Development and Qualification of an Opsonophagocytic Killing Assay To Assess Immunogenicity of a Bioconjugated Escherichia coli Vaccine.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, -30% to 30%), and total error (total error range, -65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

Evaluation of the in vitro activities of ceftobiprole and comparators in staphylococcal colony or microtitre plate biofilm assays.

The aim of this study was to evaluate the in vitro efficacy of ceftobiprole and comparator antibiotics, either alone or in combination, in staphylococcal MBEC™ (minimum biofilm eradication concentration) and colony biofilm assays at dilutions of the maximum free-drug plasma concentration attained during clinical use (fCmax). Staphylococci tested included meticillin-susceptible and meticillin-resistant Staphylococcus aureus (n=6) and Staphylococcus epidermidis (n=2). Relative to no-drug controls, after 7 days of exposure ceftobiprole concentrations from 1/4 fCmax to fCmax generally decreased CFUs in MBEC or colony biofilms of S. aureus isolates by ca. 1.5log10 to ≥2.5log10. Gentamicin reduced colony biofilm CFUs by ≥1.4log10 at these concentrations with gentamicin-susceptible isolates. Following 7 days of exposure, vancomycin and rifampicin were ineffective as single agents or in combination in the colony model, but yielded CFU decreases from 0 to 5log10 in the MBEC model. Treatment of biofilms with rifampicin for 7 days yielded rifampicin-resistant mutants, and the selection of rifampicin resistance was inhibited by co-treatment with ceftobiprole. Thus, ceftobiprole alone or in combination demonstrated promising activity against biofilms of meticillin-susceptible and -resistant staphylococci at clinically relevant concentrations. In contrast, vancomycin and rifampicin, two agents used clinically for the treatment of biofilm infections, tested separately or together gave inconsistent results and generally had little impact on cell viability.

Synergistic activity of ceftobiprole and vancomycin in a rat model of infective endocarditis caused by methicillin-resistant and glycopeptide-intermediate Staphylococcus aureus.

The therapeutic activity of ceftobiprole medocaril, the prodrug of ceftobiprole, was compared to that of vancomycin, daptomycin, and the combination of a subtherapeutic dose of ceftobiprole and vancomycin in a rat model of infective endocarditis due to methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) or glycopeptide-intermediate Staphylococcus aureus (GISA) (NRS4 and HIP 5836) strains. The minimum bactericidal concentrations of ceftobiprole, vancomycin, and daptomycin at bacterial cell densities similar to those encountered in the cardiac vegetation in the rat endocarditis model were 2, >64, and 8 µg/ml, respectively, for MRSA ATCC 43300 and 4, >64, and 8 µg/ml, respectively, for the GISA strain. Ceftobiprole medocaril administered in doses of 100 mg/kg of body weight given intravenously (i.v.) twice a day (BID) every 8 h (q8h) (equivalent to a human therapeutic dose of ceftobiprole [500 mg given three times a day [TID]) was the most effective monotherapy, eradicating nearly 5 log(10) CFU/g MRSA or 6 log(10) CFU/g GISA organisms from the cardiac vegetation and had the highest incidence of sterile vegetation compared to the other monotherapies in the endocarditis model. In in vitro time-kill studies, synergistic effects were observed with ceftobiprole and vancomycin on MRSA and GISA strains, and in vivo synergy was noted with combinations of subtherapeutic doses of these agents for the same strains. Additionally, sterile vegetations were achieved in 33 and 60%, respectively, of the animals infected with MRSA ATCC 43300 or GISA NRS4 receiving ceftobiprole-vancomycin combination therapy. In summary, ceftobiprole was efficacious both as monotherapy and in combination with vancomycin in treating MRSA and GISA infections in a rat infective endocarditis model and warrants further evaluation.

A randomised, double-blind trial comparing ceftobiprole medocaril with ceftriaxone with or without linezolid for the treatment of patients with community-acquired pneumonia requiring hospitalisation.

Community-acquired pneumonia (CAP) is a serious infection requiring hospitalisation in 20% of cases. The novel cephalosporin ceftobiprole has microbiological activity against the major bacterial pathogens causing CAP, including Streptococcus pneumoniae, Haemophilus influenzae and Klebsiella pneumoniae, as well as against Staphylococcus aureus, including meticillin-resistant S. aureus (MRSA). This was a multicentre, double-blind study in which 706 patients with CAP severe enough to require hospitalisation were randomised to ceftobiprole or to an expert-recommended course of ceftriaxone ± linezolid (comparator group). Clinical and microbiological outcomes were determined 7-14 days after completion of therapy (test-of-cure visit). For the 469 clinically evaluable patients, cure rates were 86.6% vs. 87.4% for ceftobiprole and comparator, respectively [95% confidence interval (CI) of the difference, -6.9% to 5.3%]; in the intention-to-treat (ITT) analysis of 638 CAP patients, these cure rates were 76.4% vs. 79.3%, respectively (95% CI of the difference, -9.3% to 3.6%). A typical bacterial pathogen was identified in 29% of the ITT population. Microbiological eradication rates in the 144 microbiologically evaluable patients were 88.2% and 90.8% for the respective treatment groups (95% CI of the difference, -12.6% to 7.5%). Both study drugs were well tolerated, with but a minority of patients requiring premature discontinuation due to an adverse event (6% in the ceftobiprole group and 4% in the comparator group). The overall incidence of treatment-related adverse events was higher in the ceftobiprole group, primarily owing to differences in rates of self-limited nausea (7% vs. 2%) and vomiting (5% vs. 2%). In summary, ceftobiprole was non-inferior to the comparator (ceftriaxone ± linezolid) in all clinical and microbiological analyses conducted, suggesting that ceftobiprole has a potential role in treating hospitalised patients with CAP. [ClinicalTrials.gov identifier: NCT00326287].

Longitudinal survey of carbapenem resistance and resistance mechanisms in Enterobacteriaceae and non-fermenters from the USA in 2007-09.

BACKGROUND: Antibiotic resistance is problematic in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, and is often associated with serious infections. Carbapenems are often one of the few remaining therapeutic options, so it is important to monitor carbapenem activity against these pathogens and to identify resistance mechanisms. METHODS: Carbapenem susceptibilities were determined for 14 359 Enterobacteriaceae, 3614 P. aeruginosa and 994 A. baumannii from the USA (2007-09). Klebsiella pneumoniae with doripenem MICs ≥2 mg/L (n = 88), and P. aeruginosa (n = 452), A. baumannii (n = 349) and other enterics (n = 13) with doripenem MICs ≥4 mg/L were screened for carbapenem resistance mechanisms. RESULTS: Doripenem/meropenem and imipenem susceptibilities for Enterobacteriaceae were >99% and 89%, respectively. Doripenem susceptibility (2007-09) for P. aeruginosa was 87.4%-84.1%; comparable to meropenem and higher than imipenem. For A. baumannii, doripenem susceptibility (2007-09) was 63%-58.2%; lower than imipenem and meropenem. Resistant K. pneumoniae had KPC and lacked porins OmpK35/OmpK36. In 2009, 3.4% of all K. pneumoniae possessed KPC. Five other enterics and one P. aeruginosa possessed KPC. Resistance mechanisms in P. aeruginosa were loss of porin OprD (90%), efflux (55%) and elevated AmpC activity (25%). Acquired carbapenemases OXA-23/-24 were present in 48% of resistant A. baumannii. VIM metallo-ß-lactamases were present in three P. aeruginosa and one A. baumannii isolates. CONCLUSIONS: Doripenem and meropenem were more active than imipenem against Enterobacteriaceae and P. aeruginosa from the USA. Carbapenem resistance mechanisms included serine carbapenemases, elevated AmpC activity, efflux and porin deficiencies occurring mostly in P. aeruginosa. Metallo-ß-lactamases were found in <0.1% of isolates.

In vitro antibacterial activities of JNJ-Q2, a new broad-spectrum fluoroquinolone.

JNJ-Q2, a novel fluorinated 4-quinolone, was evaluated for its antibacterial potency by broth and agar microdilution MIC methods in studies focused on skin and respiratory tract pathogens, including strains exhibiting contemporary fluoroquinolone resistance phenotypes. Against a set of 118 recent clinical isolates of Streptococcus pneumoniae, including fluoroquinolone-resistant variants bearing multiple DNA topoisomerase target mutations, an MIC(90) value for JNJ-Q2 of 0.12 microg/ml was determined, indicating that it was 32-fold more potent than moxifloxacin. Against a collection of 345 recently collected methicillin-resistant Staphylococcus aureus (MRSA) isolates, including 256 ciprofloxacin-resistant strains, the JNJ-Q2 MIC(90) value was 0.25 microg/ml, similarly indicating that it was 32-fold more potent than moxifloxacin. The activities of JNJ-Q2 against Gram-negative pathogens were generally comparable to those of moxifloxacin. In further studies, JNJ-Q2 exhibited bactericidal activities at 2x and 4x MIC levels against clinical isolates of S. pneumoniae and MRSA with various fluoroquinolone susceptibilities, and its activities were enhanced over those of moxifloxacin. In these studies, the activity exhibited against strains bearing gyrA, parC, or gyrA plus parC mutations was indicative of the relatively balanced (equipotent) activity of JNJ-Q2 against the DNA topoisomerase target enzymes. Finally, determination of the relative rates or frequencies of the spontaneous development of resistance to JNJ-Q2 at 2x and 4x MICs in S. pneumoniae, MRSA, and Escherichia coli were indicative of a lower potential for resistance development than that for current fluoroquinolones. In conclusion, JNJ-Q2 exhibits a range of antibacterial activities in vitro that is supportive of its further evaluation as a potential new agent for the treatment of skin and respiratory tract infections.

Molecular characterisation of meticillin-resistant Staphylococcus aureus isolates from two ceftobiprole Phase 3 complicated skin and skin-structure infection clinical trials.

Meticillin-resistant Staphylococcus aureus (MRSA) isolates from two worldwide ceftobiprole Phase 3 clinical trials for the treatment of complicated skin and skin-structure infections were characterised by clonality, staphylococcal cassette chromosome mec (SCCmec) type and the presence of Panton-Valentine leukocidin (PVL). PVL was predominantly found in US isolates (196/231 vs. 13/110 non-US isolates). SCCmec type IV was the most common (253/329) owing to the predominance of clone USA300 in isolates from the USA (197/226). In Europe, SCCmec type III was the most prevalent (30/74). Ceftobiprole minimum inhibitory concentrations (MICs) ranged from 0.25 microg/mL to 4 microg/mL, with MICs

Sporadic occurrences of fluoroquinolone-resistant Streptococcus pneumoniae in the United States: longitudinal analysis of institutions from the New England and West South Central regions during the TRUST 4-9 (2000-2005) Surveillance Studies.

Twenty-six institutions in New England and 24 institutions in West South Central regions participating in the Tracking Resistance in the United States Today (TRUST) 4-9 surveillance studies (2000-2005) were monitored for levofloxacin-resistant Streptococcus pneumoniae to determine if resistance was sporadic or persistent. Levofloxacin was used as a representative of the respiratory fluoroquinolones. Levofloxacin-resistant isolates were identified in 8 of the 26 New England institutions and in 11 of the 24 West South Central institutions during the surveillance period. Resistant isolates were recovered in consecutive years from 3 institutions: 1 each in Massachusetts, Oklahoma, and Texas. In total, 34 levofloxacin-resistant isolates (14 from New England, 20 from the West South Central region) were identified over the 6-year period. Two of these isolates from an institution in Connecticut and 2 from an institution in Oklahoma had the same serotype, pulsed-field gel electrophoresis pattern, and quinolone resistance-determining region (QRDR) mutations. States with elevated pneumococcal levofloxacin resistance rates, compared with the national average, did not maintain this status in consecutive years. Based on data from the same institutions over 6 years, levofloxacin resistance among US pneumococci has been sporadic, nonclonal, and rare.

In vitro activity of ceftobiprole against pathogens from two phase 3 clinical trials of complicated skin and skin structure infections.

In phase 3 clinical trials for ceftobiprole treatment of complicated skin and skin structure infections, 1,219 gram-positive and 276 gram-negative aerobic baseline pathogens were identified. Ceftobiprole inhibited all staphylococcal isolates, including methicillin-resistant strains, at MICs of

Results of a double-blind, randomized trial of ceftobiprole treatment of complicated skin and skin structure infections caused by gram-positive bacteria.

Ceftobiprole is the first broad-spectrum cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA) to be assessed in late-stage clinical trials. As a pivotal step in the clinical development of ceftobiprole, a multicenter, global, randomized, double-blind trial was conducted to compare the efficacy of ceftobiprole to that of vancomycin in patients with complicated skin and skin structure infections (cSSSIs) caused by gram-positive bacteria. The primary objective was to assess noninferiority on the basis of the cure rates 7 to 14 days after the completion of therapy in patients administered ceftobiprole 500 mg every 12 h or vancomycin 1 g every 12 h. Of 784 patients randomized, 282 receiving ceftobiprole and 277 receiving vancomycin were clinically evaluable. Of these patients, 93.3% treated with ceftobiprole and 93.5% treated with vancomycin were cured (95% confidence interval of difference, -4.4%, 3.9%). The cure rates for patients with MRSA infections were 91.8% (56/61) with ceftobiprole treatment and 90.0% (54/60) with vancomycin treatment (95% confidence interval of difference, -8.4%, 12.1%). At least one adverse event (AE) was reported by 52% of the ceftobiprole-treated patients and 51% of the vancomycin-treated patients. The most common AEs reported by the ceftobiprole-treated patients were nausea (14%) and taste disturbance (8%). Discontinuation of the study drug because of treatment-emergent AEs occurred in 4% (n = 17) of the ceftobiprole-treated patients and 6% (n = 22) of the vancomycin-treated patients. The results of this trial support the use of ceftobiprole as an effective and well-tolerated treatment option for patients with cSSSIs caused by a spectrum of gram-positive bacteria.

Parallel synthesis of potent, pyrazole-based inhibitors of Helicobacter pylori dihydroorotate dehydrogenase.

The identification of several potent pyrazole-based inhibitors of bacterial dihydroorotate dehydrogenase (DHODase) via a directed parallel synthetic approach is described below. The initial pyrazole-containing lead compounds were optimized for potency against Helicobacter pylori DHODase. Using three successive focused libraries, inhibitors were rapidly identified with the following characteristics: K(i) < 10 nM against H. pylori DHODase, sub-microg/mL H. pylori minimum inhibitory concentration activity, low molecular weight, and >10 000-fold selectivity over human DHODase.

Effects of metronidazole, tetracycline, and bismuth-metronidazole-tetracycline triple therapy in the Helicobacter pylori SS1 mouse model after 1 day of dosing: development of an H. pylori lead selection model.

We evaluated the effect of optimized doses and dosing schedules of metronidazole, tetracycline, and bismuth-metronidazole-tetracycline (BMT) triple therapy with only 1 day of dosing on Helicobacter pylori SS1 titers in a mouse model. A reduction of bacterial titers was observable with 22.5 and 112.5 mg of metronidazole per kg of body weight (as well as BMT) given twice daily and four times daily (QID). Two hundred milligrams of tetracycline per kilogram, given QID, resulted in only a slight reduction of H. pylori titers in the stomach. We argue that optimization of doses based on antimicrobial drug levels in the animal and shortened (1 or 2 days) drug administration can be used to facilitate early evaluation of putative anti-H. pylori drug candidates in lieu of using human doses and extended schedules (7 to 14 days), as can be deduced from the results seen with these antimicrobial agents.