Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic infections and antibiotic treatment failure1–3. In Escherichia coli, toxin/antitoxin (TA) modules have been linked to persister formation4–6. The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance into stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers is associated with a 100–1000 fold increase in the likelihood of survival to antibiotic challenge. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.

Heparin, known for its anticoagulant activity, is commonly used in catheter locks. Staphylococcus aureus, a versatile human and animal pathogen, is commonly associated with catheter-related bloodstream infections and has evolved a number of mechanisms through which it adheres to biotic and abiotic surfaces. We demonstrate that heparin increased biofilm formation by several S. aureus strains. Surface coverage and the kinetics of biofilm formation were stimulated, but primary attachment to the surface was not affected. Heparin increased S. aureus cell-cell interactions in a protein synthesis-dependent manner. The addition of heparin rescued biofilm formation of hla, ica, and sarA mutants. Our data further suggest that heparin stimulation of biofilm formation occurs neither through an increase in sigB activity nor through an increase in polysaccharide intracellular adhesin levels. These finding suggests that heparin stimulates S. aureus biofilm formation via a novel pathway.

It is postulated that, in addition to cell density, other factors, such as the dimensions and diffusional characteristics of the environment, could influence quorum sensing (QS) and induction of genetic reprogramming. Modeling studies predict that QS may operate at the level of a single cell, but, due to experimental challenges, the potential benefits of QS by individual cells remain virtually unexplored. Here we report a physical system that mimics isolation of a bacterium, such as within an endosome or phagosome during infection, and maintains cell viability under conditions of complete chemical and physical isolation. For Staphylococcus aureus, we show quorum sensing and genetic re-programming to occur in a single isolated organism. Quorum sensing allows S. aureus to sense confinement and to activate virulence and metabolic pathways needed for survival. To demonstrate the benefit of confinement-induced quorum sensing to individuals, we showed quorum sensing bacteria to have significantly greater viability over non-QS bacteria.

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpaclacI system, and the arabinose-inducible P BAD promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter::gfp uvr fusion carried on a shuttle plasmid, we demonstrated that dose-dependant tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.

In Staphylococcus aureus, the sigB operon codes for the alternative sigma factor B and its regulators that enable the bacteria to rapidly respond to environmental stresses via redirection of transcriptional priorities. However, a full model of B regulation in S. aureus has not yet emerged. Earlier data has suggested that mazEF, a toxin-antitoxin (TA) module immediately upstream of the sigB operon, was transcribed with the sigB operon. Here we demonstrate that the promoter P mazE upstream of mazEF is essential for full B activity and that instead of utilizing autorepression typical of TA systems, sigB downregulates this promoter, providing a negative-feedback loop for sigB to repress its own transcription. We have also found that the transcriptional regulator SarA binds and activates P mazE . In addition, P mazE was shown to respond to environmental and antibiotic stresses in a way that provides an additional layer of control over sigB expression. The antibiotic response also appears to occur in two other TA systems in S. aureus, indicating a shared mechanism of regulation.The ability of the major human pathogen Staphylococcus aureus to respond to challenging biotic (e.g., the nares of healthy individuals) (27) and abiotic environments (e.g., catheters, clothing, and doorknobs) (45) has promoted its persistence and transmission within hosts. As a result, S. aureus is now endemic to many hospitals and has become the leading cause of postoperative surgical infections (46). Furthermore, these adaptive responses have contributed to the sharp increase in S. aureus skin and soft tissue infections seen in nonrisk populations in the community (26). The ability of S. aureus to survive adverse host conditions is mediated by the alternative sigma factor B , which redirects RNA polymerase to transcribe genes involved in environmental stress responses (22 Immediately upstream of the rsbUVW-sigB operon, which codes for B and its regulators, are two small genes, mazF (SA1873) and mazE (SAS0167) (see Fig. 1E), which similar to their Escherichia coli namesakes (1), encode a toxin-antitoxin (TA) system (17). TA systems are common among prokaryotes and typically consist of a labile antitoxin that binds and inactivates a more stable toxin (18). Cellular proteases (e.g., Lon and ClpP) normally degrade these antitoxins, ensuring toxin activation if antitoxin production ever becomes interrupted. Starvation and antibiotic exposure are the most well-described stimuli of TA systems, leading to hypotheses that TA systems could be used as either a form of bacterial programmed cell death or as a way to selectively shut down the bacterium's metabolism (1, 18). Other hypotheses have laid out possible roles of TA systems in stabilizing nearby sections of the chromosome, in phage resistance, or in mediating the emergence of persister cells (33).In the present study, we expand the existing model of B regulation in S. aureus to include the transcriptional contribution from P mazE . We describe how the P mazE impacts B activity and how its activati...