Device-associated infections involving biofilm remain a persistent clinical problem. We recently reported that four methicillin-resistant Staphylococcus aureus (MRSA) strains formed biofilm independently of the icaADBC-encoded exopolysaccharide. Here, we report that MRSA biofilm development was promoted under mildly acidic growth conditions triggered by the addition of glucose to the growth medium. Loss of sortase, which anchors LPXTG-containing proteins to peptidoglycan, reduced the MRSA biofilm phenotype. Furthermore introduction of mutations in fnbA and fnbB, which encode the LPXTG-anchored multifunctional fibrinogen and fibronectin-binding proteins, FnBPA and FnBPB, reduced biofilm formation by several MRSA strains. However, these mutations had no effect on biofilm formation by methicillin-sensitive S. aureus strains. FnBP-promoted biofilm occurred at the level of intercellular accumulation and not primary attachment. Mutation of fnbA or fnbB alone did not substantially affect biofilm, and expression of either gene alone from a complementing plasmid in fnbA fnbB mutants restored biofilm formation. FnBP-promoted biofilm was dependent on the integrity of SarA but not through effects on fnbA or fnbB transcription. Using plasmid constructs lacking regions of FnBPA to complement an fnbAB mutant revealed that the A domain alone and not the domain required for fibronectin binding could promote biofilm. Additionally, an A-domain N304A substitution that abolished fibrinogen binding did not affect biofilm. These data identify a novel S. aureus biofilm phenotype promoted by FnBPA and FnBPB which is apparently independent of the known ligandbinding activities of these multifunctional surface proteins.

Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/ PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucoseinduced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillinsensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolate's genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development.

Staphylococcus aureus clinical isolates are capable of producing at least two distinct types of biofilm mediated by the fibronectin-binding proteins (FnBPs) or the icaADBC-encoded polysaccharide intercellular adhesin (PIA). Deletion of the major autolysin gene atl reduced primary attachment rates and impaired FnBPdependent biofilm production on hydrophilic polystyrene in 12 clinical methicillin-resistant S. aureus (MRSA) isolates but had no effect on PIA-dependent biofilm production by 9 methicillin-susceptible S. aureus (MSSA) isolates. In contrast, Atl was required for both FnBP-and PIA-mediated biofilm development on hydrophobic polystyrene. Here we investigated the role of Atl in biofilm production on hydrophilic polystyrene. The alternative sigma factor B , which represses RNAIII expression and extracellular protease production, was required for FnBP-but not PIA-dependent biofilm development. Furthermore, mutation of the agr locus enhanced FnBP-dependent biofilm development, whereas a sarA mutation, which increases protease production, blocked FnBP-mediated biofilm development. Mutation of sigB in MRSA isolate BH1CC lowered primary attachment rates, in part via reduced atl transcription. Posttranslational activation or inhibition of Atl activity with phenylmethylsulfonyl fluoride and polyanethole sodium sulfonate or mutation of the Atl amidase active site interfered with lytic activity and biofilm development. Consistent with these observations, extracellular DNA was important for the early stages of Atl/FnBP-dependent biofilm development. Further analysis of atl regulation revealed that atlR encodes a transcriptional repressor of the major autolysin and that an atlR::Tc r mutation in BH1CC enhanced biofilm-forming capacity. These data reveal an essential role for the major autolysin in the early events of the FnBP-dependent S. aureus biofilm phenotype.

A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA.