Colistin resistance beyond carbapenems: molecular epidemiology of multi-drug resistant Klebsiella pneumoniae from an Italian hospital

Abstract

Purpose: Multidrug resistant (MDR) Klebsiella pneumoniae (Kp) is a worldwide concern worsened by colistin resistance emergence. In this study, molecular epidemiology of clinical MDR Kp strains is described, assessing clonal relationships and resistance genes profiles. Prevalence of Extended-Spectrum Beta-Lactamase (ESBL) blaCTX-M, blaTEM and blaSHV genes was evaluated, as well as that associated to carbapenems (blaKPC, blaGES, blaVIM, blaIMP, blaNDM, blaOXA-48) and colistin resistance (mcr-1,2,3,4 and mgrB). Methods & Materials: Twenty-six Kp cultures were collected from “A.Cardarelli” hospital in Molise Region (Central Italy), 57.7% from Intensive Care Unit (ICU). The Minimum Inhibitory Concentration (MIC) was assessed by BD Phoenix™. Genotyping was achieved through Pulsed-Field Gel Electrophoresis (PFGE) with XbaI and MultiLocus Sequence Typing (MLST). PCRs for resistance genes detection and mgrB sequencing were performed. Results: Most (n = 20, 77%) strains were carbapenems resistant (imipenem, meropenem, ertapenem), and 53% (n = 14) to colistin. A higher prevalence of blaKPC (100%) and blaSHV (96.2%) was found compared with blaTEM (88.5%), blaVIM (69.0%) and blaCTX-M (7.7%). While none colistin-resistant (col-R) strain carried mcr-1,2,3,4 variants, 42.8% (n = 6) had an Insertion Sequence (IS) in mgrB, identified as IS5-like (5/14, 36%) and ISKpn14 (1/14, 7%). The prevalent Sequence Type was ST512 (50%), followed by ST101 (38.5%) and ST307 (11.5%). PFGE detected 12 clusters and 18 pulsotypes (95% similarity), and was more discriminating than MLST (Simpson's Index 95% vs 61%). An ICU outbreak, during November 2014-January 2015, included five ST512 strains, blaTEM/blaSHV/blaVIM/blaKPC positive, and col-R due to mgrB IS5-like. Conclusion: Molecular epidemiology study identified an outbreak caused by poor compliance with hygienic standards because of reduced personnel during Christmas holidays. Although ST258 is the most prevalent in Italy, half strains were typed as ST512 that represents its monoallelic variant. Our results confirm the endemic blaKPC distribution and blaTEM/blaSHV as the prevalent ESBLs in Kp hospital outbreaks. According to epidemiological data, plasmid mcr variants were not found in col-R strains. Conversely, IS elements prevalence in mgrB explain resistance in about half col-R strains, and was in line with Italian data. Further investigations are needed to identify mgrB mutations in IS negative col-R strains.