Staphylococcus aureus ( S. aureus ) is a frequent culprit in implant-associated infections and employs many virulence factors to escape killing by the host immune system. The specific immune evasion strategies used by small aggregates of S. aureus on a surface, precursors to mature biofilm, are still relatively unknown. Time-lapse confocal microscopy was leveraged to quantify interactions between S. aureus aggregates and human neutrophils in vitro and identify specific mechanisms of resistance to neutrophil killing. Surface-associated wild-type S. aureus rapidly formed small biofilm aggregates when grown in human serum. Conversely, aggregation was inhibited when the SaeR/S two-component gene regulatory system was deleted. Wild-type aggregates began to show individual and population-level resistance to neutrophil killing upon reaching sizes of approximately 50 to 75 µm ² , whereas Δ sae clusters failed to reach these sizes and were readily cleared. Aggregation of Δ sae strains was impaired by serum complement, and this inhibition required complement proteins C3 and factor B, but not C4 or C5, suggesting that this activity primarily occurs at the level of the alternative pathway. Several complement-inhibiting genes regulated by SaeR/S were identified that collectively facilitate biofilm aggregate formation in human, but not murine serum. Finally, aggregation of two related opportunistic pathogens, Staphylococcus epidermidis and Enterococcus faecalis , was inhibited by serum. These data demonstrate a function of serum complement, the ability to inhibit bacterial aggregation, that is potently blocked by S. aureus through the production of multiple complement-interfering proteins that are regulated by the SaeR/S system.

The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18β-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D ¹H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell–cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics.

Staphylococcus aureus (S. aureus) is the leading cause of surgical site infections (SSIs) and is capable of biofilm growth on implanted foreign devices. The use of surgical irrigation solutions has become a common strategy to combat bacterial contamination events that occur during surgery. Despite their antimicrobial activity, SSI rates remain consistent, suggesting that low-level contamination persists. In these cases, circulating neutrophils must traffic from the blood to contamination sites to aid in bacterial clearance. The influence of irrigation solutions on neutrophils’ ability to engage with bacteria has not been explored. The effects of three commonly used irrigation solutions: Xperience (sodium lauryl sulfate), Irrisept (chlorhexidine gluconate), and Betadine^® (povidone-iodine) on nascent S. aureus biofilms alone and in the presence of human neutrophils were assessed at manufactured and diluted concentrations. All three solutions, at a 10% dilution, inhibited bacterial growth as demonstrated by culture assays and confocal video microscopy of bacterial aggregate formation. The effects of 10% dilutions of each of these solutions on neutrophil membrane integrity (by flow cytometry and propidium iodide staining) and motility (by confocal video microscopy of neutrophil track length) were investigated with differing outcomes for each irrigation solution. At this concentration only Irrisept preserved neutrophil membrane integrity and motility. Together, this study examines an overlooked aspect of surgical irrigation solutions by investigating their impact on innate immunity and highlights the feasibility of formulations wherein solution effectiveness is complemented by neutrophil function to reduce risks of infection.

Staphylococcus aureus (S. aureus) is a prominent Gram-positive bacterial pathogen that expresses numerous cytotoxins known to target human polymorphonuclear leukocytes (PMNs or neutrophils). These include leukocidin G/H (LukGH, also known as LukAB), the Panton–Valentine leukocidin (PVL), γ-hemolysin A/B (HlgAB), γ-hemolysin B/C (HlgBC), leukocidin E/D (LukED), α-hemolysin (Hla), and the phenol-soluble modulin-α peptides (PSMα). However, the relative contribution of each of these cytotoxins in causing human PMN lysis is not clear. In this study, we used a library of cytotoxin deletion mutants in the clinically relevant methicillin-resistant S. aureus (MRSA) isolate LAC (strain ST8:USA300) to determine the relative importance of each for causing human PMN lysis upon exposure to extracellular components as well as following phagocytosis. Using flow cytometry to examine plasma membrane permeability and assays quantifying lactose dehydrogenase release, we found that PVL was the dominant extracellular factor causing human PMN lysis produced by USA300. In contrast, LukGH was the most important cytotoxin causing human PMN lysis immediately following phagocytosis with contributions from the other bicomponent leukocidins only observed at later time points. These results not only clarify the relative importance of different USA300 cytotoxins for causing human PMN destruction but also demonstrate how two apparently redundant virulence factors play distinctive roles in promoting S. aureus pathogenesis.

Influenza infections typically present mild to moderate morbidities in immunocompetent host and are often resolved within 14 days of infection onset. Death from influenza infection alone is uncommon; however, antecedent influenza infection often leads to an increased susceptibility to secondary bacterial pneumonia. Bacterial pneumonia following viral infection exhibits mortality rates greater than 10-fold of those of influenza alone. Furthermore, bacterial pneumonia has been identified as the major contributor to mortality during each of the previous four influenza pandemics. Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pyogenes are the most prevalent participants in this pathology. Of note, these lung pathogens are frequently found as commensals of the upper respiratory tract. Herein we describe influenza-induced host-changes that lead to increased susceptibility to bacterial pneumonia, review virulence strategies employed by the most prevalent secondary bacterial pneumonia species, and highlight recent findings of bacterial sensing and responding to the influenza infected environment.