Abstract

Salmonella is a major foodborne pathogen found in various environmental sources, with the rise of antibiotic resistance posing a growing public health concern. This study isolate, identify, and characterize Salmonella spp. from milk, beef, and chicken meat to determine antibiotic resistance patterns and assess public health implications. A total of 120 samples were collected from different markets of Savar and Gazipur Sadar of Bangladesh. Presumptive identification of Salmonella isolates was performed by culturing on selective media, Gram’s staining and confirmed by polymerase chain reaction (PCR) targeting the genus-specific primer of invA. Bivariate analysis to assess the strong and significant correlations between the pairs of any of two antibiotic-resistant Salmonella spp. On the basis of cultural characteristics, the overall occurrence of Salmonella spp. was 20% in milk, 10% in beef and 25% in chicken meat. Furthermore, amplification of invA gene revealed the occurrence as 63.63%. Among then 62.5% in milk, 75% in beef and 60% in chicken meat, respectively. The antimicrobial resistance pattern was determined by disc diffusion method. Among the antibiotics used in this study highest level of sensitivity were shown in streptomycin 100%, imipenem 100% and meropenem 80% in milk samples whereas highly resistance obtained to oxytetracycline 66.66% in beef and 83.33% in chicken meat. Notably, 20%, 33.33% and 16.66% isolates of milk, beef, and chicken meat, respectively were multidrug-resistant. Salmonella contamination in raw milk, beef and chicken poses a public health risk in the study area. Proper hygiene measures, awareness among sellers and judicious control use of antibiotic at the farm level are crucial to reducing contamination and antibiotic resistance.

1. Introduction

Foodborne diseases are a persistent global health challenge, affecting developing countries where food hygiene and regulatory oversight remain inadequate ¹. Salmonella spp. is one of the most significant bacterial pathogens, causes of foodborne illness, responsible for an estimated 93.8 million infections and 155,000 deaths annually ². It is a Gram-negative, facultative anaerobic member of the Enterobacteriaceae family comprising two species, S. bongori and S. enterica, with the latter being the most clinically relevant to humans and animals ³.

Animal-derived foods such as milk, beef, and chicken are nutritious but also serve as important reservoirs of Salmonella transmission to humans ^{4, 5, 6}. In Bangladesh, food safety concerns are compounded by poor hygienic practices, inadequate meat processing, and lack of effective regulation ^{7, 8}. Outbreaks linked to contaminated poultry and raw milk have been widely reported, with prevalence ranging from 6.79% to 97.6% in chicken meat and varying detection rates in beef and dairy products worldwide ^{9, 10, 11, 12}.

Beyond its epidemiological burden, Salmonella represents a growing antimicrobial resistance (AMR) threat. It is among the most common multidrug-resistant (MDR) pathogens, contributing to treatment failure, prolonged hospitalization, and increased mortality ^{13, 14}. Inappropriate antimicrobial use in livestock production-often without veterinary oversight-accelerates the emergence of resistance ^{15, 16, 17}. Resistant strains can disseminate through direct contact, food consumption, or environmental contamination, creating a One Health challenge ^{18, 19}.

Previous studies have reported high resistance rates of Salmonella isolates to tetracyclines, beta-lactams, fluoroquinolones, and aminoglycosides in both humans and food-producing animals ^{7, 17, 19, 20, 21}. The detection of MDR Salmonella-defined as resistance to three or more antimicrobial classes is particularly concerning ²². Molecular studies indicate that resistance is often linked to transferable genes, enabling horizontal dissemination across bacterial populations ^{23, 24}.

Although Salmonella prevalence has been investigated in Bangladesh ^{18, 25, 26} integrated studies addressing both phenotypic resistance and molecular confirmation of isolates across multiple food sources remain scarce. Existing reports often focus on either cultural detection or antibiograms without systematically linking these findings to molecular identification of virulence or resistance determinants ⁸. This knowledge gap limits understanding of MDR Salmonella in the local food chain.

The present study was therefore conducted to (i) determine the prevalence of Salmonella spp. in raw milk, beef, and chicken meat collected from local markets in Bangladesh, (ii) confirm isolates by molecular detection targeting the invA gene, and (iii) assess antimicrobial resistance profiles and MDR patterns. This work provides comprehensive data for veterinary and public health sectors, with implications for antimicrobial stewardship, food safety policy, and consumer health protection.

2. Materials and Methods

A total of 120 samples were collected from Gazipur Sadar and Savar of Dhaka, Bangladesh. The samples included raw milk (n = 40), beef (n = 40), and chicken meat (n = 40). All samples were collected aseptically and transferred to the pathology laboratory at Gonoshasthaya Samaj Vittik Medical College Hospital, Savar, Dhaka-1344, Bangladesh for microbiological analysis.

Twenty-five grams of meat or 25 mL of milk was homogenized in 225 mL buffered peptone water (BPW; HiMedia, India) and incubated at 37 °C for 24 h. Aliquots were streaked onto MacConkey agar and Salmonella–Shigella (SS) agar and incubated at 37 °C for 24-48 h. Colonies with characteristic morphology (smooth, round, black-centered on SS agar) were subjected to Gram staining for confirmation.

Antibiotic susceptibility was determined by Kirby-Bauer disc diffusion on Mueller-Hinton agar (HiMedia, India), following CLSI guidelines ²⁸. A 0.5 McFarland suspension of each isolate was inoculated, and commercial discs of streptomycin (10 µg), amoxicillin (30 µg), cefoxitin (30 µg), cefuroxime (30 µg), ceftriaxone (30 µg), doxycycline (30 µg), oxytetracycline (30 µg), clindamycin (2 µg), meropenem (10 µg), and imipenem (10 µg) were applied. After 18–24 h incubation at 37 °C, inhibition zones were measured and interpreted as susceptible, intermediate, or resistant according to ²⁸.

Multidrug resistance (MDR) was defined as resistance to three or more antibiotic classes ²². The MAR index was calculated using the formula:

Results are interpreted following guidelines of the Clinical and Laboratory Standards Institute (CLSI) 2021.

Data were analyzed using IBM SPSS Statistics v25.0 (Chicago, IL, USA). Descriptive statistics were used to calculate prevalence with 95% confidence intervals (CI). Chi-square tests assessed differences in prevalence between sample types, with p<0.05 considered statistically significant. Correlation of resistance patterns was evaluated using Pearson’s correlation coefficient.

3. Results

A total of 120 samples were analyzed for Salmonella contamination, with an overall culture-positive occurrence of 18.33%. In Gazipur Sadar, Salmonella was detected in 25% of milk samples, 5% of beef samples and 30% of chicken meat samples. In Savar, the prevalence was 15% in milk, 15% in beef and 20% in chicken meat (Table 2). Chicken meat exhibited the highest occurrence of Salmonella spp., where 30% of samples from Gazipur Sadar and 20% from Savar. The lowest occurrence was observed in beef which was collected from Gazipur Sadar (5%), with a statistically significant association (p = 0.00004) (Table 2).

All culture positive Salmonella spp. isolates were confirmed by PCR targeting the invA gene. Among all culture-positive 22 Salmonella spp., 14 isolates were found positive to contain invA gene and a positive band appeared at 211 bp after PCR amplification (Figure 2). On the basis of PCR amplification of invA gene positive isolates of Salmonella spp. from the collected samples, 14/22 (63.63%, CI: 42.95-80.26) were found (Figure 1).

The sample-wise occurrence of Salmonella spp. in milk was 4/5 (80%, CI: 37.55-98.97), beef was 1/1(100%, CI: 5.12-100) and chicken meat was 3/6 (50%, CI: 18.76-81.23), respectively in Gazipur Sadar. Conversely, 1/3 (33.33%, CI: 1.70-88.15), 2/3 (66.66%, CI: 11.84-98.29), and 3/4 (75%, CI: 30.06-98.71) Salmonella spp. was found in milk, beef, and chicken meat, respectively in Savar (Figure 1).

In this research a total of 10 commercially available antibiotics were utilized. In milk samples, the highest levels of sensitivity were observed for streptomycin and imipenem (100%), followed by doxycycline, amoxicillin, and meropenem (80%). In contrast, oxytetracyline, cefoxitin, and cefuroxime demonstrated higher resistance rates, with resistance levels of 80%, 60%, and 40%, respectively. Additionally, in beef samples, Salmonella spp. obtained the highest number of resistances found in amoxicillin and oxytetracyline 66.66% whereas 100% sensitive against streptomycin, imipenem, and meropenem. Salmonella spp. showed highly sensitive against streptomycin, imipenem, merooenem and cefuroxime 100% whereas highest resistant pattern was found against oxytetracyline 83.33% in chicken meat samples, respectively (Figure 3).

These patterns of resistance and sensitivity are consistent with the bivariate analysis's statistical conclusions, in which a strong and positive significant correlation was observed between clindamycin (CD) and oxicillin amoxicillin (AMC) (PCC = 0.645, p = 0.013. Clindamycin (DA) and oxytetracycline (O) exhibited a strong negative correlation (PCC = -0.782, p = 0.001). A moderate positive correlation (PCC = 0.548, p = 0.043) was found between cefuroxime (CX) and cefoxitin (FOX). No significant correlations were observed for doxycycline (DA), ceftriaxone (CTX), and imipenem (IPM) with other antibiotics (Table 3).

Among the Salmonella isolates from milk samples, 40.00% (2/5) exhibited MDR, with two distinct resistance patterns. One isolate (20.00%) showed resistance to cefoxitin (FOX), cefuroxime (CXM), and oxytetracycline (O) (MAR index 0.3). Another isolate (20.00%) exhibited resistance to amoxicillin (AMC), cefoxitin (FOX), oxytetracycline (O), cefuroxime (CXM), clindamycin (CD), and doxycycline (DA) (MAR index 0.6) (Table 4).

Among the beef-derived Salmonella isolates, 33.33% (1/3) demonstrated MDR, showing resistance to amoxicillin (AMC), cefoxitin (FOX), ceftriaxone (CTR), oxytetracycline (O), and clindamycin (CD) (MAR index 0.5). In chicken meat samples, 16.66% (1/6) of Salmonella isolates were identified as MDR. The resistance pattern included amoxicillin (AMC), cefoxitin (FOX), ceftriaxone (CTR), clindamycin (CD), and doxycycline (DA) (MAR index 0.5). These findings indicate higher MDR prevalence in milk samples (40%) compared to beef (33.33%) and chicken meat (16.66%) (Table 4).

4. Discussion

The present study provides critical insights into the prevalence, molecular confirmation, and antimicrobial resistance (AMR) patterns of Salmonella spp. isolated from raw milk, beef, and chicken meat in Bangladesh. Our findings indicate an overall culture-based prevalence of 18.33%, with PCR confirmation of the invA gene in 63.63% of isolates. These results reinforce the role of animal-derived foods as significant reservoirs of foodborne Salmonella and underscore the escalating challenge of antimicrobial resistance in the country.

The prevalence observed in this study is consistent with reports from Bangladesh and other low and middle-income countries, where the food supply chain is often compromised by inadequate hygiene and regulatory oversight. ⁷ reported Salmonella prevalence of 16% in milk and meat samples in Bangladesh, which aligns closely with our findings. Similarly, ²⁵ detected Salmonella in 14% of eggs and egg trays, highlighting the widespread presence of this pathogen across different animal products.

Our study found the highest occurrence in chicken meat (30% in Gazipur Sadar and 20% in Savar), supporting global evidence that poultry is the primary reservoir of Salmonella ^{5, 9}. In contrast, beef samples showed the lowest prevalence (5–15%), which may reflect differences in animal husbandry practices, slaughterhouse hygiene, and post-harvest handling. Compared to international reports, our prevalence rates are somewhat lower than the 36–40% reported in Ethiopia ¹² and Iraq ¹⁰, but higher than some industrialized settings where stricter food safety measures are implemented ³.

PCR confirmation of the invA gene further strengthens our findings, as this gene is a highly conserved marker associated with Salmonella virulence and invasion capacity ^{11, 23}. The gene was detected in 80% of milk, 100% of beef, and 60% of chicken isolates, highlighting the pathogenic potential of these strains and their risk to consumer health.

The AMR profiles in our study reveal a disturbing trend. High resistance was observed against oxytetracycline (80% in milk, 66.6% in beef, and 83.3% in chicken meat), amoxicillin, and cefoxitin, consistent with earlier findings in Bangladesh ^{20, 29}. Tetracyclines and beta-lactams remain widely used in livestock production in the region, often as growth promoters or for prophylactic purposes without veterinary oversight ¹⁶. This misuse likely explains the elevated resistance rates documented here.

In contrast, isolates demonstrated complete sensitivity to imipenem and streptomycin, and high sensitivity to meropenem, corroborating reports from other LMICs ^{13, 19}. These findings suggest that carbapenems remain effective against Salmonella in this context, though their use in veterinary practice is restricted. The resistance patterns identified here mirror global concerns that excessive reliance on older antibiotics has reduced their efficacy, while last-resort antimicrobials remain effective but should be preserved to mitigate the risk of future resistance ³⁰.

Our study detected multidrug-resistant (MDR) isolates in 40% of milk, 33.33% of beef, and 16.66% of chicken samples, with MAR indices ranging from 0.3 to 0.6. The higher MDR prevalence in milk compared to meat products is concerning, given the frequent consumption of raw or inadequately boiled milk in many Bangladeshi households ³¹. Previous studies in Bangladesh ²⁶ and other countries ^{24, 32} similarly report MDR prevalence ranging between 20–60%, reinforcing that Salmonella has developed substantial resistance reservoirs within the food chain.

The identified MDR patterns often involved resistance to amoxicillin, cefoxitin, oxytetracycline, and doxycycline, consistent with the presence of transferable resistance determinants such as tet, bla, and qnr genes reported globally ^{23, 24}. Our correlation analysis further suggested co-selection of resistance phenotypes, a finding in agreement with previous research demonstrating horizontal gene transfer as a key driver of AMR dissemination ^{33, 35}. The detection of MDR Salmonella in animal-derived foods has critical public health implications. Resistant infections are associated with prolonged illness, treatment failure, and increased hospitalization costs ^{14, 34}. Furthermore, the transmission of resistant strains from food animals to humans exemplifies the interconnectedness of human, animal, and environmental health, reinforcing the need for a One Health approach ^{6, 18}.

Our findings highlight the urgent need for stronger antimicrobial stewardship, improved food safety regulation, and robust AMR surveillance in Bangladesh. Interventions should focus on restricting non-therapeutic antimicrobial use in livestock, promoting farm-level hygiene, and raising consumer awareness about the risks of consuming raw or undercooked animal products. This study provides integrated cultural, molecular, and phenotypic resistance data across multiple food sources, which is relatively scarce in the Bangladeshi context. However, limitations include the relatively small sample size and the lack of molecular characterization of specific resistance genes, which would have provided deeper insights into genetic mechanisms underlying MDR.

5. Conclusion

Salmonella is a major bacterial pathogen in animal and poultry and also considered as one of the major foodborne bacterial pathogens causing human illness worldwide. Antibiotic susceptibility testing revealed alarming levels of resistance, particularly to oxytetracycline, cefoxitin, and amoxicillin, suggesting unregulated antibiotic use in livestock production. The high prevalence of multidrug-resistant (MDR) Salmonella isolates posed by antimicrobial-resistant foodborne pathogens. The strong correlation between resistances to multiple antibiotic classes indicates the potential for co-selection and horizontal gene transfer. Given these findings urgent interventions are needed to mitigate the public health risks associated with foodborne Salmonella infections. Strengthened food safety policies, enhanced surveillance of antimicrobial resistance and the promotion of responsible antibiotic use in veterinary medicine are essential to controlling the spread of MDR Salmonella.

ACKNOWLEDGMENTS

Authors Contribution

SSS, SNS, MTI, SB, SCS, BSS, and MRA performed sample collection, sample processing, data collection, laboratory work, and interpretation. MNB, SB, PPG, TA, and MAN performed the data analysis, statistical analysis, visualization, and literature searches. MMH and PPG made critical comments on the manuscript. HS supervised the present study. All authors contributed to, reviewed, and approved the final manuscript.

References