During 2021-2023, we obtained MRSA isolates from 2 clinical laboratories in 2 regions of the Czech Republic. We included in our study only unique-patient isolates for that period. All 103 MRSA isolates had limited patient information (specimen type and region) and were confirmed as mecA-MRSA by PCR targeting the SA-442 species-specific fragment and the mecA gene (6,7). We assessed CC398 affiliation and presence of the lukF/lukS-PV gene by using PCR (8,9). We performed phenotypic detection of antibiotic resistance by using the disk-diffusion method and interpreted the results according to guidelines provided by the European Committee on Antimicrobial Susceptibility Testing version 14.0 (10). We extracted DNA by using the NucleoSpin Microbial DNA isolation kit (Machery-Nagel, https://www.mn-net.com). Library preparation and whole-genome sequencing were outsourced to Eurofins (Stade, Germany), where Illumina NovaSeq6000 technology (https://www.illumina.com) was used. Reads were quality trimmed and de novo assembled by using Shovill v1.0.4 (https://github.com/tseemann/shovill), and we assessed assembly quality by using QUAST v5.0.2 (https://quast.sourceforge.net).
We performed typing by using MLSTFinder v2.0.9 and spaTyper (Genomic Epidemiology Center, http://www.genomicepidemiology.org) and identified resistance and virulence genes by using ResFinder 4.1 and VirulenceFinder v2.0.3 (Genomic Epidemiology Center) (identity ˃95%) and confirmed resistance genes by using CARD 3.2.9. (https://card.mcmaster.ca). We characterized the genetic environment of transposon Tn554 by using Bakta 1.9.1 (https://bakta.computational.bio). To compare sequences, we used the National Center for Biotechnology Information (NCBI) BLASTn tool (https://blast.ncbi.nlm.nih.gov).
We constructed a single-nucleotide polymorphism-based phylogeny by using Roary as previously published (6) (Roary v3.13.0, Gubbins v2.4.1, and snp-dists v0.7.0; https://github.com) on all CC398 PVL-positive isolates retrieved from the RefSeq database (https://www.ncbi.nlm.nih.gov/refseq) as of March 2024 and from selected publications of interest from which data were not retrieved in the RefSeq database (1,4,5,11,12). We used iTOL v6 (https://itol.embl.de) to visualize phylogenetic trees. Raw data of the sequenced strains are available in GenBank (accession no. PRJNA1095719).
We tested 103 human MRSA isolates from the Czech Republic; 5 (4.9%) isolates were identified as CC398 and PVL positive and 8 (7.8%) as CC398 and PVL negative. All 5 PVL-positive isolates came from abscess swab samples; the average patient age was 27 years (range 18-45 years). Three patients were from outside the Czech Republic (2 from Asia, 1 from Ukraine), and the other 2 were Czech nationals with no known travel history. All 5 isolates showed identical phenotypes: resistant to cefoxitin, clindamycin, erythromycin, and tetracycline. Four isolates belonged to ST1232 with spa-type t034, and the fifth isolate was ST9181 (a single-locus variant of ST1232) with spa-type t571. spa-types t571 and t034 are closely related. All isolates carried the SCCmec type V(5C2).
We analyzed the ermA and ant (9)-Ia genes as part of the Tn554 transposon on the PVL-positive isolates, which includes transposition-related genes tnpA, tnpB, and tnpC with the resistance genes oriented in opposite directions (Figure). We identified the ermA gene by using ResFinder (sequence identity 95%) and CARD (sequence identity 85%). Alignment with the ResFinder reference (EU348758) revealed 21 nt differences compared with the reference from Streptococcus suis. BLASTn and BLASTp analyses showed 100% identity/coverage at nucleotide and protein levels for ermA variants in PVL-positive ST1232 genomes, explaining the undetected ermA gene in some MRSA ST1232 studies despite reported phenotypic resistance (Appendix).
We constructed a single-nucleotide polymorphism-based phylogenetic tree (Appendix Figure), incorporating PVL-positive isolates from the RefSeq database and major publications. We also included PVL-negative human and animal genomes from clades I, II-GOI, and IIa-GOI, positioning the 5 isolates from the Czech Republic within evolutionary pathways of related isolates. The ST1232 genomes, including those from the Czech Republic, formed a subclade within II-GOI, emerging since 2013 as the main PVL-positive CC398 carriers.