| 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
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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. |