Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.     

Cholera toxin gene polymerase chain reaction for detection of non-culturable Vibrio cholerae O1

Cholera enterotoxin is a major antigenic determinant for virulence of Vibrio cholerae O1 which can enter into a viable but non-culturable (N-C) state, not detectable by conventional culture methods, yet remain capable of producing enterotoxin and potentially pathogenic. PCR was applied in the current study to detect the chilera toxin (ctx) gene of N-C cells, thus eliminating the necessity of culture. Sets of oligonucleotide primers were designed, based on the ctxAB operon of V. cholerae O1, to detect the presence of the ctx gene. DNA from both culturable and N-C cells of V. cholerae O1 was amplified by PCR using sets of primers flanking 302-, 564- and 777-bp fragments of the ctx gene. The PCR method employed was capable of detecting the ctx gene in N-C V. cholerae in aquatic microcosms and in diarrheal stool samples from three patients who had distinct clinical symptoms of cholera but were culture-negative for V. cholerae O1 and non-O1 and enterotoxigenic Escherichia coli. Forty cycles of a two-step reaction (30 s each at 94 and 60°C) were optimal and more time efficient than a three-step PCR described previously. The procedure, from the point of heating microcosms or broth culture samples to observation on gels, requires < 4 h to complete.J.A.K. Hasan, A. Huq, M.A.R. Chowdhury, and R.R. Colwell are with the Department of Microbiology, University of Maryland, College Park, MD, USA. M. Shahabuddin is with the National Institute of Health. Bethesda, MD, USA. L. Loomis is with New Horizons Diagnostics Corporation, Columbia, MD, USA.     

A Potential Epidemic Factor from the Bacteria, <Emphasis Type="Italic">Vibrio cholerae WO7</Emphasis>

Certain species of Vibrio cholerae have evolved mechanisms to become pathogenic to humans, with the potential to cause a severe life-threatening diarrheal disease, cholera. Cholera can emerge as explosive outbreaks in the human population. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. The present study has been carried out on a novel toxin purified from V. cholerae W07, an epidemic cholera strain devoid of cholera toxin gene (ctx). A modified method of purification improved purification fold as well as yield of this toxin. Heating was found to be the essential and sufficient condition for dissociation of the two subunits (58 kDa and 40 kDa) of this toxin (pI 5.2). The 40-kDa subunit of the purified toxin was identified as the carbohydrate binding subunit. This toxin was found to induce apoptosis in HEp-2 cells. Thus, the WO7 toxin seems to have potential importance in the pathogenesis of disease associated with Vibrio cholerae WO7.     

Toxigenic Vibrio cholerae from environmental sources associated with the cholera outbreak after ‘AILA’ cyclone in West Bengal,India

Aims: West Bengal experienced a devastating storm named ‘AILA’ in its coastal and southern districts. We attempted to understand the transmission dynamics emphasizing on potable water to detect the presence of toxigenic strains of Vibrio cholerae, followed by the natural devastation. Methods and Results: A total of 33 water samples (from tap, tube well and ponds) were analysed. From them, 11 (33·3%) samples were found to be contaminated with V. cholerae, among which 5 (45%) isolates were V. cholerae O1 biotype Ogawa. Antibiogram profile reveals that most of the V. cholerae O1 isolates were highly sensitive against fluroquinolone group of antibiotics, but less sensitive against cefuroxime (50%), cefotaxime (40·9%), ceftriaxone (38·63%), trimethoprim (37·3%), streptomycin (29·2%) and furazolidon (4·54%). Three (36%) V. cholerae isolates were found to be ctxB positive (2 ctxB classical). Conclusions: Potable water plays a crucial role in cholera transmission. Natural disasters like the reported one aided with feacal–oral contamination enhance the possibilities of drinking water contamination. Significance and Impact of the Study: The application of the modified technique, making use of the enrichment subsequently followed by culture and PCR, will help us to detect the presence of toxigenic V. cholerae contamination in different aquatic environment. Moreover, natural extremes have a direct role in increase of salinity level, followed by higher predominance of V. cholerae along with their toxicity development in terms of genetic modification.     

Comparison of a Reversed Passive Latex Agglutination and a Polymerase Chain Reaction for Identification of Cholera Toxin Producing Vibrio cholerae O1

Production of cholera toxin (CT) in AKI medium and conservation of CT gene (ctx) of 49 strains of Vibrio cholerae O1 were compared by reversed passive latex agglutination (RPLA) and polymerase chain reaction (PCR). The production of CT agreed with conservation of the ctx in 48 out of the 49 strains. Ten strains were positive, and 38 strains were negative by both methods. Only one strain was negative in RPLA and positive in PCR. This suggested that the combination of AKI-SW and RPLA is comparable to PCR to identify CT-producing V. cholerae O1.     

Successful Application of Enzyme-Labeled Oligonucleotide Probe for Rapid and Accurate Cholera Diagnosis in a Clinical Laboratory

Two cholera cases were diagnosed using an enzyme-labeled oligonucleotide probe (ELONP) hybridization test for detection of cholera toxin gene (ctx) in a clinical laboratory at Osaka Airport Quarantine Station. The ELONP test with suspicious colonies of Vibrio cholerae O1 grown on TCBS or Vibrio agar plates gave positive result for ctx within 3 hr. We also tried to apply the ELONP test for direct detection of ctx in their stool and their non-selective culture. Specimens from Case #1, which contained 5.9 × 10⁵ CFU/g of V. cholerae O1 in the stool, cultured for 7–8 hr or longer in alkaline peptone water or Marine broth at 37C, became positive for ctx. On the other hand, specimens from Case #2, which contained 8.7 × 10⁸ CFU/ml (of V. cholerae O1 in the stool), gave positive result in this stool itself without any further culture. These data suggest that the ELONP test provides successfully a more rapid and accurate means of identifying “toxigenic” V. cholerae O1 in a clinical laboratory.     

Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand

Background

Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time.

Methods/Findings

A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB) and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect Vibrio seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009–2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1–2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area.

Conclusions

MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.     

Isolation,identification, and characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from the Danube River in Slovakia

The occurrence of Vibrio cholerae, an important aquatic pathogen, was assessed in the surface water of the Danube River near Bratislava. The isolates were distinguished by biochemical tests and grouped by ARDRA to three clusters corresponding to three species (V. cholerae, Vibrio metschnikovii, and Aeromonas spp.). The identification of V. cholerae was confirmed by multiplex PCR using primer pairs targeted to ompW gene (membrane protein), ctxA gene (toxicity gene), and toxR gene (regulatory gene). None from the isolated V. cholerae from surface water contained ctxA gene; seven of them possessed toxR gene. Serotyping of V. cholerae isolates with polyvalent O antiserum and O/139 antiserum was negative. All isolates of V. cholerae were susceptible to chloramphenicol, rifampicin, tetracycline, variable to ampicillin, and resistant to kanamycin and streptomycin.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.     

Susceptibility to Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh

Background

Despite recent progress in understanding the molecular basis of Vibrio cholerae pathogenesis, there is relatively little knowledge of the factors that determine the variability in human susceptibility to V. cholerae infection.

Methods and Findings

We performed an observational study of a cohort of household contacts of cholera patients in Bangladesh, and compared the baseline characteristics of household members who went on to develop culture-positive V. cholerae infection with individuals who did not develop infection. Although the vibriocidal antibody is the only previously described immunologic marker associated with protection from V. cholerae infection, we found that levels of serum IgA specific to three V. cholerae antigens—the B subunit of cholera toxin, lipopolysaccharide, and TcpA, the major component of the toxin–co-regulated pilus—also predicted protection in household contacts of patients infected with V. cholerae O1, the current predominant cause of cholera. Circulating IgA antibodies to TcpA were also associated with protection from V. cholerae O139 infection. In contrast, there was no association between serum IgG antibodies specific to these three antigens and protection from infection with either serogroup. We also found evidence that host genetic characteristics and serum retinol levels modify susceptibility to V. cholerae infection.

Conclusions

Our observation that levels of serum IgA (but not serum IgG) directed at certain V. cholerae antigens are associated with protection from infection underscores the need to better understand anti–V. cholerae immunity at the mucosal surface. Furthermore, our data suggest that susceptibility to V. cholerae infection is determined by a combination of immunologic, nutritional, and genetic characteristics; additional factors that influence susceptibility to cholera remain unidentified.     

Molecular diversity of CTX prophage in Vibrio cholerae

Aims: The objective of this study was to investigate the molecular diversity of CTX genetic element within toxigenic Vibrio cholerae genomes and to determine the genetic diversity of V. cholerae population collected in a 6‐year period (2004–2009) in Iran. Methods and Results: The results of mismatch amplification mutation assay (MAMA)‐PCR and sequencing showed cytosine nucleotide in positions 203 and 115 in all 50 El Tor V. cholerae strains, which is the same as classical ctxB sequence. One strain yielded amplicons with both El Tor and classical biotype primers in MAMA‐PCR indicative of presence of two copies of CTX phages with different genotypes (rstR^ET ctxB^class and rstR^ET ctxB^ET) integrated within the genome of this isolate, which suggested the integration of two different CTX phages at different occasions or point mutation in one copy of CTX. Sequencing and PCR analysis indicated the presence of hybrid CTX genotype (rstR^ET ctx^class) in 70·6% of the isolates; however, only El Tor RS1 phage has been integrated in flanking to the CTX phages with different genotypes. Conclusions: Enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and ribosomal gene spacer‐PCR (RS‐PCR) showed a relatively homogenous population in different years. Our findings indicate that sequence analysis of RS and ctxB regions has more discriminative power than restriction‐based methods. Significance and Impact of the Study: Investigating the molecular diversity of CTX prophage among V. cholerae strains helps to establish a new valuable database of genetic information about isolates, which is of great importance for epidemiologic studies in Iran and other countries encountering cholera epidemics.     

Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.     

wbeT sequence typing and IS1004 profiling of Vibrio cholerae isolates

Aims: To investigate the molecular basis for serotype variation in Vibrio cholerae O1 and the genetic relatedness amongst different serotypes isolated from 2004 to 2008 in Iran. Methods and Results: Despite the presence of all three serotypes of V. cholerae O1 (Ogawa, Inaba and Hikojima) in Iran in the last decade, the Inaba strains have been the dominated serotype. Sequence analysis of wbeT determined only a single substitution of G for A at position 295 in all Inaba strains resulting in a replacement of serine to proline. No difference was found in the copy numbers and profile of IS1004 between the classical and El Tor V. cholerae O1 strains, supporting the clonality amongst the isolates obtained over 5 years in Iran. In addition, Southern blots of HpaII‐digested chromosomal DNAs of our Ogawa and Inaba isolates showed the presence of an incomplete copy of IS1004 for all isolates. Conclusions: IS1004 profiling can be a reliable method for analysis of clonal dissemination of V. cholerae. The results indicated that specific point mutation at a particular position within the wbeT of V. cholerae O1 strains in Iran may occur which, in turn, may result in serotype switching. Significance and Impact of the Study: Understanding the molecular basis for serotype conversion of V. cholerae and their genetic relatedness could give insights for the incoming cholera epidemic prediction and control.     

Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae

Summary The B subunit (CTB) of cholera toxin (CT) can be used as a carrier protein for conjugate vaccines designed to elicit antipolysaccharide antibodies. A defined medium, AGM4, was designed to grow a high-producing mutant of Vibrio cholerae expressing only the B subunit of CT: V. cholerae 0395-NI. AGM4 contains four amino acids, asparagine, glutamic acid, arginine and serine, salts and a trace element solution. The carbon source is glucose. The fermentations performed in AGM4 indicated that CTB production paraleled the growth of the organism but that there was a maximal release of CTB during the stationary phase. There was a clear optimum of productivity at pH 8.0 and 30°C. The pH had an influence on CTB production and not only on its release. Analysis of the amino acids present in the medium showed a correlation between their consumption rates and CTB productivity. Offprint requests to: J. Shiloach     

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera-like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non-O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor-specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.     

Molecular Analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 Strains: Clonal Relationships between Clinical and Environmental Isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

Demonstration of viable but nonculturable Vibrio cholerae O1 in fresh water environment of India using ciprofloxacin DFA-DVC method

Aim: To demonstrate the presence of culturable and nonculturable viable pathogenic Vibrio cholerae O1 in fresh water environments of a cholera‐endemic region in India. Methods and Results: Conventional culture and ciprofloxacin DFA–DVC were utilized to investigate the existence of V. cholerae O1. We isolated pathogenic culturable V. cholerae O1 from water samples collected from cholera‐affected areas. No culturable V. cholerae O1 was isolated from water and plankton samples from natural fresh water bodies. Ciprofloxacin was used for DFA–DVC as V. cholerae O1 are 100% resistant to nalidixic acid in our region. The viable but nonculturable O1 cells were demonstrated in 2·21 and 40·69% samples from natural water bodies and cholera‐affected areas, respectively. Conclusion: Vibrio cholerae O1 VBNC could be demonstrated using modified DFA–DVC technique. Ciprofloxacin is preferable to nalidixic acid for DVC in view of existing high‐level resistance to nalidixic acid in cholera‐endemic areas. Significance and Impact of the study: We endorse that for public health surveillance, cholera outbreak investigation and disease control water samples in addition to culture should be tested for V. cholerae using DFA–DVC.