To Örebro University

Background: Increasing levels of multi-antibiotic resistant bacteria are ound in the environment, causing serious concerns for treatment of infectious diseases. his increase is believed to be due to release of antibiotic resistant bacteria and election pressure resulting from pharmaceuticals in the environment.

Objectives: We evaluated the presence of multi-antibiotic resistant fecal ndicator bacteria from the surface waters of a recipient river and lake downstream of he wastewater treatment plant (WWTP) in Sweden.

Methods: Surface waters from Svartån river and Hjälmaren lake in Sweden were ampled in 2010 and 2011. The waters were analyzed for fecal indicator bacteria Escherichia coli, enterococci) by membrane filtration and selective agar plating. E. coli nd enterococci were evaluated by Etest for resistance to tetracycline, chloramphenicol, alidixic acid, trimethoprim-sulfamethoxazole, ciprofloxacin, cefotaxime, ceftazidime, eropenem, imipenem, ampicillin, vancomycin, gentamycin and streptomycin.

Results: The highest concentration of E. coli and enterococci were found in vartån river at Naturens Hus closest site downstream of the WWTP. Tetracycline resistance as the most prominent in both fecal indicator bacteria. Over the two years, there was 42% (13/31) and 24% (7/29) multi-antibiotic resistant (≥2 antibiotics) E. coli and nterococci, respectively. Furthermore, we identified one ESBL and one AmpC hyperproducing . coli in 2010 and vancomycin (vanA) resistant E. faecium in 2011.

Conclusions: The presence, of multi-antibiotic resistant strains of fecal ndicator organisms in regions considered predominantly clean, is of great concern. While t currently may not be a major threat in the region, it is demonstrating the accelerating incidence and spread of antibiotic resistance worldwide.

We examined the prevalence of fecal indicator bacteria (coliform E. coli and enterococci) for water quality (World Health Organization, 2011, European Enviroment Agency, 2012) and antibiotic resistant E. coli in lake Mälaren from surface water in Björnö Island at Vinterviken bay and in untreated incoming water to Mälarenergi, drinking water treatment plant (DWTP) in Västerås, Sweden. Our studies have demonstrated that the water quality of environmental water was ‘excellent‘ in both surface water at Bjornö in Vinterviken bay and in the untreated incoming water to the DWTP. However, one multi-antibiotic resistant and one antibiotic resistant strain were isolated from the surface waters. Hence antibiotic resistant strains can persist even in low population of fecal bacterial contamination in the waters. Pharmaceutical compounds in the environment are believed to provide a selective pressure for the development of antibiotic resistance in bacteria (Tenover, 2006, Alekshun and Levy, 2007, Mazumdar et al., 2006). Our study demonstrated that pharmaceutical compounds persisted in lake waters with detectable concentrations as others have shown (Godfrey et al., 2007, Loos et al., 2009). Caffeine, naproxen, hydrochlorothiazide, metoprolol and tramadol were present at detectable levels. To study the effect of contaminants on microorganisms, qPCR method was chosen due to its rapid, sensitive and quantitative analyses of gene expression. Thus a qPCR array was designed, to determine the effect of environmental waters on E. coli isolates. Specific genes that were responsible for antibiotic resistance, virulence factors and stress responses were selected. E. coli CFT073 was treated with either sub-MIC levels (0.1 μg/ml) of tetracycline in LB medium or incoming untreated waters to DWTP in LB medium and compared to controls. Gene expression was determined using qPCR. No significant difference in gene response was observed after treatment with sub-MIC of tetracycline or environmental waters. Pharmaceutical compounds which contaminated the water did not appear to exert a significant gene response in the pathogenic E. coli. Pharmaceutical contamination in the water can promote human and animal health risks however the effect of long-term exposure is yet unknown (European Environment Agency, 2011, Wennmalm and Gunnarsson, 2005, Triebskorn et al., 2007). The antibiotic resistant strains likely originated from the WWTP rather than the selective pressure due to pharmaceutical pollutants in the water.