Multicenter prospective study on the prevalence of colistin resistance in escherichia coli: Relevance of mcr-1-positive clinical isolates in Lombardy, Northern Italy

Background: The emergence of the plasmid-mediated colistin resistance mechanism in Escherichia coli has raised concern among public health experts as colistin is a last-line antimicrobial resort. The primary aim of the study was to investigate the prevalence of this resistance trait in E. coli isolates circulating in the Lombardy region, Northern Italy. The presence of mcr-type genes and their genetic relationship were also studied. Materials and methods: A prospective study was performed during a 4-month period (May to August, 2016) in six acute care Hospitals. Consecutive nonduplicate clinical isolates of E. coli from any type of clinical specimen, with the exception of rectal swabs, were included in the study. Isolates that exhibited MIC values for colistin >2 mg/L were further investigated. Bacterial identification was obtained by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Amplification of mcr-type genes (-1 to -5 variants) and microarray analysis were accomplished. Repetitive sequence-based PCR (Rep-PCR) and multilocus sequence typing (MLST) analysis were used for genotyping. Results: Overall, 3,902 consecutive E. coli isolates (2,342 from outpatients, 1,560 from inpatients) were evaluated during the study period. Of them, 18/3,902 (0.5%), collected from 4/6 centers, showed resistance to colistin. These isolates were mostly obtained from urine of both outpatients (n=12) and inpatients (n=6). Colistin MIC values ranged from 4 to 8 mg/L. The mcr-1 gene was detected in 10/18 isolates (7 from outpatients, 3 from inpatients). Rep-PCR and MLST analysis revealed the presence of nine different clusters. Further mcr-type genes were not detected. Conclusion: Resistance to colistin in E. coli clinical isolates appears low in our geographic area. With regard to mcr-1-positive isolates, they accounted for approximately 50% of colistin-resistant E. coli isolates, thus representing a relevant resistance mechanism in this context. Although overall limited, the presence of mcr-1 determinant in our region should not be ignored and great concern should be given to the continuous surveillance

Occurrence of extended spectrum \u3b2-lactamases, KPC-Type, and MCR-1.2-producing enterobacteriaceae from wells, river water, and wastewater treatment plants in Oltrep\uf2 Pavese area, Northern Italy

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepo Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-beta-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 bla(CTX-M)-type was identified in 19/30 isolates. In further two E. coli strains, a bla(CTX-M-1) gene co-existed with a bla(SHv)-type ESBL determinant. A bla(SHv-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 bla(DHA)-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 lncX4 plasmid (33.303 bp in size) from a stream of Oltrepo 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

Emergence of a VIM-1 MBL and CTX-M-15 ESbL-producing Klebsiella pneumoniae clone from acute and rehabilitation hospitals in Italy.

We report the emergence of VIM-1 MBL and CTX-M-15-producing K. pneumoniae isolates collected from patients at two acute care hospitals (I.R.C.C.S. "S. Matteo" and "Casa Sollievo della Sofferenza" Hospital) and a long-term rehabilitation facility in Northern Italy (I.R.C.C.S. "S. Maugeri"). Between February 2007 and October 2008, 30 K. pneumoniae strains showing decreased susceptibility to carbapenems were collected. PCR and sequencing experiments revealed the presence of blaVIM-1 gene in 14/30 isolates. All the above isolates carried the blaSHV-5 determinant as well; interestingly, 8/14 VIM positive isolates were also CTX-M-1- like producers. VIM-1 positive strains were present in all hospitals. PFGE genomic profiles of the 14/30 isolates showed that 2 different clones, A and B, were responsible for outbreaks. The coexistence in the same bacterial cell of compatible plasmids carrying epidemiologically important emerging resistance genes, such as MBLs and CTX-Ms, is worrisome since it could predict the generation and spread of pan-resistant bacteria and the consequent treatment option limitations that can lead to significant morbidity and mortality. Control measures should be applied to detect MBL-producing strains and to contrast the vertical and plasmidic diffusion of carbapenem-resistant K. pneumoniae in acute care and rehabilitation facilities