Insight in methicillin-resistant Staphylococcus aureus (MRSA) biofilms: identification of key determinants in biofilm formation of highly pathogenic and globally successful MRSA clones
16 mei 2019
UAntwerp - Campus Drie Eiken - Building O - Auditorium O5 - Universiteitsplein 1 - 2610 WILRIJK (route: UAntwerpen, Campus Drie Eiken
Sarah De Backer
Prof S. Malhotra & Prof H. Goossens
PhD defence Sarah De Backer - Faculty of Medicine and Health Sciences (Presentation in English)
In addition to multidrug resistance (methicillin-resistant Staphylococcus aureus, MRSA) and presence of virulence-specific factors, the success of S. aureus is also attributable to prolonged persistence of infections linked to the formation of sessile matrix-encased microcolonies termed biofilms. These biofilms are notoriously difficult to eradicate, with sessile populations being up to 1000-fold more resistant than their planktonic counterparts. Despite the important role of S. aureus biofilms in disease, our comprehension of the molecular mechanisms promoting biofilm formation is incomplete. Recent studies of S. aureus biofilms hint that the extracellular matrix consists of proteins, DNA and/or polysaccharide (polysaccharide intercellular adhesin, PIA/PNAG). However, it has become evident that clinical MRSA isolates are not only dependent on PIA/PNAG for biofilm formation. Protein-mediated biofilm formation has emerged as another option to PIA/PNAG, and many surface adhesins, such as Bap, Spa, FnBPA, FnBPB, and SasG have been implicated in this divergent biofilm mechanism.
We aim to dissect the fundamental aspects underlying the pathogenic success of S. aureus/MRSA. Three highly pathogenic, globally successful and prolific biofilm forming MRSA clones, USA300-UAS391, EMRSA-15 H-EMRSA-15 and ST239-SCCmecIII EU_ST239_16, were used as a model to look for commonalities in global MRSA biofilm formation. MRSA transcriptional signatures associated with in vitro planktonic and biofilm phenotypes were identified by means of differential gene expression analysis. Additionally, in order not to overlook other important genes with non-significant up-regulation in the transcriptome analysis, screening of a high-throughput USA300-UAS391 transposon library revealed a large number of mutants displaying changes in biofilm formation capacity. Interesting ‘hits’ were functionally validated for biofilm formation in both static and shear flow in vitro biofilm assays. The relevance of these mutations was also further confirmed by transduction experiments to look for similar phenotypic effects and complementation to demonstrate restoration of the wild type phenotype when a functional protein was present. As such, fatty acid kinase A (fakA), intermediates of the TCA-cycle (fumC, sdhA and sdhB), and staphylococcal surface protein X (sasX) were marked as important determinants contributing to the pathogenic success of MRSA.
The delineation of factors that define virulence in the entire range of infectious MRSA strains will permit the rational design of anti-biofilm drugs that can be combined with conventional antibiotics, thus restoring efficacy the latter show to staphylococci in a non-biofilm status. Interestingly, this research opens up the possibility of a single target affecting two key aspects of MRSA pathogenesis; β-lactam resistance and biofilm formation.