Evaluation of antibiotic resistance dissemination by wastewater treatment plant effluents with different catchment areas in Germany

Abstract

The study quantified the abundances of antibiotic resistance genes (ARGs) and facultative pathogenic bacteria (FPB) as well as one mobile genetic element in genomic DNA via qPCR from 23 different wastewater treatment plant (WWTP) effluents in Germany. 12 clinically relevant ARGs were categorized into frequently, intermediately, and rarely occurring genetic parameters of communal wastewaters. Taxonomic PCR quantifications of five FPB targeting Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and enterococci were performed. The WWTPs differed in their catchment areas being impacted by hospitals, food processing companies, or housing areas only. The total discharges of the analyzed ARGs and FPB were found to cluster independently of the sizes of the WWTPs with a maximum difference of two log units within one cluster. Initially, quantitative data evaluations revealed no significant difference between ARG categories and WWTP catchment areas. More distinct correlations became obvious with a Pearson correlation approach, where each single taxonomic marker is compared to each ARG target. Here, increased correlation of FPB (i.e. E. coli, K. pneumoniae, P. aeruginosa, and enterococci) with clinically relevant ARGs of the category of rarely occurring resistance genes (bla_NDM-1, vanA) was found in WWTP effluents being influenced by hospital wastewaters.

Figure 1

Daily discharge of the total amount of investigated ARGs (circles) and FPB (squares) by differently sized WWTPs (n = 23). Each circle and square represent an average value of four independent sampling periods during 2018. Deviations are listed in SI Tables 1–3.

Figure 2

Abundance of frequent (sul1, ermB, bla_TEM, tetM), intermediate (bla_CTX-M32, bla_OXA48, bla_CTX-M15, bla_CMY-2), and rare (mecA, bla_NDM-1, mcr-1, vanA) antibiotic resistance genes in cluster C (only communal WWTP effluents), cluster F (food production-impacted WWTP effluents), and cluster H (hospital-impacted WWTP effluents) displayed as cell equivalents per daily WWTP discharge volume. Each WWTP was sampled four times, and the significance was determined using the two-tailed non-parametric Mann-Whitney U test. Edges between two different bars represent the median. The bars themselves represent the upper (p = 0.75) and the lower (p = 0.25) quantiles. Error bars illustrate the maximum and minimum abundances.

Figure 3

Daily discharge of facultative pathogenic bacteria in C) communal WWTP effluents (n = 11), F) food-producing impacted WWTP effluents (n = 4), and H) hospital-impacted WWTP effluents (n = 8). Displayed are the median values of each individual facultative pathogenic bacterium, as well as the median values of the total FPB for each WWTP effluent cluster. Deviations are listed in SI Tables 1–3.