Occurrence of Extended Spectrum β-Lactamases, KPC-Type, and MCR-1.2-Producing from Wells, River Water, and Wastewater Treatment Plants in Oltrepò Pavese Area, Northern Italy.

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to ( = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of -type was identified in 19/30 isolates. In further two strains, a gene co-existed with a -type ESBL determinant. A gene was detected in two isolates of ( = 1) and ( = 1), while any ESBL determinant was ascertained in seven strains. A -type gene was detected in a cefoxitin resistant from a stream. Interestingly, two strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of ST10 on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs and ESBLs/carbapenemase-producing strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.