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

Authors : Sreedhara H.G, Mohan N.K

DOI : 10.18231/2581-4761.2019.0010

Volume : 5

Issue : 1

Year : 2019

Page No : 41-46

Introduction

Table 1: PCR test results for O1rfb gene, CTX–A, tcp–A El Tor and tcp–A Classical

	O1rfb gene	ctx–A	tcp–A El Tor	tcp–A Classical
No of isolates tested	18	46	46	46
Positive	18	38	38	Nil
Negative	Nil	°8 (NAG Vibrio)	°8 (NAG Vibrio)	46

 

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Fig. 1: ctxA-tcpA multiplex PCR results with 16 V.cholerae isolates. M denotes 1° °bp DNA ladder. NC denotes negative control. ctxA is of 3 °1 bp while tcpA is of 472 bp.

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Fig. 2: O1rfb PCR results with 16 V.cholerae isolates. M denotes 1° °bp DNA ladder. NC denotes negative control. O1 rfb is of 192 bp.

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Fig. 3: RAPD fingerprints of the 18 isolates of V.cholerae with PB1 primer

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Fig. 4: RAPD fingerprints of the 16 isolates of V.cholerae with PB1 primer

Table 3: Antibiotic resistance pattern of isolates for key antibiotics

Table 4: The prevalence of resistance pattern of the isolated V. cholerae °1 strains during the study period

Out of total 620 stool samples received in the laboratory during the study period, V. cholerae was isolated in 74 stool samples, giving an isolation rate of 11.94%. Many studies throughout India reported V cholera ogawa as commonest serotype with isolation rate varying from 5.79% to 56.6%.^[1],^[3],^[7],^[14],^[15]Mandal J et al reported 5.79% isolation rate with 97.4% V. cholerae ogawa and remaining Inaba serotypes.^[14] Chatterjee S et al reported 10.12% isolation rate and all are El tor V. cholerae ogawa serotypes.^[3] Misra M reported 34.95% isolation rate of El tor V. cholerae Ogawa (76.8%), Inaba (1.52%) and Hikojima (2.02%).^[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 co- regulated 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 P et al who reported 94.3%.^[20]100% resistance with Furazolidone was noted with study by Das S et al^[1] & Garg P et al.^[20] But resistance to Furazolidone in our study was 90.70% and varied degree of resistance was noted with a number of studies like Palewar MS et al,88%^[21] and Chander J et al, 45.4%^[22]In the present study, 25.68% of isolates were resistant to Tetracycline and 24.39% resistant to Doxycycline. Resistance to tetracycline in other studies were as low as Mandal J et al, 18.6%^[14]and Chander J et al, 15.4%.^[22]Few other studies had higher percentage of resistance like Misra M et al, 31.1%^[15]and Devnikar AV et al, 38.1%.^[23]Resistance with Co-trimaxazole (94.59%) and Ampicillin (97.30%) were in higher range with majority of the studies including ours.100% resistance to Ampicillin, reported by Das S et al^[1] and Garg P et al.^[20] But few other studies reported very low degree of resistance like Roy S et al, 26.8% ^[11] and Misra M et al, 15.2%.^[15] Similarly a study by Devnikar AV et al reported a lower resistance with Co-trimoxazole (42.86%).^[23]

Ciprofloxacin resistance in this study was 18.92%. Similar degree of resistance was noted with Garg P et al (18.9%) ^[20]. Das S et al^[1] (30.4%) and Misra M et al^[15](37.9%) reported higher percentage resistance compared to number of other studies which reported Kutar BMRNS et al, 12.6%^[5], Mandal J et al, 4.2%^[14] and Chander J et al, 8.33%^[22] resistance. Among representative Cephalosporins of 2^nd generation, Ceftriaxone had 10.81% resistance. But a number of studies reported no resistance with cephalosporins.^[1],^[21],^[22],^[23] But study by Misra M et al reports resistance to Ceftriaxone as high as 34.9%.^[15]

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.

Acknowledgment

The authors are thankful to Dr.Subarna Roy; senior scientist and Deputy Director, Regional Medical research center, Belgaum for technical support during our study. He helped in our study by confirming isolates with doubtful identity and antibiogram and in molecular biology tests like PCR for ctx, tcp, O1rfb and RAPD.

Conflicts of Interest: None.

 

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