In-silico insights of ESBL variants and tracking the probable sources of ESBL-producing Escherichia coli in a small-scale poultry farm.

Commercial broiler farms face challenges of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli transmitted from both vertical and horizontal routes. Understanding the dynamics of ESBL-E. coli transmission in compromised biosecurity settings of small-scale rural poultry farms is essential. This study aimed to elucidate the probable transmission pathways of ESBL-E. coli in such settings, employing phylogenetic analysis and molecular docking simulations to explore the catalytic properties of ß-lactamase variants. Sampling was conducted on a small-scale poultry farm in West Bengal, India, collecting 120 samples at three intervals during the broiler production cycle. E. coli isolates underwent resistance testing against eight antimicrobials, with confirmation of ESBL production. Genotypic analysis of ESBL genes and sequencing were performed, alongside molecular docking analyses and phylogenetic comparisons with publicly available sequences. Among 173 E. coli isolates, varying resistance profiles were observed, with complete resistance to cefixime and high resistance to amoxicillin and tetracycline. The incidence of ESBL-E. coli fluctuated over the production cycle, with dynamic changes in the prevalence of blaCTX-M-type and blaSHV-type genes. Phylogenetic analysis indicated partial clonal relationships with human clinical strains and poultry strains from the Indian subcontinent. Molecular docking confirmed the catalytic efficiencies of these ESBL variants. The study highlights probable vertical transmission of ESBL-E. coli and emphasizes drinking water as a potential source of horizontal transmission in small-scale poultry farms. Strict biosecurity measures could prevent the spread of antimicrobial-resistant bacteria in birds and their products in a small scale poultry farm.

Docking analysis of circulating CTX-M variants in multi-drug resistant, beta-lactamase and biofilm-producing E. coli isolated from pet animals and backyard livestock.

The generation of antimicrobial-resistant bacteria largely depends on the use of antimicrobials not only in humans but also in pet animals and livestock. The present study was conducted to detect the occurrence of beta-lactamase and biofilm-producing- E.coli in healthy pet and backyard livestock. The study also intended on molecular docking experiments to confirm the nature of the catalytic mechanism in ß-lactamase enzymes, encoded by the various blaCTX-M genotypes and phylogenetic analysis to reveal clonal relationship of the animal origin E. coli isolates with human clinical strains. The rectal swabs were collected from healthy dogs (n = 254), cats (n = 108), sheep (n = 119) and goats (n = 143) in India. In total 247 (76.47%) E. coli strains were identified as ESBL producers. The possession of ESBL-producers was significantly more (p < 0.05) in pets than in the backyard livestock. Most of the strains possessed blaCTX-M-15 like clones. E. coli strains possessing blaCTX-M-15.2, blaCTX-M-157, blaCTX-M-181 and blaCTX-M-218 like clones, isolated from pets were not reported earlier. The study detected 56.65% of E. coli strains as moderate or strong biofilm producers possessing biofilm-associated genes (csgA, rcsA, rpoS, sdiA). ESBL-producing E. coli showed phenotypical resistance to tetracycline (93.1%), azithromycin (89.8%), ampicillin (84.2%), cefotaxime (80.9%), doxycycline (82.5%), co-trimoxazole (80.9%), ampicillin/cloxacillin (76.9%). The CTX-M variants obtained in this study were modelled by the SWISS-MODEL and verified. Ligand having minimum binding energy, show the highest affinity of ß-lactamases for cefotaxime and cefpodoxime. The Gibbs free energy release for all 14 different complex ranges between -6.9 (CTX-M-15.2+cefpodoxime) to -5.3 (CTX-M-218+cefpodoxime) Kcal/mol. Phylogenetic analysis of the animal origin ESBL-E. coli strains revealed a partial clonal relationship with the clinical isolates of local human patients. The present study described the significant presence of biofilm and ß-lactamase producing, multi-drug resistant E. coli in pet animals having public health importance.

Companion Animals Emerged as an Important Reservoir of Carbapenem-Resistant Enterobacteriaceae: A Report from India.

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) are perceived as a serious public-health threat world-wide. Despite sporadic reports, no systemic study has been carried out on CRE in companion animals in Indian subcontinent. In total, 237 canine specimens collected from five veterinary polyclinics in and around Kolkata were analyzed for isolation, antimicrobial resistance profiling and molecular characterization of carbapenem-resistant (CR) E. coli. Of the 29 CR isolates, 19 were identified as metallo-ß-lactamase producers (MP-CRE) and 10 as metallo-ß-lactamase non-producers (MNP-CRE). Eleven of them were extended spectrum ß-lactamase and/or AmpC type ß-lactamase producers and harboured fluoroquinolone-, tetracycline-, sulfonamide- and aminoglycoside-resistant genes. Beside uropathogenic virulence determinants, they carried the adhesion factors mediating biofilm production which was remarkably higher in 6 MP-CRE and one MNP-CRE isolates. Although the CRE were of diverse origin including the healthy and the diseased dogs, these were more frequently isolated from canine pyometra. The MP-CRE harboured plasmids of IncF and IncA/C types. Phylo-type B1 was observed in 38% of the CR isolates, followed by A0 in 31% and rest were attributed to A1 and D1. The Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) revealed that these isolates were genetically diverse and constituted of a heterogenous population. Detection of CRE in pet dogs despite the fact that carbapenems are not used in animals in India emphasizes the need for active surveillance to identify the transmission and dynamics of such pathogens in companion animals.

Edible Mushrooms as Functional Ingredients for Development of Healthier and More Sustainable Muscle Foods: A Flexitarian Approach.

Consumers are increasingly interested in nutritious, safe and healthy muscle food products with reduced salt and fat that benefit their well-being. Hence, food processors are constantly in search of natural bioactive ingredients that offer health benefits beyond their nutritive values without affecting the quality of the products. Mushrooms are considered as next-generation healthy food components. Owing to their low content of fat, high-quality proteins, dietary fibre and the presence of nutraceuticals, they are ideally preferred in formulation of low-caloric functional foods. There is a growing trend to fortify muscle food with edible mushrooms to harness their goodness in terms of nutritive, bioactive and therapeutic values. The incorporation of mushrooms in muscle foods assumes significance, as it is favourably accepted by consumers because of its fibrous structure that mimics the texture with meat analogues offering unique taste and umami flavour. This review outlines the current knowledge in the literature about the nutritional richness, functional bioactive compounds and medicinal values of mushrooms offering various health benefits. Furthermore, the effects of functional ingredients of mushrooms in improving the quality and sensory attributes of nutritionally superior and next-generation healthier muscle food products are also highlighted in this paper.

Application of nanoemulsion-based approaches for improving the quality and safety of muscle foods: A comprehensive review.

Recently, there has been growing interest in implementing innovative nanoscience-based technologies to improve the health, safety, and quality of food products. A major thrust in this area has been to use nanoemulsions because they can easily be formulated with existing food ingredients and technologies. In particular, oil-in-water nanoemulsions, which consist of small oil droplets (<200 nm) dispersed in water, are being utilized as delivery systems for various hydrophobic substances in foods, including nutrients, nutraceuticals, antioxidants, antimicrobials, colors, and flavors. In this article, we focus on the application of nanoemulsion-based delivery systems for improving the quality, safety, nutritional profile, and sensory attributes of muscle foods, such as meat and fish. The article also critically reviews the formulation and fabrication of food-grade nanoemulsions, their potential benefits and limitations in muscle food systems.

Dragon fruit (Hylocereus undatus) peel as antioxidant dietary fibre on quality and lipid oxidation of chicken nuggets.

In this study, the efficacy of dragon fruit peel (DFP) powder as antioxidant dietary fibre (ADF), at two different concentrations (T1-1.5% and T2-3.0%), on quality improvement and susceptibility to lipid oxidation of chicken nuggets during 20 days of refrigerated storage was assessed. DFP, rich in dietary fibre (56.91%) with higher insoluble dietary fibre, phenolics (36-39 mgGAE/100 g) content and possessing good radical scavenging activity as well as reducing power, contained 10.36% protein, 4.48% fat and 2.34% ash. HPLC analysis revealed presence of high concentrations of gallic and ferulic acid, among the phenolics. Incorporation of DFP in nuggets although decreased the pH but improved emulsion stability as well as cooking yield and had higher protein, ash and lower fat content. Further, the treated nuggets had significantly (p < 0.05) higher dietary fibre and total phenolics content than control. Incorporation of DFP decreased the hardness, gumminess and chewiness and improved (p < 0.05) the products' redness values. Sensory evaluation of the products revealed significant improvement in the appearance score and non-significant (p > 0.05) increase in the scores of other attributes compared to control samples. DFP significantly decreased lipid peroxidation, odour scores and microbial load in chicken nuggets during 20 days of storage period. From the study, it could be deduced that DFP rich in bioactive components had positive influence on the nutritional quality of chicken nuggets and could also be used as ADF in muscle food without affecting the quality and acceptability of products.

Virulence repertoire, characterization, and antibiotic resistance pattern analysis of Escherichia coli isolated from backyard layers and their environment in India.

This study was undertaken to observe the prevalence, serogroup, avian pathogenic Escherichia coli (APEC)-associated virulence gene, randomly amplified polymorphic DNA (RAPD) pattern, and antibiotic resistance genes of E. coli in backyard layers and their environment in India. From the 360 samples of healthy layers and their environment, 272 (75.5%) E. coli were isolated. The majority (28.67%) of them were untypeable. Among the studied virulence genes (papC, tsh, iucC, astA), 52 (14.32%) isolates were found to possess astA, including the isolates from the drinking water of the birds (4/272, 1.47%). These strains belonged to 18 different serogroups. Most of the isolates were typeable by RAPD and they produced different patterns. Phenotypic resistance of the isolates was most frequently observed to erythromycin (95.83%), chloramphenicol (87.52%), and cotrimoxazole (78.26%). None of the isolates was found to possess extended-spectrum beta-lactamases (bla(TEM), bla(SHV), bla(CTX-M) or quinolone resistance (qnrA) genes by PCR. The present study was the first attempt in India to assess APEC distribution in backyard poultry production.

Stray Dogs (Mongrels) Are Potent Reservoir of Drug-Resistant Pathogens: A Study in Peri-Urban Areas of Kolkata, India.

This study depicts the drug-resistance and phylogenomic characteristics of 365 Escherichia coli (EC) and 76 Klebsiella pneumoniae (KP) isolated from stray dogs (293) in and around Kolkata, India. Initial screening found 59 isolates, including 48 E. coli and 11 KP multidrug resistant, which included 33 extended-spectrum ß-lactamase, 41 AmpC ß-lactamase and 18 metallo-ß-lactamase producers carrying blaNDM-1 (11) and blaNDM-5 (7) genes. Majority of them had the resistant genes such as blaCTX-M (33), blaTEM (18), blaSHV (4), blaOXA (17), blaFOX (2), blaDHA (2), blaCITM (15), blaCMY-2 (13), blaGES (2) and blaVEB (2), qnrS (15), qnrB (3), aac-6'-Ib-cr (14), tetA (26), tetB (14), sul-1 (25), armA (2) and rmtB (6), in addition to adherence genes such as csgA (33), fimA (27), fliC (13), sdiA (33), rcsA (38), and rpoS (39). They also carried plasmid of diverse replicon types of which IncFIA and FIB were the most frequent. Phylogrouping categorized most of the MDR E. coli in phylogroup A (20), B1 (14), and B2 (6). Enterobacteriaceae repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) showed genetic diversity of multidrug resistant isolates irrespective of their origin, resistance, and virulence types, differentiating the EC in five clades (A-E) and KP in four clades (A-D). As these stray dogs, which had no history or scope of previous antimicrobial therapy, were found to have contracted potential antimicrobial resistance pathogens, the role of environment in spread of such pathogens and further possibility of human infections cannot be ruled out.

Immunotoxic and genotoxic potential of arsenic and its chemical species in goats.

The study investigated the immunotoxic and genotoxic effect of arsenic and its different species on goats. It was found that arsenic causes haematological crisis. Histopathological changes in spleen and reduced serum immunoglobulin G level without any changes in formazan production in arsenic-treated animals indicated that arsenic is toxic to the humoral immune system. Increased caspase-3 production and higher number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive bone marrow cells along with oligonucleosomal DNA fragmentation on agarose gel suggested apoptosis induction by arsenic in the bone marrow cells of goat. Total arsenic concentration in the plasma, bone marrow, and spleen of the exposed group was, respectively, 1.22 ± 0.11, 2.20 ± 0.21, and 3.39 ± 0.14 ppm. Speciation study revealed that arsenite and organoarsenic were the major arsenic species in these samples, suggesting their role in immunotoxic and genotoxic potential in goats.

Molecular Characterization of Methicillin-Resistant Staphylococci from the Dairy Value Chain in Two Indian States.

Bovine milk and milk products may contain pathogens, antimicrobial resistant bacteria, and antibiotic residues that could harm consumers. We analyzed 282 gram-positive isolates from milk samples from dairy farmers and vendors in Haryana and Assam, India, to assess the prevalence of methicillin-resistant staphylococci using microbiological tests, antibiotic susceptibility testing, and genotyping by PCR. The prevalence of genotypic methicillin resistance in isolates from raw milk samples was 5% [95% confidence interval, CI (3-8)], with 7% [CI (3-10)] in Haryana, in contrast to 2% [CI (0.2-6)] in Assam. The prevalence was the same in isolates from milk samples collected from farmers [5% (n = 6), CI (2-11)] and vendors [5% (n = 7), CI (2-10)]. Methicillin resistance was also observed in 15% of the isolates from pasteurized milk [(n = 3), CI (3-38)]. Two staphylococci harboring a novel mecC gene were identified for the first time in Indian dairy products. The only SCCmec type identified was Type V. The staphylococci with the mecA (n = 11) gene in raw milk were commonly resistant to oxacillin [92%, CI (59-100)] and cefoxitin [74%, CI (39-94)], while the isolates with mecC (n = 2) were resistant to oxacillin (100%) only. All the staphylococci with the mecA (n = 3) gene in pasteurized milk were resistant to both oxacillin and cefoxitin. Our results provided evidence that methicillin-resistant staphylococci occur in dairy products in India with potential public health implications. The state with more intensive dairy systems (Haryana) had higher levels of methicillin-resistant bacteria in milk.

Analyses of Extended-Spectrum-ß-Lactamase, Metallo-ß-Lactamase, and AmpC-ß-Lactamase Producing Enterobacteriaceae from the Dairy Value Chain in India.

The consumption of milk contaminated with antibiotic-resistant bacteria poses a significant health threat to humans. This study aimed to investigate the prevalence of Enterobacteriaceae producing ß-lactamases (ESBL, MBL, and AmpC) in cow and buffalo milk samples from two Indian states, Haryana and Assam. A total of 401 milk samples were collected from dairy farmers and vendors in the specified districts. Microbiological assays, antibiotic susceptibility testing, and PCR-based genotyping were employed to analyze 421 Gram-negative bacterial isolates. The overall prevalence of ß-lactamase genes was 10% (confidence interval (CI) (7-13)), with higher rates in Haryana (13%, CI (9-19)) compared to Assam (7%, CI (4-11)). The identified ß-lactamase genes in isolates were blaCMY, blaMOX, blaFOX, blaEBC, and blaDHA, associated with AmpC production. Additionally, blaCTX-M1, blaSHV, and blaTEM were detected as ESBL producers, while blaVIM, blaIMP, blaSPM, blaSIM, and blaGIM were identified as MBL producers. Notably, Shigella spp. were the dominant ß-lactamase producers among identified Enterobacteriaceae. This study highlights the presence of various prevalent ß-lactamase genes in milk isolates, indicating the potential risk of antimicrobial-resistant bacteria in dairy products. The presence of ß-lactam resistance raises concern as this could restrict antibiotic options for treatment. The discordance between genotypic and phenotypic methods emphasizes the necessity for comprehensive approaches that integrate both techniques to accurately assess antibiotic resistance. Urgent collaborative action incorporating rational and regulated use of antibiotics across the dairy value chain is required to address the global challenge of ß-lactam resistance.

Prevalence, molecular fingerprinting and drug resistance profile of enterovirulent Escherichia coli isolates from free-ranging yaks of Tawang district, Arunachal Pradesh, India.

Of 273 samples (rectal swab) collected from free-ranging yaks of Tawang district, Arunachal Pradesh, 42 Shiga toxin-producing Escherichia coli (STEC), six enteropathogenic E. coli (EPEC) and 27 enterotoxigenic E. coli (ETEC) strains were isolated. All the STEC and EPEC strains were further investigated for respective stx variants (for STEC only) and additional putative virulence factors. The 27 ETEC strains were also screened for characteristic enterotoxin gene(s) and colonization factors. Occurrence of ETEC was significantly (p < 0.05) higher in the diarrheic yaks and yaks of less than 1 year of age. Majority of enterovirulent E. coli isolates were resistant to amikacin, azithromycin, chloramphenicol, colistin, doxycycline, furazolidone, nalidixic acid, nitrofurantoin, streptomycin and tetracycline. Dendrogram, constructed with molecular fingerprinting profiles obtained from RAPD (Randomly Amplified Polymorphic DNA) and ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR, placed the isolates in different clusters irrespective of their serotypes, virulence gene and drug resistance pattern. Collectively, the study indicates that yaks, being a potential reservoir of multidrug resistant STEC and EPEC, may represent significant risk to public health in this region. Higher recovery of ETEC isolates from yaks with diarrhea points out that ETEC may be a major determinant for repeated occurrence of diarrhea in yaks.

One Health approaches adapted in low resource settings to address antimicrobial resistance.

Inter-disciplinary collaborations are now considered as key factors for integrated health system strengthening. Its application in the domain of One Health needs more milestones to achieve. Other than the human health sector, the antimicrobials are used in food animals and aquaculture for therapy, prophylaxis and growth promotion which significantly contributes to the development of antimicrobial resistance. It is the high time to develop a sustainable collaboration between the concerned sectors of One Health for a resilient health system. The domain of One Health not only mitigates the emergence of antimicrobial resistance but also helps in realizing the surveillance and epidemiology of zoonotic diseases, and the control of public health emergencies such as COVID-19. The review identified the key One Health strategies adapted by India, the exemplary low resource settings, to address antimicrobial resistance and zoonosis.

Antimicrobial Resistance Pattern, Clustering Mechanisms and Correlation Matrix of Drug-Resistant Escherichia coli in Black Bengal Goats in West Bengal, India.

A cross-sectional study covering four agro-climatic zones of West Bengal, India, was carried out to understand the risk-factors, antimicrobial resistance mechanism and clustering of the resistance characteristics of Escherichia coli isolated from healthy (170) and diarrhoeic (74) goats reared under intensive (52) and semi-intensive (192) farming practices. Of the 488 E. coli isolates, the majority, including the extended spectrum (n: 64, 13.11%) and AmpC ß-lactamase (ACBL) (n: 86, 17.62%) producers, were resistant to tetracycline (25.2%), followed by enrofloxacin (24.5%), cefotaxime (21.5%) and amikacin (20.5%). Statistical modelling revealed that the isolates from diarrhoeic animals (p < 0.001) are likely to be more ACBL-positive than those from the healthy counterparts. Similarly, cefotaxime (p < 0.05) and enrofloxacin-resistance (p < 0.01) were significantly higher in diarrhoeic goats and in goats reared intensively. The isolates (n = 35) resistant to multiple drugs revealed the presence of ß-lactamase [blaCTXM-1-(21), blaSHV-(7), blaTEM-(3), blaCMY-6-(1), blaCITM-(3)]; quinolone [qnrB-(10), qnrS-(7), aac(6')-Ib-cr-(3)]; tetracycline [tetA-(19), tetB-(4)] and sulphonamide resistance determinants [sul1-(4)]; multiple plasmids, especially those belonging to the IncF and IncI1 replicon types; and active acrAB efflux pumps. Further, two isolates harbored the carbapenem resistance (blaNDM-5) gene and eight were strong biofilm producers. This first ever study conducted to unravel the status of AMR in goat farming reveals that not only the intensive farming practices but also certain clinical ailments such as diarrhoea can increase the shedding of the drug-resistant isolate. The emergence of multi-drug resistant (MDR) E. coli in goats, particularly those that are carbapenem resistant, is a cause for concern that indicates the spread of such pathogens even in the livestock sub-sector generally considered as naive.

Exploring broilers and native fowls of Andaman and Nicobar Islands as a source of ß-lactamase-producing Enterobacteriaceae even with limited anthropogenic activities and docking-based identification of catalytic domains in novel ß-lactamase variants.

Objectives: The present study was conducted to detect the occurrence of ß-lactamase and biofilm-producing Escherichia coli, Salmonella, and Klebsiella in broilers and native fowl reared in the Andaman and Nicobar Islands, India. The study also included molecular docking experiments to confirm the nature of the catalytic domains found in the ß-lactamase variants obtained and to reveal the clonal relationship of the isolates with human clinical strains from the database. Materials and methods: A total of 199 cloacal swabs were collected from five poultry breeds/varieties (broiler, Vanraja, Desi, Nicobari, and layer) in three districts of the Andaman and Nicobar Islands. E. coli, Salmonella enterica, and Klebsiella pneumoniae were isolated by standard techniques and confirmed by PCR. Phenotypical ß-lactamase producers were identified by a double-disc test. The genes (bla CTX, bla SHV, bla TEM , and bla AmpC) were screened, and selected sequences of ß-lactamase variants were submitted to DDBJ. Homology modeling, model validation, and active site identification of different ß-lactamase variants were done by the SWISS-MODEL. Molecular docking was performed to identify the catalytic domains of the ß-lactamase variants. The selected ß-lactamase sequences were compared with the Indian ESBL sequences from human clinical strains in NCBI-GenBank. Results: In total, 425 Enterobacteriaceae strains were isolated from the collected samples. Klebsiella pneumoniae (42.58%) was found to be the most prevalent, followed by Salmonella enterica (30.82%) and E. coli (26.58%). The phenotypical antibiogram of all 425 isolates showed the highest resistance against oxytetracycline (61-76%) and the lowest against gentamicin (15-20%). Phenotypical production of ß-lactamase enzymes was observed in 141 (33.38%) isolates. The isolation rate of ß-lactamase producing E. coli, Salmonella enterica, and Klebsiella pneumoniae was significantly higher (p < 0.05) in the birds reared in the South Andaman district (25.6, 17.5, and 18.7%, respectively) than in Nicobar (11.5, 7.6, 7.1%, respectively). Genotyping of the ß-lactamase-producing isolates revealed the maximum possession of bla TEM, followed by bla SHV and bla CTX - M. The nucleotide sequences were found to be similar with bla CTX - M-15, bla SHV - 11, bla SHV - 27, bla SHV - 228, bla TEM - 1, and bla AmpC in BLAST search. Distribution of studied biofilm-associated genes in Enterobacteriaceae strains from different varieties of the birds revealed that the layer birds had the maximum possession, followed by Vanraja, Desi, broilers, and Nicobari fowls. The phylogenetic analysis of selected sequences revealed a partial clonal relationship with human clinical strains of the Indian subcontinent. Molecular docking depicted the Gibbs free energy release for 10 different macromolecules (proteins) and ligand (antibiotic) complexes, ranging from -8.1 (SHV-27 + cefotaxime) to -7 (TEM-1 + cefotaxime) kcal/mol. Conclusion and relevance: The study revealed ß-lactamase variants circulating in the fowl population of the Andaman and Nicobar Islands (India), even in remote places with low anthropogenic activity. Most of the strains possessed bla TEM - 1, followed by bla CTX - M-15. Possession of bla SHV - 11, bla SHV - 27, and bla SHV - 228 in poultry Enterobacteriaceae strains was not reported earlier from any part of the world. The phylogenetic analysis revealed a partial clonal relationship of ß-lactamase sequences with the human clinical strains isolated from the Indian subcontinent.

Comparing the Effectiveness of Different Approaches to Raise Awareness About Antimicrobial Resistance in Farmers and Veterinarians of India.

Background: Antimicrobial resistance (AMR) is a global public health threat. The indiscriminate use of antibiotics in the animal health sector contributes to increasing rates of AMR and studies involving dairy farmers in India have found knowledge levels regarding antibiotics and AMR to be very low. The purpose of this study was to assess different methods to raise awareness and knowledge about AMR and antibiotic use among dairy farmers, paravets (veterinary assistants), and veterinarians. Materials and Methods: The study was conducted in September-December of 2018 in some parts of Haryana, Assam, Karnataka, and West Bengal. It had two parts: an intervention meeting (September-October 2018) which consisted of focus group discussions (FGD) with farmers, key informant interviews (KII) with veterinary professionals along with distribution of information packages, and then a follow-up survey (November-December 2018). The villages were randomly allocated to either one of the four intervention approaches (1-FGD/KII and information package on AMR; 2-FGD/KI and information on animal health; 3- FGD/KII and information package on animal health plus information on AMR; or 4- only the FGD/KII). A follow-up survey was done to assess the effect of interventions. Results: In total, 274 dairy farmers and 51 veterinary professionals (21 veterinarians and 30 paravets) participated in the follow-up survey. Many of the farmers and veterinary professionals who participated in the follow-up survey had been part of the intervention meetings. The average knowledge score of farmers was 7.8. It was found that the knowledge score was higher amongst farmers who had participated in the intervention meetings (p < 0.05), had received intervention approach 2 (p = 0.03) or approach 3 (p = 0.01), and amongst female farmers (p = 0.03) compared to male. The veterinary professionals had good knowledge but lacked interest in training the farmers about antimicrobial resistance. Conclusion: Our research demonstrated that a higher percentage of farmers and veterinary professionals who attended the intervention meeting had improved knowledge. Dairy farmers should be regularly educated on antibiotic usage and how to avoid misusing them. Also, veterinary experts should be provided with tools and strategies to educate farmers on the use of antimicrobials.

Virulence repertoire of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic Escherichia coli (ETEC) from diarrhoeic lambs of Arunachal Pradesh, India.

A total of 107 faecal samples were collected from diarrhoeic lambs of high altitude terrains (2,000 to 5,000 m above the mean sea level) of Tawang and West Kameng districts of Arunachal Pradesh, India. Total 234 Escherichia coli were isolated and further subjected to PCR for the study of virulence repertoire characteristics of Shiga toxin-producing E. coli (STEC) and enterotoxigenic E. coli (ETEC). Out of the 234 isolated E. coli, 32% were found positive for STEC, and 9% were carrying virulence gene for ETEC. The isolated STEC serogroups were O159, O127, O120, O113, O60, O30, O25, O8 and O2. Of all the 74 STEC strains, PCR showed that 18% isolates carried stx ( 1 ), 26% possessed stx ( 2 ) and 47% produced positive amplicon for both. Other virulent attributes like intimin (eaeA), enterohaemolysin (ehxA) and STEC auto-agglutinating adhesin (saa) were present in 18%, 43% and 44% of the isolates, respectively. The isolated ETEC serogroups were O172, O170, O159, O146, O127, O120, O113, O86, O75, O60, O30, O25, O8, O2, OR and OUT. Of the 22 ETEC-positive isolates, 23%, 18% and 4.5% possessed the gene only for LT, STa and STb, respectively, whereas 54% carried genes for both LT and STb. Some serogroups of E. coli like O159, O127, O120, O113, O60, O30, O25, O8 and O2 possessed genes for both Shiga toxin and enterotoxin. This study is the first report of ETEC isolation from diarrhoeic lambs in India. The moderately high proportion of STEC and ETEC in the diarrhoeic lambs implicated that these animals are important reservoir of STEC and ETEC. This is really a grave concern for the 'brokpas' and nomads (shepherds) who share a close relationship with this animals for their livelihood. This study also indicates that ETEC may be a major cause for frequent diarrhoeal episodes in lambs of this region.

Characterization of Multidrug-Resistant Biofilm-Producing Escherichia coli and Klebsiella pneumoniae in Healthy Cattle and Cattle with Diarrhea.

This study describes comparative occurrence and characterization of multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae (KP) in healthy cattle (HC) and cattle with diarrhea (DC) in India. During 2018-2020, 72 MDR isolates, including 35 E. coli (DC: 27; HC 8) and 37 K. pneumoniae (DC: 34; HC: 3), from 251 rectal swabs (DC: 219; HC: 32) were investigated for extended-spectrum beta-lactamase (ESBL), AmpC type ß-lactamase and carbapenemase production, antimicrobial susceptibility profile, biofilm production, and efflux pump activity. Fifty-five MDR isolates were ESBL producers (ESBLPs) (DC: 50; HC: 5) and ESBLPs from DC were coresistant to multiple antibiotics. The blaCTX-M gene (50) was the most frequently detected ß-lactamases followed by blaAmpC (22), blaTEM1 (13), blaCMY-6 (6), blaOXA1 (5), blaPER (2), blaDHA, and blaFOX and blaSHV12 (1 each). Plasmid-mediated quinolone resistance determinants qnrB, qnrS, qnrA, and qepA were detected in 18, 16, 2, and 3 isolates, respectively. Twenty three isolates revealed mutation in gyrA and parC genes. Tetracycline-resistance markers tetA, tetB, tetC, and tetE were detected in 33, 10, 3, and 2 isolates, respectively. Only one of the 41 imipenem-resistant isolates harbored blaNDM-5 and two were colistin-resistant. Altogether, 20 MDR isolates were strong biofilm producers and 19 harbored different virulence factors. This is the first ever report from India on the presence of MDR Enterobacteriaceae with resistance to even last-resort antimicrobials in the bovine diarrhea.

Understanding Antibiotic Usage on Small-Scale Dairy Farms in the Indian States of Assam and Haryana Using a Mixed-Methods Approach-Outcomes and Challenges.

The use and misuse of antibiotics in both humans and animals contributes to the global emergence of antimicrobial resistant (AMR) bacteria, a threat to public health and infection control. Currently, India is the world's leading milk producer but antibiotic usage within the dairy sector is poorly regulated. Little data exists reflecting how antibiotics are used on dairy farms, especially on small-scale dairy farms in India. To address this lack of data, a study was carried out on 491 small-scale dairy farms in two Indian states, Assam and Haryana, using a mixed method approach where farmers were interviewed, farms inspected for the presence of antibiotics and milk samples taken to determine antibiotic usage. Usage of antibiotics on farms appeared low only 10% (95% CI 8-13%) of farmers surveyed confirmed using antibiotics in their dairy herds during the last 12 months. Of the farms surveyed, only 8% (6-11%) had milk samples positive for antibiotic residues, namely from the novobiocin, macrolides, and sulphonamide classes of antibiotics. Of the farmers surveyed, only 2% (0.8-3%) had heard of the term "withdrawal period" and 53% (40-65%) failed to describe the term "antibiotic". While this study clearly highlights a lack of understanding of antibiotics among small-scale dairy farmers, a potential factor in the emergence of AMR bacteria, it also shows that antibiotic usage on these farms is low and that the possible role these farmers play in AMR emergence may be overestimated.

Elucidating the resistance repertoire, biofilm production, and phylogenetic characteristics of multidrug-resistant Escherichia coli isolated from community ponds: A study from West Bengal, India.

This study details about the phenotypic and molecular characteristics of multidrug-resistant (MDR) Escherichia coli in the fresh community pond water (n = 257) collected from three districts of West Bengal, India. In total, 57 isolates were MDR of which 38 emerged as extended spectrum and 7 as AmpC-type ß-lactamase producers in phenotypic assay. Among ß-lactamase genes, blaCTXM-1was predominant (87.71%) followed by blaAmpC (77.2%) and blaTEM-1 (22.8%). Six MDR strains carried metallo-ß-lactamase (MBL, blaNDM-1) gene. Tissue culture plate assay confirmed strong biofilm (SP) production in four MDR and one non-MDR isolates. In PCR-based replicon typing (PBRT), multiple plasmids of diverse replicon types (Frep, FIB, I1, FIA, K/B, HI1, and Y) were identified. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)-based phylogenetic analysis revealed a high degree of genetic divergence among the MDR isolates. Multiplex PCR-based phylogrouping categorized 11 isolates as virulent (B2/D/F), which carried blaCTXM-1 gene and three had blaNDM-1 gene. Relative transcriptional activity of AcrAB efflux pump was significantly elevated among the SP and MBL producers. The presence of MDR E. coli isolates, particularly those resistant to carbapenem, in pond water used for daily domestic and household work, is a cause of concern as these pathogens may sneak into human food chain causing life-threatening infections. PRACTITIONER POINTS: Multidrug-resistant biofilm producing E. coli isolated from community pond water. A few of them were carbapenem-resistant and belonged to virulent (B2/D) types. Expression of AcrAB efflux pumps was found significantly elevated among biofilm producers and carbapenem-resistant population.