Mixed-biofilm natural transformation assay reveals the presence of staphylococci in human environments that can transfer SCC <i>mec</i> to <i>Staphylococcus aureus</i>

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is an important pathogen that causes healthcare-, community-, and livestock-associated infections. The methicillin resistance gene <i>mecA</i> is embedded in the mobile genetic element termed Staphylococcal Cassette Chromosome (SCC<i>mec</i>). SCC<i>mec</i> is shared among staphylococci inhabiting human and animal hosts, which are recognized epidemiologically as the genetic reservoir of SCC<i>mec</i>. However, the ability of diverse methicillin-resistant staphylococci (MRS) to serve as SCC<i>mec</i> donors for <i>S. aureus</i> has not been tested experimentally. Here, we investigated the ability of 157 MRS isolates from pets, meat, livestock, and humans to transfer SCC<i>mec</i> to methicillin-sensitive <i>S. aureus</i> strains using a recently developed natural transformation protocol in mixed biofilms. We found that 25 out of 157 isolates were able to transfer SCC<i>mec</i> to <i>S. aureus</i>. The most effective donor species were <i>S. epidermidis</i> (~33% of the tested isolates), <i>S. felis</i> (40%), and <i>S. capitis</i> (30%). Isolates from meat and livestock (collected in Vietnam and Thailand) had lower transfer rates of SCC<i>mec</i> (5% and 3%, respectively), compared to human and pet isolates from Japan (35% and 25%, respectively). The SCC<i>mec</i> transfer depended on site-specific integration/excision mediated by an intact <i>attB</i> site, which is recognized by the SCC recombinase Ccr. Our study experimentally demonstrates the presence of SCC<i>mec</i> donors in our living environments, highlighting the importance of specific staphylococcal species.IMPORTANCEHow MRSA emerges has long been the pivotal question regarding the ever-increasing burden of antimicrobial resistance (AMR) issues for over half a century. Extensive research efforts in bacteriology, epidemiology, genome biology, and healthcare fields have led to the common understanding that SCC<i>mec</i> is transmitted among distinct staphylococcal species. However, global efforts to provide empirical evidence for intercellular SCC<i>mec</i> transmission have yielded limited results. We recently established the mixed-biofilm transformation assay to evaluate intercellular and interspecies SCC<i>mec</i> transmission. This novel assay system allows us to gain insight into the question "How MRSA emerges," and here, we provide the first experimental results about the potential donor species and habitats. This is the first report to show the ability of staphylococci from distinct sources to transfer SCC to <i>S. aureus</i>. Moreover, the new finding of <i>S. felis</i> as an effective donor that is not commensal to humans reinforces the importance of the One Health concept.