A synthetic congener modeled on a microbicidal domain of thrombin- induced platelet microbicidal protein 1 recapitulates staphylocidal mechanisms of the native molecule

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal alpha-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains for the following quantitative evaluations: staphylocidal efficacy; comparative MIC; membrane permeabilization (MP) and depolarization; and DNA, RNA, and protein synthesis. Although the proteins had similar MICs, RP-1 caused significant killing of ISP479C (<50% survival), correlating with extensive MP (>95%) and inhibition of DNA and RNA synthesis (>90%), versus substantially reduced killing of ISP479R (>80% survival), with less MP (55%) and less inhibition of DNA or RNA synthesis (70 to 80%). Interestingly, RP-1-induced protein synthesis inhibition was equivalent in both strains. RP-1 did not depolarize the cell membrane and caused a relatively short postexposure growth inhibition. These data closely parallel those previously reported for tPMP-1 against this strain set and exemplify how synthetic molecules can be engineered to reflect structure-activity relationships of functional domains in native host defense effector molecules.     

Susceptibility to thrombin-induced platelet microbicidal protein is associated with increased fluconazole efficacy against experimental endocarditis due to Candida albicans

Platelet microbicidal proteins (PMPs) are believed to be integral to host defense against endovascular infection. We previously demonstrated that susceptibility to thrombin-induced PMP 1 (tPMP-1) in vitro negatively influences Candida albicans virulence in the rabbit model of infective endocarditis (IE). This study evaluated the relationship between in vitro tPMP-1 susceptibility (tPMP-1s) or resistance (tPMP-1r) and efficacy of fluconazole (FLU) therapy of IE due to C. albicans. Candida IE was established in rabbits with either tPMP-1s or tPMP-1r strains. Treatment groups received FLU (100 mg/kg/day) intraperitoneally for 7 or 14 days; control animals received no therapy. At these time points, cardiac vegetations, kidneys, and spleens were quantitatively cultured to assess fungal burden. At both 7 and 14 days and in all target tissues, the extent of candidal clearance by FLU was greater in animals infected with the tPMP-1s strain than in those infected with the tPMP-1r strain. These differences were statistically significant in the spleen and kidney. Corroborating these in vivo data, FLU (a candidastatic agent), in combination with tPMP-1, exerted an enhanced fungicidal effect in vitro against tPMP-1s and tPMP-1r C. albicans, with the extent of this effect greatest against the tPMP-1s strain. Collectively, these results support the concept that tPMP-1 susceptibility contributes to the net efficacy of FLU against C. albicans IE in vivo, particularly in tissues in which platelets and tPMP-1 likely play significant roles in host defense.     

Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action.

Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP-1. Reconstitution of Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.     

In vitro antibacterial activities of platelet microbicidal protein and neutrophil defensin against Staphylococcus aureus are influenced by antibiotics differing in mechanism of action

Thrombin-induced platelet microbicidal protein-1 (tPMP-1) and human neutrophil defensin-1 (HNP-1) are small, cationic antimicrobial peptides. These peptides exert potent in vitro microbicidal activity against a broad spectrum of human pathogens, including Staphylococcus aureus. Evidence suggests that tPMP-1 and HNP-1 target and disrupt the bacterial membrane. However, it is not yet clear whether membrane disruption itself is sufficient to kill the bacterium or whether subsequent, presumably intracellular, events are also involved in killing. We investigated the staphylocidal activities of tPMP-1 and HNP-1 in the presence or absence of pretreatment with antibiotics that differ in their mechanisms of action. The staphylocidal effects of tPMP-1 and HNP-1 on control cells (no antibiotic pretreatment) were rapid and concentration dependent. Pretreatment of S. aureus with either penicillin or vancomycin (bacterial cell wall synthesis inhibitors) significantly enhanced the anti-S. aureus effects of tPMP-1 compared with the effects against the respective control cells over the entire tPMP-1 concentration range tested (P < 0.05). Similarly, S. aureus cells pretreated with these antibiotics were more susceptible to HNP-1 than control cells, although the difference in the effects against cells that received penicillin pretreatment did not reach statistical significance (P < 0.05 for cells that received vancomycin pretreatment versus effects against control cells). Studies with isogenic pairs of strains with normal or deficient autolytic enzyme activities demonstrated that enhancement of S. aureus killing by cationic peptides and cell wall-active agents could not be ascribed to a predominant role of autolytic enzyme activation. Pretreatment of S. aureus cells with tetracycline, a 30S ribosomal subunit inhibitor, significantly decreased the staphylocidal effect of tPMP-1 over a wide peptide concentration range (0.16 to 1.25 microgram/ml) (P < 0.05). Furthermore, pretreatment with novobiocin (an inhibitor of bacterial DNA gyrase subunit B) and with azithromycin, quinupristin, or dalfopristin (50S ribosomal subunit protein synthesis inhibitors) essentially blocked the S. aureus killing resulting from exposure to tPMP-1 or HNP-1 at most concentrations compared with the effects against the respective control cells (P < 0.05 for a tPMP-1 concentration range of 0.31 to 1.25 microgram/ml and for an HNP-1 concentration range of 6.25 to 50 microgram/ml). These findings suggest that tPMP-1 and HNP-1 exert anti-S. aureus activities through mechanisms involving both the cell membrane and intracellular targets.     

Rapid bactericidal activity of daptomycin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus peritonitis in mice as measured with bioluminescent bacteria

The rising rates of antibiotic resistance accentuate the critical need for new antibiotics. Daptomycin is a new antibiotic with a unique mode of action and a rapid in vitro bactericidal effect against gram-positive organisms. This study examined the kinetics of daptomycin's bactericidal action against peritonitis caused by methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in healthy and neutropenic mice and compared this activity with those of other commonly used antibiotics. CD-1 mice were inoculated intraperitoneally with lethal doses of MSSA (Xen-29) or MRSA (Xen-1), laboratory strains transformed with a plasmid containing the lux operon, which confers bioluminescence. One hour later, the animals were given a single dose of daptomycin at 50 mg/kg of body weight subcutaneously (s.c.), nafcillin at 100 mg/kg s.c., vancomycin at 100 mg/kg s.c., linezolid at 100 mg/kg via gavage (orally), or saline (10 ml/kg s.c.). The mice were anesthetized hourly, and photon emissions from living bioluminescent bacteria were imaged and quantified. The luminescence in saline-treated control mice either increased (neutropenic mice) or remained relatively unchanged (healthy mice). In contrast, by 2 to 3 h postdosing, daptomycin effected a 90% reduction of luminescence of MSSA or MRSA in both healthy and neutropenic mice. The activity of daptomycin against both MSSA and MRSA strains was superior to those of nafcillin, vancomycin, and linezolid. Against MSSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than nafcillin or linezolid. Against MRSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than vancomycin or linezolid. The rapid decrease in the luminescent signal in the daptomycin-treated neutropenic mice underscores the potency of this antibiotic against S. aureus in the immune-suppressed host.     

Beta-lactams versus glycopeptides in treatment of subcutaneous abscesses infected with Staphylococcus aureus.

The antibacterial efficacies of the beta-lactam antibiotics nafcillin and cefazolin were compared with those of the glycopeptide antibiotics vancomycin and teicoplanin in rats with subcutaneous abscesses infected with methicillin-susceptible Staphylococcus aureus. Animals were treated with antibiotics or diluent for 3 or 7 days. Rats receiving any antibiotic treatment, with the exception of teicoplanin-treated animals at day 7, had lower bacterial counts in their abscesses than did controls at days 3 and 7. Rats in the nafcillin and cefazolin treatment groups had lower bacterial counts in their abscesses than did rats in the vancomycin and teicoplanin treatment groups at days 3 and 7. The beta-lactam antibiotics were more effective therapy than the glycopeptide antibiotics in rats with subcutaneous abscesses infected with methicillin-susceptible S. aureus. In vitro, animal, and clinical data comparing beta-lactam and glycopeptide treatment of S. aureus infections were reviewed along with the resulting treatment recommendations.     

Thrombin-induced platelet microbicidal protein susceptibility phenotype influences the outcome of oxacillin prophylaxis and therapy of experimental Staphylococcus aureus endocarditis

We previously showed that in vitro susceptibility profiles of Staphylococcus aureus to thrombin-induced platelet microbicidal protein 1 (tPMP-1) impacted the outcome of vancomycin treatment in experimental infective endocarditis. In this same model, treatment with oxacillin (a more rapid staphylocidal agent than vancomycin) enhanced the clearance of both tPMP-1-susceptible and -resistant cells from vegetations. The extent of clearance was greater for tPMP-1-susceptible cells.     

Enhancement of the Effects of Anti-Staphylococcal Antibiotics by Aminoglycosides

The in vitro activity of seven anti-staphylococcal antibiotics alone and in combination with four aminoglycoside antibiotics against 35 clinical isolates of Staphylococcus aureus from blood cultures of patients with endocarditis or septicemia was studied. The combination of nafcillin-gentamicin or nafcillin-tobramycin when compared with nafcillin alone killed significantly more S. aureus at 6 h for 33 of 35 isolates. A significant decrease in viable colony-forming units at 24 and 48 h was demonstrated for a smaller number of isolates. Using the agar dilution antibiotic susceptibility test, significant enhancement of activity was demonstrated against almost all isolates when cephalothin, cefazolin, or clindamycin was combined with gentamicin, tobramycin, or kanamycin. The combination of nafcillin or vancomycin with gentamicin, tobramycin, or kanamycin also showed enhanced activity against the majority of isolates. Oxacillin or methicillin when combined with the aminoglycosides showed enhancement of activity against the least number of isolates. Streptomycin was the least effective of the four aminoglycoside antibiotics studied.     

Low-level resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein 1 in vitro associated with qacA gene carriage is independent of multidrug efflux pump activity

Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.     

In vitro evaluation of the antibiotic lock technique (ALT) for the treatment of catheter-related infections caused by staphylococci

OBJECTIVES: To investigate appropriate antimicrobial agents, the optimal concentration and treatment duration for the antibiotic lock technique (ALT) to treat catheter-related infections caused by Staphylococcus epidermidis and Staphylococcus aureus. METHODS: We evaluated the bacterial killing activity of vancomycin, teicoplanin, ciprofloxacin, rifampicin, cefazolin, gentamicin, nafcillin and erythromycin against biofilms formed by two strains of S. aureus and two strains of S. epidermidis. The effectiveness of the antibiotic lock was assayed after exposure to antibiotics (1, 5 and 10 mg/mL) for 1, 3, 5, 7, 10 or 14 days using an in vitro model of biofilms on polyurethane film. RESULTS: The biofilms were completely sterile after exposure to vancomycin (5 mg/mL) for 5 days and teicoplanin (5 and 10 mg/mL) for 7 days. Ciprofloxacin and rifampicin (both 5 mg/mL) achieved eradication of the biofilms of both staphylococcal species more rapidly than vancomycin or teicoplanin. Significant biofilm eradication was not achieved with cefazolin, nafcillin, gentamicin and erythromycin at any of the time exposures examined. CONCLUSIONS: The data suggest that 5 mg/mL vancomycin, ciprofloxacin or rifampicin can eradicate S. epidermidis and S. aureus biofilms within 5 days. These findings warrant prospective clinical trials for the evaluation of the clinical efficacy of ALT for less than 10 days.     

Comparative activity of CGP 31608, nafcillin, cefamandole, imipenem, and vancomycin against methicillin-susceptible and methicillin-resistant staphylococci.

The activity of CGP 31608 for 53 strains of Staphylococcus aureus and 48 strains of S. epidermidis, both methicillin susceptible and resistant, was compared with that of vancomycin, nafcillin, cefamandole, and imipenem. Microdilution MICs were determined in Mueller-Hinton broth with or without 2.5% NaCl at an inoculum of 3 x 10(5) CFU/ml with a 20-h, 37 degrees C incubation. The MICs of imipenem and CGP 31608 for methicillin-resistant strains were lower than the MICs of nafcillin or cefamandole for these strains; these differences diminished in the presence of 2.5% NaCl. Subpopulations were detected in strains of methicillin-resistant S. aureus and S. epidermidis that were resistant to all the beta-lactam antibiotics tested at 5 micrograms/ml. This resistant subpopulation produced progeny that were uniformly resistant to high concentrations of each of the beta-lactams.     

Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells

Most antibiotics with bactericidal activity require that the bacteria be actively dividing to produce rapid killing. However, in many infections, such as endocarditis, prosthetic joint infections, and infected embedded catheters, the bacteria divide slowly or not at all. Daptomycin is a lipopeptide antibiotic with a distinct mechanism of action that targets the cytoplasmic membrane of gram-positive organisms, including Staphylococcus aureus. Daptomycin is rapidly bactericidal against exponentially growing bacteria (a 3-log reduction in 60 min). The objectives of this study were to determine if daptomycin is bactericidal against nondividing S. aureus and to quantify the extent of the bactericidal activity. In high-inoculum methicillin-sensitive S. aureus cultures in stationary phase (10(10) CFU/ml), daptomycin displayed concentration-dependent bactericidal activity, requiring 32 micro/ml to achieve a 3-log reduction. In a study comparing several antibiotics at 100 microg/ml, daptomycin demonstrated faster bactericidal activity than nafcillin, ciprofloxacin, gentamicin, and vancomycin. In experiments where bacterial cell growth was halted by the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or erythromycin, daptomycin (10 microg/ml) achieved the bactericidal end point (a 3-log reduction) within 2 h. In contrast, ciprofloxacin (10 microg/ml) did not produce bactericidal activity. Daptomycin (2 microg/ml) remained bactericidal against cold-arrested S. aureus, which was protected from the actions of ciprofloxacin and nafcillin. The data presented here suggest that, in contrast to that of other classes of antibiotics, the bactericidal activity of daptomycin does not require cell division or active metabolism, most likely as a consequence of its direct action on the bacterial membrane.     

Combinations of lysostaphin with beta-lactams are synergistic against oxacillin-resistant Staphylococcus epidermidis

Oxacillin-resistant Staphylococcus aureus is rapidly killed by the endopeptidase lysostaphin, and the addition of beta-lactam antibiotics provides synergistic killing. We investigated the possibility that beta-lactams given in combination with lysostaphin would improve the activity of lysostaphin against oxacillin-resistant Staphylococcus epidermidis (ORSE), which is normally less susceptible to lysostaphin. Checkerboard synergy testing was performed for lysostaphin given in combination with oxacillin against 10 ORSE isolates for which the lysostaphin MICs were > o r= 8 microg/ml. The fractional inhibitory concentration index ranged from 0.0234 to 0.2656, indicating synergy, which was confirmed in growth curve experiments. In the rabbit model of experimental aortic valve endocarditis using an ORSE strain, the combination of lysostaphin and nafcillin was as effective as vancomycin alone and significantly better than lysostaphin or nafcillin alone. We conclude that beta-lactam antibiotics given in combination with lysostaphin are synergistic against many strains of ORSE.     

Low-level methicillin resistance in strains of Staphylococcus aureus.

Two strains of Staphylococcus aureus expressing borderline or low-level methicillin resistance by one or more in vitro test methods were examined for resistance in vivo and for biochemical and genetic markers of methicillin resistance. In vivo, nafcillin was equally effective against experimental aortic valve endocarditis in rabbits, regardless of whether they were infected by a fully susceptible or a low-level-resistant strain. Resistance did not emerge during therapy. For the more resistant of the two low-level-resistant strains, methicillin was as effective as nafcillin. Nafcillin was ineffective against endocarditis caused by a truly methicillin-resistant strain, and resistance emerged on therapy. The low-level-resistant strains did not produce the low-affinity penicillin-binding protein 2a that is associated with methicillin resistance and did not contain DNA that hybridized with probes that recognized the methicillin resistance determinant. Low-level resistance in S. aureus is a phenomenon that is biochemically and genetically distinct from true methicillin resistance. These strains actually are susceptible to beta-lactam antibiotics. The clinical problem posed by these strains is not a therapeutic one but, instead, one of how to differentiate them from those that are truly methicillin resistant.     

Teicoplanin versus nafcillin and vancomycin in the treatment of experimental endocarditis caused by methicillin-susceptible or -resistant Staphylococcus aureus.

In rabbits with experimentally induced endocarditis, the efficacy of teicoplanin compared favorably both with that of nafcillin for infection by a methicillin-susceptible strain of Staphylococcus aureus and with that of vancomycin for infection by a methicillin-resistant strain of S. aureus. In a 4-day treatment regimen, teicoplanin was as effective as either nafcillin or vancomycin in eliminating organisms from aortic valve vegetations in the respective infection. In a 10-day regimen for methicillin-resistant S. aureus endocarditis, both teicoplanin and vancomycin sterilized the vegetations of some rabbits, but the relapse rate was high for both. These results justify further investigation into the role of teicoplanin for the treatment of serious infections caused by S. aureus.     

In vitro antimicrobial activity of aztreonam alone and in combination against bacterial isolates from pediatric patients

We examined 134 pediatric clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and gram-positive cocci for susceptibility to aztreonam alone and in combination with seven other antibiotics. All 98 gram-negative isolates were susceptible to aztreonam with similar inhibitory and bactericidal activity. Combinations of aztreonam with cefoxitin, ampicillin, or clindamycin were generally indifferent or additive. Synergism was occasionally seen against enteric organisms with aztreonam plus cefoxitin or clindamycin. Combinations of tobramycin and aztreonam were synergistic (62%) against P. aeruginosa; aztreonam plus piperacillin or ticarcillin was additive. Aztreonam did not affect the activity of nafcillin against Staphylococcus aureus, or of ampicillin against species of Streptococcus group B or D. Antagonism was seen only with aztreonam plus cefoxitin against Enterobacter species, but not at clinically significant concentrations. Several combinations of antibiotics with aztreonam should be appropriate for initial therapy of infections in children without major risks of antibacterial antagonism.     

Coagulase-negative staphylococci resistant to beta-lactam antibiotics in vivo produce penicillin-binding protein 2a.

Strains of coagulase-negative staphylococci were tested for in vivo resistance in a rabbit model of prophylaxis of endocarditis. Regimens of nafcillin, cefazolin, cefamandole, and vancomycin were compared for efficacy in the prevention of infection caused by two methicillin-resistant strains and a susceptible strain. For the two resistant strains, vancomycin was the most effective drug tested. All regimens were effective against the susceptible strain. The two strains for which prophylaxis with beta-lactam antibiotics failed produced a beta-lactam antibiotic-inducible penicillin-binding protein (PBP) that comigrated in sodium dodecyl sulfate-polyacrylamide gels with the low-affinity PBP 2a that is associated with methicillin resistance in strains of Staphylococcus aureus. Like PBP 2a, this PBP had low binding affinity for beta-lactam antibiotics. Peptide maps after either V8 protease or chymotrypsin digestion of radiolabeled PBP 2a or silver-stained preparations were virtually identical to one another and to maps of PBP 2a from a heterogeneous and a homogeneous strain of S. aureus. Methicillin resistance in coagulase-negative staphylococci and therapeutic failure with beta-lactam antibiotics in vivo is associated with production of PBP 2a, which appears to be highly conserved structurally among different species of staphylococci.     

Ciprofloxacin therapy of experimental endocarditis caused by methicillin-susceptible or methicillin-resistant Staphylococcus aureus.

Ciprofloxacin was more effective (P less than 0.01) than either imipenem or nafcillin therapy of experimental methicillin-susceptible Staphylococcus aureus endocarditis in rabbits after 2 or 3 days of treatment. There was no significant difference between results of treatment of methicillin-susceptible S. aureus experimental endocarditis with ciprofloxacin and results with the combination of nafcillin and gentamicin. Ciprofloxacin was more effective (P less than 0.01) than vancomycin therapy of experimental methicillin-resistant S. aureus endocarditis after 3 days of treatment. After 5 days of treatment, there was no significant difference between the results of treatment of experimental methicillin-resistant S. aureus endocarditis with ciprofloxacin and results with vancomycin.     

Combinations of Vancomycin and β-Lactams Are Synergistic against Staphylococci with Reduced Susceptibilities to Vancomycin

Evidence of synergism between combinations of vancomycin and beta-lactam antibiotics against 59 isolates of methicillin-resistant staphylococci (Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus) for which vancomycin MICs ranged from 1 to 16 microg/ml were tested by broth microdilution checkerboard, disk diffusion, agar dilution, and time-kill antimicrobial susceptibility tests. The combination of vancomycin and oxacillin demonstrated synergy by all test methods against 30 of 59 isolates; no antagonism was seen. Synergy with vancomycin was also found by modified disk diffusion testing for ceftriaxone, ceftazidime, cefpodoxime, and amoxicillin-clavulanate but not for aztreonam. Evidence of synergy correlated directly with vancomycin MICs. The efficacy of vancomycin given alone and in combination with nafcillin was tested in the rabbit model of experimental endocarditis caused by three clinical isolates of glycopeptide-intermediate-susceptible S. aureus (GISA) (isolates HIP5827, HIP5836, and MU50). Two of the GISA isolates (isolates MU50 and HIP5836) were extremely virulent in this model, with 27 of 42 (64%) animals dying during the 3-day trial. Therapy with either vancomycin or nafcillin given as a single agent was ineffective for animals infected with HIP5827 or MU50. However, the combination of vancomycin and nafcillin resulted in a mean reduction of 4.52 log10 CFU/g of aortic valvular vegetations per g compared to the reduction for controls for animals infected with HIP5827 and a reduction of 4. 15 log10 CFU/g for animals infected with MU50. Renal abscesses caused by HIP5827 were sterilized significantly better with the combination of vancomycin and nafcillin than by either treatment alone. We conclude that the combination of vancomycin and beta-lactams with antistaphylococcal activity is an effective regimen for the treatment of infections with clinical strains of staphylococci which demonstrate reduced susceptibility to glycopeptides.     

Anti-clumping factor A immunoglobulin reduces the duration of methicillin-resistant Staphylococcus aureus bacteremia in an experimental model of infective endocarditis

SA-IGIV is a human polyclonal immunoglobulin containing elevated levels of antibodies specific for the fibrinogen-binding MSCRAMM protein clumping factor A (ClfA). In vitro, SA-IGIV specifically recognized ClfA that was expressed on the surface of Staphylococcus aureus and inhibited bacterial adherence to immobilized human fibrinogen by >95%. Moreover, SA-IGIV efficiently opsonized ClfA-coated fluorescent beads and facilitated phagocytosis by human polymorphonuclear leukocytes. To determine its potential therapeutic efficacy, SA-IGIV was evaluated in combination with vancomycin in a rabbit model of catheter-induced aortic valve infective endocarditis (IE) caused by methicillin-resistant S. aureus (MRSA). The combination therapy was more effective than vancomycin alone in sterilizing all valvular vegetations when used therapeutically during early (12-h) IE. The combination therapy resulted in clearance of bacteremia that was significantly faster than that of vancomycin alone in animals with well-established (24-h) IE. Therefore, in both early and well-established MRSA IE, the addition of SA-IGIV to a standard antibiotic regimen (vancomycin) increased bacterial clearance from the bloodstream and/or vegetations.