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.

This cross-sectional study, conducted from August to December 2024, investigated the antimicrobial resistance and biofilm-forming abilities of Staphylococcus aureus from clinical bovine mastitis in Chitwan, Nepal. Out of 134 California Mastitis Test-positive milk samples, 32 (23.9%) were confirmed as S. aureus by biochemical tests and species-specific nuc gene PCR. Antimicrobial susceptibility testing of the 32 isolates against 12 antibiotics revealed high resistance rates, particularly to ampicillin (78.1%), nalidixic acid (75.0%), and enrofloxacin (62.5%). The prevalence of multidrug resistance (MDR), defined as resistance to ≥3 antibiotic classes, was alarmingly high, with 27 (84.4%) isolates classified as MDR. Presumptive methicillin-resistant S. aureus (MRSA), detected via cefoxitin resistance, was identified in 14 (43.8%) isolates, all of which were also MDR. Biofilm-forming abilities were assessed qualitatively and quantitatively, with 5 (15.6%) isolates classified as strong biofilm producers. Fisher's exact test revealed no significant association between biofilm formation and overall MDR status (p > 0.05). However, a statistically significant correlation was found between strong biofilm formation and high-level MDR (resistance to ≥ 4 classes) (p < 0.05), as well as between strong biofilm formation and presumptive MRSA status (p < 0.01). These findings highlight the co-existence of high-level resistance and strong virulence phenotypes in S. aureus from bovine mastitis in Nepal, underscoring the urgent need for robust antimicrobial stewardship, enhanced surveillance, and the development of strategies to mitigate biofilm-associated treatment failures.

This study focuses on the identification and antibiogram profiles of Escherichia coli, Salmonella spp., and Staphylococcus spp. isolated from cloacal swabs of captive yearling gharials (Gavialis gangeticus) at the Gharial Breeding Center, Chitwan National Park, Nepal. A total of 28 samples were collected and processed, yielding 38 isolates comprising E. coli (46.43 %), Staphylococcus spp. (46.43 %), and Salmonella spp. (42.86 %). All three bacteria were isolated in 14.29 % of samples. Antimicrobial susceptibility testing, performed using the Kirby-Bauer disk diffusion method, demonstrated high levels of resistance to commonly used antibiotics such as ampicillin and ciprofloxacin. Multidrug resistance was prevalent, with 84.62 % of E. coli and 58.33 % of Salmonella spp. isolates classified as multidrug-resistant, while all Staphylococcus spp. isolates showed resistance to three or more classes of antibiotics underscoring critical AMR challenges. The study also revealed a significant interplay between the reptilian gut microbiota and environmental factors, suggesting shared reservoirs for bacterial transmission. The findings underscore the urgent need for antimicrobial stewardship, improved environmental management, and regular health monitoring to mitigate the risks posed by antimicrobial resistance and pathogenic bacteria in critically endangered gharials. This research contributes valuable insights into the health challenges facing captive gharials and provides a basis for developing targeted conservation strategies.

Poultry meat is Nepal's most dynamic agricultural subsector, yet food safety remains a concern as retail carcasses are frequently contaminated by antimicrobial-resistant Escherichia coli. This cross-sectional study determined the tissue-site distribution, antimicrobial resistance profile, and phenotypic ESBL production of phenotypically identified E. coli isolated from 144 retail chicken carcasses collected from 16 randomly selected meat shops (nine carcasses per shop) in Bharatpur Metropolitan City, Chitwan, Nepal, sampling the liver, muscle, and skin of each bird. Of 432 tissue samples examined, 96 E. coli isolates were recovered, giving an overall tissue-level prevalence of 22.22% and a carcass-level prevalence of 40.97% (59/144; shop-cluster-adjusted 95% CI 29.4-53.6%). Isolation was highest from liver (34.72%), followed by muscle (21.53%) and skin (10.42%); the odds of isolation were 4.17 times higher in the liver than in muscle and 12.67 times higher in the liver than in skin (p < 0.001). Susceptibility testing against seven antibiotics showed the highest resistance to nalidixic acid (82.29%) and tetracycline (71.88%), and the lowest resistance to ceftriaxone (12.50%). Multidrug resistance was detected in 91.67% (88/96; 95% CI 84; 24-96.33%) extensive drug resistance in 59.38% (57/96; 95% CI: 48.87-69.29%) and pan drug resistance in 2.08% (2/96; 95% CI: 0.25-7.32%) of isolates, with a mean MAR index of 0.63 ± 0.17, highest among liver isolates (0.68 ± 0.16). Phenotypic ESBL positivity by the combined disk test was observed in 18.75% (18/96) of isolates, most frequently from the liver (22%). As identification relied on phenotypic methods without molecular or virulence confirmation, these findings indicate that poultry may serve as a reservoir of phenotypically antimicrobial-resistant E. coli, with the liver showing the highest contamination and resistance burden, underscoring the need for targeted surveillance along Nepal's poultry supply chain.