Paul M. McNicholas

In Vitro Activities of Posaconazole, Fluconazole, Itraconazole, Voriconazole, and Amphotericin B against a Large Collection of Clinically Important Molds and Yeasts

ABSTRACT 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 MIC50 and MIC90 values for posaconazole were 0.063 μg/ml and 1 μg/ml, respectively. MIC90 values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 μg/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.

Three-Dimensional Models of Wild-Type and Mutated Forms of Cytochrome P450 14α-Sterol Demethylases from Aspergillus fumigatus and Candida albicans Provide Insights into Posaconazole Binding

ABSTRACT The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from Aspergillus fumigatus and Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in A. fumigatus and C. albicans act by interfering with the binding of the long side chain.

Mutations in Aspergillus fumigatus Resulting in Reduced Susceptibility to Posaconazole Appear To Be Restricted to a Single Amino Acid in the Cytochrome P450 14α-Demethylase

ABSTRACT To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 108 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [14C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14α-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

Application of Real-Time Quantitative PCR to Molecular Analysis of Candida albicans Strains Exhibiting Reduced Susceptibility to Azoles

ABSTRACT Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1, in a collection of 14 fluconazole-susceptible Candida albicans isolates. For each gene, average expression levels and variations within the population were determined. These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis. For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above. Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates. In contrast, MDR1, ERG11, CDR1, and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles. In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11. Select ERG11 alleles were expressed in Saccharomyces cerevisiae; all of the alleles tested conferred reduced susceptibility to fluconazole. Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility.

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.

Posaconazole Is a Potent Inhibitor of Sterol 14α-Demethylation in Yeasts and Molds

ABSTRACT Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14α-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14α-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14α-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.

Impact of Antifungal Prophylaxis on Colonization and Azole Susceptibility of Candida Species

ABSTRACT Two large studies compared posaconazole and fluconazole or itraconazole for prophylaxis in subjects undergoing allogeneic hematopoietic stem cell transplantation or subjects with acute myelogenous leukemia. To assess the impact of prophylaxis on colonization and the development of resistance in Saccharomyces yeasts, identification and susceptibility testing were performed with yeasts cultured at regular intervals from mouth, throat, and stool samples. Prior to therapy, 34 to 50% of the subjects were colonized with yeasts. For all three drugs, the number of positive Candida albicans cultures decreased during drug therapy. In contrast, the proportion of subjects with positive C. glabrata cultures increased by two- and fourfold in the posaconazole and itraconazole arms, respectively. Likewise, in the fluconazole arm the proportion of subjects with positive C. krusei cultures increased twofold. C. glabrata was the species that most frequently exhibited decreases in susceptibility, and this trend did not differ significantly between the prophylactic regimens. For the subset of subjects from whom colonizing C. glabrata isolates were recovered at the baseline and the end of treatment, approximately 40% of the isolates exhibited more than fourfold increases in MICs during therapy. Molecular typing of the C. albicans and C. glabrata isolates confirmed that the majority of the baseline and end-of-treatment isolates were closely related, suggesting that they were persistent colonizers and not newly acquired. Overall breakthrough infections by Candida species were very rare (∼1%), and C. glabrata was the colonizing species that was the most frequently associated with breakthrough infections.

Evernimicin Binds Exclusively to the 50S Ribosomal Subunit and Inhibits Translation in Cell-Free Systems Derived from both Gram-Positive and Gram-Negative Bacteria

ABSTRACT 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 inStaphylococcus aureus. Using a susceptibleEscherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli orS. 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. [14C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli andS. aureus and 50S subunits from E. colireturned 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 [14C]evernimicin to ribosomes.

Comparative in vitro activities of posaconazole, voriconazole, itraconazole, and amphotericin B against Aspergillus and Rhizopus, and synergy testing for Rhizopus

We compared the in vitro activities of posaconazole, voriconazole, itraconazole, and amphotericin B against clinical isolates of Aspergillus spp. and Rhizopus spp., and explored the in vitro interaction between posaconazole and amphotericin B against Rhizopus spp. Clinical strains of 82 Aspergillus spp. (43 Aspergillus fumigatus, 29 A. flavus, 7 A. niger, 2 A. terreus, 1 A. nidulans) and 11 Rhizopus oryzae isolates were tested in accordance with CLSI M38-A microdilution guidelines. In vitro activity of posaconazole against Aspergillus spp. was also investigated with the Etest. The combination of posaconazole and amphotericin B against R. oryzae isolates was investigated by the checkerboard methodology. Voriconazole was the most active drug in vitro against Aspergillus spp., followed by posaconazole, itraconazole, and amphotericin B, in order of decreasing activity. In studies with R. oryzae isolates, posaconazole was found to be the most potent drug followed by itraconazole and amphotericin B. Voriconazole had no meaningful activity against Rhizopus. Posaconazole Etest MICs (microg/ml) with Aspergillus spp. were found to be considerably lower than those obtained with the CLSI microdilution method (4-9 and 3-7 two-fold lower than CLSI MICs at 24 and 48 h, respectively). The interaction between posaconazole and amphotericin B was indifferent for all R. oryzae isolates tested; importantly no antagonism was observed.

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.