Population structural analysis of O1 El Tor Vibrio cholerae isolated in China among the seventh cholera pandemic on the basis of multilocus sequence typing and virulence gene profiles.

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Population structural analysis of O1 El Tor Vibrio cholerae isolated in China among the seventh cholera pandemic on the basis of multilocus sequence typing and virulence gene profiles

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Haijian Zhou a,b,1, Xuan Zhao a,1,2, Rui Wu a, Zhigang Cui a,b, Baowei Diao a, Jie Li a, Duochun Wang a,b, Biao Kan a,b,⇑, Weili Liang a,b,⇑ a National Institute for Communicable Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Chinese Centre for Disease Control and Prevention, 155, Changbai Road, Changping, Beijing 102206, People’s Republic of China b Collaborative Innovation Centre for Diagnosis and Treatment of Infectious Diseases, 866 Yuhangtang Road, Hangzhou 310003, People’s Republic of China

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Article history: Received 13 September 2013 Received in revised form 12 December 2013 Accepted 16 December 2013 Available online xxxx Keywords: Vibrio cholerae Multilocus sequence typing Virulence gene Serogroup O1 Population structure CTXU

a b s t r a c t Serogroup O1 Vibrio cholerae is the most common agents to cause epidemic and pandemic cholera disease. In this study, multilocus sequence typing (MLST) was performed on 160 serogroup O1 strains (including 42 toxigenic and 118 non-toxigenic), and the virulence/fitness gene profiles of 16 loci were further analysed for 60 strains of these. Eighty-four sequence types (STs) with 14 clonal complexes were distinguished, and 29 STs were unique. Except SD19771005, all toxigenic strains were well-separated from the non-toxigenic strains. While a group of non-toxigenic strains clustered closer to the toxigenic strains compared to the other strains. Overall the examined gene loci showed higher presence rates in the toxigenic strains compared to the non-toxigenic strains. It is worth noting that the presence rates of VPI, TLC, VSP-I and VSP-II in the non-toxigenic strains that were clustered closer to the toxigenic strains were much higher compared to the other non-toxigenic strains. Our study indicated the complex population structure of O1 strains, and parts of non-toxigenic strains are genetically more closely related to toxigenic strains than other non-toxigenic strains, suggesting that these strains may have a higher potential for infection with CTXU in the environment or host intestine and is more efficient to become new pathogenic or epidemic clones. Ó 2014 Published by Elsevier B.V.

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1. Introduction

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Cholera is a historically severe dehydrating diarrheal disease and remains a public health problem worldwide, particularly in Africa and Asia. The causative organism is Vibrio cholerae, which is divided into serogroups on the basis of the somatic O antigen. The O1 serogroup is known to be the most common strain to cause this epidemic and pandemic disease. Humans are the transient host for V. cholerae. Aquatic systems, such as river, marine, and brackish water are environmental reservoirs where the bacteria multiply in association with zooplankton (Colwell, 1996; Faruque et al., 1998). The organism is transmitted to contaminate food and water mainly via the faecal-oral route.

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⇑ Corresponding authors at: Department of Diarrheal Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Centre for Disease Control and Prevention, 155, Changbai Road, Changping, Beijing 102206, People’s Republic of China. Tel./fax: +86 010 58900744. E-mail addresses: [email protected] (B. Kan), [email protected] (W. Liang). 1 These authors contributed equally to this work. 2 Present address: Tianjing Centre for Disease Control and Prevention, Tianjing 300171, People’s Republic of China.

The evolutionary genetic relationships among V. cholerae strains have been examined using different methods. In these studies, the epidemic V. cholerae isolates form a lineage that is separate from the nonepidemic strains (Beltran et al., 1999; Byun et al., 1999; Karaolis et al., 1995; Wachsmuth et al., 1993). One common characteristic of epidemic and pandemic cholera isolates is that these strains harbour genes encoding the cholera toxin (CT) and the toxin co-regulated pilus (TCP), which are two of the most important virulence factors of V. cholerae. CT is encoded by ctxAB genes, which are integral components of the filamentous phage CTXU (Waldor and Mekalanos, 1996). The TCP biosynthesis genes are located on the Vibrio pathogenicity island 1 (VPI-1) (Karaolis et al., 1998). A number of other genes or gene clusters have also been identified predominantly among epidemic V. cholerae isolates and may play a role in the environmental fitness of the isolates and in cholera pathogenesis. These genes or gene clusters include filamentous phage RS1U (Davis et al., 2002), toxin-linked cryptic plasmid (TLC) (Rubin et al., 1998), RTX toxin gene cluster(Lin et al., 1999), hapA and hapR (Jobling et al., 1997), VPI-2 (Jermyn and Boyd, 2002), hemolysin (hlyA), genes encoding type VI secretion system (T6SS) (Pukatzki et al., 2006), Chitin-regulated pilus (ChiRP)

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Please cite this article in press as: Zhou, H., et al. Population structural analysis of O1 El Tor Vibrio cholerae isolated in China among the seventh cholera pandemic on the basis of multilocus sequence typing and virulence gene profiles. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/ j.meegid.2013.12.016

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(Meibom et al., 2004), mannose-sensitive hemolysin agglutination pilin (MSHA) (Jonson et al., 1991), Vibrio polysaccharide (VPS) (Fong et al., 2010), and Vibrio seventh pandemic island I (VSP-I) and VSP-II, which consists of VC0175 to VC0185 and VC0490 to VC0516, respectively. These two islands were found exclusively among the El Tor biotype isolates in comparative genomic studies using a V. cholerae DNA microarray (Dziejman et al., 2002). Recently study focusing on genomic diversity of 2010 Haitian cholera outbreak strains showed that CTXU, RS1U, TLC, VPI-1, VPI-2, VSP-I and VSP-II were found in almost all clinical O1 isolates but not in clinical or environmental V. cholerae non-O1/O139 isolates; meanwhile, the non-O1/O139 populations in Haiti harbor a genomic backbone similar to that of toxigenic V. cholerae O1 (Hasan et al., 2012). Revealing the virulence gene profiles and elucidating the steps and significance of virulence gene acquisition in the evolution of V. cholerae is helpful to understand the underlying phylogenetic relationships among strains. Multilocus sequence typing (MLST) is a useful molecular subtyping method that plays an important role in epidemiological and phylogeny studies. MLST has been previously used to study molecular subtyping, population structure and lateral gene transfer of V. cholerae (Keymer and Boehm, 2011; Lee et al., 2006; Octavia et al., 2013; Salim et al., 2005). In this study, we used MLST to study the population structure of O1 serogroup V. cholerae strains isolated in China among the seventh cholera pandemic and examined the presence and distribution of gene regions associated with virulence or survival and persistence in the environment among strains to elucidate the role of the gene regions involved in the emergence of epidemic isolates.

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2. Materials and methods

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2.1. Bacterial strains

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One hundred and sixty V. cholerae serogroup O1 strains, including 42 toxigenic (ctxAB-positive) and 118 non-toxigenic (ctxABnegative) strains, isolated in China were investigated in this study (Supplementary Table 1). All of the strains were isolated in different years (1961–2010) and were involved in three nationwide cholera epidemics in the 1960s, 1980s and 1990s, and the epidemic-interval periods in China. The strains were isolated using gentamicin-selective agar or thiosulfate citrate bile salt sucrose (TCBS) agar and identified using the V. cholerae diagnostic sera and biochemical tests. Additionally, six O1 strains (N16961, O395, MJ-1236, M66-2, 2010EL-1786 and IEC224) from other countries were used as reference strains in MLST analysis.

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2.2. MLST

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The DNA genomes were extracted using the DNeasy Blood & Tissue Kit (Qiagen, Dusseldorf, Germany) according to the instructions provided by the manufacturer. Seven loci (dtds, gyrB, mdh, pntA, pyrC, recA and tnaA) were used for the MLST analysis. These loci were used in the MLST schemes for other Vibrio species, such as V. parahaemolyticus and V. vulnificus (Bisharat et al., 2005; González-Escalona et al., 2008). Both chromosomes are represented with three (gyrB, mdh, recA) and four (dtds, pntA, pyrC, tnaA) genes from chromosomes I and II, respectively, and the genes were evenly distributed around the chromosomes. The primers for the seven housekeeping genes are shown in Supplementary Table 2. The PCR products were sequenced in both directions using the Big Dye cycle sequencing kit (ABI) according to the manufacturer’s instructions. Sequencing was performed on an ABI 3770 automatic sequencer. The gene sequences were analysed using the Molecular Evolutionary Genetics Analysis (MEGA) suite of programmes, ver-

sion 5.1 (Kumar et al., 2001) or BioNumerics version 5.10 software (Applied Maths, Kortrijk, Belgium). Phylogenetic gene trees were constructed using the neighbour-joining method with the Jukes– Cantor distance method. Bootstrap values were calculated for 1000 trees. Sequences of each locus were compared with each other to determine the allele numbers and MLST types (STs). The STs were analysed using eBURST to determine the presence of clonal complexes (CCs) (Feil et al., 2004). BioNumerics version 5.10 software was used to create the minimum spanning tree. In the minimum spanning tree, the founder ST was defined as the ST with the greatest number of single-locus variants. Types were represented by circles and the size of a circle indicated the number of strains with this particular type.

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2.3. Pulsed-field gel electrophoresis (PFGE)

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We used the PulseNet 1-day standardised PFGE protocol for V. cholerae (Cooper et al., 2006). Cell suspensions were placed in polystyrene tubes (Falcon; 12 75 mm), and their optical density was adjusted to 4.0–4.2 using the Densimat photometer (bioMérieux, Marcy l’Etoile, France). V. cholerae slices were digested using 20 U per slice of NotI (New England BioLabs, Ipswich, MA, USA) for 4 h at 37 °C. Electrophoresis was performed using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA, USA). Images were captured on the Gel Doc 2000 system (Bio-Rad) and converted into TIFF files for computer analysis using the BioNumerics version 5.10 software (Applied Maths, Kortrijk, Belgium). The similarity between two patterns was expressed as the Dice coefficient (SD) (Dice, 1945) and dendrogrammes were clustered and constructed using the unweighted pair group method with arithmetic averages (UPGMA).

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2.4. PCR analysis

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PCR was used to assay 60 O1 serogroup V. cholerae isolates (including 20 toxigenic and 40 non-toxigenic isolates) for the presence of 16 loci associated with virulence or survival and persistence in the environment of V. cholerae. Of the 16 virulence loci examined, 12 loci consisted of multiple genes (CTXU, VPI-1, T6SS locus, VPI-2, VSP-I, VSP-II, RTX toxin cluster, TLC, VPS, MSHA, RS1U, ChiRP) and 4 loci had single gene (hlyA, hapA, hapR, vpsR). A total of 26 primer pairs were used to determine the distribution of the 16 loci among the 60 V. cholerae isolates. Five primer pairs were used to assay for the presence of VPI-1, two primer pairs (each) were used to assay for T6SS, VSP-I, VSP-II, VPI-2, VPS, RTX, TLC, and one primer pair (each) was used to assay for the presence of CTXU, ChiRP, hlyA, hapA, hapR, RS1U, and MSHA. The PCR were prepared in a reaction volume of 50 ll with 5 ll of 10 PCR buffer (Takara, Dalian, China), 1 unit Taq polymerase (Takara), 200 lM of dNTPs (Takara), 0.4 lM of each primer set, 20 ng of the DNA template and filtered sterile water. PCR was performed with an initial denaturation step at 94 °C for 5 min followed by 33 cycles each consisting of an initial denaturation at 94 °C for 40 s followed by annealing and extension steps. The primers and corresponding annealing temperatures are listed in Supplementary Table 2.

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2.5. Data analysis

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Nei’s indices (Malorny et al., 2008) of each locus and Simpson’s diversity index (Hunter and Gaston, 1988) of the typing methods P were calculated. The formulas were as follows: Nei’s index = 1 2 (allele frequency) ; Simpson’s diversity index ðDÞ ¼ 1 P ½nj ðnj 1Þ=½NðN 1Þ, where nj is the number of strains belonging to the jth pattern, and N is the number of strains in the population.

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3. Results and discussion

3.2. Population structure of V. cholerae serogroup O1 isolates

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3.1. Nucleotide diversity at each locus and STs generated using MLST

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One hundred and sixty V. cholerae serogroup O1 strains (including 42 toxigenic and 118 non-toxigenic strains) isolated from China were analysed by MLST using sequences generated from the internal fragments of seven genes (dtds, gyrB, mdh, pntA, pyrC, recA and tnaA). The sequences were obtained for all seven genes for 160 isolates and these data were summarised in Table 1. A total of 4676 bp from the seven gene loci were sequenced in each of the 160 isolates and 450 (9.62%) polymorphic nucleotide sites were found. The internal fragments of seven genes ranged in size from 497 bp (dtdS) to 762 bp (mdh). In addition, the number of information sites observed varied per locus from 23 (pntA) to 109 (pyrC). The nucleotide diversity of each locus ranged from 0.006 to 0.056. The number of alleles observed for each locus ranged from 16 (mdh and recA) to 22 (gyrB). Nei’s index of each locus was between 0.4434 (dtdS) and 0.8461 (recA). Since the completion of this study, two MLST schemes have been applied to sets of environmental isolates (Keymer and Boehm, 2011) and non-O1/non-O139 isolates (Luo et al., 2013; Octavia et al., 2013). The reported schemes shared only four and three genes with this study, respectively, which indicated that a direct seven-gene sequence based comparison was not feasible. If the same genes were used in different studies, then the typing information of the isolates from these different studies may be compared and analysed together. Thus, a standardised MLST scheme that could be used by different laboratories for V. cholerae isolates should be established. The V. cholerae MLST scheme established by Octavia and colleague is now accessible (http://pubmlst.org/ vcholerae/) and could be used to compare data from different laboratories worldwide (Octavia et al., 2013).

For 160 Chinese isolates, 84 STs were obtained, 22 of which were represented by multiple strains, while the remaining STs were found in a single strain (Fig. 1, Supplementary Table 1). For 42 toxigenic and 118 non-toxigenic strains, 11 and 73 STs were obtained with 1–31 and 1–10 strains in each ST, respectively. ST1 was predominant which contained 31 toxigenic strains and persisted 50 years (1961–2010) in 15 different provinces. We used the definition of six out of seven shared alleles for a clonal complex (CC) and identified 14 CCs and 29 singletons (Fig. 1). The largest CC contained 39 toxigenic strains belonging to eight STs. The other 13 CCs contained 2–24 strains belonging to 2–10 STs in each CC; all of which were non-toxigenic strains. Among the six reference strains from other countries, the four seventh pandemic strains, N16961, IEC224, MJ-1236, and 2010EL-1786, had same ST with the primary Chinese toxigenic O1 strains (ST1). The pre-seventh pandemic strain, M66-2, and classical strain, O395, showed two (pyrC, tnaA) and three (pyrC, recA, tnaA) loci different to the major Chinese toxigenic O1 strains, respectively. N16961, IEC224, MJ-1236, and 2010EL-1786 were clinical isolates form different cholera-endemic areas in 1971, 1994, 1994 and 2010, respectively. These results suggested that strains belong to ST1 were widely spread or the isolates from the seventh pandemic from different locations and time periods were genetically closely related. From the sequences of the seven loci of 160 V. cholerae serogroup O1 strains, we constructed a neighbour-joining tree (Fig. 2). From the seven-gene tree groups, 41 of the 42 tested toxigenic V. cholerae O1 isolates formed an epidemic clone complex (group A1 in Fig. 2). In contrast, the non-toxigenic strains showed high polymorphism. Interestingly, one toxigenic strain (SD19771005) was clustered far away from the main toxigenic

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Table 1 Nucleotide sequence variation for each MLST locus. Locus

Fragment size (bp)

No. of alleles

Nei’s index

Nucleotide diversity

No. of polymorphic sites

No. of information sites

dtdS gyrB mdh pntA pyrC recA tnaA

497 564 762 724 715 728 686

17 22 16 19 19 16 18

0.4434 0.8044 0.7840 0.8192 0.8042 0.8461 0.8069

0.012 0.021 0.010 0.006 0.056 0.020 0.012

54 59 68 44 124 49 52

33 40 35 23 109 46 46

Fig. 1. eBURST analysis of 84 STs of 160 Vibrio cholerae O1 isolates from China. The digits out brackets in each circle indicate the STs and those in the brackets indicate the number of isolates within this particular type. The STs of toxigenic isolates were showed in bold, and that of non-toxigenic were showed in general.


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cluster. Sixteen non-toxigenic strains (group A in Fig. 2) showed closer relationships to the toxigenic strains compared to the other non-toxigenic strains (group B in Fig. 2). Among which, 12 were isolated from environmental water samples and other four were clinical isolates. A previous clonal relationship study between clinical and environmental V. cholerae isolates found that clinical isolates closely resembled environmental isolates in their genomic patterns (Singh et al., 2001). But different from our study, the environmental isolates in Singh’s study were all toxigenic. Among the clinical isolates, one non-toxigenic O1 strain was included which displayed identical or similar fingerprint to toxigenic strain with BOX-PCR, AFLP, and PFGE analysis. Although different methods used by our study and the previous one, the results of both studies suggested that non-toxigenic strains which were closely related to the toxigenic strains exist in the environment. To identify the cause of independence of SD19771005 (ST5) outside the main toxigenic group in the neighbour-joining tree (Fig. 2), a minimal spanning tree was constructed with 42 toxigenic

strains based on allelic difference rather than nucleotide sequence difference. The contribution of recombination and point mutation of each different locus between different STs were investigated (Fig. 3). Using the counting method of Feil et al. (2000) and Octavia et al. (2013), we designated difference by one base as mutation and by two or more bases as recombination. ST5 is a triple-locus variant of ST1. All three different loci between ST5 and ST1 were aroused by recombination, especially in loci pyrC and tnaA, having 46 and 27 polymorphic nucleotide sites respectively. On the contrary, ST6, another triple-locus variant of ST1, was aroused by three mutations. So the far away clustering of SD19771005 from the main toxigenic cluster in the neighbour-joining tree was aroused by recombination. The high polymorphism of non-toxigenic O1 strains and high clonality of the toxigenic O1 strains are shown in this study, which was consistent with previous studies analysing Chinese V. cholerae isolates using array-based comparative genomic hybridisation (Pang et al., 2007) and whole genome PCR scanning (Pang et al.,

Fig. 2. Neighbour-joining tree of 160 Vibrio cholerae strains isolated from China according to the 4676 bp sequence from the seven gene loci (gyrB, mdh, recA, tnaA, dtds, pntA and pyrC). The detail of the branches marked as different colors was described in the text. The tree was building by using Molecular Evolutionary Genetics Analysis (MEGA) suite of programmes, version 5.1.


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Fig. 3. Relationships of SD19771005 (ST5) and the main toxigenic group of Chinese strains (ST1) based on minimum spanning tree. Events were marked on the branches by different loci (out brackets) and number of polymorphic nucleotide sites (in the brackets). The strains in each STs could be found in Supplementary Table 1.

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2011). In these studies, the toxigenic serogroup O1 isolates formed a cluster and the non-toxigenic O1 strains dispersed in the phylogenetic trees. However, in this study, we detected parts of non-toxigenic strains that showed closer genetic relationships to toxigenic strains compared to others and one toxigenic strain was clustered together with the non-toxigenic strains. These results suggested there might be a more complex population structure of O1 strains compared to that revealed by previous studies. Because of the low polymorphism of toxigenic strains by MLST, PFGE was performed to subtype 42 toxigenic strains. The 42 toxigenic strains were divided into 32 types by PFGE with a D-value of 0.9837 (Fig. 4), showing a significant higher discriminatory power than MLST. MLST mainly detected point mutations among housekeeping genes, whereas PFGE detected the insertions and deletions of large fragments, the acquisition and loss of plasmids, homologous recombination, and point mutations in restriction enzyme sites. The higher discriminatory power of PFGE suggested that there were more types of genetic variation other than point mutations among housekeeping genes in the toxigenic serogroup O1 V. cholerae strains. As a molecular typing method, MLST was not suitable for subtyping toxigenic V. cholerae serogroup O1 strains because of its low discriminatory power. Although the MLST scheme used here could not distinguish most of toxigenic strains, it would be better to classify strains based on PFGE and sub-classify based on MLST or vice versa.

Fig. 4. PFGE cluster tree of 42 toxigenic V. cholerae strains. The year the strain was isolated, source state, MLST type and allele number of each locus are listed on the right of each strain.


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3.3. Presence of virulence regions

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Among the 160 MLST-analysed V. cholerae isolates, 60 strains consisting of 20 toxigenic and 40 non-toxigenic strains were selected and further examined for the presence of 16 loci associated with virulence using PCR assays with 26 primer pairs (Supplementary Table 2). During the choosing, both genetic and epidemiological polymorphisms of strains were considered. The selected strains belonged to different MLST types and dispersed in the neighbourjoining tree (Fig. 2) and isolated from different years and regions. Prevalence of the major virulence genes was quite asymmetric among the toxigenic and non-toxigenic strains. hlyA, mshA, vpsA and vpsR were detected in all strains, and the other examined virulence loci showed lower presence rates in non-toxigenic strains compared to the toxigenic strains (Fig. 5, Table 2). hlyA encodes hemolysin of V. cholerae, an oligomerising pore-forming toxin that

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causes lysis in erythrocytes and demonstrates cytotoxic activity. Thus is considered an important virulence factor, particularly in ctxAB-negative V. cholerae strains (Zitzer, et al., 1995). MSHA pilus is used by environmental V. cholerae to adhere to plankton or to form biofilms on abiotic surfaces (Chiavelli et al., 2001; Fong et al., 2010; Watnick et al., 1999). The genes responsible for exopolysaccharide biosynthesis (VPS) are clustered into two operons and vpsA is the first gene of operon I (Yildiz et al., 1999). vpsR is a positive transcriptional regulator, which promotes the transcription of vps genes and the formation of typical 3D biofilm structures (Yildiz et al., 2001). MSHA and VPS are important factors that play a role in biofilm formation; biofilms can act as a reservoir for V. cholerae between epidemics because of its long-term viability in biofilms, and thus contributes to the environmental fitness of the V. cholerae (Alam et al., 2007). These regions are present in nearly all V. cholerae strains, including the serogroup O1 and the serogroup

Fig. 5. Neighbour-joining tree of 60 Vibrio cholerae strains used to detect the virulence loci. The distribution of the 16 loci associated with virulence was listed on the left of the strain ID. The tree was building by using BioNumerics version 5.10 software.


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H. Zhou et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx Table 2 Presence rates of 16 regions associated with virulence among V. cholerae isolates as determined using PCR analysis in this study. Regions

CTXU VPI-1 T6SS locus HlyA RTX hapA hapR RS1U TLC VSP-I VSP-II VPI-2 ChiRP MSHA vpsA vpsR

355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398

Groups of strains All 60 strains tested (%)

20 Toxigenic strains (%)

40 Non-toxigenic strains (%)

19 Toxigenic strains (group A1 in Fig. 5) (%)

14 Non-toxigenic strains (group A in Fig. 5) (%)

26 Non-toxigenic strains (group B in Fig. 5) (%)

33.3 68.3 88.3 100.0 98.3 86.7 91.7 28.3 41.7 45.0 35.0 80.0 83.3 100.0 100.0 100.0

100.0 100.0 95.0 100.0 100.0 100.0 95.0 60.0 75.0 95.0 85.0 95.0 95.0 100.0 100.0 100.0

0 52.5 85.0 100.0 97.5 85.0 90.0 12.5 25.0 20.0 10.0 70.0 77.5 100.0 100.0 100.0

100.0 100.0 94.7 100.0 100.0 100.0 94.7 63.2 73.7 100.0 89.5 94.7 94.7 100.0 100.0 100.0

0 92.9 85.7 100.0 100.0 100.0 100.0 14.3 71.4 28.6 28.6 85.7 71.4 100.0 100.0 100.0

0 30.8 84.6 100.0 96.2 69.2 84.6 11.5 0 15.4 0 61.5 80.8 100.0 100.0 100.0

O139 and non-O1/non-O139 strains (O’Shea et al., 2004; Rahman et al., 2008). These results suggested that MSHA and VPS might be the basic needs of the V. cholerae isolates for survival as biofilms in the environment between epidemics. ChiRP and VPI-2 are two other factors that play a role in the survival of V. cholerae in the ecosystem and persist longer in the environment (Meibom et al., 2004; O’Shea et al., 2004). These isolates showed high presence rates among the O1 strains in this study, particular in the toxigenic strains. The most significant differences in prevalence between the toxigenic and non-toxigenic strains were observed in the VSP-I, VSP-II, RS1U, TLC and VPI-1 loci (Table 2, Fig. 5). In addition, 95.0%, 85.0%, 60%, 75% and 100% of the toxigenic strains had VSP-I, VSP-II, RS1U, TLC and VPI-1, respectively; however, only 20.0%, 10.0%, 12.5%, 25% and 50% of the non-toxigenic strains had VSP-I, VSP-II, RS1U, TLC and VPI-1, respectively. High coexisting correlations of VPI-1, VSP-I, VSP-II and CTXU were observed. VPI-1 encoded TCP pilus, which functioned as a CTXU receptor, a prerequisite for CTXU infection (Waldor and Mekalanos, 1996; Rahman et al., 2008). VSP-I and VSP-II were identified to exclusively present in El Tor epidemic strains and were thought to contribute to the endemic and pandemic feature of El Tor V. cholerae (Dziejman et al., 2002). In a study conducted by O’shea and colleagues, 75.0% and 66.7% of toxigenic O1 El Tor V. cholerae strains had VSP-I and VSP-II, respectively, and all non-toxigenic O1 El Tor V. cholerae strains lacked these two regions (O’Shea et al., 2004). However, in this study, we detected six and two non-toxigenic strains containing only VSP-I and VSP-II, respectively, and two strains containing both of the two pandemic islands, which suggested that there may be a horizontal transfer of VSP-I and VSP-II between the El Tor epidemic strains and the environmental no-epidemic strains. Both RS1U and TLC also showed a high coexisting correlation with CTXU in this study. This was understandable because RS1U was required for CTXU production (Davis et al., 2002; Faruque et al., 2002) and TLC flanks CTXU (Rubin et al., 1998). Overall, there was no significant difference in the prevalence of the putative accessory virulence loci, including RTX (rtxA and rtxC), T6SS locus, hapA and hapR between the toxigenic and non-toxigenic strains, from 95% of the toxigenic and 85% of the non-toxigenic isolates. The RTX genes were widely prevalent, where all of the toxigenic strains and 97.5% of the non-toxigenic strains examined carried either rtxA or rtxC or both of these genes. The T6SS genes were also common; overall 88.3% of the O1 strains tested were both vasK- and vasH-positive. In general, 86.7% and 91.7%

of the strains carried hapA and hapR, respectively. The hapA gene encodes haemagglutinin/protease (HA/protease) of V. cholerae (Hase and Finkelstein, 1991). HA/protease plays an important role in cholera pathogenesis by proteolytically activating cholera toxin A subunit (Booth et al., 1984) and the El Tor cytolysin/haemolysin (Nagamune et al., 1996), hydrolysing physiologically important proteins (Finkelstein et al., 1983), promoting mucin gel penetration, and facilitating the detachment and spread from the infected host intestinal mucosa (Silva et al., 2003). Although hapA and hapR were separated on two chromosomes, the expression of hapA is dependent on hapR, which is the global regulator of the quorum sensing system in V. cholerae. The carrying consistency was observed for hapA and hapR in this study. There were 52 strains (86.7%) carrying both hapA and hapR and only one strain lacking both genes. Except for one toxigenic strain lacking hapR, all of the strains lacking hapA and (or) hapR belonged to group B in Fig. 2. Of the 20 toxigenic strains, 11 strains contained all 16 virulence regions examined. The other nine toxigenic strains lacked one to three virulence regions examined (eight strains lacked RS1U, five strains lacked TLC, three strains lacked VSP-II, one strain lacked VSP-I, one strain lacked VPI-2, one strain lacked ChiRP, one strain lacked HapR, and one strain lacked T6SS). Interestingly, the only toxigenic strain (SD19771005) that lacked VSP-I, VSP-II and RS1U clustered together with the non-toxigenic strains and were far away from the main toxigenic branch in the neighbour-joining tree constructed using sequences of the seven loci. Gene composition variation on VPI-1 region was observed in several nontoxigenic strains; for example, the GD20061007 and HI20051037 strains contained three (acfB, aldA, tagA) out of the five genes detected within VPI-1 locus; while the LN20041017 and LN20041019 strains were positive for only tagA, which suggested that the loss and recruitment of VPI-1 may not be integral, but progressive. Furthermore, the presence rates of VPI-1, TLC, VSP-I and VSP-II in the non-toxigenic strains in group A were significantly higher compared to that in group B (Table 2). In addition, the prevalence of hapA and hapR in strains of the non-toxigenic strains in group A (Fig. 2) was also higher compared to those in group B and were more similar to those of the toxigenic strains (group A1 in Fig. 2). These findings were very interesting and consistent with the results of the MLST analysis, suggesting that the non-toxigenic strains of group A were genetically closer to the toxigenic strains and might have a higher potential to become new pathogenic or epidemic clones once capture CTXU in the environment or host


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intestine. This speculation was supported by recent study of Haiti outbreak stains. V. cholerae non-O1/O139 (non-toxigenic) populations in Haiti harbor a genomic backbone similar to that of toxigenic V. cholerae O1 and most probably serve as a reservoir for genomic and pathogenicity islands (Hasan et al., 2012).

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4. Conclusion

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The complex population structure of O1 strains was revealed using MLST analysis, and the toxigenic strains were well-separated from the non-toxigenic strains by population structure analysis, except for one strain, which was clustered far away from main toxigenic group as a result of recombination. PFGE subtyping of 42 toxigenic strains showed greater gene polymorphism compared to MLST, suggesting that there were more types of genetic variation other than point mutations and recombination among housekeeping genes in the toxigenic serogroup O1 V. cholerae strains. Virulence-related gene profile analysis revealed that the prevalence of major virulence genes was notably asymmetric among the toxigenic and non-toxigenic strains. A group of non-toxigenic strains showed closer genetic relationships to the toxigenic strains than the other non-toxigenic strains using MLST analysis. Consistently, the rates of prevalence of some environmental fitness and virulence loci (VPI-1, TLC, VSP-I, VSP-II, hapR and hapA) in these strains were much higher compared to that in the other non-toxigenic strains, suggesting that these strains may more easily evolve into new pathogenic or epidemic clones by capture CTXU in the natural environment and infected host intestine. Our results indicated that the population structure of O1 El Tor V. cholerae strains might be more complex than we had previously thought.

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5. Uncited references

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Boyd and Waldor (2002), O’Shea and Boyd (2002), Waldor et al. Q4 (1997).

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Acknowledgments

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This study was supported by the Project of the National Natural Science Foundation of China (81071410 and 81171640) and the Priority Project on Infectious Disease Control and Prevention (2012ZX10004215) from the Ministry of Health of the People’s Republic of China.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2013. 12.016.

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The seventh pandemic Vibrio cholerae O1 El Tor isolate in China has undergone genetic shifts.

Genome Sequence of Vibrio cholerae Strain O1 Ogawa El Tor, Isolated in Mexico, 2013.

Prevalence of Vibrio cholerae O1 El Tor variant in a cholera-endemic zone of Kenya.

Travel-Associated Vibrio cholerae O1 El Tor, Russia.

Structural characterization of the extracellular polysaccharide from Vibrio cholerae O1 El-Tor.

Outer membrane protein OmpW is the receptor for typing phage VP5 in the Vibrio cholerae O1 El Tor biotype.

The Resistance of Vibrio cholerae O1 El Tor Strains to the Typing Phage 919TP, a Member of K139 Phage Family.

Draft Genome Sequencing of Vibrio cholerae O1 El Tor Isolates Collected in the Russian Federation from Imported Cholera Cases.

Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012.

Draft Whole-Genome Sequence of a New Variant of Vibrio cholerae O1 El Tor Strain Isolated from a Cholera Patient in Russia.

Genome Sequence of the K139-Like Phage VcP032 Originating from the Vibrio cholerae O1 El Tor Ogawa Serotype.

Molecular insights into the evolutionary pathway of Vibrio cholerae O1 atypical El Tor variants.

Whole-Genome Sequencing of Vibrio cholerae O1 El Tor Strains Isolated in Ukraine (2011) and Russia (2014).

Whole-Genome Sequencing of a Vibrio cholerae El Tor Strain Isolated in the Imported Cholera Focus in Siberia.

Epitope differences in toxin-coregulated pili produced by classical and El Tor Vibrio cholerae O1.

Genetic Screens and Biochemical Assays to Characterize Vibrio cholerae O1 Biotypes: Classical and El Tor.

Analysis of expression of toxin-coregulated pili in classical and El Tor Vibrio cholerae O1 in vitro and in vivo.

Pathophysiological mechanisms of diarrhea caused by the Vibrio cholerae O1 El Tor variant: an in vivo study in mice.

Antibiotics resistance in El Tor Vibrio cholerae 01 isolated during cholera outbreaks in Mozambique from 2012 to 2015.

Emergence of Tetracycline Resistant Vibrio cholerae O1 Biotype El Tor Serotype Ogawa with Classical ctxB Gene from a Cholera Outbreak in Odisha, Eastern India.

Epidemiologic and Drug Resistance Pattern of Vibrio cholerae O1 Biotype El Tor, Serotype Ogawa, in the 2011 Cholera Outbreak, in Alborz Province, Iran.

Unique Clones of Vibrio cholerae O1 El Tor with Haitian Type ctxB Allele Implicated in the Recent Cholera Epidemics from Nigeria, Africa.

Clonal Dissemination of a Single Vibrio cholerae O1 Biotype El Tor Strain in Sistan-Baluchestan Province of Iran During 2013.

Anethole inhibits growth of recently emerged multidrug resistant toxigenic Vibrio cholerae O1 El Tor variant strains in vitro.