The Staphylococcus aureus cidA and lrgA genes have been shown to affect cell lysis under a variety of conditions during planktonic growth. It is hypothesized that these genes encode holins and antiholins, respectively, and may serve as molecular control elements of bacterial cell lysis. To examine the biological role of cell death and lysis, we studied the impact of the cidA mutation on biofilm development. Interestingly, this mutation had a dramatic impact on biofilm morphology and adherence. The cidA mutant (KB1050) biofilm exhibited a rougher appearance compared with the parental strain (UAMS-1) and was less adherent. Propidium iodide staining revealed that KB1050 accumulated more dead cells within the biofilm population relative to UAMS-1, indicative of reduced cell lysis. In agreement with this finding, quantitative real-time PCR experiments demonstrated the presence of 5-fold less genomic DNA in the KB1050 biofilm relative to UAMS-1. Furthermore, treatment of the UAMS-1 biofilm with DNase I caused extensive cell detachment, whereas similar treatment of the KB1050 biofilm had only a modest effect. These results demonstrate that cidA-controlled cell lysis plays a significant role during biofilm development and that released genomic DNA is an important structural component of S. aureus biofilm.autolysis ͉ extracellular DNA ͉ programmed cell death ͉ holin

Staphylococcus aureus and Staphylococcus epidermidis are the leading causes of nosocomial infections in the United States and often are associated with biofilms attached to indwelling medical devices. Despite the importance of biofilms, there is very little consensus about the metabolic requirements of S. aureus during biofilm growth. To assess the metabolic requirements of S. aureus growing in a biofilm, we grew USA200 and USA300 clonal types in biofilm flow cells and measured the extraction and accumulation of metabolites. In spite of the genetic differences, both clonal types extracted glucose and accumulated lactate, acetate, formate, and acetoin, suggesting that glucose was catabolized to pyruvate that was then catabolized via the lactate dehydrogenase, pyruvate formate-lyase, and butanediol pathways. Additionally, both clonal types selectively extracted the same six amino acids (serine, proline, arginine, glutamine, glycine, and threonine) from the culture medium. These data and recent speculation about the importance of arginine in biofilm growth and the function of arginine deiminase in USA300 clones led us to genetically inactivate the sole copy of the arginine deiminase operon by deleting the arginine/ornithine antiporter gene (arcD) in the USA200 clonal type and to assess the effect on biofilm development and pathogenesis. Although inactivation of arcD did completely inhibit arginine transport and did reduce polysaccharide intercellular adhesin accumulation, arcD mutants formed biofilms and achieved cell densities in catheter infection studies that were equivalent to those for isogenic wild-type strains.

We used a murine model of catheter-associated biofilm formation to determine whether the mutation of the staphylococcal accessory regulator (sarA) has an impact on the susceptibility of established Staphylococcus aureus biofilms to treatment with daptomycin in vivo. The experiments were done with two clinical isolates, one of which (UAMS-1) was obtained from the bone of a patient suffering from osteomyelitis, while the other (UAMS-1625) is an isolate of the USA300 clonal lineage of community-acquired methicillin (meticillin)-resistant S. aureus. UAMS-1625 had a reduced capacity to form a biofilm in vivo compared to that of UAMS-1 (P ‫؍‬ 0.0015), but in both cases the mutation of sarA limited biofilm formation compared to that of the corresponding parent strain (P < 0.001). The mutation of sarA did not affect the daptomycin MIC for either strain, but it did result in increased susceptibility in vivo in the context of an established biofilm. Specifically, daptomycin treatment resulted in the clearance of detectable bacteria from <10% of the catheters colonized with the parent strains, while treatment with an equivalent daptomycin concentration resulted in the clearance of 46.4% of the catheters colonized with the UAMS-1 sarA mutant and 69.1% of the catheters colonized with the UAMS-1625 sarA mutant. In the absence of daptomycin treatment, mice with catheters colonized with the UAMS-1625 parent strain also developed skin lesions in the region adjacent to the implanted catheter. No such lesions were observed in any other experimental group, including untreated mice containing catheters colonized with the UAMS-1625 sarA mutant.

Mutation of the staphylococcal accessory regulator (sarA) in Staphylococcus aureus limits but does not abolish the capacity of the organism to form a biofilm. As a first step toward determining whether this limitation is therapeutically relevant, we carried out in vitro studies comparing the relative susceptibility of an S. aureus clinical isolate (UAMS-1) and its isogenic sarA mutant (UAMS-929) in the specific context of a catheter-associated biofilm. The antibiotics tested were daptomycin, linezolid, and vancomycin, all of which were evaluated by using concentrations based on the MIC defined as the breakpoint for a susceptible strain of S. aureus (<1.0, <2.0, and <4.0 g/ml for daptomycin, vancomycin, and linezolid, respectively). Mutation of sarA had no significant impact on the MIC of UAMS-1 for any of the targeted antibiotics, as defined by Etest antimicrobial susceptibility testing. However, mutation of sarA did result in a significant increase in antimicrobial susceptibility to all targeted antibiotics when they were tested in the specific context of a biofilm. Additionally, whether susceptibility was assessed by using UAMS-1 or its sarA mutant, daptomycin was found to be more effective against established S. aureus biofilms than either linezolid or vancomycin.Staphylococcus aureus is a devastating human pathogen, with the death toll from invasive S. aureus infections recently having passed that from AIDS in the United States (9, 18). These infections range from acute toxemias and septic shock to more chronic infections, including osteomyelitis and infections associated with indwelling medical devices. The latter are characterized by the formation of a biofilm, which has a significant impact not only on the disease process itself but also on the ability of clinicians to effectively treat the infection. This is true because biofilm-associated infections are recalcitrant to antimicrobial therapy, irrespective of the resistance status of the offending strain or the ability to achieve what would otherwise be therapeutic serum levels of antibiotic (10,21,30). For this reason, the resolution of biofilm-associated staphylococcal infections often requires surgical debridement to remove infected tissues and/or indwelling devices (5,20).An alternative approach to the therapeutic problem of biofilm-associated infection would be the development of methods that specifically prevent or at least limit biofilm formation. This could be done by targeting either the substrate (e.g., by developing novel biomaterials that are less conducive to biofilm formation) or the bacterium (e.g., by developing novel agents capable of limiting biofilm formation). The latter approach requires the identification of those bacterial targets that are most relevant in the specific context of a biofilm. Studies focusing on the staphylococci have led to the identification of many potential targets (24). However, in almost every case, there are conflicting reports about the roles of different genes and gene products in biofilm formation (12,24,35)...

To investigate the regulatory role of traP (target of RNAIII-activating peptide) in Staphylococcus aureus, we generated traP mutations in the clinical isolates UAMS-1 and USA300. In neither case did mutation of traP affect expression of the accessory gene regulator (agr) or the ability to form a biofilm. We were also unable to confirm that mutation of traP in the prototype 8325-4 laboratory strain RN6390 results in reduced expression of agr, reduced hemolytic activity, or an altered capacity to form a biofilm.There is a growing body of literature indicating that mutation or inhibition of the Staphylococcus aureus regulatory locus designated traP (target of RNAIII-activating peptide) results in reduced expression of the accessory gene regulator (agr), a reduced capacity to form a biofilm, and reduced virulence in animal models of staphylococcal disease (3,5,6,17,23,28). The traP gene reportedly encodes a response regulator (TraP) that is triply phosphorylated as a result of the accumulation of a protein designated RAP (RNAIII-activating peptide), resulting in activation of the agr regulatory system and induction of toxin synthesis (21). A number of studies have also concluded that clinical isolates of S. aureus are responsive to RAP and that a modified peptide derivative designated RIP (RNAIIIinhibiting peptide) can be used to limit phosphorylation of TraP and thereby limit induction of agr, the capacity to form a biofilm, and virulence (1,3,12,14,24).To the extent that both RAP and RIP reportedly function by modulating the activity of TraP, studies indicating that clinical isolates are responsive to RAP and RIP would suggest that the traP regulatory system functions in the same fashion in most if not all S. aureus strains. However, the primary focus to date, particularly in terms of direct mutagenesis studies, has been on derivatives of the prototype 8325-4 laboratory strain such as RN6390, and recent studies from our laboratory have confirmed that regulatory circuits in RN6390 are different than those observed in at least some clinical isolates (9, 11). One such difference is the impact of agr on biofilm formation. Specifically, mutation of agr enhances biofilm formation in RN6390 but has little impact in clinical isolates (7,29). Since TraP reportedly functions through an agr-dependent pathway, we wanted to investigate whether similar, strain-dependent differences also existed with respect to traP. To that end, we mutated traP in two clinical isolates (UAMS-1 and USA300-0114) as well as our version of the prototype laboratory strain RN6390. The impact of mutating traP in all three strains was assessed based on biofilm formation, hemolytic activity, and production of the agr-encoded regulatory molecule RNAIII.