Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used “survival” model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.

bThe virulence and fitness in vivo of the major human pathogen Staphylococcus aureus are associated with a cell-to-cell signaling mechanism known as quorum sensing (QS). QS coordinates the production of virulence factors via the production and sensing of autoinducing peptide (AIP) signal molecules by the agr locus. Here we show, in a wax moth larva virulence model, that (i) QS in S. aureus is a cooperative social trait that provides a benefit to the local population of cells, (ii) agr mutants, which do not produce or respond to QS signal, are able to exploit the benefits provided by the QS of others ("cheat"), allowing them to increase in frequency when in mixed populations with cooperators, (iii) these social interactions between cells determine virulence, with the host mortality rate being negatively correlated to the percentage of agr mutants ("cheats") in a population, and (iv) a higher within-host relatedness (lower strain diversity) selects for QS and hence higher virulence. Our results provide an explanation for why agr mutants show reduced virulence in animal models but can be isolated from infections of humans. More generally, by providing the first evidence that QS is a cooperative social behavior in a Gram-positive bacterium, our results suggest convergent, and potentially widespread, evolution for signaling to coordinate cooperation in bacteria. Staphylococcus aureus is a major human pathogen, responsible for 1% of all hospital admissions and leading to an estimated cost of $9.5 billion per year in the United States alone (1-4). Infections caused by S. aureus range from relatively mild boils and infected skin wounds to those with high mortality rates, such as bacteremia, infective endocarditis, and toxic shock. S. aureus infections can be difficult to treat because of antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA), which are resistant to beta-lactam antibiotics. Globally, MRSA infection rates are increasing (2, 5, 6).The virulence and growth in vivo of S. aureus depend upon quorum sensing (QS) via the accessory gene regulator (agr) locus (1,7,8). Cells release a small autoinducing peptide (AIP), the accumulation of which induces the production and secretion of both more AIP and a range of virulence factors. These virulence factors include hemolysins ␣, ␤, ␦, and ␥, toxic shock syndrome toxin (TSST), enterotoxins, Panton-Valentine leukocidin (PVL), and exfoliatins A and B (8-10). At high cell densities, or when cells are enclosed in small spaces, the agr system leads to positive feedback in the production of AIP and a significant increase in the production of virulence factors (11-15).It is commonly assumed that QS is a cooperative social trait, in which the benefits of virulence factors are shared between the local population of cells and their production is coordinated across cells via QS (16, 17). However, there has been no empirical demonstration that QS in S. aureus, or any other Gram-positive bacteria, is a cooperative social trait (see Discussion). Evidence that QS...

All bacterial infections occur within a polymicrobial environment, from which a pathogen population emerges to establish disease within a host. Emphasis has been placed on prevention of pathogen dominance by competing microflora acting as probiotics. Here we show that the virulence of the human pathogen Staphylococcus aureus is augmented by native, polymicrobial, commensal skin flora and individual species acting as 'proinfectious agents'. The outcome is pathogen proliferation, but not commensal. Pathogenesis augmentation can be mediated by particulate cell wall peptidoglycan, reducing the S. aureus infectious dose by over 1,000-fold. This phenomenon occurs using a range of S. aureus strains and infection models and is not mediated by established receptor-mediated pathways including Nod1, Nod2, Myd88 and the NLPR3 inflammasome. During mouse sepsis, augmentation depends on liver-resident macrophages (Kupffer cells) that capture and internalize both the pathogen and the proinfectious agent, leading to reduced production of reactive oxygen species, pathogen survival and subsequent multiple liver abscess formation. The augmented infection model more closely resembles the natural situation and establishes the role of resident environmental microflora in the initiation of disease by an invading pathogen. As the human microflora is ubiquitous, its role in increasing susceptibility to infection by S. aureus highlights potential strategies for disease prevention.

Staphylococcus aureus is historically regarded as a non-motile organism. More recently it has been shown that S. aureus can passively move across agar surfaces in a process called spreading. We re-analysed spreading motility using a modified assay and focused on observing the formation of dendrites: branching structures that emerge from the central colony. We discovered that S. aureus can spread across the surface of media in structures that we term ‘comets’, which advance outwards and precede the formation of dendrites. We observed comets in a diverse selection of S. aureus isolates and they exhibit the following behaviours: (1) They consist of phenotypically distinct cores of cells that move forward and seed other S. aureus cells behind them forming a comet ‘tail’; (2) they move when other cells in the comet tail have stopped moving; (3) the comet core is held together by a matrix of slime; and (4) the comets etch trails in the agar as they move forwards. Comets are not consistent with spreading motility or other forms of passive motility. Comet behaviour does share many similarities with a form of active motility known as gliding. Our observations therefore suggest that S. aureus is actively motile under certain conditions.