The Resistome of ESKAPEE Pathogens in Untreated and Treated Wastewater: A Polish Case Study

Abstract

The aim of this study was to quantify ESKAPEE bacteria, genes encoding resistance to antibiotics targeting this group of pathogens, as well as integrase genes in municipal wastewater and river water. Environmental DNA was extracted from the collected samples and used in deep sequencing with the Illumina TruSeq kit. The abundance of bacterial genera and species belonging to the ESKAPEE group, 400 ARGs associated with this microbial group, and three classes of integrase genes were determined. A taxonomic analysis revealed that Acinetobacter was the dominant bacterial genus, whereas Acinetobacter baumannii and Escherichia coli were the dominant bacterial species. The analyzed samples were characterized by the highest concentrations of the following ARGs: bla_GES, bla_OXA-58, bla_TEM, qnrB, and qnrS. Acinetobacter baumannii, E. coli, and genes encoding resistance to β-lactams (bla_VEB-1, bla_IMP-1, bla_GES, bla_OXA-58, bla_CTX-M, and bla_TEM) and fluoroquinolones (qnrS) were detected in samples of river water collected downstream from the wastewater discharge point. The correlation analysis revealed a strong relationship between A. baumannii (bacterial species regarded as an emerging human pathogen) and genes encoding resistance to all tested groups of antimicrobials. The transmission of the studied bacteria (in particular A. baumannii) and ARGs to the aquatic environment poses a public health risk.

Keywords: ESKAPEE; antibiotic resistance; antibiotic resistance genes; river water; wastewater.

Figure 1

Abundance of ESKAPEE group members in the analyzed samples (genus level). The distribution of data (standard deviation) is marked by whiskers in the box plot. Sampling sites: UWW—untreated wastewater, PC—wastewater exiting from the primary clarifier, AS—wastewater from the activated sludge tank, MR—wastewater from the multifunctional reactor, TWW—treated wastewater, URW—river water collected upstream from the wastewater discharge point, DRW—river water collected downstream from the wastewater discharge point. Data regarding UWW, PC, AS, MR, and TWW correspond to the left axis, and data regarding URW and DRW correspond to the right axis.

Figure 2

Abundance of ESKAPEE group members in the analyzed samples (species level). The distribution of data (standard deviation) is marked by whiskers in the box plot. Sampling sites: UWW—untreated wastewater, PC—wastewater exiting from the primary clarifier, AS—wastewater from the activated sludge tank, MR—wastewater from the multifunctional reactor, TWW—treated wastewater, URW—river water collected upstream from the wastewater discharge point, DRW—river water collected downstream from the wastewater discharge point. Data regarding UWW, PC, AS, MR, and TWW correspond to the left axis, and data regarding URW and DRW correspond to the right axis.

Figure 3

Distribution of antibiotic resistance genes (ppm) in subsequent stages of wastewater treatment and in river water. Sampling sites: UWW—untreated wastewater, PC—wastewater exiting from the primary clarifier, AS—wastewater from the activated sludge tank, MR—wastewater from the multifunctional reactor, TWW—treated wastewater, URW—river water collected upstream from the wastewater discharge point, DRW—river water collected downstream from the wastewater discharge point.

Figure 4

Distribution of integron-integrase genes (ppm) in subsequent stages of wastewater treatment and in river water. Sampling sites: UWW—untreated wastewater, PC—wastewater exiting from the primary clarifier, AS—wastewater from the activated sludge reactor, MR—wastewater from the multifunctional reactor, TWW—treated wastewater, URW—river water collected upstream from the wastewater discharge point, DRW—river water collected downstream from the wastewater discharge point.

Figure 5

Network analysis of the correlations between variables in wastewater and river water samples. Three types of correlations were evaluated: (1) bacterial species vs. ARGs, (2) integrase genes vs. ARGs, and (3) bacterial species vs. integrase genes. All correlations (edges) in the analysis are statistically significant.