Antimicrobial resistance (AMR) is a significant global public health threat. The World Health Organization (WHO) emphasizes the need for surveillance systems in both veterinary and human medicine to combat this issue. *Escherichia coli* (*E. coli*), a common inhabitant of the gastrointestinal tracts of poultry, animals, and humans, is a major cause of foodborne illnesses. Unhygienic slaughter practices contribute to *E. coli* contamination of meat products, and many strains isolated from contaminated sources exhibit resistance to commonly used antibiotics. The overuse of antibiotics in animal agriculture is a primary driver of AMR, with resistance acquired through horizontal gene transfer or gene mutations. Multidrug-resistant (MDR) bacteria, harboring multiple drug-resistance genes, pose a severe risk to human health. Beta-lactamases, bacterial enzymes that hydrolyze the beta-lactam ring of antibiotics like penicillin and cephalosporin, are key contributors to AMR. Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases are particularly concerning due to their location on mobile genetic elements (plasmids or integrons), facilitating their spread to other bacterial cells. Food animals and retail meat act as reservoirs for ESBL and AmpC-producing *E. coli*. Bangladesh, a major poultry producer, faces challenges with underdeveloped hygienic raw food processing and a lack of AMR surveillance. The uncontrolled use of antimicrobials in food animals increases the risk of AMR emergence. This study aimed to identify and characterize *E. coli* isolates from broiler and layer chicken meat in Sylhet, Bangladesh, focusing on antibiotic resistance profiles and the presence of specific antibiotic resistance genes (ARGs), including those conferring beta-lactam resistance.

Numerous studies have documented the high prevalence of antibiotic-resistant *E. coli* in retail poultry products worldwide. Previous research has highlighted the role of unhygienic slaughter practices and the overuse of antibiotics in livestock as significant contributors to this problem. The emergence of ESBLs and AmpC beta-lactamases, which confer resistance to broad-spectrum beta-lactam antibiotics, represents a growing concern. These enzymes are often located on mobile genetic elements, allowing for rapid dissemination of resistance among bacterial populations. Studies have demonstrated the presence of ESBL-producing *E. coli* in various regions, highlighting the global nature of this threat. Data on the prevalence and genotypic characteristics of antibiotic-resistant *E. coli* in Bangladesh, however, has been limited, prompting the need for this investigation.

This study involved the collection of 600 chicken meat swabs from broiler and layer chicken farms (300 each) across four districts within the Sylhet division of Bangladesh. *E. coli* isolates were identified using a combination of Gram staining, growth characteristics on various culture media (nutrient broth, nutrient agar, MacConkey's agar, Eosin Methylene Blue agar), and biochemical tests (sugar fermentation, indole, methyl red, Voges-Proskauer, and citrate utilization tests). Molecular confirmation was performed using PCR targeting the 16S rRNA gene. Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar, according to CLSI guidelines, to determine resistance to seven commonly used antibiotics: trimethoprim-sulfamethoxazole, chloramphenicol, erythromycin, gentamicin, tetracycline, streptomycin, and ampicillin. Multidrug resistance (MDR) was defined as non-susceptibility to at least one agent in three or more different classes of antimicrobial agents. Bacterial genomic DNA was extracted using the Phenol-Chloroform Isoamyl Alcohol (PCI) method. PCR, using both uniplex and multiplex assays, was employed to detect the presence of 13 antibiotic resistance genes: *tetA* (tetracycline), *aadA1* (streptomycin), *sul1* (sulfonamide), *ereA* (erythromycin), *aac-3-IV* (gentamicin), *cmlA*, and *catA1* (chloramphenicol), and six beta-lactam resistance genes: *blaSHV*, *blaTEM*, *blaCTX-M*, *blaCTX-M-1*, *blaCTX-M-2*, and *CITM* (AmpC). Statistical analysis, including two-way ANOVA, was used to assess significant differences in resistance prevalence between broiler and layer chickens and among the four districts.

A total of 381 *E. coli* isolates (63.5% of total samples) were identified. 75.06% (286/381) of these isolates were MDR, exhibiting resistance to at least three antibiotic classes. Resistance to ampicillin, erythromycin, and tetracycline was particularly high (98.95%, 89.5%, and 85.3%, respectively). The most prevalent non-beta-lactam ARGs were *tetA* (77.17%), *sul1* (45.94%), and *aadA1* (34.65%). 13.91% (53/381) of isolates harbored beta-lactam ARGs, with the *blaSHV* gene (ESBL) being most common (10%). The AmpC gene (*CITM*) was detected in 3.93% of isolates. 50.13% (191/381) of isolates carried three or more MDR genes; the most frequent MDR profile involved sulfonamide, erythromycin, and tetracycline resistance genes. Significant correlations were found between most antimicrobial resistance phenotypes and genotypes, although some discrepancies were observed. There were no statistically significant differences in the prevalence of *E. coli* between broiler and layer chickens or among the four districts, but significant differences existed in antimicrobial resistance patterns between the two types of chickens and among the antibiotic resistance genes for specific antibiotics.

The high prevalence of MDR *E. coli* in chicken meat from retail shops in Sylhet, Bangladesh, raises significant public health concerns. The findings underscore the need for improved hygiene practices during poultry processing and stricter regulation of antibiotic use in animal agriculture. The high prevalence of resistance to commonly used antibiotics, such as ampicillin, erythromycin, and tetracycline, limits treatment options for human infections. The presence of ESBL and AmpC genes further complicates the situation. The strong correlation between phenotypic and genotypic resistance in many instances suggests that the presence of specific ARGs is a major driver of resistance. However, discrepancies between phenotype and genotype highlight the complexity of AMR and the potential role of other factors. This study demonstrates the importance of comprehensive surveillance programs to track the spread of AMR in food animals and identify potential sources of contamination. The observed resistance patterns highlight the need for the development of new antibiotics with activity against MDR and ESBL-producing bacteria.

This study revealed a high prevalence of MDR *E. coli* in raw chicken meat from Sylhet, Bangladesh, emphasizing the need for improved hygiene practices in poultry processing and responsible antibiotic stewardship. The strong correlation between phenotypic and genotypic resistance for several antibiotics highlights the importance of specific resistance genes. Future studies should focus on identifying the mechanisms responsible for resistance discrepancies and the roles of other less common AMR genes. Development of novel antibiotics is crucial, along with continued surveillance and the promotion of safe food handling practices to mitigate the risk of AMR dissemination.

The study was limited to a single geographic region in Bangladesh. The selection of antibiotics tested might not encompass the full spectrum of antimicrobial agents used in poultry farming in the region. The study did not fully investigate the genetic mechanisms responsible for phenotypic resistance in instances where the corresponding resistance genes were not detected.