|Year : 2016 | Volume
| Issue : 4 | Page : 462-470
Genomic profile of antibiotic resistant, classical ctxB positive Vibrio cholerae O1 biotype El Tor isolated in 2003 and 2005 from Puri, India: A retrospective study
T Bhotra1, MM Das1, BB Pal2, DV Singh1
1 Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, Odisha, India
2 Department of Infectious Disease Biology, Regional Medical Research Centre, Bhubaneswar, Odisha, India
|Date of Submission||03-Jun-2015|
|Date of Acceptance||29-Sep-2016|
|Date of Web Publication||8-Dec-2016|
D V Singh
Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, Odisha
Source of Support: None, Conflict of Interest: None
Objectives: To examine eight strains of Vibrio cholerae O1 isolated in 2003 and 2005 from Puri, India, for antibiotic susceptibility, presence of virulence and regulatory genes, cholera toxin (CT) production, CTX arrangement and genomic profiles. Materials and Methods: Bacterial strains were tested for antibiotic susceptibility using disc diffusion assay. Polymerase chain reaction determined the presence of antibiotic resistance, virulence and regulatory genes. To determine the type of cholera toxin subunit B (ctxB), nucleotide sequencing was performed. Southern hybridisation determined the number and arrangement of CTXΦ. Ribotyping and pulsed-field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. Results: All the eight strains, except one strain, showed resistant to nalidixic acid, sulphamethoxazole, streptomycin and trimethoprim and possessed the sullI, strB, dfrA1 and int SXT genes. All the strains carried the toxin-co-regulated pilus pathogenicity island, the CTX genetic element, the repeat in toxin and produced CT. Restriction fragment length polymorphism (RFLP) analysis showed that V. cholerae O1 possess a single copy of the CTX element flanked by tandemly arranged RS element. Nucleotide sequencing of the ctxB gene showed the presence of classical ctxB. RFLP analysis of conserved rRNA gene showed two ribotype patterns. PFGE analysis also showed at least three PFGE patterns, irrespective of year of isolations, indicating the genomic relatedness among them. Conclusion: Overall, these data suggest that classical ctxB-positive V. cholerae O1 El Tor strains that appeared in 2003 continue to cause infection in 2005 in Puri, India, and belong to identical ribotype(s) and/or pulsotype(s). There is need to continuous monitor the emergence of variant of El Tor because it will improve our understanding of the evolution of new clones of variant of V. cholerae.
Keywords: Antibiotic resistant, classical ctxB, genetic relatedness, pulsotype, ribotype, V. cholerae O1
|How to cite this article:|
Bhotra T, Das M M, Pal B B, Singh D V. Genomic profile of antibiotic resistant, classical ctxB positive Vibrio cholerae O1 biotype El Tor isolated in 2003 and 2005 from Puri, India: A retrospective study. Indian J Med Microbiol 2016;34:462-70
|How to cite this URL:|
Bhotra T, Das M M, Pal B B, Singh D V. Genomic profile of antibiotic resistant, classical ctxB positive Vibrio cholerae O1 biotype El Tor isolated in 2003 and 2005 from Puri, India: A retrospective study. Indian J Med Microbiol [serial online] 2016 [cited 2017 May 24];34:462-70. Available from: http://www.ijmm.org/text.asp?2016/34/4/462/195356
| ~ Introduction|| |
Vibrio cholerae O1, the causative agent of cholera, was classified into two biotypes “classical” and “El Tor” based on certain phenotypic and genetic traits.V. cholerae O1 strains may undergo serotype conversion or switch between Inaba and Ogawa serotypes. Several workers reported variations in toxin-co-regulated pilin A (tcpA), cholera toxin subunit B (ctxB), repeat in toxin (RTX) and haemolysin A (hlyA) genes between two biotypes., The sequences of the ctxA gene are identical in both classical and El Tor strains. However, the ctxB gene sequence of the El Tor biotype varies from the classical biotype in two nucleotides positions, resulted in differences of two amino acids., Cholera toxin (CT) is one of the major toxins responsible for causing severe diarrhoea. The emergence of the El Tor variants has been reported in Bangladesh replacing the prototype of the seventh pandemic strain of El Tor biotype belonging to multiple ribotypes. Another study reported coexistence of the classical and El Tor biotypes in 1990–1994. The hybrid CTXΦ isolates carrying El Tor rstR and classical ctxB present until 1995 were replaced by the El Tor biotype in Kolkata, India. These isolates belonged to single pulsotype  which spread to several other countries of Asia and Africa., The El Tor variants were classified into five groups; MATLAB variants categorised into three subtypes I, II and III, altered El Tor variant, Mozambique, Hybrid El Tor and Haitian variant.,, The El Tor strains carrying classical ctxB had been reported causing outbreaks during 2007–2009 in different parts of Odisha.,, Whether El Tor the variant carrying classical ctxB existed before 2007 that caused outbreaks in Puri is to be investigated.
Several molecular techniques, such as ribotyping, enterobacterial repetitive intergenic consensus fingerprinting, amplified fragment length polymorphism, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing, have been used to study relationships among clinical and pathogenic isolates of microorganisms. In this study, we examined eight strains of V. cholerae isolated from Puri, India, in 2003 and 2005 for the antibiotic susceptibility, presence of virulence and regulatory genes, ctxB allele and CTX organisation. We also determined the overall genetic relatedness and clonality among strains by using ribotyping and PFGE.
| ~ Materials and Methods|| |
We recovered eight representative 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 diarrhoeal patients from Puri in 2003 and 2005. The stool samples from the diarrhoeal patients were processed by standard methods described elsewhere  and identified by following the standard bacteriological methods. [Table 1] shows the details of source, year and place of isolation. V. cholerae O1 strains VC20 provided by GB Nair and KO194 from the laboratory stock were used as control. All strains were maintained in Tryptic Soy Broth (Difco) supplemented with 30% glycerol at 80°C or in Luria-Bertani (LB) agar stab culture at room temperature. Before the use, the identity of the cultures was confirmed by selected biochemical tests and serology.
|Table 1: Antibiotic resistance pattern and the presence of antibiotic resistance and intSXT genes in Vibrio cholerae O1 strains isolated from Puri carrying classical cholera toxin subunit B and El Tor rstR|
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Antibiotic susceptibility assay
All the V. cholerae O1 strains were tested for antimicrobial susceptibility by the method of Bauer et al. The antibiotic discs used were ampicillin (A: 10 µg), chloramphenicol (C: 30 µg), cefotaxime (Ce: 30 µg), ciprofloxacin (Cf: 5 µg), cephalexin (Ce: 30 µg), co-trimoxazole (Co: 25 µ
g), furazolidone (F: 100 µg), gentamicin (G: 10 µg), neomycin (N: 30 µg), norfloxacin (Nx: 10 µg), nalidixic acid (Na: 30 µg), polymyxin B (Pb: 50U), streptomycin (S: 10 µg), spectinomycin (Se: 100 µg), tetracycline (T: 30 µg) and trimethoprim (Tr: 5 µg) from Hi-Media Laboratories, Bombay, India. Except for furazolidone, all strains were rated as susceptible, intermediate resistant or resistant according to the interpretive criteria recommended by the Clinical and Laboratory Standards Institute.
Polymerase chain reaction assays
The presence of antibiotic resistance genes encoding for sulphamethoxazole (sullI), trimethoprim (dfr18/dfrA1) and streptomycin (strB) and the SXT genetic element was determined by tetraplex polymerase chain reaction (PCR) assay. SXT gene-positive strains V. cholerae O139 strain ATCC 51394 and V. cholerae O1 strain KO194 were used as positive controls.
Hexaplex PCR assay was employed to determine the presence of ctxA, zot, ace, tcpA, ompU and toxR genes in V. cholerae strains. Single PCR assays were used to detect the tcpI and hlyA genes,acfB gene,rstR genes  and RTX gene. A double mismatch amplification mutation assay (DMAMA)-PCR was performed using biotype specific primers Rv-cla, ctxB3 and ctxB4 for detection of Haitian and classical types of ctxB allele. The location of CTX ETΦ in the chromosome was determined by PCR described by Mohapatra et al. Oligonucleotide primer sequences were as published ,,, and purchased from GCC, New Delhi. Amplified products were separated on an agarose gel, stained with ethidium bromide and visualised in Gel-Doc-XR (Bio-Rad, USA).
The PCR was performed using forward primers of rstRET and reverse primer of ctxB to get amplicon of ~6.9 kb. This PCR product was then used as a template for nested PCR to amplify the ctxB gene of 449 bp lengths, comprising complete coding sequence. PCR fragments of the ctxB gene were purified using the QIAquick gel extraction kit (Qiagen), and both strands were sequenced at the Centre for Genome Application, New Delhi.
Assay for cholera toxin production
The ability of V. cholerae strains to produce CT in vitro was determined by GM1-ganglioside-dependent enzyme-linked immunosorbent assay as described by Svennerholm and Holmgren.
Probes and hybridisation
For the preparation of DNA blots, total cellular DNA was isolated from the overnight culture of V. cholerae strains as described by Ausubel et al. Five-microgram aliquots of DNA were digested with appropriate restriction enzymes (NEB, UK). The digested fragment was separated by electrophoresis on 0.8% agarose gels and transferred to hybond N + membrane (GE Healthcare, Buckinghamshire, UK) by Southern blotting. The ctxA gene probe was a 0.54 kb fragment amplified by PCR from V. cholerae strain 569B, and the RS1 probe was a 0.9 kb fragment amplified by PCR from the same strain. The rRNA gene probe was a 7.5 kb Bam HI fragment of pKK3535. The plasmid is a pBR322-derived plasmid containing an Escherichia coli rRNA operon consisting of one copy each of the genes coding for 5S rRNA, 16S rRNA, 23S rRNA and tRNAGlu. The probes were labelled by random priming  with random primer labelling kit (NEB, UK) and [α-32 P] deoxycytidine triphosphate (3000 Ci/mmol; BARC, Bombay, India). The blot was hybridised with the labelled probes, and autoradiograph was developed in Phosphor-Imager (Fuji Photo Film Co. Ltd., Tokyo, Japan).
Pulsed field gel electrophoresis
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  were digested with 50U 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.
| ~ Results|| |
Antibiotic susceptibility pattern
All the eight isolates of V. cholerae O1 biotype El Tor, except one strain (PU1024), showed resistance to co-trimoxazole, trimethoprim, streptomycin and nalidixic acid [Table 1]. One of the eight strains (PU382) showed additional resistant to chloramphenicol and norfloxacin while another strain (PU1023) showed resistance to cefotaxime, ciprofloxacin, furazolidone and neomycin. Strain PU1 showed additional resistance to norfloxacin, however, strain PU1024 showed resistance to co-trimoxazole, furazolidone, nalidixic acid and trimethoprim, respectively. All the strains gave positive results by PCR and amplified a portion of 626 bp of sullI, 278 bp of dfrA1, 515 bp of strB genes and 1035 bp of int SXT. However, one strain (PU1024) was negative for sullI and strB genes [Table 1].
Virulence pattern and nucleotide sequencing
Analysis of virulence and regulatory genes by hexaplex PCR assay showed that all the strains of V. cholerae harboured the ctxA, zot, ace, El Tor tcpA, ompU and toxR genes. All these strains also gave positive results for tcpI, acfB, hlyA rtx and rstC genes in the single PCR assay. This observation thus indicates that the tcpA regulatory genes, accessory colonisation factor, haemolysin, RTX and RS1 element are present in all of the isolates. A DMAMA-PCR yielded classical specific ctxB allele [Figure 1]a and [Figure 1]b which was confirmed by DNA sequencing of the ctxB amplicon. The alignment of deduced amino acid sequences was made with the ctxB sequences of reference El Tor strain N16961 and classical strain 569B. The deduced amino acid sequences of seven selected strains showed complete identity with classical reference strains possessing histidine at 39 positions (tyrosine in El Tor) and threonine at 68 positions (isoleucine in El Tor). This finding thus confirms the result obtained with DMAMA-PCR [Figure 2]. The ctxB nucleotide sequences obtained from V. cholerae O1 biotype El Tor strains were deposited in the NCBI GenBank under accession numbers HQ699885 to HQ699891. Determination of the location of CTX ETΦ in the chromosome showed that CTX ETΦ is present on the large chromosome [Figure 3]. All these strains also carried the rstRET [Figure 4] and produced CT.
|Figure 1: Ethidium bromide-stained agarose gel of double mismatch amplification mutation assay-polymerase chain reaction products obtained with Vibrio cholerae O1 El Tor strains isolated from Puri, India, using (a) Rv-cla and F4 primer (cla-ctxB) and (b) Rv-cla and F3 primer (Haiti-ctxB). Lane 1, PU1; Lane 2, PU302; Lane 3, PU375; Lane 4, PU382; Lane 5, PU389; Lane 6, PU622; Lane 7, PU1023; Lane 8, PU1024; Lane 9, Vibrio cholerae O1 classical strain 569B; Lane 10, Vibrio cholerae O1 El Tor strain N16961; Lane 11, Vibrio cholerae O1 strain DN5 (Haiti ctxB-positive strain); Lane 12, Vibrio cholerae O1 El Tor strain VC20; Lane 13, Vibrio cholerae O1 El Tor strain KO194; Lane M, 100 bp ladder (NEB, UK)|
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|Figure 2: Amino acid sequence alignment of ctxB gene fragment obtained from CTXETΦ of Vibrio cholerae O1 strains isolated from Puri, India. Dots represent identical amino acids|
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|Figure 3: Ethidium bromide-stained agarose gel of CII-polymerase chain reaction products obtained from Vibrio cholerae O1 strains isolated from Puri, India. Lane M, 1 kb DNA ladder (NEB); Lane 1, Vibrio cholerae O1 PU1; Lane 2, Vibrio cholerae O1 PU302, Lane 3, Vibrio cholerae O1 PU375; Lane 4, Vibrio cholerae O1 PU382; Lane 5, Vibrio cholerae O1 PU389; Lane 6, Vibrio cholerae O1 PU622; Lane 7, Vibrio cholerae O1 PU1023; Lane 8, Vibrio cholerae O1 PU1024; Lane 9, Vibrio cholerae O1 El Tor strain N16961; Lane 10, Vibrio cholerae O1 classical strain 569B|
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|Figure 4: Ethidium bromide-stained agarose gel of polymerase chain reaction products obtained with Vibrio cholerae O1 strains isolated from Puri using (a) rstRET and (b) rstRcla primers. Lane M, 100 bp DNA ladder; Lane 1, PU1; Lane 2, PU302; Lane 3, PU375; Lane 4, PU382; Lane 5, PU389; Lane 6, PU622; Lane 7, PU1023; Lane 8, PU1024; Lane 9, N16961; Lane 10, 569B and (c) rstRcal primers. Lane M, 100bp DNA ladder (NEB); Lane 1, PU1; Lane 2, PU302; Lane 3, PU375; Lane 4, PU382; Lane 5, PU389; Lane 6, PU622; Lane 7, PU1023; Lane 8, PU1024; Lane 9, Vibrio cholerae O1 El Tor strain N16961; Lane 10, Vibrio cholerae O1 classical strain 569B; Lane 11, Vibrio cholerae O139 strain AS207; Lane 12, Vibrio cholerae O139 strain MO10|
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Restriction fragment length polymorphism (RFLP) pattern of the CTX element was studied in the V. cholerae strains. The Hind III-digested genomic DNA hybridisation with ctxA probe gave a single band of size 22 kb in seven strains (PU1, PU375, PU382, PU398, PU622, PU1023 and PU1024) and a band of 23 kb in one strain (PU302). This result indicates that the CTX element is located on a single site in the chromosome as Hind III does not have any recognition site within the CTX element. The number of copies and arrangement of the CTX genetic element were investigated using the same probe and other restriction enzymes that cut within CTX, but not in ctxA. RFLP pattern generated with Bgl I, Bgl II and Pst I showed a single band of 9.5 kb, 7.5 kb and 6.0 kb, respectively, indicating the presence of a single copy of the CTX element. Bgl I and Hind III combined digestion produced a band of size 4.2 kb in all strains when hybridised with ctxA probe [Table 2].
|Table 2: Ribotypes, pulsotype, CTX patterns of Vibrio cholerae O1 strains isolated from Puri carrying classical cholera toxin subunit B and El Tor rstR|
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The CTX genetic element has two regions comprising 4.5 kb core region included ctxAB and a 2.4 kb region termed RS2. The hybridisation with Bgl I digested genomic DNA with RS probe gave a single band of 7.5 kb in seven isolates and 9.4 kb in one (PU375) and a single band of 22 kb in all isolates, except one strain PU302, with Pst I. This finding thus suggests that all strains carried at least one copy of the RS element. This finding was confirmed by digestion with Hind III, which also produced a single band in all strains. Combined digestion with Hind III and Bgl I and probing with RS1 also showed a single band of 7.5 kb size, except for strain PU375, which showed a band of size 23 kb. Digestion of DNA with Bgl II, which cuts within the RS1 region, showed three bands of sizes 2.7, 7.5 and 12 kb in all isolates. However, strain PU375 showed two bands of sizes 2.7 kb and 7.5 kb [Table 2]. The 2.7 kb band indicates the presence of tandemly repeated RS elements in the upstream region of the CTX element. The presence of a larger band in two strains of size 10.5 kb may be due to partial digestion of DNA with Bgl I (data not shown). The organisation of the CTX elements in the V. cholerae strains derived from results of the hybridisation obtained with ctxA and RS probes indicate that CTX genetic element is flanked by RS1, which is related to RS2 but lack rstC. Moreover, CTX organization of V. cholerae O1 strain 569B shown in [Table 1] used for comparison in this study was reported earlier.
The results of nucleotide sequencing of ctxB clearly indicated the presence of a variant of El Tor strains carrying classical ctxB in Puri prompted to look for relatedness among the V. cholerae O1 strains. RFLP in the conserved rRNA region was used to study genomic profile and relatedness among them. Bgl I is the most discriminatory restriction enzyme used for ribotyping of V. cholerae. This study was designed to ascertain whether the strains isolated in 2003 and 2005 from Puri represented an identical clone of V. cholerae. Analysis of rRNA genes with Bgl I produced two ribotype patterns among Puri strains showing 8–10 bands between 2 and 23 kb sizes [Figure 5]. Five strains of which three strains PU302, PU375, PU382 isolated in 2003 and two strains PU1023, PU1024 isolated in 2005 showed identical ribotype profile that lacked the 23 kb band, designated B-III. However, the remaining three strains of which two strains PU389, PU622 isolated in 2003 and one strains PU1 isolated in 2005 showed a distinct but identical ribotype profile that lack 10 kb band, designated ribotype B-IV. This observation thus indicates the existent of two ribotypes that caused cholera outbreaks in 2003 and 2005, respectively.
|Figure 5: Ribotype patterns of Vibrio cholerae O1 strains carrying classical ctxB and El Tor rstR isolated from Puri, India. Lane M, 1 kb DNA EcoR I-Hind III digested DNA Marker; Lane 1, Vibrio cholerae O1 El Tor strain VC20 (B-II); Lane 2, Vibrio cholerae O1 El Tor strains from Kottayam (B-III); Lanes 3–10, Vibrio cholerae O1 strains isolated from Odisha; PU1 (B-IV), PU302 (B-III), PU375 (B-III), PU382 (B-III), PU389 (B-IV), PU622 (B-IV), PU1023 (B-III) and PU1024 (B-III)|
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Pulsed-field gel electrophoresis analysis
Not I digested PFGE pattern obtained with V. cholerae strains were analysed and compared using BioNumerics software (Applied Maths, Belgium). Dice coefficient determined the similarity between strains and cluster analysis was carried out by un-weighted pair group method of the arithmetic mean. PFGE analysis of strains showed at least three groups, irrespective of the year of isolation. Like ribotypes, strains isolated in 2003 showed two closely related PFGE patterns with those of V. cholerae O1 strains isolated in 2005. One of these PFGE patterns was similar to that of El Tor strain isolated from Trivandrum in 2000 (strain KO194). However, one strain formed a distinct cluster indicating considerable variations in genomic content among them [Figure 6].
|Figure 6: Pulsed-field gel electrophoresis patterns of Not I digested product of agarose embedded genomic DNA of Vibrio cholerae O1 strains carrying classical ctxB and El Tor rstR and isolated from Puri, India along with dendrogram analysis using BioNumerics software (Applied Maths, Belgium). Pulsed-field gel electrophoresis analysis showed three distinct clusters for strains possessing classical ctxB (genotype-1), suggesting considerable relatedness in genomic content among them|
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| ~ Discussion|| |
Until the emergence of non-O1 V. cholerae designated O139 Bengal in 1992, O1 biotype El Tor was considered the only causative agent of epidemic cholera.,, The O1 strains examined in this study showed antibiotic susceptibility pattern similar to those Ogawa strains isolated from different parts of India., Although isolates from 2005 showed resistance to furazolidone, strains isolated in 2003 were sensitive to this antibiotic. This finding is similar to those who reported furazolidone resistance in V. cholerae O1 El Tor strains isolated during the cholera outbreak in May to June 1993 in different parts of Odisha. Existence of resistance to co-trimoxazole, trimethoprim and streptomycin suggested the possibility of the presence of int SXT that integrates into the chromosome by a site-specific mechanism independent of recA. The extensive use of co-trimoxazole, used in combination, may have provided additional selective pressure for the sporadic appearance of SXT-positive strains. The tetraplex PCR results confirmed the presence of int SXT and other antibiotic resistance genes commonly associated with the SXT element. However, one strain PU1024, although showed resistance to co-trimoxazole and trimethoprim amplified dfrA1 gene but failed to amplify sulII gene in tetraplex PCR indicating other mechanisms for the antibiotic resistance. This observation is similar to those who also reported resistance to antibiotics and presence of int SXT and resistance genes in V. cholerae strains.,
The result of the PCR assay, southern hybridisation and nucleotide sequencing showed that all isolates possess virulence and regulatory genes, rstRET and CTX element. Results of nucleotide sequencing of ctxB gene showing the presence of classical ctxB allele among O1 strains indicate the existence of variant of O1 strains before 2007 in Puri. Several workers also reported the presence of classical ctxB in V. cholerae isolated from India and elsewhere.,,,,, Recently, newer clones of V. cholerae possessing Haitian ctxB have been reported causing epidemic in Haiti was isolated in Kolkata, India, However, this Haitian ctxB was first reported in an isolate from Odisha. This finding is similar to those who also reported that V. cholerae O1 strains possessed CTX ETΦ located on the large chromosome, TCP pathogenicity Island and the RTX genetic cluster and produced CT.,,, These characteristics suggests that these isolates had potential to cause cholera. The number and arrangement of the CTX genetic element is known to vary in toxigenic strains of V. cholerae.,, The differences in CTX prophage arrangement is known to affect the regulation and production of CT. However, in this study, we found the presence of a single copy of CTX prophage with two tandemly arranged RS elements upstream in all the strains. This organisation of CTX element is similar to V. cholerae strains isolated in Kerala. In contrast, these isolates differ in CTX organisation from other V. cholerae strains isolated elsewhere in India prior to 2000. and other part of the world.,
Ribotyping is a useful tool to monitor and analyse the spread of the pathogenic strains in disease surveillance.,,,, Based on results of ribotyping, it can be interpreted that the V. cholerae O1 El Tor might have evolved from parental strains or clones., Serotype conversion occurs as a result of selection due to the pressure of lytic phages and immune response during cholera infection., The mutation rate in this biotype is high enough to explain worldwide differences. Seasonal epidemics of cholera are often known to associate with the emergence of new epidemic clones replacing existing clones of V. cholerae.,,,, The ribotype designation (Pattern I–IV) followed those of Dalsgaard et al. and other investigators.,, From this study, it is clear that V. cholerae O1 strains belonging to ribotypes B-III and B-IV which appeared in 2003 continue to exist and cause cholera outbreaks in 2005 in Puri. The ribotype B-IV which was first reported from Trivandrum in 2000 was found in isolates from Puri. It seems ribotype IV may have originated from V. cholerae O1 El Tor Ogawa strains isolated as far back as 2000 in Trivandrum, Southern India. Similarly, ribotype B-III was reported from cholera outbreaks in different parts of India,, and two North Central Districts of Bangladesh  indicating that latter strains may have emerged from the prevalent Ogawa strains. Recently, V. cholerae O1 belonging to ribotype III and IV was reported from the water of the river Ganga in Varanasi, India. These observations thus suggests that these ribotypes may have present in the aquatic environment prior to causing cholera outbreaks.
Similar to ribotypes, PFGE analysis showed the existence of three pulsotypes among 2003 and 2005 isolates indicating heterogeneity among them. However, the strains of V. cholerae that was isolated in 2003 showed similar to closely related pulsotype(s) with those strains isolated in 2005 from Puri. This observation thus suggests that pulsotypes of V. cholerae that appeared in 2003 continue to cause infection in 2005 in Puri. These findings are similar to those who reported the existence of identical pulsotypes of variant or hybrid O1 causing an outbreak of cholera in India.,,
Overall, these data suggest classical ctxB-positive V. cholerae O1 El Tor strains that appeared in 2003 continue to cause infection in 2005 in Puri, India, and belong to identical ribotype(s) and/or pulsotype(s). There is continuous need to monitor the emergence of variant of El Tor because it will improve our understanding of the evolution of new clones of variant of V. cholerae.
| ~ Acknowledgements|| |
The study, in part, was supported by fund contributed by the Department of Biotechnology, New Delhi to Institute of Life Sciences, Bhubaneswar, and Indian Council of Medical Research, New Delhi to Regional Medical Research Centre, Bhubaneswar. MMD and TB are grateful to Department of Biotechnology, New Delhi, for providing Senior Research Fellowships. We thank Sarbari Acharya for providing technical help during the initial study. The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The part of this work was presented in the 9th Convention of Biotech Research Society (India) at Patiala, India, 21–23 November 2012.
Financial support and sponsorship
The study was supported by the Department of Biotechnology, New Delhi, and the Indian Council of Medical Research, New Delhi.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Safa A, Nair GB, Kong RY. Evolution of new variants of Vibrio cholerae
O1. Trends Microbiol 2010;18:46-54.
Colwell RR, Huq A, Chowdhury MA, Brayton PR, Xu B. Serogroup conversion of Vibrio cholerae
. Can J Microbiol 1995;41:946-50.
Singh DV, Matte MH, Matte GR, Jiang S, Sabeena F, Shukla BN, et al.
Molecular analysis of Vibrio cholerae
O1, O139, non-O1, and non-O139 strains: Clonal relationships between clinical and environmental isolates. Appl Environ Microbiol 2001;67:910-21.
Olsvik O, Wahlberg J, Petterson B, Uhlen M, Popovic T, Wachsmuth IK, et al
. Use of automated sequencing of polymerase chain reaction-generated amplicons to identify three types of cholera toxin subunit B in Vibrio cholerae
O1 strains. J Clin Microbiol 1993;31:22-5.
Popovic T, Fields PI, Olsvik O. Detection of cholera toxin genes. In: Wachsmuth IK, Blake PA, Olsvik O, editors. Vibrio cholerae
and Cholera: Molecular to Global Perspectives. Washington, DC: ASM Press; 1994. p. 41-52.
Nair GB, Faruque SM, Bhuiyan NA, Kamruzzaman M, Siddique AK, Sack DA. New variants of Vibrio cholerae
O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh. J Clin Microbiol 2002;40:3296-9.
Raychoudhuri A, Patra T, Ghosh K, Ramamurthy T, Nandy RK, Takeda Y, et al.
Classical ctxB in Vibrio cholerae
O1, Kolkata, India. Emerg Infect Dis 2009;15:131-2.
Naha A, Pazhani GP, Ganguly M, Ghosh S, Ramamurthy T, Nandy RK, et al.
Development and evaluation of a PCR assay for tracking the emergence and dissemination of Haitian variant ctxB
in Vibrio cholerae
O1 strains isolated from Kolkata, India. J Clin Microbiol 2012;50:1733-6.
Nguyen BM, Lee JH, Cuong NT, Choi SY, Hien NT, Anh DD, et al.
Cholera outbreaks caused by an altered Vibrio cholerae
O1 El Tor biotype strain producing classical cholera toxin B in Vietnam in 2007 to 2008. J Clin Microbiol 2009;47:1568-71.
Safa A, Sultana J, Dac Cam P, Mwansa JC, Kong RY. Vibrio cholerae
O1 hybrid El Tor strains, Asia and Africa. Emerg Infect Dis 2008;14:987-8.
Goel AK, Jain M, Kumar P, Bhadauria S, Kmboj DV, Singh L. A new variant of Vibrio cholerae
O1 El tor causing cholera in India. J Infect 2008;57:280-1.
Hasan NA, Choi SY, Eppinger M, Clark PW, Chen A, Alam M, et al.
Genomic diversity of 2010 Haitian cholera outbreak strains. Proc Natl Acad Sci U S A 2012;109:E2010-7.
Pal BB, Khuntia HK, Samal SK, Kar SK, Patnaik B. Epidemics of severe cholera caused by El Tor Vibrio cholerae
O1 Ogawa possessing the ctxB
gene of the classical biotype in Orissa, India. Int J Infect Dis 2010;14:e384-9.
Kumar P, Thomas S. Classical ctxB
gene in Vibrio cholerae
O1 and O56 serogroups from Kerala, South India. J Med Microbiol 2011;60(Pt 4):559-60.
Khuntia HK, Pal BB, Samal SK, Kar SK. Rapid spread of Vibrio cholerae
O1 El Tor variant in Odisha, Eastern India, in 2008 and 2009. J Clin Microbiol 2013;51:1909-12.
Popovic T, Bopp C, Olsvik O, Wachsmuth K. Epidemiologic application of a standardized ribotype scheme for Vibrio cholerae
O1. J Clin Microbiol 1993;31:2474-82.
Rivera IG, Chowdhury MA, Huq A, Jacobs D, Martins MT, Colwell RR. Enterobacterial repetitive intergenic consensus sequence and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae
O1, O139 and non-O1 strains. Appl Environ Microbiol 1995;61:2898-904.
Jiang SC, Louis V, Choopun N, Sharma A, Huq A, Colwell RR. Genetic diversity of Vibrio cholerae
in Chesapeake Bay determined by amplified fragment length polymorphism fingerprinting. Appl Environ Microbiol 2000;66:140-7.
Cameron DN, Khambaty FM, Wachsmuth IK, Tauxe RV, Barrett TJ. Molecular characterization of Vibrio cholerae
O1 strains by pulsed-field gel electrophoresis. J Clin Microbiol 1994;32:1685-90.
Kotetishvili M, Stine OC, Chen Y, Kreger A, Sulakvelidze A, Sozhamannan S, et al
. Multilocus sequence typing has better discriminatory ability for typing Vibrio cholerae
than does pulsed-field gel electrophoresis and provides a measure of phylogenetic relatedness. J Clin Microbiol 2003;41:2191-6.
World Health Organization. Manual for Laboratory Investigations of Acute Enteric Infections, Programme for Control of Diarrhoeal Diseases. Genva, Switzerland: World Health Organization; 1987.
Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.
CLSI. Performance Standard for Antimicrobial Susceptibility Testing. 20th
Information Supplement. Vol. 30. Document M100-S20, Wayne, PA; 2010.
Ramachandran D, Bhanumathi R, Singh DV. Multiplex PCR for detection of antibiotic resistant genes and SXT element: Application to characterize Vibrio cholerae
. J Med Microbiol2007;5:3346-51.
Singh DV, Isac SR, Colwell RR. Development of a hexaplex PCR assay for rapid detection of virulence and regulatory genes in Vibrio cholerae
and Vibrio mimicus
. J Clin Microbiol 2002;40:4321-4.
Rivera IN, Chun J, Huq A, Sack RB, Colwell RR. Genotypes associated with virulence in environmental isolates of Vibrio cholerae
. Appl Environ Microbiol 2001;67:2421-9.
Faruque SM, Asadulghani, Saha MN, Alim AR, Albert MJ, Islam KM, et al.
Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae
for susceptibility to CTXPhi: Molecular basis for origination of new strains with epidemic potential. Infect Immun 1998;66:5819-25.
Bhattacharya T, Chatterji S, Maiti D, Bhadra RK, Takeda Y, Nair GB, et al
. Molecular analysis of the rstR
genes of the CTX prophages integrated in the small chromosomes of environmental Vibrio cholerae
non-O1, non-O139 strains. Environ Microbiol2006;8:526-34.
Chow KH, Ng TK, Yuen KY, Yam WC. Detection of RTX toxin gene in Vibrio cholerae
by PCR. J Clin Microbiol 2001;39:2594-7.
Mohapatra SS, Ramachandran D, Mantri CK, Singh DV. Characterization of the genetic background of Vibrio cholerae
O1 biotype El Tor serotype Inaba strains isolated in Trivandrum, Southern India. J Med Microbiol 2007;56(Pt 2):260-5.
Mantri CK, Mohapatra SS, Singh DV. Effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae
O139 strains. Infect Genet Evol 2010;10:925-30.
Svennerholm AM, Holmgren J. Identification of Escherichia coli
heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM1-ELISA). Curr Microbiol 1978;1:19-23.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al
. Short Protocols in Molecular Biology. 3rd
ed. New York: John Wiley and Sons; 1995.
Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-17.
Fields PI, Popovic T, Wachsmuth K, Olsvik O. Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae
O1 strains from the Latin American cholera epidemic. J Clin Microbiol 1992;30:2118-21.
Qu M, Xu J, Ding Y, Wang R, Liu P, Kan B, et al.
Molecular epidemiology of Vibrio cholerae
O139 in China: Polymorphism of ribotypes and CTX elements. J Clin Microbiol 2003;41:2306-10.
Brosius J, Ullrich A, Raker MA, Gray A, Dull TJ, Gutell RR, et al.
Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli
. Plasmid 1981;6:112-8.
Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13.
Cooper KL, Luey CK, Bird M, Terajima J, Nair GB, Kam KM, et al.
Development and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio cholerae
. Foodborne Pathog Dis 2006;3:51-8.
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al.
Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9.
Mekalanos JJ. Duplication and amplification of toxin genes in Vibrio cholerae
. Cell 1983;35:253-63.
Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 1996;272:1910-4.
Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada T, et al.
Emergence of novel strain of Vibrio cholerae
with epidemic potential in southern and eastern India. Lancet 1993;341:703-4.
Rivas M, Toma C, Miliwebsky E, Caffer MI, Galas M, Varela P, et al.
Cholera isolates in relation to the eighth pandemic. Lancet 1993;342:926-7.
Sharma C, Nair GB, Mukhopadhyay AK, Bhattacharya SK, Ghosh RK, Ghosh A. Molecular characterization of Vibrio cholerae
O1 biotype El Tor strains isolated between 1992 and 1995 in Calcutta, India: Evidence for the emergence of a new clone of the El Tor biotype. J Infect Dis 1997;175:1134-41.
Chaicumpa W, Srimanote P, Sakolvaree Y, Kalampaheti T, Chongsa-Nguan M, Tapchaisri P, et al.
Rapid diagnosis of cholera caused by Vibrio cholerae
O139. J Clin Microbiol 1998;36:3595-600.
Garg P, Nandy RK, Chaudhury P, Chowdhury NR, De K, Ramamurthy T, et al.
Emergence of Vibrio cholerae
O1 biotype El Tor serotype Inaba from the prevailing O1 Ogawa serotype strains in India. J Clin Microbiol 2000;38:4249-53.
Niyogi SK, Mondal S, Sarkar BL, Garg S, Banerjee D, Dey GN. Outbreak of cholera due to Vibrio cholerae
O1 in Orissa state. Indian J Med Res 1994;100:217-8.
Waldor MK, Tschäpe H, Mekalanos JJ. A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae
O139. J Bacteriol 1996;178:4157-65.
Kumar P, Wilson PA, Bhai R, Thomas S. Characterization of an SXT variant Vibrio cholerae
O1 Ogawa isolated from a patient in Trivandrum, India. FEMS Microbiol Lett 2010;303:132-6.
Taneja N, Mishra A, Sangar G, Singh G, Sharma M. Outbreaks caused by new variants of Vibrio cholerae
O1 El Tor, India. Emerg Infect Dis 2009;15:352-4.
Bhadra RK, Roychoudhury S, Banerjee RK, Kar S, Majumdar R, Sengupta S, et al.
Cholera toxin (CTX) genetic element in Vibrio cholerae
O139. Microbiology 1995;141(Pt 8):1977-83.
Dalsgaard A, Mortensen HF, Mølbak K, Dias F, Serichantalergs O, Echeverria P. Molecular characterization of Vibrio cholerae
O1 strains isolated during cholera outbreaks in Guinea-Bissau. J Clin Microbiol 1996;34:1189-92.
Faruque SM, Abdul Alim AR, Roy SK, Khan F, Nair GB, Sack RB, et al.
Molecular analysis of rRNA and cholera toxin genes carried by the new epidemic strain of toxigenic Vibrio cholerae
O139 synonym Bengal. J Clin Microbiol 1994;32:1050-3.
Mukhopadhyay AK, Garg S, Mitra R, Basu A, Rajendran K, Dutta D, et al.
Temporal shifts in traits of Vibrio cholerae
strains isolated from hospitalized patients in Calcutta: A 3-year (1993 to 1995) analysis. J Clin Microbiol 1996;34:2537-43.
Faruque SM, Albert MJ, Mekalanos JJ. Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae
. Microbiol Mol Biol Rev 1998;62:1301-14.
Faruque SM, Ahmed KM, Abdul Alim AR, Qadri F, Siddique AK, Albert MJ. Emergence of a new clone of toxigenic Vibrio cholerae
O1 biotype El Tor displacing V. cholerae
O139 Bengal in Bangladesh. J Clin Microbiol 1997;35:624-30.
Mohaptra SS, Mantri CK, Bhotra T, Singh DV. Characteristics of Vibrio cholerae
O1 isolated from water of the River Ganga, Varanasi, India. Indian J Med Microbiol 2015;33:507-15.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]