To Örebro University

Extensive use of antibiotics both from human-medicine and veterinary sources are believed to provide selective pressure on bacteria that leads to an increase in antibiotic resistance in environmental waters. Contamination of antibiotic resistant microbes will raise human health risks. Escherichia coli are Gram negative bacilli that belong to the coliform group. E. coli are used as fecal indicators organism (FIO) to determine microbial contamination and water quality. We aimed to investigate the prevalence of antibiotic resistant bacteria in Swedish inland waters and determine the response of uropathogenic E. coli to the environmental waters. Samples were collected in different locations near Örebro Sweden at 4 different time points during 2010-2011. Waters were filtered and FIO were isolated using selective medium. The highest numbers of FIO were detected for both E. coli and enterococci in the river Svartån near the effluent from the wastewater treatment plant (WWTP). Over the two years, 42% and 24% of the antibiotic resistant strains were multi-drug resistant (MDR) E. coli and enterococci, respectively. In addition, 15% of MDR E. coli were extended spectrum beta-lactamase producing and AmpC overproducing strains. A vancomycin resistant E. faecium was also identified. Tetracycline resistance was the most common in FIO isolates. Our study suggests that WWTP distributed FIO and antibiotic resistant bacteria. In a second study we analyzed for the presence of various pharmaceutical residues from lake Mälaren in Västerås Sweden. Some pharmaceutical compounds were present at detectable levels but were removed by the drinking water treatment plant. Quantitative PCR was performed to investigate the effects on genes focused on antibiotic resistance, virulence factors and stress response. Forty one-gene array was developed and tested using tetracycline treatment or environmental water. No significant difference was found when compared to controls in the gene expression profile of bacteria grown in medium prepared with sub-MIC of tetracycline or environmental waters. We concluded that the pharmaceutical levels detected did not exert any significant effects on the E. coli strain tested. From this study, we conclude that MDR bacteria may actually persist in environmental waters in what is considered as a clean urban region. Pharmaceutical pollutants in the inland water did not exert a significant effect on the E. coli, suggesting that MDR strains are released in the effluent of the WWTP rather than induced through selective pressure by the pharmaceuticals contamination.

The release of antibiotics and pharmaceuticals into environmental waters contribute to the increasing risk of antibiotic resistant bacteria. The spread of antibiotic resistant bacteria in the environment increases the health risks to the community. Enterococci are fecal indicator bacteria (FIB) in aquatic environments for determining water quality. In order to study enterococcal distribution and their response to environmental waters, we first screened for fecal indicator bacteria and their antibiotic resistance. Samples were collected from different locations of inland waters near Örebro city, Sweden at 4 time points during 2010 and 2011. Waters were filtered and the bacteria were cultured on selective media. We observed that the distribution of fecal indicator bacteria was higher at Svartån at Naturens Hus (≤705 CFU/100 ml for enterococci and ≤5867 CFU/100 for E. coli) near the effluent of the wastewater treatment plant (WWTP) than other locations tested. The eastern side of Hjälmaren lake, Storhjälmaren, had the lowest number of FIB (0 CFU/100 ml for enterococci and ≤2 CFU/100 ml for E.coli). Isolated E. coli, E. faecalis and E. faecium were evaluated for antibiotic resistance. We observed that ≤18% of E. coli environmental isolates and 12% of E. faecium and E. feacalis isolates were resistant to antibiotics during 2010 and 2011. Fifteen percent of these were multi antibiotic resistant (MAR) enterococci in 2010 and 31% in 2011. Tetracycline resistance was the most widespread antibiotic resistance found in FIB insolates. Extended spectrum β-lactamase expressing E. coli strains were found to also be MAR. Vancomycin and imipenem resistance was found in E. faecium isolate. Our results suggest that WWTP contributes to the distribution of FIB and antibiotic resistance. Secondly we aimed to evaluate the cellular responses of human and bacterial cells in environmental waters. We found that the pro-inflammatory response (IL-1β and TNF-α) of THP-1 cell was significantly higher in Svartån at Naturens Hus downstream of WWTP than the other locations. Based on this we evaluated E. feacalis responses to the same water. There were no statistical significant changes in gene response found in E. feacalis isolates, suggesting that environmental waters contain unidentified substances can effect on human cells responses but not bacteria. In this report we conclude that transferring of MAR strains in the environmental waters were increased annually in enterococci and E. feacalis did not initiate a response to the unknown substances that are present in river.