Multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli in rescued Sloth bears (Melursus ursinus), India.

The study reports the multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli (CRE) isolated from rescued sloth bear (Melursus ursinus), India. Non-duplicate faecal samples from 21 adult rescued sloth bears were collected at once during 2015-2016 and processed for isolation of E. coli and antibacterial susceptibility pattern. From 21 samples, 45 E. coli were isolated and on phenotypic screening, 23 were MDR, 17 were ESBL producers, and five were carbapenem-resistant (CR). Three E. coli isolates (6.67%, 3/45) showed no resistance, however 42 isolates (93.33%, 42/45) exhibited resistant to at least one antibiotics. The MDR isolates carried beta-lactamase, chloramphenicol, aminoglycosides, tetracycline, fluroquinolone, and sulphadimidine resistance genes. All the phenotypic ESBL producing isolates harbored blaCTX-M genes. On genotypic screening, three CRE (60.0%, 3/5) were positive for blaNDM carbapenemase gene and efflux pump-mediated carbapenem resistance was detected in two CRE isolates (40.0%, 2/5) which were negative for carbapenemase genes. The CRE isolates (n = 5) also co-harbored AMR genes like blaTEM-1, blaAmpC, qnrA, qnrB, qnrS, tetA, tetB and sulI. Virulence screening of the resistant isolates detected the presence of Stx1(n = 1), Stx2 (n = 3), eaeA (n = 4) and hlyA (n = 3) genes. Plasmid incompatibility (Inc) typing revealed that two isolates harboured blaNDM-5 gene on Incl1 and one isolate on IncF plasmid. Apart from the NDM gene, the plasmids also carried tetracycline, beta-lactamase and quinolone resistance genes. The plasmid multilocus sequence typing (pMLST) of the E. coli Incl1 plasmid showed the Sequence Type (ST) 297. This appears to be the first report of MDR, ESBL producing and blaNDM-5 genes on Incl1 and IncF plasmids from rescued sloth bear.

Cross-sectional study on rotavirus A (RVA) infection and assessment of risk factors in pre- and post-weaning piglets in India.

Rotavirus A (RVA) infections are known to retard the piglets' growth and minimize the profit to the pig farming community. Between August 2014 and July 2017, in a cross-sectional study, we surveyed 13 organized pig farms located in the eight states of India representing northern, north-eastern and southern regions, to identify the risk factors associated with RVA infection in pre- and post-weaning piglets. Faecal samples (n = 411) comprising of non-diarrhoeic (n = 320) and diarrhoeic (n = 91) were collected and screened for RVA infection using VP6 gene-based RT-PCR. RVA positivity of 52.5% (168/320) in non-diarrhoeic and 59.3% (54/91) in diarrhoeic piglets was noticed. Further, 53.3% (120/225) and 54.8% (102/186) of the samples from pre- and post-weaned samples were positive for RVA, respectively. To note, no statistically significant association was noticed between RVA infection, health and weaning status. Additionally, a questionnaire-based survey was conducted to identify the risk factors for RVA infections in piglets. The analysis revealed that good ventilation (OR 0.2, 95% CI 0.15-0.39), use of deep well water (OR 0.2, 95% CI 0.13-0.43) and feeding of commercial feed (OR 0.3, 95% CI 0.18-0.41) were associated with reduced risk of RVA infection compared with poor ventilation, use of shallow well water and feeding of own milled feed, respectively. Contrarily, mixed farms (OR 2.1, 95% CI 1.26-3.37), use of heater or cooler (OR 5.9, 95% CI 3.74-9.30), sheds in different elevation (OR 2.5, 95% CI 1.20-5.01) and weekly and occasional use of disinfectant for surface cleaning (OR 1.8, 95% CI 1.12-2.96) were associated with higher RVA infection. Mitigating the risk factors might help in better health management of piglets and increase the economic return to pig farming community in the country.

Risk factor analysis, antimicrobial resistance and pathotyping of Escherichia coli associated with pre- and post-weaning piglet diarrhoea in organised farms, India.

A cross-sectional study was conducted from 2014 to 2017 in 13 organised pig farms located in eight states of India (Northern, North-Eastern and Southern regions) to identify the risk factors, pathotype and antimicrobial resistance of Escherichia coli associated with pre- and post-weaning piglet diarrhoea. The data collected through questionnaire survey were used to identify the risk factors by univariable analysis, in which weaning status, season, altitude, ventilation in the shed, use of heater/cooler for temperature control in the sheds, feed type, water source, and use of disinfectant, were the potential risk factors. In logistic regression model, weaning and source of water were the significant risk factors. The piglet diarrhoea prevalence was almost similar across the regions. Of the 909 faecal samples collected (North - 310, North-East - 194 and South - 405) for isolation of E. coli, pathotyping and antibiotic screening, 531 E. coli were isolated in MacConkey agar added with cefotaxime, where 345 isolates were extended spectrum ß-lactamase (ESBL) producers and were positive for blaCTX-M-1 (n = 147), bla TEM (n = 151), qnrA (n = 98), qnrB (n = 116), qnrS (n = 53), tetA (n = 46), tetB (n = 48) and sul1 (n = 54) genes. Multiple antibiotic resistance (MAR) index revealed that 14 (2.64%) isolates had MAR index of 1. On the virulence screening of E. coli, 174 isolates harboured alone or combination of Stx1, Stx2, eaeA, hlyA genes. The isolates from diarrhoeic and post-weaning samples harboured higher number of virulence genes than non-diarrhoeic and pre-weaning. Alleviating the risk factors might reduce the piglet diarrhoea cases. The presence of multidrug-resistant and ESBL-producing pathogenic E. coli in piglets appears a public health concern.

Epidemiology of carbapenem-resistant Escherichia coli and first report of blaVIM carbapenemases gene in calves from India.

A cross-sectional study on six dairy farms was conducted to ascertain the occurrence of carbapenem-resistant Escherichia coli in calves. Two-hundred and seventy-nine isolates of E. coli were recovered from 90 faecal samples from apparently healthy (45) and diarrhoeal (45) calves. The isolates were screened for phenotypic susceptibility to carbapenems and production of metallo ß-lactamase, as well as five carbapenemase resistance genes by PCR, and overexpression of efflux pumps. Eighty-one isolates (29.03%) were resistant to at least one of three carbapenem antibiotics [meropenem (23.30%), imipenem (2.15%) and ertapenem (1.43%)], and one isolate was positive for the blaVIM gene which was located on an Incl1 plasmid of a novel sequence type (ST 297) by multilocus sequence typing. The majority (83.95%) of isolates had an active efflux pump. Calves housed on concrete floors were approximately seven times more likely to acquire meropenem-resistant isolates than those housed on earthen floors (95% CI 1.27-41.54). In India, carbapenem drugs are not used in food animal treatment, hence carbapenem-resistant strains in calves possibly originate from the natural environment or human contact and is of public health importance. To our knowledge, this is the first report of blaVIM carbapenemases gene in calves from India.

Molecular characterisation of blaOXA-48 carbapenemase-, extended-spectrum ß-lactamase- and Shiga toxin-producing Escherichia coli isolated from farm piglets in India.

OBJECTIVES: The aim of this study was to characterise carbapenemase-, extended-spectrum ß-lactamase (ESBL)- and Shiga toxin-producing Escherichia coli isolated from farm piglets in India. METHODS: Faecal samples (n=741) from 10 organised pig farms, including non-diarrhoeic (n=546) and diarrhoeic (n=195) piglets, were processed for isolation of carbapenem-resistant and ESBL-producing E. coli. RESULTS: A total of 27 and 243 isolates were phenotypically confirmed as carbapenem-resistant and ESBL-producers, respectively. The meropenem minimum inhibitory concentration (MIC) of carbapenem-resistant isolates ranged from 8-128µg/mL. On genotypic screening of the 27 carbapenem-resistant isolates, 3 isolates were positive for the blaOXA-48 carbapenemase gene; no other carbapenemase genes were detected. The 243 ESBL-producing isolates were positive for blaCTX-M-1 (n=135), qnrA (n=92), qnrB (n=112), qnrS (n=49), tetA (n=42), tetB (n=45) and sul1 (n=43). The Shiga toxin virulence markers stx1 and stx2 were detected in 41 and 38 of the 243 phenotypic ESBL-producing isolates, respectively. Multilocus sequence typing (MLST) of blaOXA-48-positive E. coli isolates showed ST10- and ST5053-like sequence types. CONCLUSION: This is the first report on the presence of blaOXA-48-carrying E. coli in piglets in India, which pose a potential risk to public health.

Spatial molecular epidemiology of carbapenem-resistant and New Delhi metallo beta-lactamase (blaNDM)-producing Escherichia coli in the piglets of organized farms in India.

AIM: A cross-sectional study was conducted in 10 government-organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli. METHODS AND RESULTS: In this study, fecal sample (n = 673) from non-diarrheic (n = 501) and diarrheic (n = 172) piglets were processed for isolation of carbapenem resistant E. coli. Of 673, E. coli isolate (n = 112) was genotyped for confirming the carbapenem resistance and associated virulence factors. Of the 112 isolates, 23 were phenotypically resistant to carbapenem and 8 were carrying the New Delhi metallo beta-lactamase (blaNDM) gene. The carbapenem-resistant isolates also produced extended spectrum beta-lactamases and were multidrug resistant. The PCR-based pathotyping revealed the presence of stx1, stx2, eae and hlyA genes. The enterobacterial repetitive intergenic consensus PCR dendrogram analysis of the isolates yielded three distinct clusters. The statistical analysis revealed no association between carriages of carbapenem-resistant E. coli in different breed of piglets however, location, sex, health status of piglets and age showed significant difference. The spatial analysis with SaTScan helped in identification of carbapenem-resistant clusters. CONCLUSIONS: The presence of carbapenem resistant E. coli isolates with virulence genes in the piglet poses a potential public health risk through possible access and spread via the food chain and environment. Efflux pump may also play an important role in carbapenem resistance in piglet E. coli isolates. Furthermore, identification of risk factors in relation to spatial clusters will help in designing preventive strategies for reducing the risk of spread of carbapenem resistant bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: 1. Piglets harbor carbapenem resistant E. coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India.