Randomized, Double-Blind, Placebo-Controlled, Single-Ascending-Dose Study of the Penetration of a Monoclonal Antibody Combination (ASN100) Targeting Staphylococcus aureus Cytotoxins in the Lung Epithelial Lining Fluid of Healthy Volunteers.

ASN100 is a novel antibody combination of two fully human IgG1(κ) monoclonal antibodies (MAbs), ASN-1 and ASN-2, which neutralize six Staphylococcus aureus cytotoxins, alpha-hemolysin (Hla) and five bicomponent leukocidins. We assessed the safety, tolerability, and serum and lung pharmacokinetics of ASN100 in a randomized, double-blind, placebo-controlled single-dose-escalation first-in-human study. Fifty-two healthy volunteers were enrolled and randomized to receive either ASN-1, ASN-2, a combination of both MAbs (ASN100), or a corresponding placebo. Thirty-two subjects in the double-blind dose escalation portion of the study received ASN-1 or ASN-2 at a 200-, 600-, 1,800-, or 4,000-mg dose, or placebo. Eight subjects received both MAbs simultaneously in a 1:1 ratio (ASN100) at 3,600 or 8,000 mg, or they received placebos. Twelve additional subjects received open-label ASN100 at 3,600 or 8,000 mg to assess the pharmacokinetics of ASN-1 and ASN-2 in epithelial lining fluid (ELF) by bronchoalveolar lavage fluid sampling. Subjects were monitored for 98 days (double-blind cohorts) or 30 days (open-label cohorts) for safety assessment. No dose-limiting toxicities were observed, and all adverse events were mild and transient, with only two adverse events considered possibly related to the investigational product. ASN100 exhibited linear serum pharmacokinetics with a half-life of approximately 3 weeks and showed detectable penetration into the ELF. No treatment-emergent anti-drug antibody responses were detected. The toxin neutralizing potency of ASN100 in human serum was confirmed up to 58 days postdosing. The favorable safety profile, ELF penetration, and maintained functional activity in serum supported the further clinical development of ASN100.

Allele- and tir-independent functions of intimin in diverse animal infection models.

Upon binding to intestinal epithelial cells, enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium trigger formation of actin pedestals beneath bound bacteria. Pedestal formation has been associated with enhanced colonization, and requires intimin, an adhesin that binds to the bacterial effector translocated intimin receptor (Tir), which is translocated to the host cell membrane and promotes bacterial adherence and pedestal formation. Intimin has been suggested to also promote cell adhesion by binding one or more host receptors, and allelic differences in intimin have been associated with differences in tissue and host specificity. We assessed the function of EHEC, EPEC, or C. rodentium intimin, or a set of intimin derivatives with varying Tir-binding abilities in animal models of infection. We found that EPEC and EHEC intimin were functionally indistinguishable during infection of gnotobiotic piglets by EHEC, and that EPEC, EHEC, and C. rodentium intimin were functionally indistinguishable during infection of C57BL/6 mice by C. rodentium. A derivative of EHEC intimin that bound Tir but did not promote robust pedestal formation on cultured cells was unable to promote C. rodentium colonization of conventional mice, indicating that the ability to trigger actin assembly, not simply to bind Tir, is required for intimin-mediated intestinal colonization. Interestingly, streptomycin pre-treatment of mice eliminated the requirement for Tir but not intimin during colonization, and intimin derivatives that were defective in Tir-binding still promoted colonization of these mice. These results indicate that EPEC, EHEC, and C. rodentium intimin are functionally interchangeable during infection of gnotobiotic piglets or conventional C57BL/6 mice, and that whereas the ability to trigger Tir-mediated pedestal formation is essential for colonization of conventional mice, intimin provides a Tir-independent activity during colonization of streptomycin pre-treated mice.

Associations between the genotypes of Staphylococcus aureus bloodstream isolates and clinical characteristics and outcomes of bacteremic patients.

We investigated associations between the genotypic and phenotypic features of Staphylococcus aureus bloodstream isolates and the clinical characteristics of bacteremic patients enrolled in a phase III trial of S. aureus bacteremia and endocarditis. Isolates underwent pulsed-field gel electrophoresis, PCR for 33 putative virulence genes, and screening for heteroresistant glycopeptide intermediate S. aureus (hGISA). A total of 230 isolates (141 methicillin-susceptible S. aureus and 89 methicillin-resistant S. aureus [MRSA]) were analyzed. North American and European S. aureus isolates differed in their genotypic characteristics. Overall, 26% of the MRSA bloodstream isolates were USA 300 strains. Patients with USA 300 MRSA bacteremia were more likely to be injection drug users (61% versus 15%; P < 0.001), to have right-sided endocarditis (39% versus 9%; P = 0.002), and to be cured of right-sided endocarditis (100% versus 33%; P = 0.01) than patients with non-USA 300 MRSA bacteremia. Patients with persistent bacteremia were less likely to be infected with Panton-Valentine leukocidin gene (pvl)-constitutive MRSA (19% versus 56%; P = 0.005). Although 7 of 89 MRSA isolates (8%) exhibited the hGISA phenotype, no association with persistent bacteremia, daptomycin resistance, or bacterial genotype was observed. This study suggests that the virulence gene profiles of S. aureus bloodstream isolates from North America and Europe differ significantly. In this study of bloodstream isolates collected as part of a multinational randomized clinical trial, USA 300 and pvl-constitutive MRSA strains were associated with better clinical outcomes.

Microbiological and genotypic analysis of methicillin-resistant Staphylococcus aureus bacteremia.

In a recent landmark trial of bacteremia caused by methicillin-resistant Staphylococcus aureus (MRSA) isolates, vancomycin MICs were >or=1 microg/ml for only 16% of the isolates, and accessory gene regulator (agr) function as measured by delta-hemolysin activity was absent or reduced in only 28.1% of the isolates. This clinical study did not capture a population of MRSA isolates predictive of vancomycin treatment failure.

Effects of prior effective therapy on the efficacy of daptomycin and ceftriaxone for the treatment of community-acquired pneumonia.

OBJECTIVE: We sought to compare daptomycin with ceftriaxone for the treatment of patients with community-acquired pneumonia (CAP). METHODS: Two phase-3 randomized, double-blind trials that enrolled adult patients hospitalized with CAP were conducted. Patients received intravenous daptomycin (4 mg/kg) or ceftriaxone (2 g) once daily for 5-14 days. Aztreonam could be added for patients with gram-negative infections. Clinical responses at the test-of-cure visit among patients in the intent-to-treat and clinically evaluable populations were the primary efficacy end points. RESULTS: After combining data from the trials, the intent-to-treat population included 413 daptomycin-treated patients and 421 ceftriaxone-treated patients, and the clinically evaluable population included 369 daptomycin-treated patients and 371 ceftriaxone-treated patients. In the intent-to-treat population, the clinical cure rate among daptomycin-treated patients with CAP was 70.9%, compared with 77.4% among ceftriaxone-treated patients (95% confidence interval for the difference between cure rates, -12.4% to -0.6%). In the clinically evaluable population, the clinical cure rate was lower among daptomycin-treated patients (79.4%) than among ceftriaxone-treated patients (87.9%; 95% confidence interval for the difference between cure rates, -13.8% to -3.2%). A posthoc analysis revealed that, among those who had received up to 24 h of prior effective therapy, cure rates were similar among daptomycin-treated (90.7%) and ceftriaxone-treated patients (88.0%; 95% confidence interval for the difference between cure rates, -6.1% to 11.5%). CONCLUSIONS: Daptomycin is not effective for the treatment of CAP, including infections caused by Streptococcus pneumoniae and Staphylococcus aureus. The observation that as little as 24 h of prior effective therapy may impact clinical outcome suggests that trials to evaluate CAP treatment may need to exclude patients who have received any potentially effective therapy before enrollment.

Detection of daptomycin-nonsusceptible strains using the Neo-Sensitab prediffusion method.

Two prediffusion methods with daptomycin (DAP) Neo-Sensitabs were evaluated against a challenge set of 30 Staphylococcus aureus isolates and 30 enterococci. DAP Neo-Sensitabs were prediffused for either 8 or 20 h on Mueller-Hinton agar. Inhibition zones were plotted versus Etest MIC values determined on the same prediffused agar. A generalization to the genus level of the manufacturer's suggested Neo-Sensitabs breakpoints for staphylococci and enterococci was used to interpret the results. DAP-susceptible and DAP-nonsusceptible enterococci, Enterococcus faecium in particular, were not reliably discriminated using a 20-h prediffusion method and the manufacturer's suggested breakpoints. Further development of this testing methodology, such as changing the format to a susceptibility screen followed by a confirmatory MIC, is needed to accurately categorize the DAP susceptibility of enterococcal isolates. Prediffusion for either 8 or 20 h with DAP Neo-Sensitabs discriminated between susceptible and nonsusceptible S. aureus with minimal errors. Both prediffusion methods also detected changes in MIC values between isogenic pairs of susceptible and nonsusceptible S. aureus. These results suggest that a multisite evaluation of either prediffusion method with DAP Neo-Sensitabs against a larger collection of S. aureus is warranted.

In vitro activity of daptomycin against clinical isolates with reduced susceptibilities to linezolid and quinupristin/dalfopristin.

An initiative was taken to determine the in vitro activity of daptomycin against 85 Gram-positive isolates with reduced susceptibilities to linezolid and quinupristin/dalfopristin. Daptomycin had potent activity against all strains, with a Staphylococcus spp. minimum inhibitory concentration (MIC) < or =2 microg/mL and an Enterococcus spp. MIC < or =8 microg/mL. Resistance to linezolid and quinupristin/dalfopristin appears to be independent of reduced susceptibility to daptomycin.