Origin of Vibrio parahaemolyticus O3-K6 pandemic clone

Origin of Vibrio parahaemolyticus O3:K6pandemic clone

Guoxiang Chao a ,b ,1,Fang Wang a ,Xiaohui Zhou a ,Xinan Jiao a ,?,Jinlin Huang a ,Zhiming Pan a ,Liping Zhou b ,Xiaoqin Qian b

a Yangzhou University,Yangzhou,225009,China

b

Yangzhou Center for Disease Control and Prevention,Yangzhou,225002,China

a b s t r a c t

a r t i c l e i n f o Article history:

Received 4November 2010

Received in revised form 26December 2010Accepted 16January 2011

Keywords:

Vibrio parahaemolyticus Ancestor strain

Mutilocus sequence typing (MLST)Pandemic clonal complex (CC)

O3:K6pandemic clone of Vibrio parahaemolyticus has caused outbreaks in coastal countries since 1996.Mutilocus sequence typing (MLST)is an important tool to trace the source and analysis the evolution of bacteria.Based on MLST,the ?rst pandemic clonal complex (CC)of V.parahaemolyticus has been con ?rmed.In this study,57pandemic strains,27pathogenic strains (tdh or trh positive)and 36nonpathogenic strains isolated from China were analyzed with MLST.Forty-seven unique sequence types,one clonal complex (CC)and one doublet (D)were identi ?ed by eBURST and https://www.360docs.net/doc/e92966963.html, corresponded to not only the known O3:K6pandemic clone (including ST-3,ST-192,ST-227)but nonpathogenic clone (including ST-3,S-T2,ST-196,ST-220,ST-226).ST-3was the founder of the complex.STs of the isolates were not inevitably associated with the presence or number of the accessory genes or the serotypes of the isolates.The ancestor strain of O3:K6pandemic clone was originated from an environmental nonpathogenic O3:K6,ST-3strain.The pandemic O3:K6clone was developed from this strain in approximately 1996by laterally transferring large fragments of genes including systematic functional genes and genomic islands.

?2011Elsevier B.V.All rights reserved.

1.Introduction

Vibrio parahaemolyticus is one of the important foodborne pathogens in recent years.Infections and outbreaks caused by V.parahaemolyticus O3:K6pandemic clone have been observed throughout the world since 1996(Nair et al .,2007).The occurrence of pandemic strains was mainly due to acquiring systemic virulence genes,such as tdh ,toxRS/new ,MTase (VP0384),VP2905,HU-a and genomic islands (VPaI-1–VPaI-5,VPaI-7),especially VPaI-1and VPaI-5,which increased the ability of V.parahaemolyticus to spread to and infect humans (Hurley et al .,2006;Boyd et al .,2008;Chao et al.,2009,2010).

The molecular subtyping techniques including arbitrarily primed PCR,pulsed-?eld gel electrophoresis,ribotyping,and direct genome restriction enzyme analysis,which usually demonstrate a high degree of diversity for V.parahaemolyticus ,provide very limited information on the genetic relatedness of strains and very limited value in elucidating the evolution of the clonal complex of V.parahaemolyticus or its relationship to other V.parahaemolyticus strains or marine bacteria (González-Escalona et al.,2008).Multilocus sequence typing (MLST)is based on sequence analysis of chosen housekeeping (HK)genes and was conceived as an important tool to trace source and analyze the

relationship of evolution for clinical microbiologists and epidemiologists.MLST generates data that can be used for evolutionary and population studies of bacteria,regardless of their diversity,population structure (Urwin and Maiden,2003).Seven HK genes have been used for MLST of V.parahaemolyticus .These seven genes include recA (RecA protein),dnaE (DNA polymerase III,alpha subunit),and gyrB (DNA gyrase,subunit B)from chromosome I and dtdS (threonine 3-dehydrogenase),pntA (transhydrogenase alpha subunit),pyrC (dihydro-orotase),and tnaA (tryptophanase)from chromosome II (González-Escalona et al.,2008).MLST has con ?rmed the ?rst pandemic clonal complex (CC)of V.parahaemolyticus (González-Escalona et al.,2008).MLST database of V.parahaemolyticus was publicly accessible on the Internet (https://www.360docs.net/doc/e92966963.html,/vparahaemolyticus ).

In this study,120V.parahaemolyticus strains were analyzed by MLST https://www.360docs.net/doc/e92966963.html,bining with our previous studies on O3:K6clone systemic virulence genes and seven genomic islands (Chao et al .,2009,2010),we investigated the MLST types of V.parahaemolyticus in Chinese Mainland,analyzed the relationship between STs and pandemic,pathogenic,nonpathogenic strains,and explored the ancestor strain of O3:K6pandemic clone,which originated from Asia causing global epidemic foodbrone outbreaks (Nair et al .,2007).2.Materials and methods

2.1.Bacterial strains

Among 120V.parahaemolyticus strains,44strains were from food samples (11pandemic strains,5tdh positive strains,4trh positive

International Journal of Food Microbiology 145(2011)459–463

?Corresponding author at:Yangzhou University,88South University Ave.,Yangzhou 225009,China.Tel.:+8651487971136.

E-mail addresses:cgx1816@https://www.360docs.net/doc/e92966963.html, (G.Chao),jiao@https://www.360docs.net/doc/e92966963.html, (X.Jiao).1

Yangzhou Center for Disease Control and Prevention,36Yanfu East Road,Yangzhou 225002,China.Tel.:+86514

87314713.

0168-1605/$–see front matter ?2011Elsevier B.V.All rights reserved.doi:

10.1016/j.ijfoodmicro.2011.01.022

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j o u r n a l ho m e p a g e :w w w.e l s ev i e r.c o m /l o c a t e /i j fo o dm i c r o

strains)and76strains from clinical samples(46pandemic strains,14 tdh positive strains,4trh positive strains).These isolates were temporally(collected from2005to2009)and geographically diverse [collected from Jiangsu(81strains),Zhejiang(22strains),Shanghai (15strains),Guangdong(1strain)and Fujian(1strain),China].Two ATCC strains,ATCC3847(tdh+,O4:K11)and ATCC7802(trh+,O1:K1), were used in this study.

2.2.DNA extraction

Bacterial DNA was extracted using the TIANamp Bacteria DNA kit in accordance with the manufacturer's instructions(Tiangen,Beijing,China).

2.3.Systemic virulence genes and seven genomic islands

The systemic virulence genes[tdh,toxRS/new(both of them are positive for pandemic strains),orf8,HU-a,MTase(VP0394,in VPaI-1), VP2905(in VPaI-5)]and seven genomic islands(VPaI-1–VPaI-7)of120 strains were described in our previous studies(Chao et al.,2009,2010).

2.4.MLST

2.4.1.PCR ampli?cation and sequencing

PCR ampli?cation was carried out using primers described on the V. parahaemolyticus MLST website(https://www.360docs.net/doc/e92966963.html,/vparahaemolyticus). All PCR products consisted of a single band and were cleaned using the AxyPrep PCR puri?cation kit(AxyPrep Biosicience,Hangzhou China).PCR products were sequenced in both directions by Applied Biosystems3730 (South San Francisco,CA)with primers M13F and M13R.DNA sequences were individually inspected and manually assembled.The alignments of these sequences were determined using BioEdit(Hall,1999).Numbers for alleles and sequence types(STs)were assigned according to the database created for V.parahaemolyticus(https://www.360docs.net/doc/e92966963.html,/vparahaemolyticus). If sequences could not be unassigned to STs,the sequences and their electropherograms were submitted to the Curator,Najorl González-Escalona to assign new STs.

2.4.2.Assignment to clonal complexes

The program eBURST v3.0was used to identify the different clonal complexes(https://www.360docs.net/doc/e92966963.html,).The most restrictive group de?-nition was used to de?ne the clonal complexes,i.e.,at least six of the seven alleles had to be identical to be included in the same group or clonal complex(Feil et al.,2004).Two different STs are considered single-locus variant(SLV)when they differ from each other at a single locus.Double-locus variants(DLVs)are any two different STs differing in two loci.

2.4.

3.Phylogenetic analysis

Minimum-evolution(ME)trees for each locus and for the concatenated sequences of each ST were constructed by Mega4 software.The nucleotide diversity of each locus and their respective standard errors were determined using Mega4as described elsewhere (https://www.360docs.net/doc/e92966963.html,/perl/mlstanalyse/mlstanalyse.pl?site=pubmlst).

3.Results

3.1.STs and serotypes,systematic virulence genes and genomic islands

Among the120V.parahaemolyticus strains,47different STs were identi?ed by MLST among115strains.Five strains were untyped because one locus of each strain cannot be ampli?ed,sequenced and untyped(Supplemental Data).Among47STs,6STs included2–53 isolates,while39STs contained single isolates.ST-3was the most frequent type containing54isolates.Other STs were represented by two or more than two strains:ST-8(6isolates),ST-120(5isolates), ST-189(4isolates),ST-199(3isolates),ST-192(2isolates).The STs of ATCC3847and ATCC7802were ST-224and ST-1,respectively (Supplemental Data).

Among the57pandemic strains,52strains belonged to ST-3.There were10serotypes with the main serotype of O3:K6(36strains).Two pandemic strains belonged to ST-192,and the serotype was O1:K26 and O1:KUT.One isolate was ST-227with the serotype of O3:K6.Two pandemic strains[V439(O4:K68),V522(O1:K36)]were not typed because of the unampli?ed gyrB locus,but V439was SLV of ST3.All pandemic strains harbored all or part of systematic functional genes (toxRS/new,tdh,orf8,HU-a,VP2905,MTase)and genomic islands (VPaI-1–VPaI-5,VPaI-7)(Supplemental Data)(Chao et al.,2009, 2010).Among5O3:K6nonpathogenic strains that harbored VPaI-3 (three of them harbored VPaI-2),both V197and V220belonged to ST-3,V094belonged to ST-2,V019belonged to ST-196,V548belong to ST-220.Another nonpathogenic strain V588(O1:K33)harboring VPaI-3belonged to ST-266.Among19tdh+pathogenic isolates,4 O1:K56isolates pertained to ST-8;5isolates pertained to ST-120,and the serotypes were O3:K6(3isolates),O3:KUT(1islolate),O3:K29(1 isolate),respectively.Four isolates were ST-189and their serotypes were O3:KUT,O3:K8,O4:K8and O4:K68,respectively.The remaining ?ve isolates were ST-62(O4:K37),ST-66(O3:K58),ST-188(O3:K29) and ST-216(O3:K6),respectively.Two O3:K6strains(V384and V505)were untyped due to the unampli?ed recA locus.Among8trh+ pathogenic isolates,3isolates were ST-199(O4:K68);the remaining ?ve isolates were ST-123(O1:K56),ST-125(O1:KUT),ST-126(O1: KUT),ST-130(O10:K71)and ST-222(O1:KUT),respectively(Supple-mental Data).

3.2.Clonal complex

The49STs(including two STs of ATCC strains)generated in this data set were separated by eBURSTv3.0into one clonal complex(CC), one doublet(D),and41singletons(S)(Fig.1).In this study,CC included53pandemic strains and6environmental non-pathogenic strains,which were comprised of6different STs(ST-3,ST-2,ST-192, ST-220and ST-227,ST-266).ST-3was de?ned by eBURST as the ancestral type or founder for this clonal complex.The doublet(D) contained ST-120and ST-188.The singletons did not belong to any of the clonal complexes or other groups identi?ed in this study.

Among the?ve untyped strains,pandemic strain V439(O4:K68) belonged to CC because it was SLV of ST-3.Strain V522had some relation with CC because it had four locus identical to ST-3.Two tdh+, O4:K8strains(V384and V505)were SLVs of ST-189(Supplemental Data).Strain V064might be singleton.

3.3.Clustering and phylogenetic analysis

The ME tree was generated from the concatenated sequences of the seven loci of the49STs.Generally,eBURST and ME tree analysis were consistent,but ME tree gave better resolution and uncovered some phylogenetic relationships among groups or singletons that were not observed or resolved by eBURST(Fig.2).CC and D based on eBURST also formed individual clusters in the ME tree(Fig.2).Additionally,ST-196was highly related to ST-3and was also a member of CC in ME tree. ST-196was a DLV of ST-3.The unrelated ST-8and ST-224by eBURST belonged to the same cluster in the ME tree.Similar cases were observed for ST-123and ST-125(both trh+),ST-49and ST-221,ST-225 and ST-127,ST-218and ST-222,ST-126and ST-190,ST-66and ST-219 (the later?ve couples were all nonpathogenic,brown squares in Fig.2) (Fig.2).

4.Discussion

MLST scheme has been applied to a geographically diverse panel of V.parahaemolyticus strains for investigation of population structure and determination of the in?uence of recombination/mutation on the clonal

460G.Chao et al./International Journal of Food Microbiology145(2011)459–463

expansion of this species (González-Escalona et al.,2008).Applying MLST,González-Escalona et al.found that the O3:K6pandemic clone was the clone complex CC and con ?rmed the ?rst reported pandemic spread of V.parahaemolyticus .CC corresponded to the pandemic strains with four different STs (ST-3,ST-42,ST-27,and ST-51).ST-3was de ?ned by eBURST as the ancestral type or founder for this clonal complex and unequivocally established the clonal relationship of the pandemic complex (González-Escalona et al.,2008).

In this study,one clonal complex CC of V.parahaemolyticus was also identi ?ed in 120strains,which corresponded to the CC identi ?ed by González-Escalona et al .(González-Escalona et al.,2008).This CC included not only the reported pandemic clone strains (ST-3,ST-192,ST-227)but also the nonpathogenic strains (ST-3,ST-2,ST-196,ST-220,ST-226),which we designated as “nonpathogenic clone.”ST-3was also the founder of this CC.D was another clonal complex including ST-120and ST-188,of which all the strains were tdh positive.Meanwhile,ME tree shifted partial pathogenic strains and partial nonpathogenic strains into clonal complexes.Additionally,numerous singletons were observed,demonstrating the high dis-criminatory capability of this scheme and the extent of diversity within V.parahaemolyticus (González-Escalona et al.,2008).

Bacterial genomes are composed of two distinct parts:the “core ”genes and the “accessory ”genes.The core genome includes all genes that encode essential metabolic housekeeping and informational processing functions.Accessory genes have also been called “dispens-able ”genes or “plastic ”genome,because they can be deleted from the genome without an appreciable loss of ?tness being observed in the laboratory environment;in pathogenic species these deletions are often associated with a reduction in virulence (Hacker and Carniel,2001;Cooper and Feil,2004).Such genes are also commonly clustered in the genome to form genomic islands or islets and are typically recognized by an atypical G +C content (Lawrence and Ochman,2002).In the case of V.parahaemolyticus ,virulence genes (tdh ,trh ),systemic functional genes and genomic islands all belong to accessory genes.These genes were analyzed in our previous studies (Chao et al.,2009,2010).By analyzing the relationship between STs and accessory genes,we found that the STs of isolates were not inevitably associated with the presence or absence of accessory genes,the number of accessory genes,or the serotype.There are pandemic group and nonpathogenic group in CC,pandemic strains and nonpathogenic strains in ST-3,pathogenic strains and nonpathogenic strains with different serotypes in ST-8.

According to comparisons within clones of Feil et al.(clones in which every isolate shares seven out of seven loci were identical),the genomes in clones,both in terms of gene content and nucleotide variation and,at least in a freely recombining population,are likely to have shared a common ancestor only decades prior to sampling (Feil,2004).In this study,?rstly,two nonpathegenic foodbrone strains (V197,V220)and most of pandemic strains all belonged to ST-3.Secondly,in addition to six nonpathogenic strains (2ST-3,1ST-2,1ST-196,1ST-220,1ST-266)in CC,all the rest were pandemic strains,which not only harbored toxRS/new ,tdh ,but also included complete or partial systematic functional genes,and genomic islands (VPaI-1–VPaI-5,VPaI-7).The common characteristic between nonpathogenic and pandemic strains was to posses VPaI-3.It can be seen that the alleles played a decisive role in genetic patterns and typing of population in CC,whereas whether to have systematic virulence genes and genomic islands was not the major factor.Thirdly,the main serotype of pandemic strains was O3:K6,nonpathogenic strains such as ST-3,ST-2,ST-196,and ST-220in CC were O3:K6,indicating that O3:K6serotype was the prerequisite for the formation of O3:K6pandemic strains,around 1996.We concluded from these results that the ancestor strain of O3:K6pandemic clone originated from an O3:K6,ST-3,environmental nonpathogenic strain.

Our previous studies have shown that nonpathogenic O3:K6strains harboring VPaI-3was the intermediate strains during

the

Fig.1.V.parahaemolyticus “population snapshot ”obtained using eBURST v3.Two groups were de ?ned using stringent criteria (6/7shared alleles).In CC group,six were identi ?ed as clonal complexes,and their predicted clonal ancestor,ST-3,is shown in blue.STs that are SLVs of each other are shown connected by black lines.DLV STs are shown connected by aqua lines.The sizes of the circles are relative to the number of strains in the ST.

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transition from nonpathogenic to pandemic strains (Chao et al.,2010).The acquirement of VPaI-3was the ?rst step to form pandemic strains,then VPaI-2.It was interesting that STs of all tdh +and trh +pathogenic strains did not have any association with STs in pandemic clone CC.This suggested that pandemic clones were not originated from tdh +(embedded in VPaI-7)or trh +strains,and the VPaI-7was not the island obtained after VPaI-3and VPaI-2.As VPaI-1,VPaI-5play great role in forming pandemic clone,so VPaI-7was obtained next to VPaI-1and VPaI-5;our previous studies showed that VPaI-4may be the last genomic island obtained by pandemic clones (Chao et al.,2010).Based on the environmental strain (O3:K6,ST-3),the pandemic O3:K6clone was formed around 1996by lateral transfer large fragments of genes to obtain systematic virulence genes and genomic islands.In our study,environmental nonpathogenic strains ST-3,ST-2,ST-196,ST-220,and ST-266formed nonpathogenic CC,so CC did exist in

environment all the time.On the other hand,pandemic strains ST-3,ST-192,ST-227just formed in decades.

Based on the analysis above,we understood the source of O3:K6pandemic clone,but we do not know which and what factors trigger the development of O3:K6pandemic clone.We are also uncertain of the formation mechanism of serovariants and many extrinsic factors leading to this event.All this needs to be further investigated.

Up to now,there existed 14STs in CC including ST-3,ST-2,ST-27,ST-42,ST-51,ST-71,ST-72,ST-192,ST-196,ST-211,ST-220,ST-227,ST-266,and ST-305(released on https://www.360docs.net/doc/e92966963.html,/perl/mlstanalyse/mlstanalyse.pl?site=pubmlst ).In this study,the strains within CC had not only difference in dnaE (ST-196)but also variability in gyrB (ST-192),recA (ST-220),dtdS (ST-196),pyrC (ST-227)and tnaA (ST-266).Thus,these diversities formed new members of the clonal complex.These members were not previously published in the MLST website,suggesting that these genes are evolving more rapidly in the pandemic clonal complex in Chinese Mainland.The MLST data for V.parahaemolyticus showed that a range of recombination rates was present in this species,and genetic diversity in V.parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation (González-Escalona et al.,2008).V.parahaemolyticus popu-lation structure follows the “epidemic ”model of clonal expansion in bacteria,where more adapted clonal complexes emerge from a background of highly recombinogenic bacteria (Feil,2004).In this article,ST-2,ST-192,ST-227,ST-196,ST-220simultaneously emerged in clonal complex as SLV(s)or double-locus variants (DLVs)of ST-3,which provided a robust evidence for this conclusion.

Two untyped tdh +strains (V384and V505)were all serotype O4:K68,in which the sequences of recA allele were detected,while the other six alleles were the same with those in four ST-189strains;therefore,both the two untyped isolates and the ST-189isolates should be a clonal complex.Three clinical strains (ST-88,allele pro ?le:11,48,43,48,26,48,26)isolated from Peru in 1995and 1996were SLVs of ST-189.Interestingly,also in Peru,eight strains obtained during 1996–2006were almost identical to the two untyped isolates in this study,and all had a truncated recA ,which indicated that these strains (including ST-88,ST-189,and some untyped strains in China and Peru)had the same genetic ancestor (the information about strains of Peru was provided by Ph.D.Najorl González-Escalona,FDA,USA).The results displayed the robust function of MLST in source tracing and molecular epidemiology.

It is interesting that ME tree shifted partial nonpathogenic strains into clonal complexes (?ve couples,brown squares in Fig.2).Just as the O3:K6pandemic clone emerging from nonpathogenic CC around 1996,the next pandemic clone might also emerge from one of these clonal complexes.

5.Conclusion

Our results showed that O3:K6pandemic strains from Chinese Mainland had the same source with the international pandemic strains and belonged to the same CC with the founder ST-3.V.parahaemolyticus STs were not inevitably associated with the presence and the number of systemic virulence gene and the https://www.360docs.net/doc/e92966963.html, existed not only in pandemic clone but in nonpathogenic clone.The ancestor strain of O3:K6pandemic clone was originated from a O3:K6,ST-3,environmental nonpathogenic strain,as a result of recombination by lateral transfer large fragments of virulence genes from other vibrios .This ancestor strain ?rst obtained VPaI-3,followed by access to other accessory genes,and then formed O3:K6pandemic strains.In this study,there were some new members of CC,indicating that housekeeping genes are evolving more rapidly in the pandemic clonal complex.

Supplementary materials related to this article can be found online at doi:10.1016/j.ijfoodmicro.2011.01.022

.

Fig.2.An ME tree was constructed using the concatenated sequences of the seven loci of each of the 49STs obtained in this study.In red CC circle,except for 6STs observed by eBURST,ST-196also belongs to CC.Red circle D is the same as D in Fig.1.Two blue circles represent groups of parts of pathogens (tdh +or trh +)that did not exist in Fig.1.Brown squares represent groups of parts of nonpathogenic strains that did not exist in Fig.1,which might be the origin of new potential pandemic clone(s).Bootstrap values over 50%are shown in the branches.

462G.Chao et al./International Journal of Food Microbiology 145(2011)459–463

Acknowledgements

This work was supported by grants from National Key Technology R&D Program(2009BADB9B01),Natural Sciences Foundation of Jiangsu Province(BK2008011),Natural Science Foundation of Jiangsu Higher Education Institutions of China(2009KJA230001),and Natural Sciences Foundation of Jiangsu Province(BK2010350).We would like to thank those who kindly provided us with the V.parahaemolyticus strains used in this study.We also thanked Ph.D.Najorl González-Escalona for assistance.

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