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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 4  |  Page : 507-515
 

Characteristics of Vibrio cholerae O1 isolated from water of the River Ganga, Varanasi, India


Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, Odisha, India

Date of Submission27-May-2014
Date of Acceptance06-May-2015
Date of Web Publication16-Oct-2015

Correspondence Address:
D V Singh
Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.167327

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 ~ Abstract 

Background: Vibrio cholerae is an autochthonous inhabitant of fresh and brackish water and estuarine system. Investigation of V. cholerae from the River Ganga seems important to find variation in CTX arrangement and genomic diversity. Objectives: To investigate V. cholerae O1 strains for the presence of virulence and regulatory genes, variation in number and organisation of the pre-CTXΦ and/or CTXΦ, and for the genomic diversity. Materials and Methods: Polymerase chain reaction (PCR) was used to detect virulence and regulatory genes, type of rstR and location of CTXΦ on the chromosome. Southern hybridisation was conducted to see the number and arrangement of pre-CTXΦ and CTXΦ. Ribotyping and pulsed-field gel electrophoresis were used to find genetic relatedness. Results: Seven strains gave positive results by PCR for the gene encoding for ctx A, zot, ace, tcp A (El Tor), omp U, and tox R, except one strain that was negative for the ctx A. Three strains were positive for the tcp A (El Tor), omp U and tox R genes. Determination of CTX organisation showed that among the ctx-positive strains, four harboured two copies of CTXETΦ arranged in tandem and two harboured one copy of CTXETΦ, and one ctx-negative strain harboured only one copy of pre-CTXETΦ. Pulsotype and ribotype analysis showed existence of at least three pulsotype and ribotypes indicating diversity in genomic content among them. Conclusion: This study thus indicates that multiple clones (ribotypes/pulsotypes) of V. cholerae O1 carrying pre-CTXΦ and/or CTXΦ and ctx-negative strains were present in the water of the River Ganga, Varanasi, India.


Keywords: CTX typing, genomic diversity, polymerase chain reaction, Vibrio cholerae, virulence genes


How to cite this article:
Mohaptra S S, Mantri C K, Bhotra T, Singh D V. Characteristics of Vibrio cholerae O1 isolated from water of the River Ganga, Varanasi, India. Indian J Med Microbiol 2015;33:507-15

How to cite this URL:
Mohaptra S S, Mantri C K, Bhotra T, Singh D V. Characteristics of Vibrio cholerae O1 isolated from water of the River Ganga, Varanasi, India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Sep 23];33:507-15. Available from: http://www.ijmm.org/text.asp?2015/33/4/507/167327



 ~ Introduction Top


Vibrio cholerae is an autochthonous inhabitant of fresh and brackish water and estuarine system.[1] These Vibrio species not only survive in riverine, estuarine, and coastal waters around the world, but also lives in association with crustacean copepods and the aquatic plants, either in viable and culturable or in the viable but non-culturable state.[2],[3],[4] Based on somatic antigen, V. cholerae are classified into more than 200 serogroups but only strains belonging to O1 and O139 serogroups have been reported causing epidemic and pandemic cholera. The O1 serogroup is further classified into two biotypes, namely classical and El Tor. The two biotypes are distinguished from each other on the basis of their differences in several phenotypic properties [5] and genotypic properties.[6] The first six pandemics possibly originated from the Gangetic Delta of Bengal, are believed to have been caused by the classical biotype. The ongoing seventh pandemic caused by El Tor biotype of O1 serogroup started from Sulawesi island of Indonesia in 1961. Studies of V. cholerae from environmental surface water indicated that O1 vibrios and non-O1, non-O139 vibrios may survive better in the environment in association with aquatic plant.[3],[4] They also survive in association with chironomids.[7],[8]

The presence of El Tor strains with the classical biotype traits (hybrid strains or Matlab variants) and El Tor strains producing classical toxin (altered strains) have been described earlier.[9] Among altered strains, two types of CTX prophages containing classical ctxB have been reported. One is the CTX prophage harbouring classical rstR and ctxB genes as seen in Mozambique strains [10] and other is the CTX prophage containing El Tor rstR and classical ctxB. Mozambique strain contains a tandem repeat of the classical CTX prophage on the small chromosome and Vietnam strains contain classical CTX prophage on the large chromosome in addition to small chromosome.[11] Although V. cholerae O1 carrying classical ctxB and El Tor rstR have been reported from India,[12] the O1 strains having addition substitution at 20th position of classical ctxB (histidine to asparagine) caused outbreaks in Haiti,[13] which was found to exist in Kolkata.[14] The question of whether such hybrid or altered strains exist in the environment and can cause epidemic has been debated.

To determine genetic relatedness and for epidemiological studies among V. cholerae strains, several molecular techniques such as ribotyping and pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing have been successfully used.[15],[16],[17],[18],[19] Therefore, in this study, we used ribotyping and PFGE to determine overall genetic relatedness and clonality among toxigenic and non-cholera-toxigenic strains isolated from the water of the River Ganga in Varanasi, India between 1986 and 1988. These trains were also examined for the presence of virulence and regulatory genes, variation in number and organisation of the pre-CTXΦ and/or CTXΦ, and ctxB sequences.


 ~ Materials and Methods Top


Bacterial strains

We recovered 11 environmental strains of V. cholerae O1 biotype El Tor from the laboratory stock for inclusion in the study. All these strains were initially isolated from the water for the River Ganga, collected from different Ghats between 18 March 1986 and 25 October 1986 (six strains: VE41, VE42, VE43, VE44, VE45 and VE86), between 21 June 1987 and 9 August 1987 (three strains: VE46, VE47 and VE48) and between 29 July 1988 and 7 December 1988 (two strains: VE87 and VE88). The water samples from the River Ganga were processed by standard methods described elsewhere [4] and identified by following the standard bacteriological methods.[20] The V. cholerae O1 strains N16961 and VC20, provided by GB Nair and KO194 from our laboratory stock was used as controls. All strains were maintained in LB broth supplemented with 30% glycerol at −80°C and did not undergo more than two subculture prior to testing in this study.

Polymerase chain reaction assays

The presence of virulence and regulatory genes, ctxA, zot, ace, tcpA, ompU and toxR in V. cholerae was determined using hexaplex polymerase chain reaction (PCR) as described earlier.[21] PCR was further performed to detect variant of rstR genes, rstRET, rstRClass, and rstRCal, and to determine the location of CTXΦ prophage in the chromosome.[22] Amplicons were separated on 1.0–1.8% agarose gels, stained in ethidium bromide, and photographed in a gel documentation system (Bio-Rad, USA). The details of the primers purchased from GCC, New Delhi, are listed in [Table 1].
Table 1: Details of the primers used in the study

Click here to view


Probes and hybridisation

For preparation of DNA blots, chromosomal DNA was extracted from overnight culture of the V. cholerae strains by the cetyltrimethylammonium bromide method as described by Ausubel et al.[27] Approximately, 4 µg of DNA was digested with restriction enzymes, Bgl I, Bgl II, Hind III, and Pst I (NEB, Beverly, MA, USA), separated by electrophoreses in 0.8% agarose gel, and blotted onto nylon membrane by Southern blotting.[28] The gene probes used to detect CTX genetic element, RS1, ctx A. zot and rRNA, were prepared according to the method described earlier.[29] The probes were labelled by random priming [30] using a random primer labelling kit (NEB) and [α-32 P] deoxyadenosine triphosphate (3000 Ci/mmol; Bhabha Atomic Research Center, Mumbai, India). Southern blots were hybridised with labelled probes, and autoradiograph developed using a Phosphor-Imager (Fuji Photo Film, Japan).

Pulsed-field gel electrophoresis

The overnight grown culture of absorbance 1.0 at OD600 of V. cholerae O1 in LB broth was centrifuged and the pellet dissolved in cell suspension buffer. V. cholerae plugs prepared following the standard method [31] were digested with 50 U of Not I (NEB, Beverly, MA, USA). Electrophoresis was performed using clamped homogenous electric fields Mapper (Bio-Rad, USA), for 40 h 24 min with initial switch time of 2.96 s, and final switch time of 26.29 s. The gel was stained with ethidium bromide and photographed in a gel documentation system (Bio-Rad, USA). Pulsotype assignment was based on interpretation criteria of Tenover et al.[32]

Nucleotide sequencing

Polymerase chain reaction was performed to amplify ctxB genes from seven ctx-positive V. cholerae O1 biotype El Tor strains by the method described elsewhere [22] PCR fragments of ctxB gene were purified using the QIAquick gel extraction kit (Qiagen) and both strands were sequenced at The Centre for Genome Application, New Delhi. The ctxB nucleotide sequences obtained from ctx-positive V. cholerae O1 biotype El Tor strains were deposited in GenBank under accession numbers HQ405515 to HQ4055521.


 ~ Results Top


Biochemical and genetic characteristics

All tested strains when grown on thiosulfate citrate bile salt agar produced characteristic colony. All strains were positive for oxidase reaction, fermented glucose and sucrose, produced acid from KIA medium, decarboxylate lysine and ornithine, reduced nitrate to nitrite. These strains also showed chicken cell agglutination, resistant to polymyxin B and positive for Voges–Proskauer reaction and agglutinated with polyvalent O1 and monovalent Ogawa antisera. Based on biochemical and serological tests, strains were identified as V. cholerae O1 biotype El Tor serotype Ogawa. These results were confirmed by O1/O139 and ctx-PCR and tcpAET and rstRET PCR amplification.[22],[24]

Presence of virulence, regulatory and rstR genes

Seven of the 11 V. cholerae O1 strains were positive for the ctxA, zot, ace, tcpA (El Tor), ompU and toxR genes, whereas VE44, although negative for ctxA gene, was positive for zot, ace, tcpA (El Tor), ompU and toxR genes. All of the seven strains also produced cholera toxin (data not shown). Three O1 El Tor strains were negative for ctxA, zot and ace genes [Table 2] and [Figure 1]. When tested by PCR for the presence of rstR, all of the ctx-positive V. cholerae O1 strains including VE44 negative for ctxA gene showed positive results for the rstRET gene indicating the presence of El Tor type of CTXΦ prophage [Table 2] and [Figure 2]. PCR of truncated ctx- and ctx-positive strains gave expected amplicons of 0.8 kb with primers CIIF and CIIR, due to absence of the CTXΦ or pre-CTXΦ (devoid of ctxAB genes) between the traF and yciH genes indicating that El Tor CTXΦ prophage is present on the large chromosome (data not shown).
Table 2: Ribotypes, pulsotypes and virulence profiles of Vibrio cholerae O1 biotype El Tor strains isolated from the water of the River Ganga, Varanasi, India, between 1986 and 1988 and used in the study

Click here to view
Figure 1: Agarose gel electrophoresis of polymerase chain reaction (PCR) products stained with ethidium bromide obtained by hexaplex PCR with Vibrio cholerae O1 strains isolated from the water of the River Ganga, Varanasi, India. V. cholerae O1 El Tor strain KO194 was used as positive control. Strain names are given at the top of the lane and gene-specific amplicons on the left side of the lane. The molecular weight marker is a 100 bp DNA ladder

Click here to view
Figure 2: Agarose gel electrophoresis of polymerase chain reaction products stained with ethidium bromide for rstRET, gene obtained from Vibrio cholerae O1 EL Tor strains isolated from the water of the River Ganga, Varanasi, India. V. cholerae O1 El Tor strain KO194 was used as positive control. Strain names are given at the top of the lane and rstRET gene amplicon on the left side of the lane. The molecular weight marker is a 100 bp DNA ladder

Click here to view


Organisation of pre-CTXΦ and CTXΦ and ctxB sequencing

The copy number of CTX genetic element was determined after digesting DNA from V. cholerae O1 strains with restriction enzymes, Bgl I, Bgl II, Hind III and Pst I, possessing unique restriction sites within the CTX element. Southern blot hybridisation with ctxA and zot probes showed the presence of one copy of CTXΦ in two strains and two copies in four strains [Figure 3]a. Subsequent hybridisation of the same blot with RS1 probe produced multiple bands on digestion with Bgl II, indicating the presence of tandemly arranged RS element upstream of the CTXΦ. A free RS1 element was also detected in at least one strain VE45 [Figure 3]c. The results obtained after digesting DNA with different restriction enzymes and probing with ctxA, zot and RS1 are summarised in [Table 3]. We failed to get any band with strain VE48, may be due to the presence of endonuclease activity in this strain. The detection of the single band with Bgl II and Pst I but two bands with Bgl I in strain VE44 on hybridisation with zot probe indicate the presence of truncated CTXΦ [Figure 3]b. Further hybridisation with RS1 probe produced two bands indicating the presence of tandemly arranged RS element upstream of the pre-CTXΦ [Figure 3]c. Based on the hybridisation data, the organisation of copies of the CTXΦ and/or pre-CTXΦ in V. cholerae O1 strains was postulated as presented in [Figure 4].
Figure 3: Southern hybridisation patterns obtained with Vibrio cholerae O1 strains isolated from the water of the River Ganga, Varanasi, India, after digesting DNA with restriction enzymes, BglI, BglII, HindIII and PstI and probing with (a) ctxA (b) zot and (c) RS1 gene

Click here to view
Table 3: Restriction fragment length polymorphism pattern and arrangement of pre‑CTXΦ (devoid of ctxAB gene) and/or CTXΦ prophage (s) and RS element of Vibrio cholerae O1 biotype El Tor strains isolated from the water of the River Ganga, Varanasi, India

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Figure 4: Schematic representation of organisation of pre-CTXΦ (devoid of ctxAB gene) and/or CTXΦ (not to scale) deciphered by Southern hybridisation data obtained with Vibrio cholerae O1 biotype El Tor strains isolated from the water of the River Ganga, Varanasi, India. Restriction sites designations are as follows, BI: BglI; BII: BglII; H: HindIII; P: Pst

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DNA sequencing of ctxB gene amplified from CTX ETΦ of six V. cholerae O1 showed tyrosine at position 39 and isoleucine at position 68 identical to those of El Tor strain [Figure 5]. However, strain VE87 showed threonine at position 68 similar to classical strain and belong to new ctxB type. These observations thus indicate that ctx-positive V. cholerae O1 biotype El Tor strains isolated from the water of the River Ganga Varanasi, India, contain El Tor type of ctxB[9] and one strain represent a new type of ctxB, not reported earlier.
Figure 5: Amino acid sequence alignment of ctxB gene fragment obtained from CTXETΦ of toxigenic Vibrio cholerae O1 biotype El Tor strains isolated from the water of the River Ganga, Varanasi, India. Dots represent identical amino acids

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Ribotyping and pulsotype analysis

Analysis of rRNA genes with Bgl I produced five different ribotypes (patterns B-I, B-IIb, B-IIIb, B-IV and B-V) in V. cholerae O1 strains [Figure 6]. The rRNA restriction patterns were reproducible in repeated assay and consisted of 6–9 bands between 2.3 kb and 19.1 kb in size. Of the 10 isolates of V. cholerae O1, four belong to ribotype B-I, three belong to ribotype B-IIb and one each belong to B-IIIb, B-IV and B-V, respectively. This study thus suggests that diverse population of El Tor biotype of V. cholerae O1 existed in the water of the River Ganga, Varanasi, India, and that genetic recombination may be occurred in the aquatic environment giving rise the evolution of the multiple ribotypes of toxigenic V. cholerae strains.
Figure 6: Ribotyping patterns of Vibrio cholerae O1 biotype El Tor strains isolated from the water of the River Ganga, Varanasi, India. Strain names are shown above the lanes and ribotype designation below the lanes. The molecular weight marker is HindIII digestion products of λ DNA (NEB, Beverly, MA, USA)

Click here to view


Pulsed-field gel electrophoresis following Not I digestion of chromosomal DNA subtyped the V. cholerae O1 isolates into two pulsotypes designated I and II respectively [Figure 7]. Of the nine V. cholerae O1, six were pulsotype I and included sub-pulsotypes Ia through Id and three belong to type II included sub-pulsotypes IIa, IIb and IIc, respectively. No correlation was found between pulsotype and arrangement of CTX element.
Figure 7: Pulsed-field gel electrophoresis patterns of NotI digested product of agarose embedded genomic DNA of Vibrio cholerae O1 biotype El Tor strains isolated from the water of the River Ganga, Varanasi, India. Strain names are shown above the lanes and pulsotype designation below the lanes. The molecular weight marker is Saccharomyces cerevisiae DNA standard (Bio-Rad)

Click here to view



 ~ Discussion Top


The results of microbiological culture, the biochemical and serological tests primarily confirmed that the isolates from the water of the River Ganga belong to V. cholerae O1. These microbiological results were complemented by simplex and multiplex PCR that included tcpAET and rstRET and ctx A, O1 rfb and O139 rfb specific PCR.[22],[24] The result of the above data further indicates that the V. cholerae, except one isolate, were toxigenic and belong to biotype El Tor and serogroup O1. The genotypic and phenotypic tests performed in this study thus indicates that V. cholerae strains present in the River Ganga belong to serogroup O1 biotype El Tor serotype Ogawa.

V. cholerae O1 biotype El Tor are known to possess either a single or multiple copies of CTXΦ in the large chromosome, the classical biotype possess two copies of CTXΦ, one on each chromosome, respectively. However, the results of this study showed the presence of CTXΦ or truncated CTXΦ containing the El Tor rstR and El Tor ctxB on the large chromosome. Analysis of truncated CTXΦs (devoid of ctxAB) led to the concept that pre-CTXΦ phage may acquire the ctxAB genes in due course of time making V. cholerae strains toxigenic.[9] From this study, it was clear that majority of water isolates, like clinical isolates, possess virulence genes including cholera toxin encoded by a filamentous bacteriophage CTXΦ,[33] indicating that water isolates had all characteristic to cause epidemic cholera like other strains.[23] Interestingly, the presence of pre-CTXΦ in strain VE44 indicated that there could be possibility of occurrence of multiple recombination event acquiring ctxAB genes.

Ribotyping has frequently been used to determine the diversity among V. cholerae, and Bgl I was found more discriminating than other restriction enzymes used for ribotyping.[15]V. cholerae O1 belonging to multiple ribotypes,[15],[29],[26],[34] carrying similar or different CTX organisation have been reported.[35] The restriction pattern that lacked the band of 5.8, 5.9 shown by the strains represent different ribotypes. The restriction pattern produced by water isolates showing these ribotypes was reported later in clinical isolates.[29] It was known that the 2.4-kb restriction fragment is an alternative band for the 6.9-kb operon C fragments due to gain of Bgl I site in the 16S rrn gene.[36] At least one strain showed loss of the 2.4-kb band. The loss of 2.4-kb band may be due to loss of Bgl I site in the operon C. Moreover, loss of 5.8 and 5.9-kb bands in majority strains may be due to the loss of H, and I operon that occurred owing to the arrangement in tandem and recombination events. The data presented in this study indicates the possiblity of a high rate of recombination in rrn gene leading to the existence of diverse clonal population of V. cholerae O1. Since ripotypes B-I to B-IV of V. cholerae O1 strains that caused epidemics between 1990 and 2008 were present in the surface water of River Ganga, the data presented in this study indicates that simultaneous changes may have occurred or occurring in the rrn operons [36] and in other unidentified genes in the aquatic environment that might influence the prevalence and emergence of new clones of O1 strains by interacting with environmental factors. The existence of newer ribotype B-V in the water of the River Ganga suggests that this ribotype, like the clone of O1 that caused epidemic in Goa in 1998, may cause epidemic in future by combating intestinal immunity or stresses in the environmental habitats. In view of fluctuations observed in the prevalence of V. cholerae O1 relative to that of O139 in human infection [9],[10],[11] and rapid genotypic and phenotypic changes, further ecological studies are required to explain the appearance and disappearance of O1 strains and the mobility of genetic elements encoding virulence properties.

Similarly, different pulsotype carrying similar or different CTX organisation have been reported by several workers in V. cholerae.[13],[16],[25],[31],[37],[38] Overall PFGE analysis revealed that water isolates have two major banding patterns (pulsotype), I and II, pattern I has four subtypes and pattern II has three subtypes. These results reflect genetic divergence that could serve as a distinct signature for the water isolates from the River Ganga. The pulsotypes, like ribotypes, reported were causing outbreaks similar to those which reported diversity among clinical isolates.[13],[16],[25],[37],[38]

Results of this study thus suggest that multiple clones (ribotypes/pulsotypes) of V. cholerae O1 carrying pre-CTXΦ and/or CTXΦ and ctx-negative strains were present in the water of the River Ganga, Varanasi, India, indicating occurrences of genetic recombination in the aquatic environment. These findings necessitate the continuous surveillance of water and aquatic environment for the presence of V. cholerae O1 and monitoring of the transmission of the cholera infection.


 ~ Acknowledgments Top


This research was supported by the funds contributed by Department of Biotechnology, New Delhi, to Institute of Life Sciences, Bhubaneswar. Senior Research Fellowships awarded by the Council of Scientific and Industrial Research, New Delhi, India, and Institute of Life Sciences to S.S.M., C.K.M. and Junior Research Fellowship awarded by the Department of Biotechnology, New Delhi, to Tilothama Bhotra are gratefully acknowledged. Authors thank M.H.U. Turabe Fazil for providing assistance in nucleotide sequence and data analysis and Dr. B.N. Shukla of the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University for providing environmental V. cholerae strains used in the study. The part of this study was presented in the XXXV Conference of Indian Association of Medical Microbiologist at Varanasi, November 23-26, 2011.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

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