Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Eradication of Staphylococcus aureus Biofilm Infections Using Synthetic Antimicrobial Peptides.

Here, we demonstrate that antimicrobial peptides (AMPs) are an effective antibiofilm treatment when applied as catheter lock solutions (CLSs) against S. aureus biofilm infections. The activity of synthetic AMPs (Bac8c, HB43, P18, Omiganan, WMR, Ranalexin, and Polyphemusin) was measured against early and mature biofilms produced by methicillin-resistant S. aureus and methicillin-susceptible S. aureus isolates from patients with device-related infections grown under in vivo-relevant biofilm conditions. The cytotoxic and hemolytic activities of the AMPs against human cells and their immunomodulatory potential in human blood were also characterized. The D-Bac8c2,5Leu variant emerged as the most effective AMP during in vitro studies and was also highly effective in eradicating S. aureus biofilm infection when used in a CLS rat central venous catheter infection model. These data support the potential use of D-Bac8c2,5Leu, alone or in combination with other AMPs, in the treatment of S. aureus intravenous catheter infections.

Anti-biofilm activity of sub-inhibitory povidone-iodine concentrations against Staphylococcus epidermidis and Staphylococcus aureus.

Biomaterial-related infections continue to hamper the success of reconstructive and arthroplasty procedures in orthopaedic surgery. Staphylococci are the most common etiologic agents, with biofilm formation representing a major virulence factor. Biofilms increase bacterial resistance to antimicrobial agents and host immune responses. In staphylococci, production of polysaccharide intercellular adhesin (PIA) by the enzyme products of the icaADBC operon is the best understood mechanism of biofilm development, making the ica genes a potential target for biofilm inhibitors. In this study we report that the antibacterial agent povidone-iodine (PI) also has anti-biofilm activity against Staphylococcus epidermidis and Staphylococcus aureus at sub-inhibitory concentrations (p < 0.001). Inhibition of biofilm by PI correlated with decreased transcription of the icaADBC operon, which in turn correlated with activation of the icaR transcriptional repressor in Staphylococcus epidermidis. These data reveal an additional therapeutic benefit of PI and suggest that studies to evaluate suitability of PI as biomaterial coating agent to reduce device-related infections are merited.

Staphylococcus epidermidis polysaccharide intercellular adhesin induces IL-8 expression in human astrocytes via a mechanism involving TLR2.

Staphylococcus epidermidis is an opportunistic biofilm-forming pathogen associated with neurosurgical device-related meningitis. Expression of the polysaccharide intercellular adhesin (PIA) on its surface promotes S. epidermidis biofilm formation. Here we investigated the pro-inflammatory properties of PIA against primary and transformed human astrocytes. PIA induced IL-8 expression in a dose- and/or time-dependent manner from U373 MG cells and primary normal human astrocytes. This effect was inhibited by depletion of N-acetyl-beta-d-glucosamine polymer from the PIA preparation with Lycopersicon esculentum lectin or sodium meta-periodate. Expression of dominant-negative versions of the TLR2 and TLR4 adaptor proteins MyD88 and Mal in U373 MG cells inhibited PIA-induced IL-8 production. Blocking IL-1 had no effect. PIA failed to induce IL-8 production from HEK293 cells stably expressing TLR4. However, in U373 MG cells which express TLR2, neutralization of TLR2 impaired PIA-induced IL-8 production. In addition to IL-8, PIA also induced expression of other cytokines from U373 MG cells including IL-6 and MCP-1. These data implicate PIA as an important immunogenic component of the S. epidermidis biofilm that can regulate pro-inflammatory cytokine production from human astrocytes, in part, via TLR2.

Hydrogen peroxide induced repression of icaADBC transcription and biofilm development in Staphylococcus epidermidis.

Expression of the icaADBC-encoded polysaccharide intercellular adhesion by Staphylococcus epidermidis promotes biofilm formation and represents an important virulence factor in biomaterial-related infections following orthopedic surgery. Biofilm development by the pathogen can be viewed as a protective reaction to environmental stressors including osmotic stress, thermal stress, and antimicrobial chemotherapy. Oxidative stress, arising from the release of toxic oxygen radicals by polymorphonuclear cells, is encountered by bacteria entering the body perioperatively. Evasion of this and other cell-mediated immune responses by pathogenic bacteria plays an important role in the development of chronic biomaterial-related infection. Here we investigated the impact of sublethal oxidative stress induced by H2O2 (<18 mM) on S. epidermidis biofilm formation. S. epidermidis grown in brain heart infusion (BHI) media supplemented with 5 mM H2O2 or 10 mM H2O2 formed significantly less biofilm (p < 0.01 and p < 0.001, respectively) than bacteria grown in BHI alone. Consistent with this, using reverse transcription-polymerase chain reaction expression of the ica locus was also shown to be reduced by subinhibitory concentrations of H2O2. Furthermore, diminished ica operon expression correlated with increased expression of icaR, which encodes a repressor of icaADBC. Thus, these data suggest that mild oxidative stress downregulates biofilm development by S. epidermidis and may have potential in a therapeutic context.