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Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
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Short communication
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Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012
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P. Kumar a, D.K. Mishra a, D.G. Deshmukh b, M. Jain c, A.M. Zade b, K.V. Ingole b, A.K. Goel c, P.K. Yadava a,⇑ a
Jawaharlal Nehru University, New Delhi 110067, India SVN Govt. Medical College, Yavatmal, India c Defence Research and Development Establishment, Gwalior 474002, India b
a r t i c l e
i n f o
Article history: Received 21 July 2013 Received in revised form 17 March 2014 Accepted 20 March 2014 Available online xxxx Keywords: Cholera Outbreak Vibrio cholerae El Tor variant biotype ctxB7 MLST
a b s t r a c t Cholera has been a recurrent epidemic disease in human populations for the past 200 years. We present herein a comparative characterization of clinical Vibrio cholerae strains isolated from two consecutive cholera outbreaks in 2012 and associated environmental strains from western India. The clinical and toxigenic environmental isolates were identified as hybrid V. cholerae O1, serotype Ogawa, biotype El Tor carrying the variant ctxB7 allele. Partial sequences of SXT integrase from the isolates revealed 100% identity to ICEVchInd5 (Sevagram, India, 1994) and VC1786ICE (Haiti, 2013). The full clonal relationship of the strains established by RAPD, Box PCR, ERIC PCR and MLST (pyrH, recA and rpoA) analyses, and the short time between the two outbreaks, strongly supported that both outbreaks were due to a single strain. The study corroborated that faecal contamination of the potable water supply was the main reason for the first outbreak, which further spread to other areas and resulted in the second outbreak. The study concluded that the circulating El Tor variant strains of epidemic potential in the region can be a serious concern in the future. Ó 2014 Elsevier B.V. All rights reserved.
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
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1. Introduction
47
Cholera, an ancient waterborne disease, continues to be a devastating disease globally with a greater prevalence in developing nations. Vibrio cholerae is primarily an inhabitant of the aquatic environment, so water plays an important role in the transmission and epidemiology of cholera. The recent outbreak reports from India, Angola, Sudan, Zimbabwe, Nigeria and Haiti have revealed that the mortality resulting from the disease has far exceeded the World Health Organization (WHO) international target of 1% or less (Enserink, 2010). Several outbreaks and sporadic cases of cholera have been noted from time to time in India. V. cholerae O1 has two biotypes: classical (more toxigenic) and El Tor (more resistant to environmental factors). It has genetic plasticity and continues to evolve in virulence and drug resistance, thus ensuring its survival and persistence. This leads to the emergence of new hybrid or variant strains (e.g. El Tor strains have captured the toxin of the classical type), hampering disease control policies. El Tor variant strains have better colonization efficiency and are able to
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
⇑ Corresponding author. Address: Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. Tel.: +91 11 26704520; fax: +91 11 26742558. E-mail address: [email protected] (P.K. Yadava).
produce a significantly higher amount of cholera toxin (CT) in vivo or in virulence-induced conditions (Ghosh-Banerjee et al., 2010). Moreover, an El Tor variant strain emerged with a novel CT (ctxB7 allele), first reported in Orissa; later it rose in fame as Haitian ctxB (HCT) after causing a devastating epidemic in Haiti (Kumar et al., 2009; Naha et al., 2012; Kutar et al., 2013). Earlier, we noted a cholera outbreak in Yavatmal district of Maharashtra associated with HCT (Kumar et al., 2013). In the present study, we aimed to link the outbreak to equipment damage in the drinking water supply and contamination of sewer water, associated with back-to-back cholera outbreaks in the same district of Maharashtra, south-western India. We determined the molecular epidemiological traits and phylogenetic relationship of V. cholerae isolates from the two consecutive cholera outbreaks with environmental strains.
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2. Materials and methods
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2.1. Isolation and characterization of bacterial strains
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A total of 20 clinical V. cholerae isolates were recovered from randomly selected patients (unrelated individuals) during cholera outbreaks in the month of May, 2012 from Kalamb (10 isolates) and Yavatmal (10 stool samples), Maharashtra, India (see the
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http://dx.doi.org/10.1016/j.meegid.2014.03.020 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Kumar, P., et al. Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.03.020
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2.4. Genetic relatedness of the strains by DNA fingerprinting and multilocus sequence typing
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The phylogenetic relationship of the clinical and environmental isolates was analysed by enterobacterial repetitive intergenic consensus (ERIC) PCR, BOX-PCR and random amplified polymorphic DNA (RAPD) PCR (Kumar et al., 2009). The strains were further subjected to multilocus sequence typing based on pyrH, recA and rpoA genes using MEGA5 according to a previously described method (Thompson et al., 2011).
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3. Results and discussion
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3.1. Characterization of V. cholerae strains
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All the clinical and toxigenic environmental V. cholerae isolates were identified as V. cholerae O1 Ogawa. Biochemical and PCR assays confirmed the El Tor biotype. PCR analyses revealed the presence of other virulence-associated traits such as ctxAB, tcpA, Q5 zot, ace, toxR, rfbO1 and VPI genes (Table 1). The presence of toxigenic strains in environmental water is an indication of an impend-
zot+ zot+ zot+ zot+ zot+ tcp+, tcp+, tcp+, tcp+, tcp+, rfbO1+, rfbO1+, rfbO1+, rfbO1+, rfbO1+, + + + +
ctxAB+, ctxAB+, ctxAB+, ctxAB+, ctxAB+,
: negative, +: positive, +/ : weak positive, R: resistant, S: susceptible, C: classical, E: El Tor, VP: Voges Proskauer, PB: Polymyxin B; mPCR1 (multiplex PCR1), mPCR2 (multiplex PCR2) (Kumar et al., 2009); Clinical strain from Kalamb have been earlier characterized by Kumar et al. (2013).
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rtxC , ace+, hlyA , OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+
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+ + + +
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+ +/ +/ +/
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mPCR2
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OmpW+, OmpW+, OmpW+, OmpW+, OmpW+,
The presence of SXT constin was determined by PCR amplification using the primers SXT-F and SXT-R directed to the SXT element and int1-F and int1-B primers targeted for SXT integrase. The amplicon of SXT-F/SXT-R was purified and sequenced. The strains were screened for floR presence on SXT constin and the antimicrobial susceptibility test for chloramphenicol (Sjolund-Karlsson et al., 2011).
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C E E E E
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C E C C C
2.3. Analyses of SXT constin
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S R R R R
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1 1 10 10 5
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Genomic DNA was isolated from all the selected toxigenic isolates using a genomic DNA purification kit (MBI Fermentas, Vilnius, Lithuania) and screened for the presence of genetic traits associated with virulence and biotype: ompW, ctxAB, zot, rfbO1, tcp, ace, hlyA, ompU, rtx, toxR, rtxC and rstRElTor allele using PCR as described earlier (Kumar et al., 2009; Jain et al., 2011). Other virulence genes – vibrio pathogenicity island (VPI), aldA and tagA – were detected by single PCR assay. The ctxB gene was amplified from the clinical and environmental isolates and subjected to DNA sequencing and ctxB typing (Jain et al., 2011). Organization and chromosomal localization of the CTX prophage was determined by PCR using specific sets of primers reported earlier (Nguyen et al., 2009; Jain et al., 2011). Details of all the primers used in this study are given in Supplementary Table S1.
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Classical El Tor El Tor El Tor El Tor
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O1, O1, O1, O1, O1,
2.2. Screening of virulence-associated gene traits
569B N16961 Kalamb, Maharashtra Yavatmal, Maharashtra Kalamb, Maharashtra
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India, 1948 Bangladesh, 1971 Clinical/2012 Clinical/2012 Environmental/2012
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Virulence associated genotype
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mPCR1
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rtxC
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rstR
95
ctxB
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Haemolysis
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PB
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Biotype specific characteristics
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VP test
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No. of strains
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Strain type
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KML file attached as a Supplement). The patients were admitted to the Shri Vasantrao Naik Government Medical College, Yavatmal. The stool samples collected using sterile swabs were processed as described earlier (Pourshafie et al., 2007). Meanwhile, the drinking water supply to the town, which was connected to a water purification plant on the banks of Wardha River, was monitored. Water samples (sewage and potable) were collected from the outbreak-affected areas and neighbouring houses. A 1-L water sample was filtered through a 0.45-lm membrane using a vacuum pump, the membrane was enriched in alkaline peptone water for 6 h followed by plating on TCBS agar. The environmental and clinical isolates were screened for the presence of the CT gene by colony polymerase chain reaction (PCR). All the toxigenic (CT-positive) isolates (five environmental and 20 clinical) were further subjected to serotyping and biotyping (Jain et al., 2011).
Location of isolation
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Table 1 Biotype and virulence associated chracteristics.
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Origin/year of isolation
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Please cite this article in press as: Kumar, P., et al. Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.03.020
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Fig. 1. Neighbor-joining method-based phylogenetic tree construction using concatenated sequences of the pyrH, rpoA, and recA housekeeping genes. The Kimura twoparameter model was used to estimate distance for strains. A total of 2000 bootstrap replicates were chosen for analysis. Vibrio mimicus LMG 7896T was an out-group in the study. Other strains were symbolized with their place of origin and year.
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ing threat of cholera outbreak due to contamination of drinking water (Singh et al., 2001).
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3.2. ctxB genotyping and CTX prophage array
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DNA sequencing identified the ctxB7 allele carrying Asn instead of His at the 20th position in a signal peptide. The PCR amplification of the rstC gene confirmed the presence of RS1-CTX prophage array in all the isolates. It was present only on the large chromosome in the strains as indicated by PCR amplification with ch1F/ rstAR and ctxBF/ch1R primers. All the isolates belonged to Group II and contained the RS1 and CTX prophage (Fig. S1) harbouring the El Tor type of rstR and the classical type of ctxB on the large chromosome, a genetic map similar to the Vietnamese strain (Nguyen et al., 2009). Based on RS1-CTX prophage, atypical El Tor strains have been classified into two groups: Group 1 (tandem repeat of the classical CTX prophage on the small chromosome) and Group 2 (the RS1-CTX prophage with the El Tor type rstR Q3 and classical ctxB on the large chromosome) (Lee et al., 2010). The altered biotype in V. cholerae O1 El Tor (classical CT) strains has been reported from different geographical regions such as Bangladesh, Mozambique, Vietnam, Hong Kong, Japan, Zambia, India, Africa, Nigeria and Haiti (Safa et al., 2010; Marin et al., 2013). The hybrid property enhances the infection potential of the bacterium and such strains are associated with more fluid loss and a higher case fatality rate. In India, these strains were reported to cause repeated outbreaks in different parts of India including Q4 Orissa, Chennai, Hyderabad, Solapur and Assam (Goel and Jiang, 2010; Jain et al., 2011). The HCT was first reported from a large cholera outbreak in Orissa and later it was found associated with epidemics in Africa and Haiti. It was also noted in strains collected from sporadic cholera cases in Kolkata and Yavatmal; in the latter it was found to be persistent in the region after causing an outbreak in Kalamb (Kutar et al., 2013; Kumar et al., 2013).
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3.3. Analysis of SXT constin
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Partial sequences of the SXT integrase of the isolates were submitted to GenBank with Accession No. KF290492-94. The sequences were 100% identical to SXT integrase of strains from ICEVchInd5 from Sevagram (GQ463142) and Kolkata, India, and VC1786ICE from the Haiti outbreak strains (JN648379). ICEVchInd5 is a prevalent SXT element in Indian V. cholerae O1 El Tor strains and was first described in the El Tor strain collected from Sevagram (Wardha) in 1994, and a similar SXT element (ICEVchHAI1) was found in Haitian strains (Ceccarelli et al., 2011). Wardha and Yavatmal are two adjacent districts of Maharashtra state, showing that V. cholerae with an SXT element continues to prevail in the state and is disseminated to distinct geographical areas. The strains were found positive for floR present on SXT constin, conferring the
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resistance against chloramphenicol. The floR was not associated with resistance in the isolates because the minimum inhibitory concentration (MIC) for chloramphenicol was 4–8 lg/mL. The genotype (floR) with a similar phenotype has also been observed in the isolates that caused the worst epidemic in recent years in Haiti (Sjolund-Karlsson et al., 2011).
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3.4. Genetic relatedness of the strains
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Three DNA fingerprinting methods – random amplified polymorphic DNA (RAPD), Box and ERIC PCR – generated identical DNA banding patterns among the clinical and environmental strains, revealing their clonal relationships. The multilocus sequence typing (MLST) results also revealed single clusters for clinical and environmental strains along with the pandemic V. cholerae O1 El Tor strain (N16961) and strains from Haiti and Ghana. Identical DNA sequences were observed in all the strains for each of the three loci (pyrH, recA, rpoA) (Fig. 1). Only three representative strain’s concatenated nucleotide sequences, one from each group – Kalamb (VCK1201), Yavatmal (VCY1257) and environmental (VEK1235) – were included in phylogenetic tree construction. The clonality of the strains points towards a single source of origin, indicating that a single clone of V. cholerae was responsible for both outbreaks. Earlier reports established the clonal relationship of environmental strains with clinical strains, demonstrating that the environmental reservoir of V. cholerae is the cause of cholera outbreaks (Singh et al., 2001). Our previous investigations from Orissa, Hyderabad, Chennai and Solapur also revealed the involvement of a single clone in the respective outbreaks (Goel and Jiang, 2010; Jain et al., 2011; Kumar et al., 2009). The drinking water supplied to Kalamb passed through a sewer drainage system near the most severely affected houses. Leakage in the supply pipeline at the cross-point of sewer drainage near a house undergoing renovation works was observed. It might have allowed suction of sewage water into the drinking water during the supply off-period due to creation of negative pressure. The presence of clonal toxigenic strains in drinking water and associated sewage water supports this hypothesis. The faecal contamination of the potable water supply might be the major reason for the first outbreak. The strains remained persistent in the area after the first outbreak. Subsequently, the strains would have spread to other areas in the district associated with the second outbreak.
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4. Conclusions
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In the present study, all the clinical and toxigenic environmental isolates of V. cholerae O1 El Tor share their biotype with the CT variant, showing their epidemiological and environmental success. The clonality of environmental and clinical strains based on DNA
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Please cite this article in press as: Kumar, P., et al. Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.03.020
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fingerprinting and MLST analyses revealed a common origin and highlighted the role of drinking water contamination in an eruption of a cholera outbreak. The 100% identity in a partial sequence of SXT integrase with ICEVchInd5 (Sevagram, India) and VC1786ICE (Haiti) confirmed that the strains still prevail in the state and have been disseminated to other geographical regions.
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Acknowledgements
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PK is thankful to the University Grant Commission (UGC), New Delhi, India for providing Dr. DS Kothari-Postdoctoral fellowship. Research at Applied Molecular Biology Lab is also supported by Grants received from the Department of Science & Technology, Department of Biotechnology and UGC, Government of India.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2014.03. 020. These data include Google maps of the most important areas described in this article.
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References
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Ceccarelli, D., Spagnoletti, M., Bacciu, D., Cappuccinelli, P., Colombo, M.M., 2011. New V. cholerae atypical El Tor variant emerged during the 2006 epidemic outbreak in Angola. BMC Microbiol. 11, 130. Enserink, M., 2010. Infectious diseases. Haiti’s outbreak is latest in cholera’s new global assault. Science 330, 738–739. Ghosh-Banerjee, J., Senoh, M., Takahashi, T., Hamabata, T., Barman, S., Koley, H., Mukhopadhyay, A.K., Ramamurthy, T., Chatterjee, S., Asakura, M., Yamasaki, S., Nair, G.B., Takeda, Y., 2010. Cholera toxin production by the El Tor variant of Vibrio cholerae O1 compared to prototype El Tor and classical biotypes. J. Clin. Microbiol. 48, 4283–4286. Goel, A.K., Jiang, S.C., 2010. Genetic determinants of virulence, antibiogram and altered biotype among the Vibrio cholerae O1 isolates from different cholera outbreaks in India. Infect. Genet. Evol. 10, 815–819.
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Jain, M., Goel, A.K., Bhattacharya, P., Ghatole, M., Kamboj, D.V., 2011. Multidrug resistant Vibrio cholerae O1 El Tor carrying classical ctxB allele involved in a cholera outbreak in South Western India. Acta Trop. 117, 152–156. Kumar, P., Jain, M., Goel, A.K., Bhadauria, S., Sharma, S.K., Kamboj, D.V., Singh, L., Ramamurthy, T., Nair, G.B., 2009. A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, Eastern India. J. Med. Microbiol. 58, 234–238. Kumar, P., Mishra, D.K., Deshmukh, D.G., Jain, M., Zade, A.M., Ingole, K.V., Yadava P.K., 2013. Haitian variant ctxB producing Vibrio cholerae O1 with reduced susceptibility to ciprofloxacin is persistent in Yavatmal, Maharashtra, India after causing a cholera outbreak. Clin. Microbiol. Infect. Accepted. doi:10.1111/ 1469-0691.12393. Kutar, B.M., Rajpara, N., Upadhyay, H., Ramamurthy, T., Bhardwaj, A.K., 2013. Clinical isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata, India: preponderance of SXT element and presence of Haitian ctxB variant. PLoS One 8 (2), e56477. Marin, M.A., Thompson, C.C., Freitas, F.S., Fonseca, E.L., Aboderin, A.O., Zailani, S.B., Quartey, N.K., Okeke, I.N., Vicente, A.C., 2013. Cholera outbreaks in Nigeria are associated with multidrug resistant atypical El Tor and Non-O1/Non-O139 Vibrio cholerae. PLoS Negl. Trop. Dis. 7 (2), e2049. Naha, A., Pazhani, G.P., Ganguly, M., Ghosh, S., Ramamurthy, T., Nandy, R.K., Nair, G.B., Takeda, Y., Mukhopadhyay, A.K., 2012. 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. Indian J. Clin. Microbiol. 50 (5), 1733–1736. Nguyen, B.M., Lee, J.H., Cuong, N.T., Choi, S.Y., Hien, N.T., Anh, D.D., Lee, H.R., Ansaruzzaman, M., Endtz, H.P., Chun, J., Lopez, A.L., Czerkinsky, C., Clemens, J.D., Kim, D.W., 2009. 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. 47, 1568–1571. Pourshafie, M.R., Bakhshi, B., Ranjbar, R., Sedaghat, M., Sadeghifard, N., Zaemi Yazdi, J., Parzadeh, M., Raesi, J., 2007. Dissemination of a single Vibrio cholerae clone in cholera outbreaks during 2005 in Iran. J. Med. Microbiol. 56, 1615–1619. Safa, A., Nair, G.B., Kong, R.Y., 2010. Evolution of new variants of Vibrio cholerae O1. Trends Microbiol. 18, 46–54. Singh, D.V., Matte, M.H., Matte, G.R., Jiang, S., Sabeena, F., Shukla, B.N., Sanyal, S.C., Huq, A., Colwell, R.R., 2001. Molecular analysis of Vibrio cholerae O1, O139, nonO1, and non-O139 strains: clonal relationships between clinical and environmental isolates. Appl. Environ. Microbiol. 67, 910–921. Sjolund-Karlsson, M., Reimer, A., Folster, J.P., Walker, M., Dahourou, G.A., Batra, D.G., Martin, I., Joyce, K., Parsons, M.B., Boncy, J., Whichard, J.M., Gilmour, M.W., 2011. Drug-resistance mechanisms in Vibrio cholerae O1 outbreak strain, Haiti, 2010. Emerg. Infect. Dis. 17, 2151–2154. Thompson, C.C., Freitas, F.S., Marin, M.A., Fonseca, E.L., Okeke, I.N., Vicente, A.C., 2011. Vibrio cholerae O1 lineages driving cholera outbreaks during seventh cholera pandemic in Ghana. Infect. Genet. Evol. 11, 1951–1956.
Please cite this article in press as: Kumar, P., et al. Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.03.020
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No. of Pages 4, Model 5G
18 April 2014 Infection, Genetics and Evolution xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
2
Short communication
6 4 7
Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012
5 8 9 10 11 12 13 1 2 5 9 16 17 18 19 20 21 22 23 24 25 26 27 28
Q1
P. Kumar a, D.K. Mishra a, D.G. Deshmukh b, M. Jain c, A.M. Zade b, K.V. Ingole b, A.K. Goel c, P.K. Yadava a,⇑ a
Jawaharlal Nehru University, New Delhi 110067, India SVN Govt. Medical College, Yavatmal, India c Defence Research and Development Establishment, Gwalior 474002, India b
a r t i c l e
i n f o
Article history: Received 21 July 2013 Received in revised form 17 March 2014 Accepted 20 March 2014 Available online xxxx Keywords: Cholera Outbreak Vibrio cholerae El Tor variant biotype ctxB7 MLST
a b s t r a c t Cholera has been a recurrent epidemic disease in human populations for the past 200 years. We present herein a comparative characterization of clinical Vibrio cholerae strains isolated from two consecutive cholera outbreaks in 2012 and associated environmental strains from western India. The clinical and toxigenic environmental isolates were identified as hybrid V. cholerae O1, serotype Ogawa, biotype El Tor carrying the variant ctxB7 allele. Partial sequences of SXT integrase from the isolates revealed 100% identity to ICEVchInd5 (Sevagram, India, 1994) and VC1786ICE (Haiti, 2013). The full clonal relationship of the strains established by RAPD, Box PCR, ERIC PCR and MLST (pyrH, recA and rpoA) analyses, and the short time between the two outbreaks, strongly supported that both outbreaks were due to a single strain. The study corroborated that faecal contamination of the potable water supply was the main reason for the first outbreak, which further spread to other areas and resulted in the second outbreak. The study concluded that the circulating El Tor variant strains of epidemic potential in the region can be a serious concern in the future. Ó 2014 Elsevier B.V. All rights reserved.
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46
1. Introduction
47
Cholera, an ancient waterborne disease, continues to be a devastating disease globally with a greater prevalence in developing nations. Vibrio cholerae is primarily an inhabitant of the aquatic environment, so water plays an important role in the transmission and epidemiology of cholera. The recent outbreak reports from India, Angola, Sudan, Zimbabwe, Nigeria and Haiti have revealed that the mortality resulting from the disease has far exceeded the World Health Organization (WHO) international target of 1% or less (Enserink, 2010). Several outbreaks and sporadic cases of cholera have been noted from time to time in India. V. cholerae O1 has two biotypes: classical (more toxigenic) and El Tor (more resistant to environmental factors). It has genetic plasticity and continues to evolve in virulence and drug resistance, thus ensuring its survival and persistence. This leads to the emergence of new hybrid or variant strains (e.g. El Tor strains have captured the toxin of the classical type), hampering disease control policies. El Tor variant strains have better colonization efficiency and are able to
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
⇑ Corresponding author. Address: Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. Tel.: +91 11 26704520; fax: +91 11 26742558. E-mail address: [email protected] (P.K. Yadava).
produce a significantly higher amount of cholera toxin (CT) in vivo or in virulence-induced conditions (Ghosh-Banerjee et al., 2010). Moreover, an El Tor variant strain emerged with a novel CT (ctxB7 allele), first reported in Orissa; later it rose in fame as Haitian ctxB (HCT) after causing a devastating epidemic in Haiti (Kumar et al., 2009; Naha et al., 2012; Kutar et al., 2013). Earlier, we noted a cholera outbreak in Yavatmal district of Maharashtra associated with HCT (Kumar et al., 2013). In the present study, we aimed to link the outbreak to equipment damage in the drinking water supply and contamination of sewer water, associated with back-to-back cholera outbreaks in the same district of Maharashtra, south-western India. We determined the molecular epidemiological traits and phylogenetic relationship of V. cholerae isolates from the two consecutive cholera outbreaks with environmental strains.
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2. Materials and methods
79
2.1. Isolation and characterization of bacterial strains
80
A total of 20 clinical V. cholerae isolates were recovered from randomly selected patients (unrelated individuals) during cholera outbreaks in the month of May, 2012 from Kalamb (10 isolates) and Yavatmal (10 stool samples), Maharashtra, India (see the
81
http://dx.doi.org/10.1016/j.meegid.2014.03.020 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Kumar, P., et al. Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.03.020
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2.4. Genetic relatedness of the strains by DNA fingerprinting and multilocus sequence typing
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The phylogenetic relationship of the clinical and environmental isolates was analysed by enterobacterial repetitive intergenic consensus (ERIC) PCR, BOX-PCR and random amplified polymorphic DNA (RAPD) PCR (Kumar et al., 2009). The strains were further subjected to multilocus sequence typing based on pyrH, recA and rpoA genes using MEGA5 according to a previously described method (Thompson et al., 2011).
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3. Results and discussion
133
3.1. Characterization of V. cholerae strains
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134 135 136 137 138 139
All the clinical and toxigenic environmental V. cholerae isolates were identified as V. cholerae O1 Ogawa. Biochemical and PCR assays confirmed the El Tor biotype. PCR analyses revealed the presence of other virulence-associated traits such as ctxAB, tcpA, Q5 zot, ace, toxR, rfbO1 and VPI genes (Table 1). The presence of toxigenic strains in environmental water is an indication of an impend-
zot+ zot+ zot+ zot+ zot+ tcp+, tcp+, tcp+, tcp+, tcp+, rfbO1+, rfbO1+, rfbO1+, rfbO1+, rfbO1+, + + + +
ctxAB+, ctxAB+, ctxAB+, ctxAB+, ctxAB+,
: negative, +: positive, +/ : weak positive, R: resistant, S: susceptible, C: classical, E: El Tor, VP: Voges Proskauer, PB: Polymyxin B; mPCR1 (multiplex PCR1), mPCR2 (multiplex PCR2) (Kumar et al., 2009); Clinical strain from Kalamb have been earlier characterized by Kumar et al. (2013).
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rtxC , ace+, hlyA , OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+ rtxC+, ace+, hlyA+, OmpU+, toxR+
110 111
+ + + +
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+ +/ +/ +/
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mPCR2
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OmpW+, OmpW+, OmpW+, OmpW+, OmpW+,
The presence of SXT constin was determined by PCR amplification using the primers SXT-F and SXT-R directed to the SXT element and int1-F and int1-B primers targeted for SXT integrase. The amplicon of SXT-F/SXT-R was purified and sequenced. The strains were screened for floR presence on SXT constin and the antimicrobial susceptibility test for chloramphenicol (Sjolund-Karlsson et al., 2011).
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C E E E E
116
105
C E C C C
2.3. Analyses of SXT constin
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S R R R R
115
103
1 1 10 10 5
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Genomic DNA was isolated from all the selected toxigenic isolates using a genomic DNA purification kit (MBI Fermentas, Vilnius, Lithuania) and screened for the presence of genetic traits associated with virulence and biotype: ompW, ctxAB, zot, rfbO1, tcp, ace, hlyA, ompU, rtx, toxR, rtxC and rstRElTor allele using PCR as described earlier (Kumar et al., 2009; Jain et al., 2011). Other virulence genes – vibrio pathogenicity island (VPI), aldA and tagA – were detected by single PCR assay. The ctxB gene was amplified from the clinical and environmental isolates and subjected to DNA sequencing and ctxB typing (Jain et al., 2011). Organization and chromosomal localization of the CTX prophage was determined by PCR using specific sets of primers reported earlier (Nguyen et al., 2009; Jain et al., 2011). Details of all the primers used in this study are given in Supplementary Table S1.
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Classical El Tor El Tor El Tor El Tor
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O1, O1, O1, O1, O1,
2.2. Screening of virulence-associated gene traits
569B N16961 Kalamb, Maharashtra Yavatmal, Maharashtra Kalamb, Maharashtra
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India, 1948 Bangladesh, 1971 Clinical/2012 Clinical/2012 Environmental/2012
99
Virulence associated genotype
98
mPCR1
97
rtxC
96
rstR
95
ctxB
94
Haemolysis
93
PB
92
Biotype specific characteristics
90 91
VP test
89
No. of strains
88
Strain type
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KML file attached as a Supplement). The patients were admitted to the Shri Vasantrao Naik Government Medical College, Yavatmal. The stool samples collected using sterile swabs were processed as described earlier (Pourshafie et al., 2007). Meanwhile, the drinking water supply to the town, which was connected to a water purification plant on the banks of Wardha River, was monitored. Water samples (sewage and potable) were collected from the outbreak-affected areas and neighbouring houses. A 1-L water sample was filtered through a 0.45-lm membrane using a vacuum pump, the membrane was enriched in alkaline peptone water for 6 h followed by plating on TCBS agar. The environmental and clinical isolates were screened for the presence of the CT gene by colony polymerase chain reaction (PCR). All the toxigenic (CT-positive) isolates (five environmental and 20 clinical) were further subjected to serotyping and biotyping (Jain et al., 2011).
Location of isolation
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Table 1 Biotype and virulence associated chracteristics.
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Origin/year of isolation
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Please cite this article in press as: Kumar, P., et al. Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.03.020
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Fig. 1. Neighbor-joining method-based phylogenetic tree construction using concatenated sequences of the pyrH, rpoA, and recA housekeeping genes. The Kimura twoparameter model was used to estimate distance for strains. A total of 2000 bootstrap replicates were chosen for analysis. Vibrio mimicus LMG 7896T was an out-group in the study. Other strains were symbolized with their place of origin and year.
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ing threat of cholera outbreak due to contamination of drinking water (Singh et al., 2001).
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3.2. ctxB genotyping and CTX prophage array
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DNA sequencing identified the ctxB7 allele carrying Asn instead of His at the 20th position in a signal peptide. The PCR amplification of the rstC gene confirmed the presence of RS1-CTX prophage array in all the isolates. It was present only on the large chromosome in the strains as indicated by PCR amplification with ch1F/ rstAR and ctxBF/ch1R primers. All the isolates belonged to Group II and contained the RS1 and CTX prophage (Fig. S1) harbouring the El Tor type of rstR and the classical type of ctxB on the large chromosome, a genetic map similar to the Vietnamese strain (Nguyen et al., 2009). Based on RS1-CTX prophage, atypical El Tor strains have been classified into two groups: Group 1 (tandem repeat of the classical CTX prophage on the small chromosome) and Group 2 (the RS1-CTX prophage with the El Tor type rstR Q3 and classical ctxB on the large chromosome) (Lee et al., 2010). The altered biotype in V. cholerae O1 El Tor (classical CT) strains has been reported from different geographical regions such as Bangladesh, Mozambique, Vietnam, Hong Kong, Japan, Zambia, India, Africa, Nigeria and Haiti (Safa et al., 2010; Marin et al., 2013). The hybrid property enhances the infection potential of the bacterium and such strains are associated with more fluid loss and a higher case fatality rate. In India, these strains were reported to cause repeated outbreaks in different parts of India including Q4 Orissa, Chennai, Hyderabad, Solapur and Assam (Goel and Jiang, 2010; Jain et al., 2011). The HCT was first reported from a large cholera outbreak in Orissa and later it was found associated with epidemics in Africa and Haiti. It was also noted in strains collected from sporadic cholera cases in Kolkata and Yavatmal; in the latter it was found to be persistent in the region after causing an outbreak in Kalamb (Kutar et al., 2013; Kumar et al., 2013).
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3.3. Analysis of SXT constin
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Partial sequences of the SXT integrase of the isolates were submitted to GenBank with Accession No. KF290492-94. The sequences were 100% identical to SXT integrase of strains from ICEVchInd5 from Sevagram (GQ463142) and Kolkata, India, and VC1786ICE from the Haiti outbreak strains (JN648379). ICEVchInd5 is a prevalent SXT element in Indian V. cholerae O1 El Tor strains and was first described in the El Tor strain collected from Sevagram (Wardha) in 1994, and a similar SXT element (ICEVchHAI1) was found in Haitian strains (Ceccarelli et al., 2011). Wardha and Yavatmal are two adjacent districts of Maharashtra state, showing that V. cholerae with an SXT element continues to prevail in the state and is disseminated to distinct geographical areas. The strains were found positive for floR present on SXT constin, conferring the
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resistance against chloramphenicol. The floR was not associated with resistance in the isolates because the minimum inhibitory concentration (MIC) for chloramphenicol was 4–8 lg/mL. The genotype (floR) with a similar phenotype has also been observed in the isolates that caused the worst epidemic in recent years in Haiti (Sjolund-Karlsson et al., 2011).
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3.4. Genetic relatedness of the strains
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Three DNA fingerprinting methods – random amplified polymorphic DNA (RAPD), Box and ERIC PCR – generated identical DNA banding patterns among the clinical and environmental strains, revealing their clonal relationships. The multilocus sequence typing (MLST) results also revealed single clusters for clinical and environmental strains along with the pandemic V. cholerae O1 El Tor strain (N16961) and strains from Haiti and Ghana. Identical DNA sequences were observed in all the strains for each of the three loci (pyrH, recA, rpoA) (Fig. 1). Only three representative strain’s concatenated nucleotide sequences, one from each group – Kalamb (VCK1201), Yavatmal (VCY1257) and environmental (VEK1235) – were included in phylogenetic tree construction. The clonality of the strains points towards a single source of origin, indicating that a single clone of V. cholerae was responsible for both outbreaks. Earlier reports established the clonal relationship of environmental strains with clinical strains, demonstrating that the environmental reservoir of V. cholerae is the cause of cholera outbreaks (Singh et al., 2001). Our previous investigations from Orissa, Hyderabad, Chennai and Solapur also revealed the involvement of a single clone in the respective outbreaks (Goel and Jiang, 2010; Jain et al., 2011; Kumar et al., 2009). The drinking water supplied to Kalamb passed through a sewer drainage system near the most severely affected houses. Leakage in the supply pipeline at the cross-point of sewer drainage near a house undergoing renovation works was observed. It might have allowed suction of sewage water into the drinking water during the supply off-period due to creation of negative pressure. The presence of clonal toxigenic strains in drinking water and associated sewage water supports this hypothesis. The faecal contamination of the potable water supply might be the major reason for the first outbreak. The strains remained persistent in the area after the first outbreak. Subsequently, the strains would have spread to other areas in the district associated with the second outbreak.
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4. Conclusions
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In the present study, all the clinical and toxigenic environmental isolates of V. cholerae O1 El Tor share their biotype with the CT variant, showing their epidemiological and environmental success. The clonality of environmental and clinical strains based on DNA
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Please cite this article in press as: Kumar, P., et al. Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.03.020
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fingerprinting and MLST analyses revealed a common origin and highlighted the role of drinking water contamination in an eruption of a cholera outbreak. The 100% identity in a partial sequence of SXT integrase with ICEVchInd5 (Sevagram, India) and VC1786ICE (Haiti) confirmed that the strains still prevail in the state and have been disseminated to other geographical regions.
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Acknowledgements
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PK is thankful to the University Grant Commission (UGC), New Delhi, India for providing Dr. DS Kothari-Postdoctoral fellowship. Research at Applied Molecular Biology Lab is also supported by Grants received from the Department of Science & Technology, Department of Biotechnology and UGC, Government of India.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2014.03. 020. These data include Google maps of the most important areas described in this article.
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