Bacterial hosts of clinically significant beta-lactamase genes in Croatian wastewaters

Wastewater treatment plants (WWTPs) provide a suitable environment for the interaction of antibiotic resistant bacteria and antibiotic-resistance genes (ARGs) from human, animal, and environmental sources. The aim was to study the influent and effluent of two WWTPs in Croatia to identify bacterial hosts of clinically important beta-lactamase genes (blaTEM, blaVIM, blaOXA-48-like) and observe how their composition changes during the treatment process. A culture-independent epicPCR (Emulsion, Paired isolation and Concatenation Polymerase Chain Reaction) was used to identify the ARG hosts, and 16S rRNA amplicon sequencing to study the entire bacterial community. Different wastewater sources contributed to the significant differences in bacterial composition of the wastewater between the two WWTPs studied. A total of 167 genera were detected by epicPCR, with the Arcobacter genus, in which all ARGs studied were present, dominating in both WWTPs. In addition, the clinically important genera Acinetobacter and Aeromonas contained all ARGs examined. The blaOXA-48-like gene had the highest number of hosts, followed by blaVIM, while blaTEM had the narrowest host range. Based on 16S rRNA gene sequencing, ARG hosts were detected in both abundant and rare taxa. The number of hosts carrying investigated ARGs was reduced by wastewater treatment. EpicPCR provided valuable insights into the bacterial hosts of horizontally transmissible beta-lactamase genes in Croatian wastewater.