In vitro activity of tigecycline against patient isolates collected during phase 3 clinical trials for diabetic foot infections.

The in vitro activity of tigecycline and comparative antimicrobial agents was evaluated against 1828 primary baseline pathogens isolated from 844 patients enrolled in the phase 3 clinical trials investigating the efficacy of tigecycline in diabetic foot infection (DFI). The trials were global, enrolling patients in 30 countries. Tigecycline was active against the most prevalent pathogens in DFI, including Gram-positive and Gram-negative isolates of both aerobic and anaerobic bacteria with 95% of MICs < or =2 microg/mL for the entire collection. The spectrum of activity of tigecycline included important pathogens for DFI, such as Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, and Bacteroides fragilis. As reported previously, Pseudomonas aeruginosa and several pathogens in the Proteeae group were generally less susceptible to tigecycline by comparison to other Gram-negative pathogens. The excellent in vitro expanded broad-spectrum activity of tigecycline in the clinical isolates confirmed the potential utility of tigecycline for pathogens associated with DFIs.

In vitro activity of tigecycline against patient isolates collected during phase 3 clinical trials for hospital acquired pneumonia.

The in vitro activity of tigecycline was evaluated against 819 baseline pathogens isolated from 383 patients enrolled in the phase 3 clinical trial investigating the efficacy of tigecycline in hospital acquired pneumonia (HAP). The trials were global, enrolling patients in 27 countries. Tigecycline was active against the most prevalent pathogens in HAP, including gram-positive and gram-negative strains (90% of MICs ≤2 µg/mL for the entire collection). The spectrum of activity of tigecycline included important pathogens such as Staphylococcus aureus (including methicillin-resistant S. aureus), Enterococcus faecalis, Streptococcus pneumoniae, Acinetobacter baumannii/calcoaceticus complex, Escherichia coli, Klebsiella pneumonia, and Enterobacter cloacae. As reported previously, a few genera, such as Pseudomonas aeruginosa and the Proteeae, were generally less susceptible to tigecycline by comparison to other gram-negative pathogens. The excellent in vitro, expanded, broad-spectrum activity of tigecycline in the clinical isolates confirmed the potential utility of tigecycline for pathogens associated with with hospital acquired pneumonia infections.

Resistance development profiling of piperacillin in combination with the novel {beta}-lactamase inhibitor BLI-489.

OBJECTIVES: To evaluate development of resistance to the piperacillin/BLI-489 combination. METHODS: BLI-489 was used at a constant concentration of 4 mg/L. Spontaneous mutation frequency was measured on piperacillin/BLI-489-containing agar plates. Five beta-lactamase-producing strains were exposed to a serial dilution of piperacillin/BLI-489, and the highest concentration allowing growth was used to inoculate subsequent serial passage for 10 days. Mutation stability was monitored in drug-free medium for 10 days. RESULTS: Escherichia coli (OXA-3, OXA-7, ACT-1, SHV-1 or none), Salmonella enterica serovar Typhimurium (CTX-M-5), Klebsiella pneumoniae (SHV-1 and SHV-5) and Enterobacter cloacae (AmpC) had a spontaneous mutation frequency of < or =1.0 x 10(-9). Two AmpC-producing Pseudomonas aeruginosa strains had a mutation frequency of 6.52 x 10(-6) and 1.0 x 10(-7); a beta-lactamase-negative P. aeruginosa strain had a mutation frequency of 2.68 x 10(-8). The mutant prevention concentration (MPC) was < or =32 mg/L. During serial passages, the MIC increased 64- and 128-fold for S. enterica serovar Typhimurium (CTX-M-5) and E. cloacae (AmpC), respectively, to > or =512 mg/L. The MIC reverted to < or =64 mg/L after serial passages in drug-free medium. The MICs increased only 4-fold for K. pneumoniae (SHV-1 and SHV-5), E. coli (OXA-3) and E. coli (SHV-1). CONCLUSIONS: Piperacillin/BLI-489 demonstrated a low probability of spontaneous resistance development in vitro for all of the strains tested with the exception of P. aeruginosa. The MPC value for all strains was < or =32 mg/L. Resistance developed during serial passage for two of the five strains tested; however, this resistance phenotype was unstable as MIC values reverted to < or =64 mg/L after propagation in drug-free medium.

Efficacy of piperacillin combined with the Penem beta-lactamase inhibitor BLI-489 in murine models of systemic infection.

The in vivo efficacy of piperacillin in combination with the penem inhibitor BLI-489 was determined using acute lethal systemic infections in mice. On the basis of preliminary results with various ratios, a dosing ratio of 8:1 was found to be optimal for retention of enhanced efficacy. Piperacillin-BLI-489 dosed at an 8:1 ratio was efficacious against murine infections caused by class A (including extended-spectrum beta-lactamases), class C (AmpC), and class D beta-lactamase-expressing pathogens.

Establishment of in vitro susceptibility testing methodologies and comparative activities of piperacillin in combination with the penem {beta}-lactamase inhibitor BLI-489.

The novel bicyclic penem inhibitor BLI-489 has demonstrated activity as an inhibitor of class A, C, and D beta-lactamases. To determine the combination of piperacillin and BLI-489 to be used in susceptibility testing that would most accurately identify susceptible and resistant isolates, a predictor panel of beta-lactamase-producing bacteria was utilized to determine the reliability of the combination of piperacillin-BLI-489 at a constant inhibitor concentration of 2 or 4 microg/ml and at ratios of 1:1, 2:1, 4:1, and 8:1. There were a number of strains that would be falsely reported as susceptible or intermediate if tested with the ratios of 1:1 and 2:1, whereas the constant concentration of 2 microg/ml of BLI-489 and the ratio of 8:1 had a tendency to overpredict resistance. Similar MICs were obtained with piperacillin-BLI-489 in a 4:1 ratio and when BLI-489 was held constant at 4 microg/ml. Based on these results, an in vitro testing methodology employing a constant concentration of 4 microg/ml BLI-489 was used to evaluate the combination of piperacillin-BLI-489 against a larger panel of recently identified clinical isolates. Approximately 55% of all of the enteric bacilli tested were nonsusceptible to piperacillin alone (MIC > or = 32 microg/ml). However, 92% of these piperacillin nonsusceptible strains were inhibited by < or =16 microg/ml piperacillin-BLI-489; in contrast, only 66% were inhibited by < or =16 microg/ml piperacillin-tazobactam. The combination of piperacillin-BLI-489 also demonstrated improved activity compared to that of piperacillin-tazobactam against the problematic extended-spectrum beta-lactamase- and AmpC-expressing strains.

Pyrosequencing using the single-nucleotide polymorphism protocol for rapid determination of TEM- and SHV-type extended-spectrum beta-lactamases in clinical isolates and identification of the novel beta-lactamase genes blaSHV-48, blaSHV-105, and blaTEM-155.

TEM- and SHV-type extended-spectrum beta-lactamases (ESBLs) are the most common ESBLs found in the United States and are prevalent throughout the world. Amino acid substitutions at a number of positions in TEM-1 lead to the ESBL phenotype, although substitutions at residues 104 (E to K), 164 (R to S or H), 238 (G to S), and 240 (E to K) appear to be particularly important in modifying the spectrum of activity of the enzyme. The SHV-1-derived ESBLs are a less diverse collection of enzymes; however, the majority of amino acid substitutions resulting in an ESBL mirror those seen in the TEM-1-derived enzymes. Pyrosequencing by use of the single-nucleotide polymorphism (SNP) protocol was applied to provide sequence data at positions critical for the ESBL phenotype spanning the bla(TEM) and bla(SHV) genes. Three novel beta-lactamases are described: the ESBLs TEM-155 (Q39K, R164S, E240K) and SHV-105 (I8F, R43S, G156D, G238S, E240K) and a non-ESBL, SHV-48 (V119I). The ceftazidime, ceftriaxone, and aztreonam MICs for an Escherichia coli isolate expressing bla(SHV-105) were >128, 128, and >128 microg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing bla(TEM-155) were >128, 64, and > 128 microg/ml, respectively. Pyrosequence analysis determined the true identity of the beta-lactamase on plasmid R1010 to be SHV-11 rather than SHV-1, as previously reported. Pyrosequencing is a real-time sequencing-by-synthesis approach that was applied to SNP detection for TEM- and SHV-type ESBL identification and represents a robust tool for rapid sequence determination that may have a place in the clinical setting.

5,5,6-Fused tricycles bearing imidazole and pyrazole 6-methylidene penems as broad-spectrum inhibitors of beta-lactamases.

Beta-lactamases are serine- and metal-dependent hydrolases, produced by the bacteria as defense against beta-lactam antibiotics. Commercially available inhibitors such as clavulanic acid, sulbactam, and tazobactam, which are currently used in the hospital settings, have reduced activity against newly emerging beta-lactamases. Bacterial production of diverse beta-lactamases including class-A, class-C, and ESBLs has motivated several research groups to search for inhibitors with a broader spectrum of activity. Previously, several novel 6-methylidene penems bearing, [5,5] [5,6] and [5,5,5] heterocycles have been synthesized in our laboratory and were shown to be potent and broad-spectrum beta-lactamase inhibitors. As a continuation of our previous work and in order to extend the structure-activity relationships, in this paper, we describe herein the synthesis and in vitro, in vivo activities of several novel 5,5,6-fused tricyclic heterocycles attached to the 6-methylidene penem core. The compounds presented in the current paper are potent and broad-spectrum inhibitors of the TEM-1 and AmpC beta-lactamases. In combination with piperacillin, their in vitro activities showed enhanced susceptibility to class A- and C-resistant strains studied in various bacteria. Some of the newly synthesized compounds such as 12a-c were shown to have in vivo activity in the acute lethal infection model against TEM-1 producing organisms. The 5,5,6-fused heterocyclic ring cores such as 21, 25, and 35 reported here are hitherto unknown in the literature.

Occurrence of tetracycline resistance genes among Escherichia coli isolates from the phase 3 clinical trials for tigecycline.

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 > or = 8 microg/ml) and 248 (15%) were tetracycline resistant (MIC > or = 8 microg/ml) but susceptible to minocycline (MIC < or = 4 microg/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 (MIC(90), 0.5 microg/ml), regardless of the encoded tet determinant(s), were comparable to the tigecycline susceptibility of tetracycline-susceptible strains (MIC(90), 0.5 microg/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.

In vitro antibacterial activities of tigecycline and comparative agents by time-kill kinetic studies in fresh Mueller-Hinton broth.

Time-kill kinetics performed with tigecycline, in fresh MHB, demonstrated a consistent 1 to 2 log(10) CFU/ml reduction in bacterial counts against the majority of clinically relevant pathogens tested. Although classified as a bacteriostatic agent, tigecycline shows bactericidal activity against select isolates associated with serious infection. In general, vancomycin and imipenem demonstrated bactericidal activity.

A novel antibiotic bone assay by liquid chromatography/tandem mass spectrometry for quantitation of tigecycline in rat bone.

Tigecycline (Tygacil) is a first-in-class, broad spectrum antibiotic with activity against antibiotic-resistant organisms. In rats and humans, tigecycline readily distributes to bone tissue but its accuracy of quantitation via liquid chromatography/mass spectrometry (LC/MS/MS) is hindered by a low extraction recovery when using a conventional plasma extraction method. To overcome this issue, we have identified an effective extraction solvent for quantitation of tigecycline in rat bone. The current LC/MS/MS bone assay is novel, simple, and sensitive, and has a wide linear range of 50-10,000 ng/g. The assay requires homogenization of the rat bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation of the supernatant with liquid chromatography, and detection of tigecycline with tandem mass spectrometry. The incurred pooled rat bone samples obtained from rats given 3mg/kg/day of [(14)C]-tigecycline and non-radio-labeled tigecycline were analyzed with the current method. The absolute extraction recovery of the bone assay for tigecycline was 77.1%. The intra-day accuracy ranged from 91.7 to 106% with precision (CV) of 1.9-10.7%, and inter-day accuracy ranged from 96.1 to 100% with a precision of 6.3-8.7%. In addition, biological activity was demonstrated for the tigecycline extracted from incurred rat bone. This bone assay provides an important analytical tool for the determination of drug concentrations (especially, antimicrobials) in rodent bone tissues and has served as the foundation of development and validation of a similar bone assay for tigecycline in human bone tissues.

Validation and reproducibility assessment of tigecycline MIC determinations by Etest.

A multicenter study was conducted to validate Etest tigecycline compared to the Clinical Laboratory Standards Institute reference broth microdilution and agar dilution methodologies. A large collection of gram-negative (n = 266) and gram-positive (n = 162) aerobic bacteria, a collection of anaerobes (n = 385), and selected collections of nonpneumococcal streptococci (n = 369), Streptococcus pneumoniae (n = 372), and Haemophilus influenzae (n = 372) were tested. Strains with reduced susceptibility to tigecycline were used with all test methods. The Etest showed excellent inter- and intralaboratory reproducibility for all organism groups tested regardless of the test methodology. The essential agreement values with the reference method (+/-1 dilution) were >99% for the collection of gram-negative and gram-positive aerobes; >98% for the S. pneumoniae, H. influenzae, and anaerobe collections; and 100% for the group of nonpneumococcal streptococci. These results validate the performance accuracy and utility of Etest tigecycline and verify the reproducibility of this convenient predefined gradient methodology for tigecycline susceptibility determination.

Tigecycline: a case study.

The emergence of pathogenic bacteria resistant to virtually all available antibacterial agents at present has caused consternation among medical professionals, but has only intermittently raised concern among the public. This has led to a transient resurgence of interest in studying the mechanisms of resistance and in discovering and developing new antibacterial agents, but successes in the development of novel antibacterial agents have been few and far between. Although it has been known since the discovery of the tetracyclines that they are inhibitors of protein synthesis, there has been considerable recent progress on elucidating the mechanisms of action of the tetracyclines and in the enhanced understanding of the mechanisms of tetracycline resistance. In this case study, the authors discuss the discovery and development of a new class of antibacterials, which were derived from the tetracyclines, namely the glycylcyclines. This has resulted in the introduction of a new agent, tigecycline, to clinical practice. The glycylcyclines restore the antibacterial activity to levels of the earlier tetracyclines when they were first introduced, by overcoming the two major tetracycline-resistance mechanisms of efflux and ribosome protection, which promises to have a high degree of clinical utility.

Pyrazolidine-3,5-diones and 5-hydroxy-1H-pyrazol-3(2H)-ones, inhibitors of UDP-N-acetylenolpyruvyl glucosamine reductase.

A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-ones show low micromolar IC(50) values versus E. coli MurB and submicromolar minimal inhibitory concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability. None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial least squares projection to latent structures analysis shows that improving MurB potency and MIC values correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core. Docking studies using FLO and PharmDock produced several binding orientations for these molecules in the MurB active site.

Structure-activity relationship of 6-methylidene penems bearing 6,5 bicyclic heterocycles as broad-spectrum beta-lactamase inhibitors: evidence for 1,4-thiazepine intermediates with C7 R stereochemistry by computational methods.

The design and synthesis of a series of 6-methylidene penems containing [6,5]-fused bicycles (thiophene, imidazole, or pyrazle-fused system) as novel class A, B, and C beta-lactamase inhibitors is described. These penems proved to be potent inhibitors of the TEM-1 (class A) and AmpC (class C) beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. Their in vitro and in vivo activities in combination with piperacillin are discussed. On the basis of the crystallographic structures of a serine-bound reaction intermediate of 2 with SHV-1 (class A) and GC1 (class C) enzymes, compounds 14a-l were designed and synthesized. Penems are proposed to form a seven-membered 1,4 thiazepine ring in both class A and C beta-lactamases. The interaction energy calculation for the enzyme-bound intermediates favor the formation of the C7 R enantiomer over the S enantiomer of the 1,4-thiazepine in both beta-lactamases, which is consistent with those obtained from the crystal structure of 2 with SHV-1 and GC1.

In vitro antibacterial activities of tigecycline in combination with other antimicrobial agents determined by chequerboard and time-kill kinetic analysis.

OBJECTIVES: This study was undertaken to determine the interaction of tigecycline with 13 select antimicrobial agents against a wide variety of Gram-negative and Gram-positive bacterial isolates. METHODS: Antibiotic interactions were assayed using the chequerboard MIC format and selected synergistic combinations were confirmed using time-kill kinetic analysis. RESULTS: Microdilution chequerboard analysis of tigecycline in combination with amikacin, ampicillin/sulbactam, azithromycin, ciprofloxacin, colistin, imipenem, levofloxacin, piperacillin, piperacillin/tazobactam, polymyxin B, rifampicin, minocycline and vancomycin resulted in an interpretation of either no interaction or synergy. Time-kill kinetic analysis resulted in an interpretation of no interaction for all but one of the drug combinations that resulted in an interpretation of synergy by the chequerboard analysis. Antagonism was not observed for any combination when assayed by either method. CONCLUSIONS: The lack of antagonism seen with tigecycline combinations in both chequerboard and time-kill kinetic studies is an encouraging outcome, suggesting that tigecycline may prove to be effective in combination therapy as well as in monotherapy.

3,5-dioxopyrazolidines, novel inhibitors of UDP-N- acetylenolpyruvylglucosamine reductase (MurB) with activity against gram-positive bacteria.

A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 microM, 4.3 to 10.3 microM, and 6.8 to 29.4 microM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 microM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 A resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 microM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 microg/ml) and 4 (MICs, 4 to 8 microg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Pulvinones as bacterial cell wall biosynthesis inhibitors.

Pulvinones were synthesized (>180) in arrays and evaluated as inhibitors of early stage cell wall biosynthesis enzymes MurA-MurD. Several pulvinones inhibited Mur enzymes with IC(50)'s in the 1-10 microg/mL range and demonstrated antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphyloccus aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Synthesis and antibacterial activity of 9-substituted minocycline derivatives.

A number of 9-acylamino and 9-sulfonylamino derivatives of minocycline have been synthesized for structure-activity relationship studies. These compounds showed activity against both tetracycline-susceptible and tetracycline-resistant strains. Many of the 9-sulfonylamino derivatives exhibited improved antibacterial activity against a number of tetracycline- and minocycline-resistant Gram-positive pathogens.

Use of structure-based drug design approaches to obtain novel anthranilic acid acyl carrier protein synthase inhibitors.

Acyl carrier protein synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheinyl group from the coenzyme A to a serine residue in acyl carrier protein (ACP), thereby activating ACP, an important step in cell wall biosynthesis. The structure-based design of novel anthranilic acid inhibitors of AcpS, a potential antibacterial target, is presented. An initial high-throughput screening lead and numerous analogues were modeled into the available AcpS X-ray structure, opportunities for synthetic modification were identified, and an iterative process of synthetic modification, X-ray complex structure determination with AcpS, biological testing, and further modeling ultimately led to potent inhibitors of the enzyme. Four X-ray complex structures of representative anthranilic acid ligands bound to AcpS are described in detail.