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Title: Oporność na antybiotyki beta-laktamowe u bakterii bytujących w oczyszczalni ścieków w Żywcu i Jeziorze Żywieckim
Authors: Bondarczuk, Kinga
Advisor: Piotrowska-Seget, Zofia
Keywords: antibiotic resistance; resistance plasmids; next generation sequencing
Issue Date: 2017
Publisher: Katowice: Uniwersytet Śląski
Abstract: Antibiotic resistance has become a global problem that threatens contemporary medicine. The global spread of this phenomenon is mainly driven by mobile genetic elements bearing antibiotic resistance genes. It has been already established that environmental bacteria play a crucial role in the acquision of the genes among pathogens. Wastewater treatment plants has been recognised as hotspots for antibiotic-resistant bacteria and mobile genetic elements conferring antibiotic resistance. However, the fate of the bacteria, the genes and the mobile genetic elements in the environment remains uncetrain. Therefore the aim of the project was to evaluate the fate of the bacteria and the genes in final effluent receiving Żywieckie Lake (Poland). For this purpose samples of raw sewage, activated sludge and treated sewage were obtained from the wastewater treatment plant in Żywiec and samples of water and sediments from Żywieckie Lake were collected. Additional samples of water and sediments were collected from The Soła River upsteram the lake and they served as control. Plate counts were performed and culturable fraction of total heterotrophic bacteria and ampicillin-resistant bacteria were determined. The results confirmed statistically significant reduction of all studied bacterial fractions during the wastewater treatment process. The number of bacteria observed in the samples of The Soła River and Żywieckie Lake were considerably lower than in the samples collected from the wastewater treatment plant. The number of ampicillin-resistant bacteria was higher in sediment when compared to water samples what may suggest a possible accumulation of ampicillin-resistant bacteria in freshwater sediments. The biodiversity of microbial communities and antibiotic resistance genes relative abundance in treated sewage, water of the lake and the river were assessed by next generation sequencing on Illumina® platform. The metagenomic analysis revealed the highest microbial diversity in the water sample of Żywieckie Lake. On the other hand, the lowest diversity was observed in the water sample of The Soła, what was probably due to the dominance of two bacterial phyla. The microbial phyla represented in the studied metagenomes were typical of corresponding environments. The highest relative abundance of antibiotic resistance genes (ARGs/16S rRNA) was observed in treated sewage suggesting a considerable amount of the genes being released from the wastewater treatment plant. However, the lowest relative 1 abundance as well as the lowest diversity of the genes in the lake water, when compared to the other studied metagenomes, suggest rather negligible effect of the treated sewage release on antibiotic resistance spread within water microbial communities of the lake. The exogenous isolation method allowed to capture 56 resistance plasmids in E. coli cells. Using PCR-based replicon typing the majority of plasmids could not be classified to any incompatibility group. The classified replicons belonged to IncF and IncN groups. All IncF plasmids were isolated from water samples of The Soła the latter group was represented by vectors captured from lake sediments and treated sewage. Mobility testing revealed that the majority of plasmids isolated from the water samples of The Soła were conjugative, whereas among plasmids captured from other samples mobilizable vectors prevailed. Only 3 resistance vectors from the plasmid library were neither conjugative nor mobilizable. Susceptibility testing revealed that some of the plasmids conferred resistance to third generation cephalosporins and fluoroquinolones, whereas resistance to carbapenems, aminoglycosides and polymyxins was not observed. The isolation of resistance plasmids from the water samples of Żywieckie Lake failed suggesting lower resistance plasmid abundance in this environment. The results of the presented study revealed no obvious impact of the treated sewage release on the spread of antibiotic resistance among bacteria residing in water of the receiving lake. Furthermore, uncontrolled sewage dumping to the Soła River and Żywieckie Lake in the past as well as lower quality of water upstream the lake indicate rather protective role of the wastewater treatment plant in the studied ecosystem.
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