Antimicrobial Resistance Profile of Common Foodborne Pathogens Recovered from Livestock and Poultry in Bangladesh

Multidrug-resistant (MDR) foodborne pathogens have created a great challenge to the supply and consumption of safe & healthy animal-source foods. The study was conducted to identify the common foodborne pathogens from animal-source foods & by-products with their antimicrobial drug susceptibility and resistance gene profile. The common foodborne pathogens Escherichia coli (E. coli), Salmonella, Streptococcus, Staphylococcus, and Campylobacter species were identified in livestock and poultry food products. The prevalence of foodborne pathogens was found higher in poultry food & by-product compared with livestock (p < 0.05). The antimicrobial drug susceptibility results revealed decreased susceptibility to penicillin, ampicillin, amoxicillin, levofloxacin, ciprofloxacin, tetracycline, neomycin, streptomycin, and sulfamethoxazole-trimethoprim whilst gentamicin was found comparatively more sensitive. Regardless of sources, the overall MDR pattern of E. coli, Salmonella, Staphylococcus, and Streptococcus were found to be 88.33%, 75%, 95%, and 100%, respectively. The genotypic resistance showed a prevalence of blaTEM, blaSHV, blaCMY, tetA, tetB, sul1, aadA1, aac(3)-IV, and ereA resistance genes. The phenotype and genotype resistance patterns of isolated pathogens from livestock and poultry had harmony and good concordance, and sul1 & tetA resistance genes had a higher prevalence. Good agricultural practices along with proper biosecurity may reduce the rampant use of antimicrobial drugs. In addition, proper handling, processing, storage, and transportation of foods may decline the spread of MDR foodborne pathogens in the food chain.


Introduction
Foodborne illness is a key public health concern worldwide which occurs due to the ingestion of contaminated food products, mostly animal and poultry-derived food [1][2][3].

Prevalence of AMR Pathogens in Animal-Derived Food Products
E. coli, Salmonella, and Staphylococcus species were isolated from both livestock and poultry food product & by-product samples throughout the country. In addition, Streptococcus species were isolated from only livestock-source food product & by-product samples. Furthermore, Campylobacter species was only isolated from poultry food products of the Chattogram Veterinary and Animal Sciences University (CVASU) component, i.e., from the Chittagong division. The overall prevalence of E. coli, Salmonella, Staphylococcus, and Streptococcus in livestock-source food products and by-products were found to be 38.47% (554/1440), 8.26% (119/1440), 14.67% (211/1440), and 4.79% (69/1440), respectively (details of prevalence pattern are presented in Table 1). Similarly, the overall prevalence of E. coli, Salmonella, Staphylococcus, and Campylobacter in poultry food products & by-products were 51.59% (454/880), 17.61% (155/880), 21.93% (193/880), and 4.43% (39/880), respectively ( Table 2). Regardless of sources, the overall multidrug-resistant pattern of E. coli, Salmonella, Staphylococcus, and Streptococcus were found to be 88.33%, 75%, 95%, and 100%, respectively. The prevalence of E. coli was found higher in poultry food products & by-products compared with livestock (51.59% vs. 38.47%; p < 0.05). Similarly, a higher statistical association was found between the prevalence of Salmonella in livestock and poultry food products (17.61% vs. 8.26%; p < 0.05). In addition, an association was also found between the prevalence of Staphylococcus in livestock and poultry samples (21.93% vs. 14.67%; p < 0.05). On the other hand, Campylobacter was found in poultry food products & by-products collected from only the Chittagong division and the overall percentage was 4.43% (39/880).

Discussion
Antimicrobials are frequently used to treat infectious diseases in both humans and animals [29]. Recently, the overuse and misuse of antibiotics in livestock have become a great concern for public health authorities. Contrarily, because withdrawal periods before harvesting or marketing livestock products have been ignored, antibiotic residues are now another rising concern to public health [30]. The main consequence of antibiotic residues in animal-derived foods is the enhancement of the development of antimicrobial resistance. The presence of antibiotic-resistant foodborne pathogens in food may lead to gastrointestinal disorders in human beings [31]. On the other hand, antibiotic-resistant pathogens may transfer the gene to other microorganisms through vertical and horizontal transmission [29,32] resulting in the spread of AMR pathogens. Several previous studies have shown the emergence of multi-resistant bacterial pathogens from a wide variety of sources in the food chain, increasing the need for proper use of antibiotics in both the veterinary and human health sectors [33][34][35]. MDR pathogens may cause difficult-to-treat illnesses, increased mortality, and financial burden. Furthermore, infections caused by MDR pathogens are considered a major global public health crisis by the World Health Organization, as the discovery of effective antibiotics has not kept pace with the increase in bacterial antibiotic resistance [36]. The demand for high-value animal products such as milk, meat, and eggs has increased due to economic solvency, rapid urbanization, and There was a similar trend in the phenotypic resistance patterns of foodborne pathogens. The phenotypic resistance was found to be comparatively higher in the foodborne pathogens isolated from poultry than livestock-source foods and by-products (p < 0.05). The phenotypic and genotypic resistance profiles of various isolates of foodborne pathogens were shown to have a narrower range of variation and variability. The phenotypic and genotypic resistance results indicated that multidrug-resistant and ESBL-producing foodborne pathogens were prevailing in the livestock-and poultry-source food products & by-products in Bangladesh.

Discussion
Antimicrobials are frequently used to treat infectious diseases in both humans and animals [29]. Recently, the overuse and misuse of antibiotics in livestock have become a great concern for public health authorities. Contrarily, because withdrawal periods before harvesting or marketing livestock products have been ignored, antibiotic residues are now another rising concern to public health [30]. The main consequence of antibiotic residues in animal-derived foods is the enhancement of the development of antimicrobial resistance. The presence of antibiotic-resistant foodborne pathogens in food may lead to gastrointestinal disorders in human beings [31]. On the other hand, antibiotic-resistant pathogens may transfer the gene to other microorganisms through vertical and horizontal transmission [29,32] resulting in the spread of AMR pathogens. Several previous studies have shown the emergence of multi-resistant bacterial pathogens from a wide variety of sources in the food chain, increasing the need for proper use of antibiotics in both the veterinary and human health sectors [33][34][35]. MDR pathogens may cause difficult-to-treat illnesses, increased mortality, and financial burden. Furthermore, infections caused by MDR pathogens are considered a major global public health crisis by the World Health Organization, as the discovery of effective antibiotics has not kept pace with the increase in bacterial antibiotic resistance [36]. The demand for high-value animal products such as milk, meat, and eggs has increased due to economic solvency, rapid urbanization, and changing food habits of nations [37]. Foodborne pathogens can enter into the food cycle during production, processing, and marketing. Humans can get antibiotic-resistant Antibiotics 2022, 11, 1551 7 of 16 bacterial infections in many ways, including ingestion of contaminated food or contact with colonized or diseased animals, body fluids, excretions, or secretions [38]. In addition, the pathogens can cause illness due to the consumption of undercooked food and produce illnesses either by their presence or by-production of toxins, or both.
The important foodborne pathogens of animal-source foods that have been globally identified are Salmonella, Campylobacter, E. coli, and Staphylococcus [20,22], and these trends are apparent in the current study in a similar fashion. Per the previous reports from home and abroad [39][40][41][42][43][44][45], the AST results of E. coli isolated from livestock and poultry showed a wider range of resistance to penicillin (100%), tetracycline (72-100%), oxytetracycline (78-93%), sulfamethoxazole-trimethoprim (51-88%), ampicillin (89.5-100%), amoxicillin (92-95%), streptomycin (19-70%), erythromycin (89%), ciprofloxacin (50%), chloramphenicol (43-50%), gentamicin (8-28%), enrofloxacin (55%), and norfloxacin (50%). In contrast, the phenotypic resistance pattern of E. coli to various antimicrobial agents recovered from both livestock and poultry in the present study also showed a similar trend where the AST pattern of E. coli showed the highest resistance to penicillin, followed by ampicillin, amoxicillin, oxytetracycline, cloxacillin, and sulfamethoxazole-trimethoprim. Among the antibiotics, gentamicin possessed the lowest resistance percentage, which is also comparable to the previous studies stated above. E. coli is one of the major pathogenic microorganisms that may reach animal-derived foods and is an indication of contamination by manure, soil, and contaminated water [46]. E. coli are commensal bacteria, and E. coli pathotypes can cause zoonotic disease that poses a public health risk [47]. In addition, Shiga toxin-producing E. coli is associated with the development of several life-threatening infections in humans [48]. In this regard, our recently published data showed that the most common class of antimicrobials used in large animal farms were Penicillin (61.79%), Oxytetracycline (55.66%), Sulfa drug (55.66%), Streptomycin (54.72%), followed by Ciprofloxacin (51.89%), Gentamicin (43.13%), and Ceftriaxone (34.91%) [30]. These data indicate that the bacteria became more resistant to such most commonly used antimicrobials in the study area.
Staphylococcus are commensal bacteria that are normal inhabitants of the skin, nose, and mucous membranes of healthy people and animals [56,57]. However, it is also known as an opportunistic foodborne pathogen [58] that may cause several infectious diseases with Antibiotics 2022, 11, 1551 8 of 16 different degrees of severity [56]. It causes numerous infections in humans and animals [59]. The presence of S. aureus in food products is an alarming and serious threat to public health in terms of food safety when it releases toxins and causes illness [60]. Methicillin-resistant S. aureus (MRSA) has emerged due to the unnecessary use of antibiotics [61,62]. The presence of MRSA in farm animals and the potential for cross-contamination in humans have been a great concern [63].
Campylobacter are the leading cause of foodborne diarrhea in humans worldwide [64], which is mainly due to contamination of food of animal origin [65]. Campylobacter can colonize in warm-blooded animals and poultry [66]. The zoonotic nature of Campylobacter species makes it clinically and economically important worldwide [67]. It has accounted for 15% of food-related illnesses leading to hospital admissions and 6% resulting in death; about 400 million cases are reported each year due to foodborne infection [68,69]. The economic impact of Campylobacter infections is mainly related to the treatment cost, production loss, and pathogen control expenses [67].
The ESBL-producing foodborne pathogens were previously identified by researchers from a variety of sources of livestock and poultry [70][71][72]. More recent studies have shown that ESBLs-producing bacteria frequently colonized in poultry [73][74][75] and cattle [76,77]. On the other hand, tetracycline-resistant genes are commonly encoded by plasmids & transposons and are transmitted by conjugation. However, in some isolates, the corresponding gene is also found on the chromosome [78,79]. Mechanisms of resistance to tetracycline by the acquisition of the tet gene primarily include efflux pumping, ribosome protection, and enzymatic inactivation. The resistance of gram-negative bacteria to sulfonamides is associated with the presence of the sul gene, which encodes dihydropteroate synthase in a manner that the drug cannot inhibit. The sul gene has already been identified in Enterobacteriaceae, especially in the genera Escherichia and Salmonella [80]. In this regard, the present study finding showed that the sul1 and tetA genes were found in higher percentages among the foodborne pathogens compared with other resistance genes regardless of the sources of isolation of the pathogen and the type of pathogens.
To the best of our knowledge, for the first time, this study was conducted throughout the country and found that multidrug-resistant and ESBL-producing foodborne pathogens were prevailing in the livestock-and poultry-source food products & by-products in Bangladesh. However, the present study has some limitations. In this study, we did not collect the environmental samples which may be contaminated by the livestock-and poultry-source food products and by-products. In addition, the sampling area was limited in each division. Further detailed studies with larger samples size from each district of Bangladesh are needed. Details of further phenotypic & genotypic analysis in a wider range with 16S rRNA sequence profiling of these isolates would help the scientists in this field to combat AMR as well as to stop the spreading of MDR foodborne pathogens to humans.

Study Area
The study was conducted in thirty-three districts under the eight administrative divisions of Bangladesh. The seven components (educational institutes) of the project covered each division with at least four districts while the component (educational institute) Patuakhali Science and Technology University covered two divisions with nine districts. The seven components (educational institutes) of the project are the Bangladesh Agricultural University (BAU), Bangladesh Livestock Research Institute (BLRI), Rajshahi University (RU), Patuakhali Science and Technology University (PSTU), Chattogram Veterinary and Animal Sciences University (CVASU), Sylhet Agricultural University (SAU), and Haji Danesh Science and Technology University (HSTU). Figure 4 shows the study divisions followed by districts area covered by the seven components (educational institutes). The study protocol was authorized by the Animal Welfare and Experimentation Ethics Committee of the Bangladesh Agricultural University, Mymensingh, (approval number: AWEEC/BAU/2018 (17)). followed by districts area covered by the seven components (educational institutes). The study protocol was authorized by the Animal Welfare and Experimentation Ethics Committee of the Bangladesh Agricultural University, Mymensingh, (approval number: AWEEC/BAU/2018 (17)).

Sampling Design
A total of 2320 samples were collected across the country, of which 880 were taken from poultry and 1440 from large & small ruminants in thirty-three districts of eight divisions of Bangladesh by the seven components of the project. Poultry source samples were broiler meat (n = 160), layer meat (n = 160), egg (n = 240), broiler feces (n = 160), and layer feces (n = 160). All poultry sources samples were directly collected from layer and broiler farms. On the other hand, large & small ruminant samples were cattle meat (n = 160), sheep or goat meat (n = 160), buffalo meat (n = 80), cattle raw milk (n = 200), sheep or goat raw milk (n = 200), buffalo raw milk (n = 120), cattle feces (n = 200), sheep or goat feces (n = 200), and buffalo feces (n = 80). Different types of meat samples were collected for local raw meat markets; however, raw milk and feces from different animals were collected from farms. All the samples were collected in aseptic condition using sterile instruments and carefully transferred into sterile Eppendorf tubes (for raw milk) or zipper bags (for solid samples) from animal and poultry farms. Immediately after collection, samples were kept in a transport box for maintaining a 4 °C temperature. The samples were then transported to the bacteriological laboratory of the Department of Microbiology and Hygiene, Faculty of Veterinary Science, BAU, Mymensingh for microbiological analysis. According to our previous study [30], the Raosoft sample volume calculation method was used to determine the sample size with a 5% margin of error and 95% confidence level.

Conventional Culture Method
E. coli, Salmonella, Streptococcus, and Staphylococcus were targeted for the isolation from livestock whilst E. coli, Salmonella, Staphylococcus, and Campylobacter were targeted for the isolation from poultry sources following the standard procedure as applied earlier [44,45,55,[81][82][83][84][85]. Briefly, 0.5 g of each sample was inoculated in nutrient broth and incubated at 37 °C for 12 h for the initial growth of Escherichia coli, Salmonella, and Staphylococcus aureus. The cultures from nutrient broth were streaked on Eosin methylene blue (EMB) agar (Hi media, Maharashtra, India), Salmonella-Shigella (SS) agar (Hi media, Maharashtra, India), and Mannitol salt agar (MSA) (Hi media, India) plates using platinum loop for the isolation of E. coli, Salmonella, and S. aureus, respectively. Milk samples (200 µL) were also inoculated in Kenner Fecal (KF) Streptococcal broth (Hi media, India) for the initial

Sampling Design
A total of 2320 samples were collected across the country, of which 880 were taken from poultry and 1440 from large & small ruminants in thirty-three districts of eight divisions of Bangladesh by the seven components of the project. Poultry source samples were broiler meat (n = 160), layer meat (n = 160), egg (n = 240), broiler feces (n = 160), and layer feces (n = 160). All poultry sources samples were directly collected from layer and broiler farms. On the other hand, large & small ruminant samples were cattle meat (n = 160), sheep or goat meat (n = 160), buffalo meat (n = 80), cattle raw milk (n = 200), sheep or goat raw milk (n = 200), buffalo raw milk (n = 120), cattle feces (n = 200), sheep or goat feces (n = 200), and buffalo feces (n = 80). Different types of meat samples were collected for local raw meat markets; however, raw milk and feces from different animals were collected from farms. All the samples were collected in aseptic condition using sterile instruments and carefully transferred into sterile Eppendorf tubes (for raw milk) or zipper bags (for solid samples) from animal and poultry farms. Immediately after collection, samples were kept in a transport box for maintaining a 4 • C temperature. The samples were then transported to the bacteriological laboratory of the Department of Microbiology and Hygiene, Faculty of Veterinary Science, BAU, Mymensingh for microbiological analysis. According to our previous study [30], the Raosoft sample volume calculation method was used to determine the sample size with a 5% margin of error and 95% confidence level.

Conventional Culture Method
E. coli, Salmonella, Streptococcus, and Staphylococcus were targeted for the isolation from livestock whilst E. coli, Salmonella, Staphylococcus, and Campylobacter were targeted for the isolation from poultry sources following the standard procedure as applied earlier [44,45,55,[81][82][83][84][85]. Briefly, 0.5 g of each sample was inoculated in nutrient broth and incubated at 37 • C for 12 h for the initial growth of Escherichia coli, Salmonella, and Staphylococcus aureus. The cultures from nutrient broth were streaked on Eosin methylene blue (EMB) agar (Hi media, Maharashtra, India), Salmonella-Shigella (SS) agar (Hi media, Maharashtra, India), and Mannitol salt agar (MSA) (Hi media, India) plates using platinum loop for the isolation of E. coli, Salmonella, and S. aureus, respectively. Milk samples (200 µL) were also inoculated in Kenner Fecal (KF) Streptococcal broth (Hi media, India) for the initial growth of Streptococcus, then streaked on KF Streptococcal agar media for the isolation of Streptococcus. Then all the culture plates were incubated at 37 • C for 24 h. For the isolation of Campylobacter, all samples were directly inoculated on selective campylobacter base agar (Oxoid Ltd., Hampshire RG24 8PW, UK) containing antibiotics (Amphotericin B has been added to suppress the growth of yeast and fungal contaminants that may occur at 37 • C, and improved selectivity was achieved by adding cefoperazone) and 5-7% sheep blood [86]. The plates were incubated in an anaerobic jar (Oxoid™ AnaeroJar™ 2.5 L) under microaerophilic conditions with a CO 2 sachet (Thermo Scientific TM Oxoid Anaero Gen 2.5 L sachet) (10% CO 2 , 95% humidity) at 42 • C for three days. After 72 h, single characteristic (small, round, creamy-gray, or whitish) colonies from each plate were selected and inoculated in tryptic soy broth (Oxoid Ltd., Hampshire RG24 8PW, UK) and incubated at 37 • C for three days under microaerophilic conditions. The single colonies of suspected bacteria were again inoculated in selective broth and streaked on selective agar media to obtain a pure culture.

Molecular Detection
Whole genomic DNA was extracted from each pure culture by a conventional boiling method [49,85]. Following the boiling method, the DNA was measured using spectrophotometers. PCR was performed for confirmatory detection of each isolate using specific primers (Table 3) and the PCR condition for each bacterial species was used following the standard operating procedure described by different authors (Table 3).

Determination of Phenotypic Resistance Pattern
Antimicrobial susceptibility testing was performed using the Kirby Bauer disk diffusion method in adherence with the guidelines of the Clinical and Laboratory Standards Institute [91]. Briefly, fresh colonies were suspended in saline and the turbidity of the suspension was measured in comparison with the 0.5 McFarland standards (approximately 1.5 × 10 6 CFU/mL). The bacterial suspension was smeared on the surface of Mueller-Hinton (MH) agar (Oxoid Ltd., Hampshire RG24 8PW, UK) and an antibacterial disc with a disc dispenser was placed on it within 15 min and the plate was incubated at 37 • C for 24 h. The zone of inhibition adjacent to the disks was measured and compared with the breakpoints of CLSI. A number of 16 different antimicrobial disks (Hi media, India) were used for AST of all four foodborne pathogens such as penicillin (P, 10 units), ampicillin (AMP, 10 µg), amoxicillin (AMX, 30 µg), cloxacillin (COX, 5 µg), ceftriaxone (CTR, 30 µg), tetracycline (TE, 30 µg), doxycycline (DO, 30 µg), oxytetracycline (O, 30 µg), sulfamethoxazole-trimethoprim (COT, 25 µg), gentamicin (GEN, 10 µg), erythromycin (E, 15 µg), ciprofloxacin (CIP, 5 µg), streptomycin (S, 10 µg), levofloxacin (LE, 5 µg), enrofloxacin (EX, 5 µg), and neomycin (N, 30 µg). Based on their common therapeutic usage at the field level in the study areas, different antimicrobial disks for livestock and poultry sources bacteria were chosen [30]. The criteria developed by Magiorakos et al. [92] were used to define multidrug-resistant (MDR) bacteria. Susceptible, intermediate, and resistant were defined according to the new DIN EN ISO 20776-1 standard [93], which is valid worldwide. The sensitivity of a bacterial strain to a given antibiotic is said to be intermediate when it is inhibited in vitro by a concentration of this drug that is associated with an uncertain therapeutic effect [94]. The Escherichia coli ATCC 25922 strain served as a validated positive control.

Conclusions
The phenotypic and genotypic resistance profiles uncovered by the present study indicated that MDR-and ESBL-producing foodborne pathogens were prevailing in the livestock-and poultry-source food products & by-products in Bangladesh. MDR foodborne pathogens are a current public health concern worldwide including in Bangladesh. Foodborne pathogens are usually spread due to improper handling, processing, preparation, and storage of food. The unnecessary use of antimicrobials in farm practices is the main driver for the emergence of antimicrobial-resistant pathogens in the livestock and poultry value chain. Good agricultural practices, good veterinary practices, good manufacturing practices, and proper farm biosecurity are important tools to curb the development of AMR pathogens in livestock and poultry food products. Moreover, policy intervention, stakeholder awareness, motivation, training, advocacy, and mass media dissemination are the imperative pathways to combat the spread of foodborne pathogens and AMR.  (17)).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.