Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

Clarissa Pozzi,Amanda J Lohan,Pin Tong,Carolyn R Schaeffer,Brendan J Loftus,James P O'Gara,Justine K Rudkin,Ruth C Massey,Gerald B Pier,Paul D Fey,Elaine M Waters,Paul M Sullam

doi:10.1371/journal.ppat.1002626

Abstract

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.

Highlights

Infections caused by healthcare-associated Staphylococcus aureus and methicillin resistant S. aureus (MRSA) pose a major threat to hospital patients
Using clinical isolates of S. aureus, we reported that methicillin resistant S. aureus (MRSA) strains express an icaADBC-independent biofilm phenotype in vitro [3,4], which is instead dependent on the fibronectin binding proteins (FnBPA and FnBPB) and the major autolysin (Atl) [6,7]
The adaptation of MRSA to the hospital environment has apparently focused on the acquisition of antibiotic resistance and retention of biofilm forming capacity, which are likely to be more advantageous than metabolically-expensive enzyme and toxin production in immunocompromised patients with implanted medical devices offering a route to infection

Summary

Introduction

Infections caused by healthcare-associated Staphylococcus aureus and methicillin resistant S. aureus (MRSA) pose a major threat to hospital patients. Production of the exopolysaccharide polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG) synthesized and exported by proteins encoded by the icaADBC genes is common among clinical isolates of both species [2,3,4,5,6], ica-independent biofilm production has been described under in vitro conditions [1]. Using clinical isolates of S. aureus, we reported that methicillin resistant S. aureus (MRSA) strains express an icaADBC-independent biofilm phenotype in vitro [3,4], which is instead dependent on the fibronectin binding proteins (FnBPA and FnBPB) and the major autolysin (Atl) [6,7].

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