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In this study, microbial quality and antimicrobial resistance of faecal bacteria from a Portuguese river were assessed. River water samples collected upstream and downstream of a wastewater treatment plant, throughout a 3-month period, were used for the enumeration of Escherichia coli and Enterococcus spp. The highest numbers found for E. coli and enterococci were 1.1 × 10⁴ and 1.2 × 10⁴ colony forming units (CFU)/100 ml, respectively. In total, 144 isolates of E. coli and 144 of enterococci were recovered and tested for antimicrobial susceptibility; 104 E. coli and 78 Enterococcus spp. showed resistance to one or more antimicrobial drugs. Overall, 70 and 32 different resistance patterns were found for E. coli and enterococci, respectively. One E. coli showed resistance to imipenem and 29 isolates were extended spectrum β-lactamase-producers. Multidrug-resistant E. coli belonged mostly to groups A, B1 and group D. Enterococcus spp. were mostly resistant to rifampicin, tetracycline, azithromycin and erythromycin; six isolates showed resistance to vancomycin, presenting the VanA phenotype. The high levels of E. coli and enterococci and the remarkable variety of antimicrobial resistance profiles, reinforces the theory that these river waters can be a pool of antimicrobial resistance determinants, which can be easily spread among different bacteria and reach other environments and hosts.

The emergence of acquired carbapenemases is currently one of the most serious public health threats worldwide. These enzymes confer resistance to almost all ␤-lactams, including carbapenems leading to very few therapeutic options for treating patients infected by multidrug-resistant bacteria.Here we report the identification of four environmental carbapenem-resistant Escherichia coli strains, E61, E201, E202, and E203. These strains were recovered in February 2015, from the Ave river, in the north of Portugal. Water samples of 100 ml were filtered through 0.45-m-pore-size membrane filters (Millipore Corporation, USA), which were then placed on tryptone bile Xglucuronide agar (TBX) (BioKar Diagnostics, Beauvais, France) plates supplemented with imipenem (2 mg/liter). Strains growing on those selective plates were checked for carbapenemase activity by using the Carba NP test (1). Antimicrobial susceptibility testing was performed according to the standard disk diffusion method following CLSI recommendations (2) and using cation-adjusted Muller-Hinton plates (Bio-Rad, Cressier, Switzerland). The four isolates were resistant to all ␤-lactams, fluoroquinolones, and aminoglycosides (except amikacin), being susceptible only to tigecycline and colistin. Two out of the four isolates (E61 and E202) also remained susceptible to fosfomycin. MICs of carbapenems (imipenem, ertapenem, and meropenem) were performed using Etest strips (bioMérieux, La Balme-les-Grottes, France) ( Table 1). Molecular investigations were then performed by PCR using specific primers for carbapenemase genes (3) followed by sequencing (Microsynth, Balgach, Switzerland). The phylogenetic group was determined as previously described (4) and showed that isolates E201 and E203 belonged to group E, strain E61 belonged to group A, and strain E202 belonged to group F. Multilocus sequence typing performed as described previously (5) confirmed that isolates E201 and E203 belonged to the same sequence type, whereas the two other strains were clonally unrelated. PCR and sequencing analysis revealed that strains E201 and E203 harbored the bla IMP-8 gene and strains E61 and E202 harbored the bla VIM-1 and bla genes, respectively.Conjugation assays were performed in liquid medium using the azide-resistant E. coli J53 as the recipient strain. Transconjugants were selected onto Luria-Bertani agar plates supplemented with ertapenem (2 mg/liter) and azide (100 mg/liter) to detect the transfer of the carbapenemase genes. Transconjugants were obtained for three out of the four isolates, but not for strain E202 despite repeated attempts. Plasmid extractions were performed using the Kieser extraction method (6) using the environmental strains and transconjugants. Further analysis showed that the bla VIM-1 and bla IMP-8 genes were carried on a ca. 150-kb plasmid. Molecular typing of plasmids was performed using the PCR-based replicon typing (Diatheva, Fano, Italy) (7), revealing that all plasmids bearing the carbapenemase genes belonged to the IncFIB group.The failure to o...

The Ave River in northern Portugal has a history of riverbanks and water quality degradation.The river water quality was assessed by physicochemical, biological (macroinvertebrates) and microbiological (Enterococcus spp. and Escherichia coli) parameters in six locations (A-F, point A being the nearest to the source) throughout its course during a year. Epilithic biofilms were studied through polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE).Antimicrobial susceptibility testing helped with selecting isolates (n ¼ 149 E. coli and n ¼ 86 enterococci) for further genetic characterization. Pursuant to physicochemical and macroinvertebrates-based parameters, the river water was of reasonable quality according to European legislation (Directive 2000/60/EC). However, the microbiological analysis showed increased fecal contamination downstream from point C. At point D, four carbapenem-resistant E. coli isolates were recovered. Paradoxically, point D was classified as a point of 'Good Water Quality' according to macroinvertebrates results. Point F presented the highest contamination level and incidence of multidrug-resistant (MDR) isolates in the water column (13 MDR enterococci out of 39 and 33 MDR E. coli out of 97). Epilithic biofilms showed higher diversity in pristine points (A and B). Thus, biological and microbiological parameters used to assess the water quality led to divergent results; an outcome that reinforces the need for a holistic evaluation. tive 2000/60/EC (http://eur-lex.europa.eu/legal-content/EN/ TXT/PDF/?uri=CELEX:02,000L0060-20141120&qid=1506 068744209&from=EN), which provided the guidelines to be followed by all member states in order to improve the 991