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.

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.

Synergistic Improvement of Antibacterial and Osteogenic Differentiation of Thermomechanically Processed Mg-Zr-Sr-Ce Alloy: Insights into the Role of Precipitate Evolution Supported by AIMD Simulation Study.

In the present study, we have discussed the influence of forging temperature (623 K (FT623), 723 K (FT723) and 823 K (FT823)) on microstructure and texture evolution and its implication on mechanical behavior, in vitro-in vivo biocorrosion, antibacterial response, and cytocompatibility of microalloyed Mg-Zr-Sr-Ce alloy. Phase analysis, SEM, and TEM characterization confirm the presence of Mg12Ce precipitate, and its stability was further validated by performing ab initio molecular dynamic simulation study. FT723 exhibits strengthened basal texture, higher fraction of second phases, and particle-stimulated nucleation-assisted DRX grains compared to other two specimens, resulting in superior strength with comparable ductility. FT723 also exhibits superior corrosion resistance mainly due to the strengthened basal texture and lower dislocation density. All the specimens exhibit excellent antibacterial behavior with Gram-negative E. coli, Gram-positive Staphylococcus aureus, and Pseudomonas aeruginosa bacteria. 100% reduction of bacterial growth is observed within 24 h of culture of the specimens. Cytocompatibility was determined by challenging specimen extracts with the MC3T3-E1 cell lines. FT723 specimen exhibits the highest cell proliferation and alkaline phosphatase activity (ALP) because of its superior corrosion resistance. The ability of the specimens to be used in orthopedic implant application was evaluated by in vivo study in rabbit femur. Neither tissue-related infection nor the detrimental effect surrounding the implant was confirmed from histological analysis. Significant higher bone regeneration surrounding the FT723 specimen was observed in SEM analysis and fluorochrome labeling. After 60 days, the FT723 specimen exhibits the highest bone formation, suggesting it is a suitable candidate for orthopedic implant application.

Efficacy of Saccharomyces cerevisiae Fermentation Product and Probiotic Supplementation on Growth Performance, Gut Microflora and Immunity of Broiler Chickens.

Concern for global health security and the environment due to the emergence of antibiotic-resistant bacteria and antibiotic residues in meat and other livestock products has led many countries to restrict the use of antibiotics in animal feed. This experiment was performed to assess the impact of dietary supplementation of a probiotic (Bacillus subtilis) and a postbiotic (Saccharomyces cerevisiae fermentation product) on growth performance, carcass traits, blood haemato-biochemical profile, gut microflora, gut morphology, and immune response in broilers as an alternative to antimicrobials in poultry production system to minimize the effect on global health security. A total of 324 one-day-old Ven Cobb 400 broiler chicks were randomly divided into three dietary groups, each containing 12 replicated pens, and each replicate contained nine chickens. The dietary groups consisted of (1) a basal diet without any growth promoters (T1), (2) the basal diet augmented with Bacillus subtilis at 200 g/MT feed (T2), and (3) the basal diet supplemented with Saccharomyces cerevisiae fermentation product at 1.25 kg/MT feed (T3). To calculate body weight gain, all birds and residual feed were weighed on a replicated basis on days 0, 7, 14, 21, 28, 35, and 42; mortality was recorded daily. At the end of the trial (42 d), two chickens from each replicate were slaughtered for carcass traits, gut microflora, and morphology measurements. Blood samples were collected for the haemato-biochemical profile on 35 d and antibody titer on 28 d and 35 d. Feeding with SCFP (T3 group) significantly improved average daily feed intake (ADFI) and average daily gain (ADG) of chickens compared to the T1 (control) and T2 (probiotic) groups from 1 to 14 days of age. Feed conversion ratio (FCR) was significantly improved in SCFP-fed birds (T3) relative to the control (T1) over the entire experimental period. Carcass traits and blood haemato-biochemical parameters remained unaffected by any diets. However, cholesterol levels and concentrations of corticosterone were significantly lower in T3 compared to T2 and T1 groups. Total E. coli, Enterohaemorrhagic E. coli, ESBL-producing Enterobacteriaceae, and Salmonella counts were significantly lower in T2 and T3 groups compared to T1 group and Salmonella counts were lower in T3 when compared to T2. However, there was no significant difference in Lactobacillus count among treatment groups. A significant increase in villi height and villi-height-to-crypt-depth ratio (VH: CD) was observed in both T3 and T2 groups. On day 28, the T3 and T2 groups exhibited a significant increase in antibody titers against Newcastle disease virus and infectious bursal disease virus. It can be concluded that Saccharomyces cerevisiae fermentation product and Bacillus subtilis probiotic could be viable alternatives to antimicrobials in poultry production considering beneficial impacts in broilers fed an antibiotic-free diet.

Growth, haemato-biochemical, hormonal and disease characteristics in Black Bengal goats: a review.

The goats have been considered one of the noteworthy animals to provide food security and could promote socio-economic upliftment under challenging climatic scenarios in the coming decades, particularly in the tropics. Black Bengal goat (BBG) is one of the recognised native meat-type breeds of hot-humid tropics with distinguished characteristics, including superior-quality meat, excellent skin and high prolificacy. Smaller body mass, lower metabolic rate and efficient utilisation of high-fibre forages enable BBG to adapt to a wide range of harsh climates in the tropics. The BBG can maintain physiological homeostasis efficiently in terms of electrolyte profile, endocrine functions and haemato-biochemical traits in different life phases, including the gestation period, even in high-saline coastal areas of hot-humid tropics. Crossbreeding to improve its growth rate has been attempted, but the prolificacy has been decayed. This review is intended to attract global attention to the adaptive potentialities of Black Bengal goats in terms of growth and production, haemato-biochemical, endocrinological, salt tolerance and disease characteristics that could be an asset of climate-resilient agricultural farming.

Newborn care in Northern India: A study of regional and seasonal peculiarities for desired professionalism and definitive practices.

Introduction: Improvements in newborn health with study of present status, progressive perfection of practices, and data diligence for future is desirable. Methods: Study of mortality and morbidity patterns. Analyze findings for focus areas and for favourable advancements in practices. Corroborate with existing evidence and practice protocols. Setting: Tertiary care referral hospital. Protocols: Preterm babies given special attention. This includes, in addition to the WHO 10 main recommendations, meticulous monitoring and health education for care. Neonatal jaundice evaluation on the basis of risk factors and clinical monitoring. Results: A total of 1749 live, born babies (2018-2020) were studied. The neonatal mortality rate in the study was 6.29. Preterm births were 102 (5.83%), with mortality of 7.84%, that is, 8 out of 102 preterm babies, and overall 4.57 premature deaths per 1000 live births. Prematurity was the commonest cause of mortality, responsible for 8 out of 11 deaths (72.73%). Birth asphyxia incidence was 5.26% (92 babies) with a case fatality ratio of 4.35%. Newborns with severe birth asphyxia were 7, and of these 4 (all premature) had fatal outcome. The low birthweight (LBW) incidence was 13.32% with the majority (79.83%) in the 2000-2499 g range. Preventive practices for hypothermia were 100% successful. An unusual rise in incidence and severity of hyperbilirubinemia during December 2018 to February 2019 when winter was severe was noted. Conclusion: Mortality reduction requires focusing on prematurity. Meticulous monitoring and health education for them improves results. The unusual rise in incidence and severity of hyperbilirubinemia in severe winter points towards the need for vigilance. We need to be forewarned and forearmed for frequent extreme weather events.

Pharma-cartography: Navigating the complexities of antibiotic supply to rural livestock in West Bengal, India, through value chain and power dynamic analysis.

Antibiotic resistance threatens provision of healthcare and livestock production worldwide with predicted negative socioeconomic impact. Antibiotic stewardship can be considered of importance to people living in rural communities, many of which depend on agriculture as a source of food and income and rely on antibiotics to control infectious diseases in livestock. Consequently, there is a need for clarity of the structure of antibiotic value chains to understand the complexity of antibiotic production and distribution in community settings as this will facilitate the development of effective policies and interventions. We used a value chain approach to investigate how relationships, behaviours, and influences are established during antibiotic distribution. Interviews were conducted with key informants (n = 17), value chain stakeholders (n = 22), and livestock keeping households (n = 36) in Kolkata, and two rural sites in West Bengal, India. Value chain mapping and an assessment of power dynamics, using manifest content analysis, were conducted to investigate antibiotic distribution and identify entry points for antibiotic stewardship. The flow of antibiotics from manufacturer to stockists is described and mapped and two local level maps showing distribution to final consumers presented. The maps illustrate that antibiotic distribution occurred through numerous formal and informal routes, many of which circumvent antibiotic use legislation. This was partly due to limited institutional power of the public sector to govern value chain activities. A 'veterinary service lacuna' existed resulting in livestock keepers having higher reliance on private and informal providers, who often lacked legal mandates to prescribe and dispense antibiotics. The illegitimacy of many antibiotic prescribers blocked access to formal training who instead relied on mimicking the behaviour of more experienced prescribers-who also lacked access to stewardship guidelines. We argue that limited institutional power to enforce existing antibiotic legislation and guide antibiotic usage and major gaps in livestock healthcare services make attempts to curb informal prescribing unsustainable. Alternative options could include addressing public sector deficits, with respect to both healthcare services and antibiotic provision, and by providing resources such as locally relevant antibiotic guidelines to all antibiotic prescribers. In addition, legitimacy of informal prescribers could be revised, which may allow formation of associations or groups to incentivise good antibiotic practices.

Crossover-Use of Human Antibiotics in Livestock in Agricultural Communities: A Qualitative Cross-Country Comparison between Uganda, Tanzania and India.

Antibiotic use in animal agriculture contributes significantly to antibiotic use globally and is a key driver of the rising threat of antibiotic resistance. It is becoming increasingly important to better understand antibiotic use in livestock in low-and-middle income countries where antibiotic use is predicted to increase considerably as a consequence of the growing demand for animal-derived products. Antibiotic crossover-use refers to the practice of using antibiotic formulations licensed for humans in animals and vice versa. This practice has the potential to cause adverse drug reactions and contribute to the development and spread of antibiotic resistance between humans and animals. We performed secondary data analysis of in-depth interview and focus-group discussion transcripts from independent studies investigating antibiotic use in agricultural communities in Uganda, Tanzania and India to understand the practice of antibiotic crossover-use by medicine-providers and livestock-keepers in these settings. Thematic analysis was conducted to explore driving factors of reported antibiotic crossover-use in the three countries. Similarities were found between countries regarding both the accounts of antibiotic crossover-use and its drivers. In all three countries, chickens and goats were treated with human antibiotics, and among the total range of human antibiotics reported, amoxicillin, tetracycline and penicillin were stated as used in animals in all three countries. The key themes identified to be driving crossover-use were: (1) medicine-providers' and livestock-keepers' perceptions of the effectiveness and safety of antibiotics, (2) livestock-keepers' sources of information, (3) differences in availability of human and veterinary services and antibiotics, (4) economic incentives and pressures. Antibiotic crossover-use occurs in low-intensity production agricultural settings in geographically distinct low-and-middle income countries, influenced by a similar set of interconnected contextual drivers. Improving accessibility and affordability of veterinary medicines to both livestock-keepers and medicine-providers is required alongside interventions to address understanding of the differences between human and animal antibiotics, and potential dangers of antibiotic crossover-use in order to reduce the practice. A One Health approach to studying antibiotic use is necessary to understand the implications of antibiotic accessibility and use in one sector upon antibiotic use in other sectors.

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.

Pharmacokinetics of ceftriaxone-tazobactam (8:1) combination in healthy and Escherichia coli induced diarrhoeic birds.

Antibiotic-resistant Escherichia coli infection of poultry causes significant economic losses. Extended spectrum ß lactamases (ESBL) producing E. coli was inoculated in a broiler, Rhode Island Red and Haringhata Black birds orally at 56×108 c.f.u. mL-1 for induction of diarrhoea. Pharmacokinetics of ceftriaxone-tazobactam combination (8:1) was studied following a single intramuscular injection at 28.125 mg kg-1 and the combination was administered twice daily to treat such infection. Plasma concentration of both ceftriaxone persisted up to 8 h in experimental birds and maintained an approximate ratio of 8:1 with tazobactam for a period of 2 h, 0.25 h and 0.75 h, respectively in a broiler, Rhode Island Red and Haringhata Black birds. The K el was significantly lower in all experimental birds compared to healthy birds. Efficacy study was conducted in diarrhoeic birds by administration of ceftriaxone-tazobactam combination at 28.125 mg kg-1 body weight twice daily intramuscularly for three days which caused an increase in specific antibody titre in the broiler on 5th day and in Rhode Island Red birds 10th day. However, Haringhata black birds were inherently showed more resistance towards the infection. The combination of ceftriaxone and tazobactam in the ratio of 8:1 can be an effective treatment to combat ESBL producing E. coli infections.

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.

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.

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.

"If It Works in People, Why Not Animals?": A Qualitative Investigation of Antibiotic Use in Smallholder Livestock Settings in Rural West Bengal, India.

Smallholder farms are the predominant livestock system in India. Animals are often kept in close contact with household members, and access to veterinary services is limited. However, limited research exists on how antibiotics are used in smallholder livestock in India. We investigated antibiotic supply, usage, and their drivers in smallholder livestock production systems, including crossover-use of human and veterinary antibiotics in two rural sites in West Bengal. Qualitative interviews were conducted with key informants (n = 9), livestock keepers (n = 37), and formal and informal antibiotic providers from veterinary and human health sectors (n = 26). Data were analysed thematically and interpreted following a One Health approach. Livestock keepers and providers used antibiotics predominantly for treating individual animals, and for disease prevention in poultry but not for growth promotion. All providers used (highest priority) critically important antimicrobials for human health and engaged in crossover-use of human antibiotic formulations in livestock. Inadequate access to veterinary drugs and services, and a perceived efficacy and ease of dosing of human antibiotics in animals drove crossover-use. Veterinary antibiotics were not used for human health due to their perceived adverse effects. Given the extent of usage of protected antibiotics and crossover-use, interventions at the community level should adopt a One Health approach that considers all antibiotic providers and livestock keepers and prioritizes the development of evidence-based guidelines to promote responsible use of antibiotics in animals.

Multidrug-Resistant Extended-Spectrum ß-Lactamase-Producing Escherichia coli Pathotypes in North Eastern Region of India: Backyard Small Ruminants-Human-Water Interface.

A total of 648 diarrheagenic Escherichia coli (DEC) were isolated from calves (n = 219), lambs (n = 87), kids (n = 103), human (n = 193), and water (n = 46) samples. The presence of enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and shigatoxigenic E. coli (STEC) was confirmed by PCR-based detection of the Shiga toxin, intimin, hemolysin, and enterotoxin genes. All the isolates were tested for antimicrobial resistance (AMR) by disc diffusion assay. Extended-spectrum ß-lactamase (ESBL), carbapenemase, and metallo-beta-lactamase production were determined by double-disk synergy test, modified Hodge test, and combined disk test assays. AMR genes (blaTEM, blaSHV, blaCTX-M, blaCMY-2, blaNDM, blaKPC, blaVIM, and blaIMP) were detected by PCR using specific primers. Majority of the isolates from human and water exhibited resistance (>80%) against amoxicillin, ampicillin, aztreonam, cefotaxime, cefixime, gentamicin, ceftazidime, and cefalexin, and against imipenem (70.98%), doripenem (70.47%), and ertapenem (60.62%). Bovine isolates were sensitive to carbapenems. Many isolates (5.75-24.35%) from human, water, calves, kids, and lambs were multidrug resistant (MDR), with resistance against three or more classes of antimicrobials. A total of 170/648 (26.23%) isolates were classified as STEC (9.88%), EPEC (4.32%), and ETEC (12.04%). The AMR genes, including blaTEM, blaCMY2, blaCTX-M, and blaSHV were detected in the E. coli from all sources. but blaNDM and blaKPC were detected only in the isolates from human and water. Three STEC isolates from human origin possessed multiple ESBLs, carbapenemase and metallo-beta-lactamase genes reported for the first time. ESBLs producing EPEC and ETEC in lambs and kids are also reported under this study. Presence of MDR-DEC in domestic animals and common potable water poses public health concern in 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.

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.

Pharmacokinetics and Efficacy of Ceftriaxone in Staphylococcal Mastitis in Crossbred Cows Following Single Intravenous Administration.

BACKGROUND: Clinical mastitis is an important production disease of dairy animals, causing significant economic losses. OBJECTIVE: Disposition kinetics of ceftriaxone was conducted in healthy lactating and staphylococcal mastitic crossbred cows in field condition following single-dose intravenous administration of only ceftriaxone. METHODS: A single dose of ceftriaxone at 20 mg kg-1 body weight was administered intravenously through jugular vein to six clinically healthy and six mastitic crossbred cows after proper diagnosis and three mastitic cows remained untreated (positive control). Blood and milk samples were collected at 0 (pre-dosing), 5, 15, 30 min, and 1, 24, 48, 72, 96 and 120 h post drug administration and analyzed for ceftriaxone and its active metabolite (ceftizoxime) by high-performance liquid chromatography. RESULTS: Ceftriaxone achieved a peak mean plasma concentration of 131.67±1.83 µg mL-1 at 5 min, which decreased sharply until 1 h (35.56±0.44 µg mL-1) and was below detection limit at 24 h post drug administration in mastitic crossbred cows. On the other hand, ceftizoxime (active metabolite of ceftriaxone) achieved a peak level of 55.42±3.34 µg mL-1 at 72 h and could not be detected at 120 h post drug administration in the milk of those mastitic crossbred cows. The Staphylococcus aureus colony count in mastitic crossbred cows was 49.33±6.55 × 105 c.f.u./mL and the lowest colony count was achieved at 72 h with no colony at 120 h post drug administration. All the staphylococcal mastitis affected crossbred cows were cured on day 5. CONCLUSION: Ceftriaxone may prove to be effective in the treatment of staphylococcal mastitis in crossbred cows following single-dose intravenous administration at 20 mg kg-1 body weight.

Antimicrobial Resistance in Agri-Food Chain and Companion Animals as a Re-emerging Menace in Post-COVID Epoch: Low-and Middle-Income Countries Perspective and Mitigation Strategies.

Antimicrobial resistance (AMR) leads to enormous financial losses from issues such as high morbidity, mortality, man-days lost, hospital length of stay, health-care, and social costs. In humans, over prescription of antimicrobials, which is presumably higher during COVID, has been identified as the major source of selection for antimicrobial resistant bacteria; however, use of antimicrobials in food and companion animals, fish, and vegetables, and the environmental resistance gene pool, also play important roles. The possibilities of unnecessary use of antibiotics as prophylaxis during and after COVID in livestock and companion animals exist in low-and middle-income countries. A considerable loss in gross domestic product (GDP) is also projected in low-and middle-income countries (LMICs) due to AMR by the year 2050, which is further going to be reduced due to economic slowdown in the post-COVID period. Veterinary hospitals dedicated to pets have cropped up, especially in urban areas of LMICs where use of antimicrobials has also been increased substantially. The inevitable preventive habit built up during COVID with the frequent use of hand sanitizer might trigger AMR due to the presence of cross-resistance with disinfectants. In LMICs, due to the rising demand for animal protein, industrial food animal production (IFAP) is slowly replacing the small-scale backyard farming system. The lack of stringent regulations and monitoring increased the non-therapeutic use of antimicrobials in industrial farms where the persistence of antimicrobial resistant bacteria has been associated with several factors other than antimicrobial use, such as co-resistance, cross-resistance, bacterial fitness, mixing of new and old animals, and vectors or reservoirs of bacterial infection. The present review describes types of antimicrobials used in agri-food chains and companion animals in LMICs with identification of the gap in data, updated categories of prevalent antimicrobial resistant bacteria, the role of animal farms as reservoirs of resistant bacteria, and mitigation strategies, with a special focus on the pivotal strategy needed in the post-COVID period.

Efficacy evaluation of ethanolic extract of Tamarindus indica L. leaves as possible alternate therapy in septic arthritis model of rabbit.

BACKGROUND: Our previous study exhibited free radicals scavenging and antioxidant activities of ethanolic and aqueous extracts of Tamarindus indica L. leaves in chronic sodium fluoride poisoning in rats. Tamarindus indica L. seed extract was also reported to have anti-arthritic efficacy by inhibiting cartilage and bone degrading factors. Therefore, an attempt was made to evaluate the effects of ethanolic extract of Tamarindus indica L. leaves in septic arthritis. METHODS: The safety study was performed by oral dosing of ethanolic extract of the plant leaves at 2 g kg- 1 for consecutive 28 days in rabbits. Septic arthritis was induced in rabbits by single intra-articular inoculation of 104 c.f.u. of Staphylococcus aureus to the left stifle joint and was monitored by bacterial colony count, some relevant biochemical parameters and histopathological interpretation of the affected joint. For efficacy evaluation in septic arthritis, linezolid at 75 mg kg- 1 twice daily for 10 days and the ethanolic extract of Tamarindus indica L. at 500 and 1000 mg kg- 1 for consecutive 14 days were administered orally to the rabbits after 48 h of induction of arthritis. RESULTS: In sub-acute toxicity study of Tamarindus indica L. leaves ethanolic extract, no significant change between days was found for aspertate aminotransferase, alanine transaminase, alkaline phosphatase, blood urea nitrogen and creatinine compared to day 0 values of the same group. The bacterial colony count of synovial fluid following Staphylococcus aureus inoculation to left stifle joint was found to be 1.08 ± 0.47 and 1.19 ± 0.29 c.f.u. mL- 1 in ethanolic extract low dose and high dose groups respectively, on day 2 which was reduced to 0.057 ± 0.036 c.f.u. mL- 1 and nil on day 16. The test extract was also found to markedly reduce simultaneous glucose difference, total protein ratio of serum and synovial fluid, joint radius and joint narrowing. CONCLUSION: Ethanolic extract of Tamarindus indica L. leaves at 500 mg kg- 1 and 1000 mg kg- 1 produced anti-arthritic effects against S. aureus induced septic arthritis in rabbits. However, the ethanolic extract at 1000 mg kg- 1 orally for consecutive 14 days showed better effects in septic arthritis.