aa

Xia

idem

ntend as ofomb

proles of LVPCs and virulence markers were primarily used to group the strains into six distinct complexeswith different potential pathogenicity natures. The strains from each of these complexes were further

e a much more detailed discrimination of the strains. A genetic ngerprint-like

uiringwaters

International Journal of Food Microbiology 149 (2011) 143151

Contents lists available at ScienceDirect

International Journal o

sevimportant food-borne pathogens in many coastal countries (Nairet al., 2007). Infections occur mainly via the fecaloral route, althoughwound infections have been reported; ingestion of bacteria in raw orundercooked seafood (especially oysters) is the predominant cause ofinfection (Yeung and Boor, 2004). Clinical manifestations includediarrhea, abdominal cramps, nausea, vomiting, headache, fever, andchills (Yeung and Boor, 2004).

The characterized virulence determinants of V. parahaemolyticusinclude thermostable direct hemolysin (TDH), TDH-related hemolysin(TRH), and three distinct type three secretion systems (T3SS1,T3SS2, and T3SS2) (Hiyoshi et al., 2010; Makino et al., 2003;Okada et al., 2009; Park et al., 2004; Xu et al., 1994). T3SS1 and T3SS2

somal location of different strains, most likely through lateral genetransfer (Makino et al., 2003; Okada et al., 2009). As shown in theRIMD2210633 strain (Burdette et al., 2008; Hiyoshi et al., 2010;Makino et al., 2003; Park et al., 2004), T3SS1 is the major contributorto V. parahaemolyticus-induced cytotoxic activity; TDH is the soledeterminant of hemolytic activity but only partially contributes toenterotoxicity; T3SS2 is the main determinant of enterotoxicity;both T3SS1 and TDH play signicant roles in septicemia. As shown inthe TH3996 strain (Xu et al., 1994), TRH is the sole determinant ofhemolytic activity but only partially contributes to enterotoxicity,whereas T3SS2 is essential for enterotoxicity. Taken together,hemolysin TDH or TRH, T3SS1, and T3SS2 are the major de-are on chromosomes I and II, respectively (Met al., 2009). Two copies of tdh and one copypathogenicity island Vp-PAIRIMD2210633 in

Corresponding author. Tel.: +86 10 66948594.E-mail address: dongshengzhou1977@gmail.com (D

1 Xiao Xiao and Yanfeng Yan contributed equally to t

0168-1605/$ see front matter 2011 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2011.06.014and can be frequentlyents, as well as from shts. It is one of the most

the TH3996 strain (T3SS1+, tdh, T3SS2, trh+, and T3SS2+)(Okada et al., 2009). Although coming from two distinct lineages, thetwo pathogenicity islands have integrated into the same chromo-isolated frommarine and estuarine environmand shellsh dwelling in these environmenVNTRGenotyping

1. Introduction

Vibrio parahaemolyticus is a salt-reqinhabits seawater or other brackish 2011 Elsevier B.V. All rights reserved.

bacterium that naturally

RIMD2210633 (T3SS1+, tdh+, T3SS2+, trh, and T3SS2)(Makino et al., 2003). One copy each of trh and T3SS2 plus an urelocus comprise another distinct pathogenicity island Vp-PAITH3996 inakino et al., 2003; Okadaof T3SS2 constitute athe pandemic strain

terminants of hetively, and thuseach distinct asp

Serotypes ofgroups and 71 Kstrains remain ureliable for charhaemolyticus (Ha

. Zhou).his work.

l rights reserved.estimation of V. parahaemolyticus.

LVPC database of a large collection of strains established with this two-stage approach would be very useful for

identication, genotyping, origin tracing, and riskKeywords:Vibrio parahaemolyticus analyzed with VNTRs to givA novel genotyping scheme for Vibrio parvariably-presented gene clusters (LVPCs)repeats (VNTRs)

Xiao Xiao 1, Yanfeng Yan 1, Yiquan Zhang, Li Wang,Ruifu Yang, Dongsheng Zhou State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Ep

a b s t r a c ta r t i c l e i n f o

Article history:Received 9 February 2011Received in revised form 17 June 2011Accepted 18 June 2011Available online 26 June 2011

A total of 18 variably-presetandem repeats (VNTRs), anumber in 251 global strainstep genotyping approach c

j ourna l homepage: www.e lhaemolyticus with combined use of largend variable-number tandem

Liu, Lin Yang, Yafang Tan, Zhaobiao Guo,

iology, Beijing, China

d gene clusters (LVPCs) and nine previously characterized variable-numberll known virulence markers were screened for their frequency and/or copyVibrio parahaemolyticus using PCR and gel or capillary electrophoresis. A two-ining the use of LVPCs and VNTRs was established accordingly. The frequency

f Food Microbiology

i e r.com/ locate / i j foodmicromolytic, cytotoxic, and enterotoxic activities, respec-each of them has a specic role that contributes toect of pathogenesis.V. parahaemolyticus can be differentiated into 13 Otypes using the commercial antisera, although manyntypable. However, this serotyping scheme is notacterizing the epidemiological spreads of V. para-n et al., 2008). A pandemic group has been linked to

Table 1PCR targets, primers and parameters.

Genomic locus PCR target/gene

PCR primers Cycles ANTE(C)

PRSI(bp)

Name Sequences (5' to 3') Reference

PCR-screening assayVP0820 toxR toxR367F GACTTCTGACGCAATCGTTG Kim et al. (1999) 30 60 367

toxR367R ATACGAGTGGTTGCTGTCATGVPA0226 tlh tlh450F AAAGCGGATTATGCAGAAGCACTG Taniguchi et al.(1985, 1986) 30 58 450

tlh450R GCTACTTTCTAGCATTTTCTCTGCT3SS1 (VP1656-1700) vscU (VP1675) LVPC1675F ATCGTCTCTTCGGCGTTAATTC This study 30 56 717

LVPC1675R GATGTGAGTCGGGTTCGTTACVP1686 LVPC1686F ATGACAGAAGTGATGCTAGAGAC Yan et al. (2011) 30 56 704

LVPC1686R GCCAGAGGATTGAACGAGTCVPA1168 PGS-PCR PGS-1 TTCGTTTCGCGCCACAACT Okura et al. (2004) 30 63 235

PGS-2 TGCGGTGATTATTCGCGTCTVP0820 GS-PCR GS-VP.1 TAATGAGGTAGAAACA Matsumoto et al. (2000) 30 48 651

GS-VP.2 ACGTAACGGGCCTACAVPA1314 (tdhA) tdh tdh251F GGTACTAAATGGCTGACATC Tada et al. (1992) 30 55 251)VPA1378 (tdhS) tdh251R CCACTACCACTCTCATATGCLVPC01 (VP0380-0403) VP0387 LVPC0387F CTAACTCTGCCGATGCTGAC This study 30 56 690

LVPC0387R GACCTGCCGTGCCAATAAGLVPC02 (VP0634-0643) VP0643 LVPC0643F AGCGTCTTGAGTTACCTAATGC 30 56 737

LVPC0643F CAGTGGAATAGTGCGAATTGAACLVPC03 (VP1071-1095) VP1083 LVPC1083F GGCTTCTTCGGTTAGTATGTCTC 30 56 1010

LVPC1083R ATGCTGGCTTCTGATATGTTCTCLVPC04 (VP1355-1368) VP1361 LVPC1361F TGGTTGGAGATTTATGGGAGAAAG 30 56 725

LVPC1361R AAGTAGAGGCAGAACGAGGACLVPC05 (VP1386-1420) VP1401 LVPC1401F GCACGCCACTGAAGTTCTTG 30 56 1057

LVPC1401R AACGACAGATTGAGCACTTGAAGLVPC06 (VP1549-1590) VP1561 LVPC1561F CCGAGACAATACAGATCAAGTAAC 30 56 754

(ORF8) LVPC1561R TGAGTAACCATAGACCACGATTCLVPC07 (VP1771-1864) VP1823 LVPC1823F TGCGGAGGCTTCGGTATTG 30 56 656

LVPC1823R CTGACGAGAGTGTGTAGATTGTTCLVPC08 (VP2131-2144) VP2137 L02842-PVP2137F CAAGCAATACAACGCAAGGAAC 30 56 350

L02843-PVP2137R GGTGGCAGGTTCAACATATCTCLVPC09 (VP2900-2910) VP2905 L02844-PVP2905F GCCATCGCCCAGCAAATATAG 30 56 400

L02845-PVP2905R CTCCACAGCCTCATCTACATTGLVPC10 (VPA0074-0089) VPA0082 LVPCA0082F CAATCAGTCGTGTCGCCATC 30 58 880

LVPCA0082R TGTTGAATAACCGTGCCAGCLVPC11 (VPA0434-0458) VPA0447 LVPCA0447F TCAAGGCATTAGACGAGTTAGG 30 53 618

LVPCA0447R CCAAGGTCCGCTTATTGTAGGLVPC12 (VPA0713-0732) VPA0727 LVPCA0727F TCCAATTACGATGCGAGTGAAC 30 56 670

LVPCA0727R TCCATAAGCCGTGAGTGTAGGLVPC13 (VPA0887-0914) VPA0908 LVPCA0908F TTCACATCAAGCCGCCATAC 30 56 704

LVPCA0908R GTCGTCGCCTAATTCCTCACLVPC14 (VPA1194-1210) VPA1199 LVPCA1199F AGGCTGTTCTGTTCTTGTTGG 30 56 745

LVPCA1199R AATGTCGGTGTCTGCTTGAGLVPC15 (VPA1253-1270) VPA1262 LVPCA1262F GGACCTAACGGAGAACATTCATC 30 56 661

LVPCA1262R CATTGGCGGAGCGAATAAGGVp-PAIRIMD2210633 VPA1312 LVPCA1312F CACGGCACAAGCGAGAAATC 30 56 409

LVPCA1312R CCATTTGTAGCCACTCTGATCGVPA1321 LVPCA1321F GCGTGGTGGTTAGTGAATCC 30 56 561

LVPCA1321R TTGTCTTCCAAGGTGATGTGTCLVPC16(VPA1334-1370)

T3SS2 VPA1351 LVPCA1351F CCTACAGCGAGCAACAGATG Yan et al. (2011) 35 53 13641354 LVPCA1353R GCGAAGAACACCAAGTTATGAAGVPA1373 LVPCA1373F CACGCATATCCATCAGCACTTG This study 30 58 549

LVPCA1373R TGTTTCCGCCTACGCTAAGAGVPA1380 LVPCA1380F TGTGAATCTACCGACGAAGTTG 30 58 500

LVPCA1380R ACTGTCTCTGTTGCAGGAATTGLVPC17 (VPA1700-1709) VPA1706 LVPCA1706F TGATTCACTAGAGCACCATAAAGC 30 56 877

LVPCA1706R CATAGGACCAAGCAACGATAGGVp-PAITH3996 LVPC18

(RPI01-74)ure RPI11 LVPCRPI11F TCTACGGCGAAGAGGTCAAG 30 50 837locus LVPCRPI11R TCAACATATCCAAGTGCTCATCCtrh RPI21 trh250F GGCTCAAAATGGTTAAGCG Tada et al. (1992) 30 55 250

trh250R CATTTCCGCTCTCATATGCRPI25 LVPCRPI25F TCTGACCTGAGCACCAATACTG This study 30 50 513

LVPCRPI25R CGCTTCTTCACCGAACTCTAACT3SS2 vscU2 LVPCRPI48F GTCAACAGAACCTAAGAAATACCC Yan et al. (2011) 30 56 725

(RPI48) LVPCRPI48R ATTCCATCACCTCTCGCATTCRPI72 LVPCRPI72F TTGCTGCACTTGCGAATACTG This study 30 58 551

LVPCRPI72R TTGTACTTCTGAGGTGGCTAGG

MLVAVP0446 VPTR4 VPTR4F FAM-AAACGTCTCGACATCTGGATCA Harth-Chu et al. (2009) 35 58 227

VPTR4R TGTTTGGCTATGTAACCGCTCAVP2104 VP1-10 VP1-10F CGTCTTGCTCGTGAACGTAA 35 58 964

VP1-10R TCATTAAGTCAGGCGTGCTG

144 X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 143151

22% of the whole gene pool on the RIMD2210633 genome (Han et al.,2008). The gene acquisition and loss greatly promote the genetic

equ

EX-AGGTGCGAGAM-TTGAM-CATEX-AGAEX-ACTEX-AGAEX-TTG

., 20

145X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 143151the worldwide outbreaks of V. parahaemolyticus-induced diarrhea andgastroenteritis since 1996 (Nair et al., 2007). The pandemic strains arecomposed of a predominant O3:K6 serovar and its derivates, namely,O4:K68, O1:K25, O1:KUT, and O6:K18, are genetically conserved toconstitute a clonal complex (Yan et al., 2011). The presence of a toxRS/new sequence within the toxRS operon was linked to the pandemicstrains and, accordingly, a toxRS/new sequence-targeted GS-PCR wasdeveloped (Matsumoto et al., 2000). A positive detection of both tdhand toxRS/new sequence, and a negative detection of trh by PCR canreliably identify the pandemic strains (Han et al., 2008; Meador et al.,2007; Okura et al., 2003).

Various molecular typing methods, such as arbitrarily primedpolymerase chain reaction (AP-PCR), pulsed eld gel electrophoresis(PFGE), multilocus sequence typing (MLST), multi-locus variable-number tandem repeat (VNTR) analysis (MLVA), microarray-basedcomparative genome hybridization (M-CGH), have been applied todissect genetic variabilities and epidemiological spread of V. para-haemolyticus (Chowdhury et al., 2000, 2004; Gonzalez-Escalona et al.,2008; Wong et al., 2007, 1996; Yan et al., 2011). Compared to PFGEand AP-PCR, the latter three represent new generations of genotypingmethod because they are able to track accurately not only geneticdifferences but also phylogenetic relatedness of strains. M-CGH has itspotency of genomotyping based on the genome-wide determinationof gene frequency proles (Han et al., 2008; Izutsu et al., 2008), but it

Table 1 (continued)

Genomic locus PCR target/gene

PCR primers

Name S

MLVAVP2131 VPTR7 VPTR7-F H

VPTR7-R AVP2191 VP1-11 VP1-11F C

VP1-11R TVP2226 VNTR6 VPTR6-F F

VPTR6-R CVP2892 VPTR1 VPTR1F F

VPTR1R TVP2956 VPTR8 VPTR8F H

VPTR8R AVP3012 VNTR5 VPTR5F H

VPTR5R AVPA0714 VPTR3 VPTR3F H

VPTR3R AVPA1455 VP2-07 VP2-07F H

VP2-07R T

Gene IDs were derived from RIMD2210633 (Makino et al., 2003) or TH3996 (Okada et alsize (bp) of PCR product when the RIMD2210633 DNA was used as template.is a very expensive method with high technical requirements. BothMLST and MLVA are low-cost, high-resolution methods relied on afewmarkers, but they often had less potency in concisely denoting thepathogenic potentials of the strains tested.

A VNTR is composed of multiple short sequence repeats in thegenome, and often show variations in its repeat number betweenindividuals of genome.VNTRshavebeenusedextensively asmarkers fordiscrimination between bacterial strains (Lindstedt, 2005). VNTRs gavehigh resolution and reproducibility for discriminating genotypicallydiverse collections of clinical and environmental V. parahaemolyticus(Harth-Chu et al., 2009; Kimura et al., 2008), which promoted theestablishment of a rened MLVA scheme based on a collection of tenVNTR loci (Harth-Chu et al., 2009).

M-CGH is a technique by which the variably-presented DNAregions between two genomes can be analyzed by competitivehybridization to DNA microarray, which highlights the importanceof gene loss/acquisition in the microevolution of a bacterial species(Dorrell et al., 2005). The M-CGH method (Han et al., 2008; Izutsuet al., 2008) discloses very high levels of genome plasticity in thenatural populations of V. parahaemolyticus due to gene acquisition andloss. Genes that present variably in the genomes account for aboutdiversication of V. parahaemolyticus for the emergence of differentcomplexes yet each of them is related genetically and phylogeneti-cally (Han et al., 2008; Izutsu et al., 2008).

The previous M-CGH studies (Han et al., 2008; Izutsu et al., 2008)disclosed 17 large variably-presented gene clusters [LVPCs; VPC hasbeen previously abbreviated from variably-presented gene clusters(Izutsu et al., 2008)]. Vp-PAITH3996 was assigned as the 18th LVPC(Okada et al., 2009). Each LVPC, as a single DNA region containing atleast ten consecutive genes, is variably distributed within thegenomes of different V. parahaemolyticus strains. In this follow-upstudy, the above 18 LVPCs and all the known virulence markers werecollected, from which representative genes or targets were chosen forPCR-screening to check for their presence or absence within acollection of 251 strains of V. parahaemolyticus. A novel genotypingmethod was established based on the frequency proles of LVPCs andvirulence markers. The data presented here indicate that thecombined use of virulence markers, LVPCs, and VNTRs wouldbe very useful for identication, genotyping, and origin tracing ofV. parahaemolyticus.

2. Materials and methodsCycles ANTE(C)

PRSI(bp)

ences (5' to 3') Reference

TATCTACAAAGGTGGCGGAGAT 35 58 239TGTTACTTGTTCCAGACGCTGGAGAATTGGCTTA 35 58 835CCTGAAGCTGAAAACATGTCGATGGTGTTCTGTTCC 35 58 309ACTTGCTCGCTCAGGAGTAACAACGCAAGCTTGCAACG 35 58 253TCTCGCCACATAACTCAGCACATCGGCAATGAGCAGTTG 35 58 307GGTTGCTGAGCAAGCGGCTGGATTGCTGCGAGTAAGA 35 58 195CAAGGGCTGCTTCGGCGCCAGTAATTCGACTCATGC 35 58 332CTGTTCCCGTCGCTGATGATTTTGAAGCAGCGAAGA 35 48 317TGACTGCTGTCCTTGC

09). Cycles: cycles of PCR amplication; ANTE: annealing temperature (C) in PCR; PRSI:2.1. Bacterial strains

The 251 strains (S001 to S251) of V. parahaemolyticus used in thisstudy (Supplementary Table S1) included 193 isolates from patients,40 from seafood, and 18 from other environment samples. S001 toS174 had been involved in our previous M-CGH (Han et al., 2008) andMLST (Yan et al., 2011) studies. These 251 strains were isolatedbetween 1951 and 2007 from 13 countries, including China(Mainland, Hong Kong, and Taiwan), Bangladesh, India, Indonesia,Japan, Korea, Malaysia, Maldives, Philippines, Singapore, Spain,Thailand, and United States. The bacteria were grown in LB agarcontaining 2% NaCl (Amresco, Inc., Solon, OH, USA) at 37 C. Thegenomic DNA was isolated using the classical SDS (AMRESCO) lysisand phenol-chloroform extraction method (Li et al., 2009), with themethoxyethanol removal of polysaccharides that contaminate theDNA (Seidler et al., 1975). The V. parahaemolyticus-specic toxR/newsequence (Han et al., 2008; Kim et al., 1999) and tlh gene (Nordstromet al., 2007) could be detected for all the 251 strains by PCR,conrming the exact assay target of V. parahaemolyticus, as well as thegood quality of DNA templates for PCR.

146 X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 1431512.2. PCR-screening assay

The PCR-screening targets included various virulence markers andLVPCs (Table 1). Each target-specic primer pair worked well when amixture of the genomic DNAs from S175 to S178, namely,RIMD2210633, AQ3815, AQ4037 and AT4 (Kishishita et al., 1992;Makino et al., 2003; Nakaguchi et al., 2004; Nishibuchi et al., 1992,1989), was used as reference template. The bacterial genomic DNAswere arrayed in 96-well PCR plates (Axygen, Inc., Union City, CA,USA). A volume of 25 l PCR mixture contained 50 mM KCl, 10 mMTrisHCl (pH 8.0), 2.5 mM MgCl2, 0.001% gelatin, 0.1% bovine serumalbumin (Sigma Co., St. Louis, MO, USA), 100 M each of dATP, dCTP,dGTP, and dTTP (GE Healthcare Bio-Sciences Corp., Piscataway, NJ,USA), 0.1 M of each primer, 1 U Taq DNA polymerase (MBIFermentas, Burlington, ON, Canada), and 10 ng of template DNA.The parameters for amplication were as follows: 95 C for 3 min, 30or 35 cycles of 94 C for 30 s, an appropriate annealing temperaturefor 30 s, and 72 C for 1 min, and a nal extension step of 72 C for5 min. Each PCR reaction was repeated at least twice. The PCRproducts were then analyzed by 1.2% agarose gel electrophoresis withethidium bromide staining. The nal absent (0) or present (1) call ofeach PCR-screening target was assigned to each strain to generate thebinary PCR-screening dataset (Supplementary Tables S1 and S2).

2.3. MLVA

The ten VNTR loci (Table 1) for MLVA of V. parahaemolyticus hadbeen characterized previously (Harth-Chu et al., 2009). Two of them,VP1-10 and VP1-11, were amplied from each strain by PCR with theVNTR-specic primer pairs. The PCR products were analyzed by 3%agarose gel electrophoresis with ethidium bromide staining (Li et al.,2009). The DNA size markers (60 bp ladder) and amplicons from thereference strain RIMD2210633 were run as controls in the electro-phoresis for estimating the allele sizes of a test strain (Li et al., 2009).

The capillary electrophoresis assay was carried out for theremaining eight VNTR loci that were amplied from each strainwith Fam- or Hex-labeled primers (Harth-Chu et al., 2009). Thepuried and uorescently labeled PCR products were mixed withROX-500 (Applied Biosystems, Carlsbad, CA, USA) and deionizedformamide (Applied Biosystems). The denatured mixtures wereloaded into an ABI3100 sequencer (Applied Biosystems) for capillaryelectrophoresis. Each VNTR allele was identied by color and wasassigned a size by the GeneScan software version 3.7 (AppliedBiosystems).

The allele sizes, determined by agarose gel electrophoresis or bycapillary electrophoresis, were converted into the allele copy numbers(integers after rounding off) with the following equation:

R = Rc +MxMc

U

where Rc and R are the allele copy numbers in the reference(RIMD2210633) and test strains, respectively;Mc andMx are the allelesizes in the reference and test strains, respectively; and U is the numberof base pairs of the single VNTR repeat. The copy number of each VNTRallele was determined to generate the nal categorical VNTR dataset(Supplementary Table S3). A designation of 0 was given when no PCRamplication was observed at a given locus. A locus that contains bothanking sequences with no repeat unit was not observed in this study.The allelic diversity indices were calculated using the V-DICE software(http://www.hpa -bioinfotools.org.uk/cgi-bin/DICI/DICI.pl).

2.4. Clustering and genotyping analysis

The binary PCR-screening data for the frequency of virulence

markers were displayed by the TreeView tool (Eisen et al., 1998). Thebinary LVPC (Supplementary Table S2) or categorical VNTR (Supple-mentary Table S3) dataset was analyzed by BioNumerics Version 5.01(Applied Maths, Keistraat, Belgium, Germany). Clustering was carriedout subsequently by the unweighted-pair group method usingaverage linkages (UPGMA) to calculate a similarity matrix for whichthe binary Jaccard and categorical multi-state coefcients wereemployed for the LVPC and VNTR datasets, respectively. The similaritymatrix was then used to build a minimum spanning tree with asimilarity bin size of 6.0, in which the branch lengths werelogarithmically transferred.

3. Results and discussion

3.1. Identication of pandemic strains

Based on the previous genotypic denition (GS-PCR+, tdh+, andtrh) for the pandemic group, 63 of the 251 isolates tested hereinwere identied as the pandemic strains. In addition to the previouslycharacterized O3:K6, O4:K68, O1:K25, O1:KUT, and O6:K18 strains(Han et al., 2008;Meador et al., 2007; Okura et al., 2003), one O11:K36strain (S216) was also assigned to this group of pandemic strains. Ofthe 188 non-pandemic strains, one (S093) gave the GS-PCR+ result,conrming that GS-PCR alone was reliable with reservation for theidentication of pandemic strains (Han et al., 2008; Okura et al.,2003).

PGS-PCR, which is based on a 930 bp AP-PCR fragment, has beenpreviously shown to detect the pandemic strains specically (Hanet al., 2008; Okura et al., 2004). In this study, the positive PGS-PCRwasonly detected in all the 63 pandemic strains, further conrming thatthe PGS-PCR-targeted DNA marker is specic to the pandemic group.Compared with the detection of tdh, toxRS/new sequence, and trh, asingle PGS-PCR was apparently more convenient to detect thepandemic strains.

3.2. Distribution of virulence markers

Two targets from T3SS1 and six targets, each fromVp-PAIRIMD2210633and Vp-PAITH3996 (including those targeting tdh, T3SS2, trh, andT3SS2), were chosen for PCR-screening against the 251 strains (Fig. 1).The two T3SS1 genes tested were present in all 251 strains (Fig. 2),which conrmed the previous notion that T3SS1 was universallydistributed in V. parahaemolyticus (Meador et al., 2007; Noriea Iii et al.,2010; Sugiyama et al., 2008). In contrast, both Vp-PAIRIMD2210633 andVp-PAITH3996, which harbored T3SS2 and T3SS2, respectively, weredetected in portions of the tested strains (Fig. 2). The GC content ofT3SS1 resembled the average GC content of the entire genome ofRIMD2210633, whereas those of Vp-PAIRIMD2210633 and Vp-PAITH3996were obviously lower than the average one (Makino et al., 2003; Okadaet al., 2009). It was therefore speculated that the presence of T3SS1probably occurs at the speciation of V. parahaemolyticus (Makino et al.,2003), and that the lateral transfer of Vp-PAIRIMD2210633 or Vp-PAITH3996is yet a recent event of intraspecies microevolution.

Only 1.3% of the 58 nonclinical strains and 93.8% of the 193 clinicalones harbored at least one of tdh, T3SS2, trh, and T3SS2, whichindicated the close link of these virulence determinants to V.parahaemolyticus of clinical origins. Taken together, clinical isolatesof V. parahaemolyticus generally produced at least one of TDH, TRH,T3SS2 and T3SS2 in contrast to non-clinical strains, and often gavedistinct frequency proles of these virulence determinants. Thus, thedetection of all these virulence markers is needed to give a fulloverview of the pathogenic potential of the strains.

The six PCR targets from Vp-PAITH3996 gave identical frequencyproles among the 251 strains (Fig. 2), supporting the fact that thepresence of trh and that of T3SS2 were 100% correlated, and that trhmight be exclusively harbored in Vp-PAITH3996. In contrast, the

frequency proles of the six PCR targets from Vp-PAIRIMD2210633

were mosaic among the 251 strains (Fig. 3a). Notably, 26 strains thatwere positive for tdh but negative for Vp-PAIRIMD2210633 were detected(Fig. 2), indicating that tdh was not exclusively harbored in Vp-PAIRIMD2210633. These denoted the mosaic structure of Vp-PAI-RIMD2210633, rather than that of Vp-PAITH3996.

Of the 251 strains, 56% were negative for both Vp-PAIRIMD2210633and Vp-PAITH3996, whereas the remaining 44% were those thatharbored either Vp-PAIRIMD2210633 or Vp-PAITH3996 (Fig. 3b). Notably,these two pathogenicity islands were never present together in asingle strain. In contrast, the presence of both tdh and trhwas detectedwithin a small portion (10%) of the strains tested (Fig. 3c); thesestrains gave an uncommon genotype of the presence of the tdh geneand the trh-harboring Vp-PAITH3996 pathogenicity island. Thecoexistence of tdh and trh genes has already been reported in V.parahaemolyticus isolates worldwide (Roque et al., 2009), althoughthe prevalence of these strains is generally low.

3.3. LVPC-based genotyping

A total of 18 LVPCs (LVPC01 to LVPC18) were collected based onthe previous M-CGH and computational genome comparison studies(Han et al., 2008; Izutsu et al., 2008; Okada et al., 2009). There was anoverlapping of LVPCs and virulence markers; LVPC16 was essentiallythe T3SS2 that was a component of Vp-PAIRIMD2210633, whereasLVPC18 was exactly Vp-PAITH3996 that harbored trh and T3SS2. Asingle gene was chosen from each LVPC, and subjected to PCR toscreen for the presence (1) or absence (0) of each LVPCwithin the 251strains tested.

The 251 strains gave a total of 48 different LVPC frequency proles.An UPGMA-based minimum spanning tree was constructed from thebinary LVPC data (Supplementary Table S2) to give a groupingdendrogram of the 251 strains (Fig. 4a). Based on the minimumspanning tree structure in conjunction with the frequency proles of

Fig. 1. Genetic structure of V. parahaemolyticus pathogenicity islands. Genes or genomicloci underlined were subjected to PCR screening analysis. Two copies (tdhA and tdhS) oftdh were located in Vp-PAIRIMD2210633, but PCR could not discriminate between themdue their high homology; thus, data presented here only denote the presence orabsence of tdh.

147X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 143151Fig. 2. Presence or absence of pathogenicity islands and virulence markers. Each rowrepresents a strain of our collection, whereas each column except for the clinical onestands for a PCR target. For the clinical column, the black area indicates a clinical isolatefor a given strain, whereas the gray one denotes a non-clinical one. For all the othercolumns, black indicates the presence of a PCR target in a given strain, whereas absenceis indicated in gray.Fig. 3. Distribution of virulence markers. a) The six PCR targets of Vp-PAIRIMD2210633 areshown in Fig. 1. Of the 251 strains, 11% gave a mosaic distribution of the six targets,whereas the remaining 89% were with or without all six targets. b) Vp-PAIRIMD2210633and Vp-PAITH3996 were never present in a single strain. c) tdh and trh could be present in

a single strain.

virulence markers, these 251 strains could be assigned into sixcomplexes, LVPC-C1 to LVPC-C6 (Fig. 4b). In our previous M-CGHstudy (Han et al., 2008), the genome-wide gene contents of 175 ofthese 251 strains were compared using a RIMD2210633-speciccDNA microarray, which generated a similar UPGMA-based MS of the175 strains. Notably, the minimum spanning trees of LVPC (Fig. 4a)and M-CGH (Han et al., 2008) shared a very similar topologicstructure. In addition, LVPC-C1 to LVPC-C5 (Fig. 4b) generallycorresponded to the ve M-CGH complexes (C1 to C5) (Han et al.,2008), respectively. LVPC-C6 (Fig. 4b) contained only a single strainS093 (GS-PCR+, tdh, T3SS2, trh, and T3SS2), which wasassigned to neither complex in the M-CGH study (Han et al., 2008).

According to our previous M-CGH (Han et al., 2008) and MLST (Yanet al., 2011) studies, V. parahaemolyticus has at least two clinical clonalcomplexes, namely, pre-1996 old-O3:K6 clone (GS-PCR, tdh,T3SS2, trh+, and T3SS2+) and post-1996 pandemic clone (GS-PCR+, tdh+, T3SS2+, trh, and T3SS2), and a non-clinical clonalcomplex (GS-PCR, tdh, T3SS2, trh, and T3SS2). These three

major clonal complexes represent distinct well-adapted clonal isolateswith distinct frequency proles of virulence factors (i.e., differentpathogenicity natures).

All 14 LVPC-C2 strains (Fig. 4b) were the pre-1996 old-O3:K6strains (GS-PCR, tdh, T3SS2-, trh+, and T3SS2+); 12 of themwere previously characterized by MLST to constitute a clonal complex,namely the old-O3:K6 clone, whereas the remaining two were notinvolved in MLST. All 63 pandemic strains (GS-PCR+, tdh+, T3SS2+,trh, and T3SS2) tested were assigned into the LVPC-C3 complex(Fig. 4b). Moreover, 39 of them were previously characterized toconstitute another clonal complex called the pandemic clone; theremaining 24 were not involved in that study. Previous studies (Hanet al., 2008;Yan et al., 2011) denoted that S093 and similarpre-1996O3:K6 strains (GS-PCR+, tdh, T3SS2, and trh) (Okura et al., 2003),proposed as the intermediate-O3:K6 clade, was most likely aphylogenetic intermediate between the pandemic and old-O3:K6clones. In addition, the genetic relationship in the LVPC minimumspanning tree herein (Fig. 4a) supports the proposed phylogenetic

andeniche L

148 X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 143151Fig. 4. Theminimum spanning trees of the 251 strains. In the minimum spanning trees (ato a larger number of strains included. The number along each edge reects the phylogshorter phylogenic distance. a) A minimum spanning tree of the 251 strains based on t

T3SS2, trh, and T3SS2), respectively (for example, 0-0-0-1-1 for LVPC-C2). c) Another mc), each circle indicates a haplotype (node), and a larger size of the circle correspondeddistance between each neighboring node. In addition, a thicker edge corresponds to aVPC data. b) Shown are the proles of the presence (1) or absence (0) of GS-PCR (tdh,

inimum spanning tree of the same strain collection based on the VNTR data.

relationship between the old-O3:K6, pandemic, and the intermediate-O3:K6 groups.

Of the 82 LVPC-C4 strains, 93.9% were GS-PCR, tdh+, T3SS2+,trh, and T3SS2-, whereas 74.3% of the 35 LVPC-C1 members wereGS-PCR, tdh+, T3SS2, trh+, and T3SS2+ (Fig. 4b). All 56 LVPC-C5 strains (Fig. 4b) were absence of all GS-PCR, tdh, T3SS2, trh, andT3SS2, and 47 (83.9%) are of non-clinical origin. Thus, LVPC-C5 wasclosely linked to a group of non-virulent or low-virulence V.parahaemolyticus found in external environments or seafood. Incontrast, of the 194 LVPC-C1 to LVPC-C4 strains, 183 (94.3%) were ofclinical origin and 187 (96.4%) harbored at least one of tdh, T3SS2,trh, and T3SS2.

LVPC-C2 and LVPC-C3 gave only one and three LVPC frequencyproles (Fig. 4a), respectively, which conrmed the genetically clonalstructure of the old O3:K6 and pandemic groups (Han et al., 2008). In

contrast, each of LVPC-C5, LVPC-C4, and LVPC-C1 gave at least 11 LVPCfrequency proles (Fig. 4a), and thus, was genetically much moreheterogeneous relative to LVPC-C2 and LVPC-C3.

3.4. Combination of LVPC and VNTR for two-stage genotyping

A previously characterized MLVA scheme based on a collation of10 VNTR loci (Harth-Chu et al., 2009) was applied to our straincollection. The VPTR7 locus, previously characterized to have thelowest allelic diversity (Harth-Chu et al., 2009), gave negative PCRamplication for 184 of the 251 strains, and was therefore discardedfrom further assay. The copy number of each VNTR locus in each strainwas recorded to generate the categorical VNTR dataset (Supplemen-tary Table S3). All nine VNTR loci tested were polymorphic among the251 strains, but gave different allelic diversities based on their allele

Table 2Allelic diversity of the nine VNTR loci tested.

Locus HGI Condence interval No. of alleles Max (PI)

VP2-07 0.967 0.9650.969 44 0.076VPTR1 0.945 0.9410.949 32 0.108VNTR6 0.932 0.9280.937 24 0.131VPTR3 0.876 0.8680.883 14 0.227VPTR4 0.863 0.8530.872 16 0.263VPTR8 0.858 0.8500.867 17 0.235VNTR5 0.804 0.7900.818 12 0.335VP1-11 0.721 0.7120.731 8 0.327VP1-10 0.469 0.4380.501 9 0.705

HGI: Hunter and Gaston index, a measure of the variation of the number of repeats at each locus ranging from 0.0 (no diversity) to 1.0 (complete diversity). Condence interval:precision of the diversity index expressed as 95% upper and lower boundaries. Max (PI): performance index, fraction of samples that have the most frequent repeat number in thislocus (range 0.0 to 1.0).

149X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 143151Fig. 5. Theminimum spanning trees of the strains from various LVPC complexes. The 251 stracategorical VNTR datasets of the strains from each LVPC complex were then used to build theedges in the minimum spanning trees can be found in Fig. 4. Each circle represents a distinins tested hereinwere rstly assigned into six LVPC complexes, LVPC-C1 to LVPC-C6. TheUPGMA-based minimum spanning tree. The fundamental notes for circles (nodes) andct VNTR allelic prole. See text for the denotation of arrows.

150 X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 143151numbers, HunterGaston index, condence interval, and max (PI),with VP2-07 and VP1-10 having the highest and lowest allelicdiversity, respectively (Table 2). These results are consistent withthe previous observations (Harth-Chu et al., 2009).

A total of 222 distinct allelic proles of VNTRs were detected forthe 251 strains, much larger than the 48 different frequency prolesdetected for LVPCs. Thus, VNTR-based genotyping has a much higherresolution than the LVPC-based method in discriminating V. para-haemolyticus strains.

The categoricalVNTRdataset of the 251 strainswasdirectly analyzedto build a UPGMA-based minimum spanning tree (Fig. 4c). Only thestrains from LVPC-C2 or LVPC-C3 were grouped together in the VNTRminimum spanning tree (Fig. 4c), whereas those from LVPC-C1, LVPC-C4, and LVPC-C5 had a scattered distribution in the tree. In contrast, thesix LVPC complexeswere separated in the LVPCminimumspanning tree(Fig. 4a) and had good correlation with the frequency of virulencemarkers, making assessing the potential pathogenicity nature of thecorresponding complexes more convenient.

Compared with the LVPC-based genotyping method, the VNTR-based method had a very high resolution in discriminating V.parahaemolyticus strains, but direct clustering of the strains couldnot give a concise grouping structure of the strains from the point ofclinical or epidemiologic view. The LVPC-based method had arelatively low resolution in grouping the strains, but it worked wellin dividing the strains into different complexeswith distinct clinical orepidemiologic potentials.

Given these complementary disadvantages/advantages of the twomethods, a two-step genotyping approachwith combined use of LVPCand VNTRwas proposed (Fig. 5). For this purpose, PCR-screening of 18LVPCs and ve virulence markers (GS-PCR, tdh, T3SS2, trh, andT3SS2) should rst be carried out for the strains, which wouldprimarily group the strains into various complexes with differentpotential pathogenicity natures. The strains from each LVPC-basedcomplex would be further analyzed with the nine VNTRs to give amuch more detailed discrimination of the strains.

The 35 LVPC-C1 strains gave 34 VNTR allelic proles (green circlesin Fig. 5), which included 7 strains (i.e., 7 allelic proles with blackarrows) of GS-PCR, tdh, T3SS2, trh+, and T3SS2+, 2 (i.e., 2allelic proles with gray arrows) of GS-PCR, tdh, T3SS2, trh,and T3SS2, and 26 (i.e., 25 allelic proles without arrows) of GS-PCR, tdh+, T3SS2, trh+, and T3SS2+. The 14 LVPC-C2 strains(pre-1996 old O3:K6; GS-PCR, tdh, T3SS2, trh+, andT3SS2+) gave 13 VNTR allelic proles (red circles in Fig. 5). The63 LVPC-C3 strains (the post-1996 pandemic group; GS-PCR+, tdh+,T3SS2+, trh, and T3SS2) gave 55 VNTR allelic proles (bluecircles in Fig. 5). LVPC-C6 contained only a single strain S093(intermediate O3:K6; GS-PCR+, tdh, T3SS2, trh, andT3SS2) and thus, only one allelic prole (dark green circles inFig. 5). The 82 LVPC-C4 strains gave 70 VNTR allelic proles (yellowcircles in Fig. 5); they included 5 strains (i.e., 5 allelic proles withblack arrows ) of GS-PCR, tdh+, T3SS2+, trh, and T3SS2, and77 (i.e., 65 allelic proles without arrows) of GS-PCR, tdh,T3SS2, trh, and T3SS2. The 56 LVPC-C5 strains (GS-PCR,tdh, T3SS2, trh, and T3SS2) gave 49 VNTR allelic proles(sky blue circles in Fig. 5).

4. Conclusion

A scheme for grouping V. parahaemolyticus strains was character-ized herein, for which a collection of 18 LVPCs was used as markers todetect their frequency proles by an array of PCR reactions. Thisscheme was able to separate the V. parahaemolyticus populations intodistinct complexes, which had a good correlation with the mosaicfrequency of virulence markers. Given that this scheme has limitedresolution for discriminating V. parahaemolyticus strains, especially

for tracing outbreak strains, the previously characterized MLVAscheme could be further applied to give a much more detailedclassication of the strains from each LVPC-based complex. This two-stage approach could be used to construct a genetic ngerprint-likedatabase based on a large collection of global strains, which would bevery useful for identication, genotyping, origin tracing, and riskestimation of V. parahaemolyticus.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.ijfoodmicro.2011.06.014.

Acknowledgements

Financial support came from the National Natural Science Founda-tion of China (30871370), the National Basic Research Programof China(2009CB522604), and the National Key Program for Infectious Diseaseof China (2009ZX10004-103 and 2008ZX10004-009). We thank Hin-chungWong fromTaiwan SoochowUniversity, Biao Kan andXiumei Liufrom Chinese Center for Disease Control and Prevention, and MitsuakiNishibuchi from Japan Kyoto University for kindly providing bacterialstrains.

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151X. Xiao et al. / International Journal of Food Microbiology 149 (2011) 143151

A novel genotyping scheme for Vibrio parahaemolyticus with combined use of large variably-presented gene clusters (LVPCs) and variable-number tandem repeats (VNTRs)1. Introduction2. Materials and methods2.1. Bacterial strains2.2. PCR-screening assay2.3. MLVA2.4. Clustering and genotyping analysis

3. Results and discussion3.1. Identification of pandemic strains3.2. Distribution of virulence markers3.3. LVPC-based genotyping3.4. Combination of LVPC and VNTR for two-stage genotyping

4. ConclusionAcknowledgementsReferences