XerC Is Required for the Repair of Antibiotic- and Immune-Mediated DNA Damage in Staphylococcus aureus.

To survive in the host environment, pathogenic bacteria need to be able to repair DNA damage caused by both antibiotics and the immune system. The SOS response is a key bacterial pathway to repair DNA double-strand breaks and may therefore be a good target for novel therapeutics to sensitize bacteria to antibiotics and the immune response. However, the genes required for the SOS response in Staphylococcus aureus have not been fully established. Therefore, we carried out a screen of mutants involved in various DNA repair pathways to understand which were required for induction of the SOS response. This led to the identification of 16 genes that may play a role in SOS response induction and, of these, 3 that affected the susceptibility of S. aureus to ciprofloxacin. Further characterization revealed that, in addition to ciprofloxacin, loss of the tyrosine recombinase XerC increased the susceptibility of S. aureus to various classes of antibiotics, as well as to host immune defenses. Therefore, the inhibition of XerC may be a viable therapeutic approach to sensitize S. aureus to both antibiotics and the immune response.

Growth Arrest of Staphylococcus aureus Induces Daptomycin Tolerance via Cell Wall Remodelling.

Almost all bactericidal drugs require bacterial replication and/or metabolic activity for their killing activity. When these processes are inhibited by bacteriostatic antibiotics, bacterial killing is significantly reduced. One notable exception is the lipopeptide antibiotic daptomycin, which has been reported to efficiently kill growth-arrested bacteria. However, these studies employed only short periods of growth arrest (<1 h), which may not fully represent the duration of growth arrest that can occur in vivo. We found that a growth inhibitory concentration of the protein synthesis inhibitor tetracycline led to a time-dependent induction of daptomycin tolerance in S. aureus, with an approximately 100,000-fold increase in survival after 16 h of growth arrest, relative to exponential-phase bacteria. Daptomycin tolerance required glucose and was associated with increased production of the cell wall polymers peptidoglycan and wall-teichoic acids. However, while the accumulation of peptidoglycan was required for daptomycin tolerance, only a low abundance of wall teichoic acid was necessary. Therefore, whereas tolerance to most antibiotics occurs passively due to a lack of metabolic activity and/or replication, daptomycin tolerance arises via active cell wall remodelling. IMPORTANCE Understanding why antibiotics sometimes fail to cure infections is fundamental to improving treatment outcomes. This is a major challenge when it comes to Staphylococcus aureus because this pathogen causes several different chronic or recurrent infections. Previous work has shown that a lack of replication, as often occurs during infection, makes bacteria tolerant of most bactericidal antibiotics. However, one antibiotic that has been reported to kill nonreplicating bacteria is daptomycin. In this work, we show that the growth arrest of S. aureus does in fact lead to daptomycin tolerance, but it requires time, nutrients, and biosynthetic pathways, making it distinct from other types of antibiotic tolerance that occur in nonreplicating bacteria.

Human serum triggers antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus frequently causes infections that are challenging to treat, leading to high rates of persistent and relapsing infection. Here, to understand how the host environment influences treatment outcomes, we study the impact of human serum on staphylococcal antibiotic susceptibility. We show that serum triggers a high degree of tolerance to the lipopeptide antibiotic daptomycin and several other classes of antibiotic. Serum-induced daptomycin tolerance is due to two independent mechanisms. Firstly, the host defence peptide LL-37 induces tolerance by triggering the staphylococcal GraRS two-component system, leading to increased peptidoglycan accumulation. Secondly, GraRS-independent increases in membrane cardiolipin abundance are required for full tolerance. When both mechanisms are blocked, S. aureus incubated in serum is as susceptible to daptomycin as when grown in laboratory media. Our work demonstrates that host factors can significantly modulate antibiotic susceptibility via diverse mechanisms, and combination therapy may provide a way to mitigate this.

Polymyxin and lipopeptide antibiotics: membrane-targeting drugs of last resort.

The polymyxin and lipopeptide classes of antibiotics are membrane-targeting drugs of last resort used to treat infections caused by multi-drug-resistant pathogens. Despite similar structures, these two antibiotic classes have distinct modes of action and clinical uses. The polymyxins target lipopolysaccharide in the membranes of most Gram-negative species and are often used to treat infections caused by carbapenem-resistant species such as Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa. By contrast, the lipopeptide daptomycin requires membrane phosphatidylglycerol for activity and is only used to treat infections caused by drug-resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. However, despite having distinct targets, both antibiotic classes cause membrane disruption, are potently bactericidal in vitro and share similarities in resistance mechanisms. Furthermore, there are concerns about the efficacy of these antibiotics, and there is increasing interest in using both polymyxins and daptomycin in combination therapies to improve patient outcomes. In this review article, we will explore what is known about these distinct but structurally similar classes of antibiotics, discuss recent advances in the field and highlight remaining gaps in our knowledge.

Human serum induces daptomycin tolerance in Enterococcus faecalis and viridans group streptococci.

Daptomycin is a membrane-targeting lipopeptide antibiotic used in the treatment of infective endocarditis caused by multidrug-resistant Gram-positive bacteria such as Staphylococcus aureus, enterococci and viridans group streptococci. Despite demonstrating excellent in vitro activity and a low prevalence of resistant isolates, treatment failure is a significant concern, particularly for enterococcal infection. We have shown recently that human serum triggers daptomycin tolerance in S. aureus, but it was not clear if a similar phenotype occurred in other major infective endocarditis pathogens. We found that Enterococcus faecalis, Streptococcus gordonii or Streptococcus mutans grown under standard laboratory conditions were efficiently killed by daptomycin, whereas bacteria pre-incubated in human serum survived exposure to the antibiotic, with >99 % cells remaining viable. Incubation of enterococci or streptococci in serum led to peptidoglycan accumulation, as shown by increased incorporation of the fluorescent d-amino acid analogue HADA. Inhibition of peptidoglycan accumulation using the antibiotic fosfomycin resulted in a >tenfold reduction in serum-induced daptomycin tolerance, demonstrating the important contribution of the cell wall to the phenotype. We also identified a small contribution to daptomycin tolerance in E. faecalis from cardiolipin synthases, although this may reflect the inherent increased susceptibility of cardiolipin-deficient mutants. In summary, serum-induced daptomycin tolerance is a consistent phenomenon between Gram-positive infective endocarditis pathogens, but it may be mitigated using currently available antibiotic combination therapy.

A FASII Inhibitor Prevents Staphylococcal Evasion of Daptomycin by Inhibiting Phospholipid Decoy Production.

Daptomycin is a treatment of last resort for serious infections caused by drug-resistant Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus We have shown recently that S. aureus can evade daptomycin by releasing phospholipid decoys that sequester and inactivate the antibiotic, leading to treatment failure. Since phospholipid release occurs via an active process, we hypothesized that it could be inhibited, thereby increasing daptomycin efficacy. To identify opportunities for therapeutic interventions that block phospholipid release, we first determined how the host environment influences the release of phospholipids and the inactivation of daptomycin by S. aureus The addition of certain host-associated fatty acids to the growth medium enhanced phospholipid release. However, in serum, the sequestration of fatty acids by albumin restricted their availability to S. aureus sufficiently to prevent their use in the generation of released phospholipids. This finding implies that in host tissues S. aureus may be completely dependent upon endogenous phospholipid biosynthesis to generate lipids for release, providing a target for therapeutic intervention. To test this, we exposed S. aureus to AFN-1252, an inhibitor of the staphylococcal FASII fatty acid biosynthetic pathway, together with daptomycin. AFN-1252 efficiently blocked daptomycin-induced phospholipid decoy production, even in the case of isolates resistant to AFN-1252, which prevented the inactivation of daptomycin and resulted in sustained bacterial killing. In turn, daptomycin prevented the fatty acid-dependent emergence of AFN-1252-resistant isolates in vitro In summary, AFN-1252 significantly enhances daptomycin activity against S. aureusin vitro by blocking the production of phospholipid decoys, while daptomycin blocks the emergence of resistance to AFN-1252.

Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids.

Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We showed previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipids in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.