Molecular Detection of Ampicillinase blaEBCM, blaFOX and blaDHAM resistant genes in multi-drug resistant Gram-negative bacteria

Background: Multidrug-resistant (MDR) Gram-negative bacteria (GNB) harboring AmpC β-lactamase determinants represent a significant global public health threat, contributing to life-threatening infections in humans. Despite their growing prevalence, the detection of AmpC β-lactamase remains underreported in low- and middle-income countries (LMICs), where clinical microbiology laboratories often lack the resources for accurate identification. This study aimed to investigate the presence of AmpC resistance genes (blaEBC, blaFOX, and blaDHA) in MDR Gram-negative bacteria to address this critical gap. Materials and methods: Over a six-month period, 290 non-replicated urine samples were collected from hospitalized patients, yielding clinical isolates of E. coli (n=95), K. pneumoniae (n=34), and Pseudomonas species (n=17). Antibiotic susceptibility testing was performed using the Kirby–Bauer disk diffusion method, and multidrug resistance (MDR) was determined. Isolates resistant to cefoxitin were preliminarily identified as potential AmpC β-lactamase producers and further confirmed using the Cefoxitin-Cloxacillin Double Disk Synergy Test (CC-DDST). AmpC β-lactamase phenotypes were subsequently analyzed using polymerase chain reaction (PCR) to detect the presence of blaFOX-M, blaEBC-M, and blaDHA-M genes. Results: The study identified 146 MDR Gram-negative bacteria with high resistance rates to multiple antibiotic classes, including β-lactams, cephalosporins, carbapenems, anti pseudomonals, folate inhibitors, and protein synthesis inhibitors. Resistance was notably lower to macrolides (e.g., azithromycin), aminoglycosides (e.g., gentamicin), and fluoroquinolones (e.g., ciprofloxacin). Among the MDR isolates, 23 (7.9%) were confirmed as AmpC producers, with prevalence rates of 1.0% in P. aeruginosa, 1.7% in K. pneumoniae, and 5.2% in E. coli. Genetic analysis revealed the presence of FOXM (100%), EBC (95.7%), and DHAM (13.0%) genes among the MDR isolates. Conclusion: This study highlights the alarming prevalence of MDR Gram-negative bacteria with high resistance to critical antibiotic classes, particularly β-lactams, carbapenems, and cephalosporins. The widespread detection of AmpC β-lactamase genes (FOXM, EBC, and DHAM) underscores the complexity of managing MDR infections. Our findings suggest that combination therapy involving azithromycin, gentamicin, and ciprofloxacin may offer a viable treatment strategy for MDR infections. Regional variations in resistance patterns emphasize the urgent need for enhanced surveillance, routine detection of resistance mechanisms, and stricter antibiotic stewardship to combat the global threat of antimicrobial resistance.