Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001–2006

Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004–2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004–2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera.


INTRODUCTION
The aquatic bacterium Vibrio cholerae is the causative agent of cholera or cholera-like diarrhea in humans. Of the 206 serogroups identified in this species (Yamai et al., 1997), the serogroups O1 and O139 are responsible for global cholera epidemics. V. cholerae serogroup O1 is further divided into two biotypes, classical and El Tor and each has two distinct serotypes, Inaba and Ogawa. The classical biotype was associated with cholera in first six pandemics (Sack et al., 2004). The current 7th cholera pandemic is represented by V. cholerae O1 El Tor biotype, which became dominant from 1961 and gradually replaced the classical biotype from the global cholera scenario. V. cholerae O139 serogroup emerged in 1992 by replacing the El Tor biotype in the Indian subcontinent and spread to more than 14 countries in the following years (Nair et al., 1994a;Siddique et al., 1996;Ramamurthy et al., 2003). Emergence of V. cholerae O139 serogroup was thought to be the beginning of the 8th cholera pandemic considering the rapid spread of the pathogen (Nair et al., 1994b). However, after causing large cholera epidemics in 1993, the serogroup O139 disappeared abruptly from the endemic scenario ensuing resurgence of V. cholerae O1 El Tor biotype in cholera endemic regions (Sharma et al., 1997). Until late 1999, there has been periodic shift between El Tor and O139 in India and Bangladesh (Basu et al., 2000;Faruque et al., 2003a). In 2008, the incidence of V. cholerae O139 in China was 32% among cholera cases (WHO, 2009) and continued until 2012 (Zhang et al., 2014).
In V. cholerae O139, changes in the antimicrobial susceptibility patterns and arrangement of genetic elements, especially the organization of ribosomal RNA operons, location, and arrangement of cholera toxin prophages (CTX ) were reported during its emergence on several occasions (Sharma et al., 1997;Faruque et al., 2003a;Nandi et al., 2003;Chatterjee et al., 2007;Ghosh et al., 2008). Initial genetic analysis showed that emergence of V. cholerae O139 may be due to the insertion of a novel 35-kb wbf gene that encodes O139-somatic (O) antigen in a V. cholerae serogroup O22 strain or due to the loss of a 22-kb wbe region in a V. cholerae O1 that encodes the O1 antigen (Yamasaki et al., 1999). The whole genome sequence analysis by Chun et al. (2009) confirmed the above finding, i.e., substitution of the gene cluster coding for the O139 antigen took place by horizontal gene transfer but not the deletion.
Based on the amino acid changes, the B-subunits of CT have been designated into several CT-genotypes or ctxB alleles (Safa et al., 2008;Raychoudhuri et al., 2009). CT genotyping (ctxB allele) can be made using Mismatch amplification mutation assay (MAMA) PCR (Morita et al., 2008). CT genotype 1 is reported in strains of the classical biotype worldwide and in US Gulf Coast, genotype 2 is found in El Tor biotype strains from Australia, and genotype 3 is prevalent in El Tor biotype from the 7th pandemic and the Latin American epidemic strains (Olsvik et al., 1993). V. cholerae O1 El Tor isolates that produces classical CT is a newly emerged trait, which is said to be associated with the severity of the illness (Siddique et al., 2010) with a large number of cholera outbreaks Safa et al., 2008;Raychoudhuri et al., 2009). CT encoding genes of O1 and O139 serogroups is carried by a filamentous CTX , which is known to use the toxincoregulated pili (TCP) as its receptor (Waldor and Mekalanos, 1996). V. cholerae O139 harboring CTX class and CTX calc has been described based on the difference in the sequence of rstR that encodes for the repressor protein of the CTX (Faruque et al., 2003a;Bhattacharya et al., 2006;Raychoudhuri et al., 2010).
This study was undertaken to understand the phenotype and genetic changes of V. cholerae O139 isolated from sporadic hospitalized cholera cases in Delhi during [2001][2002][2003][2004][2005][2006]. The outcome of this study may be useful to comprehend the epidemiology of V. cholerae O139.

Bacterial Strains
V. cholerae O139 was isolated from cholera patients admitted at the Maharishi Valmiki Infectious Diseases Hospital, Delhi. Between 2001 and 2006, 42 isolates individually isolated strains were included in this study ( Table 1). V. cholerae O1 569B (classical biotype), N16961 (El Tor biotype), and SG-24 (serogroup O139) were used as reference strains. In the pulsedfield gel electrophoresis (PFGE), Salmonella enterica serotype Braenderup strain H9812 was used as the molecular size standard.

Bacteriology and Serotyping
V. cholerae isolates were grown on thiosulphate-citrate-bile salt-sucrose (TCBS) agar (Eiken, Tokyo, Japan) at 37 • C for 16-18 h. Typical sucrose fermenting yellow colonies was further streaked on Luria agar (LA, Difco, Detroit, MD, USA) and subsequently used in the rapid biochemical identification (Nair et al., 1987). Presumptively identified V. cholerae isolates were further confirmed by oxidase test and confirmed serologically by slide agglutination test using O1 and O139 monoclonal antibodies prepared at the National Institute of Cholera and Enteric Diseases, Kolkata, India (Garg et al., 1994;Ramamurthy et al., 1995).

Extraction of Chromosomal DNA
Modified method of Murray and Thompson (1980) was used for V. cholerae genomic DNA extraction.

Polymerase Chain Reaction (PCR) Assay
Multiplex PCRs were used for the detection of rfb genes encoding the somatic antigen of O139/O1, CT encoding gene (ctxA), and biotypes based on the allelic difference in the tcpA gene (Keasler and Hall, 1993;Hoshino et al., 1998). Simplex PCR assays with specific primers were made for the detection of rstR alleles (Bhattacharya et al., 2006). MAMA-PCR was made to detect the presence of ctxB alleles (CT genotypes) as described  previously (Morita et al., 2008). Location of CTX prophage in chromosome II was confirmed by PCR using published methods . To confirm the presence of integrative conjugative element (ICE) that carries the SXT element, two sets of primers were used in this study. Primers 10SF13 (5 ′ -TTGTGGTGGAAAGAGGGTG-3 ′ ), SXT-13 (5 ′ -CCAACAAAG AACAGTTTGACTC-3 ′ ), and ORF-16 (5 ′ -CATCTACCACTT CATAGGCAGG-3 ′ ), YND-2 (5 ′ -CAGCTTAACTCACCAAGG AC-3 ′ ) were designed using conserved right and left terminal ends of the ICE, respectively. In addition, floR, str, and dfr genes encoding chloramphenicol, streptomycin, co-trimoxazole resistance was identified using published methods (Hochhut et al., 2001). In these PCRs, V. cholerae 569B, N16961, and SG-24 were used as reference strains. PCR assays were performed using an automated thermocycler (Gene Amp PCR system 9700, Applied Biosystems, Foster City, CA).

DNA Sequencing
The 460 bp region of ctxB gene was amplified by PCR from eight representative isolates of V. cholerae O139 covering all the years (Olsvik et al., 1993). The amplified product was purified using a PCR purification kit (Qiagen, Hilden, Germany) and used directly as a template for nucleotide sequencing. Both the strands of DNA were sequenced with BigDye terminator cycle sequencing kit using an automated sequencer ABI 3700 (Applied Biosystems). The nucleotide and amino acid sequences were compared with the sequences available in the GenBank. The nucleotide sequence data generated with five representative isolates of V. cholerae O139 were submitted to the GenBank with accession numbers from GQ892075 to GQ892079.

Ribotyping
A 7.5-kb BamH1 (Fermentas, Waltham, MA, USA) fragment of plasmid pKK3535 containing the 16S and 23S rRNA genes of Escherichia coli was used as a rRNA probe (Brosius et al., 1981). Standard V. cholerae ribotyping was followed in this study (Faruque et al., 2000). Instead of radioisotope, we used chemiluminescent dye (Gene Images Alkaphos direct labeling and detection system, Amersham Biosciences, UK) in the DNA hybridization analysis.

Pulsed-Field Gel Electrophoresis (PFGE)
PFGE of V. cholerae O139 was performed as described previously for V. cholerae O1 (Cooper et al., 2006). PFGE profiles were analyzed using the BioNumerics version 4.0 software (Applied Maths, Sint Martens Latem, Belgium). The tagged image file formats were normalized by using the universal S. enterica serotype Braenderup (H9812) size standard on each gel against the reference in the database. In the dendrogram analysis, the PFGE profiles were matched using the Dice coefficient and unweighted pair group method using arithmetic averages (UPGMA). Clustering of PFGE profiles was made using 1.5% band position tolerance window and 1.5% optimization.

Identification
Conventional serology and multiplex PCRs employed in this study confirmed all the isolates as V. cholerae O139.

Analysis of Virulence Loci, ICE and Antimicrobial Resistance Encoding Genes
The O139 isolates uniformly harbored ctxA with an El Tor allele of tcpA. In the MAMA-PCR, all the isolates were identified as CT genotype 1. In addition, four isolates (37, 46, 103, and X) collected in 2001 exhibited CT genotype 3 ( Table 1). The amplified ctxB gene from eight isolates was directly sequenced. The deduced amino acid sequence analysis identified heterogeneity in the B subunit of CT. Some of the 2004 and 2005 isolates had aspartic acid (D), histidine (H), phenylalanine (F), and threonine (T) at positions 28, 39, 46, and 68, respectively in the CtxB, which is similar to the CT genotype 1 of the V. cholerae O1 classical 569B strain ( Table 2). However, the isolates representing 2001, 2004, and 2006 had amino acids alanine (A), H, F, T at positions 28, 39, 46, 68, respectively, which has been classified as CT genotype 4. This genotype was described in our previous report as genotype 5 with V. cholerae O139 isolates from Bangladesh (Bhuiyan et al., 2009). Subsequently, this was corrected in our publication in 2010 (Raychoudhuri et al., 2010). About 80% of the isolates possessed more than one allele of rstR, one being the El Tor type (rstR ET ) and the other with rstR calc type. Interestingly, three 2001 isolates (37, 46, and 103) carried all the three rstR alleles, i.e., rstR Cl , rstR ET , and rstR Calc . These isolates belonged to a new ribotype ( Table 1). ICE was present in all the V. cholerae O139 isolates as confirmed by two sets of primers. V. cholerae O139 isolated in 2001 that were resistant to chloramphenicol, streptomycin and co-trimoxazole respectively harbored floR, str, and dfr genes.

ctxA RFLP
Twenty four V. cholerae O139 isolated during 2004-2006 displayed two tandemly arranged copies of intact CTX prophages with cep, orfU, ace, zot, and ctxAB as a 23 Kb fragment ( Figure 1A). These CTX prophages were closely bordered with a 5 Kb truncated prophage (without ctxAB) as detected by the cep probe (Table 1, Figure 1A). Seven V. cholerae O139 isolated in 2001 had a single copy of CTX prophage as detected by 8 Kb ctx/cep probes (Table 1, Figure 1B). One isolate harbored two entire copies of CTX prophages as detected by ctx probe along with 3 truncated phages that were detected as three 5 Kb fragments by cep probe (Table 1, Figure 1C). Mapping could not be accomplished for 10 isolates with the applied strategy in this study.

Chromosomal Location of CTX Prophages
Three of the 2001 isolates (37, 46, and 103) carried CTX prophages on both the chromosomes, which were confirmed by PCR with specific primers for chromosome I and II of V. cholerae . In the rest of the V. cholerae O139 isolates, the CTX prophages remained in chromosome 1. To our knowledge, this is the first report indicating the presence of CTX prophages on chromosome II in V. cholerae O139.

Ribotyping
V. cholerae O139 isolates exhibited three different ribotypes (Table 1, Figure 2 1, Figure 2). These three isolates had an extra DNA band around the 2-Kb region (Figure 2). This could be the new ribotype BVIII of V. cholerae O139.

Pulsed-Field Gel Electrophoresis (PFGE)
Among the 9 2001 isolates, 8 different PFGE profiles were identified demonstrating the diversity of their genomes (Figure 3, cluster A). However, 3 isolates of 2001 belongs to ribotype BVIII were closely related in the PFGE. V. cholerae O139 isolated during 2004-2006 had similar PFGE profiles (Figure 3, cluster B), but diverged from the other isolates of 2001. A consistent correlation existed in both ribotyping and PFGE methods as most of the isolates having BII ribotype pattern were placed in clusters B. In addition, the dendrogram displayed subtypes among V. cholerae O139 isolates with the BII and BVIII ribotypes at about 97% similarity level (Figure 3).

DISCUSSION
One of the phenotypic markers used in the epidemiology of cholera is the antimicrobial susceptibility patterns. In this study, V. cholerae O139 isolates were resistant to ampicillin, furazolidone, and nalidixic acid, a trend observed in majority  (Dutta et al., 2006). The O139 isolates identified in 1992 were resistant to chloramphenicol, co-trimoxazole, and streptomycin . The reemerged V. cholerae O139 during 1996-1997 in India and Bangladesh showed susceptibility toward co-trimoxazole (Mitra et al., 1998;Faruque et al., 2003a).
In V. cholerae O1 and O139, mobile ICE that carried antimicrobial resistance genes in the variable region expressed resistance to chloramphenicol, co-trimoxazole, and streptomycin (Hochhut et al., 2001). In this study, ICE was detected in all the O139 isolates. However, only some of the 2001 isolates were resistant to chloramphenicol, streptomycin, and co-trimoxazole and harbored floR, str, and dfr. These resistance-encoding genes were not present in other isolates in the ICE variable region. Early studies conducted during the emergence of V. cholerae O139 in India showed a trend of resistance to neomycin . In this study, the O139 isolates were either susceptible or showed reduced susceptibility to neomycin. As seen in previous reports, all the V. cholerae O139 isolates remained susceptible to norfloxacin, tetracycline, and ciprofloxacin, which are used in the treatment of cholera (Basu et al., 2000).
The CT genotype of V. cholerae O1 El Tor isolates from many countries has changed from CT genotype 3 to 1 (Safa et al., 2008;Raychoudhuri et al., 2009) and such changes were detected in strains associated with large cholera outbreaks in India and Bangladesh (Kumar et al., 2009;Nguyen et al., 2009;Taneja et al., 2009). CT genotype 4 has closest homology to CT genotype 1 with a difference of only single nucleotide (nucleotide cytosine instead of adenine) at position 83 (Raychoudhuri et al., 2010). Overall, our finding matches with the observation made in V. cholerae O139 isolated during 1998, 2000, and 2002 from Bangladesh and Kolkata, respectively (Bhuiyan et al., 2009;Raychoudhuri et al., 2010). Compared to El Tor, the hybrid isolates with CT genotype 4 have caused larger cholera outbreaks with more severe clinical symptoms (Kumar et al., 2009;Nguyen et al., 2009;Taneja et al., 2009;Siddique et al., 2010).
Epidemiologically, the CTX appear to be very important as they show the genetic changes among V. cholerae O1/O139 that emerged during different periods (Faruque et al., 2000;Qu et al., 2003). In the ctxA RFLP analysis, three prophages were encountered in different years. The unusual genetic features of the three 2001 isolates of V. cholerae O139 includes identification of the new ribotype BVIII pattern, the presence of three rstR allele types, CTX prophages of the classical type, and integration of CTX prophage in both the chromosomes. Epidemiologically, the new ribotypes of V. cholerae O1/O139 has been identified along with changes in the CTX prophage or rstR allele (Faruque et al., 1997). Considering several genetic events in the past, it has been inferred that the V. cholerae O139 may have multiple origins with different progenitors (Faruque et al., 2003b;Garg et al., 2003;Qu et al., 2003).
Genesis of V. cholerae O1 El Tor from the classical biotype, the emergence of the serogroup O139, and existence of El Tor that produces classical CT suggests that the V. cholerae is in a continuous state of adaptability, resulting in generation of new serogroups and/or new variants of the same serogroup. Our results suggest that the genome of V. cholerae O139 is dynamic and has undergone several changes since its emergence in 1992. Continuous surveillance and proper monitoring of V. cholerae O139 are however needed to detect subtle genetic changes in the genomes and its implications in its epidemiology, pathogenesis and persistence. Future studies should focus on epigenetic studies to find answers to the question as to why the O139 serogroup has disappeared from cholera endemic regions despite several genetic changes.