Retrospective analysis to study trend of biotype , serotype and phage type of Vibrio cholerae isolates in South Gujarat over last 17 years

Cholera is a major infectious disease with epidemic potential, especially among communities living in congested urban slums and vast rural areas without proper sanitary facilities.[1] Cholera has re-emerged as a major infectious disease in the recent past, with a global increase in its incidence.[2] Serogroup O1 is classifi ed into two biotypes, classical and El Tor. The seventh and most recent pandemic of cholera was caused by the El Tor biotype.[1] The epidemiology of cholera showed remarkable changes soon after the introduction of El Tor biotype of Vibrio cholerae in India in 1964.[3] At present, V. cholerae O1 belonging to the El Tor biotype is most common in India. The frequency of biotype O139 has declined considerably over the past few years, which was identifi ed for time in 1992 and was responsible for many local outbreaks in India.[2] The classical biotype reported in earlier years was believed to have become extinct in the recent past. Although rapid replacement of fl uid and electrolyte using oral rehydration therapy or intravenous fl uids and the subsequent maintenance of hydration remains the mainstay of management in cholera patients, specifi c antimicrobials are given to reduce the duration and severity of diarrhea as well as to speed up the clearance of organisms from the stools.[4]


MATERIALS AND METHODS
A retrospective study was carried out in Department of Microbiology, Government Medical College, Surat.Analysis of stool samples received during last 17 years, from 1995 to 2012 in the laboratory was carried out and total isolates of V. cholerae were calculated.Ethical permission was taken from IRB. Stool samples received in the laboratory were cultured on Mac Conkey agar, nutrient agar and thiosulfate citrate sucrose bile salt (TCBS) agar.Small amount of the sample was enriched in alkaline peptone water and subculture was done after 4 h on TCBS agar.All the plates were incubated at 37°C for 24 h.Next day colonies of V. cholerae were identifi ed using colony morphology, Gram stain from colony smear, various biochemical tests such as oxidase test, catalase test and string test.Further confi rmation was done by putting all the batteries of biochemical tests including cholera red test.
For serotyping of isolates, Vibrio cholera polyvalent, Vibrio cholera Ogawa and Vibrio cholera Inaba antisera were used for agglutination (Denka Seiken Co., Ltd., Japan).Antibiotic susceptibility analysis of last 2 year isolates was done using ampicillin, ciprofl oxacin, norfl oxacin, tetracycline, doxycycline, co-trimoxazole, ceftriaxone, nalidixic acid, gentamycin and chloramphenicol by Kirby Bauer disc diffusion technique (Biogram, Microexpress-Goa).All the isolates were sent to the National Institute of Cholera and Enteric Diseases (NICED), Kolkata in nutrient agar vials in duplicate for biotyping and phage typing.

RESULT
404 isolates of V. cholerae were analyzed in the present study, which were isolated from stool samples.399 isolates (98.8%) showed agglutination with Ogawa antisera, only 5 isolates (1.2%) showed agglutination with Inaba antisera.These Inaba isolates were found during year 2006 (4) and 2007 (1) only.Rest of the years Ogawa was the predominant isolates.No discordance was found for serotyping done in the laboratory and results provided by NICED, Kolkata.Though scattered isolates of V. cholerae were seen throughout the year, 74.6% of isolates were in summer and rainy seasons (April-August).
Among the isolates, 397 isolates (98%) were of El Tor Phage type distribution according to the new scheme classifi cation is shown in Figure 4.
Antibiotic susceptibility was performed for the 2011-12 isolates by Kirby Bauer disc diffusion method.Isolates showed 70% sensitivity to ampicillin and 94% to chloramphenicol.All the isolates were sensitive to ciprofl oxacin, norfl oxacin, tetracycline and gentamycin.Only 19% sensitivity was recorded to co-trimoxazole, whereas all the isolates were resistant to nalidixic acid.

DISCUSSION
Change in epidemiology is noted with cholera also as like with other diseases.In the present study, 98% isolates were of El Tor biotype.Kanugo et al., [5] in their study has also reported that V. cholerae O1 belonging to the El Tor is the most common biotype in India and frequency of O139 biotype has been declined considerably over the past few years.Narang et al. [2] , Barve et al. [15] and Wong et al. [16] had also reported the same.Although Ogawa serotype was prevalent throughout the years, 5 Inaba serotype isolates were reported during 2006-2007 in the present study.Narang et al. [2] has also reported such a shift in 1999 and 2005.Pal et al. [6] has reported a cholera outbreak in Orissa during 2005 with V. cholerae El Tor serotype Inaba.This may be due to fl ooding in Orissa during that period.The present study area also had fl ood in 2006, during which Inaba serotype was reported.Many other studies from different part of the world have also reported association of increased isolation of Inaba serovars during fl oods.Any change in prevalent serotype should not be neglected and proper surveillance should be carried to rule out major outbreaks in the community.
Phage typing is one of the best tools to study the epidemiological aspect of isolates. [5]Sarkar et al., [7] in a study of different isolates of V. cholerae from different parts of the India has reported T-4 phage type as a predominant phage type during years 1999-2000 and 2003-2004.The fi nding was quite similar with the present study, where also peak of T-4 phage type was seen during the same period.The Sarkar et al. [7] study has reported T-27 phage type of new scheme classifi cation as a predominant phage type throughout the years in all parts of the country.The present study has also noticed the same fi nding during last 17 years.A study by Bhowmick et al. [8] has reported that T27 and T25 in particular were predominant among strains isolated from different geographical regions of India and from both Bangladesh and Brazil.The study has also reported that phage type variation was associated with tetracycline resistance in strains isolated from Kolkata.Thus, monitoring of phage type variation will help to study change in antibiotic resistance pattern in V. cholerae isolates.A study by Das et al. [4] from New Delhi, has reported 87.2% isolates of El Tor and 10% of Non O1 non O139 biotype, T 27 and T 23 phage type according to the new scheme classifi cation.The fi ndings were correlated with the present study of western India.Narang et al. [2] has also reported the same fi nding showing El Tor biotype, Ogawa serotype and T-27 being the most common phage by the new scheme classifi cation.Turbadkar et al. [9] has reported an outbreak of cholera in Mumbai, by biotype El Tor, serotype Ogawa, phage type 4 of Basu and Mukerjee and type 27 (97.5%)and type 23 (2.4%) of the new scheme classifi cation.Antibiotic resistance is a major problem in recent scenario in every organisms and Vibrio cholera is also not far from being a multidrug resistant organism.A study by Narang et al. [2] has reported 48% sensitivity to chloramphenicol and 68% to nalidixic acid, whereas that was 94% to chloramphenicol in the present study.All the isolates tested in the present study were resistant to nalidixic acid.As chloramphenicol is not being used commonly in routine since a long time, selection pressure is decreasing and increased susceptibility is recorded by isolates.Ciprofl oxacin susceptibility (100%) seen in the present study was similar to other studies by Mandal et al. [10] (97%) and Shrestha et al. [11] (100%) carried out in Puducherry and Nepal respectively.An antibiotic susceptibility result reported by Chander et al. [12] was quite similar to the present study.No association between the phage type and antimicrobial susceptibility pattern was observed in the present study.
Major drawback of the present study was that antibiotic susceptibility was studied for last 2 years only.As it was a retrospective analysis, socio-economic status, clinical presentation, clinical outcome of patients and case fatality rate were not studied.Continuous monitoring of antibiotic susceptibility should be carried out as resistance to tetracycline has been reported from some of the parts of the country. [13,14]The study will help to compare the prevalent serotype, biotype and phage type in other areas of Gujarat state.Molecular studies should be taken up to study the presence of the virulence-associated genes such as ctxA, zot, ace, tcpI and toxR to establish a correlation with biotype and virulence factors.

CONCLUSION
El Tor and Ogawa were most commonly prevalent biotype and serotype in the region, quite similar to other states of India and other neighboring countries.T-27 phage type was prevalent throughout the years.T-2 is the most common phage throughout the years except few peaks of T-4.Improved surveillance of the disease will be useful in assessing the actual burden of cholera in the country and in planning interventions appropriately.Cotrimoxazole, ampicillin and nalidixic acid were the drugs, which showed a high degree of resistance, while chloramphenicol, gentamycin, ciprofl oxacin and tetracycline showed good sensitivity in the area studied.The quick shift in resistance pattern indicates that all isolates should be subjected to susceptibility and resistance pattern should be monitored.Periodic epidemiological surveys should be carried out to study the trend of serotype, biotype and phage type in the community.All academic institutes must prepare and maintain a database of isolates so that any deviation in epidemiology can be picked up early.The isolates should be sent to a reference laboratory routinely to identify phage type and biotype.Policy-makers should target control interventions in high-risk areas including vaccines, along with improved access to safe water and adequate sanitation.

Figure 4 :
Figure 4: Distribution of isolates according to the new scheme classifi cation