The in vitro and in vivo antifungal activities of T-2307, a novel arylamidine, were evaluated and compared with those of fluconazole, voriconazole, micafungin, and amphotericin B. T-2307 exhibited broad-spectrum activity against clinically significant pathogens, including Candida species (MIC range, 0.00025 to 0.0078 g/ml), Cryptococcus neoformans (MIC range, 0.0039 to 0.0625 g/ml), and Aspergillus species (MIC range, 0.0156 to 4 g/ml). Furthermore, T-2307 exhibited potent activity against fluconazole-resistant and fluconazole-susceptible-dose-dependent Candida albicans strains as well as against azole-susceptible strains. T-2307 exhibited fungicidal activity against some Candida and Aspergillus species and against Cryptococcus neoformans. In mouse models of disseminated candidiasis, cryptococcosis, and aspergillosis, the 50% effective doses of T-2307 were 0.00755, 0.117, and 0.391 mg ⅐ kg ؊1 ⅐ dose ؊1 , respectively. This agent was considerably more active than micafungin and amphotericin B against candidiasis and than amphotericin B against cryptococcosis, and its activity was comparable to the activities of micafungin and amphotericin B against aspergillosis. The results of preclinical in vitro and in vivo evaluations performed thus far indicate that T-2307 could represent a potent injectable agent for the treatment of candidiasis, cryptococcosis, and aspergillosis.

The in vitro and in vivo activity of T-3912, a novel non-fluorinated topical quinolone, was compared with that of nadifloxacin, ofloxacin, levofloxacin, clindamycin, erythromycin and gentamicin. The in vitro activity of T-3912 against methicillin-susceptible Staphylococcus aureus, ofloxacin-resistant and methicillin-resistant S. aureus, Staphylococcus epidermidis, ofloxacin-resistant S. epidermidis, penicillin-resistant Streptococcus pneumoniae and Propionibacterium acnes was four-fold to 16 000-fold greater than that of other agents at the MIC90 for the clinical isolates. The activity of T-3912 was not influenced by grlA mutation in S. aureus, and the degree of MIC increase of T-3912 for grlA-gyrA double and triple mutants was lowest among the quinolones tested (nadifloxacin, levofloxacin and ofloxacin). The inhibitory activity of T-3912 was compared with other quinolones for DNA gyrase and topoisomerase IV of S. aureus SA113. T-3912 showed the greatest inhibitory activity for both enzymes among the quinolones tested. The isolation frequency of spontaneous mutants resistant to T-3912 was < 1.7 x 10(-9) and < 2.0 x 10(-9) for S. aureus SA113 and P. acnes JCM 6425, respectively. Furthermore, resistance to T-3912 could not be clearly detected in the 28th transfer by the serial passage method. T-3912 exhibited more potent bactericidal activity against S. aureus and P. acnes than nadifloxacin and clindamycin in a short time period. T-3912 in a 1% gel formulation showed good therapeutic activity against a burn infection model caused by S. aureus SA113, P. acnes JCM6425 and multidrug-resistant S. aureus F-2161. These results indicate that T-3912 is potentially a useful quinolone for the treatment of skin and soft-tissue infections and that its potent bactericidal activity might be able to shorten the treatment period.

T-2307, an arylamidine compound, has been previously reported to have broad-spectrum in vitro and in vivo antifungal activities against clinically significant pathogens, including Candida species, Cryptococcus neoformans, and Aspergillus species, and is now undergoing clinical trials. Here we investigated the mechanism of action of T-2307 using yeast cells and mitochondria isolated from yeast and rat liver. Nonfermentative growth of Candida albicans and Saccharomyces cerevisiae in glycerol medium, in which yeasts relied on mitochondrial respiratory function, was inhibited at 0.001 to 0.002 g/ml (0.002 to 0.004 M) of T-2307. However, fermentative growth in dextrose medium was not inhibited by T-2307. Microscopic examination using Mitotracker fluorescent dye, a cell-permeant mitochondrion-specific probe, demonstrated that T-2307 impaired the mitochondrial function of C. albicans and S. cerevisiae at concentrations near the MIC in glycerol medium. T-2307 collapsed the mitochondrial membrane potential in mitochondria isolated from S. cerevisiae at 20 M. On the other hand, in isolated rat liver mitochondria, T-2307 did not have any effect on the mitochondrial membrane potential at 10 mM. Moreover, T-2307 had little inhibitory and stimulatory effect on mitochondrial respiration in rat liver mitochondria. In conclusion, T-2307 selectively disrupted yeast mitochondrial function, and it was also demonstrated that the fungal mitochondrion is an attractive antifungal target.

Our results suggest that decreased bactericidal activity, or the in vitro PAE of carbapenems and fluoroquinolones, is related to the reduced in vivo protective effect against infection caused by high inoculum with S. aureus or P. aeruginosa.

The in vitro and in vivo activities of T-3811ME, a novel des-F(6)-quinolone, were evaluated in comparison with those of some fluoroquinolones, including a newly developed one, trovafloxacin. T-3811, a free base of T-3811ME, showed a wide range of antimicrobial spectra, including activities against Chlamydia trachomatis, Mycoplasma pneumoniae, andMycobacterium tuberculosis. In particular, T-3811 exhibited potent activity against various gram-positive cocci, with MICs at which 90% of the isolates are inhibited (MIC90s) of 0.025 to 6.25 μg/ml. T-3811 was the most active agent against methicillin-resistant Staphylococcus aureus and streptococci, including penicillin-resistant Streptococcus pneumoniae (PRSP). T-3811 also showed potent activity against quinolone-resistant gram-positive cocci with GyrA and ParC (GrlA) mutations. The activity of T-3811 against members of the familyEnterobacteriaceae and nonfermentative gram-negative rods was comparable to that of trovafloxacin. In common with other fluoroquinolones, T-3811 was highly active against Haemophilus influenzae, Moraxella catarrhalis, andLegionella sp., with MIC90s of 0.0125 to 0.1 μg/ml. T-3811 showed a potent activity against anaerobic bacteria, such as Bacteroides fragilis and Clostridium difficile. T-3811 was the most active agent against C. trachomatis (MIC, 0.008 μg/ml) and M. pneumoniae(MIC90, 0.0313 μg/ml). The activity of T-3811 againstM. tuberculosis (MIC90, 0.0625 μg/ml) was potent and superior to that of trovafloxacin. In experimental systemic infection with a GrlA mutant of S. aureus and experimental pneumonia with PRSP in mice, T-3811ME showed excellent therapeutic efficacy in oral and subcutaneous administrations.

We compared the susceptibility of six commercially available antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin, micafungin, and amphotericin B) against 133 Candida bloodstream isolates between 2008 and 2013 at Aichi Medical University Hospital. C. albicans was the most common isolate, followed by C. parapsilosis, C. glabrata, and C. tropicalis. MIC 90 s of voriconazole against C. albicans, C. parapsilosis, and C. tropicalis were the lowest and that of micafungin against C. glabrata was the lowest among the agents tested. Of the 133 isolates, two strains were identified as drug-resistant. One was a fluconazole-resistant C. glabrata strain, in which the ATP-binding cassette (ABC) transporter gene expression was upregulated. The other was a micafungin-resistant C. glabrata strain, that had 13 amino acid substitutions in FKS1 and FKS2, including a novel substitution V1342I in FKS1 hotspot 2. We also evaluated the susceptibility of T-2307, a novel class of antifungal agents used in clinical trials, against the fluconazoleand micafungin-resistant C. glabrata strain; the MICs of T-2307 were 0.0039 and 0.0078 mg/mL, respectively. In conclusion, the incidence of bloodstream infection caused by drug-resistant Candida spp. was rare from 2008 to 2013 at our hospital. Of 133 isolates, only two strains of C. glabrata were resistant to azoles or echinocandins, that upregulated the ABC transporter genes or had novel FKS mutations, respectively.

The considerably higher concentrations of T-2307 were selectively accumulated in C. albicans via transporter-mediated systems, as compared with the concentrations in rat hepatocytes. This transporter-mediated uptake of T-2307 contributes to its potent anticandidal activity.

Proteus mirabiis, Proteus vulgaris, MorganeUa morganii, Providencia rettgeri, and Providencia alcalifaciens, which were once classified into the same genus, Proteus, were studied. Cefoxitin-resistant mutants from these species were isolated, and it was confirmed that the resistance was attributed to the lack of an outer membrane protein, resulting in a significant decrease in the penetration of hydrophilic cephalosporins through the outer membrane. Comparison of the mutant strains with their parental strains in the diffusion rates of six monoanionic cephalosporins, a zwitterionic cephalosporin (cephaloridine), and a divalent anionic cephalosporin (cephalosporin C) suggested that each species had only one kind of porin protein, with molecular weights of 40,000 (Proteus mirabilis) or 37,000 (the other four species) and that the porins formed channels with cation selectivity, except for Proteus vulgaris. Porin proteins were purified from all the bacterial species except Providencia alcalifaciens, and the radius of the pores formed by the purified porins was estimated by the use of the liposome swelling assay. The pore radii were estimated to be approximately 0.59 nm (Proteus mirabilis), 0.63 nm (Proteus vulgaris), 0.58 nm (Providencia rettgeri), and 0.60 nm (M. morganiO), similar to the size of the pore radius of Escherichia coli porins.Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens were once classified into the same genus, Proteus (7), and, together with Serratia marcescens and Pseudomonas aeruginosa, are known as opportunistic pathogens. Except for Proteus mirabilis, these species show low susceptibility to many antibiotics. One of the reasons for such an intrinsic resistance may be the barrier effect of the outer membrane on antibiotic permeation. In the case of Pseudomonas aeruginosa, there is evidence suggesting that intrinsic resistance involves the outer membrane (1, 4, 32). However, the characterization of the outer membrane as the permeation route of antibiotics in the former Proteus species is incomplete. In our preliminary work (24), Proteus mirabilis N-51 was found to produce only a single major porinlike protein, with a molecular weight of 40,000. This 40K protein contributed to the bacterial susceptibility to cephalosporins and tetracycline. The present investigation was undertaken as an extension of the previous study to identify the porin proteins produced by Proteus vulgaris, M. morganii, Providencia rettgeri, and Providencia alcalifaciens and to evaluate the porin pores of these four species and of Proteus mirabilis as permeation routes for ,-lactam antibiotics. MATERIALS AND METHODSBacterial strains and P-lactamase production. Proteus mirabilis N-51, Proteus vulgaris K22-2, Providencia rettgeri RE-18, and Providencia alcalifaciens IN-06 are clinical isolates which barely produce 1-lactamase activity under usual growth conditions. M. morganii 1510/9, which is a mutant strain with lower 1-lactamase activity, was isolated from strain 151...