Molecular typing of PVL-positive Staphylococcus aureus isolates from Lithuania and genome sequencing of a local outbreak strain

Staphylococcus aureus is a major human commensal and pathogen, with the Panton-Valentine leukocidin (PVL) genes being associated with increased virulence. Rapid detection and molecular typing of such isolates are essential for effective epidemiological surveillance and infection control. Studies from Lithuania indicate that PVL is relatively common among S. aureus isolates, but detailed molecular typing data are still lacking. Clinical S. aureus isolates were collected from two hospitals in Vilnius in 2018-2019 and 2024, and from healthy volunteers between 2012 and 2020. Isolates were screened for PVL genes using real-time PCR. In addition, positive isolates harvested directly from the agar plate were tested for PVL production using an experimental lateral flow assay (LFA). Positive isolates were characterised using DNA-microarrays that facilitated the detection of resistance markers and virulence genes including PVL as well as an assignment to clonal complexes, strains and SCCmec types. Epidemiologically relevant isolates were subjected to whole-genome sequencing using Oxford nanopore technology. Out of 1296 S. aureus isolates, 124 yielded PVL-positive PCR results. 100 isolates were available for genotyping. Two PCR-positive isolates were negative by array and LFA, but PVL detection by DNA-microarray and lateral flow test (LF) showed complete concordance. Among PVL-positive isolates, 61.2% were methicillin-resistant S. aureus (MRSA). The most common PVL-MRSA strain (n = 43) was a Clonal Complex (CC) 8 MRSA that resembled the North American "USA300" strain but that lacked the arginine catabolic mobile element (ACME). This suggested an outbreak in one of the participating hospitals. Other common strains were PVL-positive CC30-MSSA, CC121-MSSA and CC8-MRSA-[IV+ACME] "USA300", while other lineages were represented by single isolates only. Whole-genome sequencing of two ACME-negative CC8-MRSA-IV isolates and of one local "USA300" isolate, as well as a comparison to international reference sequences, showed a very high degree of similarity in core genome and prophage content. The dominant PVL-MRSA strains were "USA300", indicating a possible importation from North America, and a locally emerged variant of "USA300" that lost the ACME-associated genes of its SCCmec element. These findings suggest that real-time PVL detection is essential for outbreak prevention. Given its clinical relevance, routine PVL screening -via PCR or lateral flow assays-in routine diagnostics should be seriously considered and surveillance of PVL-MRSA is urgently recommended.