Whole-genome analysis of endemic OXA-48-producing K. pneumoniae clinical isolates in Catalonia, Spain

Background: High rates of carbapenemase-producing Enterobacteriaceae have been described in Mediterranean countries. The objective of this study was to characterize OXA-48-producing K. pneumoniae (OXA-48-Kp) clinical isolates from 12 Catalonian hospitals using whole-genome sequencing (WGS).Methods: Representative isolates were selected based on MLST, PFGE and antibiotic resistance profiles. After library preparation using Nextera XT, WGS was applied to 37 isolates on a MiSeq generating 2x250 bp paired-end reads (Illumina, San Diego, USA). The reads were quality trimmed and de novo assembled using CLC Genomics Workbench v7.0.4 (CLC bio, Aarhus, Denmark). Whole-genome MLST (wgMLST) typing was performed using the scheme available in SeqSphere+ v4.0.2 (Ridom GmbH, Münster, Germany) consisting of 2,365 core-genome MLST (cgMLST) targets plus 2,526 accessory genes. A cluster alert distance threshold of 15 allele differences (cgMLST) was defined to detect closely related isolates based on retrospective analysis of well-defined outbreaks and out-group isolates with same MLST/MLVA/PFGE types. Genome assemblies were uploaded to the Center for Genomic Epidemiology to extract information on antibiotic resistance genes (ARGs) and plasmid replicons. Results: ST405 isolates (PFGE subtypes E1 to E18) had a maximum of 10 and 14 alleles difference when using cgMLST and wgMLST, respectively (Figure 1). Three paired-isolates from PFGE subtypes E1/E16, E12/E13, and E17/E18 were indistinguishable by cgMLST typing, whereas only E17/E18 were indistinguishable using wgMLST typing. ST101 isolates of PFGE subtypes A1 and A2 (different hospitals) had a 14 alleles difference by cgMLST typing, indicating they were genetically related. All isolates were positive for blaOXA-48, blaSHV-1, blaSHV-11, blaSHV-42 and blaSHV-76 genes and 78.4% contained blaCTX-M-15, blaOXA-1 and blaTEM-1 genes. All but one isolate contained plasmid L/M. All isolates carried plasmids of incompatibility group FIB and all isolates except those of PFGE profiles B, C and D, also carried FII plasmid. In addition, two isolates (ST101; A1 and A2) contained the R plasmid and two (ST405; E4 and E5) a HI1B plasmid. FIB, FII and HI1B plasmids were not amplified by PBRT PCR, whereas ColE and FIA plasmids were not found by WGS since they are not included in the PlasmidFinder database.