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1.  The antileishmanial agent licochalcone A interferes with the function of parasite mitochondria. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2742-2748.
Our previous studies have shown that licochalcone A, an oxygenated chalcone, has antileishmanial (M. Chen, S.B. Christensen, J. Blom, E. Lemmich, L. Nadelmann, K. Fich, T.G. Theander, and A. Kharazmi, Antimicrob, Agents Chemother. 37:2550-2556, 1993; M. Chen, S.B. Christensen, T.G. Theander, and A. Khrazmi, Antimicrob. Agents Chemother. 38:1339-1344, 1994) and antimalarial (M. Chen, T.G. Theander, S.B. Christensen, L. Hviid, L. Zhai, and A. Kaharazmi, Antimicrob. Agents Chemother. 38:1470-1475, 1994) activities. We have observed that licochalcone A alters the ultrastructure of the mitochondria of Leishmania promastigotes (Chen et al., Antimicrob. Agents Chemother. 37:2550-2556, 1993). The present study was designed to examine this observation further and investigate the mechanism of action of antileishmanial activity of licochalcone A. Electron microscopic studies showed that licochalcone A altered the ultrastructure of Leishmania major promastigote and amastigote mitochondria in a concentration-dependent manner without damaging the organelles of macrophages or the phagocytic function of these cells. Studies on the function of the parasite mitochondria showed that licochalcone A inhibited the respiration of the parasite by the parasites. Moreover, licochalcone A inhibited the activity of the parasite mitochondrial dehydrogenase. The inhibition of the activity of the parasite mitochondrial enzyme correlated well with the changes in the ultrastructure of the mitochondria shown by electron microscopy. These findings demonstrate that licochalcone A alters the ultrastructure and function of the mitochondria of Leishmania parasites.
PMCID: PMC163022  PMID: 8593012
2.  New semisynthetic pneumocandins with improved efficacies against Pneumocystis carinii in the rat. 
A new series of semisynthetic, water-soluble pneumocandin analogs has been found to be extremely potent against Pneumocystis carinii in an immunocompromised-rat model. These compounds are 5 to 10 times more potent than the parent natural product, pneumocandin B0 (L-688,786) (R. E. Schwartz et al., J. Antibiot. 45:1853-1866, 1992), and > 100 times more potent than cilofungin. One compound in particular, L-733,560, had a 90% effective dose against P. carinii cysts of 0.01 mg/kg of body weight when delivered parenterally (subcutaneously, twice daily for 4 days). This compound was also effective when given orally for the treatment and prevention of P. carinii pneumonia. For treating acute P. carinii pneumonia, oral doses of 2.2 mg/kg twice daily for 4 days were required to eliminate 90% of the cysts. A once-daily oral prophylactic dose of 2.2 mg/kg prevented cyst development, and a dose of 6.2 mg/kg prevented any development of P. carinii organisms (cysts and trophozoites), as determined through the use of a P. carinii-specific DNA probe (P. A. Liberator et al., J. Clin. Microbiol. 30:2968-2974, 1992). These results demonstrate that the antipneumocystis activities of the pneumocandins can be significantly improved through synthetic modification. Several of these compounds are also extremely effective against candidiasis (K. Bartizal et al., Antimicrob. Agents Chemother. 39:1070-1076, 1995) and aspergillosis (G. K. Abruzzo et al., Antimicrob. Agents Chemother. 39:860-894, 1995) in murine models, making them attractive as broad-spectrum antifungal agents.
PMCID: PMC162734  PMID: 7574523
3.  Growth inhibition of Ureaplasma urealyticum by the proton pump inhibitor lansoprazole: direct attribution to inhibition by lansoprazole of urease activity and urea-induced ATP synthesis in U. urealyticum. 
Antimicrobial Agents and Chemotherapy  1995;39(10):2187-2192.
The proton pump inhibitors (PPIs) omeprazole and lansoprazole and the acid-activated analog of lansoprazole AG-2000, which potently inhibit the urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), also inhibited the urease activities of cell-free extracts as well as intact cells of Ureaplasma urealyticum. The 50% inhibitory concentrations were between 1 and 25 microM. These compounds also inhibited the ATP synthesis induced by urea in ureaplasma cells. The 50% inhibitory concentrations for ATP synthesis were close to those for urease activity, but they were lower than those of urease inhibitors, such as acetohydroxamic acid, hydroxyurea, and thiourea. In addition, one of the metabolites of lansoprazole found in human urine, M-VI, also inhibited ureaplasmal urease activity and the ATP synthesis induced by urea at almost the same concentrations as those of lansoprazole. The inhibition of PPIs against ureaplasma urease was very similar to those against H. pylori urease, suggesting that the inhibitory mechanism against these ureases was due to the blockage of the SH residues on the cysteine of the enzyme. Omeprazole, lansoprazole, AG-2000, and M-VI inhibited the growth of U. urealyticum. Since ureaplasma urease is thought to be involved in the pathogenicity of this organism in the urogenital tract, PPIs and their analogs may be useful as chemotherapeutic agents against diseases caused by U. urealyticum.
PMCID: PMC162911  PMID: 8619564
4.  Identification of daptomycin-binding proteins in the membrane of Enterococcus hirae. 
Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, was preliminarily shown to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+ (P. Canepari, M. Boaretti, M. M. Lleó, and G. Satta, Antimicrob. Agents Chemother. 34:1220-1226, 1990). In the present study, it is shown that, along with binding bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of ionic detergents such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that five bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are therefore called daptomycin-binding proteins. In an attempt to correlate these proteins to the main inhibition observed during LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A threefold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol, and glycerophospho-kojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.
PMCID: PMC162882  PMID: 8540717
5.  In vitro pharmacodynamics of piperacillin, piperacillin-tazobactam, and ciprofloxacin alone and in combination against Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. 
The time-kill curve methodology was used to determine the pharmacodynamics of piperacillin, ciprofloxacin, piperacillin-tazobactam and the combinations piperacillin-ciprofloxacin and ciprofloxacin-piperacillin-tazobactam. Kill curve studies were performed for piperacillin, ciprofloxacin, and piperacillin-tazobactam at concentrations of 0.25 to 50 times the MICs for 13 strains of bacteria: four Pseudomonas aeruginosa, three Enterobacter cloacae, three Klebsiella pneumoniae, and three Staphylococcus aureus isolates (tazobactam concentrations of 0.5, 4, and 12 micrograms/ml). By using a sigmoid Emax model and nonlinear least squares regression, the 50% lethal concentrations and the maximum lethal rates of each agent were determined for each bacterial strain. For piperacillin-ciprofloxacin and ciprofloxacin-piperacillin-tazobactam, kill curve studies were performed with concentrations obtained by the fractional maximal effect method (R. C. Li, J. J. Schentag, and D. E. Nix, Antimicrob. Agents Chemother. 37:523-531, 1993) and from individual 50% lethal concentrations and maximum lethal rates. Ciprofloxacin-piperacillin-tazobactam was evaluated only against the four P. aeruginosa strains. Interactions between piperacillin and ciprofloxacin were generally additive. At physiologically relevant concentrations of piperacillin and ciprofloxacin, ciprofloxacin had the highest rates of killing against K. pneumoniae. Piperacillin-tazobactam (12 micrograms/ml) had the highest rate of killing against E. cloacae. Piperacillin-ciprofloxacin with relatively higher ciprofloxacin concentrations had the greatest killing rates against S. aureus. This combination had significantly higher killing rates than piperacillin (P < 0.002). For all the bacterial strains tested, killing rates by ciprofloxacin were significantly higher than those by piperacillin-tazobactam (4 and 12 micrograms/ml had significantly higher killing rates than piperacillin alone (P < 0.02 and P < 0.004, respectively). The effect of the combination of piperacillin-ciprofloxacin, in which piperacillin concentrations were relatively higher, was not statistically different from that of piperacillin alone (p > or = 0.71). The combination of ciprofloxacin-piperacillin-tazobactam achieved greater killing than other combinations or monotherapies against P. aeruginosa. The reduction in the initial inoculum was 1 to 4 logs greater with ciprofloxacin-piperacillin-tazobactam at 4 and 12 micrograms/ml than with any other agent or combination of agents. On the basis of the additive effects prevalently demonstrated in the in vitro study, the combinations of piperacillin-ciprofloxacin and piperacillin-tazobactam are rational therapeutic options. Greater killing of P. aeruginosa was demonstrated with ciprofloxacin-piperacillin--tazobactam. Since treatment failure of P. aeruginosa pneumonia is a significant problem, clinical studies are warranted.
PMCID: PMC162813  PMID: 7486906
6.  Determination of robust ocular pharmacokinetic parameters in serum and vitreous humor of albino rabbits following systemic administration of ciprofloxacin from sparse data sets by using IT2S, a population pharmacokinetic modeling program. 
Robust determination of the concentration-time profile of anti-infective agents in certain specialized compartments is often limited by the inability to obtain more than a single sample from such a site in any one subject. Vitreous humor and cerebrospinal fluid are obvious examples for which the determination of concentrations of anti-infective agents is limited. Advances in pharmacodynamics have pointed out the importance of understanding the profiles of drugs in the plasma and in specialized compartments in order to dose the drugs to obtain the best patient outcomes. Advances in population pharmacokinetic modeling hold the promise of allowing proper estimation of drug penetration into the vitreous (or other specialized compartment) with only a single vitreous sample, in conjunction with plasma sampling. We have developed a rabbit model which allows multiple samples of vitreous to be obtained without breaking down the blood-vitreous barrier. We have employed this model to test the hypothesis that robust estimates of vitreous penetration by the fluoroquinolone ciprofloxacin can be obtained from a traditional intensive plasma sampling set plus a single vitreous sample. We studied 33 rabbits which were receiving 40 mg of ciprofloxacin per kg of body weight intravenously as short infusions and from which multiple plasma and vitreous samples were obtained and assayed for ciprofloxacin content by high-performance liquid chromatography. Data were analyzed by the iterative two-stage population modeling technique (IT2S), employing the iterative two-stage program of Forrest et al. (Antimicrob. Agents Chemother. 37:1065-1072, 1993). Two data sets were analyzed: all plasma and vitreous samples versus all plasma samples and the initially obtained single vitreous sample. The pharmacokinetic parameter values identified were used to calculate the percent vitreous penetration as the ratio of the area under the concentration-time curve for the vitreous to that for the plasma. The values identified, 4% penetration for the full data set versus 3% penetration for the single vitreous sample data set, and their corresponding estimates were not statistically significantly different. We conclude that population modeling holds promise for the analysis of penetration of antimicrobiol agents into specialized spaces from which only single samples can be obtained, particularly for patients with whom robust plasma sampling can be performed.
PMCID: PMC162807  PMID: 7486900
7.  In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii. 
In order to characterize the delayed effect of clindamycin and macrolide antibiotics against Toxoplasma gondii tachyzoites (E. R. Pfefferkorn and S. E. Borotz, Antimicrob. Agents Chemother. 38:31-37, 1994), we have carefully examined the replication of parasites as a function of time after drug addition. Intracellular tachyzoites treated with up to 20 microM clindamycin (> 1,000 times the 50% inhibitory concentration) exhibit doubling times indistinguishable from those of controls (approximately 7 h). Drug-treated parasites emerge from infected cells and establish parasitophorous vacuoles inside new host cells as efficiently as untreated controls, but replication within the second vacuole is dramatically slowed. Growth inhibition in the second vacuole does not require continued presence of drug, but it is dependent solely on the concentration and duration of drug treatment in the first (previous) vacuole. The susceptibility of intracellular parasites to nanomolar concentrations of clindamycin contrasts with that of extracellular tachyzoites, which are completely resistant to treatment, even through several cycles of subsequent intracellular replication. This peculiar phenotype, in which drug effects are observed only in the second infectious cycle, also characterizes azithromycin and chloramphenicol treatment, but not treatment with cycloheximide, tetracycline, or anisomycin. These findings provide new insights into the mode of clindamycin and macrolide action against T. gondii, although the relevant target for their action remains unknown.
PMCID: PMC162776  PMID: 7492099
8.  Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain. 
Two groups of recent clinical isolates of Candida albicans consisting of 101 isolates for which fluconazole MICs were < or = 0.5 microgram/ml (n = 50) and > or = 4.0 micrograms/ml (n = 51), respectively, were compared for their susceptibilities to fluconazole, clotrimazole, miconazole, ketoconazole, and itraconazole. Susceptibility tests were performed by a photometer-read broth microdilution method with an improved RPMI 1640 medium supplemented with 18 g of glucose per liter (RPMI-2% glucose; J. L. Rodríguez-Tudela and J. V. Martínez-Suárez, Antimicrob. Agents Chemother. 38:45-48, 1994). Preparation of drugs, basal medium, and inocula was done by the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h as the lowest drug concentration at which growth was just equal to or less than 20% of that in the positive control well (MIC 80%). In vitro susceptibility testing separated azole-susceptible strains from the strains with decreased susceptibilities to azoles if wide ranges of concentrations (20 doubling dilutions) were used for ketoconazole, miconazole, and clotrimazole. By comparison with isolates for which fluconazole MICs were < or = 0.5 microgram/ml, those isolates for which fluconazole MICs were > or = 4.0 micrograms/ml were in general less susceptible to other azole drugs, but different patterns of decreased susceptibility were found, including uniform increases in the MICs of all azole derivatives, higher MICs of several azoles but not others, and elevated MICs of fluconazole only. On the other hand, decreased susceptibility to any other azole drug was never found among strains for which MICs of fluconazole were lower.
PMCID: PMC162772  PMID: 7492095
9.  Molecular characterization of the OXA-7 beta-lactamase gene. 
The OXA-7 gene, which encodes an oxacillinase, was cloned from plasmid pMG202 of Escherichia coli isolate 7181 (A. A. Medeiros, M. Cohenford, and G. A. Jacoby, Antimicrob. Agents Chemother. 27:715-719, 1985) and sequenced. The nucleotide sequence of the OXA-7 gene was closely related to that of the OXA-10 (PSE-2) gene, with a derived amino acid sequence of the OXA-7 enzyme showing greater than 95% homology with those of OXA-10 and OXA-11.
PMCID: PMC162747  PMID: 7574536
10.  Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. 
The distribution of strains producing metallo-beta-lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a metallo-beta-lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the metallo-beta-lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to < or = 8 micrograms/ml), had no detectable blaIMP gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, > or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like metallo-beta-lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.
PMCID: PMC162636  PMID: 7785978
11.  Characterization of an LysR family protein, SmeR from Serratia marcescens S6, its effect on expression of the carbapenem-hydrolyzing beta-lactamase Sme-1, and comparison of this regulator with other beta-lactamase regulators. 
Serratia marcescens S6 produces a chromosomally encoded carbapenem-hydrolyzing class A beta-lactamase, Sme-1 (T. Naas, L. Vandel, W. Sougakoff, D. M. Livermore, and P. Nordmann, Antimicrob. Agents Chemother. 38:1262-1270, 1994). Upstream from smeA we identified a second open reading frame (EMBL accession number Z30237). This encodes a 33.1-kDa protein, SmeR, which has a high degree of homology with NmcR, the LysR regulatory protein of the only other sequenced carbapenem-hydrolyzing class A beta-lactamase, NmcA from Enterobacter cloacae NOR-1. It is weakly related to AmpR of the chromosomal cephalosporinase regulatory systems described in E. cloacae, Yersinia enterocolitica, Citrobacter freundii, and Pseudomonas aeruginosa and is very weakly related to other LysR-type regulators of class A beta-lactamases. SmeR is a weakly positive regulator for Sme-1 expression in the absence of or in the presence of beta-lactam inducers. The -35 and -10 regions of smeR are in the opposite orientations and are face-to-face relative to the smeA promoter. SmeR acts similarly to NmcR and not as the AmpR regulators described for class C beta-lactamase systems. SmeR is a weak inducer in the absence or presence of beta-lactams. As was found for the AmpC-AmpR and NmcA-NmcR systems, a putative SmeR-binding site was present upstream from the beta-lactamase gene promoter regions. beta-Galactosidase activity from a smeR-lacZ translational fusion was expressed constitutively and decreased in the presence of SmeR from a coresident plasmid, suggesting that SmeR is autogeneously controlled. Finally, beta-lactams did not affect the expression of SmeR, which is the second regulator of a class A carbapenem-hydrolyzing beta-lactamase to be identified.
PMCID: PMC162596  PMID: 7793864
12.  Identification of the aminocatechol A-3253 as an in vitro poison of DNA topoisomerase I from Candida albicans. 
The aminocatechol A-3253 is active against several pathogenic fungi, including Candida albicans, Cryptococcus albidus, and Aspergillus niger. A-3253 interferes with both the in vitro biosynthesis of (1,3)-beta-glucan and the activity of topoisomerases I isolated from Candida spp. It is likely that one or more of the enzymes involved in glucan biosynthesis rather than topoisomerase I is the primary intracellular target of A-3253, since a strain of Saccharomyces cerevisiae lacking topoisomerase I is as susceptible to A-3253 as cells containing wild-type levels of topoisomerase I. However, the interaction of A-3253 with topoisomerase I in vitro is of interest since the Candida topoisomerase is more susceptible to A-3253 than is the topoisomerase I isolated from human HeLa cells. A-3253 is both a reversible inhibitor of topoisomerase I catalysis and a reversible poison of topoisomerase I, and in both reactions the fungal topoisomerase I is more susceptible than the human topoisomerase I to A-3253. In contrast, an earlier study found that the human topoisomerase I is more susceptible than the fungal topoisomerase to camptothecin (J. M. Fostel, D. A. Montgomery, and L. L. Shen, Antimicrob. Agents Chemother. 36:2131-2138, 1992). Taken together with the response to camptothecin, the greater susceptibility of the Candida topoisomerase I to A-3253 suggests that there are structural differences between the human and fungal type I topoisomerases which can likely be exploited to allow for the development of antifungal agents which act against the fungal topoisomerase and which have minimal activity against the human enzyme.
PMCID: PMC162588  PMID: 7793856
13.  Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope. 
Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethambutol, synthesis of the arabinan of lipoarabinomannan was only partially affected, demonstrating a differential effect on arabinan synthesis in the two locales. Further studies of the effect of ethambutol on cell wall biosynthesis revealed that the synthesis of galactan in the cell wall core is strongly inhibited by the drug. In addition, ethambutol treatment resulted in the cleavage of arabinosyl residues present in the mycobacterial cell wall; more than 50% of the arabinan in the cell wall core was removed from the wall 1 h after addition of the drug to growing mycobacterial cultures. In contrast, galactan was not released from the cell wall during ethambutol treatment. The natural function of the arabinosyl-releasing enzyme remains unknown, but its action in combination with inhibition of synthesis during ethambutol treatment results in severe disruption of the mycobacterial cell wall. Accordingly, ethambutol-induced damage to the cell wall provides a ready molecular explanation for the known synergetic effects of ethambutol with other chemotherapeutic agents. Nevertheless, the initial direct effect of ethambutol remains to be elucidated.
PMCID: PMC162607  PMID: 7793875
14.  Inhibitory action of lansoprazole and its analogs against Helicobacter pylori: inhibition of growth is not related to inhibition of urease. 
The proton pump inhibitors omeprazole and lansoprazole and its acid-activated derivative AG-2000, which are potent and specific inhibitors of urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), inhibited the growth of H. pylori. The growth was inhibited not only in urease-positive clinical isolates but also in their urease-negative derivatives which had no urease polypeptides. AG-1789, a derivative of lansoprazole with no inhibitory activity against H. pylori urease, also inhibited the growth of both strains even more strongly than the urease inhibitors lansoprazole and AG-2000. Furthermore, the antibacterial activity of omeprazole and lansoprazole was not affected by glutathione or dithiothreitol, which completely abolished the inhibitory activity of lansoprazole against H. pylori urease. These results indicated that the inhibitory action of these compounds against the growth of H. pylori was independent from the inhibitory action against urease.
PMCID: PMC162584  PMID: 7726537
15.  Relative importances of outer membrane permeability and group 1 beta-lactamase as determinants of meropenem and imipenem activities against Enterobacter cloacae. 
The roles of outer membrane permeability and Bush group 1 beta-lactamase activity in determining Enterobacter cloacae susceptibility to either meropenem or imipenem were investigated. A beta-lactamase-deficient strain was obtained by mutagenesis from a clinical isolate of E. cloacae, and a porin-deficient strain was selected from this mutant with cefoxitin. Both strains were transformed with the plasmid pAA20R, which contained the gene coding for the carbapenem-hydrolyzing CphA beta-lactamase, and the carbapenem permeability coefficients were measured by the Zimmermann and Rosselet technique (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). The permeability coefficient of meropenem was roughly half that of imipenem in the normally permeable strain and almost seven times lower than that of imipenem in the porin-deficient strain. In the porin-deficient strain, the virtual absence of porins caused the MICs of meropenem to increase from 8 to 16 times, while it did not affect the MICs of imipenem. Conversely, the beta-lactamase affected imipenem but not meropenem activity: meropenem showed a similar activity in the parent strain and in the beta-lactamase-deficient mutant with both a low- and high-density inoculum, whereas imipenem was 16 times less active against the parent strain when the high-density inoculum was used. It is concluded that outer membrane permeability and stability to group 1 beta-lactamase have different impacts on the activities of meropenem and imipenem against E. cloacae.
PMCID: PMC162541  PMID: 7726496
16.  Structure-activity relationships of carbapenems that determine their dependence on porin protein D2 for activity against Pseudomonas aeruginosa. 
A number of carbapenem derivatives were examined to determine the structure-activity relationships required for dependence on porin protein D2 for activity against Pseudomonas aeruginosa. As suggested by J. Trias and H. Nikaido (Antimicrob. Agents Chemother. 34:52-57, 1990), carbapenem derivatives, such as imipenem and meropenem, containing a sole basic group at position 2 of the molecule utilize the D2 channel for permeation through the outer membrane of pseudomonads; they are more active against D2-sufficient strains of P. aeruginosa. Our results indicated that carbapenems with a basic group at position 1 or 6 of the molecule did not depend on the D2 channel for activity; i.e. they were equally active against D2-sufficient and D2-deficient pseudomonal strains. However, addition of a basic group at position 1 or 6 of a carbapenem derivative already containing a basic group at position 2 resulted in its lack of dependency on the D2 pathway. Comparison between meropenem and its 1-guanidinoethyl derivative, BMY 45047, indicated that they differed in their dependence on D2; while meropenem required the D2 channel for uptake, BMY 45047 activity was independent of D2. Meropenem and BMY 45047 had similar affinities for the penicillin-binding proteins of P. aeruginosa. However, BMY 45047 and meropenem differed in the morphological changes that they induced in pseudomonal cells. While meropenem induced filamentation, BMY 45047 induced filaments only in BMS-181139-resistant mutants and not in imipenem-resistant mutants or in carbapenem-susceptible P. aeruginosa strains. These results suggested that in Mueller-Hinton medium the uptake of BMY 45047 through the non-D2 pathway is more rapid than that of meropenem through the D2 porin. In summary, the presence of a basic group at position 2 of a carbapenem is important for its preferential uptake by the D2 channel. However the addition of a basic group at position 1 or 6 of a carbapenem already containing a basic group at position 2 dissociates its necessity for porin protein D2 for activity.
PMCID: PMC162549  PMID: 7726504
17.  Multidrug resistance in Candida albicans: disruption of the BENr gene. 
The BENr gene of Candida albicans, which confers resistance on susceptible strains of Saccharomyces cerevisiae to six structurally and functionally unrelated drugs, was described recently (R. Ben-Yaacov, S. Knoller, G. Caldwell, J. M. Becker, and Y. Koltin, Antimicrob. Agents Chemother. 38:648-652, 1994). This gene bears similarity to membrane proteins encoding antibiotic resistance in prokaryotes and eukaryotes. The effect of disruption of this gene on viability and drug susceptibility was determined. The results indicate that the gene is not essential but its inactivation leads to susceptibility to three of the four drugs tested. Inactivation of this gene did not increase the susceptibility of the mutant to benomyl, suggesting that C. albicans has other mechanisms of resistance, some of which may be additional efflux pumps that confer resistance to this tubulin-destabilizing agent.
PMCID: PMC162553  PMID: 7726508
18.  A novel glycylcycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H+ antiporter. 
A novel tetracycline derivative, DMG-DMDOT [9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline] , is one of the glycylcyclines which have a broad antibacterial spectrum, including many tetracyclineresistant bacteria (R.T. Testa, P.J. Petersen, N.V. Jacobus, P.-E. Sum, V.J. Lee, and F.P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). The mechanism by which DMG-DMDOT overcomes efflux-based tetracycline resistance was investigated. Tetracycline-resistant Escherichia coli cells carrying an R plasmid encoding the tet(B) gene, which encodes the typical tetracycline efflux pump [TetA(B)] of gram-negative bacteria, were as susceptible to DMG-DMDOT as was the tetracycline-susceptible host. When mid-log-phase cells carrying the tet(B) gene were incubated with a subbactericidal concentration of DMG-DMDOT (0.5 micrograms/ml) for 2 h, a significant amount of the TetA(B) protein was detected in the cell membrane by Western blotting (immunoblotting) with an anti-carboxyl-terminal antibody, similar to the case in which tetracycline was used as the inducer, indicating that the tet repressor, TetR, can recognize DMG-DMDOT as an efficient inducer. Everted membrane vesicles prepared from cells producing the TetA(B) protein showed absolutely no transport activity for DMG-DMDOT. Furthermore, the presence of excess DMG-DMDOT had no effect on the tetracycline transport activity of the everted vesicles, indicating that DMG-DMDOT is not recognized as a substrate by the TetA(B) protein.
PMCID: PMC162518  PMID: 7695316
19.  Comparative in vitro activities of LY191145, a new glycopeptide, and vancomycin against Staphylococcus aureus and Staphylococcus-infected fibrin clots. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2832-2834.
Bactericidal activities of LY191145, an investigational glycopeptide, and vancomycin against Staphylococcus aureus were evaluated. Only LY191145 at a concentration 16-fold greater than the MIC was able to achieve 99.9% killing against methicillin-susceptible S. aureus (ATCC 25923; 8.0 h). Both agents demonstrated 99.9% killing against methicillin-resistant clinical isolate S. aureus MRSA67 over 24 h at concentrations 4-, 8-, and 16-fold greater than the MIC, but bacteria were killed at a more rapid rate by LY191145 (1.63 versus 5.02 h; P < 0.001). Against strain ATCC 25923- and MRSA67-infected fibrin clots, total reductions by LY191145 and vancomycin over 72 h were not statistically significantly different at a concentration 16 times the MIC (1.12 +/- 0.31 and 1.23 +/- 0.13 and 1.40 +/- 0.17 and 1.36 +/- 0.37 CFU/g; respectively). Increasing the drug concentration to 50 times the MIC did not alter the values significantly, and there was no statistically significant difference between the two agents. Overall, LY191145 exhibited more rapid bactericidal activity than vancomycin against S. aureus, and a concentration 16-fold greater than the MIC appears to be optimal.
PMCID: PMC163044  PMID: 8593034
20.  Clarithromycin is inactive against Mycobacterium tuberculosis. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2827-2828.
When 10% oleic acid-albumin-dextrose-catalase-enriched Mueller-Hinton agar medium was employed, the MICs of clarithromycin (CLARI) at which 50 and 90% of 12 strains of Mycobacterium tuberculosis were inhibited were 64 and > 128 micrograms/ml, respectively, which are significantly greater than the achievable peak CLARI concentrations in serum and in lung tissue in humans. In two different mouse experiments, 4 to 6 weeks of treatment with CLARI at 200 mg/kg of body weight six times weekly produced neither bactericidal nor bacteriostatic effects against M. tuberculosis. Therefore, we conclude that CLARI as a single drug is inactive against M. tuberculosis.
PMCID: PMC163042  PMID: 8593032
21.  In vitro antibacterial activity of DU-6859a, a new fluoroquinolone. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2822-2826.
The in vitro antibacterial activity of DU-6859a, a new fluoroquinolone, against a wide variety of clinical isolates was evaluated and compared with those of tosufloxacin, ofloxacin, ciprofloxacin, and sparfloxacin. DU-6859a showed potent broad-spectrum activity against gram-positive, gram-negative, and anaerobic bacteria, and its activity was greater than those of the control quinolones. By comparison of MICs at which 90% of strains are inhibited, DU-6859a had potent activity against bacteria resistant to the control quinolones. The time-killing curves of quinolones showed that the number of viable cells decreased rapidly during 2 to 4 of incubation, and regrowth was not seen even after 8 h incubation. At a concentration of four times the MIC, the frequencies of appearance of spontaneous mutants of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa resistant to DU-6859a were < or = 4.0 x 10(-9) to 1.9 x 10(-8). The 50% inhibitory concentrations of DU-6859a were 0.86 and 1.05 micrograms/ml for the supercoiling activities of DNA gyrases isolated from E. coli and P. aeruginosa, respectively. The rank order of the 50% inhibitory concentrations observed for both DNA gyrases roughly paralleled the MICs.
PMCID: PMC163041  PMID: 8593031
22.  Activities of oral antibiotics on Providencia strains isolated from institutionalized elderly patients with urinary tract infections. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2819-2821.
More than 250 Providencia strains isolated from the urine of institutionalized elderly patients were tested against cefaclor, cefuroxime, cefetamet, cefpodoxime, ciprofloxacin, and amoxicillin-clavulanic acid. Our results confirm the strong activities of expanded-spectrum oral cephalosporins against Providencia isolates, f1p4ell as the marked differences in susceptibilities among accurately identified Providencia species.
PMCID: PMC163040  PMID: 8593030
23.  Enhanced activity of the combination of penicillin G and gentamicin against penicillin-resistant viridans group streptococci. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2816-2818.
We evaluated the effects of the combination of penicillin G and gentamicin against 10 penicillin-resistant bacteremic isolates of viridans group streptococci for which the MICs of penicillin were 4 to 64 micrograms/ml. In time-kill studies, the combination resulted in more killing of eight isolates for which the MICs of penicillin were from 8 to 64 micrograms/ml than any of the antimicrobial agents tested alone. In general, clearly enhanced antimicrobial activity was observed with the combination.
PMCID: PMC163039  PMID: 8593029
24.  Levels of azithromycin and alpha-1 acid glycoprotein in serum in patients with community-acquired pneumonia. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2801-2802.
After an oral dose of 500 mg of azithromycin in patients with community-acquired pneumonia, their serum concentrations ranged between 0.06 and 0.25 mg/liter during the first 12 hours; the calculated percentages of unbound drug varied between 45 and 86%. This study shows that in these patients, the total levels of azithromycin in serum are lower than those expected and that the percentage of bound drug is clinically irrelevant.
PMCID: PMC163034  PMID: 8593024
25.  In vitro antimicrobial effect against Streptococcus pneumoniae of adding rifampin to penicillin, ceftriaxone, or 1-ofloxacin. 
Antimicrobial Agents and Chemotherapy  1995;39(12):2798-2800.
Adding rifampin to penicillin or l-ofloxacin diminished the rate at which these antibiotics killed 21 clinical isolates isolates of Streptococcus pneumoniae in vitro. A less pronounced inhibitory effect was observed when rifampin was added to ceftriaxone. Synergy was not observed for any bacterial isolate. The in vitro demonstration of indifference or antagonism using these antibiotic combinations argues against the empirical addition of rifampin to beta-lactams or fluoroquinolones in treating serious pneumococcal infections.
PMCID: PMC163033  PMID: 8593023

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