O Serogroup-Specific Touchdown-Multiplex Polymerase Chain ......ResearchArticle O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification

Research ArticleO Serogroup-Specific Touchdown-MultiplexPolymerase Chain Reaction for Detection and Identification ofVibrio cholerae O1 O139 and Non-O1Non-O139

Adisak Bhumiratana1 Achiraya Siriphap2 Nutsarin Khamsuwan3 Jednipit Borthong4

Kaknokrat Chonsin5 and Orasa Sutheinkul36

1Department of Parasitology and Entomology Faculty of Public Health Mahidol University Bangkok 10400 Thailand2Department of Microbiology and Parasitology Faculty of Medical Science University of Phayao Phayao 56000 Thailand3Department of Microbiology Faculty of Public Health Mahidol University Bangkok 10400 Thailand4Division of Bioinformatics Research Center for Zoonosis Control Hokkaido University Sapporo 001-0020 Japan5Community Health Program Faculty of Science and Technology Suratthani Rajabhat University Surat Thani 84100 Thailand6Department of Microbiology Faculty of Public Health Thammasat University Rangsit Center PathumThani 12121 Thailand

Correspondence should be addressed to Orasa Sutheinkul orasasutmahidolacth

Received 11 July 2014 Accepted 5 December 2014 Published 28 December 2014

Academic Editor Brian Shilton

Copyright copy 2014 Adisak Bhumiratana et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A novel sensitive locus-specific touchdown-multiplex polymerase chain reaction (TMPCR) which is based on two-stageamplification pertaining to multiplex PCR and conditional touchdown strategy was used in detecting and differentiating Vibriocholerae serogroups A panel of molecular marker-based TMPCR method generates reproducible profiles of V cholerae-specific(588 bp) amplicons derived from ompW gene encoding the outer membrane protein and serogroup-specific amplicons 364 bp fortheO1 and 256 bp for theO139 authentically copied from rfb genes responsible for the lipopolysaccharide biosynthesisTheTMPCRamplification efficiency yields either equally or unequally detectable duplex DNA bands of the O1 (588 and 364 bp) and O139 (588and 256 bp) or a DNA fragment of non-O1non-O139 (588 bp) while providing no false positive identifications using the genomicDNA templates of the other vibrios and Enterobacteriaceae The reciprocal analysis of two-template combinations demonstratedthat usingV choleraeO1 O139 or equally mixed O1 and O139 the TMPCR had a detection limit of as low as 100 pg of the O1 O139or non-O1non-O139 in reactions containing unequally or equally mixed gDNAs In addition the O serogroup-specific TMPCRmethod had 100 agreement with the serotyping method when examined for the serotyped V cholerae reference strains and thoserecovered from clinical samples The potential benefit of using this TMPCR tool would augment the serotyping method used inepidemiological surveillance and monitoring of V cholerae serogroups O1 O139 and non-O1non-O139 present in clinical andenvironmental samples

1 Introduction

Vibrio cholerae is a water-borne agent causing cholera andis autochthonous to aquatic habitats in coastal and estuarineecosystems to which symbiosis with zooplanktons for its sur-vival andmultiplication is related [1ndash5]Vibrio spp includingV cholerae serogroups [6] andVibrio parahaemolyticus [7 8]normally grow in the natural environments and can enter intoviable-but-nonculturable (VBNC) state The significant roles

of the toxigenic O1 and O139 serogroups of V cholerae aswell as the non-O1non-O139 serogroups have been studiedin several ways For instance the VBNC V cholerae O1 inmicrocosms or from aquatic environments are converted toculturable state through animal passage [9]The VBNCO139and non-O1non-O139 can also resuscitate when coculturedwith several animal cell lines [10 11] More recently theclassical biotype of V cholerae O1 retains viability but losesculturability when cocultured with the El Tor biotype [12]

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2014 Article ID 295421 10 pageshttpdxdoiorg1011552014295421

2 Biochemistry Research International

Thismight suggest that the emergence of the El Tor biotype ofV choleraeO1 relates to displacement of the existing classicalbiotype as the predominant cause of epidemic choleraSimilar to the toxigenic O1 and O139 serogroups that possessvirulence-association genes [13ndash18] the other non-O1 andnon-O139 serogroups recovered from aquatic environmentsor clinical specimens have also been epidemiologically linkedwith the pathogenic and epidemic potential [18ndash20] Thuspublic health surveillance and monitoring of cholera casesrequire the systems used in the national surveillance for noti-fiable diseases public health laboratory and environmentalsurveillance [21ndash24] Molecular detection techniques such asmolecular marker-based polymerase chain reaction (PCR)methods developed for probing these eccentric V choleraemicroorganisms have been so far proven useful in diagnosisand surveillance for the outbreaks or epidemic investigationsworldwide

In the Gulf of Bengal South and Southeast Asia it hasbeen suggested that such outbreaks of cholera pertainingto cross-contamination and spread of V cholerae after thepassage(s) from susceptible persons to aquatic environmentsare likely to be epidemiologically linked with the inter-connections of sanitation with poorly chlorinated waterscontamination in seafoodfood commodities and seawatersand direct fecal-oral contact among food handlers or seafoodpreprocessing plant workers [2 14ndash16 21 22 24] Standardculture methods used in routine diagnosis and surveillancefor V cholerae by public health reference laboratories dependon handling the samples of whichV cholerae culture is recov-ered Regarding thisV cholerae cultures under study investi-gation were recovered from V cholerae-contracted or sus-pected patients with acute or severe diarrhea whose stools orrectal swabs had been collectively obtained by clinical labo-ratory settings during the outbreaks over two past decadesbetween 1994 and 2010 in Central Thailand

We proposed that the genomic DNA archived fromthe preserved stock cultures could serve as the templatewhen examined for the O1 O139 and non-O1non-O139serogroups by PCR methods as this can also permit a recip-rocal detection of the O1 and O139 serogroups from clinicalspecimens associated with the past outbreaks In this studywe have successfully developed a novel sensitive specificsemiquantitative touchdown-multiplex PCR (TMPCR) fordetection anddifferentiation ofV choleraeO1O139 andnon-O1non-O139 serogroups TMPCRemploys the usefulmolec-ular markers originally derived from the DNA locus involvedin de novo O-antigen biosynthesis of V cholerae [25ndash28] andthe outer membrane protein (ompW) encoding gene specificfor V cholerae [29 30] To achieve the goal of the study weanalyzed the performance efficiency (specificity and sensi-tivity) of this O serogroup-specific TMPCR in detecting anddifferentiating theO1 O139 and non-O1non-O139 genomeswhich were empirically determined using reference strains ofV cholerae and unrelated Vibrio strains We then exploredits usefulness in differentiating the O serogroups presentin those V cholerae stock cultures originally isolated fromclinical specimens Additionally the advancement of TMPCRthat tested V cholerae present in environmental samples wasalso discussed

2 Materials and Methods

21 Bacterial Cultures and Laboratory Classification Sixty-nine V cholerae strains that served as target gDNA templatesincluded reference and wild-type strains of the O1 (includingEl TorClassical Ogawa and El TorClassical Inaba biotypes)O139 and non-O1non-O139 The 31 other bacterial strainsused as nonspecific gDNA templates included 8 Vibrio spp16 Gram-negative bacteria and 7 Gram-positive bacteria Forthe batch propagation of V cholerae the 05mL overnightculture of the O1 569B or O139 MO45 strains was inoculatedinto a 100mLLuria-Bertani (LB) broth supplementedwith 1sodium chloride (NaCl) solution and then incubated at 37∘Cfor 3 h with vigorous shakingThe cell culture with an opticaldensity of 08 was used for drop plate method and subse-quently enumerated by using plate count (PC) agar (DifcoMichigan USA or Eiken Japan) The PC agar was alsosupplemented with 1 NaCl The non-O1non-O139 strainsincluding O22 and O155 were cultured as before

Subcultures of V cholerae and other Vibrio spp weregrown on selective thiosulfate citrate bile salt sucrose (TCBS)agar (Merck Darmstadt Germany) and subsequently theywere biochemically and serologically characterized accordingto themethods described elsewhere [31 32] Briefly biochem-ical tests included triple sugar iron (TSI) motility indole-lysine (MIL) oxidase urea MR Voges-Proskauer (VP)citrate lysine ornithine arginine lactose sucrose mannosearabinose mannitol glucosegas inositol aesculin and salttolerance of 0 3 6 8 and 10 NaCl solution Allreagents were purchased from Difco Laboratory (DifcoMichigan USA)The serogroups O1 O139 and non-O1non-O139 were tested on the basis of agglutination reactionusing commercially available O serogroup-specific antisera(antiserum VcO1O139 Polyvalent SAP S amp A Reagents LabBangkok Thailand) The non-O1non-O139 was character-ized into two nonagglutinable (NAG) groups (NAG I and II)according toHeibergrsquos reaction In addition PC agarwas usedfor viable count and enumeration of all the bacterial strainstested in this study

22 V cholerae Cultures from Clinical Specimens and Serotyp-ing A total of 148 stock cultures of V cholerae that corre-sponded to 108 stools and 40 rectal swabs were collectivelyobtained from V cholerae-contracted or suspected patientswith acute or severe diarrhea during the outbreaks in CentralThailand between 1994 and 2010 By using anonymoussystem the cultures initially isolated by clinical laboratorysettings were performed on isolation source and time ofcollection and the corresponding TCBS stock cultures wereprepared All were subsequently subcultured and kept insemisolid agar medium containing 1 NaCl at the Depart-ment of Microbiology Faculty of Public Health MahidolUniversity Thailand For serotyping the O1 O139 and non-O1non-O139 the agglutination of the 148 subcultured Vcholerae isolates in individuals was blindly performed asbefore In addition only the V cholerae O1 serogroup whosesubcultures agglutinated with polyvalent O1 antiserum wascharacterized using monovalent Ogawa or Inaba antiserumFor biotyping the phenotypic testing of the strains was

Biochemistry Research International 3

Table 1 Primer sequencesa used in touchdown-multiplex PCR

Primer set Sequence (51015840 to 31015840) Tm (∘C) Direction Amplicon size (bp) ReferenceUniversal primersb

MVCO1 CAC CAA GAA GGT GAC TTT ATT GTG 68 Forward 588 Nandi et al 2000 [30]MVCO2 GAA CTT ATA ACC ACC CGC G 58 Reverse

O1-specific primersc

MVCO3 GAC TGT CAG CTG GCG GAA 58 Forward 364 This studyMVCO4 GTT GGC GTA TTA CGG TAC 54 Reverse

O139-specific primersd

MVCO5 GCG TTT ATC GCC GGT CGA C 62 Forward 256 This studyMVCO6 GTA ACT TGG TAC AAT CTC G 54 Reverse

aAll were originally derived from the nucleotide sequences (positions) available from theGenBank accession numbers bAE003853 (819612ndash820199) bCP000626(404919ndash405506) cAE003852 (264415ndash264778) cCP000627 (2789549ndash2789912) and dU47057 (902ndash1157) from the EMBL accession numbers bX51948 (221ndash808) cX59554 (18246ndash18609) cY07788 (199ndash562) and dY07786 (11878ndash12133) or from the DDBJ accession number dAB012956 (32652ndash32907)

performed using hemolysis of sheep blood agglutinationof chicken erythrocytes Voges-Proskauer and sensitivity topolymyxin B Meanwhile the genomic DNAs of the sameV cholerae isolates were prepared according to the rapidboiling method described below or using QIAamp DNAminikit (Qiagen Hilden Germany) The stock solution ofpurified gDNA extracts was initially quantified and thenstored according to the method described below

23 Genomic DNA Extraction A modified miniprep gDNAextraction method was used for both target and nontargetgDNAs as follows As mentioned above the bacterial culture(15mL) that was grown overnight at 37∘C with vigorousshaking was spun down at 12000 rpm for 1min The cellpellet was suspended in 582120583L of TE buffer (10mM Tris-HCl 1mMEDTA) pH 80The suspension was lysed with anaddition of 15 120583L 20 sodium dodecyl sulfate solution and 3120583L 20mgmLproteinaseKmixed thoroughly and then left at37∘C for 1 h A 10 120583L of 10mgmL RNase A was added mixedthoroughly and incubated at 37∘C for 30minThe 100 120583L 5Msodium chloride (NaCl) solution and 80 120583L 10 hexadecyltrimethyl ammonium bromide (CTAB) in 07M NaCl solu-tionwere addedmixed thoroughly and incubated at 65∘C for10min Then a 04x volume of chloroformisoamyl alcohol(24 1) was added mixed gently for 5min and centrifuged at12000 rpm for 5min The aqueous phase containing gDNAwas twice treatedwith an equal volume of phenol-chloroformand followed by centrifugation as before An equal volumeof isopropanol was added to recovered DNA solution mixedthoroughly and DNA pellet was centrifuged as before Afterdehydration of an extreme volume of 70 ice-cold ethanoland further centrifugation it was dissolved in TE buffer pH76

In addition using rapid boiling method the sameovernight culture was spun down at 12000 rpm for 1minThecell pellet was suspended well in 500 120583L of TE buffer pH 80and incubated at 56∘C for 30min Immediately after beingboiled at 100∘C for 10min the suspension was stood on icefor 10min and then centrifuged at 12000 rpm for 3min topellet cell debris the clear gDNA supernate was obtainedFinally the purified gDNA extracts as well as crude extracts

(boiled lysate) were qualitatively and quantitatively analyzedby spectrophotometer at a wavelength ratio of 260280 nmor by agarose gel electrophoresis The aliquots of varyingconcentration of 30 to 50120583gmL (a 260280OD greater than17) used throughout the study were kept at minus20∘C until use

24 Primer Design The homologous primer sets specificfor V cholerae serogroups (Table 1) were originally derivedfrom target DNA sequences of V cholerae O1 a 22 kb rfbDNA locus [25] and of V cholerae O139 a 46 kb rfb DNAlocus [28]These rfb gene homologs are involved in lipopoly-saccharide (LPS) biosynthesis for the O1 and O139 antigensThe rfbV gene responsible for the rhs element involved in LPSbiosynthesis was designed for V choleraeO1-specific primersto generate 364 bp amplicon The wbfR gene encodingasparagine synthetase was designated as V cholerae O139-specific primers (256 bp amplicon) The ompW gene encod-ing the outer membrane protein was used for V choleraespecies-specific or universal primers that amplify 588 bpamplicons [29 30] Analysis of their unanimous homologyat DNA and protein levels was performed on the onlineavailable BLAST Programs (httpwwwncbinlmnihgovblast) BLASTN BLASTx and BLASTP for sequence sim-ilarity algorithms The multiple sequence alignments wereanalyzed for consensus sequences using the ClustalW Pro-gram (httpwwwebiacukclustalW)The aliquots of thesesynthesized multiplex primer sets at a stock concentration of100 120583M in TE buffer pH 80 used throughout the study werestored at minus80∘C until use

25 Touchdown-Multiplex PCR Amplification Initially two-template combinations of the V cholerae O1 and O139 weretested under the simultaneous locus-specific amplificationconditions [33 34] using a 96-well MyCyclerThermal Cycler(BIORAD USA) or PCR gradient thermal cycler (Thermo-Hybraid Px2 Ashford UK) in which two combined factorssuch as concentrations of primer sets and gDNA templateswere empirically determined in a 25 120583L PCR mixture con-sisting of 1x PCR buffer (50mM KCl 15mM MgCl

2 and

10mM Tris-HCl pH 90) 200 120583M each dNTP (dATP dTTPdCTP and dGTP) 1 U of AmpliTaq (Perkin Elmer USA)or Taq DNA polymerase (Promega USA) The amplification


conditions were performed on predenaturation at 95∘C for5min and followed by 30 cycles of denaturation at 94∘C for1min and extension at 72∘C for 1min except that primerannealing temperature decrements (70 rarr 50∘C) were usedeach for 1minThe last extension was done at 72∘C for 10minFirst using a constant concentration of 1120583M each primerset reactions employed mixed O1 569B and O139 MO45gDNAs (ng) at equal ratios of 10 10 1 1 and 01 01 or atunequal ratios of 1 10 and 01 10 and vice versa Second usingmixed gDNAs (10 ng each) the primer sets (universal O1-specific O139-specific) at concentration ratios (120583M) of 1 1 11 05 05 10 02 02 05 10 10 05 05 10 02 02 10and 01 01 10 were analyzed Under these circumstancesthe primer binding specificity in amplifying target locidepended upon annealing temperature algorithms to whichprimer-template combinations were restricted These algo-rithms permitted the development of thermocycling TM-PCR protocol as described below

Finally a 25 120583L optimized TMPCR mixture was similarexcept that V cholerae O1 and O139 gDNA templates 10 ngeach and three multiplex primer sets (04 04 and 10 120583Meach resp) were used instead The optimized amplificationcondition was performed on one cycle of predenaturationat 94∘C for 5min followed by the 5 cycles with successiveannealing temperature decrements that one ∘C changed from57∘C to 53∘C in every cycle versus time increments (30 secin the first three cycles and 35 sec in the last two cycles) Thereaction was denatured at 94∘C for 1min followed by anneal-ing at this temperature algorithm and polymerization at 72∘Cfor 1min Subsequently the 20 cycles of denaturation at 94∘Cfor 1min annealing at 52∘C for 40 sec and elongation at 72∘Cfor 1min were employed The last primer extension step wasdone at 72∘C for 10min In similar fashion the purified Vcholerae O22 or O155 gDNA was used instead of the O139Either Escherichia coli gDNA template as negative control(NC) or nuclease-free water instead of gDNA template asinternal control (IC) was used in all the experiments

26 Semiquantitative TMPCR The reciprocal detection of Vcholerae O1 versus O139 was aimed at analyzing the amplifi-cation efficiency of the O serogroup-specific TMPCR undercarefully controlled experiments that utilized gDNA templateratios whether unequally or equally That is the detectablelevel of O1 gDNA could be amplified in reactions containinghigh amount of other related O139 gDNAs and vice versa

The unequally reciprocal detection of V cholerae O1 ver-sus O139 (Table 2) was done as follows In experiment A thereactions contained the amount of the 100 ngO1 gDNAversusthe amount of 10-fold serially diluted O139 gDNA (100 ngto 1 pg) or the amount ratios of the O1 O139 varied fromonefold to 105-fold and vice versa In experiment B the reac-tions contained the amount ratios of the O1 O139 gDNAs as100 ng to 1 pg 10 ng to 10 pg and 1 ng to 100 pg or the amountof the O1 as 105- 103- and 10-fold greater than the O139 andvice versa Also the amount of the 100 pg O139 gDNA wastested against the varying amounts of the O1 gDNA 100 ng50 ng 25 ng 10 ng 1 ng and 100 pg

The equally reciprocal detection of V cholerae O1 versusO139 (Table 3) was done as follows In experiment C the

Table 2The unequally reciprocal detection ofV choleraeO1 O139by the O serogroup-specific TMPCR

O1 O1390001 001 01 1 10 100

100 105 104 103 102 101 110 103 101

1 101 102

01 101 103

001 103 104

0001 105

The contents (ng) of purified O1 O139 gDNAs were used in this assay

Table 3The equally reciprocal detection ofV choleraeO1 O139 bythe O serogroup-specific TMPCR

O1 O139100 10 1 01 001 0001

100 100

10 100

1 100

01 100

001 100

0001 100


equal amounts of the O1 O139 gDNAs were tested at a serial10-fold dilution 100 ng to 1 pg For the internal controls(experiments D and E) the same amounts of the 10-foldserially diluted gDNA templates O1 or O139 were used Alsothe purifiedV choleraeO22 orO155 gDNA template was usedinstead ofO139 All experiments were performed in triplicate

27 16S rDNA Amplification For the quality control ofthe TMPCR amplification the 16s rDNA amplification thatserved as internal control reactionwas performed in triplicatewith all the reactions containing target or nontarget DNAtemplate throughout the study In addition the primers thatare specific for microbial DNA (16S rDNA locus) includedforward primer 51015840-CGGTGAAATGCGTAGAGAT-31015840 andreverse primer 51015840-TTACTAGCGATTCCGAGTTC-31015840 [35]A 25 120583L optimized PCR mixture contained 1X PCR bufferand 200120583M each dNTP as before except that forward andreverse primers Taq DNA polymerase and gDNA templatewere used at concentration of 05 120583M each 05U and 1 120583L ofundiluted template respectivelyThe optimized amplificationwas performed on predenaturing at 94∘C for 5min andfollowed by 30 cycles of denaturing at 94∘C for 45 secannealing at 55∘C for 45 sec and extension at 72∘C for 45 secFinally the last extensionwas at 72∘C for 5min In this regardthe amplicons with expected size of 663 bp were obtainedfrom all reactions containing microbial gDNA templates

28 Analysis of PCR Products One-fifth of the ampliconsmixed with 6x loading dye solution were electrophoresedthrough the 15 to 20 agarose gels at constant voltage of10Vcm in 1x TBE buffer (89mM Tris-HCl 89mM Borate


2mM EDTA) (Ameresco Ohio USA) The ethidium bro-mide- (EtBr-) stained DNA gel was used to analyze theamplicon sizes in base pairs as compared to DNA standardsizemarker (500 ng of each 100 bp ladder) (Phamacia BiotechUSA) The intensity of EtBr-stained DNA bands was visual-ized and photographed under an ultraviolet (UV)wavelengthusing DNA gel documentation (Fotodyne Hartland USA)

In addition analysis of amplification specificity sensi-tivity and fidelity of the O serogroup-specific TMPCR wasdone as follows In reactions using target gDNAs intenseEtBr-stained duplex bands were detected either equally orunequally for O1 (588 bp and 364 bp) and for O139 (588bp and 258 bp) An intense 588 bp band was detected forthe non-O1 (both NAG I and NAG II) In reactions usingnontarget gDNAs or nuclease-free water instead of DNAneither expected amplicon sizes nor nonspecific DNA bandswere observed

3 Results

Primer-template specificity binding was empirically deter-mined in reactions of which optimized PCR parameters wereanalyzed upon the amplification efficiency of three multiplexprimer sets that specifically bind to V cholerae gDNAs of theO1 O139 or O22O155 We initially optimized the separatereactions containing the gDNA content of V cholerae O1569B or O139 MO45 alone or equally mixed Most reliableamplification conditions were reproducibly performed on thereactions using a 10 ng each of V cholerae O1 569B or O139MO45 per reaction assay or equally mixed The resultingalgorithm upon annealing temperature above 55∘Cwas likelyto decrease amplification yields of the specific amplicons withexpected sizes (data not shown) All reactions containingO22or O155 gDNA putatively yielded a 588 bp DNA band (datanot shown) Optimum annealing temperatures ranged from53∘C to 57∘C It was clear that PCR factors underlying theannealing temperatures and primer set concentrations weremore likely to influence the amplification efficiency whetherthe O1 O139 or equally mixed gDNAs were used As aresult optimized multiplex PCR amplification depended onannealing temperature at 55∘C and primer set concentrationsof 04 (universal) 04 (O1-specific) and 10 (O139-specific)120583M (Figure 1)

We further employed the stringent protocol developedfor TMPCR because the multiplex PCR provided a ladder-like problem of amplification background of most reactionscontaining nontarget gDNAs Similarly the multiplex PCRyielded spurious products even in some reactions containingV cholerae O1 O139 or non-O1non-O139 gDNAs preparedby CTABphenol-chloroform extraction method or by rapidboiling method (data not shown) As a result of optimized Oserogroup-specific TMPCR with loose touchdown strategythermal cycling condition employed annealing temperaturedecrements at an interval of 57∘C to 53∘C versus timeincrements to minimize nonlagging products during initialgeometric amplification cycles As compared to the 16S rDNAamplification this proven TMPCR had 100 specificity orgave putatively positive amplifications in reactions containingV cholerae genomes but not other nontarget DNAs (Table 4)

UniversalO1-specificO139-specific

a101010

b100505

c100202

d100101

e051010

f050502

g020210

h010110

M PC

588364256

588364256

600400200

600400200

e

a b c d

f g h

Figure 1 Agarose gel electrophoresis analysis of the PCR algorithmupon annealing temperatures and primer set concentrations thathad effects on yields of three specific amplicons In a 25 120583L PCRassay using 10 ng each of purified V cholerae O1 and O139 gDNAsamplification was performed in triplicate with varying concentra-tion of three primer sets (120583M) (a to h) and annealing temperatureincrements (∘C) at 542 555 and 569The 100 bpmolecular marker(lane M) and serogroup-specific PCR amplicons as positive control(lane PC) were used for base pair size comparisons

Table 4Amplification specificity of TMPCRand 16S rDNA-specificPCR

Organism Strains tested(119899 = 100)

Specificity ()TMPCRa 16S rDNA PCRb

V cholerae O1 21 100 100V cholerae O139 16 100 100V cholerae NAG Ic 16 100 100V cholerae NAG IId 16 100 100Other vibrios 8 0 100Gram-negative bacteria 16 0 100Gram-positive bacteria 7 0 100aThe specificity () = strains showing a positive amplification of expectedamplicon sizes by TMPCR as having no cross-hybridization with negativecontrolsa total of strains tested multiplied by 100bAs for 16S rDNA-specific PCR no cross-hybridization occurred in reactionscontaining nuclease-free water or no DNAcNAG I non-O1non-O139 growing in semisolid agar in the presence ofsucrose and mannose but not arabinosedNAG II non-O1non-O139 growing in semisolid agar only supplementedwith sucrose

Of the 21 V cholerae O1 serving as target DNAs tested by theTMPCR it was clear that consistently positive amplificationswith EtBr-intense duplex DNA bands (588 and 364 bp) wereobserved in reactions containing reference strains (8) EltorInaba (5) Eltor Ogawa (4) Classical Inaba (2) and EltorOgawa (2) Similarly other 16V choleraeO139 strains yieldedthe amplified duplex DNA fragments (588 and 256 bp) whileboth NAG I and NAG II gave only a single 588 bp DNAband as expected Only the representative target and non-target gDNAs amplified by O serogroup-specific TMPCR as


M 1 2 3 4 5 6 7 8 M

588364256

600400

200

(a)

M 1 2 3 4 5 6 7 8 M

600400

200

663

(b)

Figure 2 Agarose gel electrophoresis of the amplification specificityof optimized TMPCR (a) and 16S rDNA PCR (b) using represen-tative target and nontarget gDNAs In lanes 1 to 8 amplificationreactions containedV choleraeO1V choleraeO139V choleraeNAGI V cholerae NAG II Vibrio parahaemolyticus Vibrio vulnificusEscherichia coli and nuclease-free water respectively The expectedsizes of specific amplicons (base pairs) were compared to the 100 bpstandard ladder marker (lane M)

compared to that amplified by 16S rDNA PCR are shown inFigure 2 In addition there were no differences of specificityand sensitivity of TMPCR when using V cholerae O1 O139and non-O1non-O139 gDNA templates whether purified orboiled

To analyze the amplification sensitivity of the TMPCRwe determined a reciprocal amplification pertaining to thealgorithms that used the amount of ratios of mixed Vcholerae gDNAs (O1 O139) in comparison to those reactionscontaining each gDNA template serially dilutedThe resultingTMPCR experiments in which CTABphenol-chloroformextracted V cholerae gDNA whether O1 O139 or O22O155was amplified are shown in Figure 3 As for Figure 3(a) theTMPCR could detect O serogroup-specific DNA fragmentsauthentically derived from the O1 or the O139 in reciprocalreactions The detection limit of as low as 1 ng of the O1 wasobtained in the presence of the higher amount of O139 or100-fold greater than the O1 Meanwhile the detection limitof as low as 10 ng of the O139 was obtained in the presenceof the higher amount of the O1 or 10-fold greater than theO139 Figure 3(b) shows that its detection limit was at 100 pgof the O1 or the O139 in inverse reactions containing unequalamount ratios of the 100 pg O1 to 1 ng O139 and vice versaAgain the TMPCR was likely to show detection limit of aslow as 100 pg each of gDNAs in the reaction containing equalamount ratio of the O1 to O139 It was clear to note that theTMPCR had the detection limit of as low as 100 pg gDNA ofthe O1 or O139 when examined for the equal amount ratioof the serially diluted O1 and O139 templates (Figure 3(c))

Table 5 Agreement of O serogroup-specific TMPCR for detectingand differentiating gDNAs of the O1 O139 and non-O1non-O139 serogroups archived from clinical samples (119899 = 148) usingserotyping method as reference

Source Serogroupa TMPCRompW rfbV wbf

Stool119899 = 108

O1 (119899 = 65) 65 65 0O139 (119899 = 15) 15 0 15

Non-O1non-O139 (119899 = 28) 28 0 0Total 108 65 15

Rectal swab119899 = 40

O1 (119899 = 39) 39 39 0O139 (119899 = 0) 0 0 0

Non-O1non-O139 (119899 = 1) 1 0 0Total 40 39 0

aAll laboratory settings yielded 100 concordant V cholerae cultureswhether rectal swabs or stools examined using standard serotyping methodas described in the text

Similarly it also had the same detection limit of as low as100 pg gDNA of the O1 or O139 when separately copied(Figures 3(d) and 3(e)) The amount of as low as 100 pg O22or O155 gDNA was also detected (data not shown)

Lastly we tested the amplification fidelity of O serogroup-specific TMPCR by using target gDNA samples archivedfrom the stock cultures of the 148 V cholerae clinicalisolates Overall the TMPCR exhibited 100 concordancewith serotyping method as reference whether V choleraecultures were obtained from stools or rectal swabs (Table 5)All the 108 cultures from the stools tested gave positiveTMPCR results 65 (602) of the O1 serotype concordantwith the O1 serogroup-specific TMPCR (588 and 364 bp) 15(139) of the O139 serotype concordant with the O139 sero-group-specific TMPCR (588 and 256 bp) and 28 (259) ofthe non-O1non-O139 serotype concordant with the Oserogroup-specific TMPCR (588 bp) Meanwhile all the 40cultures from the rectal swabs were also consistently positivewith the tests 39 (975) accordant with the O1 serotype andtheO1 serogroup-specificTMPCRandonly one (25) accor-dant with the non-O1non-O139 serotype and 588 bp specificTMPCR Only the representative gDNAs archived from theclinically isolated V cholerae cultures are shown in Figure 4All samples tested by O serogroup-specific TMPCR wereconsistently positive with the 16S rDNA PCR

Additionally TMPCR provided reliable testing resultswhen examined for V cholerae present in environmentalsamples such as water samples and seafood as compared tothe culture methodserotyping (Table 6) Particularly whenexamined for V cholerae present in aquatic environmentsTMPCR provided positive result forV cholerae non-O1non-O139 in a water sample whereas culture method did not

4 Discussion

Theconventional culture technique is fundamental to recoverthe V cholerae culture from clinical samples of V cholerae-contracted or cholera-like patients However the approach


1 2 3 4 5 6M 7

600400

200

NC9 10 11 12

588364256

8

(a)

600400200

588364256

1 2 3 4 5 6M 7 NC9 10 11 128

(b)

1 2 3 4 5 6M

600400

200

NC

588

364256

(c)

600400

200

1 2 3 4 5 6M NC

588

364

(d)

600400

200

1 2 3 4 5 6M NC

588

256

(e)

Figure 3 Agarose gel electrophoresis analysis of the amplification efficiency of semiquantitative TMPCR A reciprocal amplificationalgorithm of detectable levels of the O1 to O139 gDNAs (experiments (a) to (e)) was described in the text while E coli gDNA templateserving as negative control (NC) was included (a) Lanes 1 to 6 the 100 ng O1 gDNA versus a 10-fold serially diluted O139 gDNA 1 pg to100 ng and vice versa (lanes 7 to 12) (b) Lanes 1 to 3 unequal amount ratios of the O1 O139 100 ng to 1 pg 10 ng to 10 pg and 1 ng to 100 pgand vice versa (lanes 4 to 6) Lanes 7 to 12 the 100 pg O139 gDNA versus the O1 gDNA contents of varying 100 ng 50 ng 25 ng 10 ng and1 ng to 100 pg (c) Lanes 1 to 6 the equal amount ratios of 10-fold serially diluted O1 to O139 gDNAs 100 ng to 1 pg (d) Lanes 1 to 6 10-foldserially diluted O1 gDNA contents 100 ng to 1 pg (e) Lanes 1 to 6 10-fold serially diluted O139 gDNA contents 100 ng to 1 pg A 100 bp DNAladder marker (lane M) was used in comparison of the amplicons with expected size (bp)


Table 6 Advancement of TMPCR for V cholera detection using environmental samples

Sourcea Sample number Culture method and serotyping TMPCRO1 O139 Non-O1Non-O139 O1 O139 Non-O1Non-O139

Water samples 50 0 0 0 0 0 1Seafood 18 5 0 8 5 0 8aAll were collectively obtained during the outbreaks The individual samples enriched in broth were used in V cholerae detection and differentiation using theculture methodserotyping and TMPCR

O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139

Figure 4 Agarose gel electrophoresis of the O serogroup-specificTMPCR and 16S rDNA PCR profiles using representative Vcholerae gDNAs archived from the clinical specimens PCRproductsobtained by bothTMPCR (5120583L) and 16S rDNAPCR (5120583L) thatwereseparately performed are confined to the same lane lanes 1ndash3 the O1from stools lanes 4-5 the O1 from rectal swabs lanes 6ndash10 the O139from stools and lanes 11ndash15 the non-O1non-O139 from stools Ineach TMPCR employing the O1 O139 or non-O1non-O139 gDNAthe reliable reaction represents duplex DNA fragments (588 bp and364 bp from theO1 or 588 bp and 256 bp from theO139) and a single588 bp DNA band from the non-O1non-O139 as compared to 16SrDNA amplicons (663 bp) and 100 bp DNA ladder marker (laneM)

relies upon the ability of the V cholerae propagating in vitroculture and expressing normally phenotypic characteristicsunder physiological conditions whether morphologicallybiochemically or serologically This classical microbiologicalapproach is not just to grow viable microbial cells onto aplate count agar or in an enrichment culture medium whichconstitutes the entire nutrients essential for growth of the tar-get microbe(s) but also to incubate the propagated culturesunder conditions favoring their optimal growth Such VBNCV cholerae as well as some VBNC vibrios can exist in naturein aquatic habitats such that they are not as yet cultured inthe laboratory and hence have not been characterized usingroutine microbiological methods due to the limits of recov-ery of resuscitated cultures from environmental samplesHowever the V cholerae once recovered from those isolationsources needs to be logically analyzed to determine theirproperty whether phenotypically or genotypically

In the present study we provided the proof that all theserotypes ofV cholerae both reference strains and clinical iso-lates were consistently positive with the O serogroup-specificTMPCR method showing the concordance of serotypesand O serogroup-specific genotypes In other words theTMPCR method that employed the primer sets specific forthe O1 O139 and non-O1non-O139 could simultaneouslyyield amplicons authentically derived from the target DNAsequences only in reactions containing the low amounts of

target gDNAs but not the unrelated gDNAs This impliedthat the TMPCR had high performance efficiency (specificityand sensitivity) in the detection and identification of the O1O139 and non-O1non-O139 serogroups of V cholerae ref-erence strains and clinical isolates The O serogroup-specificprimer sets did not cross-hybridize against nontarget DNAsequences neither in reactions containing target gDNA tem-plates nor in reactions containing unrelated gDNA templatesAs for the quantitatively direct determination assessmenttool the TMPCR had the detection limit of as low as 100 pgof V cholerae gDNAs of the O1 O139 or non-O1non-O139When mimic conditions of the mixed gDNAs of the O1 andO139 were analyzed the TMPCR method exhibited highsensitivity The reaction assays that employed the equally orunequally mixed O1 and O139 gDNAs could compare wellwith the findings of the reactions that used the single sourcegDNAs This might suggest that the O serogroup-specificTMPCR assay could augment the efficiency of the serotypingmethod when examined for the qualitative and quantitativeanalyses of the serotyped V cholerae cultures recovered fromany isolation sources whether clinically or environmentallyAs compared to the culture method only the TMPCR gavepositive result when target DNA of V cholerae non-O1non-O139 could be detected in a water sample in this studyThat is the TMPCR can provide the proof that V choleraenon-O1non-O139 present in a water sample is the VBNCstate This promising technique can be used as similar tothe molecular markers-based PCR methods that permit thedetection and identification of V cholerae O1 O139 andnon-O1non-O139 isolates from clinical patients or aquaticenvironments [13ndash24]

Additionally the TMPCR requires the procedures for thegDNA preparation target gene amplification and postanaly-sis of PCRproductHowever compared to that of the serotyp-ingmethod the technique becomes easier in terms of processand time required to run a lot of the sampled V choleraecultures Similar to that of other touchdownormultiplex PCRmethods developed for probing a variety of microorganisms[33 36 37] the performance efficiency of the TMPCR testedin this study was achieved by a two-stage amplificationincorporating multiplex PCR with conditional touchdownstrategies whichwere suitable for simultaneous amplificationof target DNA sequences with high fidelity and workablerateThis improvedmultiplex PCR comprises a simultaneousPCR and a specific PCR and either one or both of the ampli-fication steps are performed with a touchdown strategy ofwhich loose touchdown strategy is appliedwith a temperaturelower than the optimized annealing temperature and strin-gent touchdown strategy is applied with a temperature higher


than the optimized annealing temperature The TMPCRapproach to circumvent the problems of multiplex PCRassays has been successfully used for simultaneous amplifi-cation of target DNA sequences originally derived from theO1 O139 and non-O1non-O139 serogroups with the corre-sponding touchdown-PCR parameters

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publishing of this paper

Acknowledgments

Theauthors were grateful to the Faculty of TropicalMedicineMahidol University and the Armed Forces Research Instituteof Medical Sciences Bangkok Thailand for providing theO1 569B and O139 MO45 strains Wild strains of the O1(including El TorClassical Ogawa and El TorClassical Inababiotypes) andO139 were kindly provided by BamrasnaraduraInfectious Diseases Hospital Nonthaburi The non-O1 werefrom the Faculty of Public Health Mahidol University Otherreference strains of theO22 andO155were kindly provided byProfessor Toshio Shimada theNational Institute of InfectiousDiseases Tokyo Japan

References

[1] A Huq E B Small P AWest M I Huq R Rahman and R RColwell ldquoEcological relationships between Vibrio cholerae andplanktonic crustacean copepodsrdquo Applied and EnvironmentalMicrobiology vol 45 no 1 pp 275ndash283 1983

[2] S M Faruque M J Albert and J J Mekalanos ldquoEpidemiologygenetics and ecology of toxigenicVibrio choleraerdquoMicrobiologyandMolecular Biology Reviews vol 62 no 4 pp 1301ndash1314 1998

[3] A I Gil V R Louis I N G Rivera et al ldquoOccurrence anddistribution of Vibrio cholerae in the coastal environment ofPerurdquo Environmental Microbiology vol 6 no 7 pp 699ndash7062004

[4] G Constantin de Magny and R R Colwell ldquoCholera andclimate a demonstrated relationshiprdquo Transactions of the Amer-ican Clinical and Climatological Association vol 120 pp 119ndash128 2009

[5] C Lutz M Erken P Noorian S Sun and D McDougaldldquoEnvironmental reservoirs and mechanisms of persistence ofVibrio choleraerdquo Frontiers in Microbiology vol 4 article 3752013

[6] S Chaiyanan A Huq T Maugel and R R Colwell ldquoViabilityof the nonculturable Vibrio cholerae O1 and O139rdquo Systematicand Applied Microbiology vol 24 no 3 pp 331ndash341 2001

[7] W Baffone B Citterio E Vittoria et al ldquoRetention of virulencein viable but non-culturable halophilic Vibrio spprdquo Interna-tional Journal of FoodMicrobiology vol 89 no 1 pp 31ndash39 2003

[8] H CWong andPWang ldquoInduction of viable but nonculturablestate in Vibrio parahaemolyticus and its susceptibility to envi-ronmental stressesrdquo Journal of AppliedMicrobiology vol 96 no2 pp 359ndash366 2004

[9] M Alam M Sultana G Balakrish Nair et al ldquoViable butnonculturableVibrio choleraeO1 in biofilms in the aquatic envi-ronment and their role in cholera transmissionrdquo Proceedings of

the National Academy of Sciences of the United States of Americavol 104 no 45 pp 17801ndash17806 2007

[10] M Senoh J Ghosh-Banerjee T Ramamurthy et al ldquoConver-sion of viable but nonculturableVibrio cholerae to the culturablestate by co-culture with eukaryotic cellsrdquo Microbiology andImmunology vol 54 no 9 pp 502ndash507 2010

[11] M Senoh J Ghosh-Banerjee T Ramamurthy et al ldquoConver-sion of viable but nonculturable enteric bacteria to culturableby co-culture with eukaryotic cellsrdquoMicrobiology and Immunol-ogy vol 56 no 5 pp 342ndash345 2012

[12] S Pradhan S K Mallick and R Chowdhury ldquoVibrio choleraeclassical biotype is converted to the viable non-culturable statewhen cultured with the El Tor biotyperdquo PLoS ONE vol 8 no 1Article ID e53504 2013

[13] K Hoshino S Yamasaki A K Mukhopadhyay et al ldquoDevel-opment and evaluation of a multiplex PCR assay for rapiddetectionz of toxigenic Vibrio cholerae O1 and O139rdquo FEMSImmunology and Medical Microbiology vol 20 no 3 pp 201ndash207 1998

[14] S Chakraborty J Khanam Y Takeda and G B Nair ldquoAppli-cation of PCR for detection of toxigenic Vibrio cholerae O1 inwater samples during an outbreak of cholerardquo Transactions ofthe Royal Society of Tropical Medicine and Hygiene vol 93 no5 pp 527ndash528 1999

[15] B B Pal H K Khuntia S K Samal S S Das and G P Chho-tray ldquoEmergence of Vibrio cholerae O1 biotype El Tor serotypeInaba causing outbreaks of cholera in Orissa Indiardquo JapaneseJournal of Infectious Diseases vol 59 no 4 pp 266ndash269 2006

[16] MAlamM Sultana G BNair et al ldquoToxigenicVibrio choleraein the aquatic environment of Mathbaria Bangladeshrdquo Appliedand Environmental Microbiology vol 72 no 4 pp 2849ndash28552006

[17] I N G Rivera E K Lipp A Gil N Choopun A Huq andR R Colwell ldquoMethod of DNA extraction and application ofmultiplex polymerase chain reaction to detect toxigenic VibriocholeraeO1 and O139 from aquatic ecosystemsrdquo EnvironmentalMicrobiology vol 5 no 7 pp 599ndash606 2003

[18] I N G Rivera J Chun A Huq R B Sack and R R ColwellldquoGenotypes associated with virulence in environmental Isolatesof Vibrio choleraerdquo Applied and Environmental Microbiologyvol 67 no 6 pp 2421ndash2429 2001

[19] B Bakhshi H Mohammadi-Barzelighi M Adabi A R Lariand M R Pourshafie ldquoA molecular survey on virulence associ-ated genotypes of non-O1 non-O139 Vibrio cholerae in aquaticenvironment of Tehran IranrdquoWater Research vol 43 no 5 pp1441ndash1447 2009

[20] B Bakhshi H Mohammadi-Barzelighi A Sharifnia A Dasht-bani-Roozbehani M Rahbar and M R Pourshafie ldquoPresenceof CTX gene cluster in environmental non-O1O139 Vibriocholerae and its potential clinical significancerdquo Indian Journalof Medical Microbiology vol 30 no 3 pp 285ndash289 2012

[21] C Sharma M Thungapathra A Ghosh et al ldquoMolecularanalysis of non-O1 non-O139 Vibrio cholerae associated withan unusual upsurge in the incidence of cholera-like disease inCalcutta Indiardquo Journal of Clinical Microbiology vol 36 no 3pp 756ndash763 1998

[22] G Y Ang C Y Yu K Balqis et al ldquoMolecular evidence ofcholera outbreak caused by a toxigenicVibrio choleraeO1 El Torvariant strain in Kelantan Malaysiardquo Journal of Clinical Micro-biology vol 48 no 11 pp 3963ndash3969 2010

[23] M Alam S Nusrin A Islam et al ldquoCholera between 1991 and1997 in Mexico was associated with infection by classical El


Tor and El Tor variants of Vibrio choleraerdquo Journal of ClinicalMicrobiology vol 48 no 10 pp 3666ndash3674 2010

[24] A Mishra N Taneja and M Sharma ldquoEnvironmental andepidemiological surveillance of Vibrio cholerae in a cholera-endemic region in India with freshwater environsrdquo Journal ofApplied Microbiology vol 112 no 1 pp 225ndash237 2012

[25] A Fallarino C Mavrangelos U H Stroeher and P A Man-ning ldquoIdentification of additional genes required for O-antigenbiosynthesis in Vibrio cholerae O1rdquo Journal of Bacteriology vol179 no 7 pp 2147ndash2153 1997

[26] U H Stroeher G Parasivam B Kate Dredge and P A Man-ning ldquoNovel Vibrio cholerae O139 genes involved in lipopoly-saccharide biosynthesisrdquo Journal of Bacteriology vol 179 no 8pp 2740ndash2747 1997

[27] U H Stroeher K E Jedani and P A Manning ldquoGenetic orga-nization of the regions associated with surface polysaccharidesynthesis inVibrio choleraeO1 O139 andVibrio anguillarumO1and O2 a reviewrdquo Gene vol 223 no 1-2 pp 269ndash282 1998

[28] S Yamasaki T Shimizu K Hoshino et al ldquoThe genes respon-sible for O-antigen synthesis of Vibrio choleraeO139 are closelyrelated to those ofVibrio choleraeO22rdquoGene vol 237 no 2 pp321ndash332 1999

[29] M B Jalajakumari and P A Manning ldquoNucleotide sequenceof the gene ompW encoding a 22 kDa immunogenic outermembrane protein of Vibrio choleraerdquo Nucleic Acids Researchvol 18 no 8 p 2180 1990

[30] B Nandi R K Nandy S Mukhopadhyay G B Nair TShimada and A C Ghose ldquoRapid method for species-specificidentification of Vibrio cholerae using primers targeted to thegene of outer membrane protein Omp Wrdquo Journal of ClinicalMicrobiology vol 38 no 11 pp 4145ndash4151 2000

[31] B Heiberg ldquoThe biochemical reaction of vibriosrdquo Journal ofHygiene vol 36 pp 114ndash117 1936

[32] M T Kelly FWHickman-Brenner and J J Farmer ldquoVibriordquo inManual of Clinical Microbiology A BalowW J Hauler Jr K LHerrman H D Isenberg andH J Shadorny Eds pp 384ndash395ASM Washington DC USA 1991

[33] N C Quaglia G Normanno A Dambrosio et al ldquoMultiplex-touchdown PCR assay for the detection and genotyping ofHelicobacter pylori from artificially contaminated sheep milkrdquoJournal of Food Protection vol 68 no 10 pp 2136ndash2139 2005

[34] CWDieffenbach andG SDvekslerPCRPrimer A LaboratoryManual Cold Spring Harbor Laboratory Cold Spring HarborNY USA 1995

[35] C L Tarr J S Patel ND Puhr E G Sowers C A Bopp andNA Strockbine ldquoIdentification of Vibrio isolates by a multiplexPCR assay and rpoB sequence determinationrdquo Journal of Clini-cal Microbiology vol 45 no 1 pp 134ndash140 2007

[36] P H Nhung K Ohkusu J Miyasaka X S Sun and T EzakildquoRapid and specific identification of 5 human pathogenicVibriospecies bymultiplex polymerase chain reaction targeted to dnaJgenerdquo Diagnostic Microbiology and Infectious Disease vol 59no 3 pp 271ndash275 2007

[37] P Pechgit A Intarapuk D Pinyoowong and A BhumiratanaldquoTouchdown-touchup nested PCR for low-copy gene detec-tion of benzimidazole-susceptible Wuchereria bancrofti witha Wolbachia endosymbiont imported by migrant carriersrdquoExperimental Parasitology vol 127 no 2 pp 559ndash568 2011


Thismight suggest that the emergence of the El Tor biotype ofV choleraeO1 relates to displacement of the existing classicalbiotype as the predominant cause of epidemic choleraSimilar to the toxigenic O1 and O139 serogroups that possessvirulence-association genes [13ndash18] the other non-O1 andnon-O139 serogroups recovered from aquatic environmentsor clinical specimens have also been epidemiologically linkedwith the pathogenic and epidemic potential [18ndash20] Thuspublic health surveillance and monitoring of cholera casesrequire the systems used in the national surveillance for noti-fiable diseases public health laboratory and environmentalsurveillance [21ndash24] Molecular detection techniques such asmolecular marker-based polymerase chain reaction (PCR)methods developed for probing these eccentric V choleraemicroorganisms have been so far proven useful in diagnosisand surveillance for the outbreaks or epidemic investigationsworldwide

In the Gulf of Bengal South and Southeast Asia it hasbeen suggested that such outbreaks of cholera pertainingto cross-contamination and spread of V cholerae after thepassage(s) from susceptible persons to aquatic environmentsare likely to be epidemiologically linked with the inter-connections of sanitation with poorly chlorinated waterscontamination in seafoodfood commodities and seawatersand direct fecal-oral contact among food handlers or seafoodpreprocessing plant workers [2 14ndash16 21 22 24] Standardculture methods used in routine diagnosis and surveillancefor V cholerae by public health reference laboratories dependon handling the samples of whichV cholerae culture is recov-ered Regarding thisV cholerae cultures under study investi-gation were recovered from V cholerae-contracted or sus-pected patients with acute or severe diarrhea whose stools orrectal swabs had been collectively obtained by clinical labo-ratory settings during the outbreaks over two past decadesbetween 1994 and 2010 in Central Thailand

We proposed that the genomic DNA archived fromthe preserved stock cultures could serve as the templatewhen examined for the O1 O139 and non-O1non-O139serogroups by PCR methods as this can also permit a recip-rocal detection of the O1 and O139 serogroups from clinicalspecimens associated with the past outbreaks In this studywe have successfully developed a novel sensitive specificsemiquantitative touchdown-multiplex PCR (TMPCR) fordetection anddifferentiation ofV choleraeO1O139 andnon-O1non-O139 serogroups TMPCRemploys the usefulmolec-ular markers originally derived from the DNA locus involvedin de novo O-antigen biosynthesis of V cholerae [25ndash28] andthe outer membrane protein (ompW) encoding gene specificfor V cholerae [29 30] To achieve the goal of the study weanalyzed the performance efficiency (specificity and sensi-tivity) of this O serogroup-specific TMPCR in detecting anddifferentiating theO1 O139 and non-O1non-O139 genomeswhich were empirically determined using reference strains ofV cholerae and unrelated Vibrio strains We then exploredits usefulness in differentiating the O serogroups presentin those V cholerae stock cultures originally isolated fromclinical specimens Additionally the advancement of TMPCRthat tested V cholerae present in environmental samples wasalso discussed

2 Materials and Methods

21 Bacterial Cultures and Laboratory Classification Sixty-nine V cholerae strains that served as target gDNA templatesincluded reference and wild-type strains of the O1 (includingEl TorClassical Ogawa and El TorClassical Inaba biotypes)O139 and non-O1non-O139 The 31 other bacterial strainsused as nonspecific gDNA templates included 8 Vibrio spp16 Gram-negative bacteria and 7 Gram-positive bacteria Forthe batch propagation of V cholerae the 05mL overnightculture of the O1 569B or O139 MO45 strains was inoculatedinto a 100mLLuria-Bertani (LB) broth supplementedwith 1sodium chloride (NaCl) solution and then incubated at 37∘Cfor 3 h with vigorous shakingThe cell culture with an opticaldensity of 08 was used for drop plate method and subse-quently enumerated by using plate count (PC) agar (DifcoMichigan USA or Eiken Japan) The PC agar was alsosupplemented with 1 NaCl The non-O1non-O139 strainsincluding O22 and O155 were cultured as before

Subcultures of V cholerae and other Vibrio spp weregrown on selective thiosulfate citrate bile salt sucrose (TCBS)agar (Merck Darmstadt Germany) and subsequently theywere biochemically and serologically characterized accordingto themethods described elsewhere [31 32] Briefly biochem-ical tests included triple sugar iron (TSI) motility indole-lysine (MIL) oxidase urea MR Voges-Proskauer (VP)citrate lysine ornithine arginine lactose sucrose mannosearabinose mannitol glucosegas inositol aesculin and salttolerance of 0 3 6 8 and 10 NaCl solution Allreagents were purchased from Difco Laboratory (DifcoMichigan USA)The serogroups O1 O139 and non-O1non-O139 were tested on the basis of agglutination reactionusing commercially available O serogroup-specific antisera(antiserum VcO1O139 Polyvalent SAP S amp A Reagents LabBangkok Thailand) The non-O1non-O139 was character-ized into two nonagglutinable (NAG) groups (NAG I and II)according toHeibergrsquos reaction In addition PC agarwas usedfor viable count and enumeration of all the bacterial strainstested in this study

22 V cholerae Cultures from Clinical Specimens and Serotyp-ing A total of 148 stock cultures of V cholerae that corre-sponded to 108 stools and 40 rectal swabs were collectivelyobtained from V cholerae-contracted or suspected patientswith acute or severe diarrhea during the outbreaks in CentralThailand between 1994 and 2010 By using anonymoussystem the cultures initially isolated by clinical laboratorysettings were performed on isolation source and time ofcollection and the corresponding TCBS stock cultures wereprepared All were subsequently subcultured and kept insemisolid agar medium containing 1 NaCl at the Depart-ment of Microbiology Faculty of Public Health MahidolUniversity Thailand For serotyping the O1 O139 and non-O1non-O139 the agglutination of the 148 subcultured Vcholerae isolates in individuals was blindly performed asbefore In addition only the V cholerae O1 serogroup whosesubcultures agglutinated with polyvalent O1 antiserum wascharacterized using monovalent Ogawa or Inaba antiserumFor biotyping the phenotypic testing of the strains was
















2 and









O1 O1390001 001 01 1 10 100

100 105 104 103 102 101 110 103 101

1 101 102

01 101 103

001 103 104

0001 105



O1 O139100 10 1 01 001 0001

100 100

10 100

1 100

01 100

001 100

0001 100








3 Results




a101010

b100505

c100202

d100101

e051010

f050502

g020210

h010110

M PC

588364256

588364256

600400200

600400200

e

a b c d

f g h








M 1 2 3 4 5 6 7 8 M

588364256

600400

200

(a)

M 1 2 3 4 5 6 7 8 M

600400

200

663

(b)






Stool119899 = 108

O1 (119899 = 65) 65 65 0O139 (119899 = 15) 15 0 15

Non-O1non-O139 (119899 = 28) 28 0 0Total 108 65 15


O1 (119899 = 39) 39 39 0O139 (119899 = 0) 0 0 0

Non-O1non-O139 (119899 = 1) 1 0 0Total 40 39 0





4 Discussion



1 2 3 4 5 6M 7

600400

200

NC9 10 11 12

588364256

8

(a)

600400200

588364256

1 2 3 4 5 6M 7 NC9 10 11 128

(b)

1 2 3 4 5 6M

600400

200

NC

588

364256

(c)

600400

200

1 2 3 4 5 6M NC

588

364

(d)

600400

200

1 2 3 4 5 6M NC

588

256

(e)






O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139










Acknowledgments


References
























































2 and









O1 O1390001 001 01 1 10 100

100 105 104 103 102 101 110 103 101

1 101 102

01 101 103

001 103 104

0001 105



O1 O139100 10 1 01 001 0001

100 100

10 100

1 100

01 100

001 100

0001 100








3 Results




a101010

b100505

c100202

d100101

e051010

f050502

g020210

h010110

M PC

588364256

588364256

600400200

600400200

e

a b c d

f g h








M 1 2 3 4 5 6 7 8 M

588364256

600400

200

(a)

M 1 2 3 4 5 6 7 8 M

600400

200

663

(b)






Stool119899 = 108

O1 (119899 = 65) 65 65 0O139 (119899 = 15) 15 0 15

Non-O1non-O139 (119899 = 28) 28 0 0Total 108 65 15


O1 (119899 = 39) 39 39 0O139 (119899 = 0) 0 0 0

Non-O1non-O139 (119899 = 1) 1 0 0Total 40 39 0





4 Discussion



1 2 3 4 5 6M 7

600400

200

NC9 10 11 12

588364256

8

(a)

600400200

588364256

1 2 3 4 5 6M 7 NC9 10 11 128

(b)

1 2 3 4 5 6M

600400

200

NC

588

364256

(c)

600400

200

1 2 3 4 5 6M NC

588

364

(d)

600400

200

1 2 3 4 5 6M NC

588

256

(e)






O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139










Acknowledgments


References
















































O1 O1390001 001 01 1 10 100

100 105 104 103 102 101 110 103 101

1 101 102

01 101 103

001 103 104

0001 105



O1 O139100 10 1 01 001 0001

100 100

10 100

1 100

01 100

001 100

0001 100








3 Results




a101010

b100505

c100202

d100101

e051010

f050502

g020210

h010110

M PC

588364256

588364256

600400200

600400200

e

a b c d

f g h








M 1 2 3 4 5 6 7 8 M

588364256

600400

200

(a)

M 1 2 3 4 5 6 7 8 M

600400

200

663

(b)






Stool119899 = 108

O1 (119899 = 65) 65 65 0O139 (119899 = 15) 15 0 15

Non-O1non-O139 (119899 = 28) 28 0 0Total 108 65 15


O1 (119899 = 39) 39 39 0O139 (119899 = 0) 0 0 0

Non-O1non-O139 (119899 = 1) 1 0 0Total 40 39 0





4 Discussion



1 2 3 4 5 6M 7

600400

200

NC9 10 11 12

588364256

8

(a)

600400200

588364256

1 2 3 4 5 6M 7 NC9 10 11 128

(b)

1 2 3 4 5 6M

600400

200

NC

588

364256

(c)

600400

200

1 2 3 4 5 6M NC

588

364

(d)

600400

200

1 2 3 4 5 6M NC

588

256

(e)






O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139










Acknowledgments


References












































3 Results




a101010

b100505

c100202

d100101

e051010

f050502

g020210

h010110

M PC

588364256

588364256

600400200

600400200

e

a b c d

f g h








M 1 2 3 4 5 6 7 8 M

588364256

600400

200

(a)

M 1 2 3 4 5 6 7 8 M

600400

200

663

(b)






Stool119899 = 108

O1 (119899 = 65) 65 65 0O139 (119899 = 15) 15 0 15

Non-O1non-O139 (119899 = 28) 28 0 0Total 108 65 15


O1 (119899 = 39) 39 39 0O139 (119899 = 0) 0 0 0

Non-O1non-O139 (119899 = 1) 1 0 0Total 40 39 0





4 Discussion



1 2 3 4 5 6M 7

600400

200

NC9 10 11 12

588364256

8

(a)

600400200

588364256

1 2 3 4 5 6M 7 NC9 10 11 128

(b)

1 2 3 4 5 6M

600400

200

NC

588

364256

(c)

600400

200

1 2 3 4 5 6M NC

588

364

(d)

600400

200

1 2 3 4 5 6M NC

588

256

(e)






O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139










Acknowledgments


References










































M 1 2 3 4 5 6 7 8 M

588364256

600400

200

(a)

M 1 2 3 4 5 6 7 8 M

600400

200

663

(b)






Stool119899 = 108

O1 (119899 = 65) 65 65 0O139 (119899 = 15) 15 0 15

Non-O1non-O139 (119899 = 28) 28 0 0Total 108 65 15


O1 (119899 = 39) 39 39 0O139 (119899 = 0) 0 0 0

Non-O1non-O139 (119899 = 1) 1 0 0Total 40 39 0





4 Discussion



1 2 3 4 5 6M 7

600400

200

NC9 10 11 12

588364256

8

(a)

600400200

588364256

1 2 3 4 5 6M 7 NC9 10 11 128

(b)

1 2 3 4 5 6M

600400

200

NC

588

364256

(c)

600400

200

1 2 3 4 5 6M NC

588

364

(d)

600400

200

1 2 3 4 5 6M NC

588

256

(e)






O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139










Acknowledgments


References










































1 2 3 4 5 6M 7

600400

200

NC9 10 11 12

588364256

8

(a)

600400200

588364256

1 2 3 4 5 6M 7 NC9 10 11 128

(b)

1 2 3 4 5 6M

600400

200

NC

588

364256

(c)

600400

200

1 2 3 4 5 6M NC

588

364

(d)

600400

200

1 2 3 4 5 6M NC

588

256

(e)






O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139










Acknowledgments


References













































O1 Non-O1non-O139

600400

200

588364256

663

M 1 2 3 4 5 6 7 8 M9 10 11 12 13 14 15O139










Acknowledgments


References













































Acknowledgments


References

























































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