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.

Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids.

Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA)1,2. Although resistance is rare, treatment failure can occur in more than 20% of cases3,4 and so there is a pressing need to identify and mitigate factors that contribute to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. Wild-type S. aureus was killed rapidly by daptomycin, but Agr-defective mutants survived antibiotic exposure by releasing membrane phospholipids, which bound and inactivated the antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was inhibited by the ß-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that protects against daptomycin, which can be compromised by Agr-triggered toxin production or an existing therapeutic antibiotic.

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain.

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain.

Candidatus Rickettsia hoogstraalii in Ethiopian Argas persicus ticks.

Ethiopian soft ticks Argas persicus, hard ticks including both Amblyomma variegatum and Rhipicephalus (Boophilus) spp., and fleas were collected from livestock, traditional human dwellings, and cracks and crevices of trees. They were assessed in pools for the presence of Rickettsia using PCR-based methods. The extracted tick DNA was subjected to molecular screening for Rickettsia, which revealed 50.5% of the pooled samples to be positive for Rickettsia spp. These were then subjected to multi-gene analysis using both outer surface proteins and housekeeping genes with proven discriminatory potential. Sequencing of the citrate synthase and outer membrane genes clearly led to the identification of three distinct rickettsial species, Candidatus Rickettsia hoogstraalii in Argas persicus ticks; R. africae in hard tick pools, and R. felis in fleas. Furthermore, we demonstrated the presence of the plasmid-borne small heat-shock protein gene hsp2 in DNA from A. persicus ticks suggesting that Candidatus R. hoogstraalii carried by these ticks possess a plasmid. Unlike chromosomal gene sequences, the hsp2 gene failed to cluster with Candidatus R. hoogstraalii, instead falling into an isolated separate clade, suggesting a different origin for the plasmid.