Distribution of Antibiotic-Resistant Enterobacteriaceae Pathogens in Potable Spring Water of Eastern Indian Himalayas: Emphasis on Virulence Gene and Antibiotic Resistance Genes in Escherichia coli.

Every year millions of people die due to fatal waterborne diseases around the world especially in developing countries like India. Sikkim, a northeastern state of India, greatly depends on natural water sources. About 80% of the population of Sikkim depends on natural spring water for domestic as well as agricultural use. Recent waterborne disease outbreaks in the state raises a concerning question on water quality. In this study, we analyzed water quality especially for the detection of Enterobacteriaceae members from four districts of the state. Isolation with selective culture media techniques and taxonomic characterization of Enterobacteriaceae bacteria with 16S rRNA gene showed the prevalence of Escherichia coli (37.50%), Escherichia fergusonii (29.41%), Klebsiella oxytoca (36.93%), Citrobacter freundii (37.92%), Citrobacter amalonaticus (43.82%), Enterobacter sp. (43.82%), Morganella morganii (43.82%), Hafnia alvei (32.42%), Hafnia paralvei (38.74%), and Shigella flexneri (30.47%) in the spring water of Sikkim. Antibiotic susceptibility test (AST) showed resistance of the isolates to common antibiotics like ampicillin, amoxicillin as well as to third generation antibiotics like ceftazidime and carbapenem. None of the isolates showed resistance to chloramphenicol. E. coli isolated from spring water of Sikkim showed presence of different virulence genes such as stx1 (81.81%), elt (86.66%), and eae (66.66%) along with resistance gene for ampicillin (CITM) (80%), quinolones (qnrB) (44.44%), tetracycline (tetO) (66.66%), and streptomycin (aadA1) (66.66%). The data indicates a high incidence rate of multiple antibiotic resistant enteric bacteria in the spring water of Sikkim. Additionally, the presence of enteric bacteria in the water samples indicates widespread fecal contamination of the spring water.

High prevalence of carbapenemase, AmpC ß-lactamase and aminoglycoside resistance genes in extended-spectrum ß-lactamase-positive uropathogens from Northern India.

OBJECTIVES: This study investigated the occurrence of extended-spectrum ß-lactamase (ESBL) genes coexisting with carbapenemase, AmpC and aminoglycoside resistance gene in uropathogens in India. METHODS: Antimicrobial susceptibility testing was performed by disk diffusion. Antimicrobial resistance genes were detected by multiplex PCR. RESULTS: Of 1516 consecutive urine samples, 454 (29.9%) showed significant bacteriuria with a single micro-organism, predominantly Escherichia coli (n=343), followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the uropathogens, 61 ESBL-producers were identified containing blaCTX-M-15 (n=32), blaCTX-M-15+blaOXA-2 (n=15), blaCTX-M-15+blaOXA-2+blaTEM-1 (n=6), blaOXA-2 (n=5), blaOXA-2+blaSHV-76 (n=1), blaTEM-1+blaSHV-76 (n=1) and blaTEM-1 (n=1). All ESBL genes were located on horizontally transferable plasmids of incompatibility types HI1, I1, FIA+FIB, FIA and Y. Among the 61 ESBL-producers, 59 harboured carbapenemase genes, including blaNDM-5 (n=48), blaNDM-5+blaOXA-48 (n=5), blaNDM-5+blaIMP (n=5) and blaNDM-5+blaIMP+blaVIM (n=1). ESBL-producing uropathogens also harboured 16S rRNA methylase genes, including rmtB (n=9), rmtA (n=4), rmtC (n=1) and armA (n=1). ESBL-positive isolates also contained AmpC genes, including blaCIT (n=8) and blaDHA-1 (n=1). Imipenem and gentamicin had the lowest resistance rates against the uropathogens. CONCLUSION: This is the first report showing the high prevalence of carbapenemases in ESBL-positive isolates in this area. Regular surveillance for such resistance mechanisms will be useful for health personnel to treat infections by these multidrug-resistant pathogens.

Physicochemical Parameters and Alarming Coliform Count of the Potable Water of Eastern Himalayan State Sikkim: An Indication of Severe Fecal Contamination and Immediate Health Risk.

Continuous decline in potable water sources has raised serious concerns over human health. Developing countries are the most affected in this regard due to a lack of proper hygiene maintenance. Sikkim, an Eastern Himalayan state with mountains as the predominant topological features, harbors several perennial natural springs. Spring water is the primary source of potable water for the population in four districts of the state viz. East, West, North and South. Recent outbreaks of water-borne diseases and the relative lack of scientific studies on its potential correlation with the water quality of the area have educed this study. Physicochemical parameters of springs, community reservoirs, and household water were analyzed by ICP-MS and multi probe meter. Using the membrane filtration method, the microbial quality of the water samples during different seasons was assessed, primarily evaluating the presence of fecal indicators viz. Escherichia coli, total coliform and Enterococcus. The seasonal risk category of the water sources was also determined. Most of the physicochemical parameters of the spring water were within the permissible limits of WHO standards. However, water from four districts was recorded with traces of toxic heavy metals like mercury (0.001-0.007 mg/l), lead (0.001-0.007 mg/l), and selenium (0.526-0.644 mg/l), which are above the permissible limits of WHO. All the spring water samples were categorized as Mg-HC O 3 - type and can be predicted as shallow fresh ground water based on the piper analysis. Microbial confirmatory testing indicated severe fecal contamination of water sources with high counts of total coliform (TC), Escherichia coli (EC) and Enterococcus (EN). The highest level of TC was recorded from West Sikkim (37.26 cfu/100 ml) and the lowest in North Sikkim (22.13 cfu/100 ml). The highest level of contamination of E. coli and Enterococcus was found in East Sikkim (EC = 8.7 cfu/100 ml; EN = 2.08 cfu/100 ml) followed by South Sikkim (EC = 8.4 cfu/100 ml; EN = 2.05 cfu/100 ml). There was a significant positive correlation between the contamination levels of the spring water and the community reservoir tank. As far as the seasonal variation is concerned, the rainy season showed the most contamination with coliform correlating with a high incidence of different water-borne diseases (East = 86%; West = 100%; South = 100%; North = 80%).

Prevalence of antibiotic resistance in commensal Escherichia coli among the children in rural hill communities of Northeast India.

Commensal bacteria are the representative of the reservoir of antibiotic resistance genes present in a community. The usage of antibiotics along with the demographic factors is generally associated with an increase in antibiotics resistance in pathogens. Northeast (NE) India is untapped with regard to antibiotic resistance prevalence and spread. In the current study, the prevalence of antibiotic-resistant commensal Escherichia coli in pre-school and school-going children (n = 550, 1-14 years old) from the rural areas of the state of Sikkim-an NE Indian state, with respect to associated demographic factors was investigated. A total of 550 fecal E. coli isolates were collected during July 2015 to June 2017. A structured questionnaire was used to collect data to ascertain the potential factors associated with the carriage of antibiotic resistance E. coli among the children. Statistical analysis along with a logistic regression identified potential external factors affecting the observed antibiotic resistance pattern. The data indicated a high prevalence of resistance to common antibiotics like ampicillin (92%), ceftazidime (90%), cefoxitin (88%), streptomycin (40%) and tetracycline (36%), but no resistance to chloramphenicol. The resistance to the combination of penicillin and quinolone group of antibiotics was observed in fifty-two percent of the isolates. A positive correlation between the harboring of antibiotics resistant E. coli with different demographic factors was observed such as, with children living in nuclear family (vs joint family 63.15%, OR 0.18, 95% CI:0.11-0.28, p < 0.01), below higher secondary maternal education (vs college graduates 59.27% OR 0.75, 95% CI:0.55-1.02, p < 0.02). A close association between different demographic factors and the high prevalence of antibiotic-resistant commensal E. coli in the current study suggests a concern over rising misuse of antibiotics that warrants a future threat of emergence of multidrug-resistant pathogen isolates.

Escherichia coli encoding blaNDM-5 associated with community-acquired urinary tract infections with unusual MIC creep-like phenomenon against imipenem.

OBJECTIVES: Carbapenemase-producing Escherichia coli are a major clinical concern. The current study aimed to identify NDM-5-producing E. coli associated with community-acquired urinary tract infections (UTIs) co-harbouring extended-spectrum ß-lactamases (ESBLs) and showing a phenomenon of imipenem minimum inhibitory concentration (MIC) creep. METHODS: A total of 973 urine samples were collected from females aged between 18-49 years diagnosed with UTI in Northeast India (June 2014-July 2016). Isolates were identified by standard microbiological methods. The presence of blaNDM and ESBL genes was determined by PCR and sequencing. PCR-based replicon typing was performed. Plasmid stability of all ß-lactamase-producers and their transformants was analysed by serial passage, and the MIC creep phenomenon was analysed by studying revertants. RESULTS: Among 34 blaNDM-5-positive E. coli isolates, 21 (61.8%) co harboured blaCTX-M-15, followed by multiple combinations of genes. This study revealed diverse plasmid types (HI1, I1, FIA+FIB, FIA and Y). The strains showed progressive plasmid loss after 31 passages. Most if the isolates had MICs of 0.5µg/mL and 1µg/mL to imipenem, ertapenem and meropenem. However, on studying the MIC creep phenomenon, the MIC was found to be elevated from 0.5µg/mL to 64µg/mL and from 1µg/mL to 128µg/mL. Analysis of revertants shows that the MIC of most NDM-positive isolates was reduced to 16µg/mL after the 30th serial passage. CONCLUSION: This study observed a unique phenotype of NDM-producers that has not been reported previously. The observed phenomenon poses a global threat as these pathogens may evade phenotypic screening by routine laboratories.

The First Report of Phenotypic and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Uropathogens in Sikkim and Darjeeling Hills of India.

Extended-spectrum ß-lactamase (ESBL)-producing bacteria are a global health threat both in hospital and in community settings. The emergence of these organisms poses major difficulty in treating infections. This study was carried out to assess major ESBL-producing uropathogens in female patients of Sikkim and Darjeeling by phenotypic and genotypic methods. Out of 1,516 urine samples, 454 uropathogens were isolated with a prevalence rate of 29.94%. Among them, Escherichia coli (74.3%) was the predominant type followed by Klebsiella pneumoniae (20.1%), Pseudomonas aeruginosa (2.4%), and Proteus mirabilis (1.98%). Four different ESBL genes were detected in 63 isolates, which included CTX-M (n = 32), CTX-M+OXA-2 (n = 15), CTX-M-15+OXA-2+TEM (n = 6), OXA-2 (n = 5), TEM+CTX-M-15 (n = 2), TEM+OXA-2+SHV-76 (n = 2), and TEM (n = 1). All ESBL genes (bla genes) were found on a plasmid, which was mostly of HI1, I1, FIA+FIB, FIA, and Y types and was horizontally transferable. Among all ESBL genes, blaCTX-M-I5 group was the most prevalent. The study of urinary tract infection (UTI) caused by ESBL-producing bacteria needs to be studied in other high-altitude parts of India to understand the actual burden of UTI in the female.

Prevalence of gastrointestinal disease and its associated risk factors in Sikkim and Darjeeling Districts.

The gastrointestinal disease accounts for a large number of deaths in several parts of the world. Gastrointestinal infection has been an emerging problem in Sikkim and Darjeeling District and also in other parts of our country. To study the prevalence and to explore the risk factors associated with gastrointestinal diseases in Sikkim and Darjeeling District. The present study is the population based descriptive type cross sectional study. The study design was based on random selection among 100 individuals from different areas of Sikkim and Darjeeling district of West Bengal. Questionnaire based anonymous feedback system was followed to collect the data. The data were analyzed using statistical tool and the relative risk was calculated. Total 65 (65%) cases of gastrointestinal disease were found in 100 individuals out of which 24 were males and 41 were females. Cases of diarrhea, gastroenteritis, dysentery, food poisoning, amoebiosis and enterocolitis was 34, 18, 3, 3, 1 and 0% respectively. The statistical analysis reveals that a gastrointestinal disease is more prevalent in females as compared to males and in the age group between 15 and 25 years. The various associated risk factors for gastrointestinal disease which was observed during the study were frequency of diet, diet type, consumption of spicy food, fermented food, smoking, consumption of alcohol, consumption of fruits available in market and an inappropriate sanitary condition.