Karen E. Beenken

Global Gene Expression in Staphylococcus aureus Biofilms

We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.

Mutation of sarA in Staphylococcus aureus Limits Biofilm Formation

ABSTRACT Mutation of sarA resulted in a reduced capacity to form a biofilm in six of the eight Staphylococcus aureus strains we tested (UAMS-1, UAMS-601, SA113, SC-01, S6C, and DB). The exceptions were Newman, which formed a poor biofilm under all conditions, and RN6390, which consistently formed a biofilm only after mutation of agr. Mutation of agr in other strains had little impact on biofilm formation. In every strain other than Newman, including RN6390, simultaneous mutation of sarA and agr resulted in a phenotype like that observed with the sarA mutants. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation.

Strain-dependent differences in the regulatory roles of sarA and agr in Staphylococcus aureus

ABSTRACT The accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are central regulatory elements that control the production of Staphylococcus aureus virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in rsbU that results in a phenotype resembling that of a sigB mutant (I. Kullik et al., J. Bacteriol. 180:4814–4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of S. aureus. To address this issue, we generated mutations in the sarA and agr loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of sarA in the cna-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of agr had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased cna transcription. Without exception, mutation of sarA resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of agr had the opposite effect. Although mutation of sarA resulted in a slight reduction in fnbA transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both sarA and agr mutants. While mutation of sarA in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the sarC transcript in RN6390, but there was no difference in the overall pattern of sar transcription or the production of SarA. Although mutation of sarA resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of sarA and agr by using RN6390 are not always representative of the events that occur in clinical isolates of S. aureus.

Multiplex PCR Protocol for the Diagnosis of Staphylococcal Infection

ABSTRACT We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding toStaphylococcus-specific regions of the 16S rRNA genes), (ii) distinguish between S. aureus and the coagulase-negative staphylococci (CNS) (based on amplification of theS. aureus-specific clfA gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the mecA gene). The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistantS. aureus isolates, 23 oxacillin-sensitive S. aureus isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS). No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample. The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles. Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing. Of those, one was identified as a CNS species by PCR and S. aureus by conventional methods. We also identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods. The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes.

Staphylococcus aureus Collagen Adhesin Contributes to the Pathogenesis of Osteomyelitis

To evaluate the role of the Staphylococcus aureus collagen-binding adhesin (Cna) in bone and joint infection, we generated a cna mutant in S. aureus UAMS-1, a strain that was originally isolated from the bone of a patient suffering from osteomyelitis. The mutant (UAMS-237) was unable to bind collagen but bound fibronectin at levels comparable to UAMS-1. The relative virulence of UAMS-1 and UAMS-237 was assessed using a murine model of acute hematogenous osteomyelitis. Specifically, 10(8) colony-forming units (cfu) were introduced into the bloodstream of NIH-Swiss mice via tail-vein injection. After 2 weeks, the left hind limb was harvested and examined histologically for evidence of osteomyelitis and septic arthritis. Osteomyelitis developed in 14 of 20 mice (70%) infected with UAMS-1, but only 1 of 20 (5%) infected with UAMS-237 (p < 0.001). In contrast, septic arthritis was observed in 12 of 20 mice (60%) infected with UAMS-1 and 14 of 20 (70%) infected with UAMS-237 (p < 0.75). These results indicate that Cna is not required to establish joint infection, but does make an important contribution to the ability of S. aureus to establish infection in bone through hematogenous spread.

Epistatic Relationships Between sarA and agr in Staphylococcus Aureus Biofilm Formation

Background The accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) play opposing roles in Staphylococcus aureus biofilm formation. There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci. Methodology/Principal Findings We generated isogenic sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to form a biofilm in the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard. Conclusions and Significance The results we report suggest that inhibitors of sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.

Ellagic acid derivatives from Rubus ulmifolius inhibit Staphylococcus aureus biofilm formation and improve response to antibiotics.

Background Biofilms contribute to the pathogenesis of many forms of Staphylococcus aureus infection. Treatment of these infections is complicated by intrinsic resistance to conventional antibiotics, thus creating an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections. Methodology/Principal Findings This study demonstrates that a botanical natural product composition (220D-F2) rich in ellagic acid and its derivatives can limit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility. The source of this composition is Rubus ulmifolius Schott. (Rosaceae), a plant used in complementary and alternative medicine in southern Italy for the treatment of skin and soft tissue infections. All S. aureus clonal lineages tested exhibited a reduced capacity to form a biofilm at 220D-F2 concentrations ranging from 50–200 µg/mL, which were well below the concentrations required to limit bacterial growth (530–1040 µg/mL). This limitation was therapeutically relevant in that inclusion of 220D-F2 resulted in enhanced susceptibility to the functionally-distinct antibiotics daptomycin, clindamycin and oxacillin. Testing with kidney and liver cell lines also demonstrated a lack of host cell cytotoxicity at concentrations of 220D-F2 required to achieve these effects. Conclusions/Significance These results demonstrate that extract 220D-F2 from the root of Rubus ulmifolius can be used to inhibit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility without toxic effects on normal mammalian cells. Hence, 220D-F2 is a strong candidate for development as a botanical drug for use in the prevention and treatment of S. aureus biofilm-associated infections.

Factors contributing to the biofilm-deficient phenotype of Staphylococcus aureus sarA mutants.

Mutation of sarA in Staphylococcus aureus results in a reduced capacity to form a biofilm, but the mechanistic basis for this remains unknown. Previous transcriptional profiling experiments identified a number of genes that are differentially expressed both in a biofilm and in a sarA mutant. This included genes involved in acid tolerance and the production of nucleolytic and proteolytic exoenzymes. Based on this we generated mutations in alsSD, nuc and sspA in the S. aureus clinical isolate UAMS-1 and its isogenic sarA mutant and assessed the impact on biofilm formation. Because expression of alsSD was increased in a biofilm but decreased in a sarA mutant, we also generated a plasmid construct that allowed expression of alsSD in a sarA mutant. Mutation of alsSD limited biofilm formation, but not to the degree observed with the corresponding sarA mutant, and restoration of alsSD expression did not restore the ability to form a biofilm. In contrast, concomitant mutation of sarA and nuc significantly enhanced biofilm formation by comparison to the sarA mutant. Although mutation of sspA had no significant impact on the ability of a sarA mutant to form a biofilm, a combination of protease inhibitors (E-64, 1-10-phenanthroline, and dichloroisocoumarin) that was shown to inhibit the production of multiple extracellular proteases without inhibiting growth was also shown to enhance the ability of a sarA mutant to form a biofilm. This effect was evident only when all three inhibitors were used concurrently. This suggests that the reduced capacity of a sarA mutant to form a biofilm involves extracellular proteases of all three classes (serine, cysteine and metalloproteases). Inclusion of protease inhibitors also enhanced biofilm formation in a sarA/nuc mutant, with the combined effect of mutating nuc and adding protease inhibitors resulting in a level of biofilm formation with the sarA mutant that approached that of the UAMS-1 parent strain. These results demonstrate that the inability of a sarA mutant to repress production of extracellular nuclease and multiple proteases have independent but cumulative effects that make a significant contribution to the biofilm-deficient phenotype of an S. aureus sarA mutant.

Role of sarA in the pathogenesis of Staphylococcus aureus musculoskeletal infection.

ABSTRACT We recently demonstrated that mutation of sarA in clinical isolates of Staphylococcus aureus results in a phenotype that is distinct by comparison to sarA mutants generated in the laboratory strain RN6390 (J. S. Blevins, K. E. Beenken, M. O. Elasri, B. K. Hurlburt, and M. S. Smeltzer, Infect. Immun. 70:470-480, 2002). This raises the possibility that studies demonstrating that RN6390 sarA mutants are attenuated do not accurately reflect the role of sarA in the pathogenesis of staphylococcal disease. To test this hypothesis, we used a murine model of musculoskeletal infection to assess the virulence of sarA and agr mutants generated in a clinical isolate of S. aureus (UAMS-1). By using this model, we confirmed that mutation of sarA and/or agr results in a reduced capacity to cause both septic arthritis and osteomyelitis.

Impact of sarA on Daptomycin Susceptibility of Staphylococcus aureus Biofilms In Vivo

ABSTRACT 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.