AUTHOR=Palmieri Mattia , D’Andrea Marco Maria , Pelegrin Andreu Coello , Mirande Caroline , Brkic Snezana , Cirkovic Ivana , Goossens Herman , Rossolini Gian Maria , van Belkum Alex 

TITLE=Genomic Epidemiology of Carbapenem- and Colistin-Resistant Klebsiella pneumoniae Isolates From Serbia: Predominance of ST101 Strains Carrying a Novel OXA-48 Plasmid

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.00294

DOI=10.3389/fmicb.2020.00294

ISSN=1664-302X

ABSTRACT=<p><italic>Klebsiella pneumoniae</italic> is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Carbapenem resistance is frequent, and colistin represents a key molecule to treat infections caused by such isolates. Here we evaluated the antimicrobial resistance (AMR) mechanisms and the genomic epidemiology of clinical <italic>K. pneumoniae</italic> isolates from Serbia. Consecutive non-replicate <italic>K. pneumoniae</italic> clinical isolates (<italic>n</italic> = 2,298) were collected from seven hospitals located in five Serbian cities and tested for carbapenem resistance by disk diffusion. Isolates resistant to at least one carbapenem (<italic>n</italic> = 426) were further tested for colistin resistance with Etest or Vitek2. Broth microdilution (BMD) was performed to confirm the colistin resistance phenotype, and colistin-resistant isolates (<italic>N</italic> = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, <italic>N</italic> = 38), CG258 (ST437, <italic>N</italic> = 4; ST340, <italic>N</italic> = 1; ST258, <italic>N</italic> = 1) and CG17 (ST336, <italic>N</italic> = 1). <italic>mcr</italic> genes, encoding for acquired colistin resistance, were not observed, while all the genomes presented mutations previously associated with colistin resistance. In particular, all strains had a mutated MgrB, with MgrB<sup>C28S</sup> being the prevalent mutation and associated with ST101. Isolates belonging to ST101 harbored the carbapenemase OXA-48, which is generally encoded by an IncL/M plasmid that was no detected in our isolates. MinION sequencing was performed on a representative ST101 strain, and the obtained long reads were assembled together with the Illumina high quality reads to decipher the <italic>bla</italic><sub>OXA–</sub><sub>48</sub> genetic background. The <italic>bla</italic><sub>OXA–</sub><sub>48</sub> gene was located in a novel IncFIA-IncR hybrid plasmid, also containing the extended spectrum β-lactamase-encoding gene <italic>bla</italic><sub>CTX–M–15</sub> and several other AMR genes. Non-ST101 isolates presented different MgrB alterations (C28S, C28Y, K2<sup>∗</sup>, K3<sup>∗</sup>, Q30<sup>∗</sup>, adenine deletion leading to frameshift and premature termination, IS<italic>5</italic>-mediated inactivation) and expressed different carbapenemases: OXA-48 (ST437 and ST336), NDM-1 (ST437 and ST340) and KPC-2 (ST258). Our study reports the clonal expansion of the newly emerging ST101 clone in Serbia. This high-risk clone appears adept at acquiring resistance, and efforts should be made to contain the spread of such clone.</p>