Evaluating Signature Mutations and Phenotypic Antibiotic Resistance in Wastewater Bacteria

The persistence of antibiotic resistance in bacteria poses a major global health challenge, with wastewater treatment plants (WWTPs) increasingly recognized as hotspots for both antibiotic-resistant bacteria and resistance genes (ARGs). This study investigated genetic mutations and phenotypic resistance profiles of bacterial isolates from three WWTPs in Windhoek, Namibia. Bacterial isolates were obtained through culture-based methods, identified using Gram staining and the Vitek system, and tested for antibiotic susceptibility via the Kirby–Bauer disk diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. ARGs were detected using direct polymerase chain reaction (PCR). Key isolates included Escherichia coli, Klebsiella pneumoniae, and Citrobacter freundii, with PCR confirming the presence of the gyrA gene linked to fluoroquinolone resistance. Antimicrobial susceptibility testing revealed resistance to ampicillin and tetracycline, and one isolate resistant to ciprofloxacin. Despite the detection of gyrA, most isolates remained susceptible to ciprofloxacin, suggesting these mutations may influence susceptibility to other fluoroquinolones rather than confer direct resistance. No amplification was observed for tetA and blaTEM genes. These findings reveal that WWTPs act as critical reservoirs and dissemination hubs for antibiotic-resistant bacteria and ARGs, emphasizing the urgent need for sustained genomic surveillance to curb their environmental and public health impact.