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Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is a frontline antibiotic for treating methicillin-resistant Staphylococcus aureus and other Gram-positive bacterial infections. However, vancomycin use has led to the increasing prevalence of bacterial strains with reduced susceptibility to vancomycin. Here, we show that unsaturated fatty acids act as potent vancomycin adjuvants to rapidly kill a range of Gram-positive bacteria, including vancomycin-tolerant and resistant populations. The synergistic bactericidal activity relies on the accumulation of membrane-bound cell wall intermediates that generate large fluid patches in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. Our findings provide a natural therapeutic option that enhances vancomycin activity against difficult-to-treat pathogens, and the underlying mechanism may be further exploited to develop antimicrobials that target recalcitrant infection.

The blood-clotting protein fibrin(ogen) plays a critical role in host defense against invading pathogens, particularly against peritoneal infection by the Gram-positive microbe Staphylococcus aureus. Here, we tested the hypothesis that direct binding between fibrin(ogen) and S. aureus is a component of the primary host antimicrobial response mechanism and prevention of secondary microbe dissemination from the peritoneal cavity. To establish a model system, we showed that fibrinogen isolated from FibγΔ5 mice, which express a mutant form lacking the final 5 amino acids of the fibrinogen γ chain (termed fibrinogenγΔ5), did not support S. aureus adherence when immobilized and clumping when in suspension. In contrast, purified wildtype fibrinogen supported robust adhesion and clumping that was largely dependent on S. aureus expression of the receptor clumping factor A (ClfA). Following peritoneal infection with S. aureus USA300, FibγΔ5 mice displayed worse survival compared to WT mice coupled to reduced bacterial killing within the peritoneal cavity and increased dissemination of the microbes into circulation and distant organs. The failure of acute bacterial killing, but not enhanced dissemination, was partially recapitulated by mice infected with S. aureus USA300 lacking ClfA. Fibrin polymer formation and coagulation transglutaminase Factor XIII each contributed to killing of the microbes within the peritoneal cavity, but only elimination of polymer formation enhanced systemic dissemination. Host macrophage depletion or selective elimination of the fibrin(ogen) β2-integrin binding motif both compromised local bacterial killing and enhanced S. aureus systemic dissemination, suggesting fibrin polymer formation in and of itself was not sufficient to retain S. aureus within the peritoneal cavity. Collectively, these findings suggest that following peritoneal infection, the binding of S. aureus to stabilized fibrin matrices promotes a local, macrophage-mediated antimicrobial response essential for prevention of microbe dissemination and downstream host mortality.

ABSTRACT Interactions between Staphylococcus aureus and the host immune system can have significant impacts on antibiotic efficacy, suggesting that targeting and modulating the immune response to S. aureus infection may improve antibiotic efficacy and improve infection outcome. As we’ve previously shown, high levels of reactive oxygen species (ROS), associated with an M1-like proinflammatory macrophage response, potently induce antibiotic tolerance in S. aureus. Although the proinflammatory immune response is critical for initial control of pathogen burden, recent studies demonstrate that modulation of the macrophage response to an anti-inflammatory, or M2-like, response facilitates resolution of established S. aureus skin and soft tissue infections, arthritis, and bacteremia. Here, we evaluated the impact of host-directed immunosuppressive chemotherapeutics and anti-inflammatory agents on antibiotic efficacy against S. aureus. IMPORTANCE Staphylococcus aureus is the leading cause of hospital-acquired infections in the United States with high rates of antibiotic treatment failure. Macrophages represent an important intracellular niche in experimental models of S. aureus bacteremia. Although a proinflammatory macrophage response is critical for controlling infection, previous studies have identified an antagonistic relationship between antibiotic treatment and the proinflammatory macrophage response. Reactive oxygen species, produced by macrophages during respiratory burst, coerce S. aureus into an antibiotic tolerant state, leading to poor treatment outcome. Here, we aimed to determine the potential of host-directed immunomodulators that reduce the production of reactive oxygen species to improve antibiotic efficacy against intracellular S. aureus.

Summary: Staphylococcus aureus is a leading human pathogen that frequently causes relapsing infections. The failure of antibiotics to eradicate infection contributes to infection relapse. Host-pathogen interactions have a substantial impact on antibiotic susceptibility and the formation of antibiotic tolerant cells. In this study, we interrogate how a major S. aureus virulence factor, α-toxin, interacts with macrophages to alter the microenvironment of the pathogen, thereby influencing its susceptibility to antibiotics. We find α-toxin-mediated activation of the NLRP3 inflammasome induces antibiotic tolerance. Induction of tolerance is driven by increased glycolysis in the host cells, resulting in glucose limitation and ATP depletion in S. aureus. Additionally, inhibition of NLRP3 activation improves antibiotic efficacy in vitro and in vivo, suggesting that this strategy has potential as a host-directed therapeutic to improve outcomes. Our findings identify interactions between S. aureus and the host that result in metabolic crosstalk that can determine the outcome of antimicrobial therapy.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is highly prevalent in U.S. cystic fibrosis (CF) patients and is associated with worse clinical outcomes in CF. These infections often become chronic despite repeated antibiotic therapy. Here, we assessed whether bacterial phenotypes, including antibiotic tolerance, can predict the clinical outcomes of MRSA infections. MRSA isolates (n = 90) collected at the incident (i.e., acute) and early infection states from 57 patients were characterized for growth rates, biofilm formation, hemolysis, pigmentation, and vancomycin tolerance. The resistance profiles were consistent with those in prior studies. Isolates from the early stage of infection were found to produce biofilms, and 70% of the isolates exhibited delta-hemolysis, an indicator of agr activity. Strong vancomycin tolerance was present in 24% of the isolates but was not associated with intermediate vancomycin susceptibility. There were no associations between these phenotypic measures, antibiotic tolerance, and MRSA clearance. Our research suggests that additional factors may be relevant for predicting the clearance of MRSA. IMPORTANCE Chronic MRSA infections remain challenging to treat in patients with cystic fibrosis (CF). The ability of the bacterial population to survive high concentrations of bactericidal antibiotics, including vancomycin, despite lacking resistance is considered one of the main reasons for treatment failures. The connection between antibiotic tolerance and treatment outcomes remains unexplored and can be crucial for prognosis and regimen design toward eradication. In this study, we measured the capacity of 90 MRSA isolates from CF patients to form vancomycin-tolerant persister cells and evaluated their correlation with the clinical outcomes. Additionally, various traits that could reflect the metabolism and/or virulence of those MRSA isolates were systematically phenotyped and included for their predictive power. Our research highlights that despite the importance of antibiotic tolerance, additional factors need to be considered for predicting the clearance of MRSA.

Hydrogen peroxide, povidone-iodine, and chlorhexidine are antiseptics that are commonly added to irrigants to either prevent or treat infection. There are little clinical data available that demonstrate efficacy of adding antiseptics to irrigants in the treatment of periprosthetic joint infection after biofilm establishment. The objective of the study was to assess the bactericidal activity of the antiseptics on S. aureus planktonic and biofilm. For planktonic irrigation, S. aureus was exposed to different concentrations of antiseptics. S. aureus biofilm was developed by submerging a Kirschner wire into normalized bacteria and allowing it to grow for forty-eight hours. The Kirschner wire was then treated with irrigation solutions and plated for CFU analysis. Hydrogen peroxide, povidone-iodine, and chlorhexidine were bactericidal against planktonic bacteria with over a 3 log reduction (p < 0.0001). Unlike cefazolin, the antiseptics were not bactericidal (less than 3 log reduction) against biofilm bacteria but did have a statistical reduction in biofilm as compared to the initial time point (p < 0.0001). As compared to cefazolin treatment alone, the addition of hydrogen peroxide or povidone-iodine to cefazolin treatment only additionally reduced the biofilm burden by less than 1 log. The antiseptics demonstrated bactericidal properties with planktonic S. aureus; however, when used to irrigate S. aureus biofilms, these antiseptics were unable to decrease biofilm mass below a 3 log reduction, suggesting that S. aureus biofilm has a tolerance to antiseptics. This information should be considered when considering antibiotic tolerance in established S. aureus biofilm treatment.

Bacterial biofilms, often associated with chronic infections, respond poorly to antibiotic therapy and frequently require surgical intervention. Biofilms harbor persister cells, metabolically indolent cells, which are tolerant to most conventional antibiotics. In addition, the biofilm matrix can act as a physical barrier, impeding diffusion of antibiotics. Novel therapeutic approaches frequently improve biofilm killing, but usually fail to achieve eradication. Failure to eradicate the biofilm leads to chronic and relapsing infection, is associated with major financial healthcare costs and significant morbidity and mortality. We address this problem with a two-pronged strategy using 1) antibiotics that target persister cells and 2) ultrasound-stimulated phase-change contrast agents (US-PCCA), which improve antibiotic penetration.We previously demonstrated that rhamnolipids, produced by Pseudomonas aeruginosa, could induce aminoglycoside uptake in gram-positive organisms, leading to persister cell death. We have also shown that US-PCCA can transiently disrupt biological barriers to improve penetration of therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could improve treatment of methicillin resistant S. aureus (MRSA) biofilms. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically improved biofilm killing. This novel treatment strategy has the potential for rapid clinical translation as the PCCA formulation is a variant of FDA-approved ultrasound contrast agents that are already in clinical practice and the low-pressure ultrasound settings used in our study can be achieved with existing ultrasound hardware at pressures below the FDA set limits for diagnostic imaging.