Emergence of multiple drug resistance Vibrio cholerae O 1 in East Delhi

Introduction: Considering the changing geographical and temporal occurrence of Vibrio cholerae, there is a continuing need to monitor the strain characteristics and antibiotic resistance patterns of this pathogen. The present study was conducted to document the changing biology of V. cholerae isolates in and around Delhi, India, and the development of antibiotic resistance. Methodology: A total of 1,424 stool samples or rectal swabs from patients with acute secretory diarrhoea admitted to Guru Teg Bahadur Hospital, Delhi, between January 2007 and December 2009 were processed using standard bacteriological methods. Strains identified as V. cholerae were further subjected to serogrouping, phage typing and antimicrobial susceptibility testing. Minimum inhibitory concentration (MIC) of gentamicin and tetracycline was determined. Results: V. cholerae was isolated in 242/1,424 (17.0%) specimens. Of these, the majority were V. cholerae O1 serotype (98.3%) and serovar Ogawa. The drugs to which V. cholerae O1 isolates showed high levels of resistance were nalidixic acid, furazolidone, and cotrimoxazole throughout the study period, whereas strains were usually susceptible to chloramphenicol and cefotaxime. In 2007, there was a sudden increase of resistance to gentamicin and tetracycline, followed by a slow reversal to previous levels in subsequent years. The phage typing pattern (Basu and Mukherjee scheme) showed a dominance of phage type 2 throughout the study period. Conclusion: The importance of reporting all cases of V. cholerae, should be greatly emphasized, with the ultimate goal of understanding the constantly changing resistance patterns of this pathogen.


Introduction
Vibrio cholerae has been recognised as one of the common causes of bacterial diarrhoea throughout the developing world.Epidemics of cholera caused by toxigenic V. cholerae O1 and V. cholerae O139 represent a major public health problem [1].The dynamic of its transmission is complex.Two serotypes of V. cholerae O1, Ogawa and Inaba, have been demonstrated to interconvert, owing to an antigenic shift in the O antigen lipopolysaccharide [2].At Guru Teg Bahadur (GTB) Hospital, except for a few scattered reports of V. cholerae O1 Inaba in 1998 and 1999, V. cholerae O1 Ogawa (80%) was the predominant serovar until 2003.During 2004 and 2006, the frequency of isolation of V. cholerae O1 Inaba strains steadily increased [3].The changing pattern of the antibiogram has also raised concerns about the treatment of cholera with antibiotics.The emergence of multiple antibiotic-resistant (MAR) isolates of V. cholerae [4] has added a new dimension to the variability in pathogenicity and potential virulence in precipitating diarrhoeal illness.Considering the changing geographical and temporal pattern of V. cholerae, there is a continuing need to monitor the characteristics and antibiotic resistance patterns of this pathogen.This study was conducted to document the changing biology and antibiotic trends of V. cholerae isolates in and around Delhi.
from each patient were streaked on bile salt agar (BSA) and MacConkey agar and enriched into alkaline peptone water (APW) with subsequent plating.
Phage typing was done at the Vibrio Phage reference centre (NICED Kolkata, India), following the conventional Basu and Mukherjee's method and the new phage typing scheme [10,11].
The drugs to which V. cholerae O1 isolates showed high level resistance throughout the study period were nalidixic acid, furazolidone and cotrimoxazole, whereas strains were usually susceptible to chloramphenicol and cefotaxime.The resistance pattern of V. cholerae O1 to these drugs did not show any change since 2001, as had been reported previously [3].Until 2006, V. cholerae O1 was fully susceptible (100%) to gentamicin and tetracycline.However, in 2007, there was sudden rise of resistance to these antibiotics (Table 1), followed by a slow reversion to previous levels in subsequent years.All gentamicin-tetracycline resistant strains had the same resistance patterns and were sensitive to chloramphenicol and cefotaxime alone, while resistant to ciprofloxacin, nalidixic acid, furazolidone and cotrimoxazole.MIC 50 and MIC 90 of V. cholerae for gentamicin were 16 μ/ml and 64 μg/ml respectively, while for tetracycline they were 8 μg/ml and 32 μg/ml respectively, as depicted in Table 2.
All the Hikojima isolates were sensitive to the tested drugs except cotrimoxazole.
The phage typing pattern (Basu and Mukherjee scheme) of V.cholerae O1 isolates showed 100% clustering of phage type 2 throughout the study period.With the new typing scheme, which was more discriminatory, the clusters were distributed among phage T 26 and T 27 (95%

Discussion
Epidemic cholera spread from the Indian subcontinent to the rest of the world including America, Africa and Europe by 1950, though it ultimately remained in Asia.The ease and speed of travel, which has increased the number of travellers, contributed to the re-emergence of V. cholerae globally.Annual outbreaks of cholera are a regular occurrence in India.A high population density along with open drains and poor sanitation provide an optimal niche for the survival, sustenance and transmission of V. cholerae.
This study was an attempt to highlight the divergence of V. cholerae O1 isolates and the subsequent emergence of drug resistance during different time periods.The data demonstrate that the complexities of V. cholerae convertants, which predominate at different times with different strains, are presumably mediated via the host immune response.
At GTB Hospital, V. cholerae O1 Ogawa (80%) was the predominant isolate until 2003 [3].However, between 2004 and 2006, V. cholerae O1 Inaba (60%) was isolated in large numbers [3] and, from 2007 onwards, V. cholerae O1 Ogawa (90%) again became the predominant isolate and remained as such until 2009.This evident change to Inaba occurred after an interval of several years [3], which was followed by a re-emergence of Ogawa strains in subsequent years.Such unequal reciprocal interconversion perhaps occurs due to immune pressure in the population or antimicrobial selection pressure resulting in the mutation of the wbe (rfb) gene [2].Thus serovar flux can be considered a strategy for the persistence and/or survival, and the temporality of such a flux is a reflection of the evolutionary pressure on the pathogen.
In India, cholera patients are treated with antibiotics along with a correction of dehydration status.Therefore, it becomes important to analyze the trends of antibiotic resistance among the clinical strains of V. cholerae.However, unlike other bacterial infections, little is reported about resistance patterns of V. cholerae, as laboratories do not routinely test susceptibility to different classes of antimicrobial agents.The emergence of multiple antibiotics resistant (MAR) V. cholerae with epidemic outbreaks has been reported in neighbouring Bangladesh [4,12].The antibiotic resistance patterns of epidemic strains isolated from Bangladesh have documented reduced susceptibility to ampicillin, furazolidone, neomycin, streptomycin [13].High-level resistance to choramphenicol, ampicillin, ciprofloxacin, neomycin, nalidixic acid and norfloxacin has been reported from Kolkata [14].Outbreaks due to plasmid-mediated multiple drug resistant strains have also been associated with not only V. cholerae O1, but also with rough strain and non-V.cholerae O1.These strains demonstrated resistance to a spectrum of antibiotics, including chloramplenicol, tetracycline, ampicillin, sulphamethoxazole-trimethoprim, and streptomycin.Resistance determinants to aminoglycisides (kanamycin, gentamycin, neomycin) have also been reported in such strains [15,16].
In the present study, V.cholerae O1 isolates showed 100% resistance against furazolidone, nalidixic acid and cotrimoxazole, suggesting that these drugs have a negligible role in the treatment of cholera.In contrast, most of the isolates were sensitive to chloramphenicol (98.3%) and cefotaxime (91.4%), which can be used as an alternative.We have reported previously about fluroquinolone resistance in children suffering from cholera in 2005 [17].In subsequent years, ciprofloxacin resistance has largely remained between 30-40% [3].All V. cholerae O1 isolates were sensitive to gentamicin and tetracycline in our area for the past several years.However, in 2007, 41.3% and 58.6% of isolates developed resistance against gentamicin and tetracycline, respectively.The resistance to various antibiotics with a rapid shift, as reported in this study, is consistent with many reports indicating an enhanced mobility in genetic elements conferring resistance to V. cholerae strains.This makes it imperative that all isolates be constantly subjected to susceptibility testing and resistance patterns to each antibiotic be monitored.
Multiple drug resistance in V. cholerae can be attributed to either spontaneous mutation or to the horizontal transfer of resistance genes between members of gut coliform or other co-existing microflora and Vibrio [19].The microcosm allows the existence of V. cholerae in non-culturable but viable conditions.During their human or animal passage, the sequence of events, which triggers a series of events and exchange of genetic material among the gut flora leading to rapid spread of drugresistant phenotypes both within and between bacterial species, remains to be defined [15,19].Thus selection of such drug-resistant clones can lead to seasonal epidemics of cholera with the emergence of new clones replacing the existing clones.
Phage typing still remains a useful tool in the study of the epidemiology of cholera and is a recognized scheme that has been extensively developed and evaluated by the authors' reference laboratory, NICED, Kolkata, India.The results of phage typing were consistent with the overall countrywide epidemiological data, [20] which report type 27 to be the predominant type.The present findings corroborated previous studies [20,21].
In conclusion, a sudden increase in antibiotic resistance as reported here cannot be ignored; future epidemics in the MAR serotypes may be imminent.
The current study underscores the substantial mobility in the genetic element of V. cholerae and emphasises the need for continuous monitoring to ensure awareness of the changing traits of these capricious pathogens.The importance of reporting all cases should be emphasized, with the ultimate goal being a thorough understanding of the constantly changing and obscure epidemiology and resistance patterns of V. cholerae.

Figure 1 .
Figure 1.Distribution of serotypes and serovars of V. cholerae isolated from stool samples