Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance.

35Small colony variants (SCVs) of Staphylococcus aureus typically lack a functional electron transport chain 36 and cannot produce virulence factors such as leukocidins, hemolysins or the anti-oxidant staphyloxanthin. 37Despite this, SCVs are associated with persistent infections of the bloodstream, bones and prosthetic 38 devices. The survival of SCVs in the host has been ascribed to intracellular residency, biofilm formation and 39 resistance to antibiotics. However, the ability of SCVs to resist host defences is largely uncharacterised. To 40 address this, we measured survival of wild-type and SCV S. aureus in whole human blood, which contains 41 high numbers of neutrophils, the key defense against staphylococcal infection. Despite the loss of 42 leukcocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menquinone 43 biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in human blood 44 or the presence of purified neutrophils. Supplementation of the culture medium of the heme-auxotrophic 45 SCV with heme, but not iron, restored growth, hemolysin and staphyloxanthin production, and sensitivity 46 to the oxidative burst. Since Enterococcus faecalis is a natural heme auxotroph and cause of bloodstream 47 infection, we explored whether restoration of the electron transport chain in this organism also affected 48 survival in blood. Incubation of E. faecalis with heme increased growth and restored catalase activity, but 49 resulted in decreased survival in human blood via increased sensitivity to the oxidative burst. Therefore, 50 the lack of functional electron transport chains in SCV S. aureus and wild-type E. faecalis results in reduced 51 growth rate but provides resistance to a key immune defence mechanism. 52 53

Graphical abstract

To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection. IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

Staphylococcus aureus is a common cause of both superficial and invasive infections of humans and animals. Despite a potent host response and apparently appropriate antibiotic therapy, staphylococcal infections frequently become chronic or recurrent, demonstrating a remarkable ability of S. aureus to withstand the hostile host environment.