Conventional approaches to MRSA virulence assessments have predominantly focused on putative virulence determinants individually, using “molecular Koch's postulates” (16
). Such studies have provided valuable insights into the contributions of specific virulence factors to MRSA pathogenesis, yet an important theme that has emerged from these efforts is that no single virulence determinant alone is sufficient to enable full virulence of MRSA. In this study, we are, to our knowledge, the first to employ a combinatorial strategy to assess the impact of phenotypic mosaics that potentially enable an MRSA PB outcome in the context of endovascular infections. The use of clinical MRSA isolates from a recent multinational clinical trial allowed us to exclude possible geographic, single-center, and/or clonality biases that may have substantially limited previous investigations.
A number of interesting observations emerged from the present investigation. First, an important factor in the pathogenesis of S. aureus
infection is the ability of the microbe to circumvent clearance by platelet-mediated mechanisms, principally via resistance to killing by platelet HDPs (e.g., tPMPs) secreted in response to agonists generated at endovascular infection sites (58
). Reduced killing by tPMPs in vitro
has been correlated with enhanced in vivo
virulence and less efficacy of antibiotic therapy (including vancomycin) in a rabbit endocarditis (IE) model in several previous studies from our laboratory (9
). In addition, S. aureus
bloodstream strains isolated from endovascular infections were significantly less susceptible to killing by tPMPs in vitro
than bacteremia strains from other clinical syndromes (e.g., soft tissue abscesses), demonstrating the potential importance of relative tPMP resistance in clinical settings (1
). Of note, in both these prior experimental models and clinical investigations, a “biologic breakpoint” utilizing the same 2-h killing assay appeared to emerge. Thus, a survival profile in the range of >40% in this assay correlated well with (i) an endovascular source for bacteremia, (ii) more virulent infection in IE models, and(iii) reduced responsiveness to antimicrobial prophylaxis and therapy in these same models. Interestingly, in the present study, we observed an impressive statistical relationship between reduced tPMP-induced killing in vitro
and the PB outcome, with the mean in vitro
survival profile also greater than 40% for such strains. Finally, two recent clinical studies demonstrated a similar correlation between relative tPMP resistance and PB-RB outcomes in strains from both IE and non-IE infections (23
). Similarly, reduced in vitro
susceptibilities to hNP-1 correlated with the PB outcome in our study, in accordance with our previous findings (56
). We have postulated that relative resistances to such PMN HDPs may foster PB by favoring MRSA survival within PMN-rich abscesses (e.g., within the kidneys or spleen). Taken together, these data indicate a potential role for relative resistance to tPMPs and hNP-1 as a surrogate biological marker for the PB outcome.
Second, the ability of bacteria to bind to matrix ligands and certain host cells is felt to be an important virulence factor in S. aureus
endovascular infections (56
). In the present study, we were surprised to find no significant differences in fibronectin-binding profiles between the PB and RB strain sets. This is in contrast to our previous study, in which the PB outcome was associated with better fibronectin binding (56
). Interestingly, a recent study showed that S. aureus
isolates at either extreme of fibronectin-binding capacity were equally capable of establishing IE in an experimental model (61
). Even though the ability to bind fibronectin is crucial for inducing endocarditis (48
), the extent of the binding seems to play a minor role in IE induction and in persistence. In parallel to fibronectin binding, no significant difference in endothelial cell binding profiles was noted when the PB and RB cohorts were compared. The fibronectin-binding phenotype and/or expression profiles of genes involved in fibronectin binding may differ substantially between the in vitro
conditions we tested and in vivo
infection conditions. In addition, the ability to invade and survive inside endothelial cells might be of more importance for persistence in the host than adhesion. Also, the extent of the proinflammatory response induced by the bacteria might play a role. Studies are ongoing in our laboratory to address these questions.
Last, the extensive literature correlating indwelling endovascular devices, biofilm formation on such devices, and S. aureus
bacteremia led us to examine the ability of the PB versus RB cohorts to form biofilms in vitro
). Importantly, multiple linear and logistic regression data analysis showed that combining the extent of biofilm formation with reduced susceptibility to HDPs provided a phenotypic mosaic that was significantly correlated with both the PB outcome and increased days of bacteremia. Since biofilm formation restricts the access of many conventional antimicrobial agents (e.g., vancomycin) to sessile bacterial communities (12
), it is reasonable to postulate that biofilm also mitigates the exposure of such colonies to HDPs generated at endovascular infection sites. Another interesting finding of our study is the overrepresentation of the sspA
gene, encoding the V8 protease, in the PB strain cohort (100%) compared with the RB strain cohort (89%). V8 protease has been reported to be required for biofilm dispersal mechanisms, which in turn can play a role in colonizing new sites and favor recalcitrant infections (2
Different factors, such as the production of PIA/PNAG by icaADBC
operon-encoded enzymes (7
); extracellular DNA (eDNA) (30
); and several surface proteins, including Bap, SasG, FnBPs, and Spa (5
), have all been shown to be involved in S. aureus
biofilm formation. In accordance with previous findings (35
), we found that biofilm formation in our tested MRSA isolates depended more on the protein content than on the PIA/PNAG content. Biofilms of most of the isolates tested also contained eDNA, a recently described and important S. aureus
biofilm component (30
). Interestingly, our RB isolate biofilm matrices seemed to contain more PIA/PNAG than those of the PB isolates tested. Thus, the biofilm “component type” might be another useful marker for distinguishing PB from RB isolates. Further investigations are ongoing in our laboratory to investigate this notion.
We recognize that there are some important limitations in the present study. First, we assessed a relatively small number of PB-RB strains and phenotypes, affording a substantial chance for statistical bias. We also realize that in vitro
testing of HDPs in sublethal concentrations much lower than would be encountered in vivo
may also present limitations. We are currently examining larger PB-RB strain cohorts and additional phenotypic biomarkers to circumvent some of these issues. To complement our in vitro
findings, in vivo
studies are ongoing in our laboratories using an experimental IE model and selected isolates from our current MRSA strain cohort. These investigations are designed to address the issues of whether “PB” isolates are intrinsically more virulent than “RB” MRSA isolates or whether PB outcomes are relevant only during antimicrobial therapy. The outcomes from our previous experimental IE studies in other PB-RB strain collections would predict that the latter concept is more valid than the former concept (56
We recognize that there is no clearly defined days of bacteremia “breakpoint” to distinguish PB and RB outcomes. As in our prior studies of PB and RB outcomes in other strain cohorts, we utilized the ≥7 days definition of PB for several reasons. First, our decision to define “persistent bacteremia” as ≥7 days is based on well-accepted definitions in the peer-reviewed literature (23
). Second, the definition is consistent with our own clinical experiences. The clinical signs and symptoms of patients with bacteremia lasting ≥7 days are typically quite different from those of patients with shorter bacteremic durations, even those with complicated infection (e.g., febrile morbidity). Thus, while far from perfect, our definition of PB is well accepted and allows investigators a reasonable yardstick for studying this fascinating clinical entity.
Collectively, our data strongly support our overall hypothesis that PB isolates have specific and combinatorial pathogenic “signatures” that are independent of conventional antimicrobial susceptibility. Importantly, the signatures that are predominant in one PB strain cohort (e.g., fibronectin binding [56
]) may not be contributory in others, such as in our current strain set. Overall, defining common and consensus PB signatures among large well-defined strain collections might eventually be useful to identify patients at higher risk for PB to optimize anti-MRSA therapeutic strategies.