Takashi Fukuoka

New Model of Oropharyngeal Candidiasis in Mice

ABSTRACT We established a straightforward murine model of oropharyngeal candidiasis. Mice were immunosuppressed with cortisone acetate, anesthetized, and then inoculated by placing cotton wool balls saturated with Candida albicans sublingually for 2 h. A prolonged, reproducible infection was induced. This model may be useful for antifungal screening or pathogenesis studies.

Genetic Basis for Differential Activities of Fluconazole and Voriconazole against Candida krusei

ABSTRACT Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14α-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.

Contribution of Candida albicans ALS1 to the Pathogenesis of Experimental Oropharyngeal Candidiasis

ABSTRACT We investigated the contribution of Candida albicans ALS1, which encodes a candidal adhesin, to the pathogenesis of experimental murine oropharyngeal candidiasis. Our results indicate that the ALS1 gene product is important for the adherence of the organism to the oral mucosa during the early stage of the infection.

In Vitro and In Vivo Antibacterial Activities of CS-023 (RO4908463), a Novel Parenteral Carbapenem

ABSTRACT CS-023 (RO4908463, formerly R-115685) is a novel 1β-methylcarbapenem with 5-substituted pyrrolidin-3-ylthio groups, including an amidine moiety at the C-2 position. Its antibacterial activity was tested against 1,214 clinical isolates of 32 species and was compared with those of imipenem, meropenem, ceftazidime, ceftriaxone, ampicillin, amikacin, and levofloxacin. CS-023 exhibited a broad spectrum of activity against gram-positive and -negative aerobes and anaerobes, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, penicillin-resistant Streptococcus pneumoniae (PRSP), β-lactamase-negative ampicillin-resistant Haemophilus influenzae, and Pseudomonas aeruginosa. CS-023 showed the most potent activity among the compounds tested against P. aeruginosa and MRSA, with MICs at which 90% of isolates tested were inhibited of 4 μg/ml and 8 μg/ml, respectively. CS-023 was stable against hydrolysis by the β-lactamases from Enterobacter cloacae and Proteus vulgaris. CS-023 also showed potent activity against extended-spectrum β-lactamase-producing Escherichia coli. The in vivo efficacy of CS-023 was evaluated with a murine systemic infection model induced by 13 strains of gram-positive and -negative pathogens and a lung infection model induced by 2 strains of PRSP (serotypes 6 and 19). Against the systemic infections with PRSP, MRSA, and P. aeruginosa and the lung infections, the efficacy of CS-023 was comparable to those of imipenem/cilastatin and vancomycin (tested against lung infections only) and superior to those of meropenem, ceftriaxone, and ceftazidime (tested against P. aeruginosa infections only). These results suggest that CS-023 has potential for the treatment of nosocomial bacterial infections by gram-positive and -negative pathogens, including MRSA and P. aeruginosa.

Synthesis and antimycobacterial activity of capuramycin analogues. Part 1: substitution of the azepan-2-one moiety of capuramycin

Capuramycin analogues with a variety of substituents in place of the azepan-2-one moiety were synthesized from A-500359E and were tested for their translocase I inhibitory activity and in vitro antimycobacterial activity. Phenyl-type moieties were found to be effective substituents for capuramycin analogues.

Synthesis and antimycobacterial activity of capuramycin analogues. Part 2: acylated derivatives of capuramycin-related compounds.

Acylated derivatives of capuramycin and A-500359A were synthesized and tested for antimycobacterial activity. Compound 20 having a decanoyl group showed very potent activity.

[43] In vitro and in Vivo models of bacterial biofilms

Publisher Summary This chapter focuses on several methods that have been developed in the laboratory to form bacterial biofilm models in vitro and in vivo , as well as several methods developed to investigate the nature of biofilm bacteria. Ideal properties sought in an in vitro bacterial biofilm model are uniform film formation, uniform reproducibility, and the ability to be investigated quantitatively. However, the ability to mimic a highly complicated in vivo condition is desirable in an in vitro model. There have been relatively few reports on in vivo models of biofilm growth. One of the most difficult challenges to overcome is to devise a durable infection model. Generally, it takes several days for infected bacteria to form biofilms in an infected tissue or organ, and it takes several days longer to investigate the therapeutic effect of a drug after the biofilm mode of growth of the infected bacteria has been established. Ideally, the infection should continue in a stable condition for more than 7–10 days. Respiratory tract infections, urinary tract infections, infective endocarditis, foreign body infections, and so forth are typical infectious diseases in which the biofilm mode of bacterial growth is sometimes characteristic. Therefore, these diseases should be taken into account in making a useful in vivo experimental biofilm infection model.

Increase in susceptibility of Pseudomonas aeruginosa to carbapenem antibiotics in low-amino-acid media.

The in vitro susceptibility of Pseudomonas aeruginosa PAO1 to carbapenem antibiotics, such as CS-533, was influenced by various concentrations of basic amino acids, i.e., L-lysine, L-histidine, and L-arginine, in agar media. P. aeruginosa PAO1 showed higher susceptibility to carbapenems in minimal medium than it did in rich media such as Mueller-Hinton II agar. The susceptibility was decreased by the addition of a basic amino acid to the minimal medium, whereas it was influenced less by other amino acids. The susceptibility of PAO1 to cephalosporins, piperacillin, quinolones, and gentamicin was not influenced by the addition of a basic amino acid to the minimal medium. A significant change in susceptibility to carbapenems by the addition of a basic amino acid was not observed with D2 protein-deficient mutants of PAO1. Clinical isolates of P. aeruginosa also showed an increase in susceptibility in minimal medium. L-Lysine in minimal medium did not have any influence on the production of D2 protein, beta-lactamases, or penicillin-binding proteins of PAO1 or on the chemical degradation of CS-533. These results strongly indicate that the increase in susceptibility of P. aeruginosa to carbapenems relates to less competition with basic amino acids for permeation through the D2 protein channel of P. aeruginosa. Images

Potent In Vitro Activity of Tomopenem (CS-023) against Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

ABSTRACT Tomopenem (formerly CS-023) is a novel 1β-methylcarbapenem with broad-spectrum coverage of gram-positive and gram-negative pathogens. Its antibacterial activity against European clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was compared with those of imipenem and meropenem. The MICs of tomopenem against MRSA and P. aeruginosa at which 90% of the isolates tested were inhibited were 8 and 4 μg/ml, respectively, and were equal to or more than fourfold lower than those of imipenem and meropenem. The antibacterial activity of tomopenem against MRSA was correlated with a higher affinity for the penicillin-binding protein (PBP) 2a. Its activity against laboratory mutants of P. aeruginosa with (i) overproduction of chromosomally coded AmpC β-lactamase; (ii) overproduction of the multidrug efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN; (iii) deficiency in OprD; and (iv) various combinations of AmpC overproduction, MexAB-OprM overproduction, and OprD deficiency were tested. The increases in the MIC of tomopenem against each single mutant compared with that against its parent strain were within a fourfold range. Tomopenem exhibited antibacterial activity against all mutants, with an observed MIC range of 0.5 to 8 μg/ml. These results suggest that the antibacterial activity of tomopenem against the clinical isolates of MRSA and P. aeruginosa should be ascribed to its high affinity for PBP 2a and its activity against the mutants of P. aeruginosa, respectively.

In vitro and in vivo activities of CS-758 (R-120758), a new triazole antifungal agent

ABSTRACT The activity of CS-758 (R-120758), a new triazole antifungal agent, was evaluated and compared with those of fluconazole, itraconazole, and amphotericin B in vitro and with those of fluconazole and itraconazole in vivo. CS-758 exhibited potent in vitro activity against clinically important fungi. The activity of CS-758 against Candida spp. was superior to that of fluconazole and comparable or superior to those of itraconazole and amphotericin B. CS-758 retained potent activity against Candida albicans strains with low levels of susceptibility to fluconazole (fluconazole MIC, 4 to 32 μg/ml). Against Aspergillus spp. and Cryptococcus neoformans, the activity of CS-758 was at least fourfold superior to those of the other drugs tested. CS-758 also exhibited potent in vivo activity against murine systemic infections caused by C. albicans, C. neoformans, Aspergillus fumigatus, and Aspergillus flavus. The 50% effective doses against these infections were 0.41 to 5.0 mg/kg of body weight. These results suggest that CS-758 may be useful in the treatment of candidiasis, cryptococcosis, and aspergillosis.