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R S Hare

Publications and source records attributed to R S Hare.

At least 19 recordsLinked to original sources
PubMed2001

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.

Aminoglycosides↗
PubMed2001

In vitro Gram-positive antimicrobial activity of evernimicin (SCH 27899), a novel oligosaccharide, compared with other antimicrobials: a multicentre international trial.

The antimicrobial activity of evernimicin (formerly SCH 27899), a novel oligosaccharide antimicrobial of the everninomicin class, was evaluated against four groups of Gram-positive pathogens: (i) Streptococcus pneumoniae (n = 1452); (ii) methicillin- or oxacillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MR-CoNS; n = 1427); (iii) enterococci (n = 1517); and (iv) non-pneumococcal streptococci (n = 1388), using the Etest method at each study centre throughout Eastern and Western Europe, Scandinavia, South Africa, Turkey and North America. Comparative MICs were determined for a variety of reference compounds, including vancomycin, quinupristin/dalfopristin, chloramphenicol, penicillin, ampicillin, oxacillin, ceftriaxone and ciprofloxacin. Evernimicin was highly active against all strains tested, with MIC90 values < or = 1.0 mg/L, ranging from 0.047 mg/L against S. pneumoniae to 1.0 mg/L against MRSA/MR-CoNS and enterococci. Compared with the reference agents, the MIC90 of evernimicin were lower against all species. Against MRSA and MR-CoNS the MIC90s of evernimicin, quinupristin/dalfopristin and vancomycin (the three most active agents) were 1.0, 1.5 and 3.0 mg/L, respectively. Against all species tested, the relative activities and spectra of these agents were: evernimicin > vancomycin > quinupristin/dalfopristin. The Etest proved to be reliable and reproducible, despite occasional interpretive difficulties caused by observer inexperience. Quality control results were excellent among the 33 participant sites. The results of this in vitro, multicentre, multinational study demonstrate that evernimicin possesses high antimicrobial activity against Gram-positive organisms that compares favourably with established antibacterial treatments and newer agents such as quinupristin/dalfopristin. Further clinical investigations of everninomicin class compounds appear warranted.

Aminoglycosides↗
PubMed2001

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.

Alleles↗
PubMed2001

Genetic footprinting in bacteria.

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.

Bacteriophage lambda↗
PubMed2000

Evernimicin (SCH27899) inhibits a novel ribosome target site: analysis of 23S ribosomal DNA mutants.

Spontaneous mutants of susceptible clinical and laboratory isolates of Streptococcus pneumoniae exhibiting reduced susceptibility to evernimicin (SCH27899; MIC, 0.5 to 4.0 mg/liter) were selected on plates containing evernimicin. Four isolates that did not harbor mutations in rplP (which encodes ribosomal protein L16) were further analyzed. Whole chromosomal DNA or PCR products of the 23S ribosomal DNA (rDNA) operons from these mutants could be used to transform the susceptible S. pneumoniae strain R6 to resistance at frequencies of 10(-5) and 10(-4), respectively, rates 10- to 100-fold lower than that for a single-allele chromosomal marker. The transformants appeared slowly (48 to 72 h) on selective medium, and primary transformants passaged on nonselective medium produced single colonies that displayed heterogeneous susceptibilities to evernimicin. A single passage on selective medium of colonies derived from a single primary transformant homogenized the resistance phenotype. Sequence analysis of the 23S rDNA and rRNA from the resistant mutants revealed single, unique mutations in each isolate at the equivalent Escherichia coli positions 2469 (A --> C), 2480 (C --> T), 2535 (G --> A), and 2536 (G --> C). The mutations map within two different stems of the peptidyltransferase region of domain V. Because multiple copies of rDNA are present in the chromosome, gene conversion between mutant and wild-type 23S rDNA alleles may be necessary for stable resistance. Additionally, none of the characterized mutants showed cross-resistance to any of a spectrum of protein synthesis inhibitors, suggesting that the target site of evernimicin may be unique.

Alleles↗
PubMed2000

Mutations in ribosomal protein L16 conferring reduced susceptibility to evernimicin (SCH27899): implications for mechanism of action.

A clinical isolate of Streptococcus pneumoniae (SP#5) that showed decreased susceptibility to evernimicin (MIC, 1.5 microgram/ml) was investigated. A 4,255-bp EcoRI fragment cloned from SP#5 was identified by its ability to transform evernimicin-susceptible S. pneumoniae R6 (MIC, 0.03 microgram/ml) such that the evernimicin MIC was 1.5 microgram/ml. Nucleotide sequence analysis of this fragment revealed that it contained portions of the S10-spc ribosomal protein operons. The nucleotide sequences of resistant and susceptible isolates were compared, and a point mutation (thymine to guanine) that causes an Ile52-Ser substitution in ribosomal protein L16 was identified. The role of this mutation in decreasing susceptibility to evernimicin was confirmed by direct transformation of the altered L16 gene. The presence of the L16 mutation in the resistant strain suggests that evernimicin is an inhibitor of protein synthesis. This was confirmed by inhibition studies using radiolabeled substrates, which showed that the addition of evernimicin at sub-MIC levels resulted in a rapid decrease in the incorporation of radiolabeled isoleucine in a susceptible isolate (SP#3) but was much less effective against SP#5. The incorporation of isoleucine showed a linear response to the dose level of evernimicin. The incorporation of other classes of labeled substrates was unaffected or much delayed, indicating that these were secondary effects.

Amino Acid Sequence↗
PubMed2000

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.

Aminoglycosides↗
PubMed2000

Efficacy of SCH27899 in an animal model of Legionnaires' disease using immunocompromised A/J mice.

The efficacy of SCH27899, a new everninomicin antibiotic, against replicative Legionella pneumophila lung infections in an immunocompromised host was evaluated using a murine model of Legionnaires' disease. A/J mice were immunocompromised with cortisone acetate and inoculated intratracheally with L. pneumophila serogroup 1 (10(5) CFU per mouse). At 24 h postinoculation, mice were administered either SCH27899 (6 to 60 mg/kg [MPK] intravenously) or a placebo once daily for 5 days, and mortality and intrapulmonary growth of L. pneumophila were assessed. In the absence of SCH27899, there was 100% mortality in L. pneumophila-infected mice, with exponential intrapulmonary growth of the bacteria. In contrast, administration of SCH27899 at a dose of > or =30 MPK resulted in > or =90% survival of infected mice, which was associated with inhibition of intrapulmonary growth of L. pneumophila. In subsequent studies, the efficacy of SCH27899 was compared to ofloxacin (OFX) and azithromycin (AZI). Administration of SCH27899, OFX, or AZI at a dose of > or =30 MPK once daily for 5 days resulted in > or =85% survival of infected mice and inhibition of intrapulmonary growth of the bacteria. However, L. pneumophila CFU were recovered in lung homogenates following cessation of therapy with all three antibiotics. These studies demonstrate that SCH27899 effectively prevents fatal replicative L. pneumophila lung infection in immunocompromised A/J mice by inhibition of intrapulmonary growth of the bacteria. However, in this murine model of pulmonary legionellosis, SCH27899, like OFX and AZI, was bacteriostatic.

Aminoglycosides↗
PubMed2000

In vitro and in vivo activities of SCH 56592 (posaconazole), a new triazole antifungal agent, against Aspergillus and Candida.

SCH 56592 (posaconazole), a new triazole antifungal agent, was tested in vitro, and its activity was compared to that of itraconazole against 39 Aspergillus strains and to that of fluconazole against 275 Candida and 9 Cryptococcus strains. The SCH 56592 MICs for Aspergillus ranged from </=0.002 to 0.5 microg/ml, and those of itraconazole ranged from </=0.008 to 1 microg/ml. The SCH 56592 MICs for Candida and Cryptococcus strains ranged from </=0. 004 to 16 microg/ml, and those of fluconazole ranged from </=0.062 to >64 microg/ml. SCH 56592 showed excellent activity against Aspergillus fumigatus and Aspergillus flavus in a pulmonary mouse infection model. When administered therapeutically, the 50% protective doses (PD(50)s) of SCH 56592 ranged from 3.6 to 29.9 mg/kg of body weight, while the PD(50)s of SCH 56592 administered prophylactically ranged from 0.9 to 9.0 mg/kg; itraconazole administered prophylactically was ineffective (PD(50)s, >75 mg/kg). SCH 56592 was also very efficacious against fluconazole-susceptible, -susceptible dose-dependent, or -resistant Candida albicans strains in immunocompetent or immunocompromised mouse models of systemic infection. The PD(50)s of SCH 56592 administered therapeutically ranged from 0.04 to 15.6 mg/kg, while the PD(50)s of SCH 56592 administered prophylactically ranged from 1.5 to 19.4 mg/kg. SCH 56592 has excellent potential for therapy against serious Aspergillus or Candida infections.

Animals↗
PubMed1998

Synthesis and antibacterial activity of 2-alkoxy penems.

The phosphite mediated Oxalimide cyclization reaction was extended to 4-dithiocarbonates of N-oxalyl-2-azetidinones to synthesize 2-alkoxy penems 3. In general, the in vitro antibacterial potency of compounds 3 was weak compared to the highly potent 2-alkylthiopenems 2.

Anti-Bacterial Agents↗
PubMed1997

The most frequent aminoglycoside resistance mechanisms--changes with time and geographic area: a reflection of aminoglycoside usage patterns? Aminoglycoside Resistance Study Groups.

The aminoglycoside resistance mechanisms revealed by two surveys in Europe and other countries have been compared to those revealed in earlier studies. Mechanisms have become more complex in all bacterial groups. In Providencia, Serratia, Pseudomonas, Acinetobacter, and Staphylococcus species isolates, genus-specific mechanisms were very common, and it was not possible to see differences between different geographic areas. In other Enterobacteriaceae, the increasing complexity of mechanisms was most often caused by combinations of gentamicin-modifying enzymes with AAC(6')-I, which acetylates amikacin but not gentamicin. The occurrence of these combinations varied by geographical region and among hospitals. The frequency of these combinations correlated with aminoglycoside usage in either the geographical regions or in individual hospitals. These broad-spectrum combinations occurred most frequently in Citrobacter, Enterobacter, and Klebsiella species but also occurred in Escherichia, Morganella, Proteus, Salmonella, and Shigella species. Often the only clinically available aminoglycoside that retained its normal activity was isepamicin.

Acinetobacter↗
PubMed1997

Cloning and characterization of an aminoglycoside 6'-N-acetyltransferase gene from Citrobacter freundii which confers an altered resistance profile.

A novel gene encoding a 6'-N-aminoglycoside acetyltransferase, aac(6')-In, has been cloned and sequenced from Citrobacter freundii 13996-19, a clinical isolate from Venezuela. This gene mediates resistance to amikacin, 2'-N-ethylnetilmicin, isepamicin, kanamycin, netilmicin, and tobramycin. The aac(6')-In gene is 573 nucleotides in length and encodes a putative protein of 190 amino acids. AAC(6')-In is most closely related to AAC(6')-Im and AAC(6')-Ie, demonstrating 64.4% and 62.3% similarity, respectively, at the protein level, suggesting these proteins share a common ancestor. The aac(6')-In flanking sequences demonstrated homology to integron- and transposon-related elements which are often found associated with resistance determinants. Hybridization studies performed with an intragenic probe specific for aac(6')-In indicate that this gene is prevalent within Venezuela but has not been observed outside of the country. Furthermore, the aac(6)-In gene was found in 10 different species of gram-negative bacteria.

Acetyltransferases↗
PubMed1995

Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa.

A novel gene encoding an aminoglycoside 3-N-acetyltransferase, which confers resistance to gentamicin, astromicin, and sisomicin, was cloned from Pseudomonas aeruginosa Stone 130. Its sequence was determined and found to show considerable similarity to an aac(3)-I gene previously cloned from R plasmids from Enterobacter, Pseudomonas, and Serratia spp. We have designated the genes from the R plasmids and this work aac(3)-Ia and aac(3)-Ib, respectively. The two aac(3)-I genes share 74% nucleotide identity, and their deduced protein products are 88% similar. These data suggest that the genes derive from a common ancestor. Homology between the flanking sequences of both aac(3)-I genes and other resistance determinants known to reside in integron environments was also observed. Intragenic probes specific for either aac(3)-Ia or aac(3)-Ib were used in hybridization studies with a series of gentamicin-, astromicin-, and sisomicin-resistant clinical isolates. Of 59 clinical isolates tested, no isolates hybridized with both probes, 30 (51%) hybridized with the aac(3)-Ia probe, 12 (20%) hybridized with the aac(3)-Ib probe, and 17 (29%) did not hybridize with either probe. These data suggest the existence of at least one other aac(3)-I gene.

Acetyltransferases↗
PubMed1995

The changing nature of aminoglycoside resistance mechanisms and the role of isepamicin--a new broad-spectrum aminoglycoside. The Aminoglycoside Resistance Study Groups.

Aminoglycoside resistance mechanisms from recent studies were compared with those found in earlier studies in the USA and Europe for three pathogen groups. Among Citrobacter-Enterobacter-Klebsiella, four single mechanisms (AAc(3)-II, AAC(3)-I, ANT(2")-I and AAC(6')-I were found in all studies, but the most recent studies showed a significant increase in combinations of AAC(6')-I with the other common mechanisms. Since AAC(6')-I confers resistance to tobramycin, netilmicin and amikacin, combinations of it with the other gentamicin modifying enzymes conferred broad-spectrum resistance to all clinically available aminoglycosides except isepamicin. Similar changes occurred in Escherichia-Morganella-Proteus-Salmonella-Shigella except that the frequency of combinations was much lower and two additional single mechanisms - AAC(3)-IV and permeability - were also found frequently. Among aminoglycoside-resistant Pseudomonas, three mechanisms, AAC(6')-II, ANT(2")-I and permeability, were always common and remained common. However, combinations of the three mechanisms with each other and with other mechanisms were more common in the recent surveys. Different genes which produce different proteins with the same aminoglycoside-modifying activity are now known. The results of hybridisation studies with two aac(3)-I, 2 aac(6')-II and 4 aac(6')-I gene probes are presented. The most commonly occurring genes were: aac(3)-Ia, aac(3)-IIa, aac(6')-IIa, aac(6')-Ib and, in Serratia, aac(6')-Ic. The activity of isepamicin against amikacin resistant strain which produce AAC(6')-I can be related to differences in the structure of these two similar aminoglycosides at Position 3". Amikacin may form a stable complex with AAC(6')-I enzymes via binding interaction at Position 3 and 3". Isepamicin, which has a secondary amino group at Position 3", may only be able to interact at Position 3 and enzyme-isepamicin complexes are likely to be less stable.

Acetyltransferases↗
PubMed1994

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

Amino Acid Sequence↗
PubMed1993

Sch 39304, a new antifungal agent: oral and topical treatment of vaginal and superficial infections.

Sch 39304 is a new broad spectrum triazole antifungal agent that is active, orally and topically, against superficial Trichophyton mentagrophytes and vaginal Candida albicans infections. Sch 39304 was compared to fluconazole (FLZ) in a T. mentagrophytes infection model in guinea pigs. Following topical administration, Sch 39304 (0.125%, twice daily, 10 days), was 5-8-fold more effective than FLZ, based on culture and lesion score results. Following oral administration, Sch 39304 (2.5 mg kg-1, once daily, 10 days) produced a dramatic reduction in lesion scores and was 20-fold more active than FLZ; however, due to the length of time it takes for the drugs to reach the infected area of the skin and eradicate the infections, most animals remained culture positive with both drugs. Sch 39304 was also compared with FLZ in a vaginal C. albicans infection in hamsters. Following oral administration (4 days), Sch 39304 (1.6 mg kg-1), cured all hamsters and was 4-fold more active than FLZ. In addition, Sch 39304 as a single oral dose (10 mg kg-1) also cured all hamsters. When treatment was intravaginal (8 days), Sch 39304 was again more active than FLZ (2-fold), and also micronazole (8-fold), with 100% of the hamsters cured at concentrations as low at 0.025%.

Administration, Oral↗