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.

Background: Rabies remains a fatal yet preventable zoonotic disease in Nepal, particularly in the Terai region where human-animal conflict is frequent. This study analyzes the epidemiological patterns of animal bites and evaluates the timeliness and completeness of Post-Exposure Prophylaxis (PEP) administration in Kapilvastu District to guide local public health interventions. Methods: A retrospective analysis was conducted using data from the anti-rabies vaccine register of Kapilvastu Hospital from 1 June 2022 to 31 May 2023. All recorded animal bite victims ( N = 2,853) were included. Descriptive statistics were used to characterize demographics and exposure patterns. Multivariable logistic regression was performed to identify factors associated with delayed PEP initiation (> 24 h after exposure). Results: The study recorded 2,853 animal bite cases, predominantly males (64.5%) and children aged 0–10 years (31.4%). Dogs were responsible for the vast majority of bites (91.6%). While the PEP completion rate for the full three-dose intradermal regimen was high (93.6%), a critical delay in treatment initiation was observed; only 14.1% of victims received their first dose on the day of exposure. Multivariate analysis revealed that victims residing in municipalities outside the district headquarters had significantly higher odds of delaying treatment (AOR: 1.48; 95% CI: 1.22–1.80). Adolescents (11–20 years) were also more likely to delay PEP compared to younger children (AOR: 1.35; 95% CI: 1.07–1.70), whereas victims with high-risk bites (head/neck/hand) sought care more promptly (AOR: 0.77; 95% CI: 0.62–0.95). Conclusions: Although vaccine coverage and completion rates in Kapilvastu are commendable, the dangerous delay in initial treatment remains a critical gap in rabies prevention. Public health interventions must specifically target adolescents and remote communities to emphasize the urgency of immediate PEP, regardless of wound severity or distance to healthcare facilities.

Livestock drinking water distribution systems represent a critical but understudied interface in the epidemiology of antimicrobial resistance. While engineered for production, these systems frequently function as unintended bioreactors where biofilms protect pathogens and facilitate horizontal gene transfer. Following PRISMA and SWiM guidelines, we systematically searched four databases (MEDLINE, Scopus, AGRIS, PubAg) through November 2025 for primary research on antimicrobial resistance in livestock water biofilms. Eligible studies underwent risk-of-bias assessment using JBI tools. Due to substantial methodological heterogeneity in sampling and assays, data were synthesized narratively to characterize resistance prevalence and reservoir dynamics. The synthesis reveals that DWDS biofilms harbor distinct microbial communities compared to transient planktonic or fecal inputs. Critically, these matrices sustain critical priority traits, including multidrug efflux pumps ( adeF ) in swine systems, plasmid-mediated colistin ( mcr-1 to mcr-5 ) and carbapenemase ( blaNDM ) genes. Evidence indicates that standard disinfection protocols often fail to eliminate established biofilms, allowing rapid recolonization by resistant populations within days of treatment. These findings suggest that farm water infrastructure acts as a persistent reservoir for genetic resistance traits, capable of reseeding animal cohorts despite distinct production cycles. We identify a critical surveillance blind spot and conclude that current One Health surveillance strategies should expand beyond bulk water testing to include targeted biofilm sampling. Effective mitigation requires engineering solutions and enzymatic treatments specifically designed to disrupt the protective matrix, thereby closing a significant gap in on-farm biosecurity.

Tuberculosis (TB) control in Nepal faces persistent challenges due to underdiagnosis and increasing drug resistance. This study assessed the diagnostic performance of the Xpert MTB/RIF assay compared with conventional smear microscopy and estimated the prevalence of rifampicin-resistant TB (RR-TB) in Biratnagar, a high-burden urban center. Between March and September 2023, 509 clinical specimens from TB suspects were analyzed by Ziehl-Neelsen (ZN) staining, fluorescence microscopy (FM), and Xpert MTB/RIF. Using Xpert as the operational reference, ZN and FM exhibited overall sensitivities of 55.9% (89/159) and 68.5% (109/159), respectively. In paired sputum-only analyses ( n = 446; sputum Xpert positives = 145), ZN and FM sensitivities were 60.0% (87/145) and 70.3% (102/145), respectively; sputum specificity was 100% for both methods. Xpert detected 31.2% (159/509) positive cases—78.7% more than ZN and 45.9% more than FM. Rifampicin resistance was identified in 5.03% (8/159) of Xpert-positive samples and was strongly associated with prior treatment ( P < 0.001); RR-TB occurred in 66.7% (6/9) of retreatment cases vs 1.3% (2/150) of new cases. Xpert increased detection in paucibacillary and extrapulmonary specimens. These findings support expanded use of rapid molecular testing, but results should be interpreted cautiously because culture/speciation was not performed. IMPORTANCE: The spread of drug-resistant tuberculosis (TB) is a growing public health threat in Nepal. Using a rapid molecular test in Biratnagar, we simultaneously diagnosed TB and identified rifampicin resistance. We found rifampicin-resistant TB in >5% of Xpert-positive patients, higher than national averages for new cases, and a markedly elevated risk among previously treated patients. Rapid detection of resistance permits earlier initiation of appropriate therapy and can reduce onward transmission.