Increased levels of multiresistant bacteria and resistance genes after wastewater treatment and their dissemination into lake geneva, Switzerland

Abstract

At present, very little is known about the fate and persistence of multiresistant bacteria (MRB) and their resistance genes in natural aquatic environments. Treated, but partly also untreated sewage of the city of Lausanne, Switzerland is discharged into Vidy Bay (Lake Geneva) resulting in high levels of contamination in this part of the lake. In the present work we have studied the prevalence of MRB and resistance genes in the wastewater stream of Lausanne. Samples from hospital and municipal raw sewage, treated effluent from Lausanne's wastewater treatment plant (WTP) as well as lake water and sediment samples obtained close to the WTP outlet pipe and a remote site close to a drinking water pump were evaluated for the prevalence of MRB. Selected isolates were identified (16S rRNA gene fragment sequencing) and characterized with regards to further resistances, resistance genes, and plasmids. Mostly, studies investigating this issue have relied on cultivation-based approaches. However, the limitations of these tools are well known, in particular for environmental microbial communities, and cultivation-independent molecular tools should be applied in parallel in order to take non-culturable organisms into account. Here we directly quantified the sulfonamide resistance genes sul1 and sul2 from environmental DNA extracts using TaqMan real-time quantitative PCR. Hospital sewage contained the highest load of MRB and antibiotic resistance genes (ARGs). Wastewater treatment reduced the total bacterial load up to 78% but evidence for selection of extremely multiresistant strains and accumulation of resistance genes was observed. Our data clearly indicated pollution of sediments with ARGs in the vicinity of the WTP outlet. The potential of lakes as reservoirs of MRB and potential risks are discussed.

Keywords: antibiotics; aquatic; environment; pollution; qPCR; sediment; sewage.

Figure 1

Map of the study area and sampling sites. Effluents of Lausanne’s wastewater treatment plant (WTP) are discharged via pipe 700 m off shore at 30 m depth (STEP, Station d’épuration, Swiss coordinates: 534672/ 151540). 3.2 km to the southwest, lake water is withdrawn for Lausanne’s drinking water supply (DP, Swiss coordinates: 531748/150195). Water was sampled at all points, sediment was sampled at STEP and DP.

Figure 2

Total viable counts, normalized to sample volume or mass, on agar media without antibiotics. An asterisk (*) indicates less than 10 CFU ml⁻¹. n.d., no colonies detected. Error bars indicate standard deviation of triplicate platings of the same sample.

Figure 3

(A) Total gene copy numbers and flow-cytometric cell counts, normalized to sample volume. (B) Relative abundance of sulfonamide resistance gene copy numbers normalized to 16S rRNA gene copies. Error bars indicate standard deviation from triplicate real-time PCR amplification of the same sample.

Figure 4

(A–C) Total viable counts, normalized to sample volume or mass, in the presence of (A) Sul/Trm/Str, (B) Cla/Tet, and (C) Nor/Cef. (D–F) Abundance, relative to total viable counts without antibiotics, in the presence of (D) Sul/Trm/Str, (E) Cla/Tet, and (F) Nor/Cef. The insert in (F) shows abundance of selected samples at an adjusted scale. An asterisk (*) indicates less than 10 CFU ml⁻¹ in (A–C) and less than 1% relative abundance in (D–F). n.d., no colonies detected. Error bars indicate standard deviation of triplicate platings of the same sample.

Figure 5

Percentage of bacterial isolates from HOS, WTPin, and WTPout that were classified as resistant to the listed antibiotics.

Figure 6

Comparison of percentages of bacteria inheriting more than six (“highly multiresistant”) and more than eight resistances (“extremely multiresistant”) at the three sampling sites HOS, WTPin, and WTPout.

Figure A1

CTD-profile from the Vidy Bay, Lake Geneva above the STEPoutlet, recorded on April 7, 2011.