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Seven wastewater treatment plants (WWTPs) with different population equivalents and catchment areas were screened for the prevalence of the colistin resistance gene mcr-1 mediating resistance against last resort antibiotic polymyxin E. The abundance of the plasmid-associated mcr-1 gene in total microbial populations during water treatment processes was quantitatively analyzed by qPCR analyses. The presence of the colistin resistance gene was documented for all of the influent wastewater samples of the seven WWTPs. In some cases the mcr-1 resistance gene was also detected in effluent samples of the WWTPs after conventional treatment reaching the aquatic environment. In addition to the occurrence of mcr-1 gene, CTX-M-32, blaTEM, CTX-M, tetM, CMY-2, and ermB genes coding for clinically relevant antibiotic resistances were quantified in higher abundances in all WWTPs effluents. In parallel, the abundances of Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli were quantified via qPCR using specific taxonomic gene markers which were detected in all influent and effluent wastewaters in significant densities. Hence, opportunistic pathogens and clinically relevant antibiotic resistance genes in wastewaters of the analyzed WWTPs bear a risk of dissemination to the aquatic environment. Since many of the antibiotic resistance gene are associated with mobile genetic elements horizontal gene transfer during wastewater treatment can't be excluded.

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.

The occurrence of new chemical and microbiological contaminants in the aquatic environment has become an issue of increasing environmental concern. Thus, wastewater treatment plants (WWTPs) play an important part in the distribution of so-called new emerging pathogens and antibiotic resistances. Therefore, the daily loads released by the WWTP were calculated including a model system for the distribution of these loads within the receiving water body. UV-, as well as ozone-treatment in separate or in combination for wastewater treatment were under investigation aiming at the reduction of these loads. Here, the impact of these treatments on the DNA integrity via antibody staining and PCR efficiencies experiments were included. All three facultative pathogenic bacteria [enterococci (23S rRNA), Pseudomonas aeruginosa (ecfX), and Escherichia coli (yccT)] and seven clinically relevant antibiotic resistance genes (ARGs) (mecA (methicillin resistance gene), ctx-M32 (β- lactame resistance gene), ermB (erythromycine resistance gene), blaTEM (β- lactame resistance gene), sul1 (sulfonamide resistance gene), vanA (vancomycin resistance gene), and intI1 (Integrase1 gene) associated with mobile genetic elements were detected in wastewaters. Different reduction efficiencies were analyzed during advanced wastewater treatments. ARGs were still found to be present in the effluents under the parameters of 1.0 g ozone per g dissolved organic carbon (DOC) and 400 J/m2, like ctx-M32, ermB, blaTEM, sul1, and intI1. Especially UV radiation induced thymidine dimerization which was analyzed via antibody mediated detection in the metagenome of the natural wastewater population. These specific DNA alterations were not observed during ozone treatment and combinations of UV/ozone treatment. The dimerization or potential other DNA alterations during UV treatment might be responsible for a decreased PCR efficiency of the 16S rRNA amplicons (176, 490, and 880 bp fragments) from natural metagenomes compared to the untreated sample. This impact on PCR efficiencies was also observed for the combination of ozone and UV treatment.

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Conventional wastewater treatment is not sufficient for the removal of hygienically relevant bacteria and achieves only limited reductions. This study focuses on the reduction efficiencies of two semi-industrial ultrafiltration units operating at a large scale municipal wastewater treatment plant. In total, 7 clinically relevant antibiotic resistance genes, together with 3 taxonomic gene markers targeting specific facultative pathogenic bacteria were analysed via qPCR analyses before and after advanced treatment. In parallel with membrane technologies, an ozone treatment (1 g ozone/g DOC) was performed for comparison of the different reduction efficiencies. Both ultrafiltration units showed increased reduction efficiencies for facultative pathogenic bacteria and antibiotic resistance genes of up to 6 log units, resulting mostly in a strong reduction of the bacterial targets. In comparison, the ozone treatment showed some reduction efficiency, but was less effective compared with ultrafiltration due to low ozone dosages frequently used for micro-pollutant removal at municipal wastewater treatment plants. Additionally, metagenome analyses demonstrated the accumulation of facultative pathogenic bacteria, antibiotic resistance genes, virulence factor genes, and metabolic gene targets in the back flush retentate of the membranes, which opens further questions about retentate fluid material handling at urban wastewater treatment plants.

A multidisciplinary concept has been developed to compare advanced wastewater treatment processes for their efficacy of eliminating micropollutants and pathogens. The concept is based on (i) the removal/formation of selected indicator substances and their transformation products (TPs), (ii) the assessment of ecotoxicity via in vitro tests, and (iii) the removal of pathogens and antibiotic resistant bacteria. It includes substances passing biological wastewater treatment plants regulated or proposed to be regulated in the European Water Framework Directive, TPs formed in biological processes or during ozonation, agonistic/antagonistic endocrine activities, mutagenic/genotoxic activities, cytotoxic activities, further activities like neurotoxicity as well as antibiotics resistance genes, and taxonomic gene markers for pathogens. At a pilot plant, ozonation of conventionally treated wastewater resulted in the removal of micropollutants and pathogens and the reduction of estrogenic effects, whereas the in vitro mutagenicity increased. Subsequent post-treatment of the ozonated water by granular activated carbon (GAC) significantly reduced the mutagenic effects as well as the concentrations of remaining micropollutants, whereas this was not the case for biofiltration. The results demonstrate the suitability of the evaluation concept to assess processes of advanced wastewater treatment including ozonation and GAC by considering chemical, ecotoxicological, and microbiological parameters.