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

Mattia Palmieri; Marco Maria D'Andrea; Andreu Coello Pelegrin; Caroline Mirande; Snezana Brkic; Ivana Cirkovic; Herman Goossens; Gian Maria Rossolini; Alex van Belkum

doi:10.3389/fmicb.2020.00294

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

Front Microbiol. 2020 Feb 21:11:294. doi: 10.3389/fmicb.2020.00294. eCollection 2020.

Authors

Affiliations

¹ bioMérieux, Data Analytics Unit, La Balme-les-Grottes, France.
² Department of Biology, University of "Tor Vergata", Rome, Italy.
³ Department of Medical Biotechnologies, University of Siena, Siena, Italy.
⁴ bioMérieux, R&D Microbiology, La Balme-les-Grottes, France.
⁵ Institute for Laboratory Diagnostics Konzilijum, Belgrade, Serbia.
⁶ Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia.
⁷ Laboratory of Medical Microbiology, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.
⁸ Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy.
⁹ Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Abstract

Klebsiella pneumoniae 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 K. pneumoniae isolates from Serbia. Consecutive non-replicate K. pneumoniae clinical isolates (n = 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 (n = 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 (N = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, N = 38), CG258 (ST437, N = 4; ST340, N = 1; ST258, N = 1) and CG17 (ST336, N = 1). mcr 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^C28S 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 bla _OXA- ₄₈ genetic background. The bla _OXA- ₄₈ gene was located in a novel IncFIA-IncR hybrid plasmid, also containing the extended spectrum β-lactamase-encoding gene bla _CTX-M-15 and several other AMR genes. Non-ST101 isolates presented different MgrB alterations (C28S, C28Y, K2^∗, K3^∗, Q30^∗, adenine deletion leading to frameshift and premature termination, IS5-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.

Keywords: K. pneumoniae; ST101; Serbia; WGS; blaOXA–48; colistin; mgrB.