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1.  Impact of Vancomycin on sarA-Mediated Biofilm Formation: Role in Persistent Endovascular Infections Due to Methicillin-Resistant Staphylococcus aureus 
The Journal of Infectious Diseases  2014;209(8):1231-1240.
Background. Staphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation.
Methods. Two healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub–minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model.
Results. All sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model.
Conclusions. These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation.
PMCID: PMC3969550  PMID: 24403556
sarA; biofilm formation; MRSA endocarditis
2.  Genome Sequences of Sequence Type 45 (ST45) Persistent Methicillin-Resistant Staphylococcus aureus (MRSA) Bacteremia Strain 300-169 and ST45 Resolving MRSA Bacteremia Strain 301-188 
Genome Announcements  2014;2(2):e00174-14.
Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (positive blood cultures after ≥7 days) represents a challenging subset of invasive MRSA infections. The comparison of genome sequences of persistent (300-169) and resolving (301-188) MRSA bacteremia isolates with similar genetic background (sequence type 45 [ST45]) will help us to better understand underlying mechanisms of persistent MRSA bacteremia.
PMCID: PMC3953194  PMID: 24625873
3.  Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat 
Endovascular infections, including endocarditis, are life-threatening infectious syndromes1–3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4–6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.
PMCID: PMC3471301  PMID: 22711072
Infection; Issue 64; Immunology; Staphylococcus aureus; endocarditis; animal model; methicillin resistance; MRSA; rat
4.  Reduced Vancomycin Susceptibility in an In Vitro Catheter-Related Biofilm Model Correlates with Poor Therapeutic Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus 
Staphylococcus aureus is the most common cause of endovascular infections, including catheter sepsis and infective endocarditis (IE). Vancomycin (VAN) is the primary choice for treatment of methicillin-resistant S. aureus (MRSA) infections. However, high rates of VAN treatment failure in MRSA infections caused by VAN-susceptible strains have been increasingly reported. Biofilm-associated MRSA infections are especially prone to clinical antibiotic failure. The present studies examined potential relationships between MRSA susceptibility to VAN in biofilms in vitro and nonsusceptibility to VAN in endovascular infection in vivo. Using 10 “VAN-susceptible” MRSA bloodstream isolates previously investigated for VAN responsiveness in experimental IE, we studied the mechanism(s) of such in vivo VAN resistance, including: (i) VAN binding to MRSA organisms; (ii) the impact of VAN on biofilm formation and biofilm composition; (iii) VAN efficacy in an in vitro catheter-related biofilm model; (iv) effects on cell wall thickness. As a group, the five strains previously categorized as VAN nonresponders (non-Rsp) in the experimental IE model differed from the five responders (Rsp) in terms of lower VAN binding, increased biofilm formation, higher survival in the presence of VAN within biofilms in the presence or absence of catheters, and greater biofilm reduction upon proteinase K treatment. Interestingly, sub-MICs of VAN significantly promoted biofilm formation only in the non-Rsp isolates. Cell wall thickness was similar among all MRSA strains. These results suggest that sublethal VAN levels that induce biofilm formation and reduce efficacy of VAN in the in vitro catheter-associated biofilms may contribute to suboptimal treatment outcomes for endovascular infections caused by “VAN-susceptible” MRSA strains.
PMCID: PMC3591927  PMID: 23295925
5.  Phenotypic and Genotypic Characteristics of Persistent Methicillin-Resistant Staphylococcus aureus Bacteremia In Vitro and in an Experimental Endocarditis Model 
The Journal of infectious diseases  2009;199(2):201-208.
Persistent MRSA bacteremia (PB) represents an important subset of Staphylococcus aureus infections and correlates with poor clinical outcomes.
We profiled relevant in vitro phenotypic and genotypic characteristics of MRSA isolates from 39 persons with bacteremia (21 had PB and 18 had resolving bacteremia [RB]). We also compared the intrinsic virulence and responsiveness to vancomycin of selected PB and RB strains in an experimental endocarditis model (IE).
PB and RB isolates differed significantly with regard to several in vitro characteristics that are believed to impact endovascular infections. PB isolates exhibited significantly more resistance to the cationic defensin hNP-1, enhanced membrane fluidity, and substantially greater adhesion to fibronectin, fibrinogen, and endothelial cells. Genotypically, PB isolates had higher frequency of SCCmec II, CC30, and spa 16; and higher rates of agr type III, cap8, tst-1, and cna carriage. Finally, a prototypic PB strain was more resistant to vancomycin treatment in the infective endocarditis model than a RB comparator strain, despite equivalent virulence profiles.
Our findings indicate that PB isolates may have specific virulence signatures that distinguish them from RB isolates. These data suggest that methods might be developed to identify patients at higher risk for PB in real-time, thereby optimizing the effectiveness of anti-MRSA therapeutic strategies.
PMCID: PMC2827482  PMID: 19086913
6.  Disparity in the In Vitro versus In Vivo Regulation of Fibronectin-Binding Proteins by 2 Global Regulators, saeRS and sigB, in Staphylococcus aureus 
Fibronectin-binding protein A plays an important role in Staphylococcus aureus endovascular infections. We characterized the sigB-saeRS-fnbA expression network with fibronectin binding in vitro and in an experimental infective endocarditis (IE) model using parental strains RN6390 and SH1000 and their respective isogenic saeRS mutants. In contrast to the in vitro data, there was no influence of saeRS on fnbA expression in the IE model, yet ex vivo fibronectin binding was reduced in saeRS mutants. Moreover, as opposed to the in vitro findings, sigB appeared to have a positive rather than a negative effect on saeRS expression within cardiac vegetations.
PMCID: PMC2814518  PMID: 19807278
7.  Site-Specific Mutation of the Sensor Kinase GraS in Staphylococcus aureus Alters the Adaptive Response to Distinct Cationic Antimicrobial Peptides 
Infection and Immunity  2014;82(12):5336-5345.
The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ΔgraS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.
PMCID: PMC4249274  PMID: 25287929
8.  MgrA Activates Expression of Capsule Genes, but Not the α-Toxin Gene in Experimental Staphylococcus aureus Endocarditis 
The Journal of Infectious Diseases  2013;208(11):1841-1848.
Background. Staphylococcus aureus produces numerous virulence factors but little is known about their in vivo regulation during an infection.
Methods. The production of capsule and α-toxin, and the expression of their respective genes, cap5 and hla, were analyzed by comparing CYL11481 (derivative of Newman) and its isogenic regulatory mutants in vitro. The temporal expression of cap5 and hla and the regulatory genes in vivo was carried out using a rat infective endocarditis model.
Results. In vitro analyses showed that capsule was positively regulated by MgrA, Agr, Sae, ArlR, and ClpC, and negatively by CodY and SbcDC. The α-toxin was positively regulated by MgrA, Agr, Sae, ArlR, and SbcDC but negatively by ClpC and CodY. In vivo analyses showed that cap5 expression correlated best with mgrA expression, whereas hla expression correlated best with sae expression. Mutation in mgrA drastically reduced cap5 expression in vivo.
Conclusions. Our results suggest that, in vitro, Agr is the most important regulator for capsule and α-toxin production, as well as for cap5 transcription, but SaeR is the most critical for hla transcription. However, in vivo, MgrA is the major transcriptional regulator of capsule, but not α-toxin, whereas saeR expression correlates best with hla expression.
PMCID: PMC3814835  PMID: 23901087
Staphylococcus aureus; virulence; infective endocarditis model; mgrA; capsule; α-toxin
9.  Role of the LytSR Two-Component Regulatory System in Adaptation to Cationic Antimicrobial Peptides in Staphylococcus aureus 
Many host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; the Staphylococcus aureus LytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is the lrgAB operon, which, along with the related cidABC operon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set of S. aureus strains: (i) UAMS-1, (ii) its isogenic ΔlytS and ΔlrgAB mutants, and (iii) plasmid-complemented ΔlytSR and ΔlrgAB mutants. The ΔlytS strain displayed significantly increased in vitro susceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the ΔlrgAB strain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that although lytSR positively regulates transcription of lrgAB, increased HDP/CAP susceptibilities in the ΔlytS mutant were lrgAB independent. Further, parental UAMS-1 (but not the ΔlytS mutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note, lytSR-dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression of mprF and dlt, or CM fluidity. The ΔlytS strain (but not the ΔlrgAB mutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that the lytSR TCRS plays an important role in adaptive responses of S. aureus to CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.
PMCID: PMC3719743  PMID: 23733465
10.  Role of the Serine-Rich Surface Glycoprotein Srr1 of Streptococcus agalactiae in the Pathogenesis of Infective Endocarditis 
PLoS ONE  2013;8(5):e64204.
The binding of bacteria to fibrinogen and platelets are important events in the pathogenesis of infective endocarditis. Srr1 is a serine-rich repeat glycoprotein of Streptococcus agalactiae that binds directly to the Aα chain of human fibrinogen. To assess the impact of Srr1 on the pathogenesis of endocarditis due to S. agalactiae, we first examined the binding of this organism to immobilized human platelets. Strains expressing Srr1 had significantly higher levels of binding to human platelets in vitro, as compared with isogenic Δsrr1 mutants. In addition, platelet binding was inhibited by pretreatment with anti-fibrinogen IgG or purified Srr1 binding region. To assess the contribution of Srr1 to pathogenicity, we compared the relative virulence of S. agalactiae NCTC 10/84 strain and its Δsrr1 mutant in a rat model of endocarditis, where animals were co-infected with the WT and the mutant strains at a 1∶1 ratio. At 72 h post-infection, bacterial densities (CFU/g) of the WT strain within vegetations, kidneys, and spleens were significantly higher, as compared with the Δsrr1 mutant. These results indicate that Srr1 contributes to the pathogenesis of endocarditis due to S. agalactiae, at least in part through its role in fibrinogen-mediated platelet binding.
PMCID: PMC3662765  PMID: 23717569
11.  Telavancin in Therapy of Experimental Aortic Valve Endocarditis in Rabbits Due to Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus 
Antimicrobial Agents and Chemotherapy  2012;56(11):5528-5533.
A number of cases of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains that have developed daptomycin resistance (DAP-R) have been reported. Telavancin (TLV) is a lipoglycopeptide agent with a dual mechanism of activity (cell wall synthesis inhibition plus depolarization of the bacterial cell membrane). Five recent daptomycin-susceptible (DAP-S)/DAP-R MRSA isogenic strain pairs were evaluated for in vitro TLV susceptibility. All five DAP-R strains (DAP MICs ranging from 2 to 4 μg/ml) were susceptible to TLV (MICs of ≤0.38 μg/ml). In vitro time-kill analyses also revealed that several TLV concentrations (1-, 2-, and 4-fold MICs) caused rapid killing against the DAP-R strains. Moreover, for 3 of 5 DAP-R strains (REF2145, A215, and B2.0), supra-MICs of TLV were effective at preventing regrowth at 24 h of incubation. Further, the combination of TLV plus oxacillin (at 0.25× or 0.50× MIC for each agent) increased killing of DAP-R MRSA strains REF2145 and A215 at 24 h (∼2-log and 5-log reductions versus TLV and oxacillin alone, respectively). Finally, using a rabbit model of aortic valve endocarditis caused by DAP-R strain REF2145, TLV therapy produced a mean reduction of >4.5 log10 CFU/g in vegetations, kidneys, and spleen compared to untreated or DAP-treated rabbits. Moreover, TLV-treated rabbits had a significantly higher percentage of sterile tissue cultures (87% in vegetations and 100% in kidney and spleen) than all other treatment groups (P < 0.0001). Together, these results demonstrate that TLV has potent bactericidal activity in vitro and in vivo against DAP-R MRSA isolates.
PMCID: PMC3486568  PMID: 22890759
13.  In vitro Endothelial Cell Damage is Positively Correlated with Enhanced Virulence and Poor Vancomycin Responsiveness in Experimental Endocarditis due to Methicillin Resistant Staphylococcus aureus 
Cellular microbiology  2011;13(10):1530-1541.
The pathogenesis of Staphylococcus aureus infective endocarditis (IE) is postulated to involve invasion and damage of endothelial cells (ECs). However, the precise relationships between S. aureus – EC interactions in vitro and IE virulence and treatment outcomes in vivo are poorly defined. Ten methicillin-resistant S. aureus (MRSA) clinical isolates previously tested for their virulence and vancomycin responsiveness in an experimental IE model were assessed in vitro for their hemolytic activity, protease production, and capacity to invade and damage ECs. There was a significant positive correlation between the in vitro EC damage caused by these MRSA strains and their virulence during experimental IE (in terms of bacterial densities in target tissues; P < 0.02). Importantly, higher EC damage was also significantly correlated with poor microbiologic response to vancomycin in the IE model (P < 0.001). Interestingly, the extent of EC damage was unrelated to a strain's ability to invade ECs, hemolytic activity and protease production, or β-toxin gene transcription. Inactivation of the agr locus in two MRSA strains caused ∼20% less damage as compared to the corresponding parental strains, indicating that a functional agr is required for maximal EC damage induction. Thus, MRSA-induced EC damage in vitro is a unique virulence phenotype that is independent of many other prototypical MRSA virulence factors, and may be a key biomarker for predicting MRSA virulence potential and antibiotic outcomes during endovascular infections.
PMCID: PMC3173605  PMID: 21777408
14.  The Staphylococcus aureus Two-Component Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial Peptides 
Infection and Immunity  2012;80(1):74-81.
The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, the mechanism(s) by which GraRS is induced, regulated, and modulated remain undefined. In this study, we used two well-characterized MRSA strains (Mu50 and COL) and their respective mutants of graR and vraG (encoding the ABC transporter-dependent efflux pump immediately downstream of graRS), and show that (i) the expression of two key determinants of net positive surface charge (mprF and dlt) is dependent on the cotranscription of both graR and vraG, (ii) reduced expression of mprF and dlt in graR mutants was phenotypically associated with reduced surface-positive charge, (iii) this net reduction in surface-positive charge in graR and vraG mutants, in turn, correlated with enhanced killing by a range of CAPs of diverse structure and origin, including those from mammalian platelets (tPMPs) and neutrophils (hNP-1) and from bacteria (polymyxin B), and (iv) the synthesis and translocation of membrane lysyl-phosphatidylglycerol (an mprF-dependent function) was substantially lower in graR and vraG mutants than in parental strains. Importantly, the inducibility of mprF and dlt transcription via the graRS-vraFG pathway was selective, with induction by sublethal exposure to the CAPs, RP-1 (platelets), and polymyxin B, but not by other cationic molecules (hNP-1, vancomycin, gentamicin, or calcium-daptomycin). Although graR regulates expression of vraG, the expression of graR was codependent on an intact downstream vraG locus. Collectively, these data support an important role of the graRS and vraFG loci in the sensing of and response to specific CAPs involved in innate host defenses.
PMCID: PMC3255649  PMID: 21986630
15.  Divergent Responses of Different Endothelial Cell Types to Infection with Candida albicans and Staphylococcus aureus 
PLoS ONE  2012;7(6):e39633.
Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.
PMCID: PMC3382135  PMID: 22745797
16.  Relationship of agr Expression and Function with Virulence and Vancomycin Treatment Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus ▿ 
Antimicrobial Agents and Chemotherapy  2011;55(12):5631-5639.
The accessory gene regulator (agr) locus has been shown to be important for virulence in several animal models of Staphylococcus aureus infection. However, the role of agr in human infections, and specifically in antibiotic treatment, is controversial. Interestingly, agr dysfunction has been associated with reduced vancomycin responses. To systematically investigate the role of agr in virulence and treatment outcome in the context of endovascular infection, 10 well-characterized vancomycin-susceptible methicillin-resistant S. aureus (MRSA) bloodstream isolates (5 agr-I [clonal complex 45, or CC45] and 5 agr-II [CC5]) were studied for (i) agr function, (ii) RNAIII transcriptional profiles, (iii) agr locus sequences, (iv) intrinsic virulence and responses to vancomycin therapy in an experimental infective endocarditis (IE) model, and (v) in vivo RNAIII expression. Significant differences in agr function (determined by delta-hemolysin activity) correlated with the time point of RNAIII transcription (earlier RNAIII onset equals increased agr function). Unexpectedly, four MRSA strains with strong delta-hemolysin activities exhibited significant resistance to vancomycin treatment in experimental IE. In contrast, five of six MRSA strains with weak or no delta-hemolysin activity were highly susceptible to vancomycin therapy in the IE model. agr sequence analyses showed no common single-nucleotide polymorphism predictive of agr functionality. In vivo RNAIII expression in cardiac vegetations did not correlate with virulence or vancomycin treatment outcomes in the IE model. Inactivation of agr in two strains with strong delta-hemolysin activity did not affect virulence or the in vivo efficacy of vancomycin. Our findings suggest that agr dysfunction does not correlate with vancomycin treatment failures in this experimental IE model in two distinct MRSA genetic backgrounds.
PMCID: PMC3232782  PMID: 21968365
17.  Efficacy of NZ2114, a Novel Plectasin-Derived Cationic Antimicrobial Peptide Antibiotic, in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus ▿ 
Antimicrobial Agents and Chemotherapy  2011;55(11):5325-5330.
Cationic antimicrobial peptides (CAPs) play important roles in host immune defenses. Plectasin is a defensin-like CAP isolated from the saprophytic fungus Pseudoplectania nigrella. NZ2114 is a novel variant of plectasin with potent activity against Gram-positive bacteria. In this study, we investigated (i) the in vivo pharmacokinetic and pharmacodynamic (PK/PD) characteristics of NZ2114 and (ii) the in vivo efficacy of NZ2114 in comparison with those of two conventional antibiotics, vancomycin or daptomycin, in an experimental rabbit infective endocarditis (IE) model due to a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591). All NZ2114 regimens (5, 10, and 20 mg/kg of body weight, intravenously [i.v.], twice daily for 3 days) significantly decreased MRSA densities in cardiac vegetations, kidneys, and spleen versus those in untreated controls, except in one scenario (5 mg/kg, splenic MRSA counts). The efficacy of NZ2114 was clearly dose dependent in all target tissues. At 20 mg/kg, NZ2114 showed a significantly greater efficacy than vancomycin (P < 0.001) and an efficacy similar to that of daptomycin. Of importance, only NZ2114 (in 10- and 20-mg/kg regimens) prevented posttherapy relapse in cardiac vegetations, kidneys, and spleen, while bacterial counts in these target tissues continued to increase in vancomycin- and daptomycin-treated animals. These in vivo efficacies were equivalent and significantly correlated with three PK indices investigated: fCmax/MIC (the maximum concentration of the free, unbound fraction of a drug in serum divided by the MIC), fAUC/MIC (where AUC is the area under the concentration-time curve), and f%T>MIC (%T>MIC is the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions), as analyzed by a sigmoid maximum-effect (Emax) model (R2 > 0.69). The superior efficacy of NZ2114 in this MRSA IE model suggests the potential for further development of this compound for treating serious MRSA infections.
PMCID: PMC3195053  PMID: 21859940
18.  Combinatorial Phenotypic Signatures Distinguish Persistent from Resolving Methicillin-Resistant Staphylococcus aureus Bacteremia Isolates ▿  
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.
PMCID: PMC3028773  PMID: 21098242
19.  A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors 
PLoS Pathogens  2011;7(7):e1002112.
GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspBBR), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspBBR structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspBBR-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.
Author Summary
The binding of bacteria to human platelets is thought to be important for development of infective endocarditis, a life-threatening infection of the cardiovascular system. Streptococcus gordonii is a leading cause of endocarditis. This pathogen uses a protein called GspB to attach to carbohydrates on human platelets. While this binding interaction appears to be mediated by a specific, contiguous domain within GspB, little is known about the molecular details of the interaction between GspB and the carbohydrate receptors on its human host. We therefore determined the crystal structure of the region of GspB that binds to platelet carbohydrates, both alone and in complex with a synthetic carbohydrate receptor. Using this structure as a guide, we were able to produce three strains of S. gordonii that lacked the ability to bind to platelet carbohydrates. One of these isogenic variants was studied more in-depth and lacked the ability to bind to human platelets in vitro and was reduced in virulence when tested in vivo. These studies provide the first structural information detailing the molecular interactions between any serine-rich repeat adhesin and its host receptor, and identify how different, related adhesins may have evolved different specificities for host receptors.
PMCID: PMC3131266  PMID: 21765814
20.  Daptomycin-Oxacillin Combinations in Treatment of Experimental Endocarditis Caused by Daptomycin-Nonsusceptible Strains of Methicillin-Resistant Staphylococcus aureus with Evolving Oxacillin Susceptibility (the “Seesaw Effect”)▿  
In vivo development of daptomycin resistance (DAPr) among Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA) strains, in conjunction with clinical treatment failures, has emerged as a major problem. This has raised the question of DAP-based combination regimens to enhance efficacy against such strains. We studied five recent DAP-susceptible (DAPs)/DAPr clinical MRSA strain pairs obtained from patients who failed DAP monotherapy regimens, as well as one DAPs/DAPr MRSA strain pair in which the resistant strain was generated by in vitro passage in DAP. Of note, we identified a DAP-oxacillin (OX) “seesaw” phenomenon in vitro in which development of DAPr was accompanied by a concomitant fall in OX resistance, as demonstrated by 3- to 4-fold decreases in the OX MIC, a susceptibility shift by population analyses, and enhanced early killing by OX in time-kill assays. In addition, the combination of DAP and OX exerted modest improvement in in vitro bactericidal effects. Using an experimental model of infective endocarditis and two DAPs/DAPr strain pairs, we demonstrated that (i) OX monotherapy was ineffective at clearing DAPr strains from any target tissue in this model (heart valve, kidneys, or spleen) and (ii) DAP-OX combination therapy was highly effective in DAPr strain clearances from these organs. The mechanism(s) of the seesaw effect remains to be defined but does not appear to involve excision of the staphylococcal cassette chromosome mec (SCCmec) that carries mecA.
PMCID: PMC2916313  PMID: 20547804
21.  Enhanced expression of dltABCD is associated with development of daptomycin nonsusceptibility in a clinical endocarditis isolate of Staphylococcus aureus 
The Journal of infectious diseases  2009;200(12):1916-1920.
Using isogenic clinical bloodstream Staphylococcus aureus strains from a patient with relapsing endocarditis, we investigated transcriptional profiles of mprF and dlt genes in the context of cell surface charge and daptomycin nonsusceptibility. As in prior studies, a point mutation within mprF was observed in the daptomycin-nonsusceptible strain. However, neither the transcriptional profile of mprF, nor membrane phospholipid analyses were compatible with the anticipated mprF gain-in-function phenotype. In contrast, we demonstrated enhanced dlt expression coincident with increased positive surface charge and reduced daptomycin binding.
PMCID: PMC2779839  PMID: 19919306
Staphylococcus aureus; daptomycin; cationic antimicrobial peptide (CAP); mprF; dlt
22.  Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen 
PLoS Pathogens  2010;6(8):e1001047.
The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis.
Author Summary
The binding of bacteria to human platelets is thought to be a central event in the development of endocarditis (a life-threatening cardiovascular infection). We have previously found that platelet binding by Streptococcus mitis is mediated by surface components encoded by a bacteriophage contained within the host bacterium. We now show that lysin (an enzyme of bacteriophage origin) contributes to platelet binding via its direct interaction with fibrinogen on the platelet surface. Lysin bound to purified fibrinogen in vitro, and this interaction specifically involved the Aα and Bβ chains of fibrinogen. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the gene encoding lysin gene resulted in a significant reduction in binding to fibrinogen by S. mitis, as well as a major reduction in virulence, as measured by a rat model of endocarditis. These results indicate that lysin is a multifunctional protein, representing a new class of fibrinogen-binding molecules. Lysin is localized to the bacterial surface via its interaction with cell wall choline, where it then can bind fibrinogen directly. Cell surface lysin apparently also contributes to the development of endovascular infections via its previously unrecognized fibrinogen binding activity.
PMCID: PMC2920869  PMID: 20714354
23.  Tricarboxylic Acid Cycle-Dependent Attenuation of Staphylococcus aureus In Vivo Virulence by Selective Inhibition of Amino Acid Transport▿  
Infection and Immunity  2009;77(10):4256-4264.
Staphylococci are the leading causes of endovascular infections worldwide. Commonly, these infections involve the formation of biofilms on the surface of biomaterials. Biofilms are a complex aggregation of bacteria commonly encapsulated by an adhesive exopolysaccharide matrix. In staphylococci, this exopolysaccharide matrix is composed of polysaccharide intercellular adhesin (PIA). PIA is synthesized when the tricarboxylic acid (TCA) cycle is repressed. The inverse correlation between PIA synthesis and TCA cycle activity led us to hypothesize that increasing TCA cycle activity would decrease PIA synthesis and biofilm formation and reduce virulence in a rabbit catheter-induced model of biofilm infection. TCA cycle activity can be induced by preventing staphylococci from exogenously acquiring a TCA cycle-derived amino acid necessary for growth. To determine if TCA cycle induction would decrease PIA synthesis in Staphylococcus aureus, the glutamine permease gene (glnP) was inactivated and TCA cycle activity, PIA accumulation, biofilm forming ability, and virulence in an experimental catheter-induced endovascular biofilm (endocarditis) model were determined. Inactivation of this major glutamine transporter increased TCA cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in the endocarditis model in terms of achievable bacterial densities in biofilm-associated cardiac vegetations, kidneys, and spleen. These data confirm the close linkage of TCA cycle activity and virulence factor production and establish that this metabolic linkage can be manipulated to alter infectious outcomes.
PMCID: PMC2747957  PMID: 19667045
24.  Factors Influencing Time to Vancomycin-Induced Clearance of Nonendocarditis Methicillin-Resistant Staphylococcus aureus Bacteremia: Role of Platelet Microbicidal Protein Killing and agr Genotypes 
Vancomycin susceptibility, the accessory gene global regulator (agr) genotype and function, staphylococcal cassette chromosome (SCC) mec type, and susceptibility to cationic thrombin-induced platelet microbicidal protein 1 (tPMP-1) have been individually predictive of duration of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. This investigation evaluated the interrelationship of these factors with time to clearance of MRSA bacteremia during vancomycin therapy in patients without endocarditis.
Vancomycin minimum inhibitory concentration and in vitro killing, agr function (δ-hemolysin activity), agr group, SCCmec type, and survival in tPMP-1 killing assays were determined for 29 MRSA bacteremia isolates.
Increased resistance to tPMP-1 killing was observed with agr group III MRSA (P =.025) and MRSA with reduced or absent agr function (P =.023). The median time to clearance of MRSA bacteremia was earlier for agr group III (3 days) versus group I (10.5 days) or II (15 days) (P =.001). In multivariate analysis, agr group II, reduced tPMP-1 killing in vitro, and prior vancomycin exposure were significant independent predictors of longer MRSA bacteremia duration.
Specific genotypic, phenotypic, and clinical parameters appear to correlate with persistent MRSA bacteremia. The interrelationship of these and other factors probably contributes to vancomycin-mediated clearance of MRSA bacteremia.
PMCID: PMC2819315  PMID: 20001853
25.  Regulation of mprF in Daptomycin-Nonsusceptible Staphylococcus aureus Strains▿  
We used a well-characterized isogenic set of clinical bloodstream Staphylococcus aureus strains to study (i) regulation of mprF-mediated phosphatidylglycerol lysinylation in the contexts of in vitro daptomycin (DAP) nonsuceptibility and (ii) the role of mprF mutation in endovascular virulence. We observed a correlation between increased expression of a mutant mprF gene and reduced in vitro DAP susceptibility. There were no detectable fitness differences between strains in experimental infective endocarditis.
PMCID: PMC2687189  PMID: 19289517

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