Genetic Relatedness of Multidrug Resistance and Biofilm Production Genes in C. perfringens Isolates from Broilers

Clostridium perfringens remains a leading etiological agent of necrotic enteritis in broiler chickens, as control efforts are increasingly challenged by multidrug resistance (MDR) and biofilm formation. We characterized the genetic determinants of antibiotic resistance and biofilm production in 36 pathogenic C. perfringens type A isolates (24% of 150 samples) recovered from broilers exhibited diarrhea and necrotic enteritis. All isolates harbored the alpha-toxin (cpa) and enterotoxin (cpe) genes; 75% carried the pore-forming netB toxin gene. Phenotypic assays demonstrated that 69.4% of isolates produced biofilm, of which 84% were MDR. Antimicrobial susceptibility profiling revealed highest resistance rates against chloramphenicol (80.5%), penicillin (80.5%), and oxytetracycline (77.7%), with universal sensitivity to vancomycin and ofloxacin. PCR screening detected tetracycline resistance gene tetK in 100% of isolates, β-lactamase gene bla in 62.5%, and macrolide resistance gene ermB in 37.5%. Biofilm-associated loci appB and proA were ubiquitous (100%), whereas iolD and ripH occurred in 62.5% and 50% of isolates, respectively. Statistical correlation analysis revealed a statistically significant association between MDR and biofilm gene carriage (r = 0.994; p < 0.05), suggesting co-selection and potential linkage on mobile genetic elements. These findings clarify the genetic interactions that contribute to antibiotic resistance in biofilms formed by C. perfringens . They highlight the urgent need for responsible antimicrobial use and the development of alternative prevention strategies, such as targeted vaccines and probiotic treatments, to reduce the incidence of necrotic enteritis in high-density poultry farming.