Biology subjects

Ledger, E. V.

Publications and source records attributed to Ledger, E. V..

6 recordsLinked to original sources

bioRxiv2026

Human serum triglycerides promote Staphylococcus aureus biofilm formation and antibiotic tolerance

Biofilm formation and antibiotic tolerance are major contributors to the persistence of Staphylococcus aureus infections, yet how the host environment affects these phenotypes remains poorly understood. Here, we show that incubation in human serum primes S. aureus to form robust biofilms and tolerate vancomycin and daptomycin, last resort antibiotics for the treatment of antibiotic-resistant staphylococcal infections. Mechanistically, we demonstrate that the staphylococcal Geh lipase is essential for serum-induced biofilm formation by liberating glycerol from host lipids, which is then used to promote increased synthesis of D-alanylated wall teichoic acids, driving biofilm development. Inhibition of the Geh lipase or wall teichoic acid synthesis markedly reduces biofilm formation and restores antibiotic susceptibility, highlighting clinically achievable strategies to inhibit host-induced biofilm formation and prevent the associated antibiotic tolerance. Together, our findings reveal a host-driven mechanism of biofilm-associated antibiotic tolerance in S. aureus and provide rational targets for therapeutic intervention.

microbiology↗

bioRxiv2025

Expression of mecA reduces the daptomycin susceptibility of Staphylococcus aureus

Staphylococcus aureus is a leading cause of bacteraemia and infections caused by methicillin-resistant strains (MRSA) are especially challenging to treat. MRSA strains are resistant to front-line beta-lactams due to PBP2a, a low-affinity penicillin-binding protein encoded by mecA. Daptomycin is used to treat MRSA infections but is not always effective and is associated with high rates of morbidity and mortality. Understanding why daptomycin fails is crucial to devising strategies to improve treatment outcomes. Here, using a panel of clinical bacteraemia isolates, we show that MRSA strains are less susceptible to daptomycin than methicillin-susceptible (MSSA) strains. This difference in susceptibility was due to mecA and was independent of any changes in surface properties previously associated with altered daptomycin susceptibility. Instead, using a mecA transposon mutant, we found that a lack of this gene led to higher activity of the Agr quorum sensing system, resulting in an increased release of the phenol-soluble modulin toxins. Increased levels of these surfactant-like toxins prevented daptomycin from being inactivated by lipids released by the bacteria, leading to enhanced susceptibility to the antibiotic. Additionally, the clinical MRSA strains tested produced lower levels of toxins than the MSSA strains and inactivated daptomycin to a greater extent, explaining their reduced susceptibility. Expression of mecA in clinical MSSA strains reduced toxin production, increasing daptomycin inactivation and thereby enhancing survival. Together, these results demonstrate that mecA does not only affect beta-lactam susceptibility but also compromises the efficacy of the last resort antibiotic daptomycin.

microbiology↗

bioRxiv2022

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 analogue HADA. Inhibition of peptidoglycan accumulation using the antibiotic fosfomycin resulted in a >10-fold 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 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.

microbiology↗

bioRxiv2022

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 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 sensitise 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 susceptibility of S. aureus to ciprofloxacin. Further characterisation 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 defences. Therefore, the inhibition of XerC may be a viable therapeutic approach to sensitise S. aureus to both antibiotics and the immune response.

microbiology↗

bioRxiv2021

Human serum triggers antibiotic tolerance in Staphylococcus aureus

Staphylococcus aureus is a frequent cause of bloodstream infections. Treatment can be challenging, even when isolates appear to be drug susceptible, with high rates of persistent and relapsing infection. This is particularly the case with infections caused by methicillin resistant S. aureus (MRSA) strains, which are resistant to frontline antibiotics. To understand how the host environment influences treatment outcomes in MRSA infections, we studied the impact of human serum on staphylococcal susceptibility to daptomycin, an antibiotic of last resort. This revealed that serum triggered a very high degree of tolerance to daptomycin, as well as several other classes of antibiotics and antimicrobial peptides, including gentamicin, nitrofurantoin, vancomycin, nisin and gramicidin. Serum-induced daptomycin tolerance was due to two independent mechanisms. Firstly, the host defence peptide LL-37 present in serum induced tolerance by triggering the staphylococcal GraRS two component system. This led to increased cell wall accumulation that reduced access of daptomycin to its membrane target. Secondly, GraRS-independent changes to the membrane resulted in increased cardiolipin abundance that also contributed to daptomycin tolerance. When both mechanisms were blocked, serum exposed S. aureus cells were as susceptible to daptomycin as bacteria growing in laboratory media. These data demonstrate that host factors can significantly modulate antibiotic susceptibility via diverse mechanisms, which may in turn contribute to treatment failure. The inhibition of serum-induced cell wall accumulation by fosfomycin reduced tolerance, suggesting that this antibiotic may form a useful combination therapy with daptomycin.

microbiology↗

bioRxiv2018

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 hypothesised 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 influenced the release of phospholipids and 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 implied that in host tissues S. aureus is likely to 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 summary, AFN-1252 significantly enhances daptomycin activity against S. aureus by blocking the production of phospholipid decoys, whilst daptomycin blocks the emergence of resistance to AFN-1252.

microbiology↗