Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160 000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.Increasing reports of antibiotic resistance involving opportunistic gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, have emphasized the critical need for the development of antimicrobial compounds with novel modes of action. Coenzyme A (CoA), an essential cofactor for maintaining life, is used in a multitude of biochemical reactions. In most bacteria, CoA is synthesized from pantothenic acid (vitamin B 5 ) in 5 steps (5), with the first step being the phosphorylation of pantothenate by pantothenate kinase (CoaA). Although this pathway also exists in eukaryotes, in most cases there is no sequence homology between the prokaryotic and eukaryotic CoA biosynthetic enzymes (7,9,12,18,24,27). Thus, there is the potential for developing highly specific inhibitors of bacterial CoA enyzmes.Unlike the case for other biosynthetic pathways of bacteria, the genes involved in CoA biosynthesis are not organized as operons. This has delayed the identification of the enzymes responsible for CoA synthesis, even though the intermediate chemical steps have been known since the 1960s (1). With the recent identification of the Escherichia coli genes encoding the enzymes CoaBC and CoaE, the entire pathway is now known for this organism (9,10,13,19,21). Interestingly, the gene coaA, which encodes the first enzyme in the pathway, has no homolog in the complete genome sequences of the S. aureus strains Mu50 and N315 (11).Cloning and purification of S. aureus CoaA. Initially, the coaA gene sequences in S. aureus strains Mu50 and N15 (GenBank accession numbers BA000017 and BA000018, respectively) were identified through searches of the ERGO comparative genomic database (previously WIT) (http://ergo .integratedgenomics.com/ERGO/) (8). We cloned the S. aureus RN4220 coaA gene and overexpressed it using standard techniques (4, 17). S. aureus RN4220 coaA was amplified by PCR, introducing an NdeI site at the start codon and an XhoI site after the stop codon, and cloned into pSTBlue1 using the Perfectly Blunt Cloning kit. The gene was excised by digestion with NdeI and XhoI and ligated into similarly digested pET28a. The final construct encoded the N-terminal six-His-tagged S. aureus CoaA.Tuner (DE3) cells were transformed with this construct and grown at 37°C in Luria-Bertani medium-50-g/ml kanamycin. Protein expression was induced by 500 M isopropylthio-␤-Dgalactoside, and cells were harvested 3 h postinduction. The cell pellet was resuspended and sonicated, and cell debris was removed by centrifugation. The supernatant was subjected to Ni-chelating column chromatography followed by a HiTrap Q Sepharose ion exchange column. Enzyme identity was confirmed through N-terminal sequencing and matrix-assisted...

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 ...

The aim of this study was to characterize quinolone resistance mechanisms in strains of Streptococcus pneumoniae with increased MICs of ofloxacin. These strains were also tested for their susceptibility to a battery of quinolone antimicrobial agents, including gemifloxacin. Of the S. pneumoniae isolates used, 27 were susceptible to ofloxacin, 18 intermediate and 48 resistant (ofloxacin MIC <4, 4 and >4 mg/L, respectively). In general, the ofloxacin-susceptible strains had no amino acid substitutions in GyrA, GyrB, ParC or ParE. Moderate increases in MIC were associated with substitutions in the quinolone resistance-determining region (QRDR) of ParC, while the highest MICs were found for strains that also had substitutions in the QRDR of GyrA. The most common substitutions were Ser79-->Phe in ParC and Ser81-->Phe in GyrA. Other substitutions were identified within the QRDR of ParC and outside the QRDR of ParC and ParE; these did not appear to affect susceptibility. The effects of antimicrobial efflux pumps were studied by determining MICs of a range of quinolones in the presence and absence of reserpine, an inhibitor of Gram-positive efflux pumps. Our results indicated that high-level resistance, caused entirely by efflux, was seen in a minority of ofloxacin-resistant S. pneumoniae strains. Testing the susceptibility of quinolone-resistant strains to gemifloxacin, ciprofloxacin, norfloxacin, ofloxacin and trovafloxacin revealed that gemifloxacin was least affected by this large variety of resistance mechanisms and was the only quinolone with MICs of < or =0.5 mg/L for all strains in this study. These results suggest that gemifloxacin is highly potent against S. pneumoniae and may also be effective against strains resistant to other quinolones.

This study investigated the in vitro potency of the novel quinolone agent gemifloxacin (SB-265805), in comparison with other quionolones, beta-lactams, macrolides and trimethoprim- sulphamethoxazole, against a panel of common respiratory pathogens. This panel comprised recent clinical isolates of Streptococcus pneumoniae (n = 347), Haemophilus influenzae (n = 256) and Moraxella catarrhalis (n = 184). Overall, the quinolones were highly active against H. influenzae and were the most potent agents against M. catarrhalis. Gemifloxacin was the most potent quinolone tested against all three species and was four- to 512-fold more potent against pneumococci than trovafloxacin, grepafloxacin, levofloxacin, ciprofloxacin, ofloxacin, gentamicin, cefuroxime, penicillin, ampicillin, clarithromycin, azithromycin or trimethoprim- sulphamethoxazole. Against 19 ofloxacin-intermediate and 52 ofloxacin-resistant strains of S. pneumoniae, gemifloxacin retained activity, and was the only agent tested with MICs of < or =0.5 mg/L. The results of this study demonstrate the excellent in vitro antibacterial activity of gemifloxacin against pathogens commonly associated with respiratory tract infections and suggest that gemifloxacin has significant potential in the treatment of such infections, including those caused by pneumococci considered resistant to other quinolones.