Isolation and molecular characterization of multidrug-resistant *Escherichia coli* from chicken meat

Antimicrobial resistance is a growing public health concern, and food animals can act as reservoirs of resistant bacteria and resistance genes. Escherichia coli inhabits the gastrointestinal tract of poultry and can contaminate meat during slaughter and processing, with many isolates showing resistance to commonly used antibiotics. Beta-lactamase enzymes, including ESBLs (e.g., TEM, SHV, CTX-M) and AmpC, are often plasmid-encoded and transferable. In Bangladesh, poultry production is extensive, yet hygienic processing and antimicrobial resistance surveillance are limited, raising concerns about resistant E. coli in the food chain. This study investigated E. coli from broiler and layer chicken meat in the Sylhet division to determine prevalence, antimicrobial resistance profiles, the presence of non-beta-lactam and beta-lactam resistance genes, and the correlation between resistance phenotypes and genotypes.

The authors outline prior evidence that poultry and retail meat can harbor ESBL- and AmpC-producing E. coli and that these genes are frequently plasmid-borne, facilitating horizontal transfer (TEM, SHV, OXA, CMY, CTX-M families). International studies have reported varying prevalence of resistance and ARGs in poultry-associated E. coli, with notable detection of CTX-M and TEM in several countries. In Bangladesh, reports suggest high resistance rates among poultry-derived E. coli, but comprehensive surveillance and genotypic characterization remain limited. The background motivates assessing both phenotypic resistance and ARG distribution, including non-beta-lactam genes (tetA, sul1, aadA1, ereA, cmlA, catA1, aac-3-IV) and beta-lactamase genes (ESBLs and AmpC).

Ethics: Animal handling complied with Bangladesh legislation; protocols approved by ethics committees at Sylhet Agricultural University and the National Institute of Biotechnology. Sampling and identification: 600 swabs from retail broiler (n=300) and layer (n=300) chicken meat were collected from four districts in Sylhet division (Sylhet, Moulavibazar, Sunamganj, Habiganj). E. coli identification used Gram staining, culture on nutrient agar, MacConkey’s agar, and Eosin Methylene Blue agar, with biochemical tests (sugar fermentation, indole, MR, VP, citrate). Molecular confirmation targeted E. coli 16S rRNA by PCR. In total, 381 isolates (197 broiler, 184 layer) were confirmed for further analyses. Antimicrobial susceptibility testing: Kirby-Bauer disc diffusion on Mueller-Hinton agar following CLSI guidelines. Antibiotics tested (disc content): trimethoprim-sulfamethoxazole (23.75 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), tetracycline (30 µg), streptomycin (10 µg), ampicillin (10 µg). E. coli ATCC 25922 served as control. Resistant and intermediate were grouped as non-susceptible. MDR definition: non-susceptible to at least one agent in three or more antimicrobial classes (excluding broad-spectrum penicillins without β-lactamase inhibitor). DNA extraction: Isolates were grown overnight in nutrient broth at 37 °C; genomic DNA extracted by phenol-chloroform-isoamyl alcohol (PCI) method; yield and purity assessed by NanoDrop. PCR detection of ARGs: All 381 isolates screened for seven non-beta-lactam and six beta-lactam ARGs via two uniplex and two multiplex PCR assays. Non-beta-lactam targets: tetA (tetracycline), aadA1 (streptomycin), sul1 (sulfonamide), ereA (erythromycin), cmlA and catA1 (chloramphenicol), aac-3-IV (gentamicin). Beta-lactam targets: ESBL genes blaTEM, blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaSHV; AmpC gene CITM. Primer sequences, amplicon sizes, and annealing temperatures are provided (Table 1). Thermal cycling conditions were specified for each set. Amplicons were resolved on 1.5% agarose gels stained with ethidium bromide; 100 bp DNA ladder used. Positive controls: E. coli O157:K88ac:H19, CAPM 5933 and E. coli O159:H20, CAPM 6006; negative control: distilled water. Statistical analysis: Resistance data expressed as percentages/frequencies. Two-way ANOVA without replication assessed differences among antibiotics and between broiler vs layer, and among districts; P<0.05 considered significant. Analyses performed with GraphPad Prism v6.

- E. coli prevalence: 381/600 (63.5%) meat swabs positive; broiler 197/300 (65.67%), layer 184/300 (61.33%). No significant difference between broiler and layer (P=0.06) or among districts (P=0.37). - Phenotypic resistance (n=381): ampicillin 98.95% (broiler 100%, layer 97.82%), erythromycin 89.50% (broiler 83.24%, layer 96.19%), tetracycline 85.30% (broiler 81.21%, layer 89.67%), streptomycin 70.86% (broiler 60.91%, layer 81.52%), trimethoprim-sulfamethoxazole 54.33% (broiler 51.26%, layer 57.60%), chloramphenicol 49.86% (broiler 53.29%, layer 46.19%; P=0.0003), gentamicin 27.55% (broiler 27.91%, layer 27.17%). Overall MDR (≥3 classes) 75.06% (broiler 78.17%, layer 71.73%). - Non-beta-lactam ARG prevalence overall: tetA 77.17%, sul1 45.94%, aadA1 34.65%, ereA 31.23%, aac-3-IV 24.67%, cmlA 22.05%, catA1 7.09%. Significant variation among genes (P=0.0001); no significant broiler vs layer difference overall (P=0.42). District-wise, cmlA prevalence differed among districts and was higher in broiler; other genes showed no significant district or type differences. - MDR gene carriage (non-beta-lactam classes): 191/381 (50.13%) carried ≥3 ARGs. Distribution: 3 genes 38.32% (broiler 40.11%, layer 36.41%), 4 genes 11.55% (broiler 9.13%, layer 14.13%), 5 genes 0.26% (broiler 0%, layer 0.54%). Significant differences among MDR gene categories (P=0.01); no overall difference between broiler and layer (P=0.83), though the 4-gene category differed by type (P=0.01). - Beta-lactam genes: 53/381 (13.91%) harbored ESBL and/or AmpC genes. SHV detected in 38/381 (10.0%); AmpC CITM in 15/381 (3.93%). No detections of TEM, CTX-M, CTX-M-1, or CTX-M-2. By type: broiler SHV 12.18%, CITM 4.56% (total ESBL/AmpC 16.75%); layer SHV 7.61%, CITM 3.26% (total 10.87%). Significant differences among beta-lactam gene types (P=0.001); no overall difference between broiler and layer (P=0.24). District-wise, SHV prevalence was higher in broiler than layer (P=0.01); no significant district effect for SHV (P=0.07) or CITM (P>0.05). - Phenotype-genotype correlations: Significant positive correlations for most antibiotic-ARG pairs. Strongest observed between gentamicin resistance and aac-3-IV in layer isolates (r≈0.791, P<0.001); also notable for broiler gentamicin vs aac-3-IV (r^2=0.610, P<0.001), tetracycline vs tetA (broiler r^2=0.483, P<0.001; layer r^2=0.243, P<0.001), and streptomycin vs aadA1 (broiler r^2=0.275, P<0.001). Non-significant correlations included chloramphenicol phenotype vs catA1 in broiler (r^2=0.018, P=0.067) and erythromycin phenotype vs ereA in layer (r≈0.001, P=0.672).

The study addressed the burden and genetic basis of antimicrobial resistance in E. coli contaminating retail chicken meat in Bangladesh. High prevalence of E. coli and elevated resistance to commonly used antimicrobials (ampicillin, erythromycin, tetracycline, streptomycin) indicate substantial selective pressure and potential risk to consumers. The widespread detection of tetA, sul1, and aadA1 supports the role of specific ARGs in mediating resistance, with significant phenotype-genotype correlations for several antibiotic classes (e.g., gentamicin-aac-3-IV, tetracycline-tetA, streptomycin-aadA1). Detection of ESBL (SHV) and AmpC (CITM) genes, though at lower frequencies, underscores the presence of beta-lactam resistance mechanisms within the food chain. Differences among gene prevalences and MDR gene counts, as well as the observed mismatches between phenotypes and the limited ARG panel, suggest additional resistance mechanisms (other ARGs, mutations, co-selection) not captured here. Collectively, findings reinforce the public health relevance of prudent antimicrobial use in poultry, improved hygiene during slaughter and processing, and the need for robust surveillance and control strategies in Bangladesh to limit dissemination of MDR and beta-lactamase-producing E. coli.

E. coli was detected in 63.5% of 600 chicken meat swabs from the Sylhet division. Most isolates showed high resistance to ampicillin, erythromycin, and tetracycline, with 75.06% exhibiting multidrug resistance. Half of the isolates carried three to five non-beta-lactam MDR genes, and 13.91% harbored ESBL (SHV) and/or AmpC (CITM) beta-lactamase genes. Significant correlations between resistance phenotypes and corresponding ARGs were observed for several antibiotic classes. These results highlight an urgent need for rational antibiotic use in livestock, improved food handling and processing practices, and strengthened surveillance. Future work should expand ARG panels (including additional macrolide, chloramphenicol, sulfonamide, and beta-lactamase variants), incorporate quantitative susceptibility testing and sequencing to resolve phenotype-genotype discrepancies, and assess antibiotic usage patterns in poultry production to inform interventions.

- Geographic scope limited to four districts within one division of Bangladesh; generalizability to other regions may be limited. - Only seven non-beta-lactam and selected beta-lactamase genes were screened; other resistance determinants and mechanisms (e.g., additional macrolide or chloramphenicol genes, efflux pumps, target mutations) were not assessed, contributing to phenotype–genotype mismatches observed. - Phenotypic testing used disc diffusion; minimum inhibitory concentrations and molecular typing were not performed. - Cross-sectional sampling of retail meat swabs does not capture temporal trends or on-farm antibiotic usage; data on antimicrobial use in chickens in Bangladesh were not available. - ESBL panel did not detect TEM or CTX-M variants in this cohort despite reports elsewhere, which may reflect regional differences or detection limits.