Combinatorial Phenotypic Signatures Distinguish Persistent from Resolving Methicillin-Resistant Staphylococcus aureus Bacteremia Isolates

Abstract

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) (positive blood cultures after >7 days of therapy) represents a clinically challenging subset of invasive MRSA infections. In this investigation, we examined the potential correlation of specific virulence signatures with PB versus resolving MRSA bacteremia (RB) (negative blood cultures within 2 to 4 days of therapy) strains. Thirty-six MRSA isolates from patients enrolled in a recent multinational clinical trial were studied for (i) susceptibility to host defense cationic peptides (HDPs) (i.e., thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide 1 [hNP-1]); (ii) adherence to host endovascular ligands (fibronectin) and cells (endothelial cells); and (iii) biofilm formation. We found that PB isolates exhibited significantly reduced susceptibilities to tPMPs and hNP-1 (P < 0.001 and P ‫؍‬ 0.023, respectively). There was no significant association between the PB outcome and fibronectin binding, endothelial cell binding, or biofilm formation (P ‫؍‬ 0.25, 0.97, and 0.064 versus RB strains, respectively). However, multiple logistic regression analysis revealed that the PB outcome was significantly associated with the combination of reduced susceptibilities to HDPs and extent of biofilm formation (P < 0.0001). Similar results were obtained in a second analysis using days of bacteremia as a continuous outcome, showing that reduced HDP susceptibilities and increased biofilm formation cocontributed to predict the duration of bacteremia. Our data indicate that PB isolates have specific pathogenic signatures independent of conventional antimicrobial susceptibility. These combinatorial mosaics can be defined and used to prospectively distinguish PB from RB strains in advance and potentially to predict ultimate clinical outcomes.Staphylococcus aureus is a leading cause of bacteremia and infective endocarditis (IE) throughout the industrialized world (18,22,42,54). A growing proportion of these bloodstream infections are due to methicillin-resistant Staphylococcus aureus (MRSA), which is associated with worse clinical outcome, longer hospitalization, and higher net cost than similar infections caused by methicillin-susceptible S. aureus (MSSA) (3,6,14,15,26,49,52,60). Persistent-bacteremia (PB) outcomes comprise 20 to 30% of all episodes of MRSA bacteremia and are especially relevant to endovascular infections (13,22,23,31). Why some MRSA bacteremia strains persist while others resolve (RB) despite similar baseline clinical and microbiologic characteristics and identical medical and surgical therapeutic strategies is poorly understood.In two recent studies, we investigated S. aureus virulence factors that could potentially differentiate PB from RB strains in the context of endovascular infections (23, 56). We initially studied PB or RB isolates from the Duke University Medical Center and delineated several in vitro parameters that appeared to distinguish these two strain cohorts, including enhanced matrix ligand and e...

Reduced Vancomycin Susceptibility in anIn VitroCatheter-Related Biofilm Model Correlates with Poor Therapeutic Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus

“…The 10 MRSA bloodstream study isolates are listed in Table 1; these strains have been described in detail previously (15)(16)(17). All 10 strains had VAN MICs within the susceptible range (0.5 to 1.0 g/ml).…”

“…These compositional differences have been described to contribute functionally and structurally to the organization of the biofilm (2,32). Therefore, relative differences in the stabilities of the biofilms formed by our MRSA study strains were determined as a function of specific biochemical components, using carbohydrate, protein, or DNA dispersal agents (16,33). In addition, the impact of sub-MIC levels of VAN on such differences was assessed.…”

“…In brief, the supernatants of 18-h-old biofilms generated by MRSA in the presence or absence of 0.5ϫ the MIC of VAN were replaced by fresh medium supplemented with either (i) 10 mM so- dium metaperiodate (Alfa Aesar, Ward Hill, MA) to assess relative carbohydrate content, (ii) 100 g/ml protease K (Acros Organics) to assess relative protein content, or (iii) 140 U/ml RNase-free DNase I (TaKaRa Bio Inc., Shiga, Japan) to assess relative DNA content. All incubations were for 2 h at 37°C, as detailed elsewhere (16,33). Media without the above supplements served as respective negative controls.…”

“…In this regard, Hsu et al (14) recently reported that sub-MIC levels of VAN can promote MRSA biofilm formation in vitro, a process that may have clinical significance. Thus, the degree of biofilm formation under static conditions was determined for our 10 MRSA strains in the presence or absence of 0.5ϫ the MIC of VAN, as described previously (14,16). Briefly, MRSA strains from fresh blood TSB agar plates were adjusted to a density of 0.5 McFarland standard and diluted 1:10 into brain heart infusion (BHI) medium supplemented with 0.5% glucose (BHIG; containing ϳ10 7 CFU/ml).…”

Infective Endocarditis

“…The most convincing data on the effect of biofilm formation in native valve IE comes from experimental studies in S. aureus IE. A series of studies over the past decade have linked the ability of S. aureus strains to produce biofilms in vitro and their ability to cause clinically 'persistent' methicillin-resistant S. aureus (MRSA) bacteraemia in humans (defined as >7 days of positive blood cultures despite presence of vancomycin-susceptible isolates and adequate vancomycin treatment regimens) 58,59 . Of interest, clinically-persistent MRSA bacteraemia strains produce significantly more biofilm in vitro when exposed to subinhibitory concentrations of vancomycin as compared to clinically-resolving MRSA isolates 58 .…”