Enterobacter cloacae isolates were all <1 g/ml, and there were only 8 isolates (1.3%) among these 617 clinical isolates with MIC values of >8 g/ml. In the second set, the MIC values of S-649266 were <4 g/ml against 109 strains among 116 KPC-producing and class B (metallo) carbapenemase-producing strains. In addition, S-649266 showed MIC values of <2 g/ml against each of the 13 strains that produced other types of carbapenemases such as SME, NMC, and OXA-48. The mechanisms of the decreased susceptibility of 7 class B carbapenemase-producing strains with MIC values of >16 g/ml are uncertain. This is the first report to demonstrate that S-649266, a novel siderophore cephalosporin, has significant antimicrobial activity against Enterobacteriaceae, including strains that produce carbapenemases such as KPC and NDM-1.

The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus / Lactobacillus / Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI (Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.

S-649266 is a novel antibiotic with potent in vitro activity against a range of non-fermenting Gram-negative bacteria, including MDR strains.

Determining the genetic characteristics of Staphylococcus aureus is important for better understanding of the global and dynamic epidemiology of this organism as we witness the emergence and spread of virulent and antibiotic-resistant clones. We genotyped 292 S. aureus isolates (105 methicillin resistant and 187 methicillin susceptible) using a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and SCCmec typing. In addition, S. aureus isolates were tested for the presence of the Panton-Valentine leukocidin (PVL) genes. Isolates were recovered from patients with uncomplicated skin infections in 10 different countries during five phase III global clinical trials of retapamulin, a new topical antibiotic agent. The most common methicillin-resistant clone had multilocus sequence type 8, pulsed-field type USA300, and SCCmec type IV and possessed the PVL genes. This clone was isolated exclusively in the United States. The most common PVLpositive, methicillin-susceptible clone had multilocus sequence type 121 and pulsed-field type USA1200. This clone was found primarily in South Africa and the Russian Federation. Other clones were found at lower frequencies and were limited in their geographic distribution. Overall, considerable genetic diversity was observed within multilocus sequence type clonal complexes and pulsed-field types.

All sampled PVL genes appear to share a recent common ancestor and spread via a combination of clonal expansion and horizontal transfer. US isolates harbor a variant of PVL that is strongly associated with MRSA infections. Protein modeling reveals that this variant may have functional significance. We propose a hypothesis for the origin of USA300.

The in vitro activity of retapamulin was determined and compared to that of topical and community antibiotics. The MIC 90 s of retapamulin against Staphylococcus aureus and Streptococcus pyogenes were 0.12 g/ml and 0.016 g/ml, respectively. Retapamulin has a low propensity to select resistance and produces an in vitro postantibiotic effect.The emergence and spread of antibiotic resistance in hospital and community pathogens has significantly eroded the utility of established antibiotics, a problem that has been widely publicized and poses a serious threat to public health worldwide (6,8). Novel mechanism antibiotics are needed to address rising resistance to established classes of both systemic and topical agents. Resistance has developed to two of the most commonly used topical antibiotics, fusidic acid and mupirocin, and thus dictates the need for novel mechanism topical agents for managing the treatment of bacterial skin infections.Retapamulin {mutilin 14-(exo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl-sulfanyl)-acetate} is a novel semisynthetic pleuromutilin that was discovered by GlaxoSmithKline as part of a program to identify novel compounds with the appropriate balance of drug developability and microbiological attributes to be formulated and developed as a topical antibiotic. In this work, we describe the initial microbiology evaluation that led to the selection of retapamulin for development.The isolates evaluated were obtained from the GlaxoSmithKline culture collection (Collegeville, PA). The panel of methicillin-resistant Staphylococcus aureus isolates (based on oxacillin MICs) was supplemented with 32 isolates obtained from International Health Management Associates (Schaumburg, IL). Broth microdilution, agar dilution, and cidality experiments were performed using the CLSI recommended procedures (3, 4). In the absence of approved quality control limits for retapamulin and specific comparator antibiotics, correlation with previous internal testing of these compounds was used. Modifications were made to the standard broth microdilution method (pH, inoculum density, serum) to identify potential factors that might influence the in vitro activity of retapamulin. Antibiotics were obtained as follows: retapamulin, amoxicillin, and mupirocin were from GlaxoSmithKline Pharmaceuticals, Harlow, United Kingdom; bacitracin, cefaclor, and fusidic acid were from Sigma Chemical Co., St. Louis, MO; levofloxacin and azithromycin were deformulated by GlaxoSmithKline Pharmaceuticals, Harlow, United Kingdom. The postantibiotic effect (PAE) of retapamulin and mupirocin were determined using a filtration method (staphylococci) or a dilution method (Streptococcus pyogenes) as previously described (7). The spontaneous frequencies of resistance in S. aureus and S. pyogenes were determined by plating the test isolate on agar containing 4ϫ and 10ϫ MIC of retapamulin and mupirocin. Detection of resistant colonies was performed 48 h after inoculation. The development of resistance was determined by daily passage of the test isolates ...

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Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.