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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.

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 β-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 β-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 0.5 μg/mL and 1 μg/mL to imipenem, ertapenem and meropenem. However, on studying the MIC creep phenomenon, the MIC was found to be elevated from 0.5 μg/mL to 64 μg/mL and from 1 μg/mL to 128 μg/mL. Analysis of revertants shows that the MIC of most NDM-positive isolates was reduced to 16 μg/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.

Increasing population, rapid urbanization and climate change have immensely affected the freshwater sources around the world. The continuous decline in the number of natural potable water sources raises serious concern about the overall health of the human population. Developing countries are the most affected in this regard due to lack of proper hygienic maintenance protocols. Sikkim an Eastern Himalayan state with mountains as predominant topological features, harbors several natural spring water (SW). These spring waters are the primary source of potable water for the population in the four districts of the state viz. East (E), West (W), South (S) and North (N). Several incidences of water-borne diseases and the relative lack of scientific evaluation reports on the water quality of the area have educed this study. Lack of any standard filtration and purification practice among the population is one of the prime factors for the outspread of different waterborne pathogen in the state. The people of the state mostly use boiling as a dominant method of water purification, while only a small percentage of people in the West district were found to use modern standard purification system (W = 30%). The rainy season was found to be the major contributor of different diseases (E = 86%; W=100%; S=100%; N=80%) and statistical analysis of the fecal coliforms of the different season also indicated a significant difference at p < 0.05. There was no statistical significance among the physicochemical parameter of the SW but surprisingly the water from the four districts was recorded with traces of highly toxic heavy metals like mercury/WHO limit (0.001-0.007mg/l/0.001) as well as lead/WHO limit (0.001-0.007mg/l/0.05) and selenium/WHO limit (0.526-0.644 mg/l/0.01) which was above the WHO permissible limit. Piper analysis showed that water was dominated by cation sodium ion (Na+) and bicarbonate (HCO3-) anion and the water can be categorized as Mg-HCO3- type. Pairwise Pearson correlation showed a significant correlation between Electrical conductivity and TDS (r = 0.998/1.00) as well as alkalinity and turbidity (0.993/1.00). The microbial confirmatory test showed severity in fecal contamination with high counts of Total Coliform (TC), Escherichia coli (EC) and Enterococcus (EN). Highest TC was recorded from W (37.26/ml) and lowest in N (22.13/ ml) in spring water. Highest contamination of Escherichia coli and Enterococcus was found in E (EC = 8.7/ml; EN = 2.08/ml) followed by S (EC = 8.4/ml and EN = 2.05/ml). It was found that community reservoir (CR) tank was more contaminated than SP, which indicates the negligence in maintenance and fecal contamination during transportation to the reservoir. Though household water was least contaminated compared to CR and SP, but it fails in WHO standard criteria for drinking water. These results indicate an immediate health risk of the resident of the state and which needs to be taken care of sooner as possible by protecting the important potable sources with required policies and regulations. Keywords: Sikkim, Springs, Community reservoir, Household water, pH, TDS, Alkalinity, E. coli, Enterococcus, Total coliform, Correlation coefficient.

Objectives This study investigated the occurrence of extended-spectrum β-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.

Extended-spectrum β-lactamase (ESBL) producing bacteria acts as a serious threat, and its co-existence with other antibiotic resistant gene makes the clinical scenario worse nowadays. Therefore in this study, we investigated the occurrence of ESBL genes coexisting with carbapenem, AmpC and aminoglycoside resistance gene in uropathogens. Out of 1516 urine samples, 454 showed significant bacteriuria with a prevalence rate of 29.94 %. Escherichia coli (n=340) were found to be the most predominant uropathogen followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the total uropathogens, sixty-three ESBL-producers were identified which included blaCTX-M-15 (n=32), followed by blaCTX-M-15 + blaOXA-2 (n=15), blaCTX-M-15 + blaOXA-2 + blaTEM (n=6), blaOXA-2 (n=5), blaOXA-2 + blaSHV-76 (n=1), blaTEM+SHV-76 (n= 1) and blaTEM (n=1). All ESBL genes were found on plasmid incompatibility types: HI1, I1, FIA+FIB, FIA and Y and were horizontally transferable. Among 63 ESBL-producers, 59 isolates harboured carbapenem-resistant genes which included blaNDM-5 (n=48), blaNDM-5 + blaOXA-48 (n=5), blaNDM-5 + blaIMP (n=5) and blaNDM-5 + blaIMP + blaVIM (n=1). The ESBL producing uropathogens also harbored 16S rRNA methylase genes which included rmtB (n=9), rmtA (n=4), rmtC (n=1) and ArmA (n=1) followed by AmpC genes which includes CIT (n=8) and DHA-1 (n=1) genes. Imipenem and gentamicin were found to be more effective. We speculating, this is the first report showing the prevalence of multidrug-resistant uropathogens in this area demanding regular surveillance for such resistance mechanisms which will be useful for health personnel to treat ESBL infection and its co-existence with another antibiotic resistance gene.

Water quality of a region directly corroborates with the health index of people. People in the Himalayan hills mainly depend upon the spring water for potability. To determine the microbial ecology of the spring waters of Sikkim, the variable region of 16S rRNA has been sequenced using Illumina MiSeq. Phylum wise annotation showed the East and North district are mostly dominated byProteobacteria(41% and 35.80%), whereas West and South district is dominated byPlanctomycetes(38.46%) andVerrucomicrobia(33%). The consistent dominance phyla in the all the four districts wereBacteriodetes(34-24%) which was highest dominancy in North district and lowest in wets district. Genus wise distribution showed the abundance ofBrevifolis, Flavobacterium, Verrucomicrobia subdivision3, Emticica, Cytophaga, Prosthecobacter, Planctomycetes, Varivorax, Arcicella, Isosphera, Sedimunibacteriumetc. The East district showed highest dominancy of genusEmticiciawhereasPlanctomycetesin the West district. The North district was mainly dominated by genusArcicellaandBrevifollisin the South district. North on the antonymous showed totally different sets of microbial diversity. North district showed an abundance ofArcicella, Planctomycetes, SchlerensiaandAzohydromonas. The heat map produced by Bray Curtis distance method produced three clusters which showed the close relationship between West and East district microbiome that further related to South district. The sample of North district formed out group that showed different community structure from other three districts. The principle component analysis was showed that the east and South district samples are closely related and distantly correlated to the west Sikkim, but the North district showed completely different microbial community. The canonical correspondence analysis showed correlation between bacterial diversity and hydrochemistry and it was found that the bacterial diversity was influenced by the concentration of different metallic ions like sodium, calcium, barium and iron. This is a first report from the Eastern Himalayan region of India and it largely enhances our knowledge about the microbial structure of potable spring water of Eastern Himalayan. This study is useful for Government of India as well as the state government to adopt the different strategic treatment procedures to improve the quality of water that is supplied to the community resides in the Himalayan regions and solely dependent on this untreated spring water.

Extended-spectrum β-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.

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 β-lactamases (ESBLs) and showing a phenomenon of imipenem minimum inhibitory concentration (MIC) creep.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 blaAmong 34 blaThis 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.

Commensal bacteria are the representative of the reservoir of antibiotic resistance genes present in a community. Merely a few community-based studies on the prevalence of antibiotic resistance in commensal bacteria have been conducted so far in Southeast Asia and other parts of India. Northeastern India is still untapped regarding the surveillance of antibiotic-resistant genes and prevalence in commensal bacteria. In the present work, the prevalence of antibiotic resistance in commensal Escherichia coli was investigated along with the associated demographic factors in pre-school and school going children in rural areas of Sikkim. A total of 550 fecal E. coli isolates were obtained from children of the age 1-14 years living in different villages at various altitudes of Sikkim from July 2015 to June 2017. Standard antibiotic susceptibility testing of these isolates was performed. A structured questionnaire was designed to study the factors associated with carriage of antibiotic resistance in commensal E. coli isolates among children. Descriptive statistics analysis and a logistic regression model were used to identify the effect of external factors on antibiotic resistance pattern. High prevalence of resistance was found against commonly used antibiotics ampicillin (92%), ceftazidime (90%), cefoxitin (88%), streptomycin (40%) and tetracycline (36%) among the samples examined in our present study. No resistance to chloramphenicol was recorded. Fifty-two percent of the isolates were resistant to the combination of penicillin and quinolone group of antibiotics. Children living in nuclear families showed higher incidence of resistance to ampicillin (63.15%, OR 0.18,95% CI:0.11 – 0.28, pE. coli against the commonly used antibiotics among children in the study area. A close association between different demographic factors and the pattern of carriage of antibiotic-resistant isolates was observed suggesting a concern over misuse of antibiotics and warrants a future threat of emerging multidrug resistant isolates.

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.

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.