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

Abstract

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.

Keywords: 16S rRNA; Croatia; antibiotic-resistance genes; bacteria; epicPCR; wastewater.

Figure 1.

Outline of the research methods used in this study.

Figure 2.

Bacterial composition of wastewater from Zagreb and Varazdin WWTPs in influent (IN1-3) and effluent (EF1-3) based on analysis of 16S rRNA amplicon sequences. Top 20 families with the highest relative abundance are presented.

Figure 3.

Principal coordinate analysis plot of the relative abundance of bacteria in wastewater samples using the Bray–Curtis dissimilarity index. The significant difference of all sample types from each other was confirmed with permutational multivariate analysis of variance.

Figure 4.

Number of genera detected by epicPCR carrying antibiotic-resistance genes studied. The genes bla_OXA-48-like (in the effluent of the Varazdin WWTP) and bla_VIM (in the influent of the Zagreb WWTP) had the highest number of hosts.

Figure 5.

Bacterial genera detected by 16S rRNA amplicon sequencing. The size of the circle indicates relative abundance. Only genera with a relative abundance of ≥0.5% are shown. If the investigated antibiotic-resistance gene was detected in a particular genus by epicPCR, it is marked with a full circle, otherwise the circle is empty.