Molecular analyses of motility and chemotaxis genes and their organization in Borrelia burgdorferi.

Motility and chemotaxis have been considered as important candidates for the study of virulence in many pathogenic spirochetes, including B. burgdorferi. Little is known about the underlying mechanisms of motility and chemotaxis. In this study, we have carried out an extensive search for the motility and chemotaxis genes in B. burgdorferi. The unique technique of semi-random PCR chromosome walking was developed and used to clone several large motility and chemotaxis gene clusters. Together with the previously reported flaB and flgE genes, 38 motility and chemotaxis related genes (more than 36 kb) were identified. These genes demonstrated extensive homology with their bacterial counterparts, especially with those from Treponema and Bacillus subtilis. Motility and chemotaxis genes constitute four major clusters on the chromosome of B. burgdorferi. Transcript analyses using RT-PCR and primer extension revealed that at least five operons were present in these motility and chemotaxis clusters; they were flgB, flgK, flaB, flaA/che, and fliD operons. The conserved {dollar}\\sigma\\sp{lcub}70{rcub}{dollar}-like promoters were involved in all of these operons, which is distinct to those of other bacteria. These results indicate that although the motility and chemotaxis genes in B. burgdorferi are well conserved, the transcriptional regulation of flagellar gene expression in B. burgdorferi is different from those of other bacteria. To further analyze these motility and chemotaxis genes, we overexpressed several motility and chemotaxis proteins in E. coli. Serological analyses using recombinant motility and chemotaxis proteins (FlgE, FliG, FliI, CheY and FlaA) indicate that except for FlgB and FlgE, these proteins were not immunodominant antigens during infection. Thus, they are not good candidates for diagnostic purposes. In order to further study the functions of these genes, we made an extensive effort to develop a gene inactivation system. Several different antibiotic constructs (gyrB-coumermycin resistant gene, cat and kanamycin resistant genes) were inserted into several motility genes. Unfortunately, so far none of these constructs worked successfully in B. burgdorferi. This study is the first important step toward the achievement of a better understanding of the genetics of spirochetal motility and chemotaxis

Comparative Pharmacokinetics of Ceftaroline in Rats, Rabbits, and Monkeys following a Single Intravenous or Intramuscular Injection ▿

This study assessed the pharmacokinetic profiles for intramuscular and intravenous ceftaroline treatment for rats, rabbits, and monkeys. Ceftaroline, a novel cephalosporin with broad-spectrum activity against Gram-positive and Gram-negative pathogens, demonstrated favorable pharmacokinetic profiles following intramuscular administration in all 3 animal species, comparable to the levels for intravenous dosing. The areas under the plasma concentration-time curve obtained after intramuscular administration were increased in rats and similar in rabbits and monkeys, compared with the levels obtained after intravenous dosing (129%, 7.29%, and 12.7% greater in rats, rabbits, and monkeys, respectively). The data reported here support the development of an intramuscular formulation for ceftaroline

Activity of cephalosporin CXA-101 (FR264205) against Pseudomonas aeruginosa and Burkholderia cepacia group strains and isolates

International audienceTwenty-five years after its introduction, ceftazidime remains the most active cephalosporin against . Nevertheless, resistance arises by upregulation of AmpC β-lactamase, by efflux or, less often, via acquisition of additional β-lactamases. Mutational resistance is especially prevalent among cystic fibrosis (CF) isolates. We examined the activity of a novel oxyimino-aminothiazolyl cephalosporin, CXA-101 (FR264205), against strains with defined resistance mechanisms as well as against multiresistant clinical CF isolates of and . Minimum inhibitory concentrations (MICs) of CXA-101 were determined by the Clinical and Laboratory Standards Institute agar dilution method and were 0.25–0.5mg/L for ‘typical' strains without acquired resistance compared with 1–2mg/L for ceftazidime. MICs of CXA-101 were 0.5–2mg/L and 4mg/L, respectively, for isolates with upregulated efflux or total AmpC derepression compared with 2–16mg/L and 32–128mg/L for ceftazidime. Full activity was retained against OprD mutants resistant to imipenem. Substantive resistance (MICs ≥ 32mg/L) arose for transconjugants with PER, VEB and OXA extended-spectrum β-lactamases and for metallo-β-lactamase producers, with reduced susceptibility (MIC=8mg/L) for transconjugants with OXA-2, OXA-3 and NPS-1 enzymes. MICs of CXA-101 were 2- to 16-fold below those of ceftazidime for multiresistant from CF patients, but ranged up to >128mg/L; values for from CF resembled those for ceftazidime

Phase 2 Study of Ceftaroline versus Standard Therapy in Treatment of Complicated Skin and Skin Structure Infections▿

Ceftaroline, the bioactive metabolite of ceftaroline fosamil (previously PPI-0903, TAK-599), is a broad-spectrum cephalosporin with potent in vitro activity against multidrug-resistant gram-positive aerobic pathogens, including methicillin-resistant Staphylococcus aureus. A randomized, observer-blinded study to evaluate the safety and efficacy of ceftaroline versus standard therapy in treating complicated skin and skin structure infections (cSSSI) was performed. Adults with cSSSI, including at least one systemic marker of inflammation, were randomized (2:1) to receive intravenous (i.v.) ceftaroline (600 mg every 12 h) or i.v. vancomycin (1 g every 12 h) with or without adjunctive i.v. aztreonam (1 g every 8 h) for 7 to 14 days. The primary outcome measure was the clinical cure rate at a test-of-cure (TOC) visit 8 to 14 days after treatment. Secondary outcomes included the microbiological success rate (eradication or presumed eradication) at TOC and the clinical relapse rate 21 to 28 days following treatment. Of 100 subjects enrolled, 88 were clinically evaluable; the clinical cure rate was 96.7% (59/61) for ceftaroline versus 88.9% (24/27) for standard therapy. Among the microbiologically evaluable subjects (i.e., clinically evaluable and having had at least one susceptible pathogen isolated at baseline), the microbiological success rate was 95.2% (40/42) for ceftaroline versus 85.7% (18/21) for standard therapy. Relapse occurred in one subject in each group (ceftaroline, 1.8%; standard therapy, 4.3%). Ceftaroline exhibited a very favorable safety and tolerability profile, consistent with that of marketed cephalosporins. Most adverse events from ceftaroline were mild and not related to treatment. Ceftaroline holds promise as a new therapy for treatment of cSSSI and other serious polymicrobial infections

Affinity of the New Cephalosporin CXA-101 to Penicillin-Binding Proteins of Pseudomonas aeruginosa▿

CXA-101, previously designated FR264205, is a new antipseudomonal cephalosporin. The objective of this study was to determine the penicillin-binding protein (PBP) inhibition profile of CXA-101 compared to that of ceftazidime (PBP3 inhibitor) and imipenem (PBP2 inhibitor). Killing kinetics, the induction of AmpC expression, and associated changes on cell morphology were also investigated. The MICs for CXA-101, ceftazidime, and imipenem were 0.5, 1, and 1 μg/ml, respectively. Killing curves revealed that CXA-101 shows a concentration-independent bactericidal activity, with concentrations of 1× the MIC (0.5 μg/ml) producing a >3-log reduction in bacterial load after 8 h of incubation. Live-dead staining showed that concentrations of CXA-101 as low as 0.5× the MIC stopped bacterial septation and induced an intense filamentation, which is consistent with the documented high affinity of PBP3. CXA-101 was found to be a potent PBP3 inhibitor and showed affinities ≥2-fold higher than those of ceftazidime for all of the essential PBPs (1b, 1c, 2, and 3). Compared to imipenem, in addition to the obvious inverse PBP2/PBP3 affinities, CXA-101 showed a significantly higher affinity for PBP1b but a lower affinity for PBP1c. Furthermore, CXA-101, like ceftazidime and in contrast to imipenem, was found to be a very weak inducer of AmpC expression, consistent with the low PBP4 affinity documented

In Vitro Profiling of Ceftaroline against a Collection of Recent Bacterial Clinical Isolates from across the United States▿

This study evaluated the in vitro activity of ceftaroline, a novel cephalosporin with broad-spectrum activity against gram-negative and -positive pathogens, against 4,151 recent clinical isolates collected in the United States. Ceftaroline was very potent against bacteria found in community- and hospital-acquired infections, including methicillin-resistant Staphylococcus aureus, multidrug-resistant Streptococcus pneumoniae, and common Enterobacteriaceae spp

Antimicrobial Activity and Spectrum of PPI-0903M (T-91825), a Novel Cephalosporin, Tested against a Worldwide Collection of Clinical Strains

PPI-0903M is a novel N-phosphono-type cephalosporin active against oxacillin-resistant staphylococci and many other gram-positive organisms. This study evaluated the in vitro activity and spectrum of PPI-0903M against 1,478 recent clinical isolates collected from 80 medical centers (22 countries). PPI-0903M demonstrated broader in vitro activity against gram-positive bacteria, particularly against multidrug-resistant staphylococci and streptococci of current clinical concern, than currently available extended-spectrum cephalosporins while maintaining similar activity against gram-negative pathogens