Molecular characterization, antimicrobial resistance profiling, and biofilm analysis of Salmonella isolates from dead-in-shell embryonated eggs

Abstract

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.

Introduction

Salmonellosis is a zoonotic food-borne enteric infection that has great economic importance in the livestock market, especially in the poultry industry (Li et al., 2020). In 2019, invasive non-typhoidal Salmonella caused an estimated 594,000 incident cases and 79,000 deaths globally, while non-invasive non-typhoidal Salmonella accounted for approximately 73.9 million cases and 61,600 deaths (World Health Organization and Institute for Health Metrics and Evaluation, 2019). Egg-associated outbreaks represent a potential route of Salmonella contamination in the human food chain. A recent global review reports that between 200 million and 1 billion Salmonella infections occur annually worldwide, with about 93 million gastroenteritis cases and 155,000 deaths. Approximately 85 % of these are foodborne, many linked to eggs (Lamichhane et al., 2024).

Salmonella is a gram-negative, rod-shaped, non-capsulated, and non-spore-forming facultative anaerobe belonging to the Enterobacteriaceae. Most serovars are motile and peritrichous, except Salmonella Pullorum and Salmonella Gallinarum, and more than 2600 serovars have been identified so far (Xin et al., 2021). A variety of Salmonella serovars can infect poultry, entering hatcheries either through vertical transmission to progeny or through horizontal spread via cross-contamination within flocks (Yang et al., 2019). The vertical transmission is primarily due to the colonization of the reproductive tract, particularly the ovaries and oviducts of poultry. Once established, it may remain for several years, leading to embryonic mortality and threatening both animal and public health (Liu et al., 2022).

The emergence of multi-drug resistant (MDR) Salmonella resistant to clinically critical antimicrobial agents complicates treatment and prophylaxis. This increases infection-related morbidity and mortality, while also escalating healthcare and economic costs (Gong et al., 2013). Biofilm formation by bacteria such as Salmonella is an important virulence factor that facilitates evasion of host immunity and resists antibiotics penetration. Thus, a serious concern for public health and the food industry (Harrell et al., 2021).

The molecular characterization of virulence genes provides insight into pathogenic potential of the organism. Invasion A (invA) serves as a marker gene for detecting Salmonella spp., and plays a crucial role in host cell invasion by targeting the intestinal epithelial cells (Mthembu et al., 2019). The Salmonella plasmid virulence genes (spvC and spvB1) have a pivotal role in intracellular survival, proliferation, and modulation of host immunity (Sun et al., 2020, Zuo et al., 2020). Furthermore, the aggregative fimbriae A (agfA) gene contributes to the production of fimbriae during bacterial colonization and is associated with biofilm production in Salmonella (Yoo et al., 2013).

The poultry industry is an integral component of Nepal’s agricultural economy, particularly in Chitwan, which serves as a major hub for poultry production. Despite the importance of the poultry sector, a research gap exists, as limited studies have focused on Salmonella from dead-in-shell embryonated eggs. Therefore, this study was conducted to determine the prevalence, antimicrobial resistance (AMR) profiles, biofilm-forming capacity, and presence of key virulence genes (invA, spvC, spvB1, and agfA) in Salmonella. Our investigation is crucial for developing effective control strategies, as it addresses the challenges posed by AMR and biofilms and emphasizes the economic and public health impacts of salmonellosis linked to poultry hatcheries in Nepal.

Materials and methods

Study design, area, and sample collection

A cross-sectional study was conducted in Chitwan, Nepal, from August 2024 to December 2024, with sample collection focused on hatcheries located within Bharatpur Metropolitan. Purposive sampling was carried out to select samples, targeting embryonated eggs exhibiting high mortality, to investigate Salmonella’s role in hatchery losses. This was achieved by collaborating with hatchery managers to identify specific incubator batches that were experiencing higher-than-average embryonic mortality based on historical performance records and candling results. A total of 210 dead-in-shell embryonated eggs were obtained from 15 commercial hatcheries, with 14 samples collected from each hatchery. On the 21st day of incubation, only the embryos identified as dead were selected for the study, while excluding eggs that were cracked or where the embryos had piped the shell but failed to hatch. Samples were collected twice weekly and transported to the laboratory, using sterile Ziplock bags under refrigerated conditions.

Eggs were disinfected using 70 % ethyl alcohol and subsequently wiped with sterile tissue paper before breaking the shells. The unhatched embryos were aseptically extracted using sterile forceps and scissors, placed in a sterile Petri dish, and dissected to expose the internal organs for further analysis. Sterile dry swabs were used to prepare impression smears from selected tissues of the dead-in-shell embryos, including the yolk sac and liver. The swabs were transferred into sterile test tubes containing 10 mL of Buffered Peptone Water (BPW) to facilitate pre-enrichment and incubated at 37°C for 24 h.

Salmonella isolation and phenotypic identification

Isolation and identification of Salmonella were carried out in accordance with the procedures outlined in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (WOAH, 2024). Following pre-enrichment, 1 mL of BPW suspension was transferred to 10 mL of Selenite Cystine (SC; Hope Biol) broth for enrichment and incubated at 42°C for 24 h. A loopful of enriched suspension was streaked in MacConkey agar and incubated at 37°C for 24 h for isolation of Salmonella spp.. Presumptive Salmonella colonies appeared as colorless or pale colonies on MacConkey agar and were subjected to Gram staining, which revealed Gram-negative rods. The isolates were further characterized using a panel of biochemical tests, including KOH, catalase, oxidase, citrate utilization, triple sugar iron, methyl red, Voges-Proskauer, and hydrogen sulphide production. Motility, indole, and lysine decarboxylase properties were evaluated in motility indole lysine medium (HiMedia). Biochemically confirmed isolates were streaked onto Xylose Lysine Deoxycholate Agar (XLD) (HiMedia), where development of red colonies with black centers occurred at 18-24 h of incubation at 37°C. Pure cultures were obtained by repeated streaking on XLD agar and subsequently inoculated into nutrient broth. Cultures were incubated overnight at 37°C and stocked in 40 % glycerol the next day.

Antimicrobial susceptibility testing (AST) and resistance profiling

The antibiotic susceptibility was evaluated using the Kirby-Bauer disk diffusion method on Mueller-Hinton Agar (MHA; HiMedia), following the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2024). Pure colonies were inoculated in brain heart infusion broth (HiMedia) and incubated at 37°C for 4 h. The turbidity was adjusted to standard 0.50 McFarland, and 100 µl of the suspension was spread onto MHA plates. Antibiotic discs commonly available in Nepal were selected and aseptically placed on the plates (Table 1).

Table 1.

List of antibiotics used and their disc potency for antimicrobial susceptibility testing of Salmonella isolates from dead-in-shell embryonated eggs in Chitwan, Nepal.

Name of antibiotics	Disc potency
Ampicillin	10 µg
Ceftriaxone	30 µg
Gentamicin	10 µg
Chloramphenicol	30 µg
Trimethoprim/Sulfamethoxazole	25 µg
Levofloxacin	5 µg
Doxycycline	30 µg
Ciprofloxacin	5 µg
Cephalexin	30 µg
Amikacin	30 µg
Piperacillin/Tazobactam	110 µg
Nalidixic Acid	30 µg

The resistance profiles were interpreted by calculating the multiple antibiotic resistance (MAR) index, which is calculated as the ratio of the number of antibiotics to which an isolate was resistant (a) to the total number of antibiotics tested (b). Isolates having a MAR index ≥ 0.2 were considered to originate from a high-risk contamination source (Krumperman, 1983), while MAR < 0.2 indicated low-risk sources (Chitanand et al., 2010). In this study, an organism is labeled MDR if it exhibits resistance to at least one agent in three or more antibiotic classes (Magiorakos et al., 2012).

Biofilm formation assay

The biofilm-forming ability of Salmonella isolates was quantified using the crystal violet-based microtiter plate assay (Stepanović et al., 2007). Confirmed colonies were inoculated into 1 ml Trypticase Soy Broth (TSB) supplemented with 1 % glucose and incubated at 37°C for 24 hrs. Cultures were diluted 1:100 by transferring 10 μl of the culture into freshly prepared TSB. 200 μl of the diluted broth was inoculated into a 96-well flat-bottomed sterile polystyrene microplate. The uninoculated TSB served as the negative control. After incubation (37°C, 24 h), the contents of each well were carefully aspirated using a pipette placed at the lowest corner of the well. The wells were washed four times with 0.2 ml of phosphate-buffered saline. Subsequently, the wells were stained with 1 % crystal violet solution. Excess stain was removed by washing with deionized water, and the plates were air-dried for 30 min. To dissolve the adherent biofilm, 150 μl of an ethanol-acetone solution (80:20) was added to each well. The plate was incubated for 10-15 min to allow complete solubilization of the dye (Grakh et al., 2022).

The optical density (OD) of the wells was measured at 570 nm using a microplate reader. The results were interpreted based on categorization as no biofilm production (0), weak (+ or 1), moderate (++ or 2), and strong biofilm production (+++ or 3), using the cutoff value (ODc, Table 2), calculated as:

Table 2.

Interpretation of biofilm production of Salmonella isolates from dead-in-shell embryonated eggs in Chitwan, Nepal, based on optical density (OD) measurements.

Optical density range	Interpretation
OD ≤ ODc	No
ODc < OD ≤ 2 × ODc	Weak
2 × ODc < OD ≤ 4 × ODc	Moderate
4 × ODc < OD	Strong

ODc = Mean OD of blank wells + (3 × SD of blank wells), where the mean OD represents the average of the uninoculated wells and SD is the standard deviation of the blank wells.

Molecular characterization of virulence genes

The genomic DNA was extracted from Salmonella isolates using a modified boiled cell method (Pui et al., 2011). Cultured Salmonella colonies were inoculated into 1 mL of Luria-Bertani broth and incubated at 37°C for 24 h. The bacterial culture was then centrifuged at 15,000 x g for 3 min, and the resulting supernatant was discarded. The pellet was resuspended in 500 µL of nuclease-free water, heated at 100°C for 10 min, and then rapidly cooled to 4°C for 10 min. Following this, the samples were centrifuged at 15,000 x g for 3 min. The supernatant, which contained the genomic DNA, was then transferred to a fresh Eppendorf tube and stored at −20°C until PCR amplification. The concentration and purity of the DNA isolated from the Salmonella culture were quantified spectrophotometrically (Quawell, UV-Vis Spectrophotometer Q5000) at 260 and 280 nm, with acceptable ratios ranging from 1.6-2.

The PCR amplification of four virulence genes (invA, spvC, spvB1, and agfA) was carried out using Bio-Rad T100™ Thermal Cycler (Bio-Rad, USA). Each 10 μL reaction mixture contained 5 μL Master Mix (Invitrogen), 1 μL forward primer, 1 μL reverse primer, 1 μL DNA template, and 2 μL nuclease-free water (Ambion, REF: AM9932). PCR was performed to amplify the virulence genes of Salmonella. Detailed information on the primers (Macrogen, Inc., Seoul, South Korea), including their melting temperatures (Tm) and amplicon sizes, is presented in Table 3. The cycling conditions included pre-denaturation at 95°C for 5 min and 40 cycles of denaturation at 95°C for 30 s, gene-specific annealing temperatures based on their optimization, followed by initial elongation at 72°C for 30 s and a final elongation at 72°C for 10 min.

Table 3.

Primers used for molecular characterization of Salmonella isolates from dead-in-shell embryonated eggs in Chitwan, Nepal.

Target genes	Sequence (5-3)	Tm(°C)	Amplicon size (bp)	References
invA (Invasive protein)	F-GTGAAATTATCGCCACGTTCGGGCAA R-TCATCGCACCGTCAAAGGAACC	66 63	284	(Yanestria et al., 2019)
spvC (Salmonella plasmid virulence)	F-CGGAAATACCATCTACAAATA R-CCCAAACCCATACTTACTCTG	50 55	669	(Crăciunaş et al., 2012)
spvB1 (Salmonella plasmid virulence)	F-CTATCAGCCCCGCACGGAGAGCAGTTTTTA R-GGAGGAGGCGGTGGCGGTGGCATCATA	69 73	717	(Deng et al., 2021)
agfA (Aggregative fimbriae)	F-TCCACAATGGGGCGGCGGCG R-CCTGACGCACCATTACGCTG	70 61	350	(Naidoo et al., 2022)

The PCR products that were amplified were separated using 1.5 % agarose gels prepared in 0.5X TBE (Tris-Borate-EDTA) with 2 µl ethidium bromide (EtBr). Five microliters of PCR product, mixed with loading dye, were loaded alongside a 100 bp DNA ladder. The gel electrophoresis was performed at 90 V for 60 min, and DNA bands were visualized under a UV transilluminator (Platinum Q9, Uvitec Cambridge).

Statistical analysis

All data were entered in Microsoft Excel 2020 and analyzed in RStudio 4.3.3.2. Prevalence rates were calculated as percentages with 95 % confidence intervals. The association between the presence of the agfA gene and the categorization of biofilm production (strong vs. non-strong) was assessed using Fisher’s exact test. A P-value of < 0.05 was considered statistically significant for all tests.

Results

In the present study, out of a total of 210 samples collected, 24 (11.42 %) Salmonella isolates were analyzed in dead-in-shell chick embryos across fifteen hatcheries in Bharatpur. The isolates were initially identified with the help of cultural characteristics, followed by biochemical tests and further confirmed by molecular detection methods.

AST revealed high levels of resistance to several clinically important antibiotics, with near-universal resistance observed for cephalexin (100 %) and ampicillin (95.83 %) (Table 4). In general, trimethoprim-sulfamethoxazole, gentamicin, chloramphenicol, and levofloxacin were the active antimicrobials indicated against Salmonella spp. The average MAR index was 0.51. Among these isolates, 95.83 % of isolates had a MAR index of 0.2 or greater, and 8.33 % (2/24) showed resistance to all of the antibiotics tested (MAR index of 1). The distribution of MDR phenotypes among the isolates varied, with the largest group (29.17 %) showing resistance to six different classes of antibiotics (Table 5).

Table 4.

Antimicrobial resistance patterns of 24 Salmonella isolates from dead-in-shell embryonated eggs in Chitwan, Nepal.

Name of Antibiotics	Total sensitive	Total intermediate	Total resistance
Ampicillin	0 (0 %)	1 (4.17 %)	23 (95.83 %)
Ceftriaxone	6 (25 %)	9 (37.50 %)	9 (37.50 %)
Gentamicin	14 (58.33 %)	4 (16.67 %)	6 (25 %)
Chloramphenicol	14 (58.33 %)	4 (16.67 %)	6 (25 %)
Trimethoprim/ Sulfamethoxazole	16 (66.67 %)	1 (4.17 %)	7 (29.17 %)
Levofloxacin	14 (58.33 %)	3 (12.50 %)	7 (29.17 %)
Doxycycline	11 (45.83 %)	1 (4.17 %)	12 (50 %)
Ciprofloxacin	8 (33.33 %)	6 (25 %)	10 (41.67 %)
Cephalexin	0 (0 %)	0 (0 %)	24 (100 %)
Amikacin	6 (25 %)	6 (25 %)	12 (50 %)
Piperacillin/ Tazobactam	1 (4.17 %)	13 (54.17 %)	10 (41.67 %)
Nalidixic Acid	1 (4.17 %)	1 (4.17 %)	22 (91.67 %)

Data reflects the number and percentage of Salmonella isolates (n=24) categorized by their susceptibility to each antibiotic.

Table 5.

Distribution of multidrug-resistance phenotypes among Salmonella Isolates from dead-in-shell embryonated eggs in Chitwan, Nepal.

S.N.	Number of antimicrobial classes of resistance	MDR isolates	MDR isolates in percentage (%)
1	1	0	0.00
2	2	4	16.67
3	3	6	25.00
4	4	5	20.83
5	5	2	8.33
6	6	7	29.17

Biofilm formation, assessed via crystal violet assay, was observed in 70.83 % (17/24) of Salmonella isolates, with 41.67 % (10/24) classified as weak producers and 16.67 % (4/24) as strong producers (Table 6). A significant association between strong biofilm production and the agfA gene was found (P = 0.00198, Fisher’s exact test) (Table 7).

Table 6.

Categorization of biofilm production (none, weak, moderate, or strong) by 24 Salmonella isolates from dead-in-shell embryonated eggs.

S.N.	Biofilm category	Number of isolates(n=24)	Percentage (%)
1	No	7	29.17
2	Weak	10	41.67
3	Moderate	3	12.50
4	Strong	4	16.67

Table 7.

Association between agfA gene presence and strong biofilm production in Salmonella isolates.

S.N.	Category	agfA (+ve)	agfA(-ve)	P-value
1	Strong biofilm	3	1
2	No strong biofilm	0	20	0.00198

In this study, four virulence genes were amplified using PCR techniques. All 24 Salmonella isolates (100 %) were positive for the invA gene (Fig. 1), which is suggested as a biomarker for confirmation of Salmonella spp. from dead-in-shell chick embryos (Li et al., 2020; Zhao et al., 2021). Similarly, 12 isolates (50%) carried the virulence gene spvC (Fig. 2), 8 isolates (33.33%) harbored spvB1 (Fig. 3), and 3 isolates (12.5%) possessed the agfA gene (Fig. 4), exhibiting varying degrees of virulence among isolates.

Fig. 1.

Agarose gel electrophoresis (1.5 %) of PCR products for the invA gene (284 bp). Lane 1: 100 bp DNA ladder; Lane 5: Negative control (nuclease-free water); Lanes 2-4 and 6-14: positive isolates.

Fig. 2.

Agarose gel electrophoresis (1.5 %) of PCR products for the spvC gene (669 bp). Lane 1: 100 bp DNA ladder; Lane 5: Negative control (nuclease-free water); Lanes 2-4 and 6-14: positive isolates.

Fig. 3.

Agarose gel electrophoresis (1.5 %) of PCR products for the spvB1 gene (717 bp). Lane 1: 100 bp DNA ladder; Lane 5: Negative control (nuclease-free water); Lanes 2-4 and 6-10: positive isolates.

Fig. 4.

Agarose gel electrophoresis (1.5 %) of PCR products for the agfA gene (350 bp). Lane 1: 100 bp DNA ladder; Lane 3: Negative control (nuclease-free water); Lanes 2, 4-5 : positive isolates.

Discussion

The high prevalence of Salmonella in dead-in-shell embryonated eggs highlights a significant obstacle for the poultry industry in Nepal. Salmonella contamination can severely undermine overall productivity in the hatchery. It reduces the chick quality and hatchability, which can threaten the economy of major poultry hubs of the country. The Salmonella prevalence observed in this study aligns closely with reported rates of Egyptian (10 %) (Barac et al., 2024) and Pakistani hatcheries (12.5 %) (Shehata, 2019). A similar study in Chitwan has reported a 9 % prevalence in poultry farm environmental samples, possibly because sampling was conducted in the dry season (Sharma et al., 2021). This is lower than our findings, which were obtained during the monsoon period. Conversely, a 26.2 % prevalence in Chitwan’s poultry carcasses suggests post-hatchery contamination amplification (Shrestha et al., 2017).

Several factors may account for differences in prevalence rates, and hatchery hygiene is often a primary driver. In Chitwan, smallholder-dominated hatcheries may lack consistent biosecurity protocols, unlike larger operations in industrialized countries (Dhakal et al., 2025). The inadequate sanitation of incubators or egg-handling surfaces facilitates the persistence of Salmonella, particularly through biofilms. This can be noted in our study, which showed significant biofilm-forming Salmonella isolates. In addition, our focus on dead-in-shell embryos may capture higher Salmonella loads compared to environmental swabs, which dilute contamination across surfaces. Nepal’s monsoon season overlaps with our sampling period (August-December). So, seasonal effects such as increading humidity during the monsoon, can potentiate microbial proliferation, which is unlike the dry conditions (Cruz-Paredes et al., 2021). These factors suggest that prevalence rates are context-specific, requiring tailored interventions.

The high AMR in Salmonella isolates has limited the treatment options for poultry infections. The challenges imposed by AMR are critical in the poultry industry. With pronounced resistance to commonly available antibiotics in Nepal, there is a greater concern to find a viable agent for controlling Salmonellosis in the poultry sector. The elevated MAR index suggests extensive antibiotic exposure in poultry production, often resulting from overuse for both therapeutic and prophylaxis purposes (Shakir et al., 2021). This can stem from the common practice of injudicious antibiotic use in Asia, where antimicrobials are often used widely without proper prescription or consideration for withdrawal period (Ghimire et al., 2023). Further, a study conducted in Nepal has reported MDR ranging from 45 to 46 % in Salmonella isolates from 20 poultry farms, with amikacin being the most sensitive antibiotic and ciprofloxacin and trimethoprim-sulfamethoxazole showing the highest resistance (Pal et al., 2022), which is consistent with the resistance patterns observed in our samples. These discrepancies in AMR reflect the differences in sample sources, antibiotic usage practices, or regional variations in AMR patterns.

Biofilms are microbial communities surrounded by self-produced extracellular polymeric substances adhered to biotic or abiotic surfaces. This polymeric matrix serves as a protective shield against harsh environmental stresses, antibiotics, and disinfectants (Sharma et al., 2023). The persistence of isolates via biofilm formation makes it quite difficult to eradicate them from a hatchery environment. The strong biofilm producers have exacerbated the contamination, creating a resilient environment in the hatchery. These are linked with the presence of the agfA gene, which enhances the ability to withstand environmental stress as it is often associated with promoting biofilm stability on surfaces (Mashayekh et al., 2022). Also, the consistent presence of spvC, spvB1, and universal invA underscores the potent pathogenicity of Salmonella in embryos. These genes contribute to increased systemic infection and disease severity in poultry (Alarcón Navas et al., 2024). They likely drive effective establishment of infection in hatchery settings, further confirming the plasmid-mediated virulence potential of these isolates. They also promote strong biofilm formation, aiding the persistence and stabilization of Salmonella on hatchery equipment (Dlamini et al., 2024). The findings contrast with the much higher prevalence of the agfA gene reported in Brazil (96%) compared with 12.5% in this study (Borges et al., 2018). This could be attributed to regional variations in farming practices, biosecurity measures, genetic diversity of Salmonella strains, and differing selective pressures from antimicrobial usage (Lamichhane et al., 2024).

Association between strong biofilm formation and agfA gene in the studied isolates

While, the agfA gene was significantly associated with strong biofilm phenotype(P=0.00198), its overall prevalence was relatively low (12.5 %) compared to the high proportion of isolates capable of forming biofilms (70.83 %). This suggests that biofilm formation in this Salmonella population is primarily driven by alternative genetic pathways. Other genetic determinants, such as those in the csg (curli-specific gene) operon, which regulate curli fimbriae and cellulose production, play a crucial role in promoting adhesion and biofilm structure in the absence of agfA (Tursi and Tükel, 2018). These findings indicate complex and multifactorial regulation in Salmonella and indicate that multiple genetic mechanisms contribute to its persistence and resilience within hatchery environments. Therefore, future studies should aim to characterize the full repertoire of biofilm-associated genes in these isolates to develop more effective eradication strategies.

Public health implications and control challenges

MDR Salmonella contamination in eggs and hatched chicks via the hatchery environment can pose severe health risks to vulnerable populations in Nepal. Young children, the elderly, and immunocompromised patients are more susceptible to severe foodborne illness (World Health Organization, 2008). They may present with diarrhea, fever, and abdominal cramps, which can lead to hospitalization in severe cases (Keestra-Gounder et al., 2015). It can strain healthcare resources, particularly in rural settings. It is further complicated by the limited availability of drugs due to AMR, which prolongs the illness duration (Kumar et al., 2025).

Despite existing food safety frameworks, there is weak enforcement and a lack of regular monitoring of food safety policies in Nepal. On top of this, limited consumer awareness is another prime concern. It has hindered the application of effective control strategies for Salmonella contamination in the poultry sector (Subedi et al., 2025a). The informal poultry market is predominant with minimal hygiene oversight. It facilitates the distribution of contaminated eggs and hatched chicks to unaware farmers and consumers, escalating the public health risks. Also, routine surveillance for Salmonella in poultry and human populations is inadequate. This results in underreporting and limited understanding of disease burden in the local context (Osti et al., 2017; Subedi et al., 2025b). With the limited availability of routine data, partnering with regional laboratories could explain the surveillance and vastly improve the data quality, which is beneficial to future studies. Moreover, a unified data-sharing system between veterinary hospitals and regulatory bodies would also enhance monitoring efforts.

The porous border between Nepal and neighbouring countries, particularly India, has facilitated the unregulated movement of poultry products and day-old chicks. This cross-border trade often occurs without sanitary checks, substantially increasing the risk of introduction and dissemination of MDR Salmonella strains across the country (Sharma et al., 2021). Additionally, the lack of formal training and biosecurity awareness among the hatchery workers and farmers further amplify this challenge. Many stakeholders are unaware of basic hygienic handling and disinfection protocols essential in poultry operations, which contribute to pathogen persistence in the environment. So, strengthening the disease prevention and control would require coordinated cross-border surveillance and targeted workforce capacity-building programs.

Strategic recommendations for Salmonella control

With the rise of resistant strains, several promising approaches have been presented as promising alternatives for antimicrobial properties. Given the high prevalence of MDR (83.33 % of isolates) and robust biofilm formation (70.83 % of isolates) observed in this study, non-antibiotic strategies such as bacteriophage therapy are particularly relevant, as phages can effectively penetrate protective biofilms and lyse resistant bacterial strains (Lin et al., 2017). Probiotics can introduce beneficial bacteria in order to improve poultry gut health and competitively inhibit Salmonella colonization. It can reduce reliance on traditional antibiotics while improving gut health (Naeem and Bourassa, 2025). Furthermore, organic acid disinfectants like lactic acid have been shown to effectively control Salmonella in poultry hatcheries. These compounds can disrupt Salmonella biofilm on equipment and reduce environmental contamination (Bai et al., 2022). Integrating these measures into poultry management practices can significantly reduce the burden of antimicrobial resistance while maintaining flock health and productivity.

As a proactive measure, vaccination in poultry flocks can effectively mitigate the colonization of Salmonella (Siddique et al., 2024). Successful vaccination programs against Salmonella in several developed countries can be a valuable lesson to Nepal. It can improve poultry health and enhance food safety standards which aligns with the global trend of sustainable poultry production. However, challenges of cost, infrastructure, and farmer education should also be taken into consideration. The effectiveness of local disinfectants should be routinely and rigorously evaluated (Hansson et al., 2025) and appropriate measures should be taken to ensure their ability to combat MDR and biofilm-forming Salmonella, which is prevalent in Nepalese hatcheries.

Limitation and future directions

One limitation of this study is the absence of serological typing of the isolates. Although serotyping remains an important tool for epidemiological surveillance and source attribution, we focused on molecular characterization by profiling virulence genes associated with invasion, systemic infection, and biofilm formation. There is a need for longitudinal studies to reveal the prevalence and resistance profile by season. Advanced molecular tools like whole genome sequencing for gene detection would allow detailed identification of resistance genes along with their mechanism (Chrzastek et al., 2025). Specific studies on ESBL genes (bla_TEM, bla_CTX-M, bla_SHV, and bla_OXA) and biofilm genes (adrA, csgD) in Salmonella from dead-in-shell eggs are needed to address the increasing threats. It is advisable to enforce strict antibiotic stewardship programs as per the WHO global action plan (World Health Organization, 2015) for AMR in order to curb the alarming resistance. Routine sampling of hatchery environments and poultry products is equally important for early detection of Salmonella (National Advisory Committee On Microbiological Criteria For Foods, 2024).

Conclusions

This study highlights the dual threat of MDR and biofilm-forming Salmonella in Nepalese poultry hatcheries. The dead-in-shell embryonated eggs are identified as a critical reservoir for persistence and transmission of resistant bacteria. The findings provide a baseline for future studies aimed at controlling MDR Salmonella in poultry hatcheries. The presence of virulence genes alongside AMR reinforces the potential for severe and sustained infections. Beyond economic losses, these strains represent a serious public health risk, as antibiotic-resistant infections can easily spread through the food chain. This study sets the stage for a broader effort to address the burning issue of AMR and promote sustainable and safe poultry production in Nepal.

Availability of supporting data

Datasets generated for this study are available on request to the corresponding author.

Ethics statement

This study was reviewed and approved by the thesis advisory committee of the Agriculture and Forestry University, Chitwan, Nepal, prior to its initiation. Informed consent was obtained from participating poultry farm owners, and no individual identifiers of farms or owners were disclosed. All data were anonymized before analysis to ensure confidentiality. No interventions or experimental procedures were performed on live animals during the study.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Swagat Khanal: Writing – original draft, Methodology, Formal analysis, Investigation, Writing – review & editing. Himal Luitel: Supervision, Resources, Project administration, Validation. Sujan Adhikari: Writing – review & editing, Writing – original draft, Investigation, Validation. Aakash Marasini: Writing – review & editing, Software, Formal analysis, Investigation. Rebanta Kumar Bhattarai: Resources, Project administration, Formal analysis.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.