| Summary: | <i>Acinetobacter</i> species have been found in a variety of environments, including soil, food, plants, hospital environments and water. <i>Acinetobacter baumannii</i> is an opportunistic and emerging waterborne pathogen. It has been implicated in several nosocomial infections that demonstrate resistance to commonly administered antibiotics. We investigated phenotypic antibiotic resistance (PAR) and relevant antibiotic resistance genes (ARGs) in <i>A. baumannii</i> isolated from three freshwater resources in the Eastern Cape Province, South Africa; <i>A. baumannii</i> (410) was confirmed by the <i>recA</i> and <i>gyrB</i> genes of 844 suspected <i>Acinetobacter</i> species in the water samples. The PAR of the confirmed isolates was assessed using a panel of 11 antibiotics by the disc diffusion method, while ARGs were investigated in isolates exhibiting PAR. The <i>A. baumannii</i> isolates were resistant to piperacillin-tazobactam (11.2%), ceftazidime (12%), cefotaxime (18.8%), cefepime (8.8%), imipenem (2.7%), meropenem (4.15%), amikacin (2.4%), gentamicin (8.8%), tetracycline (16.8%), ciprofloxacin (11%) and trimethoprim/sulfamethoxazole (20.5%). For multidrug resistance (MDR), two isolates were resistant to all antibiotics and 28 isolates were resistant to imipenem and meropenem. Moreover, β-lactamases <i>bla</i><sub>TEM</sub> (64.4%) and <i>bla</i><sub>OXA-51</sub> (28.70%) as well as sulphonamides <i>sul1</i> (37.1%) and <i>sul2</i> (49.4%) were common ARGs. Overall, PAR and ARGs had positive correlations (<i>r</i>) in all rivers. Detection of MDR-<i>A. baumannii</i> in freshwater resources could be linked to possible wastewater discharge from the nearby animal farms, indicating potential implications for public health.
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