Detection of ESBL-producing Gram-negative bacteria from surface and ground-waters in the Po Valley

Background: The aim of the study was both to inspect the occurrence of Extended Spectrum β- Lactamases (ESBLs)-producing Gram-negative bacteria in the Po Valley wells, streams and treatment plants and to assess epidemiological strain relatedness. Material/methods: A total of 193 water samples from 11 wells, 5 streams and 3 treatment plants were collected once a month during the period December 2014 - September 2015. Ninety-two out of 193 (47.7%) water samples were from streams, 82/193 (42.5%) were from wells and 19/193 (9.8%) were from treatment plants. A quantity of 100 ml of water from wells and 1 ml from streams/treatment plants, was filtered using 0.22µm membranes. The filters were placed on Plate Count Agar (PCA), McConkey Agar (MCA) and selective MCA containing cefotaxime 8mg/L (MCA+CTX). Identification and antimicrobial susceptibility profiles of bacteria from MCA+CTX were obtained by MicroScan/A4 System (Beckman Coulter) and interpreted according to EUCAST 2015 guidelines. Double disk ESBLs confirmatory test using piperacillin-tazobactam, CTX, ceftazidime, cefepime, aztreonam togheter with PCR targeting blaCTX-M/TEM/SHV-type genes and PFGE were performed. Results: Seventy out of 92 (76%), 11/19 (58%) and 33/82 (40.24%) water samples obtained from streams, treatment plants and wells yielded positive bacterial growth on MCA+CTX, respectively. A total of 99 (44.8%) Enterobacteriaceae, 38 (17.2%) Pseudomonas spp., 35 (16%) Acinetobacter spp., 29 (13%) Vibrio fluvialis, 15 (6.8%) Aeromonas hydrophila and 5 (2.2%) other Gram-negative bacteria were identified. Double-disk test results confirmed as ESBL-positive 47 Enterobacteriaceae, 17 being Yersinia enterocolitica, 16 Escherichia coli, 11 Klebsiella spp., two Enterobacter spp. and one Leminorella spp.. All Y. enterocolitica strains showed clinical resistance to tetracycline and amoxicillin/clavulanate (80%), ciprofloxacin (33.3%), chloramphenicol (20%), amikacin and gentamicin (13.3%). Thirteen/16 (81.25%) E. coli and 7/11 (63,6%) Klebsiella spp. isolates resulted MDR, showing resistance to β-lactams and tetracycline (100%). Ciprofloxacin resistance was ascertained in 81.8% ESBL-positive E. coli and 50% of ESBL-Klebsiella spp. isolates respectively. Amikacin resistance was present in a lower extent in both the above species, being 8.2% and 16.6% respectively. Twenty out of 47 ESBL-positive Enterobacteriaceae were blaCTX-M-type (18/20) or blaSHV-type (2/20) positive by PCR. blaCTX-M-type genes were present in 12 E. coli and 6 Klebsiella spp., while a blaSHV-type gene was detected in single strain of both the species. PCR identified blaCTX-M genes of CTX-M-1- (84%) and CTX-M-9- group (16%). PFGE on 13 ESBL-positive strains chosen as representative, showed clonal diversity both between the 10 E. coli and the three K. pneumoniae isolates. Conclusions: Here we report a high occurrence of ESBL-enterobacteria, especially from surface waters. Due to the extensive use of surface water in the Po valley for both agricultural irrigation and watering animals, the epidemiological data on environmental well-known human opportunistic bacteria appear particularly worrisome.