Molecular Characterization and Antimicrobial Resistance Profiling of Clostridium perfringens Isolated from Poultry: Identification of Multidrug-Resistant Toxinotype G Lineages.

BACKGROUND: The occurrence of Clostridium perfringens carrying the Toxinotype G profile (netB+), coupled with the acquisition of critically important antimicrobial resistance determinants, represents a growing concern within the One Health framework. This study investigated the virulence profiles, phenotypic antimicrobial resistance, and metallo-β-lactamase (MBL) genetic markers among C. perfringens isolates recovered from commercial poultry production systems in Egypt. METHODS: Tissue samples were collected from 100 diseased birds (broilers, layers, and native Baladi chickens) presenting with clinical signs of necrotic enteritis across 15 commercial flocks in three districts. Confirmed isolates were evaluated for toxin gene profiles (cpa, cpb, etx, itx, netB) and accessory virulence factors (pfoA, cpb2, colA, tpeL). Antimicrobial susceptibility profiles against a standardized 16-drug panel representing 11 therapeutic classes were determined using Minimum Inhibitory Concentrations (MICs). PCR was used to detect β-lactamase markers (blaOXA-1, blaIMP-1, blaVIM-2). Firth's penalized logistic regression was applied to assess breed-associated flock patterns, and index strain C3 (selected from a case with severe acute pathognomonic lesions) underwent 16S rRNA and multi-gene sequence analysis. RESULTS: An overall isolation rate of 19.0% (19/100) was observed, with broiler-associated flock types demonstrating a significantly higher isolation rate (40.9%) compared to native Baladi (11.1%) and layer (16.7%) flocks (p = 0.016). Toxinotype G dominated among the isolates (57.9%, 11/19), frequently co-harboring accessory toxin markers (pfoA, cpb2, tpeL, colA). Genotypic sequencing of index strain C3 confirmed high sequence conservation across 16S rRNA (PZ362497), netB (PZ371503), blaIMP-1 (PZ372954), and blaVIM-2 (PZ372953). All isolates exhibited multidrug-resistant (MDR) phenotypes based on MIC breakpoints, with four isolates (21.1%) displaying a pan-drug resistant (PDR) profile across all 16 tested agents. Strong positive correlations (p < 0.01, rho > 0.60) were identified among blaIMP-1, blaVIM-2, and blaOXA-1 markers, while accessory toxin genes correlated significantly (p < 0.05) with resistance to ampicillin, clindamycin, and carbapenems. CONCLUSION: Our findings demonstrate the presence of multidrug-resistant, NetB-producing C. perfringens lineages acting as reservoirs for MBL carbapenemase markers within poultry production systems. Co-occurrence of virulence and resistance determinants highlights the need for continuous surveillance, improved biosecurity, and non-antibiotic mitigation strategies to curb the dissemination of these resistant clostridial lineages.