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. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 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 in-silico 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 Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX-M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX-M-1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV-12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae 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 mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae 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.

Department of Biomedical and Clinical Sciences Romeo and Enrica Invernizzi Pediatric Research Center University of Milan Milan Italy

Department of Earth and Environment Sciences University of Pavia Pavia Italy

Faculty of Medicine Charles University Plzen Czechia

Molecular Virology Unit Microbiology and Virology Department Fondazione IRCCS Policlinico San Matteo Pavia Italy

Unit of Microbiology and Clinical Microbiology Department of Clinical Surgical Diagnostic and Pediatric Sciences University of Pavia Pavia Italy

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Baquero F., Martínez J. L., Cantón R. (2008). Antibiotics and antibiotic resistance in water environments. Curr. Opin. Biotechnol. 19, 260–265. 10.1016/j.copbio.2008.05.006 PubMed DOI

Berglund B. (2015). Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with antibiotics. Infect. Ecol. Epidemiol. 5:28564. 10.3402/iee.v5.28564 PubMed DOI PMC

Biswas S., Brunel J. M., Dubus J. C., Reynaud-Gaubert M., Rolain J. M. (2012). Colistin: an update on the antibiotic of the 21st century. Expert Rev. Anti Infect. Ther. 8, 917–934. 10.1586/eri.12.78 PubMed DOI

Bonomo R. A. (2017). β-Lactamases: a focus on current challenges. Cold Spring Harb. Perspect. Med. 7:a025239. 10.1101/cshperspect.a025239 PubMed DOI PMC

Bouki C., Venieri D., Diamadopoulos E. (2013). Detection and fate of antibiotic resistant bacteria in wastewater treatment plants: a review. Ecotoxicol. Environ. Saf. 91, 1–9. 10.1016/j.ecoenv.2013.01.016 PubMed DOI

Carattoli A. (2009). Resistance plasmid families in Enterobacteriaceae. Antimicrob. Agents Chemother. 53, 2227–2238. 10.1128/AAC.01707-08 PubMed DOI PMC

Colombo S., Arioli S., Neri E., Della Scala G., Gargari G., Mora D. (2017). Viromes as genetic reservoir for the microbial communities in aquatic environments: a focus on antimicrobial-resistance genes. Front. Microbiol. 8:1095. 10.3389/fmicb.2017.01095 PubMed DOI PMC

Cristina M. L., Sartini M., Ottria G., Schinca E., Cenderello N., Crisalli M. P., et al. (2016). Epidemiology and biomolecular characterization of carbapenem-resistant Klebsiella pneumoniae in an Italian hospital. J. Prev. Med. Hyg. 3, 149–156. 10.15167/2421-4248/jpmh2016.57.3.639 PubMed DOI PMC

Davies J., Davies D. (2010). Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. Rev. 74, 417–433. 10.1128/MMBR.00016-10 PubMed DOI PMC

Di Pilato V., Arena F., Tascini C., Cannatelli A., Henrici De Angelis L., Fortunato S., et al. . (2016). mcr-1.2, a new mcr variant carried on a transferable plasmid from a colistin-resistant KPC carbapenemase-producing Klebsiella pneumoniae strain of sequence type 512. Antimicrob. Agents Chemother. 60, 5612–5615. 10.1128/AAC.01075-16 PubMed DOI PMC

Eckert C., Gautier V., Saladin-Allard M., Hidri N., Verdet C., Ould-Hocine Z., et al. . (2004). Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob. Agents Chemother. 48, 1249–1255. 10.1128/AAC.48.4.1249-1255.2004 PubMed DOI PMC

Frazão M. R., Andrade L. N., Darini A. L. C., Falcão J. P. (2017). Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years. Braz. J. Infect. Dis. 21, 477–480. 10.1016/j.bjid.2017.04.006 PubMed DOI PMC

Guyomard-Rabenirina S., Dartron C., Falord M., Sadikalay S., Ducat C., Richard V., et al. . (2017). Resistance to antimicrobial drugs in different surface waters and wastewaters of Guadeloupe. PLoS ONE 12:e0173155. 10.1371/journal.pone.0173155 PubMed DOI PMC

Hansen F., Olsen S. S., Heltberg O., Justesen U. S., Fuglsang-Damgaard D., Knudsen J. D., et al. . (2014). Characterization of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark. Microb. Drug Resist. 20, 316–324. 10.1089/mdr.2013.0157 PubMed DOI

Jarlier V., Nicolas M. H., Fournier G., Philippon A. (1988). Extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev. Infect. Dis. 10, 867–878 10.1093/clinids/10.4.867 PubMed DOI

Li A., Yang Y., Miao M., Chavda K. D., Mediavilla J. R., Xie X., et al. (2016). Complete sequences of mcr-1-harboring plasmids from extended-spectrum-β-lactamase- and carbapenemase-producing Enterobacteriaceae. Antimicrob. Agents. Chemother. 7, 4351–4354. 10.1128/AAC.00550-16 PubMed DOI PMC

Liu Y. Y., Wang Y., Walsh T. R., Yi L. X., Zhang R., Spencer J., et al. . (2016). Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect. Dis. 16, 161–168. 10.1016/S1473-3099(15)00424-7 PubMed DOI

Machado E., Coque T. M., Cantón R., Sousa J. C., Silva D., Ramos M., et al. . (2009). Leakage into Portuguese aquatic environments of extended-spectrum-β-lactamase-producing Enterobacteriaceae. J. Antimicrob. Chemother. 63, 616–618. 10.1093/jac/dkn510 PubMed DOI

Marti E., Huerta B., Rodríguez-Mozaz S., Barceló D., Jofre J., Balcázar J. L. (2014). Characterization of ciprofloxacin-resistant isolates from a wastewater treatment plant and its receiving river. Water. Res. 61, 67–76. 10.1016/j.watres.2014.05.006 PubMed DOI

Olaitan A. O., Morand S., Rolain J. M. (2016). Emergence of colistin-resistant bacteria in humans without colistin usage: a new worry and cause for vigilance. Int. J. Antimicrob. Agents 47, 1–3. 10.1016/j.ijantimicag.2015.11.009 PubMed DOI

Ovejero C. M., Delgado-Blas J. F., Calero-Caceres W. (2017). Spread of mcr-1-carrying Enterobacteriaceae in sewage water from Spain. J. Antimicrob. Chemother. 72, 1050–1053. 10.1093/jac/dkw533 PubMed DOI PMC

Pagani L., Dell'Amico E., Migliavacca R., D'Andrea M. M., Giacobone E., Amicosante G., et al. . (2003). Multiple CTX-M-type extended-spectrum β-lactamases in nosocomial isolates of enterobacteriaceae from a hospital in Northern Italy. J. Clin. Microbiol. 41, 4264–4269. 10.1128/JCM.41.9.4264-4269.2003 PubMed DOI PMC

Perilli M., Bottoni C., Pontieri E., Segatore B., Celenza G., Setacci D., et al. (2013). Emergence of bla(KPC-3)-Tn4401a in Klebsiella pneumoniae ST512 in the municipal wastewater treatment plant and in the university hospital of a town in central Italy. J. Glob. Antimicrob. Resist. 1, 217–220. 10.1016/j.jgar.2013.07.002 PubMed DOI

Perilli M., Dell'Amico E., Segatore B., de Massis M. R., Bianchi C., Luzzaro F., et al. . (2002). Molecular characterization of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian nationwide survey. J. Clin. Microbiol. 40, 611–614. 10.1128/JCM.40.2.611-614.2002 PubMed DOI PMC

Pilla G., Sacchi E., Ciancetti G. (2007). Hydrochemical and isotopic groundwater investigation in the Oltrepo region (Po valley, Northern Italy), in Proceedings of Symposium: “Advances in Isotope Hydrology and its Role in Sustainable Water Resources Management (HIS-2007),” (Vienna: IAEA, Proceedings Series; IAEA–CN−151/41; ), 49–58.

Poirel L., Kieffer N., Brink A., Coetze J., Jayol A., Nordmann P. (2016). Genetic features of MCR-1-producing colistin-resistant Escherichia coli isolates, South Africa. Antimicrob. Agents Chemother. 60, 4394–4397. 10.1128/AAC.00444-16 PubMed DOI PMC

Poppe C., Martin L., Muckle A., Archambault M., McEwen S., Weir E. (2006). Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada. Can. J. Vet. Res. 70, 105–114. PubMed PMC

Rasheed J. K., Jay C., Metchock B., Berkowitz F., Weigel L., Crellin J., et al. . (1997). Evolution of extended-spectrum beta-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia. Antimicrob. Agents Chemother. 41, 647–653. PubMed PMC

Schwartz T., Kohnen W., Jansen B., Obst U. (2003). Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiol. Ecol. 43, 325–435. 10.1111/j.1574-6941.2003.tb01073.x PubMed DOI

Tenover F. C., Arbeit R. D., Goering R. V., Mickelsen P. A., Murray B. E., Persing D. H., et al. . (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239. PubMed PMC

Wang Y., Tian G. B., Zhang R., Shen Y., Tyrrell J. M., Huang X., et al. . (2017). Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study. Lancet Infect. Dis. 17, 390–399. 10.1016/S1473-3099(16)30527-8 PubMed DOI

Watkinson A. J., Micalizzi G. R., Bates J. R., Costanzo S. D. (2007). Novel method for rapid assessment of antibiotic resistance in Escherichia coli isolates from environmental waters by use of a modified chromogenic agar. Appl. Environ. Microbiol. 73, 2224–2229. 10.1128/AEM.02099-06 PubMed DOI PMC

Wellington E. M., Boxall A. B., Cross P., Feil E. J., Gaze W. H., Hawkey P. M., et al. . (2013). The role of the natural environment in the emergence of antibiotic resistance in gram-negative bacteria. Lancet Infect. Dis. 13, 155–165. 10.1016/S1473-3099(12)70317-1 PubMed DOI

Yigit H., Queenan A. M., Anderson G. J., Domenech-Sanchez A., Biddle J. W., Steward C. D., et al. (2001). Novel carbapenem-hydrolyzing b-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45, 1151–1161. 10.1128/AAC.45.4.1151-1161.2001 PubMed DOI PMC

Zanotto C., Bissa M., Illiano E., Mezzanotte V., Marazzi F., Turolla A., et al. . (2016). Identification of antibiotic-resistant Escherichia coli isolated from a municipal wastewater treatment plant. Chemosphere 164, 627–633. 10.1016/j.chemosphere.2016.08.040 PubMed DOI

Zarfel G., Lipp M., Gürtl E., Folli B., Baumert R., Kittinger C. (2017). Troubled water under the bridge: screening of river mur water reveals dominance of CTX-M harboring Escherichia coli and for the first time an environmental VIM-1 producer in Austria. Sci. Total Environ. 593–594, 399–405. 10.1016/j.scitotenv.2017.03.138 PubMed DOI

Zurfuh K., Poirel L., Nordmann P., Nüesch-Inderbinen M., Hächler H., Stephan R. (2016). Occurrence of the plasmid-borne mcr-1 colistin resistance gene in extended-spectrum-β-lactamase-producing Enterobacteriaceae in river water and imported vegetable samples in Switzerland. Antimicrob. Agents. Chemother. 60, 2594–2595. 10.1128/AAC.00066-16 PubMed DOI PMC

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