Genetic engineering of Staphylococcus haemolyticus: overcoming restriction-modification barriers and targeting virulence genes.

Staphylococcus haemolyticus is an emerging multidrug-resistant nosocomial pathogen noted for robust biofilm formation and complex restriction-modification (RM) systems that hinder genetic manipulation. These barriers have severely limited mechanistic studies into its pathogenesis and immune evasion. Here, we report the development of a molecular toolbox that enables precise genomic engineering of clinical S. haemolyticus isolates. Using PacBio Single-Molecule Real-Time and bisulfite sequencing, we defined the complete genomes and methylomes of nine isolates, generating a functional readout of the active RM defences present in each strain. Among the RM systems identified, a Type II (PDLC03279) and a Type III (PDL3649/PDLC03643) system were significantly overrepresented in clinical isolates, suggesting a potential role in adaptation to host or hospital-associated environments. To bypass these RM barriers, we implemented a dual strategy: first, applying SyngenicDNA-based approaches to eliminate RM target motifs from genetic tools and second, engineering a surrogate Escherichia coli strain (JMC4) to mimic conserved S. haemolyticus methylation patterns. These tools significantly enhanced transformation efficiency and enabled targeted knockout of four putative virulence genes (sraP, secA2, capA and capI) as well as allelic exchange of the native capsule operon with the corresponding region from a non-encapsulated isolate. To our knowledge, this is the first report of precise genomic modifications in S. haemolyticus. The establishment of robust molecular tools for transformation and genome editing lays a foundation for future functional studies of virulence and host adaptation in this resilient opportunistic pathogen.