Background: The threat of methicillin-resistant Staphylococcus aureus (MRSA) exists globally and has been listed as a priority pathogen by the World Health Organization. One of the sources of MRSA emergence is livestock and its products, often raised in poor husbandry conditions. There are limited studies in Nepal to understand the prevalence of MRSA in dairy animals and its antimicrobial resistance (AMR) profile. A cross-sectional study was conducted in Chitwan, one of the major milk-producing districts of Nepal, from February 2018 to September 2019 to estimate the prevalence of MRSA in milk samples and its AMR profile. The collected milk samples (n = 460) were screened using the California Mastitis Test (CMT) and positive samples were subjected to microbiological analysis to isolate and identify S. aureus. Polymerase Chain Reaction (PCR) was used to identify the presence of the mecA gene and screen for MRSA. Results: In total, 41.5% (191/460) of milk samples were positive in the CMT test. Out of 191 CMT positive milk samples, the biochemical tests showed that the prevalence of S. aureus was 15.2% (29/191). Among the 29 S. aureus isolates, 6.9% (2/29) were identified as MRSA based on the detection of a mecA gene. This indicates that that 1.05% (2/191) of mastitis milk samples had MRSA. The antibiotic sensitivity test showed that 75.9% (22/29) and 48.3% (14/29) S. aureus isolates were found to be sensitive to Cefazolin and Tetracycline respectively (48.3%), whereas 100% of the isolates were resistant to Ampicillin. In total 96.6% (28/29) of S. aureus isolates were multidrug-resistant (MDR). Conclusions: This study revealed a high prevalence of S. aureus-mediated subclinical mastitis in dairy herds in Chitwan, Nepal, with a small proportion of it being MRSA carrying a mecA gene. This S. aureus, CoNS, and MRSA contaminated milk poses a public health risk due to the presence of a phenotype that is resistant to very commonly used antibiotics. It is suggested that dairy herds be screened for subclinical mastitis and treatments for the animals be based on antibiotic susceptibility tests to reduce the prevalence of AMR. Furthermore, future studies should focus on the Staphylococcus spp. to explore the antibiotic resistance genes in addition to the mecA gene to ensure public health.

Background and Aim: Antimicrobials are extensively used in poultry production for growth promotion as well as for the treatment and control of diseases, including avian pathogenic Escherichia coli (APEC). Poor selection, overuse, and misuse of antimicrobial agents may promote the emergence and dissemination of antimicrobial resistance (AMR) in APEC. This study aimed to assess antimicrobial susceptibility patterns and detect antibiotic resistance genes (ARGs) in APEC isolated from clinical cases of colibacillosis in commercial broiler, layer, and breeder chickens. Materials and Methods: A total of 487 APEC were isolated from 539 across 300 poultry farms in various regions of Nepal. antimicrobial susceptibility patterns was determined using the Kirby-Bauer disk diffusion and broth microdilution methods. The index of AMR, such as multiple antibiotic resistance (MAR) index, resistance score (R-score), and multidrug resistance (MDR) profile, were determined. Polymerase chain reaction was employed to detect multiple ARGs and correlations between phenotypic and genotypic resistance were analyzed. Results: The prevalence of APEC was 91% (487/539). All of these isolates were found resistant to at least one antimicrobial agent, and 41.7% of the isolates were resistant against 8–9 different antimicrobials. The antibiogram of APEC isolates overall showed the highest resistance against ampicillin (99.4%), whereas the highest intermediate resistance was observed in enrofloxacin (92%). The MAR index and R-score showed significant differences between broiler and layers, as well as between broiler breeder and layers. The number of isolates that were R to at least one agent in three or more antimicrobial categories tested was 446 (91.6%) and were classified as MDR-positive isolates. The ARGs were identified in 439 (90.1%) APEC isolates, including the most detected mobilized colistin resistance (mcr1) which was detected in the highest (52.6%) isolates. Overall, resistance gene of beta-lactam (blaTEM), mcr1, resistance gene of sulphonamide (sul1) and resistance gene of tetracycline (tetB) (in broiler), were detected in significantly higher than other tested genes (p < 0.001). When examining the pair-wise correlations, a significant phenotype-phenotype correlation (p < 0.001) was observed between levofloxacin and ciprofloxacin, chloramphenicol and tetracycline with doxycycline. Similarly, a significant phenotype-genotype correlation (p < 0.001) was observed between chloramphenicol and the tetB, and colistin with blaTEM and resistance gene of quinolone (qnrA). Conclusion: In this study, the current state of APEC AMR in commercial chickens is revealed for the first time. This information is useful for the clinical management of disease as well as the creation of policies and guidelines to lower AMR in Nepal’s commercial chicken production. Keywords: antibiotic resistance gene, multiplex PCR, colibacillosis, multiple antibiotic resistance index, mcr1, commercial chicken.

Avian pathogenic Escherichia coli (APEC) is a major bacterial pathogen responsible for the most widespread form of colibacillosis, resulting in substantial economic losses within the poultry sector and posing a potential public health risk. From July to September 2021, our study investigated the antibiotic resistance pattern of Escherichia coli (E. coli) and the presence of virulence-associated genes (iucD, iutA, iss, and ompT) linked to APEC using 105 broiler meat samples comprising liver, thigh, and breast muscle, in Chitwan, Nepal. E. coli was isolated and identified by culturing samples on MacConkey's agar, Eosin-methylene blue (EMB) agar and performing different biochemical tests. Antibiotic resistance patterns of E. coli were determined by the Kirby-Bauer disc diffusion method. Following the isolation of E. coli, the molecular detection of APEC was performed using conventional polymerase chain reaction (PCR). Out of the 105 samples analyzed, 61 (58.1 %) tested positive for E. coli. In antibiotic susceptibility test (AST), gentamicin and tetracycline exhibited the highest resistance rates, with 90.2 % and 67.2 %, respectively and 29.5 % of the E. coli isolates displayed multidrug-drug resistance. Out of 61 confirmed E. coli isolates, iutA was detected in 47 (77.0 %) samples, iucD in 46 (75.4 %), iss in 53 (86.8 %), and ompT in 39 (63.9 %) samples. This study reports the occurrence of MDR E. coli in meat samples, together with virulence genes associated with APEC which poses a public health threat. Continuous surveillance is vital for monitoring APEC transmission within poultry farms, coupled with efforts to raise awareness of food safety among consumers of broiler meat.

In this study, chopping board meat samples collected from meat shops of Bharatpur Metropolitan City, Nepal, were screened for the presence of Escherichia coli (E. coli), with a special emphasis on the identification of extended-spectrum β-lactamase (ESBL)-producing strains. Representatives from the meat shops were also interviewed to understand the sanitary status and hygienic practices. E. coli bacteria were detected in one third (33/99) of the meat samples, while none of the samples had ESBL-producing strains. While 60.6% (60/99) of the meat shop personnel wore protective clothing, 15.15% (15/99) used gloves, and only 5.05% (5/99) had separate equipment for cleaning the viscera of animals. This study highlights the need for the regular screening of meat samples to identify pathogenic bacteria such as E. coli and for improvements in the sanitary status and hygienic practices of retail meat shops in Bharatpur Metropolitan City, Nepal.

This study investigates the molecular characterization, antimicrobial resistance (AMR) profiling, and biofilm formation of Salmonella isolates from dead-in-shell embryonated eggs collected from 15 hatcheries in Bharatpur, Chitwan. A total of 210 samples were analyzed using cultural, biochemical, and molecular techniques, including PCR for virulence gene detection. The study revealed a prevalence rate of 11.42 %, with isolates demonstrating significant AMR, particularly to cephalexin (100 %), ampicillin (95.83 %), and nalidixic acid (91.67 %). Moreover, 83.33 % (n = 20) of the isolates were classified as multidrug-resistant (MDR), with a mean multiple antibiotic resistance index of 0.51. Biofilm formation analysis indicated that 70.83 % of the isolates could produce biofilms, with 41.67 % classified as weak producers and 16.67 % as strong producers. Molecular analysis identified key virulence genes, including invA (100 %), spvC (50 %), spvB1 (33.33 %), and the biofilm-associated gene agfA (12.5 %), emphasizing their roles in pathogenicity, systemic infections, and biofilm formation. Our findings highlight the critical public health risks posed by MDR Salmonella, which can persist in the food chain and compromise human and animal health. This research underscores the need for enhanced biosecurity in hatcheries, judicious use of antimicrobials, and periodic monitoring of bacterial resistance and virulence factors to mitigate the economic and health impacts of Salmonella infections in poultry production systems.