Research Article Investigation of Biofilm Forming Ability in …downloads.hindawi.com/journals/tswj/2013/378492.pdf · 2019-07-31 · coagulase negative Staphylococcus (CNS) ... was

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 378492 9 pageshttpdxdoiorg1011552013378492

Research ArticleInvestigation of Biofilm Forming Ability in StaphylococciCausing Bovine Mastitis Using Phenotypic and Genotypic Assays

Samah F Darwish1 and Hanaa A E Asfour2

1 Biotechnology Research Unit Animal Reproduction Research Institute (ARRI) Giza Egypt2 Department of Mastitis and Neonatal Diseases Animal Reproduction Research Institute (ARRI) Giza Egypt

Correspondence should be addressed to Samah F Darwish samahtarekhotmailcom

Received 4 August 2013 Accepted 5 September 2013

Academic Editors M Fernandez H Omoe and L Visai

Copyright copy 2013 S F Darwish and H A E Asfour This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A total of 40 S aureus and 68 coagulase negative Staphylococcus (CNS) isolates from bovine subclinical mastitis were investigatedfor their ability to form biofilm as one of the most important virulence factorsUsing Congo Red Agar (CRA) method 32535 and 325 of S aureus strains were strong intermediate and negative biofilm producers while in CNS the percentages were295 426 and 279 respectively By microtiter plate (MTP) method 525 275 and 20 of S aureus isolates were strongmoderate and weak biofilm producers while in CNS the percentages were 44 309 and 192 respectively Indian ink stainingwas used to detect the EPS layer of biofilm producers All isolates were screened for presence of biofilm related genes eno icaAicaD and bap In S aureus isolates the positive rates of eno icaA icaD and bap genes were 75 15 625 and 25 while inCNS were 926 59 471 and 44 respectively The eno gene had the highest rate while the bap gene had the lowest ratePresence of icaA and icaD genes was not always correlated with biofilm production This study demonstrated high prevalence ofStaphylococcus biofilm producers among bovinemastitis in EgyptTherefore attentionmust be paid toward implementation of newways for effective treatment of such infections

1 Introduction

Bovine mastitis caused by S aureus and coagulase negativeStaphylococci (CNS) remains a substantial problem for milkproducers worldwide The pathogenesis of Staphylococcusmastitis is attributed to a combination of extracellular factorsand properties such as adherence and biofilm formation [1 2]

Biofilm is an exopolysaccharide a slime matrix aroundmultiple layers of cells The ability of Staphylococci to formbiofilms is one of the virulence factors that facilitate theadherence and colonization of Staphylococci on the mam-mary gland epithelium also contributing to the evasion ofthe immunological defences and to the difficulty of pathogeneradication leading to recurrent or persistent infections[3ndash8]

Staphylococcus biofilm formation mechanisms are com-plex and include the participation of many kinds of pro-teins and so many genes are involved It is consideredto be a two-step process Firstly the bacteria adhere to a

surface mediated by a capsular antigen namely capsularpolysaccharideadhesin (PSA) Then the bacteria multiplyto form a multilayered biofilm which was associated withproduction of polysaccharide intercellular adhesin (PIA)Theintercellular adhesion (ica) locus consisting of the genesicaADB and C encodes the proteins mediating the synthesisof PIA and PSA in staphylococcal species [1 9]

Monitoring the biofilm forming ability of Staphylococcicausingmastitis and the genes involved in it may provide newideas or strategy for the prevention or the effective treatmentof bovine mastitis According to our knowledge in Egyptresearches on biofilm only began recently andmainly focusedon Staphylococcus clinical isolates from human sources [1011] So the aim of the present study was to investigatethe biofilm forming ability of Staphylococcus strains caus-ing bovine mastitis using different phenotypic methodsMoreover the detection of some biofilm related genes inthese strains and their involvement with biofilm will beevaluated

2 The Scientific World Journal

2 Material and Methods

21 Bacterial Isolates The investigation was carried out on108 Staphylococcus isolates recovered from bovine subclinicalmastitic milk The isolates were identified by their culturalcharacteristics on blood agar mannitol salt agar and BairdParker agar media microscopic appearance in Gram stainedpreparations positive catalase reaction hemolysin and coag-ulase test (tube method) with rabbit plasma and biochemicalanalysis according to Quinn et al [12]

22 Biofilm Formation Assays

221 Phenotypic Analysis

(1) Detection of Slime Production by Congo Red Agar MethodSlime production was evaluated by cultivation of all Staphylo-coccus isolates on Congo Red Agar (CRA) plates as describedbyMathur et al [13] (2006) Briefly CRAplateswere preparedusing Tryptic Soy agar containing 008 Congo red (Sigma)The inoculated CRA plates were incubated at 37∘C in aerobicconditions for 24 h followed by storage at room temperaturefor 48 h [4] Isolates were interpreted according to theircolony phenotypes [14] Black colonies with dry consistencyand rough surface and edges were considered a positiveindication of slime production while both black colonieswith smooth round and shiny surface and red colonies ofdry consistency and rough edges and surface were consideredas intermediate slime producers Red colonies with smoothround and shiny surface were indicative of negative slimeproduction

(2) Detection of Biofilm by Microtiter Plate (MTP) MethodAll Staphylococcus isolates were grown overnight at 37∘C aspure cultures on blood agar Groups of three single colonieswere inoculated in 3mL Tryptone Soya broth Suspensionswere incubated for 24 h at 37∘C and then diluted at 1 40in a fresh TSB (2ndash7 times 107 cfumL) using 05 MacFarlandstandard tube This dilution was used as the inoculum inthe microtiter plate test Microtiter plate test was performedaccording to Dubravka et al [14] and Stepanovic et al[15] For each Staphylococcus isolate 200120583L aliquots ofprepared suspension were inoculated into four wells of the96-well tissue culture plates (Nunclon Delta Nunc RoskildeDenmark) Each culture plate included a negative controlfour wells with TSB The plates were incubated at 37∘Cfor 24 h Afterwards content of each well was removed byaspiration and the wells were rinsed three times with 250120583Lsterile physiological saline The plates were dried in invertedposition The attached bacteria were fixed for 15 minutes atroom temperature by adding 200120583L volumes of methanolinto each well The plates were stained with 160 120583L aqueoussolution of crystal violet 05 (Crystal Violet Fluka) for 15minutes at room temperature Following staining the plateswere rinsed under running water until there was no visibletrace of stain The stain bound to bacteria was dissolved byadding 160 120583L of 95 ethanol The optical density (OD) ofeach well was measured using a microplate ELISA reader(119864max USA) at 570 nm Cut-off OD (ODc) is defined as

three standard deviations above the mean OD of the negativecontrol Strains were interpreted as follows

(1) nonbiofilm producers (OD le ODc)(2) weak biofilm producers (ODc lt OD le 2 timesODc)(3) moderate biofilm producers (2 times ODc lt OD le 4 times

ODc)(4) Strong biofilm producers (4 timesODc lt OD)

(3) Detection of Exopolysaccharide Production (EPS) PossibleEPS-producing isolates were identified via their mucoid orropy appearance EPS production was assessed by Indian ink(ArtMaterial SA China) staining of wetmount preparationof each strain after overnight growth on CRA and examinedunder light microscope [16 17] Appearance of a transparenthalo zone around the Staphylococcus cells denoted the exis-tence of an organized EPS structure

23 Genotypic Analysis Four biofilm related genes wereanalyzed by simplex PCR assays to detect the presence of icaA(intercellular adhesion gene A) icaD (intercellular adhesiongene D) bap (encoding biofilm associated protein) and eno(encoding laminin binding protein) in all Staphylococcusisolates First crude DNA of the tested strains was extractedusing a rapid boiling procedure according to Reischl et al[18] Two to 5 loops of Staphylococcus isolates taken from thebrain heart infusion agar plate were collected and suspendedin 200120583L of lysis buffer comprised of 1 Triton X-100 05Tween 20 10mM Tris-HCl (pH 80) and 1mM EDTA Afterboiling for 10min the suspension was centrifuged for 2minto sediment bacterial debris The supernatant was aspiratedfrom which 5 120583L was used directly for PCR amplificationBecause of nonavailability of positive controls for the fourgenes and to exclude any false negative results the DNA ofall isolates was firstly examined by amplification of 16S rRNAgene using Staphylococcus genus specific primers The namesof target genes nucleotide sequences of primers referencesannealing temperature and amplicon sizes are shown inTable 1 The amplification cycles were carried out in a PT-100 Thermocycler (MJ Research USA) With the exceptionof specific annealing temperatures mentioned in Table 1 thereaction condition was optimized to be 94∘C for 4min asinitial denaturation followed by 40 cycles of 94∘C for 60seconds annealing for 60 seconds and 72∘C for 60 secondsA final extension step at 72∘C for 10min was followedPCR condition was optimized using a total volume of 25120583Lreaction mixtures which contained 5120583L of DNA as template25 pmol of each primer and 1X of PCR master mix (DreamTaq Green PCR Master Mix Fermentas Life Science) PCRproducts were analyzed by electrophoresis in 15 agarosegel in 05X TBE buffer containing ethidium bromide andvisualized under a UV transilluminator

24 Statistical Analysis Data were presented as count andpercentage Sensitivity and specificity of CRA method werecalculated according to Ilstrup [22] using 2 times 2 table andMTPmethod as gold standardThe kappa coefficient test was

The Scientific World Journal 3

Table 1 Primers used in the study their nucleotide sequence annealing temperatures amplicon sizes and their references

Gene targeted Primer sequence 51015840-31015840(references) Annealing temperature ∘C Amplicon size bp

16S rRNAGTA GGT GGC AAG CGTTAT CCCGC ACA TCA GCG TCA GMonday and Bohach [19]

55 228

icaACCTAACTAACGAAAGGTAGAAGATATAGCGATAAGTGCVasudevan et al [4]

49 1315

icaDAAACGTAAGAGAGGTGGGGCAATATGATCAAGATACVasudevan et al [4]

49 381

EnoACGTGCAGCAGCTGACTCAACAGCATYCTTCAGTACCTTCTristan et al [20]

55 302

BapCCCTATATCGAAGGTGTAGAATTGGCTGTTGAAGTTAATACTGTACCTGCCucarella et al [21]

60 971

Table 2 Biofilm formation in S aureus and CNS isolates according to CRA method

Species (no)No () of isolates

Positive result Intermediate result Negative result Total positiveDry black Smooth black Dry red Smooth red

S aureus (40) 13 (325) 8 (20) 6 (15) 13 (325) 27 (675)CNS (68) 20 (295) 19 (279) 10 (147) 19 (279) 49 (721)Total (108) 33 (306) 27 (25) 16 (148) 32 (296) 76 (704)

Table 3 Biofilm formation in S aureus and CNS isolates accordingto MTP method


Strong Moderate Weak Non Totalpositive


also used to determine the agreement between the resultsobtained by the CRA method

3 Results

31 Bacterial Isolation and Identification The 108 Staphylo-coccus isolates were identified to be 40 S aureus and 68 CNS

32 Biofilm Formation

321 Slime Production on Congo RedAgar (CRA) The resultsof biofilm production by S aureus and CNS using CRAmethod are demonstrated in Table 2 About 704 of theisolates were positive for biofilm production with varieddegree Out of 40 S aureus strains 325 35 and 325were strong intermediate and negative biofilm producersrespectively Among the 68 CNS strains 295 426

and 279 were strong intermediate and negative biofilmproducers respectively Totally from the 108 Staphylococcusisolates 33 (306) 27 (25) 16 (148) and 32 (296)were dry black smooth black dry red and smooth redrespectively Morphology of all types of colonies is showedin Figure 1

322 Biofilm Formation by MTP Method As shown inFigure 2 andTable 3 approximately 963 of the isolates werebiofilm producers with MTP method although productionlevel varied The biofilm production of S aureus isolates(100) was slightly higher than that of CNS isolates (941)In the biofilm positive S aureus strains 525 of isolates werestrong producers while 275 and 20 were moderate andweak respectively Out of the biofilm positive CNS isolates44 309 and 192 were strong moderate and weakrespectively

Comparison of results obtained by CRA method versusthat of MTP method is declared in Table 4 Out of 32 biofilmnegative Staphylococcus isolates by CRA method 28 isolateswere positive by MTP method but with different degreesof production (4 strong 8 moderate 16 weak) and only4 CNS isolates were true negative by both methods CRAmethod showed little correlation with MTP assay where only76 (704) of the isolates were positive by both methodskappa coefficient 0167 slight agreement The sensitivity andspecificity of CRA method versus MTP method as goldstandard were calculated to be 731 and 100 respectively


(a) (b)

(c) (d)

Figure 1 Screening of biofilm (slime) producer Staphylococci using Congo red agar plate method ((a) dry black colonies (b) smooth blackcolonies (c) dry red colonies (d) smooth red colonies)

Weak Moderate Strong None

Figure 2 2 Microtiter plate method showing none strong moder-ate and weak biofilm producers differentiated by crystal violet stainin 96-well tissue culture plate

323 Detection of EPS Production All the 108 Staphylococcusisolates except the 4 biofilm negative CNS showed a distincthalo transparent zone surrounding the grapes linked cellssimilar to a capsule representing EPS layer (Figure 3)

33 Genotypic Analysis of Biofilm Related Genes All the fourbiofilm related genes eno icaA icaD and bap could bedetected in both S aureus and CNS isolates under study

Table 4 CRAmethod versus MTP method for detection of biofilmformation by S aureus and CNS isolates

Species CRA No MTP methodStrong Moderate Weak Non

S aureus

Dry black 13 12 1 0 0Smooth black 8 5 2 1 0

Dry red 6 1 5 0 0Smooth red 13 3 3 7 0

Total 40 21 11 8 0

CNS



Total 68 30 21 13 4

(Figure 4) but with varied prevalence (Figure 5) In S aureusisolates the positive rates of eno icaA icaD and bap geneswere 75 15 625 and 25 respectively (Table 5 andFigure 5)The prevalence of eno icaA IcaD and bap genes inCNS isolates were 926 59 471 and 44 respectively(Figure 5 andTable 5)Theprevalence of biofilm related genesvaried between S aureus and CNS The results indicated thatthe eno gene had the highest rate in CNS (926) and Saureus (75) followed by icaD gene which was detected in


Table 5 Association between results of MTP method and positive amplification of biofilm related genes in S aureus and CNS isolates

Species MTP No +eno +icaA +icaD +bapNo No No No

S aureus

Strong 21 17 81 5 24 13 62 0 0Moderate 11 9 82 1 9 9 82 0 0Weak 8 4 50 0 0 3 375 1 125None 0 0 0 0 0 0 0 0 0Total 40 30 75 6 15 25 625 1 25

CNS


Figure 3 Morphology of some of Staphylococcus isolates asobserved after staining with Indian ink The arrows pointed tocapsular-like EPS (light microscopy times100)

625 of S aureus and 471 of CNS The prevalence of icaAgene was low versus that of icaD gene The bap gene wasdetected in only 4 isolates one S aureus isolate (25) and3 CNS isolates (44)

4 Discussion

Biofilms are related to pathogenicity and it has been proposedthat Staphylococcus biofilms are major causes of recurrentand chronic mastitis in dairy cattle [23] Numerous authorsstated that bacteria in a biofilm are more resistant to antibi-otics than in their planktonic form [24 25]

In this study slime production was examined quali-tatively depending on colony morphology of 108 bovinemastitis isolates of genus Staphylococci (S aureus and CNS)produced on Congo red agar Some differences betweenresearches were apparent with respect to interpretation ofCRA test results In that respect both bright black colonies[26] and black colonies [8 27] were considered as a positiveresult However Cucarella et al [5] described the dry crys-talline surface (rough colonymorphology) as a positive resultdisregarding the color (black or pink) Such discrepancywhen interpreting the results may possibly be due to the factthat the test itself was not originally designed for investigatingS aureus isolates as reported by Freeman et al [28] In thisinvestigation according to Dubravka et al [14] isolates that

formed blackrough colonies were recorded as strong slimeproducing whereas isolates forming redsmooth colonieswere described as nonslime producersThe smooth black anddry red colonies were considered as indeterminate result Asdisplayed in Table 2 slime production was detected in 33(306) isolates that produced characteristic black colonies ofdry crystalline consistencywhile 32 isolates (296) producedsmooth red colonies Also 27 isolates (25) produced blackcolonies with smooth shiny surfaces were interpreted as inde-terminate result Furthermore 16 isolates (148) producedred colonies with dry rough consistency were described asindeterminate result Totally 76 isolates (704) were foundto be slime producers with variable degrees

MTP method was reported to have high specificitysensitivity and positive predictive value [13] As shown inTable 3 using MTP method 963 of the isolates werebiofilm producers with variable production levels This highprevalence agreed with that reported by Seo et al [29] whonoted that approximately 80of their isolates produced slimewith the MTP method but with variable degrees of pro-duction On the contrary lower prevalence rates of biofilmproducers among S aureus and S epidermidis bovinemastitisisolates were also reported to be 2941 [30] and 375 [8]Comparison of results obtained by CRA method versus thatof MTP method was declared in Table 4 Out of 32 biofilmnegative Staphylococcus isolates (13 S aureus and 19 CNS) byCRA method 28 isolates were positive by MTP method butwith different degrees of production (4 strong 8 moderateand 16 weak) while only 4 CNS isolates were negative byboth methods Congo red agar (CRA) method showed littlecorrelation with MTP method where only 76 (704) of theisolates were positive by both methods with kappa coefficient0167 (slight agreement) The sensitivity and specificity ofCRA method versus MTP method as gold standard werecalculated to be 731 and 100 respectively AlthoughCRA method was easy to perform and less time consuminghowever our findings confirm what was reported by Mathuret al [13] that CRA method cannot be recommended fordetection of biofilm formation by Staphylococci alone

Difference between results of CRAandMTPmethods canbe attributed to the fact that phenotypic expression of biofilm


M 1 2 3 4 5 6 7 8 N

(a)

M 1 2 3 4 5 6 7 8 9 10 N

(b)

M 1 2 3 4 5 6 7 8 9 10 N

(c)

M 1 2 3 4 5 6 7 8 9 N

(d)

Figure 4 Agarose gel electrophoresis of PCR products stained with ethidium bromide (a) eno gene (302 bp) M 100 bp ladder DNAmarker1ndash8 positive samples N negative control (b) icaA gene (1315 bp) M 1 kb plus DNAmarker 2 3 5 7 8 positive samples 1 4 6 9 10 negativesamples N negative control (c) icaD gene (381 bp) M 100 bp ladder DNA marker 2ndash5 7ndash10 positive samples 1 6 negative samples Nnegative control (d) bap gene (971 bp) M 100 bp ladder DNA marker 1 3 positive samples 2 4ndash9 negative samples N negative control

100

()

80

60

40

20

0

S aureus CNS

enoicaA

icaDbap

Figure 5 Prevalence of biofilm related genes in S aureus and CNSisolated from bovine mastitis milk

formation is highly sensitive to in vitro conditions and hencecan be detected variably by different methods Also bothtests measure the same phenomenon but in different waysCRA has been used as indirect indicator of polysaccharideproduction [16 31]

Additionally Indian ink was used to stain wet prepara-tions of Staphylococcus isolates all the 108 isolates except 4biofilm negative CNS showed distinct halo transparent zonesdenoting to EPS layer surrounding the cells (Figure 3) Thesize of the hallo zones enlarged with the ability of the isolatesto produce biofilms This result proposed the use of Indianink staining as a rapid screening test for biofilm formationwhen other methods are not feasible

As combination of phenotypic and genotypic methodswould be recommended for identifying biofilm producingstrains the detection of 4 biofilm related genes and theirincidence in Staphylococcus isolates were investigated usingsimplex PCR reactions (Figures 4 and 5) Because of theabsence of a reference positive control for each of thestudied genes the DNA of all isolates was first examinedby Staphylococcus genus specific primers to monitor thequality of the DNA for amplification and to exclude anyfalse negative results All the 108 Staphylococcus isolatessuccessfully amplified the specific 228 bp of the 16S rRNAgene of genus Staphylococcus This amplification confirmedall the isolates to be Staphylococci and ensuring the suitabilityof the DNA for amplification

The intercellular adhesion (ica) locus consisting of thegenes icaADBC has been reported to have a potential role as avirulence factor in the pathogenesis of mastitis in ruminants[4] Among the ica genes icaA and icaD have been reportedto play a significant role in biofilm formation in S aureus


and S epidermidis [32] The prevalence rates of icaA andicaD genes were 15 and 625 in S aureus isolates and59 and 471 in CNS isolates respectively (Figure 5)Although the majority of researches reported the icaA andicaD to be nearly similar in incidence [2 4 30] our resultsagreed with Ciftci et al [33] who found that 16 and 38out of 59 strains were positive for icaA and icaD genesrespectively This difference in the prevalence rates can beattributed to variation in DNA sequences which may leadto failed amplification of the gene in some isolates leadingto false negative results [34] This was previously reportedby Simojoki et al [35] who excluded icaA and icaD resultsfrom their work because their primers although were able todetect the genes in S aureus isolates were not able to detectthe genes in known icaA and icaD positive S epidermidis

Detection of icaA and icaD was not well correlatedwith biofilm production on MTP methods (Table 5) Pres-ence of icaA or icaD negative biofilm positive isolates canbe accounted for by an ica gene independent control ofslime productionadhesionmechanism [36]On the contraryinability of Staphylococcus isolates that were positive for icaAandor icaD genes to produce biofilm in vitro can be due topoint mutation in the locus andor any other yet unidentifiedfactors that negatively regulate polysaccharide intercellularadhesion synthesis or influence biofilm formation [1] Alsosome experimental evidence supports the development ofnew clones referred as biofilm negative with both icaAand icaD genes positive [37] Additionally lacking of icaAmRNA or icaD mRNA or both was reported to explainabsence of phenotypic expression of biofilm [36] Despitediscrepancy between results of ica locus and biofilm pheno-typic expression Vasudevan et al [4] argued that a bettermethodology for biofilm detection is to screen strains forica genes in addition to CRA or MTP methods not to missthe genotypically positive phenotypically negative strains Onthe contrary Ciftci et al [33] reported that PCR targetingicaA and icaD genes may be not sufficient to detect slimeproduction and further studies targeting other genes shouldbe conducted for accurate evaluation of slime productioncharacters of S aureus strains

Among the studied genes eno encoding the laminin-binding protein was the most commonly detected gene inStaphylococcus mastitis isolates under study The prevalencerates of the eno gene were 75 and 926 in S aureus andCNS respectively (Table 5 and Figure 5) This high preva-lence agreed with that of Simojoki et al [35] who reportedthe same gene to be the most commonly detected in CNSisolates from mastitis in a percentage of 75 Also this genewas detected with the highest rate in airborne Staphylococci(83) when compared with animal isolates (56) as reportedby Seo et al [29]

The bap gene encoding the biofilm associated proteinwas the least detected gene where it was only detectedin one S aureus (25) isolate and 3 (44) CNS isolates(Table 5 and Figure 5) This very low prevalence was previ-ously reported by Cucarella et al [5] who detected the bapgene in 5 of S aureus obtained from bovine subclinicalmastitis This gene is also detected in other Staphylococcusspecies including S epidermidis S chromogenes S simulans

and S hyicus [34] Other authors did not detect the bap genein their Staphylococcus isolates [29 35 38]

Antimicrobial therapy of mastitis is based on resultsof susceptibility tests in vitro therefore new tests must beadopted to select the antibiotic of choice for treatment ofbiofilm positive strains where ordinary test selects antibioticsthat are effective only in inhibiting planktonic bacterialpopulation whereas bacteria in biofilm resist and survive thetreatment and provide materials for further growth [39]

5 Conclusions

Findings of the present study demonstrated the great abilityof both S aureus and CNS bovine mastitis isolates to formbiofilms with different degrees of production using differentmethods This must be considered as an alarming situationand so attention must be paid toward implementation of newways for effective prophylaxis control and treatment of suchinfections in the dairy farms

Conflict of Interests

The authors have no conflict of interests

Authorsrsquo Contribution

Samah F Darwish and Hanaa A E Asfour contributedequally to the work

References

[1] S E Cramton C Gerke N F Schnell W W Nichols andF Gotz ldquoThe intercellular adhesion (ica) locus is present inStaphylococcus aureus and is required for biofilm formationrdquoInfection and Immunity vol 67 no 10 pp 5427ndash5433 1999

[2] D Vancraeynest K Hermans and F Haesebrouck ldquoGenotypicand phenotypic screening of high and low virulence Staphylo-coccus aureus isolates from rabbits for biofilm formation andMSCRAMMsrdquo Veterinary Microbiology vol 103 no 3-4 pp241ndash247 2004

[3] R N Zadoks W B van Leeuwen D Kreft et al ldquoComparisonof Staphylococcus aureus isolates from bovine and human skinmilking equipment and bovine milk by phage typing pulsed-field gel electrophoresis and binary typingrdquo Journal of ClinicalMicrobiology vol 40 no 11 pp 3894ndash3902 2002

[4] P Vasudevan M K M Nair T Annamalai and K S Venki-tanarayanan ldquoPhenotypic and genotypic characterization ofbovine mastitis isolates of Staphylococcus aureus for biofilmformationrdquoVeterinaryMicrobiology vol 92 no 1-2 pp 179ndash1852003

[5] CCucarellaM A TormoC Ubeda et al ldquoRole of biofilmasso-ciated protein Bap in the pathogenesis of bovine Staphylococcusaureusrdquo Infection and Immunity vol 72 no 4 pp 2177ndash21852004

[6] L K Fox R N Zadoks and C T Gaskins ldquoBiofilm produc-tion by Staphylococcus aureus associated with intramammaryinfectionrdquo Veterinary Microbiology vol 107 no 3-4 pp 295ndash299 2005


[7] M B Melchior J Fink-Gremmels and W Gaastra ldquoCompar-ative assessment of the antimicrobial susceptibility of Staphy-lococcus aureus isolates from bovine mastitis in biofilm versusplanktonic culturerdquo Journal of VeterinaryMedicine B vol 53 no7 pp 326ndash332 2006

[8] M Oliveira R Bexiga S F Nunes et al ldquoBiofilm-formingability profiling of Staphylococcus aureus and Staphylococcusepidermidis mastitis isolatesrdquo Veterinary Microbiology vol 118no 1-2 pp 133ndash140 2006

[9] D McKenney J Hubner E Muller Y Wang D A Goldmannand G B Pier ldquoThe ica locus of Staphylococcus epidermidisencodes production of the capsular polysaccharideadhesinrdquoInfection and Immunity vol 66 no 10 pp 4711ndash4720 1998

[10] G F M Gad M A El-Feky M S El-Rehewy M A Hassan HAbolella and R M A El-Baky ldquoDetection of icaA icaD genesand biofilm production by Staphylococcus aureus and Staphy-lococcus epidermidis isolated from urinary tract catheterizedpatientsrdquo Journal of Infection in Developing Countries vol 3 no5 pp 342ndash351 2009

[11] R A Nasr H M AbuShadady and S H Hussein ldquoBiofilmformation and presence of icaAD gene in clinical isolatesof staphylococcirdquo The Egyptian Journal of Medical HumanGenetics vol 13 pp 269ndash274 2012

[12] P J Quinn B K Markey M E Carter W J Donnelly and FC Leonard Veterinary Microbiology and Microbial Disease ABlackwell Science 2002

[13] T Mathur S Singhal S Khan D J Upadhyay T Fatmaand A Rattan ldquoDetection of biofilm formation among theclinical isolates of staphylococci an evaluation of three differentscreeningmethodsrdquo Indian Journal ofMedicalMicrobiology vol24 no 1 pp 25ndash29 2006

[14] M Dubravka S Lazic V Branka P Jelena D Bugarski andS Zorica ldquoSlime production and biofilm forming ability bystaphylococcus aureus bovine mastitis isolatesrdquo Acta Veteri-naria vol 60 no 2-3 pp 217ndash226 2010

[15] S Stepanovic D Vukovic I Dakic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 pp 175ndash179 2000

[16] R Baselga I Albizu M De la Cruz E Del Cacho M Bar-beran and B Amorena ldquoPhase variation of slime productionin Staphylococcus aureus implications in colonization andvirulencerdquo Infection and Immunity vol 61 no 11 pp 4857ndash4862 1993

[17] R Bennama M Fernandez V Ladero M A Alvarez NRechidi-Sidhoum and A Bensoltane ldquoIsolation of an exopoly-saccharide-producing Streptococcus thermophilus from Alge-rian raw cow milkrdquo European Food Research and Technologyvol 234 no 1 pp 119ndash125 2012

[18] U Reischl M Pulz W Ehret and H Wolf ldquoPCR-baseddetection of mycobacteria in sputum samples using a simpleand reliable DNA extraction protocolrdquo BioTechniques vol 17no 5 pp 844ndash845 1994

[19] S R Monday and G A Bohach ldquoUse of multiplex PCR todetect classical and newly described pyrogenic toxin genes instaphylococcal isolatesrdquo Journal of Clinical Microbiology vol 37no 10 pp 3411ndash3414 1999

[20] A Tristan L Ying M Bes J Etienne F Vandenesch and GLina ldquoUse of multiplex PCR to identify Staphylococcus aureusadhesins involved in human hematogenous infectionsrdquo Journalof Clinical Microbiology vol 41 no 9 pp 4465ndash4467 2003

[21] C Cucarella C Solano J Valle B Amorena I Lasa andJ R Penades ldquoBap a Staphylococcus aureus surface proteininvolved in biofilm formationrdquo Journal of Bacteriology vol 183no 9 pp 2888ndash2896 2001

[22] D M Ilstrup ldquoStatistical methods in microbiologyrdquo ClinicalMicrobiology Reviews vol 3 no 3 pp 219ndash226 1990

[23] M B Melchior J Fink-Gremmels and W Gaastra ldquoExtendedantimicrobial susceptibility assay for Staphylococcus aureusisolates from bovine mastitis growing in biofilmsrdquo VeterinaryMicrobiology vol 125 no 1-2 pp 141ndash149 2007

[24] M BMelchior H Vaarkamp and J Fink-Gremmels ldquoBiofilmsa role in recurrent mastitis infectionsrdquo Veterinary Journal vol171 no 3 pp 398ndash407 2006

[25] A Raza G Muhammad S Sharif and A Atta ldquoBiofilm pro-ducing Staphylococcus aureus and bovine mastitis a reviewrdquoMolecular Microbiology Research vol 3 no 1 pp 1ndash8 2013

[26] S Citak O Varlik and N Gundogan ldquoSlime production andDNase activity of staphylococci isolated from rawmilkrdquo Journalof Food Safety vol 23 no 4 pp 281ndash288 2003

[27] A Jain and A Agarwal ldquoBiofilm production a marker ofpathogenic potential of colonizing and commensal staphylo-coccirdquo Journal of Microbiological Methods vol 76 no 1 pp 88ndash92 2009

[28] D J Freeman F R Falkiner and C T Keane ldquoNew methodfor detecting slime production by coagulase negative staphylo-coccirdquo Journal of Clinical Pathology vol 42 no 8 pp 872ndash8741989

[29] Y-S Seo D Y Lee N Rayamahji M L Kang and H SYoo ldquoBiofilm-forming associated genotypic and phenotypiccharacteristics of Staphylococcus spp isolated fromanimals andairrdquo Research in Veterinary Science vol 85 no 3 pp 433ndash4382008

[30] N B Dhanawade D R Kalorey R Srinivasan S B Barbuddheand N V Kurkure ldquoDetection of intercellular adhesion genesand biofilm production in Staphylococcus aureus isolated frombovine subclinical mastitisrdquo Veterinary Research Communica-tions vol 34 no 1 pp 81ndash89 2010

[31] N T Stevens C M Greene J P OrsquoGara and H HumphreysldquoBiofilm characteristics of Staphylococcus epidermidis isolatesassociated with device-related meningitisrdquo Journal of MedicalMicrobiology vol 58 no 7 pp 855ndash862 2009

[32] F Gotz ldquoMicroreview on Staphylococcus and biofilmsrdquoMolec-ular Microbiology vol 43 pp 1367ndash1378 2002

[33] A Ciftci A Findik E E Onuk and S Savasan ldquoDetec-tion of methicillin resistance and slime factor production ofStaphylococcus aureus in bovine mastitisrdquo Brazilian Journal ofMicrobiology vol 40 no 2 pp 254ndash261 2009

[34] M A Tormo E Knecht F Gotz I Lasa and J R Penades ldquoBap-dependent biofilm formation by pathogenic species of Staphy-lococcus evidence of horizontal gene transferrdquo Microbiologyvol 151 no 7 pp 2465ndash2475 2005

[35] H Simojoki P Hyvonen C Plumed Ferrer S Taponen and SPyorala ldquoIs the biofilm formation and slime producing abilityof coagulase-negative staphylococci associated with the per-sistence and severity of intramammary infectionrdquo VeterinaryMicrobiology vol 158 pp 344ndash352 2012

[36] M C Liberto G Matera A Quirino et al ldquoPhenotypicand genotypic evaluation of slime production by conventionaland molecular microbiological techniquesrdquo MicrobiologicalResearch vol 164 no 5 pp 522ndash528 2009


[37] C R Arciola L Baldassarri and L Montanaro ldquoPresenceof icaA and icaD genes and slime production in a collectionof Staphylococcal strains from catheter-associated infectionsrdquoJournal of Clinical Microbiology vol 39 no 6 pp 2151ndash21562001

[38] E Vautor G Abadie A Pont and R Thiery ldquoEvaluationof the presence of the bap gene in Staphylococcus aureusisolates recovered from human and animals speciesrdquoVeterinaryMicrobiology vol 127 no 3-4 pp 407ndash411 2008

[39] M Fenton R Keary O McAuliffe R Paul Ross O rsquoMahonyJ and A Coffey ldquoBacteriophage-derived peptidase CHAPKeliminates and prevents Staphylococcal biofilmsrdquo InternationalJournal of Microbiology vol 2013 Article ID 625341 8 pages2013

Submit your manuscripts athttpwwwhindawicom

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Microbiology


2 Material and Methods

21 Bacterial Isolates The investigation was carried out on108 Staphylococcus isolates recovered from bovine subclinicalmastitic milk The isolates were identified by their culturalcharacteristics on blood agar mannitol salt agar and BairdParker agar media microscopic appearance in Gram stainedpreparations positive catalase reaction hemolysin and coag-ulase test (tube method) with rabbit plasma and biochemicalanalysis according to Quinn et al [12]

22 Biofilm Formation Assays

221 Phenotypic Analysis

(1) Detection of Slime Production by Congo Red Agar MethodSlime production was evaluated by cultivation of all Staphylo-coccus isolates on Congo Red Agar (CRA) plates as describedbyMathur et al [13] (2006) Briefly CRAplateswere preparedusing Tryptic Soy agar containing 008 Congo red (Sigma)The inoculated CRA plates were incubated at 37∘C in aerobicconditions for 24 h followed by storage at room temperaturefor 48 h [4] Isolates were interpreted according to theircolony phenotypes [14] Black colonies with dry consistencyand rough surface and edges were considered a positiveindication of slime production while both black colonieswith smooth round and shiny surface and red colonies ofdry consistency and rough edges and surface were consideredas intermediate slime producers Red colonies with smoothround and shiny surface were indicative of negative slimeproduction

(2) Detection of Biofilm by Microtiter Plate (MTP) MethodAll Staphylococcus isolates were grown overnight at 37∘C aspure cultures on blood agar Groups of three single colonieswere inoculated in 3mL Tryptone Soya broth Suspensionswere incubated for 24 h at 37∘C and then diluted at 1 40in a fresh TSB (2ndash7 times 107 cfumL) using 05 MacFarlandstandard tube This dilution was used as the inoculum inthe microtiter plate test Microtiter plate test was performedaccording to Dubravka et al [14] and Stepanovic et al[15] For each Staphylococcus isolate 200120583L aliquots ofprepared suspension were inoculated into four wells of the96-well tissue culture plates (Nunclon Delta Nunc RoskildeDenmark) Each culture plate included a negative controlfour wells with TSB The plates were incubated at 37∘Cfor 24 h Afterwards content of each well was removed byaspiration and the wells were rinsed three times with 250120583Lsterile physiological saline The plates were dried in invertedposition The attached bacteria were fixed for 15 minutes atroom temperature by adding 200120583L volumes of methanolinto each well The plates were stained with 160 120583L aqueoussolution of crystal violet 05 (Crystal Violet Fluka) for 15minutes at room temperature Following staining the plateswere rinsed under running water until there was no visibletrace of stain The stain bound to bacteria was dissolved byadding 160 120583L of 95 ethanol The optical density (OD) ofeach well was measured using a microplate ELISA reader(119864max USA) at 570 nm Cut-off OD (ODc) is defined as

three standard deviations above the mean OD of the negativecontrol Strains were interpreted as follows

(1) nonbiofilm producers (OD le ODc)(2) weak biofilm producers (ODc lt OD le 2 timesODc)(3) moderate biofilm producers (2 times ODc lt OD le 4 times

ODc)(4) Strong biofilm producers (4 timesODc lt OD)

(3) Detection of Exopolysaccharide Production (EPS) PossibleEPS-producing isolates were identified via their mucoid orropy appearance EPS production was assessed by Indian ink(ArtMaterial SA China) staining of wetmount preparationof each strain after overnight growth on CRA and examinedunder light microscope [16 17] Appearance of a transparenthalo zone around the Staphylococcus cells denoted the exis-tence of an organized EPS structure

23 Genotypic Analysis Four biofilm related genes wereanalyzed by simplex PCR assays to detect the presence of icaA(intercellular adhesion gene A) icaD (intercellular adhesiongene D) bap (encoding biofilm associated protein) and eno(encoding laminin binding protein) in all Staphylococcusisolates First crude DNA of the tested strains was extractedusing a rapid boiling procedure according to Reischl et al[18] Two to 5 loops of Staphylococcus isolates taken from thebrain heart infusion agar plate were collected and suspendedin 200120583L of lysis buffer comprised of 1 Triton X-100 05Tween 20 10mM Tris-HCl (pH 80) and 1mM EDTA Afterboiling for 10min the suspension was centrifuged for 2minto sediment bacterial debris The supernatant was aspiratedfrom which 5 120583L was used directly for PCR amplificationBecause of nonavailability of positive controls for the fourgenes and to exclude any false negative results the DNA ofall isolates was firstly examined by amplification of 16S rRNAgene using Staphylococcus genus specific primers The namesof target genes nucleotide sequences of primers referencesannealing temperature and amplicon sizes are shown inTable 1 The amplification cycles were carried out in a PT-100 Thermocycler (MJ Research USA) With the exceptionof specific annealing temperatures mentioned in Table 1 thereaction condition was optimized to be 94∘C for 4min asinitial denaturation followed by 40 cycles of 94∘C for 60seconds annealing for 60 seconds and 72∘C for 60 secondsA final extension step at 72∘C for 10min was followedPCR condition was optimized using a total volume of 25120583Lreaction mixtures which contained 5120583L of DNA as template25 pmol of each primer and 1X of PCR master mix (DreamTaq Green PCR Master Mix Fermentas Life Science) PCRproducts were analyzed by electrophoresis in 15 agarosegel in 05X TBE buffer containing ethidium bromide andvisualized under a UV transilluminator

24 Statistical Analysis Data were presented as count andpercentage Sensitivity and specificity of CRA method werecalculated according to Ilstrup [22] using 2 times 2 table andMTPmethod as gold standardThe kappa coefficient test was





55 228


49 1315


49 381


55 302


60 971










3 Results








(a) (b)

(c) (d)








S aureus



Total 40 21 11 8 0

CNS



Total 68 30 21 13 4





S aureus


CNS




4 Discussion







M 1 2 3 4 5 6 7 8 N

(a)

M 1 2 3 4 5 6 7 8 9 10 N

(b)

M 1 2 3 4 5 6 7 8 9 10 N

(c)

M 1 2 3 4 5 6 7 8 9 N

(d)


100

()

80

60

40

20

0

S aureus CNS

enoicaA

icaDbap













5 Conclusions






References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





55 228


49 1315


49 381


55 302


60 971










3 Results








(a) (b)

(c) (d)








S aureus



Total 40 21 11 8 0

CNS



Total 68 30 21 13 4





S aureus


CNS




4 Discussion







M 1 2 3 4 5 6 7 8 N

(a)

M 1 2 3 4 5 6 7 8 9 10 N

(b)

M 1 2 3 4 5 6 7 8 9 10 N

(c)

M 1 2 3 4 5 6 7 8 9 N

(d)


100

()

80

60

40

20

0

S aureus CNS

enoicaA

icaDbap













5 Conclusions






References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)








S aureus



Total 40 21 11 8 0

CNS



Total 68 30 21 13 4





S aureus


CNS




4 Discussion







M 1 2 3 4 5 6 7 8 N

(a)

M 1 2 3 4 5 6 7 8 9 10 N

(b)

M 1 2 3 4 5 6 7 8 9 10 N

(c)

M 1 2 3 4 5 6 7 8 9 N

(d)


100

()

80

60

40

20

0

S aureus CNS

enoicaA

icaDbap













5 Conclusions






References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




S aureus


CNS




4 Discussion







M 1 2 3 4 5 6 7 8 N

(a)

M 1 2 3 4 5 6 7 8 9 10 N

(b)

M 1 2 3 4 5 6 7 8 9 10 N

(c)

M 1 2 3 4 5 6 7 8 9 N

(d)


100

()

80

60

40

20

0

S aureus CNS

enoicaA

icaDbap













5 Conclusions






References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2 3 4 5 6 7 8 N

(a)

M 1 2 3 4 5 6 7 8 9 10 N

(b)

M 1 2 3 4 5 6 7 8 9 10 N

(c)

M 1 2 3 4 5 6 7 8 9 N

(d)


100

()

80

60

40

20

0

S aureus CNS

enoicaA

icaDbap













5 Conclusions






References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusions






References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Investigation of Biofilm Forming Ability in …downloads.hindawi.com/journals/tswj/2013/378492.pdf · 2019-07-31 · coagulase negative Staphylococcus (CNS) ... was