Peter J. Petersen

In Vitro and In Vivo Activities of Tigecycline (GAR-936), Daptomycin, and Comparative Antimicrobial Agents against Glycopeptide-Intermediate Staphylococcus aureus and Other Resistant Gram-Positive Pathogens

ABSTRACT Tigecycline (GAR-936) and daptomycin are potent antibacterial compounds in advanced stages of clinical trials. These novel agents target multiply resistant pathogenic bacteria. Daptomycin is principally active against gram-positive bacteria, while tigecycline has broad-spectrum activity. When tested by the standard protocols of the National Committee for Clinical Laboratory Standards in Mueller-Hinton broth II, tigecycline was more active than daptomycin (MICs at which 90% of isolates tested are inhibited, 0.12 to 1 and 0.5 to 16 μg/ml, respectively) against staphylococcal, enterococcal, and streptococcal pathogens. Daptomycin demonstrated a stepwise increase in activity corresponding to an increase in the supplemental concentration of calcium. When tested in base Mueller-Hinton broth supplemented with 50 mg of calcium per liter, daptomycin demonstrated improved activity (MIC90s, 0.015 to 4 μg/ml). The activity of daptomycin, however, equaled that of tigecycline against the glycopeptide-intermediate Staphylococcus aureus (GISA) strains only when the test medium was supplemented with excess calcium (75 mg/liter). Tigecycline and daptomycin demonstrated in vivo efficacies against GISA, methicillin-resistant S. aureus, and methicillin-susceptible S. aureus strains in an intraperitoneal systemic murine infection model. These data suggest that tigecycline and daptomycin may offer therapeutic options against clinically relevant resistant pathogens for which current alternatives for treatment are limited.

Synthesis and structure-activity relationship of novel glycylcycline derivatives leading to the discovery of GAR-936

A number of new glycylcyclines were synthesized for structure-activity relationship study. Many of the derivatives exhibit potent, broad spectrum antibacterial activity against both tetracycline susceptible and resistant organisms. GAR-936 (TBG-MINO) shows better activity than the previously reported DMG-MINO and DMG-DMDOT.

In Vitro Activity of Tigecycline against Isolates from Patients Enrolled in Phase 3 Clinical Trials of Treatment for Complicated Skin and Skin-Structure Infections and Complicated Intra-Abdominal Infections

The in vitro activity of tigecycline was evaluated against 4913 baseline pathogens isolated from 1986 patients enrolled in 4 pivotal phase 3 clinical trials. The trials, which were conducted in 38 countries worldwide, involved patients with complicated skin and skin-structure infections or complicated intra-abdominal infections. Tigecycline was active against the most prevalent pathogens for each infection type, including gram-positive and gram-negative strains of both aerobic and anaerobic bacteria (MICs, < or =2 microg/mL for most pathogens). The spectrum of activity of tigecycline included important pathogens, such as Staphylococcus aureus (including methicillin-resistant S. aureus), Streptococcus pyogenes, Escherichia coli, Klebsiella pneumoniae, and Bacteroides fragilis. A few genera, such as Pseudomonas aeruginosa and members of the tribe Proteeae, were generally less susceptible to tigecycline than were other gram-negative pathogens. The susceptibility of the pathogens to tigecycline was similar for isolates obtained from patients enrolled in the studies of complicated skin and skin-structure infection or of complicated intra-abdominal infection. For most pathogens, the susceptibility to tigecycline was similar across all geographic regions. The excellent expanded broad-spectrum activity of tigecycline demonstrated in vitro against clinical isolates confirmed its potential utility for pathogens associated with complicated skin and skin-structure infections or complicated intra-abdominal infections.

In vitro and in vivo antibacterial activities of the glycylcyclines, a new class of semisynthetic tetracyclines.

N,N-Dimethylglycylamido (DMG) derivatives of minocycline and 6-demethyl-6-deoxytetracycline are new semisynthetic tetracyclines referred to as the glycylcyclines. The in vitro activities of the glycylcyclines were evaluated in comparison with those of minocycline and tetracycline against strains carrying characterized tetracycline resistance determinants and against 995 recent clinical isolates obtained from geographically distinct medical centers in North America. The glycylcyclines were active against tetracycline-resistant strains carrying efflux [tet(A), tet(B), tet(C), and tet(D) in Escherichia coli and tet(K) in Staphylococcus aureus] and ribosomal protection [tet(M) in S. aureus, Enterococcus faecalis, and E. coli)] resistance determinants. Potent activity (MIC for 90% of strains, < or = 0.5 microgram/ml) was obtained with the glycylcyclines against methicillin-susceptible and methicillin-resistant S. aureus, E. faecalis, Enterococcus faecium, and various streptococcal species. The glycylcyclines exhibited good activity against a wide diversity of gram-negative aerobic and anaerobic bacteria, most of which were less susceptible to minocycline and tetracycline. The activities of the glycylcyclines against most organisms tested were comparable to each other. The in vivo efficacies of the glycylcyclines against acute lethal infections in mice when dosed intravenously were reflective of their in vitro activities. The glycylcyclines had efficacies comparable to that of minocycline against infections with methicillin-susceptible and methicillin-resistant S. aureus strains, a strain carrying tet(K), and a tetracycline-susceptible E. coli strain but exceeded the effectiveness of minocycline against infections with resistant isolates, including strains harboring tet(M) or tet(B). Levels of DMG-6-deoxytetracycline in serum were higher and more sustained than those of DMG-minocycline or minocycline. Our results show that the glycylcyclines have potent in vitro activities against a wide spectrum of gram-positive and gram-negative, aerobic and anaerobic bacteria, including many resistant strains. On the basis of their in vitro and in vivo activities, the glycylcyclines represent a significant advance to the tetracycline class of antibiotics and have good potential value for clinical efficacy.

Tigecycline MIC Testing by Broth Dilution Requires Use of Fresh Medium or Addition of the Biocatalytic Oxygen-Reducing Reagent Oxyrase To Standardize the Test Method

ABSTRACT Tigecycline is a broad-spectrum glycylcycline antibiotic with activity against not only susceptible gram-positive and gram-negative pathogens but also strains that are resistant to many other antibiotics. In the process of determining quality control (QC) limits for the American Type Culture Collection reference strains for tigecycline, a number of inconsistencies in MICs were encountered which appeared to be related to the age of the Mueller-Hinton broth (MHB) medium used in the MIC testing. The objective of this study was to determine the cause of the discrepant MIC results between fresh and aged MHB. The MICs of tigecycline were determined in MHB that was either prepared fresh (<12 h old), prepared and stored at 4°C, stored at room temperature, stored anaerobically, or supplemented with the biocatalytic oxygen-reducing reagent Oxyrase. When tested in fresh media, tigecycline was 2 to 3 dilutions more active against the CLSI-recommended QC strains compared to aged media (MICs of 0.03 to 0.25 and 0.12 to 0.5 μg/ml, respectively). Media aged under anaerobic conditions prior to testing or supplemented with Oxyrase resulted in MICs similar to those obtained in fresh medium (MICs of 0.03 to 0.12 and 0.03 to 0.25 μg/ml, respectively). Time-kill kinetics demonstrated a >3 log10 difference in viable growth when tigecycline was tested in fresh or Oxyrase-supplemented MHB compared to aged MHB. High-pressure liquid chromatography analysis revealed the accumulation of an early peak (oxidative by-product of tigecycline) to be 3.5% in fresh media and 25.1% in aged media after 24 h and that addition of Oxyrase prevented the accumulation of this oxidized by-product. These results suggested that the activity of tigecycline was affected by the amount of dissolved oxygen in the media. The use of fresh MHB or supplementation with Oxyrase resulted in a more standardized test method for performing MIC tests with tigecycline.

Comparative in vitro and in vivo activities of piperacillin combined with the beta-lactamase inhibitors tazobactam, clavulanic acid, and sulbactam.

Tazobactam (YTR-830H), a novel beta-lactamase inhibitor, was compared with clavulanic acid and sulbactam for enhancement of the activity of piperacillin against beta-lactamase-producing, piperacillin-resistant clinical isolates. Piperacillin MICs were determined in media containing a fixed concentration of 2 or 4 micrograms of the inhibitors per ml. The higher concentration was generally more effective. Tazobactam was superior to sulbactam in enhancing the spectrum and potency of piperacillin. Although the calvulanic acid combination was more potent, tazobactam was effective for a similar spectrum of resistant gram-negative clinical isolates containing beta-lactamase. MICs were reduced to the susceptible range for Escherichia coli, Klebsiella pneumoniae, Proteus spp., Salmonella spp., and Shigella spp. Combinations with tazobactam and sulbactam, but not clavulanic acid, were effective against Morganella spp. Some antagonism of the activity of piperacillin was observed with clavulanic acid but not with tazobactam or sulbactam. The inhibitors were similarly effective with piperacillin against beta-lactamase-positive Staphylococcus spp. and the Bacteroides fragilis group. Piperacillin-tazobactam was more effective against a broader spectrum of gram-negative enteric bacteria than ticarcillin plus clavulanic acid was. Combinations with tazobactam or clavulanic acid had a broader spectrum of activity than combinations with sulbactam against bacteria that produce characterized plasmid-mediated enzymes of clinical significance. In particular, piperacillin with tazobactam or clavulanic acid, but not with sulbactam, inhibited TEM-1, TEM-2, and SHV-1 enzymes. In vitro activity was reflected in vivo. Tazobactam and clavulanic acid were superior to sulbactam in enhancing the therapeutic efficacy of piperacillin in mice infected with beta-lactamase-positive E. coli, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Only combinations with tazobactam and sulbactam were effective against the Morganella infection. Tazobactam has a good potential for enhancing the clinical efficacy of piperacillin.

In vitro activity of tigecycline against Staphylococcus epidermidis growing in an adherent-cell biofilm model.

ABSTRACT The activity of tigecycline against Staphylococcus epidermidis growing in an in vitro adherent-cell biofilm model was determined. Tigecycline minimum bactericidal concentrations (MBCs) ranged from 1 to 8 μg/ml for S. epidermidis growing in a biofilm of adherent cells, compared to MBCs of 0.12 to >32 μg/ml for freely growing cells. The killing activity of tigecycline against the adherent bacteria was at least fourfold better than that of vancomycin and daptomycin.

Muraymycins, novel peptidoglycan biosynthesis inhibitors: synthesis and SAR of their analogues.

A series of Muraymycin analogues was synthesized. These analogues showed excellent antimicrobial activity against gram-positive organisms. These analogues also showed excellent inhibitory activity against the target peptidoglycan biosynthesis enzyme MraY, the cell membrane associated transglycosylase responsible for the formation of Lipid II.

Identification and Analysis of Bacterial Protein Secretion Inhibitors Utilizing a SecA-LacZ Reporter Fusion System

ABSTRACT Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds forStaphylococcus aureus strain MN8 were found to be ≤128 μg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.

Occurrence of Tetracycline Resistance Genes among Escherichia coli Isolates from the Phase 3 Clinical Trials for Tigecycline

ABSTRACT Tigecycline, a member of the glycylcycline class of antibiotics, was designed to maintain the antibacterial spectrum of the tetracyclines while overcoming the classic mechanisms of tetracycline resistance. The current study was designed to monitor the prevalence of the tet(A), tet(B), tet(C), tet(D), tet(E), and tet(M) resistance determinants in Escherichia coli isolates collected during the worldwide tigecycline phase 3 clinical trials. A subset of strains were also screened for the tet(G), tet(K), tet(L), and tet(Y) genes. Of the 1,680 E. coli clinical isolates screened for resistance to classical tetracyclines, 405 (24%) were minocycline resistant (MIC ≥ 8 μg/ml) and 248 (15%) were tetracycline resistant (MIC ≥ 8 μg/ml) but susceptible to minocycline (MIC ≤ 4 μg/ml). A total of 452 tetracycline-resistant, nonduplicate isolates were positive by PCR for at least one of the six tetracycline resistance determinants examined. Over half of the isolates encoding a single determinant were positive for tet(A) (26%) or tet(B) (32%) with tet(C), tet(D), tet(E), and tet(M), collectively, found in 4% of isolates. Approximately 33% of the isolates were positive for more than one resistance determinant, with the tet(B) plus tet(E) combination the most highly represented, found in 11% of isolates. The susceptibilities of the tetracycline-resistant strains to tigecycline (MIC90, 0.5 μg/ml), regardless of the encoded tet determinant(s), were comparable to the tigecycline susceptibility of tetracycline-susceptible strains (MIC90, 0.5 μg/ml). The results provide a current (2002 to 2006) picture of the distribution of common tetracycline resistance determinants encoded in a globally sourced collection of clinical E. coli strains.