Correlation of posaconazole minimum fungicidal concentration and time kill test against nine Candida species.

OBJECTIVES: We evaluated the in vitro activity of posaconazole against nine Candida species using minimum fungicidal concentration (MFC) measurements and time-kill methods. METHODS: MFCs of posaconazole were determined for 209 clinical isolates (32 Candida albicans, 30 Candida glabrata, 21 Candida tropicalis, 29 Candida krusei, 28 Candida parapsilosis sensu stricto, 50 Candida inconspicua, 13 Candida kefyr, 3 Candida lusitaniae and 3 Candida guilliermondii) and 7 ATCC Candida strains. The following strains were tested in time-kill studies: 3 strains each of C. glabrata, C. kefyr, C. guilliermondii and C. lusitaniae; 2 C. tropicalis; 4 C. albicans; 4 C. inconspicua; 9 C. krusei; 12 C. parapsilosis; and 7 ATCC strains. RESULTS: Posaconazole was fungicidal in both MFC and time-kill experiments (at 2 mg/L within 48 h in time-kill assays) against each C. krusei, C. inconspicua and C. lusitaniae strain and was fungistatic against each C. albicans, C. glabrata, C. tropicalis and C. guilliermondii strain. For the C. parapsilosis strains, posaconazole MFCs were

Posaconazole susceptibility testing against Candida species: comparison of broth microdilution and E-test methods.

Posaconazole (POS) is a newer triazole with activity against yeasts and moulds. POS and fluconazole were tested in vitro against 32 Candida albicans, 30 C. glabrata, 21 C. tropicalis, 29 C. krusei, 28 C. parapsilosis, 50 C. inconspicua, 13 C. kefyr and 5 C. famata isolates using CLSI broth microdilution method (BMD). We compared E-test and a modified BMD using polyethylene-glycol (PEG) as solvent to the CLSI method. BMDs and E-test were performed according to CLSI and the manufacturer's instructions respectively. Geometric means of POS MICs using BMD were 0.71, 0.22 and 0.21 microg ml(-1) against C. glabrata, C. krusei and C. inconspicua, respectively, and remained below 0.1 microg ml(-1) against all other species tested. One of two C. albicans and two of three C. glabrata isolates resistant to fluconazole showed MICs above 8 microg ml(-1) to POS. The impact of using PEG instead of DMSO had only a minor effect (agreements above 95% with the exception of C. parapsilosis). E-tests read after 24 h showed good agreement with the BMD. POS exhibited excellent in vitro activity against Hungarian Candida strains. E-test showed good correlation with the CLSI method, but to facilitate the comparability of results we believe that DMSO should be used as solvent in the BMD.

In vitro activities of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts.

The in vitro activity of the novel triazole antifungal agent posaconazole (Noxafil; SCH 56592) was assessed in 45 laboratories against approximately 19,000 clinically important strains of yeasts and molds. The activity of posaconazole was compared with those of itraconazole, fluconazole, voriconazole, and amphotericin B against subsets of the isolates. Strains were tested utilizing Clinical and Laboratory Standards Institute broth microdilution methods using RPMI 1640 medium (except for amphotericin B, which was frequently tested in antibiotic medium 3). MICs were determined at the recommended endpoints and time intervals. Against all fungi in the database (22,850 MICs), the MIC(50) and MIC(90) values for posaconazole were 0.063 microg/ml and 1 mug/ml, respectively. MIC(90) values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 mug/ml. In comparative studies against subsets of the isolates, posaconazole was more active than, or within 1 dilution of, the comparator drugs itraconazole, fluconazole, voriconazole, and amphotericin B against approximately 7,000 isolates of Candida and Cryptococcus spp. Against all molds (1,702 MICs, including 1,423 MICs for Aspergillus isolates), posaconazole was more active than or equal to the comparator drugs in almost every category. Posaconazole was active against isolates of Candida and Aspergillus spp. that exhibit resistance to fluconazole, voriconazole, and amphotericin B and was much more active than the other triazoles against zygomycetes. Posaconazole exhibited potent antifungal activity against a wide variety of clinically important fungal pathogens and was frequently more active than other azoles and amphotericin B.

EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

A novel site of antibiotic action in the ribosome: interaction of evernimicin with the large ribosomal subunit.

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Effects of mutations in ribosomal protein L16 on susceptibility and accumulation of evernimicin.

Chemical mutagenesis of Staphylococcus aureus RN450 generated two strains that displayed a stable reduction (30- to 60-fold) in susceptibility to evernimicin. Cell-free translation reactions demonstrated that the resistance determinant was located in the ribosomal fraction. Compared to ribosomes isolated from a wild-type strain, ribosomes from the mutant strains displayed an 8- to 10-fold reduction in affinity for [(14)C]evernimicin. In contrast, the mutants displayed no alteration in either binding affinity or in vitro susceptibility to erythromycin. Exponential cultures of the mutant strains accumulated significantly less [(14)C]evernimicin than the wild-type strain, suggesting that accumulation is dependent on the high affinity that evernimicin displays for its binding site. Sequencing rplP (encodes ribosomal protein L16) in the mutant strains revealed a single base change in each strain, which resulted in a substitution of either cysteine or histidine for arginine at residue 51. Introduction of a multicopy plasmid carrying wild-type rplP into the mutant strains restored sensitivity to evernimicin, confirming that the alterations in rplP were responsible for the change in susceptibility. Overexpression of the mutant alleles in S. aureus RN450 had no effect on susceptibility to evernimicin, demonstrating that susceptibility is dominant over resistance.

Evernimicin binds exclusively to the 50S ribosomal subunit and inhibits translation in cell-free systems derived from both gram-positive and gram-negative bacteria.

Evernimicin (SCH 27899) is a new antibiotic with activity against a wide spectrum of gram-positive bacteria and activity against some gram-negative bacteria. Previous metabolic labeling studies indicated that evernimicin specifically inhibited protein synthesis in Staphylococcus aureus. Using a susceptible Escherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli or S. aureus, evernimicin had a 50% inhibitory concentration of approximately 125 nM. In contrast, cell-free systems derived from wheat germ and rabbit reticulocytes were inhibited only by very high levels of evernimicin. Evernimicin did not promote transcript misreading. [(14)C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli and S. aureus and 50S subunits from E. coli returned dissociation constants of 84, 86, and 160 nM, respectively. In binding experiments, performed in the presence of excess quantities of a selection of antibiotics known to bind to the 50S subunit, only the structurally similar drug avilamycin blocked binding of [(14)C]evernimicin to ribosomes.

Functional dissection of the molybdate-responsive transcription regulator, ModE, from Escherichia coli.

The product of the Escherichia coli modE gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the modA promoter in vitro and to repress expression from a modA-lacZ operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of lambda CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the moaA and dmsA promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.

Anaerobic regulation of the Escherichia coli dmsABC operon requires the molybdate-responsive regulator ModE.

Expression of the Escherichia coli dmsABC operon that encodes a molybdenum-containing DMSO/TMAO reductase is increased in response to anaerobiosis and repressed by nitrate. These changes are mediated by the transcription factors Fnr and NarL respectively. Interestingly, modC strains that are defective in molybdate uptake exhibit impaired anaerobic induction and no nitrate-dependent repression of the dmsABC operon. To determine if the molybdate-responsive transcription factor ModE is involved in this process, a set of dmsA-lacZ operon fusions were constructed and analysed. The pattern of dmsA-lacZ expression in response to anaerobiosis and nitrate addition was identical in both modC and modE strains, thus suggesting a regulatory role for ModE. In vitro studies confirmed that ModE bound the dmsA promoter at a high-affinity site typical of other E. coli ModE operator sites. Mutations in this site abolished ModE binding in vitro and displayed the same phenotype as a modE mutation. In contrast to previously characterized ModE operator sites, which either overlap or are located immediately upstream of the ModE-regulated promoter, the ModE site is centred 52.5 bp downstream of the major dmsA transcript start site. We identified a putative integration host factor (IHF) binding site in the intervening sequence, and in vitro studies confirmed that IHF bound this site with high affinity. Using himA mutants, we confirmed that IHF plays a role in the molybdate-dependent regulation of dmsA-lacZ expression in vivo. This study provides the first example in which ModE affects gene regulation in concert with another transcription factor.

Characterization of the ModE DNA-binding sites in the control regions of modABCD and moaABCDE of Escherichia coli.

The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration. Utilizing an in vivo titration assay, we localized the ModE-binding site to the start of modA transcription. This localization was further characterized using in vitro gel-shift assays and DNase I footprinting. ModE bound the wild-type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding. Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro. DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position -18 to +10, relative to the transcript start site. Gel-shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24nM. Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative -35 consensus sequence and extending from position -202 to -174, relative to the start of translation. In vivo expression of a moaA-lacZ operon fusion was stimulated twofold by ModE. However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo. These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site.

Expression of the heme biosynthetic pathway genes hemCD, hemH, hemM, and hemA of Escherichia coli.

Little is known about the control of latter steps of heme biosynthesis in Escherichia coli. In this study we examined the transcriptional regulation of genes that encode two intermediate heme pathway enzymes, porphobilinogen deaminase (hemC) and uroporphyrinogen III cosynthase (hemD), and the final enzyme of the pathway, ferrochelatase (hemH). We also reexamined the regulation of hemA and the gene located immediately upstream of hemA, hemM. The regulatory regions of hemC, hemH, hemA and hemM were fused to lacZ. The resultant operon fusions were inserted into the E. coli chromosome in single copy and expression monitored under conditions of oxygen and heme limitation. Expression of hemM appeared constitutive under the conditions tested here. In contrast, expression of hemCD, hemH and hemA were shown to be mildly regulated in response to heme availability. Thus, transcription of four of the nine genes of the E. coli heme pathway appears to be only mildly regulated in response to heme limitation.

The Escherichia coli modE gene: effect of modE mutations on molybdate dependent modA expression.

The Escherichia coli modABCD operon, which encodes a high-affinity molybdate uptake system, is transcriptionally regulated in response to molybdate availability by ModE. Here we describe a highly effective enrichment protocol, applicable to any gene with a repressor role, and establish its application in the isolation of transposon mutations in modE. In addition we show that disruption of the ModE C-terminus abolishes derepression in the absence of molybdate, implying this region of ModE controls the repressor activity. Finally, a mutational analysis of a proposed molybdate binding motif indicates that this motif does not function in regulating the repressor activity of ModE.