The Population Structure of Vibrio cholerae from the Chandigarh Region of Northern India

Background

Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.

Methodology/Principal Findings

Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters.

Conclusions/Significance

The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.     

Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of Vibrio cholerae

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTX), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPI. In order to determine the prevalence of horizontal transfer of VPI and CTX among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI⁺ strains have also acquired the CTX. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTX prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTX prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTX independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.     

Viable but Nonculturable Vibrio cholerae O1 in the Aquatic Environment of Argentina

In Argentina, as in other countries of Latin America, cholera has occurred in an epidemic pattern. Vibrio cholerae O1 is native to the aquatic environment, and it occurs in both culturable and viable but nonculturable (VNC) forms, the latter during interepidemic periods. This is the first report of the presence of VNC V. cholerae O1 in the estuarine and marine waters of the Río de la Plata and the Argentine shelf of the Atlantic Ocean, respectively. Employing immunofluorescence and PCR methods, we were able to detect reservoirs of V. cholerae O1 carrying the virulence-associated genes ctxA and tcpA. The VNC forms of V. cholerae O1 were identified in samples of water, phytoplankton, and zooplankton; the latter organisms were mainly the copepods Acartia tonsa, Diaptomus sp., Paracalanus crassirostris, and Paracalanus parvus. We found that under favorable conditions, the VNC form of V. cholerae can revert to the pathogenic, transmissible state. We concluded that V. cholerae O1 is a resident of Argentinean waters, as has been shown to be the case in other geographic regions of the world.     

Biological Activities of Lipopolysaccharide Isolated from Vibrio cholerae O139, a New Epidemic Strain for Recent Cholera in Indian Subcontinent

Biological activities of lipopolysaccharide (LPS) isolated from Vibrio cholerae O139, a new causative agent for recent cholera epidemic in Indian subcontinent, were investigated in comparison with those of LPS from O1 V. cholerae. V. cholerae O139 LPS exerted mitogenic activity, lethal toxicity and Shwartzman reaction to the same extent as those observed for O1 V. cholerae LPS, although these activities except for lethal toxicity were obviously lower than those of Salmonella typhimurium LT-2 LPS used as a reference. It was, therefore, suggested that O139 LPS does not contribute to the high infective and pathogenic potentials of the V. cholerae O139 strain as in the case of O1 V. cholerae.     

Simple Procedure for Rapid Identification of Vibrio cholerae from the Aquatic Environment

Biochemical tests commonly used to screen for Vibrio cholerae in environmental samples were evaluated, and we found that a combination of alkaline peptone enrichment followed by streaking on thiosulfate citrate bile salts sucrose agar and testing for arginine dihydrolase activity and esculin hydrolysis was an effective rapid technique to screen for aquatic environmental V. cholerae. This technique provided 100% sensitivity and ≥70% specificity.     

Prophage CTXphi genome variability and its role in alteration of Vibrio cholerae El Tor virulence characteristics

Comparative analysis of CTXphi prophage genome of 366 V. cholerae El Tor strains isolated from infected people and water was carried out using the polymerase chain reaction. Four groups of vibrios, which carry different combinations of ctxA, zot, and ace genes from core region of CTXphi prophage coding key (cholera enterotoxin) and accessory (Zot and Ace toxins) pathogenicity factors, were determined: ctxA(+) zot(-) ace(+), ctxA(-) zot(+) ace(+), ctxA(-) zot(+) ace(-), ctxA(-) zot(-) ace(+). Vibrios that had lost all tested genes were also revealed. Genomic rearrangements occurring in water environment in virulent V. cholerae strains, which acquired foreign pathogenicity genes necessary for their existence in human organism, were proposed as one of the mechanisms of formation of clones with an incomplete or no prophage. Infection process in model animals challenged with wild and isogenic strains of V. cholerae differing in the set of the phage genes (ctxA, zot, and ace) was comparatively analyzed. It was shown that variability of CTXphi prophage genome was an important factor of modification of cholera vibrios virulent characteristics. Obtained data point to usefulness of ctxA, zot, and ace phage genes detection in wild V. cholerae isolates as it could permit evaluation of their virulent potential determining the severity of the infection.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

霍乱弧菌N16961超级整合子重复序列及基因盒分析

目的:确定O1群El Tor型霍乱弧菌N16961超级整合子(SI)中霍乱弧菌重复序列(VCR)的序列特点,以及VCR和基因盒的数量及位置。方法:用局部序列比对软件BLAST将VCR参考序列与霍乱弧菌N16961的Ⅱ号染色体进行比对,用Artemis Comparison Tool查看比对结果获得比对区域的位置信息,并采用perl语言脚本获得霍乱弧菌N16961的Ⅱ号染色体VCR相应区域的序列;用全局比对软件Clustal W将上一步获得的所有VCR序列进行多序列比对,采用perl语言脚本处理比对结果获得一致性序列;用MEGA4.0软件查看多序列比对结果,并采用perl语言脚本计算各位置变异频率,据此分析霍乱弧菌N16961的Ⅱ号染色体上VCR和基因盒的特点。结果:在N16961的超级整合子中有158个VCR,其核苷酸长度为117～124 bp;其一致性序列有126个核苷酸,其中37个为保守核苷酸位点,89个为可变核苷酸位点;139个VCR与相邻的VCR之间至少有1个基因,19个VCR相互之间没有任何基因;N16961的SI中共存在146个基因盒,基因盒大小为390～5924 bp不等,每个基因盒中整合的基因数目为1～9个不等。结论:建立了SI中VCR和基因盒的分析流程,分析了SI中VCR的保守及变异位点,明确了霍乱弧菌N16961的SI中VCR和基因盒的信息,为霍乱弧菌和其他细菌中SI的研究提供了分析基础。     

Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae non O-1 with plasmid DNA by electroporation

An electroporation procedure for the plasmid-mediated transformation of the genus Vibrio was performed, as part of an effort to develop recombinant DNA techniques for genetic manipulation of the genus Vibrio. Vibrio parahaemolyticus, V. alginolyticus, and V. cholerae non O-1 (9 different strains) were transformed with 3 vector plasmids (pACYC184, pHSG398, and pBR325). The efficiency of transformation was highly dependent on three parameters: the concentration of plasmid DNA; the strength of the electric field; and the combination of plasmid DNA and recipient strain. The drug-resistance genes on the vector plasmid were expressed in the Vibrio strains.     

Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Worldwide Occurrence of Integrative Conjugative Element Encoding Multidrug Resistance Determinants in Epidemic Vibrio cholerae O1

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.     

Environment and virulence factors of Vibrio cholerae strains isolated in Argentina

AIMS: To determine the presence of Vibrio cholerae in different areas of Argentina in three sample types, to determine the composition of planktonic communities in areas at which this pathogen was detected and to characterize the virulence properties and antimicrobial resistance of the recovered environmental isolates. METHODS AND RESULTS: Water and plankton samples were collected in marine, brackish and freshwater environments. Vibrio cholerae non-O1, non-O139 was isolated in 36.1% of the samples analysed. The micro-organism was detected in freshwater but not in marine or brackish samples. No relationship was found between isolation of V. cholerae and presence of any species of plankton. All the isolates presented very similar virulence profiles by PCR, lacking ctxA and tcpA El Tor and containing hlyA (98.7%), rtxA (99.0%), toxR (98.7%) and stn-sto (1.9%). Resistance to ampicillin was found in both Tucumán (21%) and Buenos Aires isolates (45%). CONCLUSIONS: We identified two geographic areas in Argentina where V. cholerae was present: freshwaters of the rivers from Tucumán and the Río de la Plata. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of V. cholerae strains in the environment, carrying both virulence factors and resistance to antimicrobial agents, highlight the need for a continuous and active surveillance of this pathogen.     

Genome Sequence and Comparative Genomics Analysis of a Vibrio cholerae O1 Strain Isolated from a Cholera Patient in Malaysia

The genome sequence analysis of a clinical Vibrio cholerae VC35 strain from an outbreak case in Malaysia indicates multiple genes involved in host adaptation and a novel Na⁺-driven multidrug efflux pump-coding gene in the genome of Vibrio cholerae with the highest similarity to VMA_001754 of Vibrio mimicus VMA223.     

Scanning the Landscape of Genome Architecture of Non-O1 and Non-O139 Vibrio cholerae by Whole Genome Mapping Reveals Extensive Population Genetic Diversity

Historically, cholera outbreaks have been linked to V. cholerae O1 serogroup strains or its derivatives of the O37 and O139 serogroups. A genomic study on the 2010 Haiti cholera outbreak strains highlighted the putative role of non O1/non-O139 V. cholerae in causing cholera and the lack of genomic sequences of such strains from around the world. Here we address these gaps by scanning a global collection of V. cholerae strains as a first step towards understanding the population genetic diversity and epidemic potential of non O1/non-O139 strains. Whole Genome Mapping (Optical Mapping) based bar coding produces a high resolution, ordered restriction map, depicting a complete view of the unique chromosomal architecture of an organism. To assess the genomic diversity of non-O1/non-O139 V. cholerae, we applied a Whole Genome Mapping strategy on a well-defined and geographically and temporally diverse strain collection, the Sakazaki serogroup type strains. Whole Genome Map data on 91 of the 206 serogroup type strains support the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity. Interestingly, we discovered chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae. We also found pervasive chromosomal rearrangements such as duplications and indels in many strains. The majority of Vibrio genome sequences currently in public databases are unfinished draft sequences. The Whole Genome Mapping approach presented here enables rapid screening of large strain collections to capture genomic complexities that would not have been otherwise revealed by unfinished draft genome sequencing and thus aids in assembling and finishing draft sequences of complex genomes. Furthermore, Whole Genome Mapping allows for prediction of novel V. cholerae non-O1/non-O139 strains that may have the potential to cause future cholera outbreaks.     

Release of the Outer Membrane Vesicles from Vibrio cholerae and Vibrio parahaemolyticus

We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze-substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti-O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron-dense mass.     

Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.     

Population Structure and Evolution of Non-O1/Non-O139 Vibrio cholerae by Multilocus Sequence Typing

Pathogenic non-O1/non-O139 Vibrio cholerae strains can cause sporadic outbreaks of cholera worldwide. In this study, multilocus sequence typing (MLST) of seven housekeeping genes was applied to 55 non-O1/non-O139 isolates from clinical and environmental sources. Data from five published O1 isolates and 17 genomes were also included, giving a total of 77 isolates available for analysis. There were 66 sequence types (STs), with the majority being unique, and only three clonal complexes. The V. cholerae strains can be divided into four subpopulations with evidence of recombination among the subpopulations. Subpopulations I and III contained predominantly clinical strains. PCR screening for virulence factors including Vibrio pathogenicity island (VPI), cholera toxin prophage (CTXΦ), type III secretion system (T3SS), and enterotoxin genes (rtxA and sto/stn) showed that combinations of these factors were present in the clinical isolates with 85.7% having rtxA, 51.4% T3SS, 31.4% VPI, 31.4% sto/stn (NAG-ST) and 11.4% CTXΦ. These factors were also present in environmental isolates but at a lower frequency. Five strains previously mis-identified as V. cholerae serogroups O114 to O117 were also analysed and formed a separate population with V. mimicus. The MLST scheme developed in this study provides a framework to identify sporadic cholera isolates by genetic identity.     

Molecular cloning and characterization of mannitol-1-phosphate dehydrogenase from Vibrio cholerae

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a K(m) of 1.54 +/- 0.1 mM and V(max) of 320.8 +/- 7.81 micronmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37°C, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.     

Genetic Traits of Vibrio cholerae O1 Haitian Isolates That Are Absent in Contemporary Strains from Kolkata,India

The world''s worst cholera epidemic in Haiti (2010) coerced to trace the origin and dissemination of the causative agent Vibrio cholerae O1 for proper management of cholera. Sequence analysis of the Haitian strain showed several variations in the genes encoding cholera toxin B subunit (ctxB); toxin-co-regulated pilus (tcpA), repeat in toxins (rtxA), quinolone resistance-determining region (QRDR) of gyrase A (gyrA), rstB of RS element along with the change in the number of repeat sequences at the promoter region of ctxAB. Our earlier studies showed that variant tcpA (tcpA CIRS) and ctxB (ctxB7) first appeared in Kolkata during 2003 and 2006, respectively. The present study revealed that a variant rtxA was first isolated in Kolkata during 2004 and probably formed the genetic background for the emergence of the ctxB7 allele as we were unable to detect a single strain with the combination of El Tor rtxA and ctxB7. The variant gyrA was first time detected in Kolkata during 1994. The Kolkata strains contained four heptad repeats (TTTTGAT) in their CT promoter regions whereas Haitian strains carried 5 heptad repeats. Haitian strains had 3 nucleotide deletions at the rstB gene, which is a unique feature of the classical biotype strains. But the Kolkata strains did not have such deletion mutations in the rstB. Our study demonstrated the existence of some Haitian genetic traits in Kolkata isolates along with the dissimilarities in genomic content with respect to rstB and ctxAB promoter region. Finally, we conclude that Haitian variant strain may be evolved due to sequential event in the Indian subcontinent strain with some cryptic modification in the genome.