Molecular epidemiology of vibrio cholerae causing outbreaks and sporadic cholera in and around Hassan district and its antibiotic susceptibility pattern

Introduction: Aim of the study is isolation and identification of V cholerae followed by study of clonal relationship between isolates by molecular typing methods, molecular characterization by detection of genes coding for cholera toxin (CTX) & toxinco-regulated pili (TCP) and study of antibiotic susceptibility pattern of V cholerae isolates Materials and Methods: 74 V cholerae isolates from 620 stool samples were included in study. Isolation, identification and antibiogram done by using standard techniques. Clonal relationship between isolates studied by RAPD assays. Molecular characterization done by detection of CTX and TCP using multiplex PCR. Results: 74 V. cholerae 01 biotype El Tor serotype Ogawa isolated from 620 stool samples with isolation rate of 11.94%. 38 of 46 isolates were positive for CTX – A and TCP – A El Tor by multiplex PCR. All 46 isolates were negative for TCP-A classical. 38 isolates subjected to RAPD typing assays revealed 11 different RAPD types. Antibiotic sensitivity tests for the isolates revealed the presence of diverse sensitivity patterns. 97.3% of isolates were multidrug resistant (MDR). 2.7% of isolates remained sensitive to all tested antibiotics. Conclusion: V. cholerae 01 biotype El Tor serotype Ogawa is currently circulating type in our geographical region. All isolates tested positive for CTX and TCP indicating that circulating strains are pathogenic in nature. Presence of different RAPD types indicates that cholera is endemic in this region with periodic epidemics. Presence of large number of MDR isolates necessitates curbing of irrational use of antibiotics to prevent further spread of drug resistance.


Introduction
Vibrio cholerae has been recognised as one of the common causes of bacterial diarrhea throughout the developing world. Epidemics of cholera caused by toxigenic V. cholerae O1 and V. cholerae O139 are major public health problems. 1 Recent years have witnessed resurgence in global incidence of cholera cases as reported by WHO. Globally, cholera alone causes 120,000 deaths annually. Cholera is endemic in southern Asia and in several parts of Africa and Latin America with seasonal outbreaks. 2 Increase in the number of multidrug resistant pathogens have accompanied rise in case fatality rates & hampered the treatment of many infectious diseases including cholera. 2 Cases of cholera have been on the rise in India and more than doubled from 1939 cases in 2006 to 5155 in 2010. While half a century ago cholera was more of a problem of the eastern parts of India, 3 it is increasingly being reported from southern India recently. 4,5,6,7 Even though cholera is endemic with periodic epidemics in India, data regarding molecular epidemiology and antibiogram of V. cholerae isolates is scarce. Not much is known about the molecular epidemiology of the cholera in Hassan and surrounding areas. There is also scarcity of knowledge in the changing drug susceptibility pattern in V. cholerae isolated from this region over period of time. Because in depth knowledge on the epidemiology and current resistance profile is essential for implementing prevention strategies, the current research work is done in our institute.

Materials and Methods
It is an observational study done for 3 years from April 2012 to April 2015 in the Department of Microbiology, Hassan Institute of Medical Sciences, Hassan, Karnataka.
Inclusion criteria; the study group includes patients presenting with acute watery diarrhea (Three or more abnormally loose or fluid stools in the past 24 hours with or without dehydration). Criteria used to diagnose suspected cholera epidemic includes. 1. Area where disease is present: Severe dehydration or death from acute watery diarrhea in patient aged ≥ 5 years. 2. Area where Cholera is endemic: Acute watery diarrhea, with or without vomiting in a patient aged ≥ 5 years. 3. In an area where there is a cholera epidemic: Acute watery diarrhea, with or without vomiting, in any patient.
Exclusion criteri a; samples which are not properly transferred and cases with incomplete details.
Details of the cases such as age, sex and clinical features were collected by examining case sheet, contacting patient attenders & referring medical officers. Human excreta such as stool samples (preferably), vomitus or rectal swabs collected from all suspected cases before giving antibiotics & immediately transported to laboratory. Cary -Blair media used as transport media in case of delay in transportation.
Stool samples from each patient were streaked on TCBS and MacConkey agar and enriched in alkaline peptone water with subsequent plating. The isolates are identified by biochemical reactions & confirmed by serotyping using specific antisera. 8 The susceptibility of confirmed V.cholerae strains to antimicrobial agents performed on Mueller Hinton agar by Kirby Bauer disc diffusion technique with commercially available discs. 9 Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used for internal quality control.
Further molecular studies conducted at Regional Medical Research Center, Belgaum. Biotyping of V. cholerae isolates and detection of genes coding for CTX & TCP done by multiplex PCR. Clonal relationship between isolates studied by Random Amplified Polymorphic DNA (RAPD) analysis.
Multiplex PCR was performed with ctxA-tcpA primers developed by exploiting biotype-specific variation of nucleotide sequences of tcpA, responsible for the expression of the major subunit protein (TcpA) of the toxin coregulated pilus of V. cholerae. 10 Amplification of ctxA and tcpA El Tor produces bands of 301 and 472 bp, respectively. PCR was performed in a Bio-Rad iCycler in 25 ml reaction volumes. A quantity of 2.5 ml template DNA in the form of heat-treated rapid lysates from 18 hour cultures was used in PCR with 1 µM each primer, 250 µM each dNTP, 1.5 mM MgCl2 and 0.5 U Taq DNA polymerase in 10 mM Tris/HCl (pH 9.0) and 50 mM KCl. The temperature programme consisted of an initial denaturation of 5 min at 94 0 C followed by 29 cycles of 1.5 min at 94 0 C, 1.5 min at 60 0 C and 1.5 min at 72 0 C, and a final cycle of 1.5 min at 94 0 C, 1.5 min at 60 0 C and a final extension of 7 min at 72 0 C. 11 For Random amplified polymorphic DNA (RAPD) fingerprinting assay, purified genomic DNA was isolated from cultures of V. cholerae grown overnight in Luria broth (BD) following the cetyltrimethyl ammonium bromide method. 12 PB1 primer (59-GCG CTG GCT CAG-39) was employed in a RAPD fingerprinting assay with all isolates. 13 PCR was performed in a Bio-Rad iCycler in 50 ml reaction volumes. A quantity of 50 ng DNA extracted from 18 hr cultures was used in PCR with 2 mM primer, 250 mM each dNTP, 1.5 mM MgCl2 and 0.5 U Taq DNA polymerase in 10 mM Tris/HCl (pH 9.0) and 50 mM KCl. The temperature programme consisted of 1 cycle of 3 min at 97 0 C, 1 min at 40 0 C and 1 min at 72 0 C; 4 cycles of 1 min at 97 0 C, 1 min at 40 0 C and 1 min at 72 0 C; 24 cycles of 1 min at 95 0 C, 1 min at 55 0 C and 1 min at 72 0 C; and 1 cycle of 1 min at 95 0 C, 1 min at 55 0 C and 7 min at 72 0 C. PCR products were electrophoresed onto a 1% agarose gel, with gel red dye, viewed under UV light and documented in gel documentation system. 11

Results
Out of total 620 stool samples received in the laboratory during the study period, Vibrio cholerae was isolated in 74 stool samples, giving a positivity rate of 11.94%. On serotyping and biotyping, all isolates were identified as V. cholerae 01 biotype El Tor serotype Ogawa. 46 out of 82 isolates studies for ctx-A, tcp-A El Tor and tcp-A Classical by PCR. 18 isolates subjected to PCR for O1 rfb gene. PCR results for O1rfb gene, ctx-A, tcp-A El Tor and tcp-A Classical shown in table 1. 8 isolates which were negative for ctx-A and tcp-A were considered as non agglutinable (NAG) vibrio and excluded from study.    A-Ampicillin, Na-Nalidixic acid, Cootrimoxazole, T-Tetracycline, Nor-Norfloxacin, Ch-Chloramphenicol, Cef-Ceftriaxone, G-Gentamycin.
The v cholerae isolates were tested for susceptibility to 18 different antibiotics. Antibiotic sensitivity pattern for all 18 antibiotics given in table 4.  15 Das S et al reported 56.6% isolation rate of El tor V. cholerae Ogawa (85%) followed by Inaba serotype. 1 The lower isolation rate in our study may be because; most of the cases included were inpatients in tertiary care hospital and might have received antibiotics before sample collection. All the V cholera isolates were identified as V. cholerae biotype El tor serotype Ogawa suggesting that, it is currently the predominant circulating serotype in our region.
PCR done for detection of O1rfb gene on 18 out of 74 biochemically confirmed isolates (Table; 1). All 18 isolates showed the presence of a 192 bp amplicon marker for the O1rfb gene indicating that isolates belong to V cholerae subgroup O1. Multiplex PCR done for detection of genes coding for ctx-A and tcp-A for 38 V. cholerae isolates and 8 isolates of non agglutinable vibrio (Table 1). All 38 isolates confirmed as V. cholerae serotype ogawa were positive for ctx-A (301 bp amplicon marker) and toxin coregulated pilus gene tcp-A el tor (472 bp amplicon marker) and negative for tcp-A classical. This confirms that all the V. cholerae strains isolated during the outbreak were highly pathogenic with virulence markers ctx and tcp.
RAPD done on 38 isolates out of 74 V. cholerae isolated over period of 3 years. This revealed presence of 11 different patterns among 38 isolates. Reports shows that strains found in outbreaks as well as sporadic cases in present study belong to diverse genotypes. Presence of multiple different genotypes in single outbreak also noted in present study. This indicates possibility of origin of outbreak from different sources. High prevalence of cholera in particular regions (Belur and Sakaleshpur talukas), prevalence of multiple and different RAPD types in an outbreak and higher incidence in children indicates that, cholera is endemic in these regions leading to frequent outbreaks. Most of the outbreaks associated with mass gatherings with evidence of breakdown of water supply and sanitation systems with an unhygienic environment.
The RAPD fingerprinting assay of 18 V. cholerae isolates from an epidemic in Belgaum done by Roy S et al. The study revealed presence of eight different patterns among the 18 isolates. 11 A RAPD fingerprinting study of V. cholerae isolates from Talikoti and Harnal outbreaks was done by Dey S et al in the year 2014. They identified that RAPD fingerprints of these isolates were found to be identical with one particular clone out of eight different clones of V. cholerae O1 obtained from an outbreak that occurred in Belgaum in 2010. 16 Prabhu DIG et al analysed genetic similarities between environmental and clinical isolates of V. cholerae using RAPD finger printing using A5 & A18 primers. They observed that environmental isolates showed very close genetic relationship (ranging from 80.81% to 90.91%) with clinical isolates. 17 These studies indicate that RAPD finger printing is simple, rapid and reliable method for typing V. cholerae isolates, phylogenetic studies and to find out possible source of infection.
Most of the cases are reported from 3 taluks; Belur, Arasikere and Sakaleshpura. All RAPD types found to be distributed in all three regions. This could be because; they are neighboring talukas with frequent travelling of people for functions and festivals.
In cholera cases fluid and electrolytes replacement is main stay of management and antibiotics are not required in all. Use of antimicrobial agents can only reduce the duration and severity of illness. Antibiotic sensitivity tests for the isolates revealed the presence of diverse patterns, and 97.3% of isolates were multidrug resistant (MDR) with resistance to more than one antibiotic. Only 2.7% of isolates remained sensitive to all antibiotics. Among all the antibiotics tested, only Imipenem found to be effective against all the isolates. MDR cholera is being reported from India by many investigators. 1,15,18,19 This wide spectrum of resistance found in V. cholerae in this study is not very common and a cause for serious concern. Large-scale, irrational antibiotic usage among humans and animals, inadequate therapy and lack of awareness regarding antibiotic policy and antibiotic stewardship may be responsible for development of high degree of drug resistance in our country. Antibiogram pattern also found to be fluctuating over the period of study. This could be because of changes in antibiotic choice of treatment which is not determined based on scientific data.
97.29% of isolates resistant to combination of antibiotics A, Na and Co with or without resistance to other antibiotics. (Table; 3) Most common resistant pattern is A, Na, Co (66.23%) followed by A, Na, Co, T, Nor, Ch, Cef, G (10.81%) and A, Na, Co, T (6.76%).These kind of resistance patterns with resistance to more than 8 antibiotics indicates that it could be plasmid mediated drug resistance. This is a matter of grave concern as plasmid mediated resistance can spread in environment very easily.
Nalidixic acid resistance was 100% with a number of studies, 1,5,11 but our study reports lesser percentage of resistance (97.30%) similar to a study by Garg

Conclusion
V. cholerae 01 biotype El Tor serotype Ogawa is currently circulating type in our geographical region. All isolates tested positive for CTX and TCP indicating that circulating strains are pathogenic in nature. Presence of different RAPD types indicates that cholera is endemic in this region with periodic epidemics. Presence of large number of MDR isolates necessitates curbing of irrational use of antibiotics to prevent further spread of drug resistance. Resistance to commonly used antimicrobial agents which were considered as drug of choice is a major public health concern. Drug resistance itself may result in longer hospital stays for patients thereby increasing mortality, morbidity and adds economical burden on community.

Conflicts of Interest:
None.