Mikrobiologische und molekulargenetische Untersuchungen zu bakteriellen Resistenzen in Abwasserproben verschiedener Herkunft

  • Microbiological and molecular genetic studies on bacterial resistances in sewage samples of various origins

Hönings, Robert; Peltroche-Llacsahuanga, Heidrun (Thesis advisor); Blank, Lars Mathias (Thesis advisor); Dott, Wolfgang (Thesis advisor)

Aachen (2016)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2016

Abstract

Antibiotic-resistant bacteria (ARB) are a serious problem for the health of humans and animals. Antibiotic resistance genes (ARG) and ARB, which are emitted from anthropogenic sources such as clinical wastewaters and wastewater treatment plants (WWTP), as well as the high consumption of antibiotics in human and veterinary medicine, are now regarded as a serious threat to the (aquatic) environment. However, current investigations are not sufficient for a meaningful risk assessment and there is a lack of uniform methods. Therefore, different wastewaters were compared over a period of more than three years by modified microbiological and molecular genetic methods, which were originally derived from clinical diagnostics, and a comprehensive risk assessment was attempted. The concentrations of different resistant pathogens with high clinical relevance were determined and the resistance patterns of more than 4000 identified isolates were analyzed phenotypically and genotypically. The investigated wastewaters came from two municipal WWTPs, only one of which received clinical effluents, as well as the central sewer of the Aachen University Hospital and the residential area of Aachen-Kullen.The concentrations of the total number of cultivable bacteria and overall enterobacteria were nearly constant over the entire period; highest concentrations were found in the inflows of both WWTPs. Enterobacteriaceae exhibited a 10-fold lower concentration at all times. The treatment process of both WWTPs resulted in a significant reduction in the quantity of bacteria. ESBL-producing enterobacteria were also detected at all times, with the exception of the WWTP effluents, and were present in lower concentrations than the counts of overall Enterobacteriaceae. Almost all isolates were detected as CTX-M1 producers. Carbapenem-resistant bacteria (CRB) were only isolated after previous selective enrichment, whereas no CRB were detected in the effluents of both WWTPs even after enrichment. Different genotypes were detected in the examined isolates (blaOXA-48, blaNDM-1, blaKPC). VRE could be detected exclusively in the inflows of both WWTPs and the sewage of the hospital in comparable concentrations. Corresponding isolates were all identified as E. faecium with vanA genotype and phenotype. In contrast, the concentrations of S. aureus and MRSA were below the cultural detection limit at all times.The new methodology provides representative, reproducible and detailed results as well as more detailed insights into the resistance situation and potential hazards of different wastewaters so that primarily wastewaters from WWTPs and the clinical setting are actually hotspots for ARB and ARG, while the prevalence of the investigated resistant bacteria is significantly lower in domestic effluents. Furthermore, the quantities and the sources of the incoming sewage in WWTPs do not seem to have any influence on the resistance situation and risk-assessment of the inflows, since no significant differences could be observed when comparing both WWTPs. Due to the high correlation with published data from clinical iso-lates, an indirect derivation of the local resistance situation also appears to be possible via wastewaters. The three-stage clarification mechanism is also unspecific and effective for both WWTPs so that the concentrations of the investigated groups of resistant bacteria are always below the microbiological detection limit, and thus a threat to the connected aquatic environment by both WWTPs in Aachen can be regarded as very low or unlikely.