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.

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.

Analysis of the aac(3)-VIa gene encoding a novel 3-N-acetyltransferase.

Biochemical analysis (G. A. Papanicolaou, R. S. Hare, R. Mierzwa, and G. H. Miller, abstr. 152, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., 1989) demonstrated the presence of a novel 3-N-acetyltransferase in Enterobacter cloacae 88020217. This organism was resistant to gentamicin, and the MIC of 2'-N-ethylnetilmicin for it was fourfold lower than that of 6'-N-ethylnetilmicin, a resistance pattern which suggested 2'-acetylating activity. However, high-pressure liquid chromatography analysis demonstrated that the enzyme acetylated sisomicin in the 3 position. We have cloned the structural gene for this enzyme from a large (> 70-kb) conjugative plasmid present in E. cloacae. Subcloning experiments have localized the aac(3)-VIa gene to a 2.1-kb Sau3A fragment. The deduced AAC(3)-VIa protein showed 48% amino acid identity to the AAC(3)-IIa protein and 39% identity to the AAC(3)-VII protein. Examination of the 5'-flanking sequences demonstrated that the aac(3)-VIa gene was located 167 bp downstream of the aadA1 gene and was present in an integron. In addition, the aac(3)-VIa gene is also downstream of a 59-base element often seen in an integron environment. Primer extension analysis has identified a promoter for the aac(3)-VIa gene downstream of both the aadA1 gene and a 59-base element.

Increased biliary transferrin excretion following parenteral aluminium administration to rats.

Aluminium accumulates in the livers of patients receiving either parenteral nutrition or haemodialysis. When given parenterally to rats, aluminium causes cholestasis. However, the mechanism of hepatic aluminium uptake and the fate of aluminum in the liver are poorly understood. We examined the effect of parenteral aluminium administration on biliary excretion of transferrin, the major circulating aluminum-binding protein. Male Wistar rats were given parenterally aluminum 5 mg/kg/day for 1-14 days. Bile was collected for 3 hr at the end of the study period. Biliary total protein concentration and IgA/total protein were unaffected by up to 14 days of parenteral aluminium administration. However, biliary transferrin excretion increased with duration of aluminum administration up to five-fold by day 14. Biliary transferrin concentration and transferrin/total protein was higher in aluminum treated rats than controls after 7 and 14 days of study. Hepatic aluminum concentration reached a maximum after 4 days of parenteral aluminum administration, at which time serum bile acid and alanine amino transferase values were not different from controls. Since biliary transferrin is normally derived from the serum, it is likely that aluminum promotes hepatocellular uptake of transferrin and that aluminum enters the hepatocyte bound to transferrin. We postulate that transferrin may direct aluminum to intracellular sites where its toxic effects would be minimized.

Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 6'-N-acetyltransferase Ib and IIa proteins.

The aminoglycoside 6'-N-acetyltransferase [AAC(6')-I] and AAC(6')-II enzymes represent a class of bacterial proteins capable of acetylating tobramycin, netilmicin, and 2'-N-ethylnetilmicin. However, an important difference exists in their abilities to modify amikacin and gentamicin. The AAC(6')-I enzymes are capable of modifying amikacin. In contrast, the AAC(6')-II enzymes are capable of modifying gentamicin. Nucleotide sequence comparison of the aac(6')-Ib gene and the aac(6')-IIa gene showed 74% sequence identity (K. J. Shaw, C. A. Cramer, M. Rizzo, R. Mierzwa, K. Gewain, G. H. Miller, and R. S. Hare, Antimicrob. Agents Chemother. 33:2052-2062, 1989). Comparison of the deduced protein sequences showed 76% identity and 82% amino acid similarity. A genetic analysis of these two proteins was initiated to determine which amino acids were responsible for the differences in specificity. Results of domain exchanges, which created hybrid AAC(6') proteins, indicated that amino acids in the carboxy half of the proteins were largely responsible for determining specificity. Mutations shifting the specificity of the AAC(6')-Ib protein to that of the AAC(6')-IIa protein (i.e., gentamicin resistance and amikacin sensitivity) have been isolated. DNA sequence analysis of four independent isolates revealed base changes causing the same amino acid substitution, a leucine to serine, at position 119. Interestingly, this serine occurs naturally at the same position in the AAC(6')-IIa protein. Oligonucleotide-directed mutagenesis was used to construct the corresponding amino acid change, a serine to leucine, in the AAC(6')-IIa protein. This change resulted in the conversion of the AAC(6')-IIa substrate specificity to that of AAC(6')-Ib. Analysis of additional amino acid substitutions within this region of AAC(6')-Ib support the model that we have identified an aminoglycoside binding domain of these proteins.

Effects of aluminum on the liver following high-dose enteral administration to rats.

Parenteral administration of aluminum (Al) to animals can result in hepatobiliary dysfunction, including elevated total serum bile acid concentration, reduced bile flow, and reduction of mixed function oxidase activities. Despite substantial hepatic Al accumulation, biliary Al excretion is negligible. We studied the effects of enteral administration of pharmacologic doses of Al to rats in order to see if by this route Al also produced hepatobiliary dysfunction or if biliary Al excretion was enhanced following enteral administration, protecting the liver from the effects of Al. Six rats were given 100 mg/kg/day of Al for 14 days as Al citrate by duodenal cannula. Pair-fed littermate controls were given sodium citrate. Serum Al and urinary Al/creatinine were significantly higher in Al-fed rats than in controls. Liver Al was significantly increased in the Al-fed group, but very low when compared to liver Al concentration with intravenous Al administration. Biliary Al was only 2 +/- 1% of urinary Al in the experimental group. Serum bile acid concentration and bile flow were not different between groups. We conclude that Al given in pharmacologic doses is absorbed but does not accumulate in the liver. We hypothesize that a slow rate of Al absorption may not overwhelm plasma transferrin carrying capacity or renal Al excretory capacity.

Effect of acute and chronic pentylenetetrazol treatment on benzodiazepine and cholinergic receptor binding in rat brain.

The binding of [3H] diazepam and [3H] flunitrazepam in rat cerebral cortex was not altered by either acute or chronic administration of pentylenetetrazol except in rats made to convulse 30 min before sacrifice. Rats treated for up to 6 months with doses of pentylenetetrazol which are below seizure threshold in naive rats, became increasingly sensitive to the CNS stimulant effect of pentylenetetrazol as demonstrated by the development of myoclonus and convulsions during treatment periods. These effects were not correlated with any changes in benzodiazepine binding in cerebral cortex or cerebellum and [3H] quinuclidinyl benzilate binding in cerebral cortex. Acute convulsant doses of pentylenetetrazol increased benzodiazepine binding in cerebral cortex, but only in those rats which actively convulsed. Benzodiazepine and cholinergic receptors of the cortex, and benzodiazepine receptors of the cerebellum, therefore, do not appear to change with either the acute or chronic subconvulsive administration of pentylenetetrazol.