Contribution of Panton-Valentine Leukocidin in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains typically carry genes encoding Panton-Valentine leukocidin (PVL). We used wild-type parental and isogenic PVL-deletion (Δpvl) strains of USA300 (LAC and SF8300) and USA400 (MW2) to test whether PVL alters global gene regulatory networks and contributes to pathogenesis of bacteremia, a hallmark feature of invasive staphylococcal disease. Microarray and proteomic analyses revealed that PVL does not alter gene or protein expression, thereby demonstrating that any contribution of PVL to CA-MRSA pathogenesis is not mediated through interference of global gene regulatory networks. Inasmuch as a direct role for PVL in CA-MRSA pathogenesis remains to be determined, we developed a rabbit bacteremia model of CA-MRSA infection to evaluate the effects of PVL. Following experimental infection of rabbits, an animal species whose granulocytes are more sensitive to the effects of PVL compared with the mouse, we found a contribution of PVL to pathogenesis over the time course of bacteremia. At 24 and 48 hours post infection, PVL appears to play a modest, but measurable role in pathogenesis during the early stages of bacteremic seeding of the kidney, the target organ from which bacteria were not cleared. However, the early survival advantage of this USA300 strain conferred by PVL was lost by 72 hours post infection. These data are consistent with the clinical presentation of rapid-onset, fulminant infection that has been associated with PVL-positive CA-MRSA strains. Taken together, our data indicate a modest and transient positive effect of PVL in the acute phase of bacteremia, thereby providing evidence that PVL contributes to CA-MRSA pathogenesis

A Mapping of Drug Space from the Viewpoint of Small Molecule Metabolism

Small molecule drugs target many core metabolic enzymes in humans and pathogens, often mimicking endogenous ligands. The effects may be therapeutic or toxic, but are frequently unexpected. A large-scale mapping of the intersection between drugs and metabolism is needed to better guide drug discovery. To map the intersection between drugs and metabolism, we have grouped drugs and metabolites by their associated targets and enzymes using ligand-based set signatures created to quantify their degree of similarity in chemical space. The results reveal the chemical space that has been explored for metabolic targets, where successful drugs have been found, and what novel territory remains. To aid other researchers in their drug discovery efforts, we have created an online resource of interactive maps linking drugs to metabolism. These maps predict the “effect space” comprising likely target enzymes for each of the 246 MDDR drug classes in humans. The online resource also provides species-specific interactive drug-metabolism maps for each of the 385 model organisms and pathogens in the BioCyc database collection. Chemical similarity links between drugs and metabolites predict potential toxicity, suggest routes of metabolism, and reveal drug polypharmacology. The metabolic maps enable interactive navigation of the vast biological data on potential metabolic drug targets and the drug chemistry currently available to prosecute those targets. Thus, this work provides a large-scale approach to ligand-based prediction of drug action in small molecule metabolism

In Vitro Selection and Characterization of Ceftobiprole-Resistant Methicillin-Resistant Staphylococcus aureus▿ †

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to β-lactam antibiotics because it expresses penicillin-binding protein 2a (PBP2a), a low-affinity penicillin-binding protein. An investigational broad-spectrum cephalosporin, ceftobiprole (BPR), binds PBP2a with high affinity and is active against MRSA. We hypothesized that BPR resistance could be mediated by mutations in mecA, the gene encoding PBP2a. We selected BPR-resistant mutants by passage in high-volume broth cultures containing subinhibitory concentrations of BPR. We used strain COLnex (which lacks chromosomal mecA) transformed with pAW8 (a plasmid vector only), pYK20 (a plasmid carrying wild-type mecA), or pYK21 (a plasmid carrying a mutant mecA gene corresponding to five PBP2a mutations). All strains became resistant to BPR by day 9 of passaging, but MICs continued to increase until day 21. MICs increased 256-fold (from 1 to 256 μg/ml) for pAW8, 32-fold (from 4 to 128 μg/ml) for pYK20, and 8-fold (from 16 to 128 μg/ml) for pYK21. Strains carrying wild-type or mutant mecA developed six (pYK20 transformants) or four (pYK21 transformants) new mutations in mecA. The transformation of COLnex with a mecA mutant plasmid conferred BPR resistance, and the loss of mecA converted resistant strains into susceptible ones. Modeling studies predicted that several of the mecA mutations altered BPR binding; other mutations may have mediated resistance by influencing interactions with other proteins. Multiple mecA mutations were associated with BPR resistance in MRSA. BPR resistance also developed in the strain lacking mecA, suggesting a role for chromosomal genes

Efficacy of Telavancin in a Rabbit Model of Aortic Valve Endocarditis Due to Methicillin-Resistant Staphylococcus aureus or Vancomycin-Intermediate Staphylococcus aureus

The activities of telavancin and vancomycin were compared in vitro and in the rabbit model of aortic valve endocarditis against a methicillin-resistant Staphylococcus aureus strain, COL, and a vancomycin-intermediate S. aureus (VISA) strain, HIP 5836. Telavancin was bactericidal in time-kill studies at a concentration of 5 μg/ml against both COL and HIP5836. Vancomycin was bacteriostatic at 5 μg/ml and bactericidal at 10 μg/ml against COL and was bacteriostatic at 10 μg/ml against VISA strain HIP 5836. Compared to untreated controls, a twice-daily regimen of 30 mg/kg of telavancin reduced mean aortic valve vegetation titers of the COL strain by 4.7 log(10) CFU/g after 4 days of therapy and sterilized 6/11 vegetations compared to 3.4 log(10) CFU/g with 3/10 vegetations sterilized for a regimen of twice-daily vancomycin, 30 mg/kg; these differences were not statistically significant. Telavancin was significantly more effective than vancomycin in the VISA model, producing a 5.5 log(10) CFU/g reduction versus no reduction in CFU with vancomycin. In experiments comparing 2-day regimens of telavancin at 30 mg/kg and 50 mg/kg twice daily, organisms were rapidly eliminated from vegetations, but the effect was not different between the two doses. These results suggest that telavancin may be an effective treatment for endocarditis and other serious staphylococcal infections accompanied by bacteremia, including infections caused by staphylococci not susceptible to vancomycin

Comparative Efficacies of Tedizolid Phosphate, Vancomycin, and Daptomycin in a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Endocarditis

Tedizolid, the active component of the prodrug tedizolid phosphate, is a novel oxazolidinone that is approximately 4 times more active by weight than linezolid against Staphylococcus aureus in vitro. The in vivo efficacy of tedizolid phosphate (15 mg/kg body weight intravenous [i.v.] twice a day [b.i.d.]) was compared to those of vancomycin (30 mg/kg i.v. b.i.d.) and daptomycin (18 mg/kg i.v. once a day [q.d.]) in a rabbit model of aortic valve endocarditis (AVE) caused by methicillin-resistant S. aureus strain COL (infection inoculum of 10(7) CFU). Median vegetation titers of daptomycin-treated rabbits were significantly lower than those of rabbits treated with tedizolid phosphate (15 mg/kg b.i.d.) (P = 0.016), whereas titers for vancomycin-treated compared to tedizolid-treated rabbits were not different (P = 0.984). The numbers of organisms in spleen and kidney tissues were similar for all treatment groups. A dose-ranging experiment was performed with tedizolid phosphate (2, 4, and 8 mg/kg b.i.d.) compared to vancomycin (30 mg/kg b.i.d.), using a higher infecting inoculum (10(8) CFU) to determine the lowest efficacious dose of tedizolid phosphate. Tedizolid phosphate (2 mg/kg) (equivalent to 60% of the area under the concentration-time curve from 0 to 24 h (AUC0-24) for the human 200-mg dose approved by the U.S. Food and Drug Administration) was not efficacious. Tedizolid phosphate at 4 mg/kg (equivalent to 75% of the AUC0-24 for the human 400-mg dose) and 8 mg/kg produced lower vegetation titers than the control, but neither was as efficacious as vancomycin

A mecA-Negative Strain of Methicillin-Resistant Staphylococcus aureus with High-Level β-Lactam Resistance Contains Mutations in Three Genes▿

We previously generated a ceftobiprole-resistant Staphylococcus aureus strain after high inoculum serial passage of a mecA-negative variant of strain COL (R. Banerjee, M. Gretes, L. Basuino, N. Strynadka, and H. F. Chambers, Antimicrob. Agents Chemother. 52:2089-2096, 2008). Genome resequencing of this strain, CRB, revealed that it differs from its parent by five single-nucleotide polymorphisms in three genes, specifically, those encoding PBP4, a low-molecular-weight penicillin-binding protein, GdpP, a predicted signaling protein, and AcrB, a cation multidrug efflux transporter. CRB displayed resistance to a variety of β-lactams but was hypersusceptible to cefoxitin

Comparative Efficacies of Tedizolid Phosphate, Vancomycin, and Daptomycin in a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Endocarditis

Tedizolid, the active component of the prodrug tedizolid phosphate, is a novel oxazolidinone that is approximately 4 times more active by weight than linezolid against Staphylococcus aureus in vitro. The in vivo efficacy of tedizolid phosphate (15 mg/kg body weight intravenous [i.v.] twice a day [b.i.d.]) was compared to those of vancomycin (30 mg/kg i.v. b.i.d.) and daptomycin (18 mg/kg i.v. once a day [q.d.]) in a rabbit model of aortic valve endocarditis (AVE) caused by methicillin-resistant S. aureus strain COL (infection inoculum of 10(7) CFU). Median vegetation titers of daptomycin-treated rabbits were significantly lower than those of rabbits treated with tedizolid phosphate (15 mg/kg b.i.d.) (P = 0.016), whereas titers for vancomycin-treated compared to tedizolid-treated rabbits were not different (P = 0.984). The numbers of organisms in spleen and kidney tissues were similar for all treatment groups. A dose-ranging experiment was performed with tedizolid phosphate (2, 4, and 8 mg/kg b.i.d.) compared to vancomycin (30 mg/kg b.i.d.), using a higher infecting inoculum (10(8) CFU) to determine the lowest efficacious dose of tedizolid phosphate. Tedizolid phosphate (2 mg/kg) (equivalent to 60% of the area under the concentration-time curve from 0 to 24 h (AUC(0–24)) for the human 200-mg dose approved by the U.S. Food and Drug Administration) was not efficacious. Tedizolid phosphate at 4 mg/kg (equivalent to 75% of the AUC(0–24) for the human 400-mg dose) and 8 mg/kg produced lower vegetation titers than the control, but neither was as efficacious as vancomycin

Daptomycin–β-Lactam Combinations in a Rabbit Model of Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus Endocarditis

Beta-lactams enhance the in vitro activity of daptomycin against methicillin-resistant strains of Staphylococcus aureus Experiments were performed in a rabbit model of aortic valve endocarditis caused by methicillin-resistant daptomycin-nonsusceptible S. aureus strain CB5054 to determine if a cephalosporin, ceftriaxone, administered as a once-daily dose of 100 mg/kg of body weight, or a carbapenem, ertapenem, administered as a once-daily dose of 40 mg/kg, improved the efficacy of daptomycin, administered as a once-daily dose of 12 mg/kg. Daptomycin was ineffective alone in reducing organism densities compared to untreated controls in vegetations and spleen, but densities were 1.4 log10 CFU/g lower in kidney. The combination of daptomycin plus ceftriaxone or daptomycin plus ertapenem reduced bacterial densities in all tissues compared to single agents, with 0.6 to 1.0 log10 CFU/g fewer organisms in vegetations, 1.5 to 2.5 log10 CFU/g fewer organisms in spleen, and 1.8 to 2.5 log10 CFU/g fewer organisms in kidney, although differences were statistically significant only in spleen for daptomycin plus ceftriaxone and in kidney for daptomycin plus ertapenem. Drug exposures in rabbits were less than those achievable in humans, which may have limited the in vivo activity, particularly in vegetations

Ceftobiprole- and Ceftaroline-Resistant Methicillin-Resistant Staphylococcus aureus

The role of mecA mutations in conferring resistance to ceftobiprole and ceftaroline, cephalosporins with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, was determined with MRSA strains COL and SF8300. The SF8300 ceftaroline-passaged mutant carried a single mecA mutation, E447K (E-to-K change at position 447), and expressed low-level resistance. This mutation in COL conferred high-level resistance to ceftobiprole but only low-level resistance to ceftaroline. The COL ceftaroline-passaged mutant, which expressed high-level resistance to ceftobiprole and ceftaroline, had mutations in pbp2, pbp4, and gdpP but not mecA