Emergence of Tetracycline Resistant Vibrio cholerae O1 Biotype El Tor Serotype Ogawa with Classical ctxB Gene from a Cholera Outbreak in Odisha, Eastern India

In September 2010, a cholera outbreak was reported from Odisha, Eastern India. V. cholerae isolated from the clinical samples were biochemically and serologically confirmed as serogroup O1, biotype El Tor, and serotype Ogawa. Multiplex PCR screening revealed the presence of various genes, namely, ompW, ctxB, zot, rfbO1, tcp, ace, hlyA, ompU, rtx, and toxR, in all of the isolates. The isolates were resistant to co-trimoxazole, nalidixic acid, polymyxin B, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). Minimum inhibitory concentration of tetracycline decreased in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting the involvement of efflux pumps. PCR analysis confirmed the presence of class I integrons as well as SXT elements harbouring antibiotic resistance genes in all isolates. Sequencing revealed the presence of ctxB gene of classical biotype in all the isolates. The isolates harboured an RS1-CTX prophage array with El Tor type rstR and classical ctxB on the large chromosome. The study indicated that the V. cholerae El Tor variants are evolving in the area with better antibiotic resistance and virulence potential.


Introduction
Cholera is still a major health problem of global importance as evidenced by the recent outbreaks in Haiti and Zimbabwe. In 2013, a total of 47 countries from all continents reported 129 064 cases of cholera including 2102 deaths with a case fatality rate of 1.63% [1]. Toxigenic Vibrio cholerae, the causative agent of cholera, has exhibited several changing patterns in its biotype as well as in drug resistance for its better survival and infection [2,3]. Classical biotype V. cholerae strains after causing earlier six cholera pandemics were replaced by El Tor biotype strains in the seventh pandemic. The variant of V. cholerae El Tor strain possessing cholera toxin of classical biotype appeared after the emergence of O139 strain and has now become prevalent worldwide [4]. Antimicrobial susceptibility pattern has continuously changed for V. cholerae [2]. Tetracycline is an important broad spectrum antibiotic used for the prophylaxis and treatment of variety of bacterial infections including listed biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis [5]. It has been a preferred choice for control of cholera as recommended by WHO [6]. Earlier, we reported a novel mutation in ctxB gene of outbreak isolates in 2007 from Rayagada (19.09 ∘ N 83.27 ∘ E), Odisha, and later on the same allelic variation was observed in the Haitian outbreak isolates [7,8]. These isolates were multidrug resistant but sensitive to tetracycline [8]. In September-October, 2010, another cholera outbreak was observed in the same area of Rayagada, Odisha. In this communication, we report the emergence of tetracycline resistant V. cholerae from this outbreak of Eastern India.

Isolation and Identification of V. cholerae.
A total of 27 V. cholerae isolates were recovered from randomly selected patients during the cholera outbreak in the month of September, 2010, from Rayagada district of Odisha (19.09 ∘ N 83.27 ∘ E). Samples were collected using sterile rectal swabs from the patients and processed as described earlier [9]. V. cholerae isolates were identified and confirmed by using standard biochemical methods. Serological identification of the isolates was done by slide agglutination using commercially available polyvalent antiserum against V. cholerae O1 (Difco, USA). Voges Proskauer (VP) test, sheep erythrocyte haemolysis, and polymyxin B susceptibility tests were performed for determination of biotype of isolates. Other bacterial reference strains used in the study were V. cholerae O1 El Tor (ATCC 14033), V. cholerae O1 classical (ATCC 11623), and V. cholerae O139 (ATCC 51394).

Mobile Genetic Elements and Their Gene Cassettes.
The presence of class 1 integron and SXT constins was detected by PCR as described by Hochhut et al. [13]. The presence of gene cassettes for aadA2 (encoding resistance for streptomycin and spectinomycin), strA (encoding resistance for streptomycin), blaP1 (encoding resistance for -lactams), floR (encoding resistance for chloramphenicol), and dfrA1 (encoding resistance for trimethoprim) was determined by PCR as described earlier [14,15].

2.5.
Sequencing of the ctxB Gene. The ctxB gene was amplified from the isolates using primers described previously [16]. Sequencing was carried out using the same PCR primers on a 96-capillary model 3730xl system using the Big Dye Terminator kit from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The sequences were edited with SeqED program (Applied Biosystems) and were aligned with sequences of ctxB gene of reference strains of V. cholerae O1 El Tor, classical, and O139. The deduced amino acid sequences of the ctxB gene from all the strains were aligned using CLUSTAL W.
2.6. Analysis of RS1 and CTX Prophage Array. Arrangement of RS1 and CTX phage was determined by PCR. Presence of the RS1 element was confirmed by PCR using the rstC specific primers as described elsewhere [17]. The primers for El Tor , Classical , and Calcutta were used for rstR typing of RS element and for differentiation of classical and El Tor biotype of V. cholerae [18]. The location of an RS1-CTX prophage array on the large chromosome, insertion of the CTX prophage on the small chromosome, and tandem repeats of RS1 and CTX prophage on chromosome were determined by PCR as described earlier [17].

Identification of Outbreak Strains.
A total of 27 clinical strains of V. cholerae were isolated from the affected patients during cholera outbreak in Odisha, Eastern India. On the basis of biochemical tests, all the isolates were found to belong to El Tor biotype as the isolates were VP test positive, hemolytic on sheep blood agar, and resistant to polymyxin B.

Antibiotic Drug Resistance Pattern.
The antimicrobial disc diffusion test revealed that all the isolates were resistant to co-trimoxazole, nalidixic acid, polymyxin B, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and vibriostatic agent 2,4-diamino-6,7diisopropylpteridine (O/129). However, the isolates were sensitive to ampicillin, chloramphenicol, erythromycin, ciprofloxacin, and rifampicin. The MIC for selected antibiotics is given in Table 2. The presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of major facilitator superfamily (MFS) proteins in broth at subinhibitory concentration (0.5 g/mL), reduced MIC for tetracycline to eightfold (16 to 2 g/mL) and the isolates became susceptible. CCCP also reduced the MIC for trimethoprim from 1024 g/mL to 256 g/mL, whereas MIC for nalidixic acid, streptomycin, sulfamethoxazole, and ampicillin remained unaffected ( Table 2).

Prevalence of Multidrug Resistant
Elements. PCR analysis revealed the presence of class I integron as well as SXT element harbouring antibiotic resistance traits against multiple drugs in all the isolates. The isolates were positive for aadA2 (streptomycin and spectinomycin) and dfrA1 (trimethoprim)  gene cassettes but negative for blaP1 ( -lactam antibiotics) cassettes [14]. The strains were also positive for strA carried on SXT constin that confers resistance for streptomycin. However, all the isolates were negative for floR (chloramphenicol) genes.

Sequence Analysis of ctxB.
The deduced amino acid sequence analysis of CT-B from the representative isolates varied from CT-B of reference El Tor strain N16961 and O139 strain at position 39 (histidine in place of tyrosine) and position 68 (threonine in place of isoleucine) but was similar to CT-B subunit of reference classical strains 569B ( Figure 1). However, no mutation was observed at position 20, which was found in the V. cholerae isolates from the previous outbreak in 2007 (Figure 1).

Phage
Array. The cholera toxin gene ctxAB lies on the single stranded DNA of CTX phage which is a filamentous phage [20]. There is specific site for integration of CTX phage DNA into the V. cholerae chromosome. Toxigenic strains contain a genetic element RS1 which is related to CTX phage. RS1 element is found adjacent (5 or 3 ) to CTX prophage. Several types of CTX phage/RS1 arrays have been found in the epidemic V. cholerae strains [21]. In this study, rstC specific primer set rstCF/rstCR amplified the DNA, indicating the presence of the RS1 element in the chromosome. A PCR product was amplified with rstCF/cepR showing the existence of RS1-CTX prophage array. In contrast, ctxBF and rstCR primer pair did not give any amplification, indicating the absence of CTX prophage-RS1 array. No DNA fragment was amplified using the primer pairs of ctxBF/cepR and rstCF4/rstCR4, indicating the absence of tandem repeats of CTX prophage and RS1 element on the genome, respectively. Location of RS1-CTX prophage array on the large chromosome was confirmed with the PCR amplified products using Ch1F/rstAR and ctxBF/Ch1R primers. The isolates yielded PCR products using Ch2F/Ch2R primers indicating the absence of the CTX prophage or RS1 element on small chromosome. The RS1-CTX prophage array has been shown in Figure 2.

Discussion
V. cholerae O1 El Tor strains are supposed to have better adaptability in the environment and colonize more efficiently in the intestinal lumen as compared to the classical one [22]. However, cholera toxin of classical type causes greater fluid accumulation in the intestinal lumen as compared to toxin of El Tor type [23]. Therefore, hybrid biotype property of V. cholerae enhances the infection potential and adaptability in the environment showing their epidemiological successfulness. Outbreaks of such variant type strains are associated with more fluid loss and increased case fatality rate [24]. It has now become a global threat as most of the outbreak strains are associated with variant El Tor strains in India and other countries [3,7,16]. All isolates from this outbreak were identified as V. cholerae O1 biotype El Tor serotype Ogawa. The isolates harboured various toxigenic and pathogenic genes ( Table 1). The presence of ompW, ctxB, and rfbO1 genes confirmed the V. cholerae species, its toxigenicity, and group 1 antigen of the "O" side chain of the LPS (serogroup), respectively. Presence of rtxA or rtxC genes determines the biotypes of V. cholerae O1 [25,26]. All isolates in this study belonged to the El Tor biotype on the basis of the repeat in toxin gene (rtxC).
Sequencing of ctxB gene revealed that all V. cholerae O1 El Tor isolates in this study had ctxB sequence of the classical biotype. Classical biotype strains were replaced by the El Tor biotype in the seventh and current pandemic of cholera. Both biotypes of V. cholerae O1 are closely related in their Oantigen biosynthetic genes. However, the genomic structure of the CTXΦ, in which the cholera toxin genes are contained, differs between the classical and El Tor biotypes [20,27]. On the basis of genetic structures of the CTX prophage and RS1 element on each chromosome, atypical El tor strains have  been classified into two different groups [17,28]. Group 1 strains harbor a tandem repeat of the classical CTX prophage on the small chromosome and Group II strains contain the RS1 and CTX prophage with El Tor type rstR and classical ctxB on the large chromosome [28,29]. All the strains used in this study belonged to Group II and contained the RS1 and CTX prophage with El Tor type rstR and classical ctxB on the large chromosome.
Odisha is situated at the coast of Bay of Bengal and in general outbreaks in the state are linked to flooding and cyclones [10,30]. The variant El Tor strains are circulating in Odisha since 2000 and appeared in Rayagada district in 2002 but the first outbreak due to this strain was recorded in 2007 [10,31]. To the best of our knowledge, this was the first time when tetracycline resistant clone of variant El Tor strain caused an outbreak in Odisha. Recently, tetracycline resistant V. cholerae strains have been reported from several other parts of India [32,33]. Tetracycline has been a drug of choice for control of severe cases of cholera [6]. Therefore, resistance for tetracycline further curtails the list of cost effective first-line drugs for control of cholera.
All the Vibrio cholerae isolates in this study harboured class 1 integron and SXT constins. In this region, qacEΔ1 and sul1 genes provide resistance to ammonium compounds and sulphonamides, respectively. All the isolates were positive for aadA2 and dfrA1 genes, which confer resistance to streptomycin and trimethoprim, respectively. However, all the isolates were PCR negative for blaP1 involved in resistance for -lactam antibiotics ( Table 1). The SXT constin is a conjugative, self-transmissible, and integrative element that provides resistance to sulphamethoxazole, trimethoprim, chloramphenicol, and streptomycin. In this study, all isolates were susceptible to chloramphenicol and PCR negative for floR gene. In addition, the gene for streptomycin resistance (strA) was invariably present in all the isolates.
Integrons provide natural genetic engineering platforms that incorporate gene cassettes and convert them to functional genes by ensuring correct expression in bacteria. The present study showed that now V. cholerae outbreak strains carry the mobile antibiotic genetic elements and the horizontal transfer of these genes is the major cause of emergence of multidrug resistance strains. The commonly prescribed antibiotics in India for the treatment of diarrhea are fluoroquinolones (DNA damaging drugs) that induce horizontal transfer of SXT elements leading to inter-and intraspecies spread of multidrug resistance [34]. Therefore, use of such drugs should be closely monitored as most of the recent outbreak strains carry these elements [16,19,35].
MIC of tetracycline decreased eightfold in the presence of CCCP, indicating the role of efflux pumps in tetracycline resistance [36]. However, the MIC for nalidixic acid (32 g/mL) remained unaffected in the presence of CCCP indicating the involvement of some other mechanisms also for antibiotic resistance. Tetracycline resistance in Gram negative bacteria occurs due to the acquisition of genes encoding efflux pump and ribosomal protection proteins. Most of the Journal of Pathogens 5 efflux pumps belong to major facilitator superfamily (MFS), which export tetracycline from the cell and exchange a proton against the concentration gradient [37]. The isolates from this outbreak were PCR negative for the reported ribosomal protection protein Tet(M) in Vibrios (data not shown). Efflux mediated tetracycline resistance (tet genes) in Vibrios is mostly characterized in non-V. cholerae species [37]. Therefore, the detailed investigation of the mechanism is needed. In recent years, rise in antimicrobial resistance in clinical strains is a serious concern as it hampers outbreak control policies [10,19,35]. Thus, antimicrobial resistance can increase the outbreak size, duration, and case fatality rates. Continued monitoring of antimicrobial susceptibility as well as strain-tracking is important in adapting policies for cholera control at national and global levels.