Country-wide expansion of a VIM-1 carbapenemase-producing Klebsiella oxytoca ST145 lineage in Poland, 2009–2019

European Journal of Clinical Microbiology & Infectious Diseases https://doi.org/10.1007/s10096-023-04682-x ORIGINAL ARTICLE Country‑wide expansion of a VIM‑1 carbapenemase‑producing Klebsiella oxytoca ST145 lineage in Poland, 2009–2019 M. Biedrzycka1 · P. Urbanowicz1 · D. Żabicka2 · W. Hryniewicz2 · M. Gniadkowski1 · R. Izdebski1 Received: 11 August 2023 / Accepted: 10 October 2023 © The Author(s) 2023 Abstract Purpose To elucidate the role of the Klebsiella oxytoca species complex (KoSC) in epidemiology of VIM-type MBLproducing Enterobacterales in Poland. Methods The study comprised all 106 VIM-positive KoSC isolates collected by the Polish National Reference Centre for Susceptibility Testing during 2009–2019 from 60 institutions in 35 towns. All isolates were sequenced by Illumina MiSeq, followed by MinION sequencing of selected organisms. Genomes were subjected to bioinformatic analysis, addressing taxonomy, clonality, phylogeny and structural characterisation of key resistance determinants within their chromosomal and plasmidic loci. Results Among five species identified, K. oxytoca was predominant (n = 92), followed by Klebsiella michiganensis (n = 11). MLST distinguished 18 STs, with the most prevalent Klebsiella oxytoca ST145 (n = 83). The clone segregated a lineage with the In237-like integron [blaVIM-1–aacA4 genes; n = 78], recorded in 28 cities almost all over the country. The integron was located in a ~ 49–50 kb chromosomal mosaic region with multiple other resistance genes, linked to a ~ 51 kb phage-like element. The organism might have originated from Greece, and its evolution in Poland included several events of chromosomal ~ 54–258 kb deletions, comprising the natural β-lactamase blaOXY gene. A group of other isolates of various species and clones (n = 12) carried the integron In916 on self-transmissible IncA-type plasmids, effectively spreading in Italy, France and Poland. Conclusion KoSC has been one of the major VIM producers in Poland, owing largely to clonal expansion of the specific K. oxytoca–In237-like lineage. Its apparently enhanced epidemic potential may create a danger on international scale. Keywords VIM · Enterobacterales · Klebsiella oxytoca species complex · KoSC · ST145 · Poland Introduction Carbapenemase-producing Enterobacterales (CPE) are considered to be largely responsible for the current antimicrobial resistance (AMR) crisis around the world [1]. One of their critically important groups is metallo-β-lactamase (MBL) producers, including those with VIM-type enzymes. Initially, these were described in Pseudomonas spp. in the mid-1990s, followed soon by Enterobacterales [2, 3]. In * R. Izdebski 1 Department of Molecular Microbiology, National Medicines Institute, Chełmska 30/34, 00‑725 Warsaw, Poland 2 Department of Epidemiology and Clinical Microbiology, National Medicines Institute, Chełmska 30/34, 00‑725 Warsaw, Poland general, the VIM-type carbapenemases are encoded by gene cassettes usually of class 1 integrons that in enterobacteria are located on a variety of plasmids and only occasionally in the chromosome. The first successful VIM CPE country-wide spread in Europe was observed in Greece in the early 2000s, leading to endemic situation with numerous host species, VIM variants and their genetic determinants [4–6]. Soon, regional or interregional dissemination of such organisms has been reported in other European countries, including Spain, Italy or Hungary [6–9]. The first VIM-producing Enterobacterales isolate in Poland, Klebsiella pneumoniae, was confirmed in 2006 by the National Reference Centre for Susceptibility Testing (NRCST) in Warsaw [10]. Till the end of 2012, 118 VIM CPE isolates were reported, with predominance of Enterobacter spp. (n = 64; 54.2%), followed by Klebsiella oxytoca (n = 23; 19.5%) [11]. Since then, annual numbers 13 Vol.:(0123456789) European Journal of Clinical Microbiology & Infectious Diseases of VIM CPE multiplied, resulting in a total of 927 isolates from 2006 to 2019 [12]. Although surpassed by K. pneumoniae in recent years, the K. oxytoca species complex (KoSC) has remained to be a highly relevant fraction of VIM CPE, ranking third among the taxonomic groups (n = 106; 11.4%). Here, we report the genomic analysis of all VIM-producing KoSC isolates, collected from the first identification in 2009 till the end of 2019, being part of a comprehensive WGS study of all Polish VIM CPE confirmed by the NRCST in 2006–2019. Materials and methods Bacterial isolates, WGS and species identification The study comprised 106 non-duplicate VIM-producing KoSC isolates from 105 patients, collected by the NRCST during the CPE surveillance in Poland from 2009 to 2019 (Table S1) [12]. The isolates were from 60 centres in 35 cities of all 16 main administrative regions. The majority of the isolates were detected in the region Mazowieckie (n = 56; 52.8%) and often in Warsaw (n = 34; 32.1%). Around a half of the isolates were recovered from infections (n = 52; 49.1%), mainly of the urinary tract (n = 27; 25.5%) and wounds (n = 15; 14.1%), and most of the remaining ones were from carriage (n = 53; 50.0%). The KoSC isolates were tested for the carbapenemase presence by CARBA NP [13], phenotypic and PCR tests [14] and sequenced by MiSeq (Illumina, San Diego, CA, USA), with reads assembled with SPAdes 3.15.5 [15]. Four isolates, representing predominant blaVIM-carrying integron types, were subjected to long-read sequencing by MinION (Oxford Nanopore Technologies, Oxford, UK). The MiSeq and MinION hybrid assemblies were done with Unicycler v.0.4.8 [16]. Species identification was based on average nucleotide identity (ANI) scores, using FastANI v.1.32 with ≥ 95% cutoff [17] and RefSeq reference genomes. MLST, clonal and phylogenetic analyses, characterisation of blaOXYgenes MLST was performed by the mlst tool (https://github.com/ tseemann/mlst). The in-sample clonality SNP analysis was done by BioNumerics v.7.6.3 (Applied Maths NV, SintMartens-Latem, Belgium). The SNP-based phylogenetic analysis in the international context was performed with all 271 KoSC genomes available in RefSeq as of the 1st of February 2023, using Parsnp v.1.5.4 (https://github.com/marbl/ parsnp). Phylotrees were visualised by iTOL (https://itol. embl.d e). Variants of the intrinsic KoSC β-lactamase blaOXY genes were identified using the BIGSdb database (https:// bigsdb.pasteur.fr/klebsiella/). Broader blaOXY-containing 13 genomic regions were studied using the progressive Mauve algorithm [18]. Integrons with blaVIMgenes and their chromosomal and plasmid context; resistomes andantimicrobial susceptibility The composition of blaVIM-carrying integrons was characterised manually using Geneious Prime v.2022.0.1 (Biomatters, Auckland, New Zealand) and BLASTn. Chromosomal loci containing blaVIM-carrying integrons and blaOXY deletions were analysed using Mauve [18]; PHASTER [19] was applied to identify phage-like elements. Plasmid repli (...truncated)