Interaction between Streptococcus pneumoniae and Staphylococcus aureus Generates •OH Radicals That Rapidly Kill Staphylococcus aureus Strains

Streptococcus pneumoniae rapidly kills Staphylococcus aureus by producing membrane-permeable hydrogen peroxide (H2O2). The mechanism by which S. pneumoniae-produced H2O2 mediates S. aureus killing was investigated. An in vitro model that mimicked S. pneumoniae-S. aureus contact during colonization of the nasopharynx demonstrated that S. aureus killing required outcompeting densities of S. pneumoniae. Compared to the wild-type strain, isogenic S. pneumoniae Delta lctO and S. pneumoniae Delta spxB, both deficient in production of H2O2, required increased density to kill S. aureus. While residual H2O2 activity produced by single mutants was sufficient to eradicate S. aureus, an S. pneumoniae Delta spxB Delta lctO double mutant was unable to kill S. aureus. A collection of 20 diverse methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains showed linear sensitivity (R-2 = 0.95) for S. pneumoniae killing, but the same strains had different susceptibilities when challenged with pure H2O2 (5 mM). There was no association between the S. aureus clonal complex and sensitivity to either S. pneumoniae or H2O2. To kill S. aureus, S. pneumoniae produced similar to 180 mu M H2O2 within 4 h of incubation, while the killing-defective S. pneumoniae Delta spxB and S. pneumoniae Delta spxB Delta lctO mutants produced undetectable levels. Remarkably, a sublethal dose (1 mM) of pure H2O2 incubated with S. pneumoniae Delta spxB eradicated diverse S. aureus strains, suggesting that S. pneumoniae bacteria may facilitate conversion of H2O2 to a hydroxyl radical ((OH)-O-center dot). Accordingly, S. aureus killing was completely blocked by incubation with scavengers of (OH)-O-center dot radicals, dimethyl sulfoxide (Me2SO), thiourea, or sodium salicylate. The (OH)-O-center dot was detected in S. pneumoniae cells by spin trapping and electron paramagnetic resonance. Therefore, S. pneumoniae produces H2O2, which is rapidly converted to a more potent oxidant, hydroxyl radicals, to rapidly intoxicate S. aureus strains. IMPORTANCE Streptococcus pneumoniae strains produce hydrogen peroxide (H2O2) to kill bacteria in the upper airways, including pathogenic Staphylococcus aureus strains. The targets of S. pneumoniae-produced H2O2 have not been discovered, in part because of a lack of knowledge about the underlying molecular mechanism. We demonstrated that an increased density of S. pneumoniae kills S. aureus by means of H2O2 produced by two enzymes, SpxB and LctO. We discovered that SpxB/LctO-produced H2O2 is converted into a hydroxyl radical ((OH)-O-center dot) that rapidly intoxicates and kills S. aureus. We successfully inhibited the toxicity of OH with three different scavengers and detected (OH)-O-center dot in the supernatant. The target(s) of the hydroxyl radicals represents a new alternative for the development of antimicrobials against S. aureus infections.