Naresh C. Sharma

Epidemiological study of Vibrio cholerae using variable number of tandem repeats

By conventional genetic methods, including pulse-field gel electrophoresis and multilocus sequence typing, most pathogenic, cholera toxin-positive O1 and O139 isolates of Vibrio cholerae cannot be distinguished. We evaluated relationships among 173 V. cholerae isolates collected between 1992 and 2007 from different geographic areas in India by analyzing five variable number of tandem repeat (VNTR) loci. Each VNTR locus was highly variable, with between 5 and 19 alleles. eburst analysis revealed four large groups of genetically related isolates. Two groups contained genotypes of isolates with the O139 serogroup (which emerged for the first time in epidemic form in 1992), with the other two groups containing O1 strains. In subsequent analysis, it was possible to track the spread of specific genotypes across time and space. Our data highlight the utility of the methodology as an epidemiologic tool for assessing spread of isolates in both epidemic and endemic settings.

Cholera outbreaks in India.

Cholera is a global health problem as several thousands of cases and deaths occur each year. The unique epidemiologic attribute of the disease is its propensity to occur as outbreaks that may flare-up into epidemics, if not controlled. The causative bacterial pathogen Vibrio cholerae prevails in the environment and infects humans whenever there is a breakdown in the public health component. The Indian subcontinent is vulnerable to this disease due its vast coastlines with areas of poor sanitation, unsafe drinking water, and overcrowding. Recently, it was shown that climatic conditions also play a major role in the persistence and spread of cholera. Constant change in the biotypes and serotypes of V. cholerae are also important aspects that changes virulence and survival of the pathogen. Such continuous changes increase the infection ability of the pathogen affecting the susceptible population including the children. The short-term carrier status of V. cholerae has been studied well at community level and this facet significantly contributes to the recurrence of cholera. Several molecular tools recognized altering clonality of V. cholerae in relation with the advent of a serogroup or serotype. Rapid identification systems were formulated for the timely detection of the pathogen so as to identify and control the outbreak and institute proper treatment of the patients. The antimicrobials used in the past are no longer useful in the treatment of cholera as V. cholerae has acquired several mechanisms for multiple antimicrobial resistance. This upsurge in antimicrobial resistance directly influences the management of the disease. This chapter provides an overview of cholera prevalence in India, possible sources of infection, and molecular epidemiology along with antimicrobial resistance of V. cholerae.

Molecular Insights into Antimicrobial Resistance Traits of Multidrug Resistant Enteric Pathogens isolated from India

Emergence of antimicrobial resistant Gram-negative bacteria has created a serious global health crisis and threatens the effectiveness of most, if not all, antibiotics commonly used to prevent and treat bacterial infections. There is a dearth of detailed studies on the prevalence of antimicrobial resistance (AMR) patterns in India. Here, we have isolated and examined AMR patterns of 654 enteric pathogens and investigated complete genome sequences of isolates from six representative genera, which in aggregate encode resistance against 22 antibiotics representing nine distinct drug classes. This study revealed that ~97% isolates are resistant against ≥2 antibiotics, ~24% isolates are resistant against ≥10 antibiotics and ~3% isolates are resistant against ≥15 antibiotics. Analyses of whole genome sequences of six extensive drug resistant enteric pathogens revealed presence of multiple mobile genetic elements, which are physically linked with resistance traits. These elements are therefore appearing to be responsible for disseminating drug resistance among bacteria through horizontal gene transfer. The present study provides insights into the linkages between the resistance patterns to certain antibiotics and their usage in India. The findings would be useful to understand the genetics of resistance traits and severity of and difficulty in tackling AMR enteric pathogens.

Characterization of Vibrio cholerae O1 strains that trace the origin of Haitian-like genetic traits

Vibrio cholerae O1 is the etiological agent of the severe diarrheal disease cholera. The bacterium has recently been causing outbreaks in Haiti with catastrophic effects. Numerous mutations have been reported in V. cholerae O1 strains associated with the Haitian outbreak. These mutations encompass among other the genes encoding virulence factors such as the pilin subunit of the toxin-co-regulated pilus (tcpA), cholera toxin B subunit (ctxB), repeat in toxins (rtxA), and other genes such as the quinolone resistance-determining region (QRDR) of gyrase A (gyrA), rstB of RS element along with the alteration in the number of repeat sequences at the promoter region of ctxAB. Given the numerous genetic changes in those Haitian isolates, we decided to investigate the possible origins of those variations in the Indian subcontinent. Thus, we determined the genetic traits among V. cholerae O1 strains in Delhi, India. A total of 175 strains isolated from cholera patients during 2004 to 2012 were analysed in the present study. Our results showed that all the tested strains carried Haitian type tcpA (tcpACIRS) and variant gyrA indicating their first appearance before 2004 in Delhi. The Haitian variant rtxA and ctxB7 were first detected in Delhi during 2004 and 2006, respectively. Interestingly, not a single strain with the combination of El Tor rtxA and ctxB7 was detected in this study. The Delhi strains carried four heptad repeats (TTTTGAT) in the CT promoter region whereas Haitian strains carried 5 such repeats. Delhi strains did not have any deletion mutations in the rstB like Haitian strains. Overall, our study demonstrates the sequential accumulation of Haitian-like genetic traits among V. cholerae O1 strains in Delhi at different time points prior to the Haitian cholera outbreak.

Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001–2006

Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004–2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004–2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera.

Comparative performance of some pear cultivars on southern aspect of wet temperate zone in Western Himalaya

The studies were conducted at experimental farm of Temperate Horticultural Research Station, Kotkhai to compare the performance of 12 pear cultivars for their fruit quality and leaf nutritional status. Various cultivars under study differed significantly with respect to their fruit physico-chemical characteristics and leaf nutrient contents. Cultivar Red Bartlett had highest fruit length (82.37 mm, whereas, fruit diameter (76.65 mm) and fruit weight (193.50 g) were recorded significantly higher in cultivar Max Red Bartlett. Cultivar Starking Delicious exhibited highest total soluble solids (16.05°Brix), TSS/acid ratio (66.27) and minimum titratable acidity (0.28%). The highest fruit firmness (15.40 lbs/inch2) was recorded in cv. Beurre Hardy, whereas significantly higher total sugars content (5.95%) and reducing sugars content (5.03) were registered by Doyenne du Comice and Kala Hathi, respectively. The cultivars Aval Number, Max Red Bartlett and Flemish Beauty exhibited highest leaf nitrogen (2.31%), phosphorus (0.212%) and potassium (1.96%) contents, respectively. On the basis of fruit physicochemical characteristics, Red Bartlett, Max Red Bartlett, Starking Delicious, Doyenne du Comice and Beurre Hardy were found suitable for cultivation in zone III of Himachal Pradesh.