Staphylococcus aureus is a major cause of prosthetic joint infection (PJI), which is characterized by biofilm formation. S. aureus biofilm skews the host immune response toward an anti-inflammatory profile by the increased recruitment of myeloid-derived suppressor cells (MDSCs) that attenuate macrophage proinflammatory activity, leading to chronic infection. A screen of the Nebraska Transposon Mutant Library identified several hits in the ATP synthase operon that elicited a heightened inflammatory response in macrophages and MDSCs, including atpA, which encodes the alpha subunit of ATP synthase. An atpA transposon mutant (ΔatpA) had altered growth kinetics under both planktonic and biofilm conditions, along with a diffuse biofilm architecture that was permissive for leukocyte infiltration, as observed by confocal laser scanning microscopy. Coculture of MDSCs and macrophages with ΔatpA biofilm elicited significant increases in the proinflammatory cytokines interleukin 12p70 (IL-12p70), tumor necrosis factor alpha (TNF-α), and IL-6. This was attributed to increased leukocyte survival resulting from less toxin and protease production by ΔatpA biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by ΔatpA biofilm was cell lysis-dependent since it was negated by polyanethole sodium sulfanate treatment or deletion of the major autolysin, Atl. In a mouse model of PJI, ΔatpA-infected mice had decreased MDSCs concomitant with increased monocyte/macrophage infiltrates and proinflammatory cytokine production, which resulted in biofilm clearance. These studies identify S. aureus ATP synthase as an important factor in influencing the immune response during biofilm-associated infection and bacterial persistence. IMPORTANCE Medical device-associated biofilm infections are a therapeutic challenge based on their antibiotic tolerance and ability to evade immune-mediated clearance. The virulence determinants responsible for bacterial biofilm to induce a maladaptive immune response remain largely unknown. This study identified a critical role for S. aureus ATP synthase in influencing the host immune response to biofilm infection. An S. aureus ATP synthase alpha subunit mutant (ΔatpA) elicited heightened proinflammatory cytokine production by leukocytes in vitro and in vivo, which coincided with improved biofilm clearance in a mouse model of prosthetic joint infection. The ability of S. aureus ΔatpA to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a ΔatpAΔatl biofilm did not elicit heightened cytokine production. These studies reveal a critical role for AtpA in shaping the host immune response to S. aureus biofilm.

Biofilms are bacterial communities characterized by antibiotic tolerance. Staphylococcus aureus is a leading cause of biofilm infections on medical devices, including prosthetic joints, which represent a significant health care burden. The major leukocyte infiltrate associated with S. aureus prosthetic joint infection (PJI) is granulocytic myeloid-derived suppressor cells (G-MDSCs), which produce IL-10 to promote biofilm persistence by inhibiting monocyte and macrophage proinflammatory activity.

According to the NIH, 80% of chronic infections are caused by biofilm‐associated bacteria. Biofilm communities contribute to antimicrobic resistance by providing a physical extracellular barrier and exhibit metabolic diversity within bacterial subpopulations. Clinically, combination antibiotic strategies are used to provide high dosage treatments to successfully combat biofilm‐associated infections. Microscopically, biofilms of Pseudomonas aeruginosa have been visualized as mushroom‐like structures with two distinct subpopulations: a high‐metabolic “mushroom cap” outer population and a low‐metabolic “stalk” inner population. Further, the inner subpopulation consists of persister cells that are not killed by many therapies and contribute to chronicity of biofilm‐associated infections. It has been proposed that complete eradication of biofilm is achieved by targeting specific biofilm populations with antimicrobic combination of colistin and ciprofloxacin that target inner and outer subpopulations of biofilm respectively. However, colistin is sparingly used clinically because of high renal toxicity. Use of phytochemical alternatives instead of antibiotics is under assessment for treatment of biofilm‐associated infections. Extract from Azadirachta indica, or neem tree, is reported to have antimicrobic and antibiofilm effects on P. aeruginosa with low human toxicity. For these reasons, P. aeruginosa was grown (24 h) and microplates washed to remove non‐adherent cells. Remaining biofilm and cells were treated overnight with neem, colistin, ciprofloxacin or dual combinations. Finally, biofilm was removed from the substratum by scraping and aspiration, and bacterial viability was determined by colony forming unit (CFU/mL) enumeration. Results indicated that neem and ciprofloxacin in combination had an inhibitory effect (≤103 CFU/ml) similar to colistin and ciprofloxacin combination (≤102 CFU/ml) compared to growth and vehicle controls (both of which were 1012 CFU/ml). Colistin and neem combination showed no combined effect (107 CFU/ml) different from single treatment effects (108 and 1010 CFU/ml, respectively). These results suggest that neem extract targets the inner, low‐metabolic subpopulation of bacteria in biofilms and, for this reason, neem has potential to be a non‐toxic alternative to colistin for use therapeutically. When colistin or neem (that target the inner, low‐metabolic bacterial subpopulation) are used in combination with ciprofloxacin (that targets the outer, high‐metabolic subpopulation), the entirety of the biofilm may be targeted and prevent chronicity in biofilm‐associated infections.Support or Funding InformationFinancial support for BPB was received from the DeNardo Education and Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.