMV hp

transgene targeting 4 ORFs namely AC1(Rep); AC4 silencing suppressor; and the

overlapping region of the transcriptional and replication enhancers AC2/AC3, the

difference between the two being that basemutationswere introduced to the sense

arm of AMM2. CMM6 and AMM2 cassava cv. 60444 lines were subjected to

reproducible trials, and evaluated for response to virus challenge. AMM2 and CMM6

transgeniclineswereselectedandmonitoredat14,35and56dayspostinfection(dpi)

for symptomdevelopment,plantgrowthandviral load. FromtheCMM6trial3 lines,

CMM6lines2,6and10showedlowersymptomscoresandlowerviralloads,compared

to non-transgenic controls,which is associatedwith durable resistance/tolerance. The

sixAMM2lineschosenhadrelativelylowersymptoms(tolerance)comparedtothewild

type cv.60444 but there were no lines showing total resistance compared to our

resistant control line dsAC1. These lines could be of interest to farmers but more

potentialresistantortolerantlineswillbeselectedforfurtherexpandedtrials.

P29

ScreeningofEndophyticBacteriatowardstheDevelopmentofCottageIndustry:Anin

VitroStudy

LubanzaNgoma*,KeneilweMogatlanyaneandOlubukolaOlurantiBabalola

DepartmentofBiologicalSciences,SchoolofEnvironmentalandHealthSciences,FacultyofAgriculture,

ScienceandTechnology,North-WestUniversity,MafikengCampus,PrivateBagX2046,Mmabatho2735,

SouthAfrica

Discoveryofnoveltechnologywhichusebeneficialendophyticbacteriaassociatedwith

therootofSorghumbicolor,Spinaciaoleracea,andZeamayswasresearched.Totalof

23endophyticbacteriawerecharacterizedonthebasisbiochemicalanalysisandplant

growth-promotingtraits.ResultsshowedthatGramnegative(60.8%)wereisolatedmore

frequently thanGram-positivebacteria (39.1%).Approximately65.2%weremotileand

the remaining 34.7% were non-motile. Eleven isolates were able to produce indole

acetic(IAA)(0.15-2.84mgl-1).Elevenisolatesshowedtheabilitytoproduceammonium.

Hydrogencyanide(HCN)productionwasobservedin10isolates.Itwasobservedthat16

isolatessolubilized insolublephosphates inPikovskyaplates (8-60.5%).All the isolates

testedwereactiveagainstFusariumoxysporum.Therefore,followingthesetests itcan

beconcludedthat11 isolatesexhibiteddifferencesandtheyweresubjectedtopartial

16S-rDNA gene sequencing using polymerase chain reaction for phylogenetic analysis.

MEGA5.10packagewasusedto identifythefollowingisolatedbacteria:Pseudomonas

sp. (KC010520), Ochrobactrum intermedium (KC010521), Ochrobactrum intermedium

(KC010522), Ochrobactrum anthropi (KC010523), Ochrobactrum anthropi (KC010524)

OchrobactrumspTOA62,andOchrobactrumspTOA64. InoculationofZeamaysseeds

withtheidentifiedbacterialshowedagoodlevelofgermination(66%)comparedtothe

control(44%).

P30

DETERMINATIONOFFACTORSTHATINFLUENCEREPRODUCTIVECONDITIONSINCOWS

INTHERURALFARMSOFTHENGAKAMODIRIMOLEMADISTRICTOFTHENORTH

WESTPROVINCE

Molefe,K.1,Tshitshi,L.2andMwanza,M2

DepartmentofAnimalHealth,FacultyofAgricultureandTechnology,MafikengCampus,NorthWest

University,PrivateBagX2046Mmabatho2735,SouthAfrica

Reproductivedisordersincommunalfarmingremainanswerabletoeconomiclosesand

poor reproductive performance. The aim of this study was to identify factors that

influence the prevalence of reproductive conditions in cattle in the semi-arid area of

Ngaka Modiri Molema district, North West Province. This study focused on five

reproductive conditions: downer cow syndrome, dystocia, abortion, retained placenta

andvaginalprolapses.Questionnaireswereusedtocollectdatafrom65farmersduring

farmvisitsandatcommunityoutreaches.Resultsobtainedfromthesurveyshowedthat

among 65 cases of reproductive conditions encountered in this study dystocia (26.2

percent), retained placentas (23.1 percent), abortion (23.1 percent), downer cow

syndrome(20percent)andvaginalprolapses(7.7percent).Theresultsalsoindicatethe

following probabilities: body condition score (P=0.37), breed type (P=0.025), parity

(P=0.54),treatmentgiventothecow(P=0.68),cowssupplemented(P=0.21)andmedical

history (P=0.58). The conditionmost encountered in this study was dystocia and the

difference in the breed type showed to be very influential in the prevalence of these

conditions.There isaneedto implementsustainablestrategiesto improveproduction

and educate the farmer on methods that can reduce the incidences of reproductive

conditions.

P31

PROFILINGOFUNKNOWNBACTERIALCULTURESISOLATEDFROMTISSUECULTURE

PLANTINGSUSINGPHENOTYPICMICROARRAY

Naidoo,M.,Mchunu,N.P.andJuglal,S.

DepartmentofBiotechnologyandFoodTechnology,FacultyofAppliedSciences,DurbanUniversityof

Technology,P.O.Box1334,Durban4001,SouthAfrica.

FungalplantpathogenscauseconsiderabledecreasesincropyieldwithFusariumbeing

thepredominantcontaminant.Sugarcaneproduction,duetoitsnature,issusceptibleto

FusariumcontaminationthustheabilitytodistinguishbetweenvariousFusariumspecies

maybevaluableinthecontroloffungi incrops.Phenotypicprofilingisausefultool in

analysing biochemical characteristics of an organismwhich includesmetabolic activity

(carbon, nitrogen, phosphorus sources), physiological conditions (osmotic and pH

conditions)andantimicrobial resistance. If strainsof these fungalplantpathogensare

profiled,thesefindingswillbevaluableforapplicationsinthebiocontrolofcropsinthe

agricultural sector inSouthAfrica.Therefore, this studyprofiled theFusarium species,

isolatedfromsugarcane,toprovidemoreinformationthatmaybeofvalueinsugarcane

disease control. Fungal cultures collected from the South African Sugar Research

Institute, were screened usingmorphological andmicroscopy analysis. The suspected

Fusarium isolateswere identifiedusing18SrRNAandBiolog identificationsystem.The

18S rRNA and Biolog confirmed that most belonged to the Fusarium genus and two

specieswereusedforfurtheranalysisbyusingphenotypicmicroarrays.Differentplates

wereusedincluding,PM1plates(Carbonsources),PM3plates(Nitrogensources)and

PM 21 and PM 22 plates (antimicrobial compounds). Preliminary results showed that

those isolated, although isolated from a similar source and environmental conditions,

theydifferconsiderableintheirmetabolicprofiles.

P32

INVESTIGATINGBIOSURFACTANTPRODUCTIONBYSPONGE-ASSOCIATEDBACTERIA

ANDASSESSINGTHEIRPOTENTIALANTI-BIOFILMACTIVITY

Sibisi,S.1andChenia,H.Y.

DisciplineofMicrobiology,SchoolofLifeSciences,UniversityofKwaZulu-Natal(Westvillecampus),Private

BagX5400,Durban,4001,SouthAfrica.

Biofilm formation is a critical problem in human clinical infections since biofilm-

associated bacteria demonstrate increased tolerance to antimicrobial agents. Current

biofilm inhibition strategies have limited efficacy, thus biosurfactants are being

investigatedaspromisingbiofilm-dispersingagents.Theaimofthepresentstudywasto

identifybiosurfactantproductionandnovelanti-biofilmcompoundsfrommarinesponge

bacteria. Sponge-associated bacteria (SAB; n = 100), isolated from 7 South African

marine sponges, were screened for biosurfactant production using classical

biosurfactantscreeningmethods:hemolysis, lipaseproduction,dropcollapse,bacterial

adhesiontohydrocarbonandemulsificationactivityassays.Crudebiosurfactantextracts

wereobtainedbysubmergedfermentation,acidprecipitationandchloroform/methanol

extraction.Anti-biofilmactivityofselectedbiosurfactant-producingisolateextractswere

evaluated, using microtitre plate assays, against biofilm-forming Pseudomonas

aeruginosaATCC 27853, and Staphylococcus aureusATCC 43300. α and β haemolysis

activity was observed for 86.59% of SAB, ranging from 10 - 55 mm. Using the drop

collapse assay, 60.25%of the isolates showedoil collapse activity,which ranged from

partialtostrongcollapse.Marineisolateswerealsocapableofdegradingtributyrinand

19% of isolates showed activity on chromogenic plates with yellow zones indicating

liberated fattyacidsandthus lipaseproduction.SelectedSAB isolateswerecapableof

inhibiting initial adhesion and/or promoting biofilm detachment. These biosurfactants

from SAB are promising for the development of novel antifouling agentswith diverse

potentialindustrialandbiomedicalapplications.

P33

IDENTIFICATIONOFEFFLUXPUMPACTIVITYINFISH-ASSOCIATEDFlavobacteriumSPP.

ISOLATES

Shaikh.N.A.andCheniaH.Y.

DepartmentofMicrobiology,SchooloflifeSciences,UniversityofKwaZulu-Natal,PrivateBagX54001,

Durban4001,SouthAfrica.

Fish pathogens, belonging to the genera Flavobacterium, cause opportunistic human

infections, as well as being responsible for large economic losses in the aquaculture

industry worldwide. Inmultidrug-resistant (MDR) bacteria, efflux pumps that extrude

unrelated antibiotics are over-expressed before antimicrobial agents reach their

specified targets. There is limited information regarding the presence and activity of

efflux pumps in resistant fish-pathogenic bacteria of the genus Flavobacterium.

Screeningof effluxpumppresencewas carriedoutusing theethidiumbromide (EtBr)

cartwheelmethod.IsolatesdemonstratinggrowthonagarcontainingEtBrconcentration

rangeof0 to2.5mg/l, at thehighest respective concentrations,withno fluorescence

were indicative of efflux pump presence. The presence of different genes involved in

effluxpumpfmeABCwasdeterminedbyPCR.Theactivationofeffluxpumpgeneswas

studiedusingbrothmicrodilutionassayagainstthreeantimicrobialagentsinpresenceof

effluxpumpinhibitors(EPIs).Outof38isolatesand9typestrains,25isolatesand7type

strainsshowedthepresenceofeffluxpumpgenes.FmeA,FmeBandTonB2geneswere

prevalent in most of the isolates. A decrease in chloramphenicol MIC values was

observedinthepresenceofEPIs,highlightingeffluxpumpactivity.Thehighestreduction

inMIC values, a 16-fold decrease, was observed against erythromycin in presence of

PaβN.Thissuggeststhattheisolateswhichareresistanttoantimicrobialagentscanbe

susceptible when EPIs are used, as they block efflux pump activity, which results in

decreasedMICvalues.

P34

LIPOPHILICEXTRACTIVEPROFILESOFEUCALYPTUSSPECIESUSEDINDISSOLVINGPULP

PRODUCTIONINSOUTHAFRICA:EFFECTSOFSTORAGE

RamnathL.,SitholeB.andGovindenR.

DisciplineofMicrobiology,DepartmentofBiotechnology,SchoolofLifeSciences,UniversityofKwaZulu-

Natal,UniversityRoad,PrivateBagX5400,Durban,4000,SouthAfrica

Lipophilic extractives are responsible for the formation of pitch deposits during the

productionofpulpandpaperandresultinpoorpulpquality,gummingupofmachinery

and loss of millions of rands in revenue every year. The lipophilic extractives of

Eucalyptusspecies,E.grandis,E.nitens,E.dunniisitequality3(SQ3)andE.dunniiSQ4

were evaluated. Hot water and solvent extractions were carried out on fresh wood

samplesandafterstorageat-20°Cforanextendedperiodoftime.PyrolysisGC-MSwas

alsoperformedtodeterminelipophiliccontentofbeforeandaftersolventextractionof

woodsamplesstoredatroomtemperature.Ageneralincreaseinhotwaterextractives

anddecrease insolventextractiveswasobservedafterstorageat -20°C.E.dunnii SQ4

presentedwithahigheramountofhotwaterextractives(11.2%)andloweramountof

solvent extractives (0.1%) compared to SQ3 (7.5% and 0.9%, respectively). E. grandis

containedsignificantamountsofhighermolecularweight lipophilicextractivessuchas

hexadecanoicandoctadecanoicfattyacidsbeforesolventextraction.Thesefattyacids

are indicativeofpolymerised lipids.Polymerisation isthecauseof incompleteremoval

oflipidsbyextraction.Resultsindicatethatsampleswerenotfreshandtheextractives

had disappeared or transformed into higher molecular weight lipids. Surprisingly,

storageofwoodchipsamplesat-20°hadthesameeffectasthetraditionalmethodof

seasoningusedtoreduceof lipophilicextractives.Thesedataindicatethatstorageat-

20° is not effective for the preservation of woodchip chemical composition andmay

impactanalyticaltestresults.

P35

PHYSICOCHEMICALANDMICROBIOLOGICALQUALITIESOFWATERANDSOILSAMPLES

FROMGROUNDNUTOILPRODUCINGINDUSTRY

OsunlaC.A1,2,AdejoroD.O2,BelloA2.andOkohA.I1

1SAMRCMicrobialWater QualityMonitoring Centre, University of Fort Hare, Private Bag X1314, Alice

5700,SouthAfrica.2DepartmentofMicrobiology,AdekunleAjasinUniversity,AkungbaAkoko,OndoState,Nigeria

Triplicate tap and wastewater samples collected from a groundnut oil producing

industryinAkuremetropolis,SouthwestNigeriawereanalyzedforphysicochemicaland

microbiological qualities using standard procedure. Adjoining soil samples were

aseptically collectedand serially diluted for total coliformcountsusingmostprobable

number method. The physicochemical qualities of the tap water and wastewater

samples ranged as follow: pH (5.7-6.8), colour (4.98-90.0 HU), nitrite (1.6-20.0mg/l),

alkalinity(0.02-52.0mg/l),BOD(1.79-2.87mg/l)andTDS(1.59-33.0mg/l);whileforthe

soilsamplesthephysicochemicalqualitieswereasfollows(asmeans)pH(6.7),alkalinity

(32mg/l),nitrate(9.98mg/l),phosphate(15mg/l)andtotalorganiccarbon(2.74mg/l).

Totalcoliformsrangedintheorderof104to105cfu/mlfortapwater,104to105cfu/g

forthesoilsamples.Overall,thebacterialpathogensrecoveredfromthewaterandsoil

samplesinclude:Escherichiacoli,Klebsiellaspp,Pseudomonasspp,Bacillusspp,Proteus

spp, Shigella spp, Enterobacter spp, Staphylococcus aureus, Streptococcus spp and

Salmonella spp. Conclusively, high bacterial pathogens indicate poor sanitary and

unhygienic practices in the environment, and consequently pose risk to the industrial

productandthereceivingwaterbodies.

P36

STRUCTURALCHARACTERIZATIONOFTHECELL-TO-CELLMOVEMENTAND

REPLICATIONPROTEINSOFSOUTHAFRICANCASSAVAMOSAICVIRUS

Ayres,F.,Nankoo,N.,andRey,M.E.C1.

1SchoolofMolecularandCellBiology,UniversityoftheWitwatersrand,PrivateBag3,Wits,2050.

South African cassavamosaic virus (SACMV) is a bipartite begomovirus which causes

cassava mosaic disease. DNA-A encodes six genes while DNA-B encodes two. BC1 is

locatedonDNA-Banddefinedasthemovementprotein(MP)asitprovidescell-to-cell

movementofthevirus.Rep(replicationassociatedprotein),locatedonDNA-A,iscrucial

forviralreplication,butalsomanipulatesmultiplehostcellpathwaysbyinteractingwith

host proteins. It is therefore crucial to fully understand both the structures and

functionalityoftheMPandRepaswellasidentifyhost-interactingproteinsinorderto

develop strategies to limit or eliminate SACMVmovement, once the virus has gained

entry into the plant. As limited structural and physiochemical information is known

aboutBC1andRepofSACMV,theproteinstructuresneedtobeelucidated.Inorderto

successfullycharacterizetheRepandMPproteins,theORFsoftheproteinsofinterest

were cloned into the expression vector pET-32a, and co-transformed into BL21 (DE3)

withchaperonecontainingvectors.Expressionandcharacterizationofbothproteinswill

be performed and pI and structural features determined using 2D-PAGE, circular

dichroism(CD)andtryptophanfluorescence.Molecularweightwillbeconfirmedusing

sizeexclusionhighperformanceliquidchromatography(SE-HPLC).Bytheuseof2’(3’)-N-

methylanthraniloyl-ATP (MANT-ATP), ATP binding activity of Rep and BC1 will be

measured, and ligand binding pocket will be exposed through the use of 1-anilino-8-

naphthalenesulfonate(ANS).

P37

ASSESSINGTHEINFLUENCEOFMININGACTIVITIESONTHEBIO-PHYSICOCHEMICAL

QUALITYOFSURFACEANDGROUNDWATERFORHUMANUSEINTHETUBATSE

MUNICIPALITY

MathipaM.M,MasokoP.andMoganediK.L.M

Department of Biochemistry, Microbiology and Biotechnology, Faculty of Science and Agriculture,

UniversityofLimpopo,PrivatebagX1106,Sovenga,0727,SouthAfrica,

Humanactivitiessuchasminingcontaminatethewatersourcesonwhichwealldepend.Furthermore, the extent of contamination is not strictly limited to the vicinity of themines; contaminatedmaterialsmay be physically remobilized in high flow conditions.Thecurrentstudyinvestigatedtheinfluenceofminingactivitiesonthequalityofsurfaceand ground waters. The bio-physiochemical properties of ground and surface waterwere determined. The bacteriological analyses included hetrotrophic bacteria, totalcoliformsandfaecalcoliform.ThebacterialcontaminantsidentifiedincludedAeromonashydrophila, Pseudomonas putida Pseudomonas luteola, Cronobacter sakazakii,Acinetobacterhaemolyticus,Enterobactersakazakii,Pantoeaspp,Enterobactercloacae,Seratia odifera, Kocuria rocea, Streptococcus thoratens, Streptococcus agalactiae,Acinetobacterbaumanni,BordetellasppandAcinetobactercalcaceticus.Physicalqualityofall the surfaceandgroundwater sampleswere found tobecompliantwith the setstandards fordrinkingwaterbasedon theparameters: turbidity, colour,hardnessandTSS,withtheexceptionoftheHcyminestream.TheconcentrationsofZn,[SCN-]Cr,Co,Fe, Ni, Cu, SO4, H2O2, Cl2 in surface and ground waters from the Tubatse area weredetermined. The concentrations of Zn, Fe and Co detected in the water samplesexceededthenormalexpectedconcentrationsof<3.5μg/L,0.5mg/L,<0.01mg/Land<5μg/Lrespectively,Thesedimentsandsoilsamplesweredigestedwithaquaregia forCu,Cr,Fe,CoandZnanalysis.Themetalconcentrations inthesedimentswerehighinthe wet season than in the dry season. Furthermore, themetal concentrations werehigher insedimentsthanthesoilsamplescollectednearthestreams.WhentestedforcytotoxicityonC2C12cells,thelowestdetectedconcentrationofmetalsintherangesof0.5mg/Lto10mg/Lwereshowntoreducecellviabilityby25%after24hrsofexposure.Metal and chemical pollutants were more pronounced in water streams that werecreatedbywater from themining sites. Further, the profile of surfacewater bacteriahad shifted from common surface water flora to mostly pathogenic bacteria due tohumanandanimalactivitywhichincludedbathing,washingandanimaldrinking.MininghasanegativeimpactonthewaterbodiesintheTubatsemunicipality.

P38

MODELSONTHEKINETICSOFPHENOLUTILIZATIONINAMMONIUMPHOSPHATE

SUPPLEMENTEDPHENOLLADENREFINERYEFFLUENT

*CharlesEmekaObiukwu

DepartmentofMicrobiology,ImoStateUniversity,Owerri,Nigeria.

Growth of mixed cultures of phenol utilizers which included Pseudomonas sp.tww ,

Bacillus sp.rww., Citrobacter sp.tww, Klebsiella sp.rbww and Staphylococcus sp.rww

weremonitoredinbatchculturesofphenolladenrefineryeffluentssupplementedwith

differentconcentrationsofammoniumphosphate.Thenutrientsupplement(NH4)3PO4)

encouraged cell growth and phenol reduction in a concentration dependent manner

with optimum values observed at higher concentrations. After 21 days of treatment,

(NH4)3PO4)concentrationof0.05%,0.2%,0.5%and1.0%reducedphenolconcentration

of 103ppm to 4.4%, 3.7%, 0.88% and 0.83% respectively with a corresponding cell

growth of 6.3X109, 1.1X1010, 3.0X1010 and 2.6X1010 cfu/ml respectively. As phenol

concentration increased beyond 203ppm, cell growth and phenol reduction rate

decreasedatall levelsofnutrientconcentration. Intheeffluenttreatmentcontrol,the

inoculatedprocesswastewatershowednoappreciablecellgrowthorphenolreduction

at51.65ppmofphenol.Phenolconcentrationof103ppmwasreducedto50.8%witha

maximumcellgrowthof9.8X105cfu/mlafter28days.Theuninoculatedprocesswaste

watershowednocellgrowthorphenolreduction.Theexperimentaldataobtainedfrom

this study was modelled with the first order differential equation (dx/dt = ±µx)

describingtherateofdepletionofphenol(dphenI/d(time)=µpphenI)andgrowthof

biomassd(THCI)/d(time)=µtTHCI)forthedifferentnutrientconcentrations.Ataknown

(NH4)3PO4) concentration and time, phenol depletion and biomass growth rate was

predictedusingthemodel.

P39

PHYSIOLOGICALRESPONSEOFPERIOPHTHALMUSPAPILLIOASBIOMARKEROF

AQUATICECOSYSTEMPOLLUTION

*CharlesEmekaObiukwu

DepartmentofMicrobiology,ImoStateUniversity,Owerri,Nigeria.

The physiological and biochemical responses of a benthophagous organism –

Periophthalmuspapillio (mudskipper fish) to refineryeffluentpollutionwasmonitored

and used as a biomarker of environmental stress. The liver weight of the fish from

effluent polluted site was significantly higher (95% confidence limit) than those from

unpolluted site. General statistical analysis revealed a significant difference in

haemoglobin level,whitebloodcell count (WBC)andpackedcell volume (PCV)of the

fish from polluted and unpolluted sites. The activity of Aspartate aminotransferase

(AST), Alanine aminotransferase (ALT) and Alkaline phosphatase (ALP) in fish from

effluentpollutedOkrikariverdifferedsignificantly(95%)fromthoseobtainedfromthe

unpolluted Elechi creek. Similarly, there was a significantly higher activity (95%) of

phenol degrading enzymes in the liver extracts of fish from effluent polluted sites

compared to those from unpolluted sites. The elevated levels of these biochemical

indices in the fish from refinery effluent polluted Okrika river suggest that constant

dischargesoftheeffluentintotheaquaticenvironmenthavesignificantadverseeffects

ontheaquaticbiota.

P40

In-vitroANTIMICROBIALSTUDIESOFLOCALNIGERIANSPICESAGAINSTENTERIC

PATHOGENS

OBIUKWU,C.E.andNWANEKWU,K.E

DepartmentofMicrobiology,FacultyofScience,ImostateUniversity,Owerri,Imostate.

The in-vitro antimicrobial activities of five local Nigerian spices-Monodora myristica,

Tetrapleura tetraptera, Zingiber officinale, Piper guineense and Xylopia aethiopica

against enteric pathogens- Escherichia coli, Salmonella typhi, Shigella flexneri and

Staphylococcusaureuswereinvestigated.Theethanolextractshowedvarieddegreeof

activitywith zones of inhibition ranging from10mm to asmuch as 26mm for various

organisms. The minimum inhibitory concentration (MIC) and minimum bacteriocidal

concentrations(MBC)werealsodeterminedusingthebrothdilutionmethod.TheMICs

ranged from62.5µg/ml to >1000µg/ml andMBCs values in the range of 500µg/ml to

1000µg/mlfortheplantextracts.ThecrudeextractofT.tetrapterawastheleastactive,

having inhibitoryeffectsonlyagainsttwoorganismsE.coliandS.aureus.P.guineense

recorded the strongest antimicrobial activity as shown by the largest diameter of the

zonesofinhibitionexhibitedandlowestMICvalues.

P41

COMPARISONOFTHEMLSTTYPINGANDCCM-PCRASSAYWORKFLOWSFORTHE

DETERMINATIONOFTHESEQUENCETYPESOFStaphylococcusaureusISOLATES

AdelowotanAO1,3,KockMM1,2,GoolamMahomedT1andEhlersMM1,2

1DepartmentofMedicalMicrobiology,UniversityofPretoria;2NationalHealthLaboratoryService3DepartmentofMicrobiology,UniversityofLagos

The knowledge of sequence typing (ST) provides a global epidemiological profile of

bacterialpathogens. Multi locussequencetyping(MLST)wasdesignedtocharacterise

bacteria including Staphylococcus aureus on the basis of ST. The MLST method is,

however, an expensive method with a longer work flow and result interpretation

requirestraining.TheclonalcomplexM-PCR(CCM-PCR)assayisanovel,cheaperand

faster assay designed to detect the major clonal complexes (CC) of MRSA with less

trainingandhigherdiscriminatorypowerthantheMLST.Resource-limitedlaboratories

suchasfoundinAfricamayneedthisalternativeassay.Thisstudyaimstocomparethe

work flow and discriminatory power of CC M-PCR and MLST assays for the

determinationofthesequencetypesofselectedclinicalS.aureus isolatesfromNigeria

and South Africa. The STs of selected clinical S. aureus pulsotypes from Nigeria and

South Africa were initially determined using the standard MLST protocol in our

laboratory. IsolateswerethenscreenedwiththeCCM-PCRassay. Cost,durationand

easeofworkflowwererecordedandthediscriminatorypowerwascalculatedusingthe

Simpson’sindexofdiversity,basedontheSTsdetectedbythetwomethods.TheCCM-

PCRassaywaseasier,cheaperandfaster(5h)thanMLST(5to7days). Anadditional

costofR1250perisolatewasincurredforsequencingusingMLST.Thediscriminatory

power of the CCM-PCR andMLST assaywas 0.889 for the SouthAfrican isolates but

0.733and0.833respectivelyfortheNigerianisolates.RapidandcosteffectiveCCM-PCR

assaymaybeanalternativetoMLSTinroutineaswellasresource-limitedlaboratories

fordetectingthesequencetypesofS.aureus isolates,especiallyduringoutbreaksand

whenMLSTisnotaffordable.

P42

EFFECTOFTEMPERATUREANDDISTANCEONPOLIOVIRUSTITERFROM

CLINICALSPECIMENSOFACUTEFLACCIDPARALYSISCASESINNIGERIA

Idowu,A.A.1,2,ClarkeA.M.1,Akintokun,A.K.2,Adu,F.D.3,Adeniji,J.A.3andAjuwon,B.O.3

1DepartmentsofBiochemistryandMicrobiology,UniversityofFortHare,SouthAfrica2DepartmentofMicrobiology,UniversityofAgriculture,Abeokuta,Nigeria3WHOPolioLaboratory,DepartmentofVirology,CollegeofMedicine,UniversityCollegeHospital (UCH),

Ibadan,Nigeria

The effect of cold box temperature and distance on virus titers, poliovirus isolation

rate, and appearance of orphan polioviruses was investigated. Cold boxes with

stools were randomly selected and examined for internal temperature over a 7-

month period. After virus isolation inRhabdomyosarcoma (RD) cell lineRDand L20B,

titer calculations and intratypicdifferentiationwere done on isolates. Sequencing and

molecular studieswere done on the isolates periodically in theorderofarrival in the

laboratory foraperiodof30months.Seventy-one (51.1%)boxeshad the temperature

rangeof 1 –4◦C, 53 (38.1%)had 4.5 –8◦C,while 15 (10.8%)had temperaturebetween

8.5◦Cand17.0◦C.Polioviruswasisolatedfrom24(8.6%)specimensmadeupof13wild1

and 2 and 11 Sabins 1, 2, 3 with titers between 101.8 and 105.4 TCID50/100μl.

Temperature and titer were inversely proportional and statistically significant. (r =

−0.83, P < 0.05). Distance to laboratory was not significantly related (r =−0.025) to

temperature when appropriate cold box temperaturewasmaintained. Of the 18,188

acute flaccid paralysis (AFP) specimens received in the laboratory between June

2008andDecember2010,1,752 poliovirus isolates (9.6%)consisting of 480wild and

82 orphans were found. A positive correlation between the distance and orphan

viruses (r = 0.425; P = 0.027) was observed. While poliovirus titer depends on the

inside temperature of the cold box, distance to the laboratory was found to be a

predisposingfactortotheappearanceoforphanviruses.

P43

GROUPBSTREPTOCOCCUSCOLONIZATIONINPREGNANTWOMENATDR.GEORGE

MUKHARIHOSPITAL,SOUTHAFRICA

Monyama,M.C1,Bolukaoto,Y.J1,Chukwu,O.M1,Maloba,M.R.B2.Moyo,R.S3,

Nchabeleng,M2Mavenyengwa,R.T3,Lebelo,S.L1.

1DepartmentofLifeandConsumerSciences,UniversityofSouthAfrica(UNISA)Florida,SouthAfrica.2DepartmentofMicrobiologicalPathology,UniversityofLimpopo(MEDUNSA),Pretoria,SouthAfrica.3PolytechnicofNamibia,SchoolofEngineering,DepartmentofHealthSciences,Windhoek,Namibia

The aim of the study was to estimate group B streptococcus (GBS) colonization in

pregnantmothersusingselectiveenrichmentbrothandsolidmedia forculturingGBS.

Vaginalandrectal swabswerecollected from413pregnantwomenforGBScultureat

recruitmentstage.Directplatingandenrichmentbrothculturemethodswerecompared

by using the same swab samples. The swabs were cultured on colistin nalidixic agar

(CNA)plateandincubatedat37ºCandexaminedafter18-24hours.Thesampleswhich

were culture negative on a CNA agar plate were then inoculated into a Todd-Hewitt

enrichmentbrothtorecoveranyGBSpresentthatwasnotrecoveredonthesolidagar.

TheGBScolonizationrateinpregnantwomenwas30.9%(128/413).TheCNAagarplate

recovered 45.3% (58/128) of the GBS isolates whereas 54.7% (70/128) isolates were

recoveredfromTodd-Hewittbroth.Pregnantwomenofvariousageswerefoundtobe

atriskofGBScolonization.Thecolonizationratewashoweverhighestamongwomenof

25-29 age groups as compared with other age groups. Detection of group B

streptococcusimprovedwhenbothrectalandvaginalswabsarecollectedforlaboratory

analysis.ThesimultaneoususeofTodd-HewittbrothandCNAplatealso improvedthe

yieldofgroupBstreptococcus.

P44

Thesynergisticeffectsofn-hexanefractionofParkiabiglobosa(Jacq.)barkextractand

selectedstandardantibioticsonbacterialisolates.

Abioye,OE.1,2,AkinpeluDA.,2andOkoh,AI.1

1SAMRCMicrobialWaterQualityMonitoringCentre,UniversityofFortHare,PrivateBagX314,Alice57002DepartmentofMicrobiologyObafemiAwolowoUniversity,Ile-Ife,OsunState,Nigeria.

The incidence of resistance to the commonly used antimicrobial agents by microbial

pathogensdemands increasedeffort in thedevelopmentof effectivewaysof treating

infectionsanddiseases.An-hexanefractionof lyophilizedcrudebarkextractofParkia

biglobosa(Jacq.)wasprepared,andincombinationwithselectedantibioticsassayedfor

antimicrobial activity against some selected bacterial pathogens using Time-Kill assay.

ProteinleakageanalysisofthecombinedagentswasperformedusingBradfordprotein

assay.Determinationof active compoundspresent in then-hexane fractionwasdone

using Fourier Transform Infrared Spectroscopy (FTIR). While time-kill assay detected

43.33%synergy;56.67% indifferenceandnoantagonismat½ xMIC,1 xMICexhibited

55% synergy; 45% indifference and no antagonism. Protein leakages from the cells of

selectedbacteriarangedfrom1.20to256.93µg/ml.Presenceofphenylgroup,aromatic

ring and phenolic compounds in n-hexane fraction was confirmed at 2162-2020cm-1,

1605-1533cm-1and1438-1444cm-1spectrapeaksrespectively.Theobservedantibiotic-

n-hexane fraction synergistic interaction revealed improved antibacterial activity of

selected antibiotics. Hence, exploration of antibiotic-plant secondary metabolite

combination isherebyadvocated in theglobalquest forcombating infectiousdiseases

causedbymulti-drugresistantpathogens.

P45

MICROBIOLOGICALASSESSMENTOFAIRINSOMESELECTEDSAWMILLAND

FURNITUREFACTORYENVIRONMENTSINOSUNSTATE,SOUTHWESTERNNIGERIA

FadareTO.1,2,OluduroAO.2andOkohAI.1

1SAMRCMicrobialWaterQualityMonitoringCentre,UniversityofFortHare,PrivateBagX1314,Alice

5700,SouthAfrica.2DepartmentofMicrobiology,ObafemiAwolowoUniversity,Ile-Ife,OsunState,Nigeria.

We assessed themicrobiological quality of air in five sawmills and furniture factories

each, in Ikire, Ilesha, Modakeke, Ile-Ife and Osogbo environments of Osun State,

Southwestern Nigeria. Duplicate samples were collected using a button sampler,

enumeratedforheterotrophicplatetotalcountsusingstandardprocedureandprofiled

for their antibiogram. The predominant microorganisms in the study areas include

Acinetobacter sp, Arthrobacter sp. Alcaligenes sp, Aspergillus sp, Penicillium sp and

yeasts, with significant differences observed in the counts of sawmills and furniture

factories(P<0.05).Ofallthesampledsites,thehighestGramnegativebacteriacounts

was 2.62 ± 0.01 log cfu/m3 at sawmill D in Ikire and the lowest as 1.84 ± 0.02 log

cfu/m3at sawmill A in Ile- Ife. Actinomycetes population ranged from 0.69 ± 0.02 log

cfu/m3infurniturefactoriesFFAinIle-IfeandIkireto1.24±0.06logcfu/m3ofSawmillD

inIkirewhereasfungipopulationrangedfrom0.85±0.04logcfu/m3FFCofModakeke

to 1.54 ± 0.06 log cfu/m3in sawmill B of Osogbo.All the isolateswere susceptible to

nalidixic acid and ofloxacin, and high susceptibility to gentamycin (82%). Conversely,

they were variously resistant as follows: amoxicillin (70%), cotrimoxazole (35%),

tetracycline(25%)andnitrofuratoin(25%).Bacteriaandfungirecoveredfromthisstudy

indicatehighproliferationofmicrobialpathogenspresentaroundsawmillsandfurniture

factoriesandconsequentlymayposeahealthrisktowoodworkersinthestate.

P46

ESSENTIALOILCONSTITUENTSANDINVITROANTIMICROBIALACTIVITYOFTHEROOT

OFMondiawhitei(HOOK.F.)SKEELS

Gbadamosi,I.T.1,Aboaba,S.A.2andTitilawo,O.Y.3

1Department of Botany, University of Ibadan, Nigeria. 2Department of Chemistry, University of Ibadan,

Nigeria.3DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,SouthAfrica.

TherootofMondiawhitei(Hook.f.)Skeelsisusedforthetreatmentofgastro-intestinal

disorders, sexually transmitted infections, malaria, and asthma and as aphrodisiac in

Nigeria. Inthisstudy, thecompositionoftheessentialoilof therootofM.whiteiwas

analysed by GC/MS. The agar-well diffusion techniquewas used for the antimicrobial

assayof theoilagainstninepathogenicorganismsviz.Bacilluscereus;Escherichiacoli;

Candida albicans; Klebsiella pneumoniae; Pseudomonas aeruginosa; Proteus mirabilis;

Salmonella typhi; Staphylococccus aureus; and Streptococcus pyogenes. Twenty-eight

compounds representing 99.92% of the essential oil were characterised. The major

constituents of the oil were (E)-2-Hexen-1-ol (25.96%); Heptacosane (20.94%); Phytol

(15.60%); 1-Hexanal (8.94%); (E)-2-Hexenal (4.29%) and 2-Hydroxy-p-anisaldehyde

(4.21%). At 106 cfu/ml inoculum concentration, the oil was most active against

Escherichia coli (50.0mm) and Staphylococccus aureus (50.0mm) and least active on

Candida albicans (15.0 mm). The observed antimicrobial activity justifies the

ethnomedicinalusesofM.whiteiinNigeria.

P47

TheroleofdesertinsectmicrobialsymbiontsontheCarbonCycle-PhDprogressreport

Franzini,P.Z.N.,Ramond,J-B.,Ronca,S.andCowan,D.A.

CentreforMicrobialEcologyandGenomics,DepartmentofGenetics,UniversityofPretoria

Plants constitute large carbon reservoirs which release stored carbon into the

environment and atmosphere through decomposition and consumption by animals.

Many insects consume plants and their gut microbial communities are considered

essential for the degradation of the highly recalcitrant plant cell wall, therefore

contributing directly to carbon cycling. Members of the genus Pachysoma feed on

differentsubstrates;i.e.,dungandplantdetritus.Thisofferstheopportunitytocompare

themicrobial community structures of two insect species feeding on substrates with

differingcompositions.TwoPachysoma spp,P.endroydii (plantdetritus feeder)andP.

striatum (dung feeder) were collected from Namaqualand, South Africa. Whole guts

from five insects of each species were dissected and metagenomic DNA extracted.

Bacterial community structurewasdeterminedby 454 sequencingof the 16S gene.A

numberofOTUs(OperationalTaxonomicUnit;2111)wascoupledwithalowdiversity.

Onlyeightphylawererepresentedwithintheplantfeedercomparedtothefiveofthe

dung feeder. Bacterial community structures of the two Pachysoma spp. differed

significantly, suggesting differences in both the resident (naturally occurring) and

transient (from the food source) communities, possibly linked to the chemical

composition of the respective food sources. The bacterial phyla Actinobacteria,

Proteobacteria and Firmicutesweremore highly representedwithin the dung feeder,

while Bacteriodetes was more dominant within the plant feeder. A high number of

unclassified OTUs at phylum level (approximately 40%) were reported for the plant

feeder. Of the most abundant OTUs, only five are known cellulolytic and xylanolytic

bacteria.Thechemicalcompositionofanimaldungandplantdetritusvarygreatlyand

correlations between diet composition and bacterial community structure are

suggested.

P48

CLONING,PURIFICATIONANDCRYSTALLISATIONOFABI-FUNCTIONALPaenibacillus

mucilaginosusEXOGLUCANASE.

Mosina,N.L.1,Schubert,W.D.2andCowan,D.A.1

1CentreforMicrobialEcologyandGenomicsandGenomicsResearchInstituteUniversityofPretoria.2DepartmentofBiochemistry,UniversityofPretoria.

The production of various bio-products including bioethanol is initiated through the

conversion of cellulose to fermentable sugars. The bioconversion of cellulose is a

complexprocessandrequiresthesynergisticactionoftheendo-β-1,4-glucanses,exo-β-

1,4-glucanases and β-glucosidases. Enzymatic hydrolysis of cellulose proves to be a

challengingtechnologicalandeconomicalstepinbioethanolproductionwhichdemands

a robust cellulasewithahighhydrolytic efficiency. Theaimof thepresent study is to

understandthestructuralandfunctionalpropertiesofanovelbi-functionalPaenibacillus

mucilaginosus exoglucanase. The exoglucanase gene was cloned into different pET

plasmidvectorsandtransformedintochemicallycompetentEscherichiacoliBL21cells.

E.coliclonesdisplayingcellulaseactivitywereusedinproteinexpressiontrials:optimal

protein expression was at 42°C with an induction period of 8 hours. The his-tagged

exoglucanase was purified by immobilised metal affinity chromatography and gel

filtration chromatography. The 127 kDa exoglucanase displayed activity on both

carboxymethyl celluloseandavicel.Using7mg/mlprotein, crystallisationexperiments

weresetupusingcommerciallyavailablescreensbythehangingdropandsittingdrop

method.Rhombohedral-likeandneedle-likecrystalswereamongthedifferenttypesof

proteincrystalsobtained.Optimisationofthecrystallisationconditionswillyield larger

three-dimensionalcrystalssuitableforX-raydiffraction.

P49

IMMUNEDAMPENINGANDREFOCUSINGOFASAT2FOOT-AND-MOUTHDISEASE

VACCINESTRAIN

Ramulongo,T.D.1,2;Rotherham,L.S.3;Opperman,Pamela1;Theron,Jacques2;Maree,

F.F.1,2

1TransboundaryAnimalDiseasesProgramme,OnderstepoortVeterinaryInstitute,AgriculturalResearchCouncil,Onderstepoort,Pretoria,SouthAfrica2DepartmentofMicrobiologyandPlantPathology,FacultyofAgriculturalandNaturalSciences,UniversityofPretoria,Pretoria,SouthAfrica3MolecularEpidemiologyandDiagnostics,OnderstepoortVeterinaryInstitute,AgriculturalResearchCouncil,Onderstepoort,Pretoria,SouthAfrica

Risk of foot-and-mouth disease virus (FMDV) spreading to new zones is a reality, as

evidencedbytherecent incursionofSAT2serotype intoNorthAfricaandMiddle-East.

The need to understand the antigenic diversity of field strains remains essential for

engineeringofbroadlyprotectivevaccinestrains.Inthisstudy,anepitopereplacement

strategywasutilisedtorefocustheantigenicityofSAT2/ZIM/7/83vaccinestraintothe

antigenically disparate SAT2/EGY/9/12 virus using reverse genetics. The antigenic

distance of the viable epitope-replaced mutant viruses were examined by virus

neutralizationassaysusingconvalescentSAT2/ZIM/7/83andSAT2/SAR/3/04antisera.A

reduction in neutralisation titrewas seen for vEGYVP1GH&CtSAT2 (residues 135-160 and

196-216inVP1)withtheSAT2/ZIM/7/83seraandmutantvEGYVP1site3SAT2(residues43-

50 of VP1) with the SAT2/SAR/3/04 sera. A significant increase in neutralisation was

seen for vEGYVP1GH&CtSAT2 with the SAT2/SAR/3/04 sera. Antigenic profiling of the

epitope-replaced viruses with SAT2-specificmonoclonal antibodies (mAbs) resulted in

mappingof twopotentialantigenicsites.Asignificant reduction in reactivitywasseen

forvEGYVP1GH&CtSAT2withmAbGD12;however,noreductionwasobservedwiththeVP1

C-terminal(196-216)replacedmutant,indicatingthatthebindingfootprintofthismAb

maybelocatedintheVP1βG-βHloopregion.Ahighlysignificantreductioninreactivity

wasseenwithmutantvEGYVP1site3SAT2;apreviouslyidentifiedantigenicfootprintwithin

VP2.Informationgainedfromthisstudywillpavethewaytowardsbetterunderstanding

of theantigenicdeterminantsof thediverseSAT2serotypeandassist in thedesignof

engineeredvaccinesforbroadprotectionofSAT2fieldstrains.

P50

SHARKLIVER,APOTENTIALSOURCEOFANTIMICROBIALAGENTS

MrwetyanaT1,PatersonA2andClarke,AM1

1DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,SouthAfrica2SouthAfricanInstituteforAquaticBiodiversity,SAIAB,SouthAfrica

Today the growing challenge of antimicrobial resistance prevents the effective

treatment of bacterial infections. Infections that were treatable with antibiotics are

considered fatal again. This has led to the increased need for developing novel

compoundsforantimicrobialuse.Withthemarineenvironmentcomprisingmorethan

71%oftheearth’ssurface,itprovidesavastarrayforthedevelopmentofpotentialdrug

candidates, however it still remains themost underutilised biological resource. In the

present study, the liver extracts of three different shark species; Dogfish, Catfish and

Hammerheadwerescreenedforantimicrobialproperties.Theliverswerehomogenised

in60%acetonitrileand1%Triflouroaceticacidandsubjectedtodifferentcentrifugations

at 4oC. The crude extracts were tested against Helicobacter pylori ATCC 43526,

Escherichia coli 0157 ATCC 35156, Staphylococcus aureus ATCC 29213, and Bacillus

cereus ATCC 10876 using the disc diffusion method. The minimum inhibitory

concentration (MIC) of the most active extracts was determined using the broth

microdilutionmethodandfractionationwasachievedusingthinlayerchromatography.

Directbioautographywasusedtoidentifycompoundswithantimicrobialactivityonthe

thin layer chromatographic plate. Two extracts from Dogfish and Catfish were active

againsttheselectedbacterialstrainsexceptH.pyloriwithclearinhibitionzones(≥8mm).

TheMICoftheactivecrudeextractswas19.2875mg\ml.Fourbandswereobservedon

thethinlayerplateswiththeRfvaluesof0.8,0.26,0.27and0.29.ThecompoundwithRf

0.29showedantimicrobialactivity.CatfishandDogfishsharkliversarepotentialsources

ofantimicrobialagents.

P51

INTRACELLULARGASBUBBLESINYEASTS

Dithebe,K.1,Pohl,C.1,Swart,H.2,Coetsee,E.2,VanWyk,P.3,Swarts,J.4,Lodolo,E.5and

Swart,C.1,3*

1UNESCO-MIRCEN: Department of Microbial, Biochemical and Food Biotechnology, 2Department of

Physics, 3Centre forMicroscopy, 4Department of Chemistry, University of the Free State, P.O. Box 339,

Bloemfontein, 9300, South Africa, 5SABLtd Brewing Centre of Excellence, PO Box 123902, Alrode 1451,

SouthAfrica.

The yeasts’ capabilities to produce increased ethanol and carbon dioxide (CO2) are

exploitedduring fermentation tomakeproducts suchas leavenedbreadandalcoholic

beverages. During fermentation yeasts vigorously release CO2 into the surrounding

medium, thus it is expected that yeast cellswouldbe filledwith gasbubbles.Despite

fermentation being well-established, there have been no reports of intracellular gas

bubbles in yeasts. This lackof reports is considered amissing link since it is not clear

what happens to the CO2 between fermentation, when it is produced, and eventual

releasefromthecells.Thestudythereforeaimedtofindthismissinglink.Inthisstudy,

Saccharomyces pastorianus and S. cerevisiae were grown on fermentable and non-

fermentable media respectively. The presence of intracellular gas bubbles was

investigated using various microscopy techniques including light microscopy,

transmission electron microscopy (TEM), and nano scanning Auger microscopy

(NanoSAM, http://en.wikipedia.org/wiki/Auger_architectomics). Light microscopy

analysis revealed a large number of light scattering granules inside cells grown in

fermentable medium. In contrast, very few light scattering granules were observed

insidecellsgrowninnon-fermentablemedium.TEManalysisconfirmedthepresenceof

intracellulargasbubbleswhichoccupyasignificantpartoffermentingyeastcells.Light

microscopy and TEM observations of gas bubbles coincide with observations of gas

vesicles in Cyanobacteria using both techniques. NanoSAM analysis revealed an

interconnectedmazeofgasbubblesinsidethecells.TEManalysisfurtherrevealedthat

intracellular gasbubbles isnotmembrane-boundand that they compress anddeform

cellorganelles.Themissinglinkhasthereforebeenuncovered.

P52

P53

EVALUATINGERIC-PCRASAMOLECULARTYPINGMETHODOFAvibacterium

paragallinarum

HellmuthJ.E.1,BoucherC.E.2,&BraggR.R.2

1DepartmentMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,205Nelson

MandelaDrive,ParkWest,Bloemfontein,9301

Avibacterium paragallinarum is the causative agent of Infectious Coryza that occurs

primarily in chickens. This disease may lead to huge economic losses by causing a

decrease in egg production of up to 45% in multi age farms. Avibacterium

paragallinarum are serologically classified into serogroups (A, B and C), as well as

serovars (A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3 and C-4) where only some of these

serovars results in clinical symptoms. Infections canbe treatedwith vaccines,but this

requiresthecorrectserologicalclassificationofthestainscausingtheinfectiontoensure

that the correct vaccines are used, as there are no cross protection between certain

serovars. Serotyping is done by hemagglutination inhibition tests, but these tests are

highly subjective. Alternative typingmethods such as molecular techniques has been

describedandtheseincludetheuseofMultiplexPCR,RFLP(RestrictionFragmentLength

Polymorphism) PCR and ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR.

Multiplex-PCRandRFLP-PCRhastheabilitytodistinguishbetweendifferentserogoups

(A,BorC)butnotbetweenserovars.DuringthisstudytheERIC-PCRwasevaluatedasa

method todistinguishbetweendifferent serovars. The resultsof this study concluded

that the banding patterns, produced during ERIC-PCR, of field isolates comparedwith

referencestrains(ofthesameserovar)showedlittletonocorrelationtoeachother.It

can thusbe concluded that theERIC-PCR test is not a suitable test for the serological

classification of field isolates of Av. paragallinarum. Therefore alternative molecular

serotyping techniques are needed for accurate diagnosis of Avibacterium

paragallinarum

P54

FINGERPRINTSANDPREVALENCEOFMULTIDRUGRESISTANTEscherichiacoli

PATHOVARSINSELECTEDSURFACEWATERSINSOUTHWESTNIGERIA

Titilawo,O.Y.1,2*,Obi,C.L.1,2andOkoh,A.O.1,2

1SA-MRCMicrobialWaterQualityMonitoringCentre,UniversityofFortHare,Alice5700,SouthAfrica.2Applied and Environmental Microbiology Research Group, Department of Biochemistry and

Microbiology,UniversityofFortHare,Alice5700,SouthAfrica.

Despite advances in knowledge, understanding andmanagement that have ensued in

recentyears,diarrhoealdiseaseremainsthemostprevalentandimportantpublichealth

threat globally, aswell as leading causeofmorbidity andmortality in thepaediatrics.

WatersamplesfromselectedriversinOsunState,Nigeriawerecollectedandanalyzed

using standard procedures. Escherichia coli isolates (n=300) were screened for 10

virulence genes using polymerase chain reaction technique for pathotyping. The

antimicrobialsusceptibilitytestingofthepathovarswasdeterminedbythediscdiffusion

method and the resistant pathovarswere elucidated for their genotypic antimicrobial

resistancedeterminants.TheETECpathovarconstituted46%oftheisolates,followedby

UPEC(17%)andEAEC(2%).Antimicrobialsusceptibilityprofilingrevealedalltheisolates

toberesistantagainstwhile63%oftheisolateswereresistantagainstampicillin(63%).

Others resistances follow the order: amoxycillin (55%), gentamycin (41%), cefuroxin

(39%),chloramphenicol(28%)andcefepime(26%).Thedetectionratesfor13resistance

determinants screened were as follows: [sulfonamides; (sulI (10%), sulII (26%)], [β-

lactams;ampC(7%);blaTEM,(13%),blaZ (15%)],[tetracyclines(tetA(3%),tetB(1%),tetC

(4%), tetD (10%)], [phenicols; (catI (4%), catII (3%),cmIA1 (2%)]and [aminoglycosides;

(aacC2 (2%)]. One way ANOVA revealed no significant difference between the

prevalence of EPEC, EAEC, ETEC, EHEC, DAEC andNMEC strains (P ˃ 0.05)whereas a

significant difference in the prevalence of UPEC was noticed at R1 only (P ˂ 0.05).

Conclusively,thefindingssignifyhighprevalenceofmultidrugresistantE.colipathovars

inthecatchment,andconsequentlyapotentialhazardtopublichealth.

P55

ESSENTIALOILCONSTITUENTSANDINVITROANTIMICROBIALACTIVITYOFTHEROOT

OFMondiawhitei(HOOK.F.)SKEELS

Gbadamosi,I.T.1,Aboaba,S.A.2andTitilawo,O.Y.3

1DepartmentofBotany,UniversityofIbadan,Nigeria.2DepartmentofChemistry,UniversityofIbadan,Nigeria.3DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,SouthAfrica.

TherootofMondiawhitei(Hook.f.)Skeelsisusedforthetreatmentofgastro-intestinal

disorders, sexually transmitted infections, malaria, and asthma and as aphrodisiac in

Nigeria. Inthisstudy, thecompositionoftheessentialoilof therootofM.whiteiwas

analysed by GC/MS. The agar-well diffusion techniquewas used for the antimicrobial

assayof theoilagainstninepathogenicorganismsviz.Bacilluscereus;Escherichiacoli;

Candida albicans; Klebsiella pneumoniae; Pseudomonas aeruginosa; Proteus mirabilis;

Salmonella typhi; Staphylococccus aureus; and Streptococcus pyogenes. Twenty-eight

compounds representing 99.92% of the essential oil were characterised. The major

constituents of the oil were (E)-2-Hexen-1-ol (25.96%); Heptacosane (20.94%); Phytol

(15.60%); 1-Hexanal (8.94%); (E)-2-Hexenal (4.29%) and 2-Hydroxy-p-anisaldehyde

(4.21%). At 106 cfu/ml inoculum concentration, the oil was most active against

Escherichia coli (50.0mm) and Staphylococccus aureus (50.0mm) and least active on

Candida albicans (15.0 mm). The observed antimicrobial activity justifies the

ethnomedicinalusesofM.whiteiinNigeria.

P56

THEDEVELOPMENTOFAMICROBIALGROWTHPROMOTINGMIXTUREFOR

COMMERCIALASPALATHUSLINEARIS(ROOIBOS)PLANTS

Brink,C.J.,Postma,A.andJacobs,K.

DepartmentofNaturalSciences,UniversityofStellenbosch,PrivateBagX1,Matieland,7602.

Aspalathuslinearis(rooibos)isindigenoustotheCederbergregionintheWesternCape

ofSouthAfrica.Commercialrooibosplantsoftenstruggletogroweffectivelyinthefield

after germination in the nursery. Studies have been done on plant growth-promoting

microorganismstoenhanceplantgrowthandtoimproveplantimmunesystems.Unlike

chemicalfertilizers,thesemicroorganismsareenvironmentallyfriendly.Thisstudyaim

todevelopamixtureofTrichodermametabolitesand rhizobial isolates,whichcontain

plantgrowth-promotingfactorsthatwillassisttheplant inadaptingtofieldconditions

andenhanceplantgrowth. Thirty-sixrhizobialstrainswereisolatedfromrootnodules

of rooibos plants and tenTrichoderma strainswere isolated from fynbos soil. All the

isolateswereevaluated forgrowth-promoting factors. TheTrichoderma isolateswere

screened for indole acetic acid production using HPLC and the rhizobia isolates were

screened for ammonia production using Nessler’s reagent. Three isolates of both

rhizobia and Trichoderma with the highest production of plant growth-promoting

properties were used as treatments in greenhouse trials. Different combinations of

treatments were used on rooibos plants to determine their effect on plant growth.

PreliminarydatafromthetrialssuggestthatthemetabolitesoftheTrichodermaisolates

haveabiggerinfluenceonplantgrowthcomparedtothecontrolandrhizobiaisolates.

For future research, field trials should be conducted to evaluate the effect of these

microorganismsonrooibosplantsincommercialplantations.

P57

AEROBICFERMENTATIONASANALTERNATIVETOREDUCINGETHANOLLEVELSIN

WINEMAKING

Mehlomakulu,N.N.1andJolly,N.1

1ARCInfruitec-Nietvoorbij,Stellenbosch,SouthAfrica,

Theproductionofethanol inwinemakingisattributedtothefermentativemetabolism

of the wine yeast, Saccharomyces cerevisiae. Grapes are often harvested at high

ripenesslevelstotakeadvantageoftheincreasedflavourprofiles.Thisresultsinwines

withethanolcontentsabove13%.Althoughsuchethanolconcentrationsarewithinlegal

limit,thereisagrowingdemandforthereductionofethanolinwineduetoconsumer

preferences, while maintaining good wine aroma and quality. Non-Saccharomyces

yeastspresentintheinitialstagesoffermentation,canfermentgrapesugarstoethanol

andothermetabolites under aerobic conditions, providing a channel for diverting the

excess sugar away from ethanol production. The aim of the study was therefore to

investigate the reductionofethanol content inwinesusingnon-Saccharomyces yeasts

under aerobic conditions in conjunctionwith S. cerevisiae. Firstly, non-Saccharomyces

yeasts were screened for the production of ethanol under aerobic and anaerobic

conditions.Someoftheyeastsexhibitinghighconsumptionofsugarswithinthefirstfive

days of fermentation were found to produce lower ethanol concentrations and had

acceptable aroma. These yeast species were then sequentially inoculated with S.

cerevisiae under initial aerobic conditions followed by anaerobic conditions. The

fermentations carried out on laboratory-scale yielded wines with lower ethanol

concentrationscomparedtothecontrol.

P58

GASBUBBLEFORMATIONINTHEGENUSSACCHAROMYCES

DuPlooy,L.M.1,Pohl,C.H.1,Dithebe,K.1,VanWyk,P.W.J.2andSwart,C.W.1,2*

1DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeSate,P.O.Box339,

Bloemfontein,9300.2CentreforMicroscopy,UniversityoftheFreeState,P.O.Box339,Bloemfontein,9300.

Fermentation by yeasts of the genus Saccharomyces lies at the heart of many

commercialprocessesbecauseoftheirabilitytofermentveryefficiently.Itwasrecently

shown that carbon dioxide (CO2) bubbles accumulate in the cytoplasm of fermenting

yeastasaconsequenceofvigorouscarbondioxideproductionduring fermentation. In

this study,weaimed to correlate thenumberof these gasbubbleswith thedifferent

fermentation efficiencies of the eight described species in the genus. This was

performed by growing cells in fermentable media in shake flasks and recording the

number of bubbles with light and transmission electron microscopy. Further

characterisation of the bubbles was performed by viewingwith Nano scanning auger

microscopy (NanoSAM). Viewing of cells grown in non-fermentablemedia with these

techniquesisincludedascontrol.Thevolumeofgasproducedduringfermentationwas

measured.Itwasfoundthatallofthestrainsnotusedincommercialprocesses,suchas

S. mikatae and S. kudriavzevii, yielded fewer gas bubbles than strains used in

commercial processes. It is clear from these results that strains used commercially

ferments with a greater efficiency compared to non-commercial strains and thus

producesmorebubbles.

P59

INVESTIGATIONINTOTHEEFFECTOFFATTYACIDSONTHEYIELDOFROTAVIRUS

INFECTION

Sander,W.J,Pohl,C.H.,O’NeillH.G.1

1DepartmentofMicrobial,Biochemical&FoodBiotechnology,UniversityoftheFreeState,P.O.Box339,

Bloemfontein,9300,SouthAfrica.

Rotaviruses, the leading viral cause of severe diarrhoea in children and infants, are a

member of the Reoviridae, a family of dsRNA viruses.When rotaviruses infect a host

cell, the outer layer proteins of the capsid are lost and the transcriptionally active

double-layerparticlereleasesmRNA.ThemRNAservesastemplatesforviralsynthesis.

ViralproteinsandRNAsassociateincytoplasmicinclusionbodiescalledviroplasms.Two

non-structural proteins, NSP2 and NSP5, are essential in formation of viroplasms.

Inhibitionof theviroplasm, inhibit the rotavirus replicationcycle.Viroplasmsassociate

with lipids and proteins characteristic of lipid droplets (LDs), recruiting these

componentsearlyduringtheinfectioncycle.LD-associatedproteinswerealsofoundto

co-localise with viroplasms during infection and with viroplasm-like structures in

uninfectedcells. LDsarepolymorphicorganelles that store triacylglycerols, cholesterol

and cholesterol esters. Treatment with chemical inhibitors of fatty-acid synthesis

reducedrotavirusinfectivity.Itisknownthattheadditionoflongchainfreefattyacids

tocellsareabsorbedbycellsandsubsequentlyinhibitfattyacidsynthesisofthecells.In

thestudyweareinvestigatingtheeffectofsaturated(stearicacid)andunsaturated(α-

linolenicacid)fattyacidsonrotavirusinfectivity.Specifically,MA104cellswillbetreated

with fatty acids followed by infection with rotavirus SA11, the prototype rotavirus.

InfectivitywillbemonitoredbyTCID50viraltitrations.

P60

THEMICROBIALCOMMUNITIESASSOCIATEDWITHAHONEYBEE(Apismellifera)HIVE

Lawson,K.1,Allsopp,M.H.2andJacobs,K.1

1DepartmentofMicrobiology,UniversityofStellenbosch,PrivateBagX1,Matieland,7602.2ARC-PlantProtectionResearchInstitute,PrivateBagX5017,7599,Stellenbosch,SouthAfrica

Apismellifera,commonlyknownastheWesternhoneybee,providesaglobalpollination

servicewithanestimatedvalue>$200billionperyear.Currentglobaldeclinesinthese

honey bee populations has raised concerns. Much research has been done on the

microorganisms that could be involved in or causing these population declines.

Treatment of pathogenicmicroorganisms often involves the application of fungi- and

bacteriocides. However,thesetreatmentsmayhavefar-reachingeffectsonthenative

microbialcommunitiesofthehive. Todate,fewstudiesexist,characterisingthebasal

communities associated with bee hives. In this study, we aimed to characterise the

microbial communities and spatial distribution patterns ofmicroorganisms associated

withApismellifera.Itishypothesisedthattheexternalmicrobialcommunityintroduced

into the hive determines the internalmicrobial environment of the hive. Preliminary

studies suggest that there are distinct spatial patterns of microorganisms within the

hive. Data analysis has shown that the gastrointestinal tract of the bee harbours a

uniquemicrobialcommunityfardifferenttothatoftheentrance,thereforesuggesting

thepresenceofanenvironmentalselectivepressurewithinthegastrointestinaltract.It

can be seen that the bee acts as a carrier of microorganisms from the external

environment to within the bee hive. There is a distinct shift in the microbial

communities at the entrance of the hive, and the internal environment, suggesting a

large selection pressure due to micro environmental niches. This study provides

fundamentalknowledgeforfurtherstudiesinvolvedinunderstandingglobalcolonyloss.

P61

DIAGNOSTICSOFBEAKANDFEATHERDISEASEVIRUS

VanNiekerk,J.,Bragg,R.R.andBoucher,C.E.1

1DepartmentofMicrobial,Biochemical&FoodBiotechnology,UniversityoftheFreeState,P.O.Box339


Beak and feather disease virus (BFDV) belongs to the family Circoviridae. It is a viral

disease that infects psittacine (parrots) birds. The replication associated protein (Rep)

wasusedfordiagnosticpurposes.Molecularaswellasserologicaltechniqueswereused

to determine whether the virus was present in a selection of parrots. ELISA, and

immunofluorescencewereusedasserologicaltests,whichweremuchcheaperthanthe

conventionalandreal-timePCRtechniques thatwereusedasmolecular techniques. It

was found that the PCR techniquesweremore sensitive, specific and faster than the

serologicaltechniques.Alltheserologicaltestsweredoneonayeastexpressionsystem

whichexpressedthecoatproteinofBFDV,whereasthemoleculartechniquesweredone

on theRepgene,which is theconserved region in thevirus.Withoptimizationof the

serological techniques itwas found that these testswere thebetteroption touse for

diagnosticsastheyshowahistoryofexposuretothevirus.Thereforeaconclusionwas

made that a combination of both serological andmolecular tests should be used for

routinediagnosisofinfectedbirds.

P62

SUNNYSIDEUP:USINGANANTIOXIDANTPRODUCINGBACTERIUMTOENHANCE

PIGMENTATIONINEGGYOLKSOFLAYINGHENS

Conradie,T.A.1,Pieterse,E.2andJacobs,K.1

1DepartmentofMicrobiology,2DepartmentofAnimalScience,UniversityofStellenbosch,PrivateBagX1,

Matieland,7602.

Formanyyearscarotenoidshavebeenusedtomanipulatethecolourofanimalproducts

toobtainadesiredcolour.Astaxanthin,axanthophyllcarotenoid,hasstrongantioxidant

activity and colour properties that provides health benefits to humans and animals.

Some microorganisms are able to synthesise this compound naturally and show

promising applications as feed additives. This study investigated the use of an

astaxanthin producing bacterium as a possible source of pigmentation to change egg

yolkcolour. In the feeding trial, fivedifferentdietswereprepared.Thehenswere fed

daily for 7 weeks and all eggs were collected for analysis. After 5 weeks, the diets

containingtheantioxidantproducingbacteriumshowedasignificantincrease(P≤0.05)

inyolkcolourintensity.Therewasnosignificantdifferenceobservedintheheightofthe

yolkandhenweightbeforeandafterthetrial,buttherewasasignificantdifference(P≤

0.05)observedinyolkweight,eggweightandhenlayingrate.Twoweeksaftercessation

of the experimental diet, the colour intensity decreased again. This study shows

promisingresultsinusingthisbacteriumasaneffectivefeedadditiveforhens.

P63

ScreeningforMagnetotacticBacteriainMarine,FreshwaterandDesertSedimentsof

NamibiaandSouthAfrica

MaropolaM1,LefèvreC2,vanZylL2,TrindadeM2

InstituteforMicrobialBiotechnologyandMetagenomics,UniversityoftheWesternCape,Bellville,7535,

CapeTownSouthAfrica

Magnetotacticbacteria (MTB)arepolyphyletic andaquaticbacteriawith theability to

swimalongmagnetic field linesdue to theirmagnetosomeorganelles.Magnetosomes

consistofmagneticmagnetite(Fe2O4)orgreigite(S2O4)nano-crystalchains,surrounded

by a phospholipid bilayer membrane. These nano-crystals have potential for various

biotechnologicalandbiomedicalapplicationsduetotheirmagneticproperties,smallsize

(20-150nm)anduniformity.ThisstudyaimstoexplorepreviouslyuncharacterisedMTB

communities frommarine, freshwateranddesertenvironmentsofNamibiaandSouth

Africa.MicroscopyanalysesofMTB-enriched samples fromstudied sites indicated the

presence of MTB with diverse cell and nano-crystal morphologies in all marine and

freshwatersamples,butnoneweredetectedinsamplesfromhypersalinedesertpools

of theNamibiandesert.Metagenomicanalysisof the16S rRNAclone librariescreated

from marine magnetotactic enrichments show that sequences are closely related to

uncultured species and Proteobacteria (Novosphingobium sp., Sideroxydans

lithotrophicusandPropionivibrio sz-275)withsimilarMTB-likephysiologicalproperties.

Thepresenceofmagnetotacticgeneislands,tobedeterminedbyPCR-basedscreening,

willbeemployedtoconfirmwhethertheserepresenttrulymagnetotacticbacteria.

P64

THEROLEOFCYTOCHROMEP450INTHEPRODUCTIONOFPROSTAGLANDINE2BY

Saccharomycescerevisiae

Pieterse,B.,Pohl-AlbertynandC.H.,Albertyn,J1

DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,P.O.Box339,

Bloemfontein9300,SouthAfrica

Saccharomycescerevisiaeisabuddingandsaprophyticfungusthatiscommonlyusedin

thebrewing industry. It isknowntobenon-pathogenicand issupposedtobesafefor

human consumption, but recent discoveries indicated that Saccharomyces cerevisiae

produces prostaglandin E2 which was also discovered to be present in beer.

ProstaglandinE2isaninflammatoryagentwhichplaysanimportantroleinimmunityand

disease.ThiseicosanoidisproducedinpathogenicyeastssuchasCandidaalbicansand

Cryptococcus neoformans and contributes to their virulence. The pathway that

Saccharomyces cerevisiae use to produce prostaglandin E2 is still unknown. However,

ergosterol 11, a member of the cytochrome P450 family, might play a role in

prostaglandin E2 production as itwas discovered that cytochromeP450 forms part of

prostaglandin E2 production in Candida albicans. The first research step would be to

determinethepathwaythatprostaglandinE2areproducedinSaccharomycescerevisiae

and how cytochrome P450 might contribute to its production. As the production of

prostaglandinE2inSaccharomycescerevisiaeisaveryrecentdiscovery,severalresearch

studies needs to be done to determine the important role that it might have in its

pathogenicity.

P65

CHARACTERISATIONANDEVALUATIONOFNOVELSaccharomycescerevisiaeHYBRIDS

FORTHEPRODUCTIONOFAROMATICSAUVIGNONBLANCWINEPRODUCTION

RodneyHart1,2,NeilJolly1,andBonganiNdimba2

1Post-HarvestandWineTechnologyDivision,ARCInfruitec-Nietvoorbij,PrivateBagX5026,Stellenbosch,

7599,SA2NationalAgriculturalProteomicsResearch&ServicesUnit,DepartmentofBiotechnology,Universityof

theWesternCape,PrivateBagX17,ModderdamRoad,Bellville,CapeTown,7535,SA

ThewineyeastSaccharomycescerevisiaevaryintheirabilitytodevelopthefullaroma

potentialofSauvignonBlancwinedue toan inability to releaseboundaroma-inactive

metabolites which imparts fruity and tropical aroma notes during fermentation.

Furthermore, anecdotal data suggests that some wine yeast strains commercially

available intermittently exhibits undesirable characteristics e.g. volatile acidity (VA)

formationwhichimpartsvinegar-likearomas.Therefore,atrialwasundertakentoselect

andevaluatenovelARChybridyeaststrainsfortheproductionofSauvignonBlancwine

with enhanced fruity and tropical aromas, but lower VA. Hybrid yeast strains was

characterised by CHEF DNA karyotyping and MALDI-MS biotyping, and subsequently

trialled against top commercial reference wine yeasts in laboratory-scale Sauvignon

Blanc vinifications. Final wines were subjected to chemical, sensory and gas

chromatography-mass spectrometry (GC-MS) analyses. Most hybrid yeasts produced

SauvignonBlancwineshadVAsignificantlylowerthanthatproducedwiththerespective

commercialreferences.Sensorial,somehybridyeastsalsobestassociatedwithtropical

wine aroma notes. In addition, they produced Sauvignon Blancwineswith less acetic

acid,themainvolatileacid.Itisenvisionedthatpromisinghybridswillbeevaluatedona

largerscaleforproductionofaromaticSauvignonBlancwines.

P66

DETERMINATIONOFQACRESISTANTSTAPHYLOCOCCUSAUREUSSTRAINSFROM

MASTITISSAMPLES.

Kennedy,N.,Bragg,R.R.andBoucher,C.E.

Microbial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,205NelsonMandelaDrive,

Bloemfontein,9301.

Mastitis is a major problem in dairy cows and the greatest economic loss is due to

decreasedmilkproduction.Staphylococcusaureusisthemajoretiologicalagentcausing

mastitis and is often resistant to treatment with various antibiotics. Quaternary

ammonium compound (QAC) disinfectants are used to treat the teats of the cows to

control mastitis. QAC resistance genes have been found in S. aureus strains causing

mastitis. Therefore it is important to determine whether S. aureus strains associated

withmastitisstrainscontainingQACresistancegenesareresistantagainstQACsusedin

the industry.Milksamples fromcowsshowingclinicalandsub-clinicalsymptomswere

collected,screenedandcultivatedonBTAplatesandsub-cultivateduntilpurecolonies

wereobtained.S.aureusstrainsassociatedwithmastitiswereidentifiedusing16SPCR.

Omnilog data of these strains were obtained to confirm species identification. QAC

resistance geneswere screened for bymeans of PCR and real-time PCRwas used to

studytheregulationofthesegenes.MIC’swereperformedusingvariousdisinfectantsto

study the effect and determine theMIC for each S. aureus isolate. Results from this

study are presented here. Disinfectants should be used correctly and alternation

betweenvariousdisinfectantsisrecommendedinordertoeffectivelycontrolS.aureus

causingmastitis.

P67

MICROBIALCOMMUNITYECOLOGYOFTWONAMIBDESERTFAIRYCIRCLEBIOTOPES

VanderWalt,A.J.1,Ramond,J-B.1,Johnson,R.M.1,Cowan,D.A.1

1CentreforMicrobialEcologyandGenomics(CMEG),DepartmentofGenetics,NaturalSciences2,

UniversityofPretoria,Hatfield,Pretoria,0028.

Fairy Circles (FCs),which are restricted to the coastalNamibDesert zone, are circular

patches of soil devoidof vegetation surroundedby a fringeof longer grass. Since the

first report of FCs in the 1970’s, scientists have put forward a striking number of

hypotheses on their origin. These range from micro-faunal activity, soil physics to

vegetativeself-organization.Nevertheless,noneofthesehypotheseshavebeenableto

adequatelyexplainwhyFCsexist,norwhytheyoriginateordevelop. In thisstudy,we

investigated thehypothesis thatFC formation isdue tomicrobialeffects.Surfacesoils

wereobtainedfromthecentreandmatrixoffivegravelplainandfiveduneFCsduring

April2014(n=20).AfterperformingtotalDNAextraction,samplesweresequencedfor

bacterial, archaeal (16S rRNA gene)markers to determinemicrobial diversity. Results

showthatgravelplainandduneFCmicrobialcommunitiesarephylogeneticallydistinct.

WithintheduneFCs,ActinobacteriaandCrenarchaeotawerefrequentlymoreabundant

incentresoilswhereasBacteroidetesandProteobacteriaweremorecommontomatrix

soils. This pattern is less prominent in the gravel plain FCs, with only Crenarchaeota

identified more frequently in centre soils. Identification of a single microorganism

presentinboththegravelplainandduneFCs,andabsentinthegrassed‘control’areas,

willconstituteapossiblecandidatefortheformationormaintenanceoftheFCs.

P68

''BIO-PROSPECTINGASOILMETAGENOMELIBRARYFORCARBOHYDRATEACTIVE

ESTERASES''

NtombifuthiShezia,b,KonananiRashamusea,KgamaMathibaaandBrettPletschkeb

aCSIRBiosciences,CouncilforScientificandIndustrialResearch,MeiringNaudeRoad,Brummeria,

Pretoria,0001bDepartmentofBiochemistryandMicrobiology,RhodesUniversity,Grahamstown,.6140

Feruloylesterases(FAEs,EC3.1.1.73), representasubclassofcarboxylesterhydrolases

(EC3.1.1.-) that catalyse the releaseofhydroxycinnamic acids (suchas ferulic acid,p-

coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to

polysaccharides, such arabinoxylans and pectins. Hydroxycinnamic acids have

widespread potential applications due to their antimicrobial, photoprotectant, anti-

tumour and antioxidant properties as well as their use as flavour precursors. A soil

metagenomic library was constructed using fosmid based plasmid vector and

subsequentlyfunctionallyscreenedforferulicacidesterases(FAEs)usingethylferulate

as amodel substrate. A total of 59 recombinant fosmids conferring feruloyl esterase

phenotypes were identified (Hit rate1:3122) and the two fosmids that consistently

showed high FAE activities were selected for further study. Following nucleotide

sequencingandtranslationalanalysis,twoFAEencodingopenreadingframes(FAE9and

27) of approximately 274 and 322aa respectively, were identified. The amino acid

sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G

sequence motif. The two genes (FAE 9 and 27) were successfully expressed in

Escherichia coli and the purified enzymes exhibited temperature optima of 40 °C and

respective pH optima of 6.0 and 7.0. Further biochemical characterisation aimed at

examining the substrate specificities of these enzymes and their applications in the

releaseofhydroxycinnamicacidsfromselectednaturalsubstrateswillbereported.

P69

NOVELYEAST-BASEDEXPRESSIONSYSTEMFORTHEPRODUCTIONOFSUBUNIT

VACCINES

Meyburgh,C.M.,Bragg,R.R.&Boucher,C.E.


Bloemfontein,9300.

Developments in biotechnology have enabled the production of vaccines containing a

defined subunit of a pathogen. Such subunit vaccines are regarded as safer than

traditional vaccines due to risk of infection associated with whole cell vaccines.

Recombinantsubunitvaccinesareproducedbyexpressionofantigen-encodinggenesin

host cells. Here, an expression system using the yeast Yarrowia lipolytica strain po1h

transformed with a surface display plasmid and its application in subunit vaccine

developmentisdescribed.Expressionofrecombinantgenesusingthissystemresultsin

attachment of the recombinant protein to the outer cell wall of the host. The

recombinantgeneisundercontrolofagrowth-phasedependentpromoter.Integration

of theplasmid into thehost genomeoccurs at randomsites as a resultof zetabased

elementsfromYltrretrotransposoncarriedbytheplasmid,yieldinggeneticallydistinct

transformants.Toevaluatetheuseofthissystem,heterologousexpressionofsynthetic

Beakandfeatherdiseasevirus(BFDV)coatproteinintheY.lipolyticaexpressionsystem

was performed. The antigen-producing capacity of transformants was evaluated by

quantification of gene expression during early stationary phase relative to early

exponential phase using qPCR. Results indicated differences between transformants

regardingfoldchangeingeneexpressionrangingfrom1.83to2.45.Therelevancyofthis

yeast-basedexpressionsysteminthefieldofsubunitvaccinedevelopmentisevidenced

bysuccessfulproductionofrecombinant,immunogenicBFDVcoatprotein.

P70

SUBSTRATECHARACTERIZATIONANDHETEROLOGOUSEXPRESSIONOFTHE

SELF-SUFFICIENTCYP505E3FROMAspergillusterreus

Kuloyo,O.O.,Smit,M.S.,Opperman,D.J.,PohlC.H.,Albertyn,J.



FilamentousfungicarrygenesencodinganarrayofcytochromeP450(CYP450)enzymes

essential in pathogenesis, xenobiotic degradation and substrate utilization. A self-

sufficient CYP450 with terminal alkane hydroxylase activity in cell-free extracts was

reportedforthefilamentousfungusAspergillusterreus.Sequenceinformationfromthe

CYPome ofA. terreus reveals that there are only two possible self-sufficient CYP450s

withinitsgenome.Oneofthetwoself-sufficientCYP450identifiedasCYP505A19lacksa

crucialsegmentofthehemedomain;henceit isnotlikelytobeanactiveCYP450.The

secondself-sufficientCYP450,classifiedasCYP505E3,wasexpressedheterologouslyand

itssubstratespecificityinvestigatedinthisstudy.AnN-terminalvariantoftheCYP505E3

withimprovedA/Tcontents,wasconstructedtoallowtheuseofcodonmatchingwith

Escherichia colipreferences.Whole cell biotransformationswere carried outwith the

expressed CYP505E3 variant using hexadecane, pristane, naphthalene, hexylbenzene,

nonylbenzene, 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates. Soluble

CYP450recoveryaftercelldisruptionwasimprovedfrom0.26nmolg-1to0.52nmolg-1

wetweight(101%)usingaPlackett-Burmanexperimentaldesign.Hydroxylatedproducts

ofhexylbenzoicacidwereidentifiedasω-4hydroxyhexylbenzoicacidandω-2hydroxy

hexylbenzoic acid, while no hydroxylated products were produced from hexadecane.

Hexylbenzoic acid, which is a substrate of self-sufficient sub-terminal fatty acid

hydroxylases suchasCYP102A1andCYP505A1, is alsohydroxylatedbyCYP505E3.We

thereforeconcludedthatconversionofhexylbenzoicacidbyA.terreusCYP505E3might

indicatethatCYP505E3isalso,likeCYP102A1andCYP505A1,afattyacidhydroxylase.

P71

LONGRANGEPCRTODETECTHP2ANDMU-LIKEPHAGES

WITHINTHEGENOMEOFAvibacteriumparagallinarumREFERENCEISOLATES

Coetsee,E.1,Bragg,R.R1.&Boucher,C.E1.

1DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,POBox339,

Bloemfontein,9301.

Avibacterium paragallinarum is the causative agent of infectious coryza, an upper

respiratory tract disease that occurs primarily in chickens. This disease has a huge

economic impact in the poultry industry with a 10-40% decrease in egg production.

ProphagesresemblingaMu-likeandHP2-likephagehavebeenfoundintheModesto(C-

2)strainofAv.paragallinarumbymeansofwholegenomesequencing.Prophagescan

transfer new functions to the host bacterium for example, altering its virulence,

conferring resistance to antibiotics, detoxification of heavy metals, acquisition and

utilization of certain nutrients, evasion of predators or colonization of specific

environments.Duringthisstudy,longrangePCRwasusedtoscreenforthepresenceof

complete HP2-like and Mu-like phages within the genome of Av. paragallinarum

referenceisolates.FromtheresultsithasbeenobservedthattherearecompleteHP2-

like andMu-like phages present in some of the reference isolates. Therefore future

research would be to determine what effect the presence of these prophages could

haveonthevirulenceoftheAv.pagragllinarumreferenceisolates.

P72

BACTERIOPHAGELAMBDAENDOLYSINEXPRESSIONFORAVIAN

PATHOGENICEscherichiacoliTREATMENT.

Lee,J-Y1,Theron,C.W.1,Boucher,C.E.1andBragg,R.R.1

1DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,P.O.Box339,

Bloemfontein,9301.

Asantibioticresistanceincreasesandbansontheuseofprophylacticantibioticsareon

the increase, alternative treatment against bacterial infections are necessary. This

research is important for the poultry industry as current farmingmethodsmean that

poultryare fedantibioticgrowthpromotorswithoutwhich the industrywouldendure

financial lossdue todecreasedeggproductionand smallerbroiler carcasses. Interest

andresearchinbacteriophagetherapyandtheirproductsasaviabletreatmentagainst

bacteria has grown in recent years. This is due to the natural host-virus relationship

betweenbacteria andbacteriophage.Anewpossibilityof alternatives to antibiotics is

the use of bacteriophage enzymes that target the bacterial cell wall and lyse them.

These enzymes are endolysins and their mode of action is their ability to break the

bonds between thebuilding blocks of peptidoglycan composing the cell walls N-

Acetylmuramic acid(NAM) andN-acetylglucosamine(NAG) acids. The aim of this

projectwastoexpressbacteriophagelambdaendolysin(codedbygeneR')andassayit

against bacterial strains includingGram-negative andGram-positive bacteria; focusing

on Avian Pathogenic Escherichia coli (APEC). E. coli is the causative agent for

colibacillosiswhich inpoultry results indroppedeggproduction,declininggrowthand

quality of broilers and increasedmortality. The endolysin gene of lambda phagewas

successfullyclonedintoabacterialvectorexpressionsystemandidentifiedviaMS-MS.

The expressed enzymewas tested against Gram-negative and Gram-positive bacterial

strains.

P73

THEINFLUENCEOFCandidaalbicansPHENOTYPICPLASTICITYONEXPRESSIONOF

VIRULENCEFACTORS.

Mokoena,N.Z.1,Pohl,C.H.2,Motaung,T.3,Albertyn,J.4,Swart,C.W.5andSebolai,O.6

1DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,PO.Box339,


Incidencesof fungal infectionshave increased in the last fewdecades.Althoughmost

researchinthefieldofpathogenicfungiisalsoincreasing,morestudiesstillneedtobe

doneonfungalvirulence.Candidaalbicansisoneofthewell-definedopportunisticyeast

pathogen,inhabitingthehumanmicrobialflora.Initspathogenicstate,C.albicanscause

life-threateninginfectionssuchasCandidiasis,inanimmunocompromisedhost.Despite

theeffectivenessofavailableantifungaldrugsinpreventingsuchinfections,C.albicans

hasemergedwaystobecomedrugresistant.Itexpressesseveralvirulencefactorsthat

contribute topathogenesisandmaking it a successfulhumanpathogen.These factors

include phenotypic switching, germ tube production, secretion of hydrolytic enzymes

(proteinases, phospholipases and lipases), biofilm formation and prostaglandin E2

production.TheobjectiveofthepresentstudyistocultivatecellsofC.albicansclinical

isolates, WO-1 strain and NRRL Y-27077 strain on the YPD and Lee’s medium

supplemented with N-acetylglucosamine then screen for their ability to produce

different colonies. It is known thatWO-1 strain can undergowhite-opaque switching,

while NRRL Y-27077 strain undergoes white-gray-opaque-like switching. It can be

strongly suggested that white-opaque switching andwhite-gray-opaque-like switching

can increase genetic variations and contribute to the observed increased antifungal

resistanceofbiofilms.Sotheresearchwillfocusontheexpressionofdifferentvirulence

factors, including fluconazole resistance by the different cell types (i.e. white and

opaque)ofC.albicans.

P74

DIFFERENTIALMETABOLOMICSANDTRANSCRIPTOMEANALYSISOFKLUYVEROMYCES

MARXIANUSONXYLOSEANDGLUCOSEASCARBONSUBSTRATES

LetebeleP.K.,Schabort,D.W.P.andDuPreez,J.C


Bloemfontein9300

Kluyveromycesmarxianus is a Crabtree negative yeast, possessing various features of

biotechnologicalimportancewhichincludeabroadsubstrateutilisationspectrum,ahigh

growth rate, thermotolerance and secretion of lytic enzymes. This yeast is known to

assimilate but not ferment pentose sugars under oxygen-limited conditions. The

assimilationofxylosebyyeasts involvesNADPH-specificxylosereductase,whichraises

the question of how the cell synthesizes the additional NADPH required for xylose

utilization. We investigated the global differential expression of enzymes within the

central metabolism involved with xylose metabolism in K. marxianus in response to

glucose or xylose as carbon source using RNA-seq. NADPH-dependent iso-citrate

dehydrogenase II was found to be up-regulated with xylose as sole carbon source,

whereastherewasnosignificantdifferenceintheexpressionoftheoxidativeenzymes

of the pentose phosphate pathway (glucose-6-phosphate dehydrogenase and 6-

phosphogluconate dehydrogenase). The data obtained were analyzed using

bioinformaticstoolsinGalaxyandanewin-houseprogram,Reactomica,forintegrating

systemsbiologyandomics. Inadditionwequantitativelyanalyzed twenty intracellular

metabolitesusingtriple-quadLC-MS,sixteenofwhichweredetectedandfourofwhich

showedsignificantdifferencesbetweenthetwoconditions.Citratewasdownregulated

withxyloseassubstrate,whereasxylitol,glyceraldehyde-3-phosphateandarabitolwere

upregulated. Surprisingly, enzymes that were not of interest, such as those found in

pathwayssuchasthecitricacidcycleandinfattyacidbiosynthesis,wereupregulated.

The latterobservations raise furtherquestions regarding thedifferential expressionof

genesinresponsetotheutilisationofdifferentcarbonsources.

P75

HETEROLOGOUSEXPRESSIONOFASIDEROPHOREFROMAPseudoalteromonasSPP

ASSOCIATEDWITHAMARINEINVERTEBRATE

Adams,S.R.,vanZyl.L.andTrindade,M.

InstituteforMicrobialBiotechnologyandMetagenomics(IMBM),UniversityoftheWesternCape,Robert

SobukweRoad,Bellville,CapeTown,SouthAfrica

Siderophoresarelowmolecularweightmoleculeswithstrongironbindingcapabilities.

Hundredsofsiderophoreshavebeenisolatedfromterrestrialmicroorganisms,especially

frompathogenicmicroorganisms. Incontrast,onlya fewstudieshavereportedonthe

iron sequestering systems from marine microorganisms. It is predicted that these

compounds have a greater chemical diversity compared to the siderophores isolated

from terrestrial microorganisms. Siderophores have numerous applications in human

health,rangingfromantimicrobialactivity,overcomingantibioticresistance,andinthe

treatment of a range of diseases, and could therefore contribute immensely to the

pharmaceutical industry. A Pseudoalteromonas spp. isolated from a marine

Platyhelminthesspshowedanti-inflammatory,anti-fungal,anti-yeastandanti-bacterial

activity. Here we report the identification of a siderophore biosynthetic pathway

through sequencing of the bacterial genome and attempt its overexpression in

Escherichia coli and Pseudomonas aeruginosa to determine whether the compound

produced by the pathway is responsible for any of the activities expressed by this

microorganism.

P76

INCIDENCES,SPECIESDISTRIBUTIONANDANTIMICROBIALRESISTANCEOF

EnterococcusSPPISOLATEDFROMFAECALANDWATERSAMPLESFROMTHREE

COMMERCIALFARMSINAMATOLEDISTRICTSEASTERNCAPE,SOUTHAFRICA.

TanihG.N1,2AnthonyI.Okoh1,2RolandN.Ndip1,3andEzekielGreen1,2

1DepartmentsofBiochemistryandMicrobiology,UniversityofFortHare,PrivateBagX1314,Alice5700,SouthAfrica2AppliedandEnvironmentalMicrobiologyResearchGroup,DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,Alice5700EasternCape,SouthAfrica.3DepartmentofMicrobiologyandParasitology,FacultyofScience,UniversityofBuea,P.O.Box63,Buea,Cameroon.

Theincidencesofmultidrugresistantenterococcihavebecomeamajorconcern.Thisis

due their implication in hospital acquired nosocomial infections and substantial

therapeutic failure. Occurrence of antibiotic-resistant Enterococcus spp has been

associatedwiththeoveruseofantibioticsinanimalfarmsmanagementandthetransfer

of resistant genes through plasmidmediated conjugation as some of the causes. This

study seeks to determine the incidences of Enterococcus spp and their antimicrobial

resistantprofiles frombothfaecalandwatersources indairy farmlands.Twohundred

and eighty-nine rectal swabs and forty - fivewater samples from the differentwater

sources(drinkingwater,irrigationwaterandwastewater)werecollectedfromfarmA,B

andCintheAmatoledistricts.Presumptiveenterococciisolatesobtainedbyculturingon

selective media, Gram staining and oxidase test. In addition, isolates were screened

molecularly using generic specific primers targeting the tuf gene (encodingelongation

factor).Confirmedisolateswerespeciatedintosixtargetedspeciesusingspeciesspecific

primers. Antimicrobial resistance phenotype and genes which confer resistance were

evaluated.Threehundredandfiveenterococciwereconfirmed,consistingof91,107and

107fromfarmA,BandCrespectively.Species identified includesE.hirae (78.38%),E.

faecium(4.92%),E.durans(3.93%),E.faecalis(1.97%)and10.82%unidentifiedspecies.

Resistance to clinically important antimicrobials was observed with some isolates

expressing multi-drug resistance phenotype. In line with other studies, faeces are

reservoirofEnterococcusspecies.Theoccurrenceofmulti-resistantstrainsmightposea

majorhealthchallengetobothhumansandanimals.

P77

ACTINOMYCETES:KEEPINGROOIBOSPLANTSHEALTHYNATURALLY

Ontong,V.1,Prins,A.1,2,LeRoes-Hill,M.2,Kirby,B.M.1

1Institute forMicrobial Biotechnology andMetagenomics, University of theWestern Cape, Private Bag

X17,Bellville,75352Biocatalysis and Technology Biology Group, Institute of Biomedical andMicrobial Biotechnology, Cape

PeninsulaUniversityofTechnology

SouthAfrica has a long history of using fynbos species formedicinal applications and

severalfynbos-derivednaturalproductshavebeencommercialized.Rooibos(Aspalathus

linearis) tea is internationally recognized for its numerousbeneficial healthproperties

and flavour. The tea has a soothing effect on the body and anti-oxidative properties,

whichmakes it such an appealingplant to study.Whilemany studies focusingon the

plant itself have been published, studies on the microbial diversity of rooibos are

limited. Actinobacteria are abundant in soil environments, and produce a range of

bioactive compounds which can be used as either growth promoters or biocontrol

agents.Hence,theaimofthisstudywastoisolateandcharacterizebioactiveendophytic

actinobacteria from naturally growing rooibos plants. As expected relatively large

numbers of actinobacteria (170)were isolated from the roots/rhizophere, with lower

numbers (35) found in the leaves. Of these strains, the majority were found to be

endophytic Streptomyces species, although based on morphological features

Micromonospora, Gordonia and Nocardia were also isolated. Metagenomic analysis

identified that highly diverse communities were present in the rhizosphere of both

plants. A. linearis rhizosphere was dominated by Gamma-Proteobacteria (34%),

Actinobacteria(14%)andFirmicutes(7%).Antimicrobialactivitywasdetectedagainstall

teststrains,withnearly40%oftheisolatesbeingactiveagainstB.cereusand35%were

activeagainstS.aureus. Inaddition, strainswhichhadantimicrobial activityagainstE.

coliwereisolated.Thecommercialimplicationsofabioactivecompounduniquetothe

rooibosenvironment,hasthepotentialtoadvancetheindustry.

P78

CHARACTERISINGANOVELDNAPOLYMERASEFROMNAMIBIANMETAVIROMES

vanVuurenR.M.P.,vanZyl,L.,Trindade,M.

1InstituteforMicrobialBiotechnologyandMetagenomics,UniversityoftheWesternCape,Bellville,Cape

Town,7537.

TheNamibDesertisconsideredtobetheoldestdesertintheworldandischaracteristic

ofhightemperatures, lowhumidityandhighevaporationrates, lowrainfallandstrong

winds.Althoughtheseextremeconditionswouldimposeconstraintsonmostlifeforms,

microbialorganismsareabletoliveandproliferate.Theabundanceofbacteriophagesin

theenvironment,anestimated1031phageparticles,presentsthemasuniquereservoirs

ofuntappedgeneticmaterialforuseinmolecularbiologyandbiotechnology.Owingto

thedifficulties inculturingand isolatingbacteriophages, thisgenetic information isyet

tobeaccessedcompletely.However,advancesincultureindependentmethodssuchas

metagenomicsandnextgenerationsequencinghavecreatedaplatformforharvesting

the unique attributes of phage genomes. The use of phage enzymes such as DNA

polymerases, ligasesand lysozymeasmoleculartoolshasgreatly impactedthefieldof

molecular biology and biotechnology. Therefore this research seeks to identify and

characterise a novel DNA polymerase from two Namibian desert metaviromes,

generatedbyIlluminanextgenerationsequencingtechnology.Insituanalysesofthese

metaviromeshaveindicatedthepresenceofnovelDNApolymerase.Thishasledtothe

selection,isolationandpurification,andfunctionalscreeningoftwopolymerases.

P79

ELICITATIONOFSECONDARYMETABOLITEEXPRESSIONFROMMARINESPONGE

ISOLATESFROMALGOABAY,SOUTHAFRICA

Matobole,R.MandTrindade,M.

InstituteforMicrobialBiotechnologyandMetagenomics,UniversityoftheWesternCape,Bellville7535,

CapeTown,SouthAfrica

Duetotheriseinmulti-drugresistantpathogensandotherdiseasesthereisarenewed

interest in marine sponge symbionts as a rich source of pharmaceutically relevant

natural products. Theobjectiveof this studywas to investigate theeffectof different

mediaininducingsecondarymetaboliteexpressionfromacollectionofmarinesponge

isolates harvested from two sponge samples. Terminal restriction fragment length

polymorphismanalysiswasused to investigate andascertain the twomarine sponges

whichhostedthehighestmicrobialdiversitiestobeusedinculture-dependentstudies.

Employing33mediawhichincludedliquidenrichments,heattreatmentsandantibiotic

treatments,resultedintheisolationof400spongeassociatedbacterialisolatesfromthe

twomarinesponges Isodictyacompressa andHigginsiabidentifera.Usingantibacterial

overlay assays 34 dereplicated isolates showed antibacterial activity on 29 media.

BioactivitieswerealsoexhibitedagainstE.coli1699whichisgeneticallyengineeredfor

resistanceagainst52antibiotics, implyingthatsomeofthebioactivecompoundscould

benovel.The16SrRNAgenesequencesrevealedthatthemicrobialphylaisolatedfrom

the marine sponges belonged to Actinobacteria, Firmicutes, Alphaproteobacteria and

Gammaproteobacteria.Theresultsshowthatmarinespongescanhostnovelmicrobial

specieswhichmay produce novel bioactive compounds. The results also confirm that

traditional methods employing a single culture condition restricts the expression of

somebiosyntheticpathwaysofmicroorganismsandasaresultmanymetaboliteshave

yettobeidentified.

P80

DEVELOPMENTOFANISS-BASEDAVIANPATHOGENICEscherichiacoliVACCINEIN

EscherichiacoliANDYarrowialipolytica

vanderWesthuizen,W.A.1,Boucher,C.E.1,Theron,C.W.1andBragg,R.R.1

1DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,Nelson

MandelaDrive,Bloemfontein,9300,SouthAfrica

Colibacillosis isadiseasecausedbyavianpathogenicEscherichiacoli (APEC)and it isa

majorconcernfor theglobalpoultry industryas it leadstosubstantial financial losses.

Current treatment and prevention is accomplished through antibiotic therapy of the

flocks,however,multiple-antibioticresistantstrainsandbansimposedonantibioticuse

in thepoultry industryarebecomingproblematic.Alternative treatmentsare required

andonesuchrouteisthroughimprovedvaccinationtherapyasapreventativeagentin

thepoultry industry. The gene encoding for the increased serum survival (iss) protein

hasbeenidentifiedaspartoftheminimalpredictorsofAPECandithasbeenpredicted

tobepresentonthecellwallsurface,makingitanidealtargetforvaccinedevelopment.

In this study, cell wall surface display and secretion expression systemswere used in

YarrowialipolyticaandaproteinexpressionsysteminEscherichiacoli,allwiththeaimof

simpleexpressionandpurificationoftheantigenfordevelopmentofasubunitvaccine

againstAPEC.

P81

THEPHEROMONEEXPORTER,HST6P,CONTROLSSTRESSRESPONSESAND

MORPHOGENESISINC.albicans

MotaungT.E.1*,PohlC.H.1,AlbertynJ.1,EllsR.1,2,SwartC.1,SebolaiO.1

1DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,

Bloemfontein,SouthAfrica.2NationalControlLaboratoryforBiologicalProducts,UniversityoftheFree

State,Bloemfontein,SouthAfrica

Candida albicans, a commensal of humans, can be pathogenicwhen confrontedwith

specific host-niches, eachwith specificmicroenvironmental cues. In order tomaintain

plasticphenotypicpropertiesunderhostileconditions, thisyeastevolvedstress-coping

mechanisms which allow proliferation even under such conditions. In this study, we

explore the role of themembrane-bound protein, Hst6p,member of theATP binding

cassettefamilyoftransportproteins,exertedduringstress-responses.Wecharacterized

a homozygous deletion mutant, ∆∆hst6, using phenotype microarrays (PMs) and

conventionalscreens.Previously,HST6wasfoundtobesignificantlyup-regulatedduring

growth in thepresenceof thepolyunsaturatedarachidonicacid (AA).This fattyacid is

known to sensitizeC. albicans to stressors and antifungals in vitro. According to high

throughput PMs, ∆∆hst6 displayed great sensitivity to 93% of stress inducing

compounds,whilethewildtypestrainwaslessormoderatelysensitive.Consistentwith

thisdata, conventionalphenotypic assays confirmed that thismutantwas sensitive to

oxidative, osmotic, cell wall, heavy metal and heat stress. This suggests that HST6

expression controls stress responses. In addition, in the presence of excess nitrogen

sources, filamentous growth was observed for ∆∆hst6, but not the wild type strain,

suggestingnegativeregulationoffilamentousgrowthundertheseconditions.Therefore,

we propose that Hst6p controls C. albicans stress responses as well as nitrogen-

dependentmorphogenesis.

P82

ASSESSMENTOFPIGMENTEDHALOPHILCBACTERIAFORTHEIMPROVEMENTOF

REJECTBRINEEVAPORATIONRATES

Moyo,A.C.,Khumalo,L.T.P.,Silva-Castro,G.A.,Petrik,L.,Trindade,M.

InstituteofMicrobialBiotechnologyandMetagenomics(IMBM),Universityofthe

WesternCape,PrivateBagX17,Bellville7535,CapeTown,SouthAfrica

TheeMalahleniWaterReclamationPlant(EWRP)produces150m3ofconcentratedbrine

perday.Currently,theEWRPisfacingaproblemwiththerateofevaporationfromthe

pondbecausethecapacityofthepondisunabletotreatthelargevolumesofthebrine

beingproduced.Inthisstudymethylenebluedyewasassessedforitsabilitytoimprove

the rate of evaporation of the brine. A laboratory scale experiment showed a 35 %

increaseinbrineevaporationwhen200ppmofmethylenebluewasadded.Inaddition,

a biological approach to improve the evaporation rates is also being considered. The

studywilldeterminewhetherpigmentedhalophilicbacteriaareaviablealternative to

the use of methylene blue dye. Fourteen pigmented bacteria were isolated from

eMalahleni brine and the Cerebos salt ponds. The isolates were identified to be

Microbacterium oxydans, Sporosacrcina aquamarina, Arthrobacter agillis, Planococcus

maritimus,Staphylococcuscapitis,StaphylococcushominsandBacillusanthrophaeus.A.

agilis displayed good growth and pigment production (0.048 mg/g) in R2A broth

preparedwith100%brine.Thepigmentproducedwasshowntobelycopenebasedon

spectrophotometricanalysis.BasedontheresultsA.agilishasthepotentialforaffecting

an increase in the evaporation rate of the brine. An additional approach being

investigated is the recombinant expression of pigment biosynthetic pathways in

heterologous hosts. The violacein biosynthetic pathway, producing a purple pigment,

has been selected for expression in a non-pigmented autochthonous bacterium that

growswellintheEWRPbrine.

P83

CANONICALPATHWAYS,NETWORKSANDTRANSCRIPTIONALFACTORREGULATIONBY

CLINICALSTRAINSOFM.tuberculosisINPULMONARYEPITHELIALCELLS

Mvubu,N.E1.,Bishai,W.R2.,Gamieldien,J3.,Pillay,B1.,andPillay,M4.

1School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal,

Westville,3630,SouthAfrica,2DepartmentofMedicine,DivisionofInfectiousDiseases,JohnsHopkinsSchoolofMedicine,1550Orleans

St.,Baltimore,UnitedStateofAmerica,3South African National Bioinformatics Institute/ MRC Unit for Bioinformatics Capacity Development,

UniversityoftheWesternCape,Bellville7530,SouthAfrica,4MedicalMicrobiologyandInfectionControl,SchoolofLaboratoryMedicineandMedicalSciencesCollege

ofHealthSciences,UniversityofKwaZulu-Natal,719UmbiloRoad,SouthAfrica.

Limited knowledge exists on pathways, networks and transcriptional factors regulated within

epithelial cells by diverse M. tuberculosis genotypes. This study aimed to elucidate these

mechanismsinducedinA549epithelialcellsbydominantclinicalstrainsinKwaZulu-Natal,South

Africa. RNA for sequencing was extracted from epithelial cells at 48 hr post-infection with 5

strains at a multiplicity of infection of approximately 10:1. Bioinformatics analysis performed

with the RNA-Seq Tuxedo pipeline identified differentially expressed genes. Changes in

pathways,networksandtranscriptionalfactorswereidentifiedusingIngenuityPathwayAnalysis

(IPA).Theinterferonsignallingandhepaticfibrosis/hepaticstellatecellactivationpathwayswere

among the top 5 canonical pathways in all strains. Hierarchical clustering for enrichment of

cholesterolbiosynthesisand immuneassociatedpathwaysrevealedsimilarpatterns forBeijing

andUnique; F15/LAM4/KZN and F11; and, F28 andH37Rv strains, respectively. However, the

inductionoftopscoringnetworksvariedamongthestrains.Amongthetranscriptional factors,

onlyEHL,IRF7,PML,STAT1,STAT2andVDRwereinducedbyallclinicalstrains.Activationofthe

differentpathways,networksandtranscriptionalfactorsrevealedinthecurrentstudymaybean

underlying mechanism that results in the differential host response by clinical strains ofM.

tuberculosis. These molecular mechanisms elucidate novel host factors that can be used as

biomarkersforpotentialdiagnosticsandhost-directedtherapiesagainstTBinfectionscausedby

M.tuberculosisstrainsofvaryingpathogenicity.

P84

P85

ISOLATIONANDCHARACTERIZATIONOFRHIZOBACTERIAFORTHEPRODUCTIONOF

SIDEROPHORESANDTHEIRin-vitroANTAGONISTICACTIVITYONSELECTED

PHYTOPATHOGENS

L.S.Khambani1andA.I.Hassen2.

1TshwaneUniversityofTechnology(TUT),DepartmentofBiotechnologyandFoodTechnology,PrivatebagX680,Pretoria0001,SouthAfrica2AgriculturalResearchCouncil-PlantProtectionResearch(ARC-PPR),PrivatebagX134,Queenswood0121,Pretoria,SouthAfrica

Iron(Fe)isanessentialnutrientrequiredbylivingmicroorganismsandplantsbecauseit

is an important co-factor in many cellular processes and enzymes. However, its

availabilityinthesoilrhizosphereisverylimitedforbothplantsandmicroorganismsasit

forms an insoluble complex with other elements. Under such iron limited conditions

rhizosphere bacteria, particularly those referred to as plant growth promoting

rhizobacteria (PGPR), produce siderophores, lowmolecular weight proteins with high

affinity to iron (Fe+3). The siderophores bind the insoluble the iron (Fe+3) andmake it

availabletoplantsinthesoilrhizospheretherebyassistingtheirnormalfunctioningand

growth.There isvery little informationavailableon the isolation, characterizationand

roleofbacterialsiderophoresinpromotionofplantgrowthinSouthAfrica.Inthisstudy

rhizobacteria were isolated from the rhizosphere soils of various species of the

gramineae family at the Nylsvley Nature Reserve and screened for the production of

siderophores.Threehundredandthirtyfivebacterialisolateswereinitiallyisolatedand

screenedforsiderophoreproductionusingauniversalchromeazurolS(CAS)agarassay

ofwhich34showedpositiveresultforsiderophoreproduction.Thesiderophorepositive

isolateswere investigated for in-vitro growth inhibition of selected Fusarium spp that

cause root rot in various crops. Of the 345 isolates, twelve rhizobacterial isolates

inhibitedthegrowthofF.oxysporum,F.graminearumandF.verticillioideusingthedual

culture assay technique on Potato Dextrose Agar (PDA) showing significant inhibition

zones.CurrentlythebiocontrolpotentialoftheseisolatesagainstFusariumspp.isbeing

investigated in-vivo under glasshouse conditions and their species identity is being

studiedusingthe16SrRNAnucleotidesequenceanalysis.

P86

PHYLOGENETICANALYSISOFBACTERIALPOPULATIONSOFPernapernaL.INALGOA

BAY,EASTERNCAPEPROVINCE,SOUTHAFRICA:IMPLICATIONSFORPUBLICHEALTH

Famewo,E.B.1Clarke,A.M.1andAfolayan,A.J.2

1DepartmentofBiochemistryandMicrobiology,2MPEDResearchNicheArea,DepartmentofBotany,UniversityofFortHare,Alice5700,SouthAfrica.

Despite theeconomicandnutritional importanceofPernapernawidelydistributed in

marineandestuarinewaters,thepotentialrisksassociatedwithitsconsumptioncannot

be over-emphasized. Sequences of DNA that codify to 16S rRNAwere retrieved from

Pernapernathatwerecollectedbeforeandduringredtide.TheDNAswereamplifiedby

PCRwithuniversalprimersandwasvisualizedonagarosegelelectrophoresis.Targeted

ampliconsize(586bp)fromeachDNAwasfurtherpurifiedandsequencedinABI3500

XL genetic analysers. Sequences of each of the bacteriumwere aligned, evolutionary

history was inferred using neighbour-joining method and a phylogenetic tree was

constructedusingMEGA6program.Twentyeightbacterialstrainswereidentifiedinthe

samplescollectedbeforeredtide;89.30%ofwhichbelongtoPhylumActinobacteriaand

wereallGram-positive.Theremaining10.70%belongtoPhylumSpirochaetesandwere

allGram-negative.Fewbacteriastrainswere identified inthesamplescollectedduring

red tide; most of which belong to Phylum Proteobacteria (36%) and Actinobacteria

(37%). Theywere all Gram-positive except those that belong to Phylum Spirochaetes

(27%)andwereGram-negative.Thus,PernapernainhabitingAlgoaBayarereservoirsof

various bacterial populations. The number of these bacteria assessed was drastically

reduced in the samples collected during red tidewhich could be due to the effect of

algal toxins. This in turns emphasizes proper cooking to eliminate bacteria and

avoidance of consumption of Perna pernabefore and during red tide respectively, in

ordertosafeguardthehealthoftheconsumers.

P87

ISOLATIONANDIDENTIFICATIONOFPOTENTIALPROBIOTICBACTERIAFROMSOUTH

AFRICANSAANENGOATSMILK

MaketeG.1.2,AiyegoroO.A.2andThantshaM.S.1

1UniversityofPretoria,DepartmentofMicrobiologyandPlantPathology,Pretoria,0001,SouthAfrica.2AgriculturalResearchCouncil,GastrointestinalMicrobiologyandBiotechnology,Irene,0162,SouthAfrica

Identificationandfurthertaxonomicclassificationoflacticacidbacteriaisessentialnot

onlyforunderstandingtheirindividualcontributionstofermentationprocesses,butalso

to reveal their roles in industrial and therapeutic applications and to study probiotic

candidature.Inthisstudy,probioticbacteriafoundinrawgoats’milkwereisolatedand

identified.Outofatotalof34isolates,17isolatespassedtheinitialselectioncriteriaas

putative probiotics. Analyses for the biochemical properties included catalase test,

determination of growth at temperatures 10oC and 45oC and CO2 production from

glucose. Lactic acid bacteria (LAB) were identified using standard API 50CH system,

partial16S rDNAgenesequencingandmatrix-assisted laserdesorption ionization-time

mass spectrometry (MALDI-TOF MS). The seventeen isolates were identified by

phenotypic characterization as Lactobacillus plantarum (16) and Lactobacillus

rhamnosus(1).Molecularidentificationbasedonamplificationof1.5kilobaseregionof

the 16S ribosomal DNA (rDNA), identified seven of the isolates as Lactobacillus

plantarumand tenasLactobacillus pentosus.MALDI-TOFMS identified the isolates at

the species level as Lactobacillus plantarum (16) and Pediococcus acidilactici (1).

Phenotypic characterization andMALDI-TOF identified Lactobacillus plantarum as the

dominant species found in raw goats’ milk. Whereas the 16S rDNA gene sequencing

identified Lactobacillus pentosus as the dominant species. Lactobacillus plantarum

strains were identified by phenotypic characterization and MALDI-TOF MS as the

dominant LAB found in South African Saanen raw goats’ milk. The combination of

applied methods for the identification of isolates have shown that Lactobacillus

plantarumwasthedominantspeciesinrawgoats’milkandPediococcusacidilacticitoa

lesserextent.

P88

P89

EFFICACYOFPROBIOTICBACTERIAINMANAGEMENTOFPOSTWEANINGDIARRHEAL

SYNDROMEONWEANEDPIGLETS

Dlamini,Z.C.1,2,Aiyegoro,O.A.2andOkoh,A.I.1

1DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,PrivateBagX1314,Alice,5700.2AgriculturalResearchCouncil,AnimalProductionInstitute,PrivateBagX2,Irene0062.

Weaning transition is a complicated phase in pig production, piglets are weaned

between the agesof 3 - 4weeksold. This phasemay results todigestivedisturbance

whichcausesgrowthset-backandlowfeedintakeinpiglets;whichultimatelyresultsto

death in some cases. This phase is frequently linked with high occurrence of Post-

WeaningDiarrhealSyndromes(PWDs),triggeredbypotentialentericpathogensuchas

E.coli. The aim of this study is to investigate the effect of probiotic bacteria

(Lactobacillus reuteri ZJ625, Lactobacillus reuteri VB4) on occurrence of scouring in

weanedindigenousandcommercialpigs.60(30commercialand30indigenous)weaned

pigletsblockedbyweightweredived into3 treatments:dietwithantibiotic,dietwith

no-antibiotic andnoprobiotic, anddietwithprobiotic.Microbiological compositionof

their faeces was monitored and after the probiotic treatment trial, pigs were

slaughtered and ileumwas removed formicrobial count. The findings from this study

showed increased LAB count by surviving range between 10 7 to 10 9 cfu/10ml and

reducedenterobacteriacountbysurvivingrangebetween102to106cfu/10mlinboth

faecalandileumcontentsamples.Fromtheresultsinthisresearchitismostlikelythat

theseprobioticswillofferasignificantbenefitsinpigfarmingandtherebyenhancethe

pigindustry’seconomy.

P90

P91

P92

CULTUREDANDWILDDUSKYKOB(Argyrosomusjaponicus),ARESERVOIROFHUMAN

PATHOGENICVIBRIOSPECIES

FriJ.,1NdipRN1,2,AngusPJ3andClarkeAM.1

1DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,SouthAfrica2FacultyofScience,UniversityofBuea,Buea,Cameroon3SouthAfricanInstituteofAquaticBiodiversity,SAIAB,SouthAfrica

BacteriaofthegenusVibrioareindigenoustothemarineenvironmentandtemporarily

abundant inwarmcoastalwaters. Theycause infectionstohumansandcommercially

important species of crustaceans, bivalves and fish. Seafood including fish could

thereforeserveassourcesofpathogenicVibriotohumans.Theaimofthestudywasto

isolate and characterize potential human pathogenic Vibrio associated with wild and

farmed dusky kob. Dusky Kob andwater sampleswere collected from fish farms and

KariegaEstuary in theEasternCapeProvinceandanalysed. Fish tissues (skin, gill and

gut) were aseptically excised and homogenised in sterile distilled water. The

homogenisedandwatersampleswerebothenriched inalkalinepeptonewater (APW)

and sub cultured on thiosulphate citrate bile-salt (TCBS) agar. Distinct presumptive

VibriocolonieswerepurifiedandconfirmedbyPolymeraseChainReaction(PCR)using

16srRNAprimersetsforthegenusVibrio.AmultiplexPCRwithprimersspecificforV.

cholerae,V.paraheamolyticus,V.vulnificus,andV.fluvialiswascarriedtodelineatethe

isolatetospecies.Atotalof450VibrioisolateshavebeenconfirmedbyPCR.Therestof

theworkisongoingandresultswillbecommunicatedbyOctober,2015.

P93

BIOACTIVEACTINOBACTERIAASSOCIATEDWITHAspalathuslinearis

Grobbelaar,M.C.,Ontong,V.,Kirby,B.M.

InstituteforMicrobialBiotechnologyandMetagenomics,UniversityoftheWesternCape,PrivateBagX17,

Bellville,7535.

Actinobacteria is a phylumofGrampositive bacteria that are abundant in soil. These

bacteria are frequently found in close association with plant roots, where they form

mutuallybeneficialrelationshipswiththeplant.Actinobacteriaproduceawidearrayof

bioactivecompoundsincludingmetabolitesusedasbiocontrolsubstances,plantgrowth

promotersorantibiotics.RooiboswasdiscoveredbyindigenouspeopleoftheWestern

Cape and has been used as a herbal remedy for over 300 years is currently sold

worldwide as anherbal tea.As rooibos it offers awide rangeof healthbenefits from

loweringcholesteroltotreatmentofeczema.StudieshaveshownA.linearisproducesan

array of secondarymetabolites, some ofwhich can be utilized by actinobacteria that

grow in closeassociationwith theseplants. Thesebacteriamightalsoproduce similar

metabolites as the rooibos plants, andmayhave interestingmedicinal applications as

well.Thepresentstudyaimstoinvestigatebioactiveactinobacteriaassociatedwiththe

commercially valuable indigenous South African plant, Aspaluthus linearis.

Actinobacterialisolatesfromsoil(380),root(170)andleaf(35)samplesweretestedfor

biosynthetic potential against a range of human and plant pathogenic bacteria, yeast

and fungi. The presence of oxidative enzymes was also studied. Preliminary results

showedantimicrobialactivityagainstawiderangeofpathogenswithnearly60%ofsoil

isolatesbeingactiveagainstB.cereusandalmost50%activityagainstS.aureus.These

findingssuggestthatbioactiveactinobacteriaarelinkedwithrooibosplantsandthatthis

isaviableapproachtofindinguniqueactinobacteria.

P94

CHARACTERIZATIONANDPATHOGENICITYOFENTEROBACTERSPECIESASSOCIATED

WITHBACTERIALBLIGHTANDDIE-BACKOFEUCALYPTUSSEEDLINGSANDCUTTINGS

Bophela,K.N.1,Brady,C.L.2,Venter,S.N.1andCoutinho,T.A.1

1DepartmentofMicrobiologyandPlantPathology,ForestryandAgriculturalBiotechnologyInstitute

(FABI),UniversityofPretoria,Pretoria0002,SouthAfrica2CentreforResearchinBioscience,Facultyof

HealthandLifeSciences,UniversityofWestofEngland,Frenchaycampus,Bristol,UnitedKingdom

BacterialdiebackwasfirstreportedinAustraliain1974fromCorymbriacitriodora.The

causalagentwasdescribedasXanthomonascampestrispv.eucalypti(=emendedX.dyei

subsp.eucalypti).Subsequent recordsofbacterialblightanddiebackwerereported in

SouthAfricaandBrazilandthecausalagentswerePantoeaananatisandXanthomonas

axonopodis, respectively. In recent studies strains of Enterobacter and related genera

were reported to have an associationwith blight of Eucalyptus. However, only a few

recordsexist in the literatureofEnterobacter speciescausingdisease inwoodyplants.

Mostspecies in thisgenusareknownascausalagentsofnosocomialandcommunity-

acquired infectionsofhumans. In a recent study,Enterobacter isolateswereobtained

together with Pantoea spp. from diseased and healthy stem, roots and leaves of

Eucalyptus seedlingsshowingblightsymptoms.Theaimof thisstudywasto identifya

collection of isolates from this genus previously obtained from diseased Eucalyptus

seedlingsandcuttings,anddeterminewhetherornottheywerepathogensofthishost.

Identificationswerebasedonamultilocussequenceanalysis(MLSA)approachbasedon

three housekeeping genes, gyrB, infB and atpD; the 16S rRNA gene was added for

comparison and genus level identification. Based on the MLSA, three previously

describedspecies,Enterobactermori,E.soliandE.asburiae,andeightpotentiallynovel

species (MLSA groups A-H) from two genera, Enterobacter and Kosakonia, within the

Enterobacteriaceae, were identified. Isolates from all species caused typical blight

symptoms in cuttings of a susceptible Eucalyptus grandis/nitens hybrid. These results

will impact on the future production of this susceptible clone and others in forest

nurseries.

P95

SOUTHAFRICANPhotorhabdusSPP.:GENETICANDANTIBIOTICDIVERSITY

vanWyngaard,M.G.D.1andHunter,C.H.1

1DisciplineofMicrobiology,SchoolofLifeSciences,CollegeofAgriculture,EngineeringandScience,

UniversityofKwaZulu-Natal,PrivateBagX01,Scottsville,3209.

Photorhabdusspp.areentomopathogenicnematodesymbiontsknownforsynthesising

antibioticcompoundswithbiocontrolpotential.Thisresearchaimedtoestablish levels

ofdiversityamongstasubsetof20SouthAfricanPhotorhabdussp.isolatessuppliedby

theSouthAfricanSmallGrainInstitute,AgriculturalResearchCouncilculturecollection.

Phenotypic diversity evaluation using API 20E test strips in conjunction with

supplementarytestswereof limitedvaluefor isolate identificationanddifferentiation.

GenomicdiversitywasdeterminedusingRAPD-PCR,16SrRNAgeneRFLP,andpartial16S

rRNAgenesequencing.MALDI-TOF-MSwasalsoevaluatedasaproteomicapproachto

identification.Species-leveldifferentiationbetweenisolateswasachievedwithRFLPand

genesequenceanalysis.Higher levelsofdifferentiationwereachievedusingRAPD-PCR

and MALDI-TOF-MS. Data obtained suggests that the isolates were closely-related

strainsofP.luminescens,withtwoisolatesidentifiedasXenorhabdusandPseudomonas

speciesrespectively.AntimicrobialactivityofthePhotorhabdussp.isolateswasassessed

usingbioassaysagainstEscherichiacoli,Micrococcusluteus,Bacillussubtilis,Rhizoctonia

solaniandBotrytiscinerea. IsolatesexclusivelydemonstratedactivityagainsttheGram

positive bacteria. Three representative isolates were chosen for analysis of their

bioactive compounds. Compound extraction and purification was attempted using

liquid-liquid extraction of broth supernatant using ethyl acetate, and methanol

extraction of lyophilised broth supernatant. Analysis of crude and partially purified

antibioticextractswasperformedusingTLC,UPLC-ESI-MSandGC-MS.Bothextraction

methods yielded active fractions, and ESI-MS analysis determined that each isolate

producedsimilarcompounds.AdominantpeakdetectedbyUPLC-ESI-MSsuggests the

activecompoundis3,5-dihydroxy-4-isopropyistibene.

P96

ISOLATIONANDSCREENINGOFCELLULOLYTICANDXYLANOLYTICMICROORGANISMS

FROMDECAYINGLIGNOCELLULOSICBIOMASS

MmangoZ1*,NwodoU1,MabinyaL.V12,andOkohA.I2

1SAMRCMicrobialWaterQualityMonitoringCentre,UniversityofFortHare,Alice5700,SouthAfrica.2DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,PrivateBagX1314,Alice5700,

SouthAfrica

Thestudyfocusesonthe isolationandscreeningofactinomycetescollectedfromfour

layersofsawdustsamplesinMelanivillage(woodfactory)respectivelyinEasternCape

ProvinceofSouthAfrica.Top,middle,bottomand fresh shavings layerwerecollected

from the sampling location and pre-heated to selectively kill bacteria that are not

actinomycetes.Mlagarwasusedintheisolationofactinomycetesandwasincorporated

withNystatin (concentration) andNalixidic acid (concentration) in order to retard the

growthofnon-actinomycetesandfungirespectively.Atotalof68actinomyceteswere

isolatedfromallthefourdifferentlayersofsawdust.Thirtyoneoftheseisolateswere

obtained fromthemiddle layer,22bottom layer,11 fromtop layersand4 fromfresh

shavings.Twelveisolateswithmarkeddistinctcolonycharacteristicswerescreenedfor

cellulose degradation activity and 8 of these isolates were (+) for the utilization of

cellulose as sole source of carbon. Eleven isolateswere able to degrade xylan as sole

source of carbon. Cellulose and xylanase activity as indicated by zone diameter of

clearancearoundthecolonyoncelluloseandxylanGram'siodinesolutionmediawere.

Three isolates out of the whole positive isolates coded as PLY1 (Micrococcus Luteus

(Strain B),MLY10 (Micrococcus Luteus (Strain A) and TLY3 (Micrococcus yunnanensis)

showed activity against both cellulose and xylan concurrently. The high cellulose and

xylanaseactivityobtainedfromtheseisolatesisanindicationofpotentialasindustrially

relevantastheymaybehandyintheindustrialbioethanolproductionprocess.

P97

MOLECULARCOMPARISONOFINTRASPECIFICVARIATIONINTHEBACTERIALSTRAINS

RESIDENTINTHERHIZOPLANEANDRHIZOSPHEREOFBAMBARANUT(Voandzeia

subterraneaL.Thouars)

Khantsi,M.andBabalola,O.O.

MicrobialBiotechnologyGroup,DepartmentofBiologicalSciences(RoomG10,NewSci.Building,Lab

G09),FacultyofAgriculture,ScienceandTechnology,North-WestUniversity,MafikengCampus,Private

BagX2046,Mmabatho2735.

Bambaranut(VorandzeiasubterraneanL.thouars)isaseedofAfricanoriginusedlocally

as a food source. It is the 3rd most important leguminous crop south of Sahara.

Considering thebiodiversityof indigenoussoilbacteria, ithasprovendifficult tomake

anylonglastingstructuralchangestothecompositionofbacteriawithinanygivensoil-

community. The study identifies and compares the bacterial strains residents in the

rhizosphere and rhizoplane of the Bambaranut using two fingerprinting methods:

terminal restriction fragment length polymorphism and denaturing gradient gel

electrophoresis and will be characterised by the analysis of PCR-amplified 16S rRNA.

Statistical analysis may not reveal the relationship between the rhizoplane and

rhizosphere samples, but it might demonstrate an influence of plant community

composition in resident bacterial strains.Morework also aims to study the effects of

these bacteria on tolerance to, and uptake of various kinds of stresses by plants for

development of potential plant-microbe systems useful for phytoremediation of soil

environment. Their use and other innovations could prove to be an environmentally

friendly strategy to ensure sustainable agriculture growth without recourse to costly

inputs, such as irrigation, however contributing to the food security of the poorest

people.

P98

EPIDEMIOLOGICALSURVEYOFSALMONELLACONTAMINATIONONBEEFCARCASSES

INABATTOIRSANDBEEFPRODUCTSINTHENORTHWESTPROVINCE,REPUBLICOF

SOUTHAFRICA

Mohapi,M.A.Ramaili,T.,Ndou,R.V.,Mwanza,M.

Department of Animal Health, Faculty of Agriculture Science and Technology, North West University,

MafikengCampus,PrivateBagx2046,Mmabatho,2735.SouthAfrica.

Foodbornepathogens,suchasSalmonella,mayremaininabattoirfloorsandwallseven

aftercleansingandposea riskofcross-contamination fromoneprocessingday to the

next. The aim of this study is to evaluate the prevalence of Salmonella spp in beef

carcasses in the abattoirs and themain purpose of studying abattoir pathogens is to

ensure that the number ofmicro-organisms on themeat is as low as possible by the

timeitleavestheabattoirs.Forthispurposesomeknowledgeofmicro-organisms,how

theybehaveandhowtocontrolandpreventthemisneeded.Theseorganismsmaybe

transferred to theouteror inner surfaceof carcassheld in the inspectionareawhere

theyarepackedwaiting tobe inspected.Sampleswere taken fromthe insideand the

outsideof thecarcassafter inspection (n=208)at four commercial abattoirsandmeat

samples(n=72)werepurchasedaswellfromthesupermarketsandthebutcheriesinthe

NorthWest Province and tested for the presence of Salmonella usingmicrobiological

testsuchasgramstainingtest,indoletest,AIPtest,thereafterthesamplewesendto

MALDI-TOF for further analysis. Of these, 5.8% of carcass samples were positive to

Salmonella organisms and 60.4% of purchased meats samples were positive to

salmonellaorganism.Thisresultsthatareobtainedfromtheretailsupermarketsandthe

butcheriesarequiethigherthantheresultsobtainedfromotherstudies.

P99

Determinationofmicrobialcontaminantsofinfectiousdog-to-dogbitewounds

presentedatDaleBeighleVeterinaryHospital(NorthWestUniversityMafikeng

Campus)

KgomotsoGalianSetsetse,NtebalengLesaoana,TaoleRamali,LehlohonoloKgaohelo

Mfane,MoratehiMefaneandMulundaMwanza



Limitedstudyhavebeencontactedaboutmicrobialcontaminantsof infectiousdog-to-

dogbitewound in SouthAfrica, hence this study aims to correlate the infectious and

non-infectiousbitewoundfromthetimeofpresentationtoevaluation.Atotalnumber

ofSixty(60)caseswherepresentedatDaleBeighleVeterinaryHospitalofbittencanines,

thesecaninesaremostlyinjuredonthehead,neckandupperextremelyregions(e.g.tail

andlimbs)ontheirbody.Themajorityofwoundswerelacerationandpuncturewounds

andallofthemwereinfectious.Thetreatmentofthesewoundsbecomesachallengefor

practitionersas the choiceof antibiotics is vital for correctbacterial target.Allwound

were sampled for microbiological culture and identification. Swabs were cultured

aerobicallyandevaluatedforantibioticsusceptibilityusingtheKirby-Bauerdiskdiffusion

test.Atotalof10isolateswereobtained,nothingsignificantwasfound.Staphylococcus

aureusweremostprevalent compared to theStaphylococcusepidermisby theuseof

catalase and coagulase test verification and molecular, followed by other suspected

Pseudomonasspecies.TheincidentalfindingofStaphylococcusepidermiswasconfined

tobethenormalmicroflora.Theantibioticsusceptibilitystudyshowedthattetracycline

andpenicillinwherethemosteffectiveantibioticsat100%toeliminatethesebacteria

whileampicillinwasthelesssusceptibleonewith67%ofresistance.Thisstudyaimedto

studythecommonbacterialcontaminantsofcaninebitewoundspresentedintheDale

BeighleVeterinaryHospitalanddeterminetheirantimicrobialsusceptibility inorderto

advisecliniciansonthecorrectantimicrobialtouse.

P100

EMPLOYINGCONTROLLEDMULTI-STARTERFERMENTATION,ANEWPERSPECTIVEIN

YEASTDYNAMICS

Bagheri,B.,Bauer,F.F.andSetati,M.E.

DepartmentofViticultureandOenology,InstituteforWineBiotechnology,StellenboschUniversity,South

Africa

There has been a growing trend toward application of multi starter culture in wine

industries to enhance characteristics of wine aroma. Several studies in wine

fermentationshave focusedon co-inoculationofSaccharomycescerevisiaewith single

non-Saccharomyces species and have reported positive contribution of the non-

Saccharomycesyeaststotheanalyticalcompositionandsensorialprofilesofthewines

produced. The current study explored the use of a multi-species yeast consortium

comprising 7 oxidative, weakly fermentative and strongly fermentative non-

Saccharomyces yeasts, as co-inoculants in fermentation with S. cerevisiae.

Fermentations were performed in synthetic grape juice medium and the population

dynamics monitored throughout fermentation using Automated Ribosomal Intergenic

SpacerAnalysis(ARISA).SugarconcentrationwasdeterminedenzymaticallyandGC-FID

was used for analysis of major volatiles. The fermentation performed with only the

consortiumofnon-Saccharomycesyeastswasrelativelyslowandonly57%oftheinitial

sugarswereconsumedafter40days.InthepresenceofS.cerevisiaeallthesugarswere

consumed and the fermentation was complete within 21 days. Lachancea

thermotolerans, Hanseniaspora uvarum, Starmerella bacillaris, andWickerhamomyces

anomalus were consistently the persistent non-Saccharomyces yeasts in the presence

and absence of S. cerevisiae. ARISA analysis and culture-dependentmethods showed

similarpopulationtrends.Significantdifferencesinthechemicalcompositionoftheend-

point samples were observed. The study showed the potential of ARISA as a tool to

monitorfermentationsandcomplexmulti-starterculturesasfermentationinoculantsto

enhancewinearomacomplexity.

P101

Effectofdosevariationsofrabiesvaccineoncanineantibodytitreresponse

MmutleKoos,LungisileTshitshi,LehlohonoloKhaoheloMefane,LubanzaNgoma,

MoratehiMefane.



Animals are usually vaccinated for different diseases in order to develop the immune

system.Inmostcases,itisrecommendedtovaccinateacertainamountofvaccineper

animal.Duringvaccinations,thereisalwaysacertainamountwhichislostduetostress

and inexperience of the vaccinator. The serological response of puppies to live rabies

vaccine was determined. Two sets of puppies were used andwere divided into sets,

being the control group and the other being experimental group. Different doses of

vaccine were used in experimental group and control group puppies got the

recommendeddose (WHO)whileotherpuppies in the set receivedwater. Bloodwas

collected from each puppy on the day of the arrival then vaccinatedwith live rabies

vaccineandseriallybledfromday6today30with6days interval.Serumrabiesvirus

neutralizing antibodies (VNA) were measured by a modified human enzyme-linked

immunosorbentassay(ELISA)test.Amongtheexperimentalgroupdogswerevaccinated

withdifferentdoses,wherebythefirsttwopuppieswerevaccinatedwithanamountof

0.25mland0.5ml.Onesetinthecontrolgroupreceived1mlvaccineandtheotherset

acknowledged1mlofwater.Theoverallresultsshowedvariationinantibodytitrelevel.

Themostrespondedonesamongallwerethe1mldosespuppiesduetothefactthat

the immune systemwas increasing from day one until the last day compared to the

othergroupswheretheimmunityfluctuatesfromdaytoday.

P102

CRYPTOSPORIDIUMINFECTIONINSHEEPANDGOATSAROUNDMAFIKENG,SOUTH

AFRICA

Mgiba,T.MandSyakalimaM

Cryptosporidiumisoneofthemostcommoncausesofwaterbornediseaseintheworld

andthediseaseiszoonotic.Livestockarethemainsourcesofwatercontaminationwith

the pathogen so are an important risk factor in the transmission of the disease.

Cryptosporidium has beendetected in pigs around theMafikeng area in SouthAfrica;

howeverothercarrierspecieshaveneverbeeninvestigatedinthearea.Thisstudywas

carriedout to identifyotherspecieswhichmaybeplayinga role incontaminatingthe

environmentwiththepathogen.Stoolsamplesfrom93smallruminantsbothsheepand

goatswere collected from8 farms between June andAugust in 2014. From the total

numberoftheanimals56weregoatsfrom6farmsand37weresheepfrom5farms.The

sampleswere examined for the parasite using ELISA test and a prevalence of 73.21%

wasfoundingoatsand62.1%insheep.Theprevalencewashighascomparedtothose

which were found in previous studies from other countries, therefore these species

needtobehandledwithgreatcareespeciallybecausethepathogeniszoonotic.

P103

DETERMINATIONOFQUARTERLYSUBCLINICALMASTITISUSINGDIFFERENT

SCREENINGMETHODSINDAIRYCATTLE

Mutle,MLG.,Moratehi,M.andMulunda,M.



Thesubclinicalformofmastitisisimportantbecauseitis15to40timesmoreprevalent

than the clinical form, lasts longer, difficult to detect, reduces milk production, and

affectsmilk quality. The studywas conducted to assess the effectiveness of different

screeningmethodsofdetectingsubclinicalmastitisfromquarterlymilksamplesofdairy

cattle. The tests thatwere used are the Californiamastitis test (CMT), Bacteriological

Culture test, Catalase test, API 20E Test and Antibiogram test for determining the

bacterialresistance.ThestudyareawhichwastheRooigrondCorrectionalServicesmilk

parlour plays a significant role in the livelihood of inmates. Milk from cows with

subclinical mastitis can be accidentally mixed into bulk milk. This poses a threat to

human health. Therefore this study was conducted to assess the effectiveness of

different laboratory screening methods on the detection of subclinical mastitis using

quarterly milk of dairy cattle. 40 quarterly milk samples were collected from 10

randomlyselectedmilkingcowsinapopulationof90.CMTwasusedasascreeningtest

andpositiveCMTsampleswereusedinbacteriologicalculturetest.Thebacteriological

culture test used was Gram staining test. The catalase test was then used as the

biochemicaltestusingsub-culturesofthesamplesfollowedbyAPI20Etest.Antibiogram

testwas the last test. It is concluded thatCMTmustbeusedatRooigrondbearing in

mindthatitiseasilyaccessibleandeasytousebyanyperson.

P104

Serologicalprevalencestudyofbovineviraldiarrhoeaincommunalfarmsaround

Mafikeng

MosimaneKandMwanzaM

DepartmentofAnimalScience,FacultyofAgricultureandTechnology,MafikengCampus,NorthWest


Bovineviraldiarrhoeavirus(BVDV)isthemostprevalentinfectiousdiseaseofcattle.It

causes financial losses from a variety of clinical manifestations. Persistently infected

cattlearethemainsourcefortransmissionofthevirus.Theprevalenceofbovineviral

diarrhoeadiseaseofallagecattlewastestedfromageof6monthsupto4yearsofage

cattle. Farming system in positive area might be due to mixed grazing as extensive

farming system is practiced and contact with other animals is seen this can lead to

spreadofthevirus.Thiscauseofproductionratetobelowinthefarm.Theaimofthis

studywas to determine the seroprevalence of BVD in communal farms. In this study,

1087sampleswererandomlycollectedfrom13villages.Animalselectedwerebasedon

theirbodyscorecondition(1-3scaleof5).InthisstudyElisatestwasusedtodiagnose

BVDV using serum but they are also other method to detect the virus such as

Fluorescent antibody, PCR and Immunohistochemically. Results obtained showed the

presence of serological positive cases in 3 villages (Vryhof 90%, Kabe 28% and

Skoolgesiget53%)outof13.werepositivemakingtotalnumberofpositiveanimals55

outof368(15%).Bovineviraldiarrhoeaisoneofimportantdiseaseofcattleworld-wide

affectingthereproductiveperformance.

P105

Identificationofgeneralcoliformbacteriafrommilkcollectedfromlactatingcows

amongstselectedfarmsteadofMafikengvillages

Sethaiso,J.

Department of Animal Science, Faculty of Agriculture and Technology, Mafikeng Campus, North West

University,PrivateBagX2046,Mmabatho,2735,SouthAfrica

Milk being a complex biological fluid, by its nature a good growthmedium formany

micro-organisms and due to the specific production it is impossible to avoid

contaminationofmilkwithmicro-organismsthereforethemicrobialcontentofmilkisa

majorfeatureindeterminingitsquality(Rogelj,2003).Thepresenceofbacteriainmilk

cancausesomereductionintherawmilkquality(Oliveretal.,2005).Theobjectivesof

theseresearchwastodeterminethemostprobablenumber(MPN)ofcoliformbacteria

andto identify thecoliformbacteriapresent inrawmilk thatcanaffecthumanhealth

andmilkhygienearoundareasofMafikeng. In this research studynine samples (milk

werecollectedinthebulktank)ofrawmilkwerecollectedfromdifferentfarmsteadof

MogosaneVillage(Mahikengarea).Collectionwasdoneusingsterilizedbottlesand5ml

of milk was collected directly from milk containers or bulk tank, all samples were

preparedandsampled.DifferentMediassuchas(Bufferedpeptonewater,Mannitolsalt

agar,Macconkey agar, Plate count agar and Nutrient agar) were used to culture the

samples. The results obtained from these biochemical tests indicated suspected

microorganisms that include, Salmonella from XLD agar, E. coli fromMacConkey agar

PseudomonasfromnutrientagaraswellasStreptococcusandStaphylococcusfromMSA

media.

P106

CHARACTERIZATIONOFCELLDEATHCAUSEDBYDIPLODIATOXINANDDIPMATOL,

MYCOTOXINSOFStenocarpellamaydis

Masango,M.G.1,2,Ellis,C.E.3andBotha,C.J.2

1ToxicologySection,AgriculturalResearchCouncil-OnderstepoortVeterinaryInstitute(ARC-OVI),Private

BagX05,Onderstepoort,0110.2DepartmentofParaclinicalSciences,FacultyofVeterinarySciences,UniversityofPretoria,PrivateBag

X05,Onderstepoort,0110.3MolecularEpidemiologyandDiagnostics(MED),AgriculturalResearchCouncil-OnderstepoortVeterinary

Institute(ARC-OVI),PrivateBagX05,Onderstepoort,0110.

Diplodiosis, a neuromycotoxicosis of cattle and sheep grazing on mouldy maize cobs

infectedbyStenocarpellamaydis, isconsideredthelastmajorveterinarymycotoxicosis

for which the causative mycotoxin is still unknown. The current study was aimed at

characterizing the cell death observed in mouse neuroblastoma (Neuro-2a), Chinese

hamsterovary (CHO-K1)andMadin-Darbybovinekidney (MDBK) cell linesexposed to

theS.maydismetabolites,namelydiplodiatoxinanddipmatol.Theroleofapoptosis in

the cell deaths was investigated using the caspase-3/7 and Annexin V-FITC flow

cytometry assays. In addition, transmission electron microscopy (TEM) was used to

correlate the apoptosis cell death pathway observed in this study with its typical

morphologies.Thecaspase-3/7andAnnexinV-FITCflowcytometryassaysshowedthat

exposureofNeuro-2a,CHO-K1andMDBKcellsto750µMofdiplodiatoxinanddipmatol

induceda caspase-dependent apoptosis in vitro.Ultrastructurally, the twomycotoxins

inducedmitochondrial damage, cytoplasmic vacuolation and nuclear fragmentation in

the three cell lines. These findings have laid a foundation for future studies aimed at

elucidatingindetailthemechanismofactionoftheS.maydismycotoxins.

P107

ADAPTATIONTHROUGHCHROMOSOMALREARRANGEMENTSINADAPTIVELYEVOLVED

Lachanceakluyveri

NerveZhou1,AnneFriedrich2,ConcettaCompagno3,JosephSchacherer2,KrishnaB.SSwamy4,

MichaelKatz5,SamueleBottagisi1,6,WolfgangKnecht1,ZoranGojkovic5,JurePiškur1

1DepartmentofBiology,LundUniversity,Lund,Sweden

2DepartmentofGenetics,GenomicsandMicrobiology,UniversityofStrasbourg,CNRS,UMR7156,Strasbourg,France.3DepartmentofFood,EnvironmentalandNutritionalSciences,UniversityofMilan,Milan,Italy.

4InstituteofMolecularBiology,AcademiaSinica,Taipei,Taiwan

5CalsbergLaboratories,GamleCarlsbergVej10,DK-1799CopenhagenV,Denmark.

6DipartimentodiBiologiaeBiotecnologie,UniversitàdeglistudidiPavia,Pavia,Italy

Large-scalechromosomalrearrangementsareanimportantsourceofevolutionarynoveltythat

mayhavereshapedgenomesofextantyeasts.Theydramaticallyaltergenomeorganizationand

geneexpression fuelingaphenotypic leap in response toenvironmental constraints.Although

the emergence of such signatures of genetic diversity is thought to be associated with

exhaustivehumanexploitationofyeasts,lessisknownonhowtheyappearinnature.Usingan

experimentalevolutionapproach,weattemptedtoreconstructanecologicalsetupthatmimicks

a fruit-yeast-bacteria microhabitat characteristic of every autumn when ripening fruits are

lacerated by fruit-juice feeding insects. By co-culturing a poor ethanol producing yeast,

Lachanceakluyveri, andbacteria for several generations in thepresenceofexcess glucosewe

established a cross-kingdomcompetition that couldhaveexisted in nature approximately 150

million years ago. Here we report the emergence and fixation of a novel “extra-banded”

karyotype after 720 generations. Such a karyotype was associated with a significantly higher

fitnessaswellasahigherfermentativecapacitycharacterisedbyastringentlyglucoserepressed

phenotype. Phenotypic microarrays technology (PM) revealed that the emergence of new

metabolictraitsmayhavebeenselectivelybeneficial.Wholegenomesequencingrevealedthat

the“extra-banded”karyotypewasasaresultofaduplicationandtranslocationeventinvolvinga

261kbsegment.Wemodestlyspeculate thatacross-kingdomcompetitionmayhaveplayeda

role in genome evolution and diversification of yeasts. Our strategy can be reconfigured to

developindustrialstrainsinincreasinglybecomingpopularcomplexindustrialprocessessuchas

mixedculturefermentations.

P108

PRIORINFECTIONOFWHEATWITHRUSTINDUCESRESISTANCETORUSSIANWHEAT

APHID

NjomHA1,TolmayVL3,MebaloJ3,TerefeTG3,NdipRN1,2GraemeB1

1DepartmentofBiochemistryandMicrobiology,UniversityofFortHare,SouthAfrica2FacultyofScience,UniversityofBuea,63Buea,Cameroon3 ARC-SmallGrainInstitute,PrivateBagX29,Bethlehem9700,SouthAfrica.

Induced resistance could be exploited as an important tool for pest management to

curtailtheamountofinsecticidesusedforpestcontrol.Thisstudyevaluatedtheeffect

of leaf rust infection onwheat plant colonizationwith RWA. Treatments consisted of

untreatedcontrolandtest(wheatseedlingsinoculatedwithurediniosporesofPuccinia

triticina race 3SA145 and later infested with aphids at days 3, 5, 7 and 9). Aphid

populationoneachplantwasdeterminedfor21dayspostinfestation.Diseaseresponse

ininoculatedseedlingswasscoredusingthe0to4infectiontypescale.Resultsobtained

showed infection types; 1+ and 3++ for SST347 and SST356 respectively. Few aphids

chose to colonise previously infected plants at the beginning. However, the aphid

populationgrowthdoubledlaterintheexperiment.Basedontheseresults,SST347and

SST356areresistantandsusceptibletoleafrustrace3SA145respectively.Priorinfection

of the wheat plants induced resistance to RWA. However, this resistance was later

switchedoffsuggestingthatinducedoractiveresistancemaybeshortlived.

P109

P110

MARINEACTINOMYCETESASASOURCEOFNOVELANTIMICROBIALAGENTS

Visser,R.1LeRoes-Hill,M.1andKudanga,T.2

1BiocatalysisandTechnicalBiologyResearchGroup,InstituteofBiomedicalandMicrobialBiotechnology,

CapePeninsulaUniversityofTechnology,Bellville75352DepartmentofBiotechnologyandFoodTechnology,DurbanUniversityofTechnology,POBox1334,

Durban,4000.

The discovery of antibiotics by Alexandre Flemming in 1929 has been a revolutionary

breakthrough in the field of bio-medicine. However, recently there has been an

increasedemergenceofantibioticresistantbacteria,whileatthesametimeadeclinein

thenumberofantimicrobialcompoundsenteringthemarketisbeingobserved.Thishas

ledtofearthatweareenteringapost-antibioticera.Itiswell-knownthatactinomycetes

produceantibiotics.Currently,there is littleknowledgeaboutthepotentialproduction

ofantibioticsbyactinomycetes isolated fromSouthAfricanmarineenvironments.This

environment is largely unexplored and represents a vast resource of novel marine

actinomycetes and their novel bio-active compounds. In this study, thirty strains of

marine actinomycetes were screened for their ability to produce novel bio-active

compounds. The bio-activity was tested against strains known to cause diseases,

includinganEnterococcusfaecalisvancomycin-resistantstrainandamethicillin-resistant

Staphylococcus aureus strain. Actinomycetes showing bio-activity were analysed for

theirbiosyntheticpotential by targeting specific gene clusters known tobepresent in

actinomycete genomes. Crude extractswereprepared fromactinomycete cultures for

furtheranalyses,includingthedeterminationofthebio-activityoftheextractsandtheir

LC-MS profiles. Strains that showed potential for the production of novel bio-active

compounds were identified by 16S rRNA gene sequence analysis. Preliminary results

indicate that some of the marine actinomycete strains show antimicrobial activity

against Acinetobacter baumannii ATCC 19606 as well as Enterobacter cloacae subsp.

cloacaeATCCBAA-1143,bothofwhichareknowntocausenosocomialinfections.

P111

DETERMININGANTIMICROBIALPROPERTIESOFPHENOLICSDETECTEDINPEAT

SAMPLES

Weels,S1.,Kudanga,T1,2.,LeRoes-Hill,M.1,Welz,P.J.1

1BiocatalysisandTechnicalBiologyResearchGroup,InstituteofBiomedicalandMicrobialBiotechnology,

CapePeninsulaUniversityofTechnology,POBox1906,Bellville,SouthAfrica,75352DepartmentofBiotechnologyandFoodTechnology,DurbanUniversityofTechnology,POBox1334,

Durban,SouthAfrica,4000.

Peatlandsarewetlandswithathickwater-loggedorganicsoillayer(peat)whichismade

up of dead and decaying plant material. It has been observed that when domestic

wastewaterpassesthroughpeatlands,contaminantsandpathogensareremoved.This

phenomenoncouldeitherbeduetothepresenceofphenolicspossessingantimicrobial

properties, antimicrobials produced by residing microorganisms or the acidic

environmentcausedby thechemicalpropertiesof thepeat.Theworkpresentedhere

focusedonthephenolics found inpeatlands.Thephenoliccompoundswereextracted

frompeatsamplescollectedfromthreedifferentpeatlandslocatedintheWesternCape

andanalysedby liquid chromatographymass spectrometry.Theextractedcompounds

and selected commercially available phenolic compounds similar to those detected in

peatlands were tested for antibacterial activity by performing disk diffusion and

minimum inhibitory concentration (MIC) tests against eight Gram-negative and six

Gram-positive American Type Culture Collection (ATCC) test strains. Cis-isoeugenol

exhibited the greatest antimicrobial activity, while vanillin and syringol exhibited the

least.CommonMICvaluesforthesecompoundsrangedfrom2.5mg/mlto16mg/ml.

Theresultsobtainedinthisstudysuggestthatthephenolicspresentinpeatlandscould

bewhollyorpartiallyresponsibleforthepathogenremovalabilityofpeatlands.

P112

Co-expressionofsulfhydryloxidaseanddisulphideisomeraseresultinthe

productionofsolubleCRM197inEscherichiacoli

Roth,R.,Tsekoa,T.,Kwezi,L.,Pooe,O.,Stoychev,S.,VanZyl,P.,Crampton,M.

CSIRBiosciences,CSIR,MeiringNaudeRoad,Brummeria,Pretoria,0001

Cross ReactingMaterial 197 (CRM197) is a safe and effective T-cell dependent protein

carrier for polysaccharides used in the manufacture and application of multivalent

conjugate vaccines, such as the pediatric vaccine Pevnar, a multivalent vaccine given

routinelytoinfantssince2000tohelpprotectagainststrainsofPneumococcalbacteria.

It is amutant form of the diphtheria toxin, with a single base change at position 52

(glycine to glutamic acid). The current commercially available product is produced by

isolationfromculturesofrecombinantPseudomonasfluorescens,andisveryexpensive,

at ~$4,000/10mg (non-cGMP grade). Reports in the literature show that CRM197 has

successfully been expressed in E. coli, although in an insoluble form, and only in the

presence of an affinity tag. Codon-optimisationwas performed for the CRM197 gene,

and the product was successfully expressed in E. coli in a soluble form when co-

expressed with the chaperone proteins, sulfhydryl oxidase and disulphide isomerase.

Co-expression of a sulfhydryl oxidase and disulphide isomerase in E. coli has been

reportedtoaidthesolubilityindifferentinsolubly-expressedrecombinantproteins,with

these enzymes aiding in the formation of disulphide bonds. CRM197 contains two

disulphide bonds which may be critical for the production of a soluble recombinant

proteininE.coli.Theinclusionofahistidineaffinitytagallowedforpurificationofthe

soluble recombinant CRM197, although a non-tagged version was also successfully

expressed. Analysis of the purified CRM197 showed a similar structure to the P.

fluorescens-produced commercial CRM197 in terms of primary structure, with only

minimal differences observed in native, higher-order conformation. This is the first

report of the soluble production of CRM197 in E. coli and through further production

optimisationcouldprovidea lessexpensiveadjuvantforformulationofpolysaccharide

basedvaccines.

P113

ANTIMICROBIALANDANTIOXIDANTPROPERTIESOFFUNGALENDOPHYTESFROM

Kigeliaafricana

MhlangaP.F.,GovindenR.

DisciplineofMicrobiology,DepartmentofBiotechnology,CollegeofAgriculture,EngineeringandScience,

UniversityRoad,WestvillePrivateBagX54001Durban4000

Endophytes have become microorganisms of interest in the microbial chemistry

communitybecauseoftheirpotentialtoproducesignificantbioactivemolecules,which

canbeutilized in the fieldsofmedicine, agriculture and industry.Kigelia africana is a

widelyusedtraditionalmedicinalplant inAfrica. Inapreviousstudyonehundredand

eightythreefungalendophyteswereisolatedfromK.africanaleaves.Asubsetofthese

isolateswere insolidsubstrate fermentationonricemedium.The fungalbiomasswas

extracted with ethyl acetate, evaporated to dryness and reconstituted in dimethyl

sulfoxide (0.1 mg/ml) and used for determination of antimicrobial and antioxidant

activity.Candidaalbicans (ATCC90025),Aspergillus fumigatus (Acc.No.:GU992275.1)

and Paecilomyces formosus (Acc. No.: GU968673.1) were used as fungal test strains,

whilePseudomonasaeruginosa(ATCC35032),Staphylococcusaureus(ATCC43300)and

Escherichia coli (ATCC 35218) were used for testing of antibacterial activity. Isolates

ZF42, ZF32, KZ3, ZF32 and ZF46 exhibited the best activity against most of the test

strainsandthese isolateswere identifiedbymicroscopiccharacteristics.The free radical

scavenging activity of the fungal isolateswasmeasuredby2, 2’- diphenyl-1-picrylhydrazyl

(DPPH)assay.Thescavengingeffectsofthetestedisolateswereinthefollowingorder:ZF42

> KZ60 > KZ50 > KZ3. The results of the study confirm the potential of endophytic fungi

associatedwithK.africanaasstrongantimicrobialandantioxidantproducers.

P114

REVIVALANDCHARACTERIZATIONOFENDOSPORE-FORMINGBACTERIAFROMAN

ANCIENTSEDIMENTCOREOBTAINEDFROMTHEMFABENIPEATLAND,SOUTHAFRICA

Naidoo,S.andHunter,C.H.

DisciplineofMicrobiology,SchoolofLifeSciences,CollegeofAgriculture,EngineeringandScience,

UniversityofKwaZulu-Natal,PrivateBagX01,Scottsville,3209.

Aerobicendospore-formersareconsideredtobepotentiallyusefulindicatorsofclimate

and/orenvironmentalchange.Theabilitytoformendosporesallowscertainbacteriato

surviveunfavourableconditionsandremaindormantforextendedperiodsoftime.The

MfabeniPeatlandinKwaZulu-Natal,isregardedasoneoftheoldestactivepeatlandsin

SouthAfricadatingbackatleast60000years.Astudywasundertakenwiththeaimof

revivingdormantendosporesfromsectionsofanancientsedimentcorethathadbeen

carbon dated to dates ranging from 514 to 33 500 years. Sediment samples were

heatedat80oCfor15minandthensubjectedtoasequentialextractionprotocolpriorto

dilutionseriesplatingontoselectedmedia.Thebacterialdiversityamongsttherevived

endospore-formerswasthenassessedusingRep-PCRcoupledwithhighresolutionmelt

analysis(HRMA).Severalprimersetstargetingthespo0AgeneandthehypervariableV3

and V4 regions of 16S rRNA genewere evaluated for HRMA. Taxonomic ranking and

phylogeny of representative genotypes was assessed through sequence analysis of

amplified partial 16S rRNA gene fragments. A total of 2204 colonies were revived,

including 10 isolates cultured from a 33 500 year old sample. Results of this study

indicate the variations in bacterial diversity which span timeframes across varying

environmentalconditions.For futurestudiesphysiologicalprofilingofselected isolates

willbeundertakeninordertolookforpossiblemarkersofenvironmentalchange.

P115

LONGITUDINALPREVALENCEANDANTIBIOTICSUSCEPTIBILITYOFNASOPHARYNGEAL

CARRIAGEOFSTAPHYLOCOCCUSAUREUSINHEALTHYINFANTSANDTHEIRMOTHERS

INABIRTHCOHORTINTHEWESTERNCAPE

ShimaM.Abdulgader1,SamanthaAfrica1,LourensRobberts1,MarkP.Nicol1,2,Heather

Zar3

1DivisionofMedicalMicrobiology,DepartmentofClinicalLaboratorySciences,UniversityofCapeTown;2Institute of Infectious Diseases and Molecular Medicine, University of Cape Town; 3 Department of

Paediatrics and Child Health, Red Cross War Memorial Children’s Hospital, and MRC Unit on Child &

AdolescentHealth,UniversityofCapeTown

Staphylococcus aureus carriage is a risk factor for subsequent infections. Data on

carriage of S. aureus in the community in South Africa, and associated resistance

patternsarescant.Weaimedtodeterminetheprevalenceofnasopharyngealcarriage

andantimicrobialsusceptibilitypatternsofS.aureusinSouthAfricaninfantsduringthe

firstyearoflife,andtheirmothers.Nasopharyngealswabs(NP)werecollectedfrom137

mother-infantpairsatbirthandthenevery2weeksduringthefirstyearoflifeaspartof

a birth cohort study, the Drakenstein Child Health Study. Swabs were cultured for S.

aureusandantibioticsusceptibilitytestingdoneaccordingtotheClinicalandLaboratory

Standards Institute guidelines. Of 137 mothers, 15% (20) carried S. aureus in the

nasopharynx at birth.Only 2% (3/137) of infantswere colonized at birth but carriage

reached a peak at 4 weeks where 57% (79/137) had S. aureus, this subsequently

declinedto2%at1yearofage.Ofnote,noMRSAwasdetected inmothers,andonly

6/137 infants (4%) carried MRSA during the first year of life. Maternal carriage was

associatedwithinfantilecarriageatbirth.Of11MRSAisolates10wereresistanttoboth

gentamicin and clindamycin. Of 714methicillin susceptible S. aureus 78% (563) were

resistant to penicillin, 2.6% (19) to ciprofloxacin, 8% (63) to gentamicin, 2.6% (19) to

trimethoprim-Sulfamethoxazole, and 2.3% (17) to rifampicin. Nasopharyngeal carriage

of S. aureus in the Drakenstein study populationwas 15% for pregnantmothers and

variedbetween2%-57%forinfantsduringthefirstyearoflife.MRSAwasnotdetected

inmothers,andonlyinasmallproportionofinfants.

P116

THEUSEOFBACTERIALCELLSURFACEDISPLAYASABRUCELLOSISANTIGENDELIVERY

SYSTEM

SGoolab1,2,HvanHeerden2,RRoth1,CKenyon1andMCrampton1

1CSIRBiosciences,POBox395,Pretoria,SouthAfrica.2DepartmentofVeterinaryTropicaldiseases,FacultyofVeterinaryScience,UniversityofPretoria,Private

bagX04,Onderstepoort,Pretoria0110,SouthAfrica.

Brucellosis remains one of the most widespread, under-reported global zoonotic

diseases in the world. The main economically problematic species affecting both

livestockandhumans includeBrucellaabortus,B.melitensis, andB. suis. Spontaneous

abortion and infertility occurring in cattle is the consequence of bovine brucellosis

infection,whichistransmittedbyB.abortus.Currentlythesuperiorityoflive-attenuated

vaccinestostimulateaneffectiveimmuneresponsesupersedessubunitvaccinesagainst

brucellosis,yetattheexpenseofaconstantserologicalresponseduringdiagnosis,being

infectioustohumansandstimulatingabortionwhenadministeredtopregnantanimals.

Using the Escherichia coli outer membrane protein A (OmpA) display system a safe,

recombinant vaccine will be developed to encourage cell-mediated immunity and

unarguably conferprotectionduringbrucellosis infection.Aweb-based vaccinedesign

program (Vaxign) was utilized to predict efficacious vaccine targets based on reverse

vaccinology.BcellbutprimarilyTh1cellimmunitywasthereafterutilizedasparameters

toderivepotentialantigenicpeptidetraitsoftheoutermembraneproteins,Omp16and

Omp19 vaccine targets using in silico databases (IEDB and Epitopia servers).

Furthermore, in order to characterize surface exposed loop regions proteinmodelling

was performed for OmpA, Omp16 and Omp19. These surface exposed and antigenic

epitopeswill bedisplayedon the surfaceofE. coli usingOmpAas theanchorprotein

therebyrecapitulatingprotectiveimmunityusingamultipleepitopevaccineapproach.

P117

SURVIVINGTHEACIDBARRIER:RESPONSESOFVibriocholeraeO1ANDO139TO

SIMULATEDGASTRICFLUID

SinghA1,BarnardTG1

1Water and Health Research Centre, University of Johannesburg, PO Box 17011, Doornfontein, 2028,

SouthAfrica,Johannesburg,SouthAfrica

When bacteria are subjected to low acidic pHs of the gastric environment they may

entertheviablebutnonculturable(VBNC)stateofsurvival.Inthisstatebacteriacannot

beculturedonsolidmedia,stillexhibitsignsofmetabolicactivity(viability).Inthisstudy

the response of pathogenic Vibrio cholerae O1 and O139 to low pH simulated

environments of the human stomachwas evaluated for their survival by culturability

(plate count) and viability (flow cytometry- FC) assays. Bacteria were acid challenged

with simulated gastric fluid (SGF) at pH 1.5, 2.5, 3.5 and 4.5 over a period of 180

minutes.ExposuretoSGFupto120minincreasedacidtoleranceoftheVibrio’suptopH

3.5withacidchallengeoccurringatpH4.5.BacteriawereculturablefrompH2.5–4.5up

to60minSGFexposure.ThestationaryphaseculturesofVibriowereabletosurviveSGF

atallpHsinan‘injured’statewithFC.Thiscouldpossiblymeanthatthebacteriahave

enteredtheVBNCstageofsurvival.Thisisaworryingpublichealthconcernduetothe

factthatoncefavourableconditionsarise(intestines)theseVibriocanchangebacktoan

infectiousstateandcausedisease.

P118

ROOTASSOCIATEDMICROORGANISMSINCROPPRODUCTIVITY.

Oberholster,T.,Valverde,A.andCowan,D.A.

DepartmentofGenetics,CentreforMicrobialEcologyandGenomics(CMEG),UniversityofPretoria,

Pretoria,SouthAfrica,0028

Plantrootsassociatewithlargemicrobialcommunitiesandincludemicroorganismswith

plantgrowth-promotingactivities.Therefore,investigatingtherhizospheremicrobiome

of important crops will provide an indication of soil and plant health. This may

contribute to thedesignofmanagement toolswherebyoverall yield canbe improved

through the promotion of specific combinations of rhizosphericmicroorganisms. Four

commercially important crops (maize, potato, soybean and sorghum) constituted

approximately 13%, 4%, 2% and 0.2% of the total gross value of South African

agriculturalproductionfor2012/13.Atotalof162soilsamples(controlandrhizosphere)

were collected from maize, potato and soybean pivots in Mpumalanga at four time

points (pre-plant, seedling, flowering and harvesting stages). Sorghum samples (168)

werealsocollectedateachtimepointfromfieldsinLimpopoandtheFreeState(2soil

typeseach).SoilswereanalysedforpH,electricalconductivity,Na+,K+,Ca2+,Mg2+,total

phosphate, percentage nitrogen and percentage carbon. T-RFLP fingerprinting of the

fungalITSregionsand16SrRNAgeneforbacteriaandarchaeaareneededtoassessthe

change in dominant microbial taxa. Amplicon sequencing will also be performed on

selectedsamplesfromsorghum.Initialresultsindicatedthatbacterialcommunitiesfrom

themaize rhizosphere structure separately frombulk soil.Bacterial communities from

both maize and potato structured according to time point, whereas soybean

communitieshadnosignificantstructure.Sorghumcommunitiesstructuredaccordingto

timepointandsoiltype.Thissuggeststhatbacterialcommunitiesmaybedifferentially

recruited throughout thedevelopmental stagesof theplantaswellas indifferentsoil

types.

P119

PHENOTYPICSWITCHINGINCandidaalbicansINFLUENCESPROSTAGLANDINE2

PRODUCTION

ThabisoMotaung1*,CarolinaPohl1,JacobusAlbertyn1,RuanElls1,2,ChantelSwart1,

OlihileSebolai1

1DepartmentofMicrobial,BiochemicalandFoodBiotechnology,UniversityoftheFreeState,Bloemfontein,SouthAfrica2NationalControlLaboratoryforBiologicalProducts,UniversityoftheFreeState,Bloemfontein,SouthAfrica

Thehumancommensalyeast,Candidaalbicans, iscapableofswitchingfromtheyeast

tothefilamentousphaseunderphysiologicalconditionssuchas37°C,neutralpHanda

nutrient-poorenvironmentsuchasthephagosome.Insidethehost,theyeastcellsare

disseminated via the bloodstream to organswhere they switch to invasive filaments.

Under unique culture conditions, C. albicans can also switch epigenetically from the

typicalwhite, to thegreyand/or themating competentopaquephase,bothofwhich

areassociatedwithskininfections.Inthisstudy,anovelswitchingsystem,calledwhite-

gray-opaque-like(WGOL)wasfoundtoinfluenceproductionofprostaglandinE2(PGE2),

an immunomodulatory (and often pro-inflammatory) eicosanoid derived from the

polyunsaturatedfattyacid,arachidonicacid(AA).Thefourdistinctcelltypes(i.e.white,

lightpink,darkpinkandroughpink) inthisswitchingsystemcanundergoastochastic

phenotypicswitchwhensubjectedtodifferentenvironmentalcuessuchasglucose,5%

CO2,N-acetylglucosamineandphysiologicaltemperatures.Further,WGOLswitchinghas

been found to influencedifferent virulence factors suchasgerm tubeproductionand

resistance to theantifungaldrug, fluconazole. SinceC.albicans biofilmsare known to

producemorePGE2thanplanktoniccells,weanalyzedbiofilmsofWGOLcelltypesand

discovered that they can form PGE2 at 25 °C and 37 °C,with a significantly increased

productionbywhiteandroughpinkcellsatthesetemperatures,respectively.SinceAA

isenzymaticallyreleasedfromthehostphospholipidcomponentandusedbyC.albicans

toproducePGE2,itistherefore,arguedthattheabilityofWGOLswitchingtoinfluence

thisvirulencefactormayplayaroleintheabilityofC.albicanstocauseinflammationin

specifichostsites.

P120

DOESTHEVOLUMEMATTER?ANINSIGHTINTOMODELLINGANDOPTIMIZATIONOF

BIOHYDROGENPRODUCTIONACROSSSCALES

Sewsynker,Y1andGueguimKana,E.B.G2

1,2DepartmentofMicrobiology,UniversityofKwaZulu-Natal,PrivateBagX01,

Pietermaritzburg,3201.SouthAfrica.

Renew interest in biohydrogen production as a potential alternative to the depleting

fossilfuelshaspromptedresearchtowardsthisfuel.Scaleupofthisprocessrequiresthe

development of processmodels that relate the keys operational parameterswith the

hydrogenyields.Inthispaper,ResponseSurfaceMethodology(RSM)wasusedtomodel

andoptimizebiohydrogenproductionattwodifferentprocessscales(80and800mL).

Theinputvariablesconsistedof inoculumsize(10-50%),molassesconcentration(100-

300g/L)andhydraulic retention time (10-48hours)and theoutputwas thehydrogen

yield. Seventeenexperimental datawere generatedat each scale andused formodel

developmentandprocessoptimization,thusatotaloftwomodelsateachscale.Models

gaveR2valuesof0.97and0.89for80and800mLexperiments,respectively.Validation

experimentsgave inputsof34.84and32.71%% for inoculumpercentage,100g/L for

molasses concentration and 41.84 and 38.44 hours for HRT for 80 and 800 mL,

respectively. Results showed that the experimental yield of 0.99mol H2/mol sucrose

againstthepredictedvalueof1.09molH2/molsucrosefor80mland0.70molH2/mol

sucrose against the predicted yields of 0.72 mol H2/mol sucrose were obtained. The

slight variation in experimental conditions for optimal hydrogen yield across the

different scales indicates that volume does impact process optimization and scale-up.

Thus,modellingsystemssuchasRSMmaybeusedtoaccuratelymodeltherelationships

between the considered process inputs across bioprocess scales for fermentative

biohydrogenproduction.

P121

PRODUCTION,PURIFICATIONANDCHARACTERIZATIONOFPHYTASEFROM

Enterobactersp.ACSS

Chanderman,A.,Puri,A.K.,PermaulK.andSinghS.

DepartmentofBiotechnologyandFoodTechnology,DurbanUniversityofTechnology,P.O.Box1334,


Phosphorous isanessentialelementwhich isprimarily storedasphyticacid inplants.

The phytate anion chelates with metal ions, proteins and lipids, and therefore, is

regarded as an anti-nutrient. Phytases hydrolyse phytate into orthophosphates and

inositolphosphateandreleasetheessentialmetal-ions. Inthisstudy,bacterial isolates

fromdifferentgeographicalregionswerescreenedandselectedbasedontheirphytate-

hydrolysing abilities. Sevenphytaseproducing isolateswere identifiedusing 16S rRNA

sequencing.Phytaseproductionwascomparedbetweenisolatescultivatedondifferent

agro-industrial residues (wheat bran, sugarcane bagasse, orange peel and corn cobs).

Wheatbranwas thebestsourceofphytate forall isolates, followedbycorncobsand

orangepeel.AC1,AC2,AC3,AC6andAC7produced1.87U/ml,2.48U/ml,1.58U/mland

1.28 U/ml of phytase in submerged fermentation at 37°C after 24 h, respectively.

Process parameters for enhanced phytase production by the best producer,

Enterobacter sp. ACSS, were optimized using one-variable-at-a-time approach.

Application of statistical tools such as Plackett-Burman design, the steepest ascent

method,andresponsesurfacemethodologysignificantly improvedphytaseproduction

by4.6–foldinshake-flasks.Inaddition,anoverall2.9-foldincreasewasattainedinfed-

batchfermentationsina5llaboratoryfermenter.Thephytasewaspurifiedasa62kDa

protein usingDEAE-anion exchange and Superdex-size exclusion chromatography. The

enzymeisactiveinawiderangeoftemperature(40-80°C)andacidicpH(2.0-6.0)andis

activated by Ca+2, Mg+2 and Mn+2. Overall, the thermo-acid-stable phytase from

Enterobactersp.ACSSisapotentialbiocatalystofmajorindustrialinterest.

P122

SCREENINGANDPRODUCTIONOFATHERMOANDACIDSTABLEPHYTASEPRODUCER

Zininga,J.,Puri,A.K.,Permaul,K.andSingh,S.

DepartmentofBiotechnologyandFoodtechnology,DurbanUniversityofTechnology,SteveBiko

Campus,BuildingS9,Lab0

Phytase (myo-inositol-hexakisphosphatephosphohydrolase)hydrolysesphyticacid into

orthophosphatesandinositol.Thisreleaseofphosphorousbenefitsmonogastricanimals

whichareunable todigestphyticacid-containing feed, therebyegestingandexcreting

massiveamountsofphosphorousintotheenvironmentcausingphosphorouspollution.

Phytaseisthereforepreferredoverchemicalinorganicphosphatesasafeedsupplement

and in mitigating the environmental phosphorous pollution. However, in order to

withstand the high temperature during manufacturing of feed-pellets, and to remain

active inextremeconditionsprevailing in thegastricenvironment,an idealphytase in

needed. In this study, we demonstrate the isolation of thermophilic bacteria from a

compost site near Durban followed by screening for potent phytase producers with

requisite characteristics. Four phytase producers were selected and the best isolate

produced 7.9 U/ml of phytase under submerged fermentation in phytase-screening-

medium. Based on microscopic characteristics and 16S rDNA sequencing this

thermophilic isolatewas identified asBacillus ginsengihumi.Phytase production byB.

ginsengihumi was optimized by one-factor-at-a-time approach followed by further

optimization using statistical methods. This resulted in a 4.2-fold increase in phytase

production.ThephytaseisactiveoverawiderangeofpH(2.0to10.0)andtemperature

(50to90°C)showingoptimalactivityatpH4.0and70°C.Theenzymeisthermo-andpH-

stableanditshowedresistancetopepsinaction.Thisphytaseissuitableforapplication

assupplementintheanimalfeed.

P123

BATCHANDFED-BATCHPRODUCTIONOFPHYTASEFROMThermomyceslanuginosus

Makolomakwa,M.,Puri,A.K.,PermaulK.andSinghS.



Absence or inadequate levels of phytic acid (inositol hexakisphosphate) hydrolysing

enzyme,phytase,resultsinpoorgrowthofmonogastricanimals.Theundigestedfaecal

phytate contains significant amount of phosphorus that poses major environmental

problems such as water pollution and algal blooms. Phytase (myo-inositol-

hexakisphosphate phosphohydrolase) is routinely used as food and feed additive that

improves the growth and development of monogastric animals and minimizes the

environmental phosphate load. Thermostable phytases are of considerable industrial

importanceinthemultibilliondollarbiotechnologyindustryduetotheirrobustnessand

suitability to harsh processing conditions. The thermophilic compost-dwelling fungus

Thermomyceslanuginosusisanattractivesourceofvariousthermostableenzymes.The

present study focuses on statistical optimization of culture variables affecting

productionofphytasefromT. lanuginosus.Peptone, incubationtimeandtemperature

were themost significant factorsaffectingphytaseproduction fromT. lanuginosus, as

identifiedbyPlackett-Burmanndesign.Optimisationof significant factorsbyResponse

Surface Methodology resulted in an overall 9.1-fold increase in phytase production,

whichwasvalidatedinshakeflasksandina5llaboratorybioreactor.Additionally,a2.3-

fold improvement in phytase production was observed by feeding 700 g/l of glucose

after every 30 h. The optimized production parameterswill be useful for commercial

productionofphytaseusingT.lanuginosusinbatchandfed-batchcultivations.

P124

P125

P126

COMPARATIVESTUDYOFCYANATEHYDRATASEPRODUCTIONBYDIFFERENTSTRAINSOFTHERMOPHILICFUNGUSThermomyceslanuginosus

NekhumbeD.E,RanjanB,KumarS,SinghS



Cyanate isatoxiccompoundreleased intotheenvironmentfromdifferentminingand

foodindustries.Cyanateismainlyreleasedfromtheminesduringtheextractionofgold,

steeletc. into thewastewaters. Similarly, largeamountsof cyanogenicglycosidesare

releasedduringstarchproductionfromcassava,inmanyAfricancountries.Theenzyme

cyanate hydratase catalyzes the reaction of cyanate with bicarbonate to produce

ammoniaandcarbondioxide.Fungiareextremelyusefulinbiotransformationprocesses

due to the presence of robust enzymes which are potentially important in industrial

biocatalysis and biorefinery applications. Thermomyces lanuginosus, a thermophilic

fungus,isknowntoproduceindustriallyimportantenzymessuchasxylanase,amylase,

andlipase.ArecentstudyconductedinourlaboratoryonT.lanuginosusstrainSSBPhas

revealed the presence of a cyanate hydratase gene in the fungus through secretome

analysis. The present investigation therefore focuses on the production of cyanate

hydratase from three different strains of T. lanuginosus and its characterization.

Different carbon and nitrogen sources were tested for the optimization of cyanate

hydrataseproduction inshake flask fermentations. Preliminarystudieshave indicated

that the enzyme is inducible in the presence of cyanate in the production medium.

Among the three different strains tested for cyanate hydratase production, T.

lanuginosus SSBP displayed the highest enzyme titre of 10 U/ml and has also shown

broadtemperatureandpHoptima.Futurestudieswillfocusonthecharacterizationand

applicationoftheenzyme.

P127

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P129

ModeofinhibitionofTulbaghiaviolaceaonAspergillusflavus

VuyokaziBelewa,CarminitaFrostandBeneshSomai

Over the past few years there has been a steady increase in fungal infections by

opportunistic fungal pathogens such as Aspergillus and Cryptococcus especially in

immunocompromised persons. Owing to a limited arsenal of antifungal drugs, fungal

resistancetowardstheseagentshasbeguntorise.Furthermore,someantifungalagents

have exhibited toxicity and therefore there is a need for a search for newer drugs or

alternative means of treatment. Aqueous extracts of Tulbaghia violacea have been

shown to have antifungal activity against a variety of microorganisms; however their

mechanism of action has not been investigated. This study was undertaken to

investigate themode of inhibition of T. violacea aqueous plant extracts onA. flavus.

Aspergillus flavus spores treated with various concentrations of the plant extract (0-

12.5mg/ml)showedasignificantreductioninβ-(1,3)glucanproductionandinhibitionof

β-glucan synthase. Chitin production was also reduced in the presence of various

concentrationsoftheplantextract,asaresultofthe inhibitionofchitinsynthase.The

broad spectrum of antifungal activity of T. violacea aqueous extract againstA. flavus

makesitapotentialchemotherapeuticagentfortreatmentoffungalinfections.

P130

Developmentofsemi-definedmolassesasastandardisedlaboratoryyeastculture

medium

NjabuloENeneandPatrickGovender

SchoolofLifeSciences,DepartmentofBiochemistry,UniversityofKwaZulu-Natal,PrivateBagX54001,

Durban,4000,SouthAfrica

The variability in canemolassesnutrient compositionhas given rise to theneed for a

standardised laboratory medium which can substitute the use of industrially derived

molasses in fermentation studies. This was addressed by formulating a semi-defined

molassesmediumsupplementedwithadifferentnitrogensource,viz.,10g/Lofcasein

hydrolysate, peptone or yeast extract. The semi-defined molasses media and cane

molasses sourced from threeSouthAfrican-based sugarmills (Amatikulu, Felixtonand

Gledhow),weresubjectedtobatchfermentations in250mLErlenmeyerflasksat30°C

for48hourswithdifferentSaccharomycescerevisiaestrains;BY4743,Angelyeast,cream

yeast and dry yeast. Yeast fermentation profiles were monitored by measuring CO2

evolution via flask weight measurements. Sugar attenuation, glycerol and ethanol

formationprofilesweremonitoredbyHigh-PerformanceLiquidChromatography(HPLC).

The semi-defined molasses medium containing yeast extract produced yeast

fermentation profiles most similar to those attained in the fermentation of cane

molasses. The fermentations of semi-defined molasses containing casein hydrolysate

and peptone resulted in a twofold decrease in yeast fermentation activity and

significantly diminished bioethanol yields. The results attained suggest that the novel

semi-definedmolassesmediumcontainingyeastextractcanthereforebeemployedasa

standardisedlaboratoryculturemediuminyeastfermentationstudies.

P131

ADMINISTRATIONOFPROBIOTICSINPIGSANDTHEIREFFECTONWEIGHTGAIN

Langa,RLS.,Aiyegoro,OA.,Dlamini,G.andLedwaba,R.

Gastro-IntestinalMicrobiologyandBiotechnologyUnit,AgriculturalResearchCouncil,AnimalProduction

Institute,PrivateBagX02,Irene0062,Pretoria,SouthAfrica.

Thirty indigenouspiglets and thirty commercial pigletswereweaned fourweeks after

birth. The piglets were transferred to a temperature controlled pig facility which

contained single pens, randomly and individually placed in thepens. Thepigletswere

arranged into four groups: Control, antibiotic, probiotic 1 and probiotic 2. The piglets

belongingtothecontrol,probiotic1andprobiotic2,werefedadlibitum,apigweaners

dietbutthosebelongingtotheantibioticgroupwerefedadlibitum,apigweanersdiet

containing the antibiotic, namely; lincospectin. The probiotic administered were

LactobacillusreuteriVB4andLactobacillusreuteriZJ625,respectively.Pigletsfromthe

twoprobioticgroupsweredosedorally,with10mloftherespectiveprobioticbacterium

onceaweek,foraperiodof4weeks.Beforetheprobioticswereadministered,culture

sampleswere analysed for viable counts so that their viable cell quantitywas known

before administering. The piglets were weighed once per week, a day after

administering the probiotics. The average weight gain of the piglets were: Control,

4.44kg; Lactobacillus reuteri VB4, 4.40kg; Lactobacillus reuteri ZJ 625, 4.12kg and

antibiotic, 4.46kg. The results show that the probiotics did not have a direct effect in

weightgain,butcanbeuseful inotherbiologicalfunctionalsuchasenhancedimmune

response.

P132

CREATIONOFAXYLANASEDEFICIENTSTRAINOFThermomyceslanuginosusBYDNA

RECOMBINANTTECHNOLOGY

RampersadE.,PermaulK.,andSinghS.

DepartmentofBiotechnologyandFoodTechnology,DurbanUniversityofTechnology,Durban4000,

SouthAfrica

Gene knock-out using homologous recombination is a well-established technique to

study gene-function and regulation. The recently sequenced filamentous thermophile

Thermomyces lanuginosus isaxylanase-superproducerwithrobustcharacteristics.This

investigation reports creation of a xylanase deficient T. lanuginosus DSM5826 via

homologousrecombinationforexpressionofimprovedxylanasevariantsincludingother

enzymesofmajorindustrial importance.AnantibioticcassetteonpBC-Hygrowasused

tocreateagene-disruptionconstructthatwasusedintransformationexperiments.The

disruptionconstructwasamplifiedwith70bpprimerscontaining20bphygromycin5’

and3’ regions and50bp xylanaseA (xynA) fromT. lanuginosusDSM5826.Amplified

disruptionconstruct(1μg)wastransformedintoDSM5826spheroplastswith5.5kV/cm

for 10 ms. Recovered transformants were screened on Potato-dextrose-agar (PDA)

supplementedwith50μg/mlhygromycinB, followedbyconfirmationonRBB-xylan.A

single isolate which was unable to hydrolyse RBB-xylan was chosen for downstream

experiments.Additionally,stabilityoftheknock-outvariantwasconfirmedbyrepeated

sub-culturing of spores in PDB medium containing 50-1000 μg/ml of hygromycin B.

Xylanase production of the xyn deficient (xynA-) and wild-type (WT) strains were

comparedinsubmergedfermentationcontaining1.5%beechwoodxylan.Onday7,WT

strain produced 4800 U/ml whereas the xynA- strain could only produce 12 U/ml of

xylanase. Furthermore, xynA- spores were transformed with the previously-reported

thermo-alkali-stable NC38 xylanase gene which produced 200 U/ml in submerged

fermentation.ThisxynA-T.lanuginosusstraincouldbeanoveleukaryotichostofmajor

industrialsignificancefortheexpressionofseveralcommercialgenes.

P133

OXIDATIONOFPHENOLICCOMPOUNDSFROMROOIBOS(Aspalathuslinearis):

IMPROVINGANTIOXIDANTPOTENTIAL

Thabi,M.M.1,Marnewick,J.2,Kudanga,T.3,andLeRoes-Hill,M.1

1BiocatalysisandTechnicalBiologyResearchGroup,and2OxidativeStressResearchCentre,Instituteof

BiomedicalandMicrobialBiotechnology,CapePeninsulaUniversityofTechnology,POBox1906,Bellville,

7535.3DepartmentofBiotechnologyandFoodTechnology,DurbanUniversityofTechnology,POBox1334,

Durban,4000.

Rooibos is a SouthAfricanherbal teamade from the fynbosplantAspalathus linearis.

Theplantisendemictothesouth-westernCaperegionofthecountryandhaslongbeen

explored for its health benefits. It is a natural source of unique antioxidants and it is

these bio-active compounds that have caught the attention of the public, and have

driven scientists to focus on the health promoting properties of the antioxidant

compounds.Theetiologyofcertaindiseasessuchascancerandcardiovasculardiseases

has been linked to oxidative stress and it has been shown that antioxidant

phytochemical compounds could play an important role in the prevention and/or

regressionofthesediseases.Inthisstudy,thepotentialofrooibosdust,awasteproduct

generatedintheprocessingofrooibostea,hasbeenexploitedasapotentialalternative

source for these phytochemicals. The extracts from the rooibos dust was used as a

substrate in biocatalysis reactions catalysed by bacterial oxidative enzymes. The small

laccasefromStreptomycescoelicolorA3(2),theperoxidasefromStreptomycessp.strain

BSII#1 and the tyrosinase from Streptomyces pharetrae CZA14T, was produced and

purified using three different enzyme purification approaches. The purified enzymes

were used in a preliminary screen to determine their potential to oxidize the

phytochemicalsfoundinrooibos.Thispresentationwillthereforefocusontheenzymes

produced and their ability to couple rooibos phytochemicals for the production of

compoundswithanincreasedantioxidantpotential.

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Durban University of Technology - Abstracts: Poster Presentations · 2016. 1. 18. · University of the Free State Intracellular gas bubbles in yeasts P52 Ms Thobeka Mhlongwe Stellenbosch