Unveiling the characteristics of an emerging staphylococcus iugdunensis sequence type 3 clone by genomic analysis

Abstract

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus (CoNS) that was originally isolated in the 1980s after which its clinical reports began to increase, which suggested this species ranges from a skin commensal to a life-threatening pathogen. However, due to a lack of clinical concern regarding CoNS and inappropriate species identification before the development of next-generation sequencing (NGS) and the introduction of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), its epidemiological and genomic information remain poorly understood. Our group conducted the first epidemiological study of S. lugdunensis in Hong Kong and identified sequence type (ST) 3 with multidrug-resistant patterns as the predominant clone. However, the genomic profiles of mobile genetic elements, resistant genes, and virulence factors were not fully investigated, and the reasons for the ST3 expansion remain unclear at the pan-genomic level. Therefore, high-throughput sequencing platforms were used to characterise the ST3 clone in this study. Methicillin (mecA-mediated) resistance was exclusively identified in the ST3 clone by our epidemiological study. It evaluated the breakpoints recommended for disk diffusion tests and performed antibiotic selective agar tests for resistance detection in S. lugdunensis. Additionally, an accurate prediction with a 100% category agreement was achieved, using an oxacillin salt agar with 5 μl of bacterial inoculum. A collection of cefoxitin-susceptible mecA-positive S. lugdunensis ST27 and ST42 isolates was identified, suggesting a heterogeneous resistance in the non-ST3 lineages. Comparative genomics analysis between the 60 ST3 and the 65 non-ST3 isolates revealed the ST3 clone was highly clonal and emerged with the acquisition of type V SCCmec elements (46.7% vs 9.2%, P < 0.001), together with the IS257-mobilised antimicrobial-resistant genes aacA-aphD (31.7% vs 4.6%, P < 0.001), tetK (30.0% vs 10.8%, P = 0.007), and ileS2 (13.3% vs 0.02%, P = 0.0139) on plasmids. Furthermore, two ST3 isolates, carrying one additional SasX virulence factor, and three aminoglycosides-resistant genes (aacA-aphD, aphA, and aadK) on φSPβ-like bacteriophages were identified. Statistical analysis confirmed the absence of CRISPR-Cas was positively associated with the ST3 clone and acquired resistances. However, few spacers were complementary to ST3 genomes. Spacers targeting mobile genetic elements nevertheless existed in both type II and III-A systems, indicating the CRISPR-Cas systems were actively protecting S. lugdunensis from genetic invasions, albeit insufficiently based on the plasmids and bacteriophages of the ST3 clone. SCCmec elements were characterised in 36 mecA-positive S. lugdunensis genomes and classified into ten subtypes. Eight were novel, with mosaic J1 regions. Both type IV and V were related to the community-associated methicillin-resistant Staphylococcus aureus. The 18-bp direct repeats, containing the integration site sequences (ISSs) for SCC found in multiple regions implied the high potential for integration and/or excision, which also contributed to the SCCmec mosaicism. To decolonise the methicillin-resistant staphylococci, mupirocin was introduced. However, both high-level (n = 10, plasmid ileS2) and low-level mupirocin resistance (n = 3, ileS mutation V588F) cases were identified. Worse still, of the ten ST3 isolates, six were multidrug-resistant. Strikingly, three to seven resistant loci, separated by IS257s, were found on a set of large mosaic ileS2-carrying plasmids.