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.

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.

Assessment of different tests to detect methicillin resistant Staphylococcus aureus.

The heterogeneous expression of methicillin resistance in Staphylococcus aureus affects the efficiency of tests available to detect it. Not all laboratories have access to accurate molecular tests used for this purpose. This study compares the performances of four phenotypic tests used to detect methicillin resistant S. aureus (MRSA) with the mecA gene polymerase chain reaction. Two hundred thirty-seven S. aureus isolates were isolated from different patients visiting Sir Sundar Lal Hospital, Banaras Hindu University, Varanasi, India and subjected to cefoxitin and oxacillin disc diffusion tests, oxacillin minimum inhibitory concentration (MIC) test, and oxacillin screen agar test. The tests showed the following sensitivities and specificities, respectively: cefoxitin disc diffusion (98.5% and 100%), oxacillin disc diffusion (77.3% and 84.6%), oxacillin MIC (89.4% and 87.2%), and oxacillin screen agar (87.9% and 94.9%). The cefoxitin disc diffusion test can be the best method for routine detection of MRSA when molecular techniques are not available. We recommend the Clinical Laboratory Standards Institute (CLSI) cut-off point for determining cefoxitin resistance be reexamined to see if it should be revised from < or = 19 mm to < or = 20 mm.

Methicillin resistant Staphylococcus aureus: prevalence and antibiogram in a tertiary care hospital in western Nepal.

BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial and community infections. Its prevalence varies with country and with hospitals within a country. The current study estimates the prevalence of MRSA strains and investigates their antibiogram in western Nepal. METHODOLOGY: A total of 162 S. aureus strains were isolated from various clinical specimens, and antibiotic susceptibility tests were performed using disc diffusion, growth on oxacillin screen agar, and oxacillin minimum inhibitory concentration (MIC). RESULTS: One hundred and twelve (69.1%) strains were found to be MRSA, of which 37 (33.1%) were community acquired and 75 (66.9%) were hospital acquired. Of 112 MRSA strains, 45 (40.1%) were multi-drug resistant. All MRSA strains were found resistant to penicillin, and 91.9%, 87.4%, 77%, and 55.5% were resistant to amoxicillin, ampicillin, trimethoprim/sulfamethoxazole, and cephalexin, respectively. However, low resistance was observed with amikacin (19%), ciprofloxacin (26.5%), and norfloxacin (30.6%). All strains were sensitive to vancomycin. CONCLUSION: The reported rate of MRSA prevalence is alarming. Given the ability of MRSA to spread from person to person, it is necessary to adhere to rational use of antibiotics and to raise awareness among the concerned communities and tourists who visit this area.

Molecular typing of clinical Staphylococcus aureus isolates from northern India using coagulase gene PCR-RFLP.

Molecular typing of total 84 Staphylococcus aureus clinical isolates was performed using coagulase gene PCR. Out of 84 S. aureus strains total 33 different types of S. aureus strains were prevalent in this hospital and community. Types 2-7 and 9 were the most prevalent S. aureus strains accounting for more than 53% of total isolates. This technique is relatively inexpensive and is simple to perform and analyze.

High prevalence of multidrug-resistant MRSA in a tertiary care hospital of northern India.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial and community pathogen. The objectives of this study were to estimate the prevalence of multidrug-resistant MRSA strains in clinical specimens and to investigate the sensitivity pattern of these strains against various antibiotics used for treating hospitalized and out patients. Strains were identified using standard procedures, and their sensitivity pattern was investigated using such techniques as disc diffusion, minimum inhibitory concentration (MIC), and the mecA gene PCR. Among 783 isolates of S. aureus, 301 (38.44%) were methicillin-resistant, of which 217 (72.1%) were found to be multidrug-resistant. Almost all MRSA strains were resistant to penicillin, 95.68% were resistant to cotrimoxazole, 92.36% were resistant to chloramphenicol, 90.7% were resistant to norfloxacin, 76.1% were resistant to tetracycline, and 75.75% were resistant to ciprofloxacin. Vancomycin was the most effective drug, with only 0.33% of MRSA strains being resistant to it. It is concluded that antibiotics other than vancomycin can be used as anti-MRSA agents after a sensitivity test so as to preclude the emergence of resistance to it and that prevailing problems in chemotherapy will escalate unless indiscriminate and irrational usage of antibiotics is checked.

Emergence of vancomycin resistant Staphylococcus aureus (VRSA) from a tertiary care hospital from northern part of India.

BACKGROUND: Glycopeptides such as vancomycin are frequently the antibiotics of choice for the treatment of infections caused by methicillin resistant Staphylococcus aureus (MRSA). For the last 7 years incidence of vancomycin intermediate S. aureus and vancomycin resistant S. aureus (VISA and VRSA respectively) has been increasing in various parts of the world. The present study was carried out to find out the presence of VISA and VRSA in the northern part of India. METHODS: A total 1681 staphylococcal isolates consisting of 783 S. aureus and 898 coagulase negative staphylococci (CoNS) were isolated from different clinical specimens from various outpatient departments and wards. All S. aureus and 93 CoNS were subjected to MIC testing (against vancomycin, teicolplanin and oxacillin); Brain Heart Infusion (BHI) vancomycin screen agar test; disc diffusion testing, and PCR for mecA, vanA and vanB genes detection. RESULTS: Out of 783 S. aureus two S. aureus strains were found to be vancomycin and teicoplanin resistant (one strain with MIC 32 microg/ml and the other strain with MIC 64 microg/ml); six strains of S. aureus have shown to be vancomycin intermediate (two strains with MIC 16 microg/ml and four strains with MIC 8 microg/ml); and two strains with teicoplanin intermediate (MIC 16 microg/ml). One CoNS strain was resistant to vancomycin and teicoplanin (MIC 32 microg/ml), and two CoNS strains were intermediate to vancomycin and teicoplanin (MIC 16 microg/ml). All VRSA, VISA and vancomycin resistant CoNS had shown growth on BHI vancomycin screen agar (vancomycin 6 microg/ml) and were mecA PCR positive. None of these isolates have demonstrated vanA/vanB gene by PCR. CONCLUSION: The present study reveals for the first time emergence of VISA/VRSA from this part of world and indicates the magnitude of antibiotic resistance in and around the study area. The major cause of this may be unawareness and indiscriminate use of broad-spectrum antibiotics.