Prevalence of colistin resistance in Escherichia coli: mcr-1-positive clinical isolates in Lombardy, Northern Italy

Background: The recent emergence of the plasmid-mediated colistin resistance mechanism in Escherichia coli, Klebsiella pneumoniae and Salmonella spp. has raised concern among public health experts as colistin is a last-line antibiotic for the treatment of infections by carbapenem resistant Enterobacteriaceae. The aim of the study was to investigate i) the prevalence of this resistance trait in E. coli, and ii) the presence of mcr-1 and mcr-2 genes among colistin-resistant isolates in the Lombardy region, Northern Italy. Material/methods: The study was performed during a 4-month period (May-August 2016) in six acute care Hospitals. Non duplicated E. coli isolates from clinical samples of both outpatients and inpatients were included in the study (surveillance rectal swabs were excluded). Bacterial species identification was obtained by MALDI-TOF mass spectrometry. Colistin resistance (MIC > 2 mg/L) was first evaluated by routine automated systems and then confirmed by the reference broth microdilution method (EUCAST, 2016). Amplification of mcr-type genes, and microarray analysis were accomplished. Phylogroup identification, rep-PCR (DiversiLab) and PFGE (XbaI) were used for genotyping. The mcr-1-positive plasmids were characterized by PBRT kit and PCR (IncX). Results: Overall, 3902 E. coli isolates were evaluated (1560 from inpatients, 2342 from outpatients). Of them, 18/3902 (0.5%), collected from 4/6 centers, showed resistance to colistin. These isolates were obtained from both inpatients (n=6) and outpatients (n=12). In particular, hospitalized patients were from medical (3/6), rehabilitation (2/6), and surgical (1/6) wards. The great majority of colistinresistant isolates were from urine (16/18) but two of them were from blood cultures. Colistin MIC values ranged from 4 to 16 mg/L (as assessed by the reference method). The mcr-1 gene was detected in 10/18 isolates, all of which from urine cultures (7 from outpatients, 3 from inpatients). In these cases, colistin MIC values ranged from 4 to 8 mg/L. Of note, 6/10 mcr-1-positive E. coli isolates were also resistant to ciprofloxacin and trimethoprim-sulfamethoxazole, and 2 of them were also positive for the SHV-12 ESBL enzyme. All mcr-1-positive E. coli strains resulted unrelated by PFGE analysis, while rep-PCR showed eight different clusters. The identified phylogroups were A and D (in four cases each), B1 and B2 (in one case each). The incompatibility groups were IncX4 in 7/10 strains, IncHI2 in the remaining 3/10. The mcr-2 gene was never detected. Conclusions: According to most recent surveillances, our study demonstrates a low prevalence of colistin resistance among E. coli isolates also in the Lombardy region. In addition, our data show that the mcr-1 gene plays a prominent role in this context. Of particular concern, it appears the frequent detection of mcr-1 gene among isolates from urine cultures of outpatients, thus suggesting the opportunity of routine testing the antimicrobial susceptibility for colistin in these cases.