Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Summary: Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram‐positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilus‐negative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three‐dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild‐type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GAS‐mediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.

The development of chronic and recurrent Staphylococcus aureus infections is associated with the emergence of slow-growing mutants known as small-colony variants (SCVs), which are highly tolerant of antibiotics and can survive inside host cells. However, the host and bacterial factors which underpin SCV emergence during infection are poorly understood. Here, we demonstrate that exposure of S. aureus to sublethal concentrations of H ₂ O ₂ leads to a specific, dose-dependent increase in the population frequency of gentamicin-resistant SCVs. Time course analyses revealed that H ₂ O ₂ exposure caused bacteriostasis in wild-type cells during which time SCVs appeared spontaneously within the S. aureus population. This occurred via a mutagenic DNA repair pathway that included DNA double-strand break repair proteins RexAB, recombinase A, and polymerase V. In addition to triggering SCV emergence by increasing the mutation rate, H ₂ O ₂ also selected for the SCV phenotype, leading to increased phenotypic stability and further enhancing the size of the SCV subpopulation by reducing the rate of SCV reversion to the wild type. Subsequent analyses revealed that SCVs were significantly more resistant to the toxic effects of H ₂ O ₂ than wild-type bacteria. With the exception of heme auxotrophs, gentamicin-resistant SCVs displayed greater catalase activity than wild-type bacteria, which contributed to their resistance to H ₂ O ₂ . Taken together, these data reveal a mechanism by which S. aureus adapts to oxidative stress via the production of a subpopulation of H ₂ O ₂ -resistant SCVs with enhanced catalase production.

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α₅β₁ integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis. Staphylococcus aureus is a frequent cause of bacteremia and sepsis. Adhesion to and invasion of endothelial cells lining blood vessels by S. aureus can lead to colonization of the heart valves and/or dissemination into surrounding tissues and the establishment of secondary (metastatic) infections. Uptake by endothelial cells is triggered by the interaction of fibronectin-binding protein A (FnBPA) with host cell receptors called integrins via a fibronectin (Fn) bridge. FnBPA contains 11 non-identical repeats (FnBRs) that mediate binding to Fn. Previous work has shown that partial deletions in the FnBR region do not significantly affect binding to Fn or invasion of host cells in vitro. It was therefore unclear why FnBPA contains so many repeats. This work demonstrates that the number of repeats affects the amount of surface-exposed FnBPA required to trigger invasion, as well as affecting the speed of uptake. As such, the presence of multiple repeats within FnBPA facilitates adhesion and invasion even at low surface expression levels. Experiments using a murine model of sepsis demonstrate that these differences have a significant impact on virulence; S. aureus expressing FnBPA variants with no or few FnBRs are significantly less virulent compared to FnBPA with the full complement of repeats.

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 cases^3,4 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 β-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.