Efficacy of T-2307, a novel arylamidine, against ocular complications of disseminated candidiasis in mice.

OBJECTIVES: T-2307, a novel arylamidine, shows broad-spectrum activity against pathogenic fungi, including Candida albicans. Ocular candidiasis is one of the serious complications associated with Candida bloodstream infection and is known to be refractory to conventional antifungal agents. The aim of the present study was to clarify the effectiveness of T-2307 against ocular candidiasis using a mouse model. METHODS: We evaluated ocular fungal burden in mice infected with C. albicans that received treatment with antifungal agents [T-2307, liposomal amphotericin B (LAMB) or fluconazole] for 3 consecutive days. We also assessed survival rates of mice after C. albicans infection followed by treatment for 7 consecutive days. In addition, ocular T-2307 concentrations and in vitro effectiveness against C. albicans biofilm formation were evaluated. RESULTS: The ocular fungal burdens were significantly reduced after T-2307 treatment compared with the control group (no treatment received) and were comparable with those observed following treatment with LAMB or fluconazole in both early- and late-phase treatment experiments. In addition, all of the mice treated with antifungal agents survived for 3 weeks after infection, whereas mice in the control group died within 3 days. The ocular T-2307 trough concentration was maintained above the MIC in the infected mice. An in vitro biofilm inhibition experiment showed that T-2307 suppressed C. albicans biofilm formation at the sub-MIC level, which was comparable with amphotericin B. CONCLUSIONS: Given these results in experimental disseminated candidiasis, T-2307 may be an effective treatment against the complication of ocular candidiasis.

Successful treatment of invasive pulmonary aspergillosis caused by Aspergillus felis, a cryptic species within the Aspergillus section Fumigati: A case report.

Aspergillus species are a major cause of life-threatening infections in immunocompromised hosts, and the most common pathogen of invasive aspergillosis is Aspergillus fumigatus. Recently, the development of molecular identification has revealed cryptic Aspergillus species, and A. felis is one such species within the Aspergillus section Fumigati reported in 2013. We describe a case of invasive pulmonary aspergillosis caused by A. felis in a 41-year-old Japanese woman diagnosed with myelodysplastic syndrome. She presented with fever 19 days after undergoing autologous peripheral blood stem cell transplantation and was clinically diagnosed with invasive pulmonary aspergillosis. Bronchoscopy and bronchoalveolar lavage were performed for definitive diagnosis. The ß-tubulin genes of the mold isolated from the bronchoalveolar lavage fluid, and sequenced directly from the PCR products using a primer pair were found to have 100% homology with A. felis. We successfully treated the patient with echinocandin following careful susceptibility testing. To the best of our knowledge, this is the first published case reporting the clinical course for diagnosis and successful treatment of invasive aspergillosis by A. felis.

CRISPR/Cas9 Genome Editing To Demonstrate the Contribution of Cyp51A Gly138Ser to Azole Resistance in Aspergillus fumigatus.

A pan-azole-resistant Aspergillus fumigatus strain with the cyp51A mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing cyp51A with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains. Recombinant strains showed increased susceptibility via the replacement of Ser138 by glycine. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using CRISPR/Cas9 genome editing.

In vitro and in vivo antifungal activities of T-2307, a novel arylamidine, against Cryptococcus gattii: an emerging fungal pathogen.

Objectives: T-2307, a novel arylamidine, exhibits potent broad-spectrum activities against the majority of fungal pathogens. In this study, the antifungal activity of T-2307 against Cryptococcus gattii was evaluated in comparison with those of amphotericin B, fluconazole and voriconazole in vitro and in vivo . Methods: The MICs for 15 clinical isolates were determined according to CLSI guidelines and time-kill studies were performed using C. gattii YF2784. In a murine model for intranasal pulmonary infection caused by C. gattii YF2784, the test compounds were administered once daily for 7 days from 2 h or 14 days post-infection. The viable counts in the lungs and brain were determined at 21 days post-infection. Results: The MIC range, MIC 50 , MIC 90 and geometric mean MIC of T-2307 were 0.0078-0.0625, 0.0313, 0.0625 and 0.0394 mg/L, respectively. The MIC of T-2307 was significantly lower than those of fluconazole, voriconazole and amphotericin B. T-2307 showed concentration-dependent fungicidal activity at 4 times the MIC or higher. Administration of T-2307 at 2 mg/kg/day, amphotericin B at 1 mg/kg/day and fluconazole at 160 mg/kg/day from 2 h post-infection significantly reduced viable counts in the lungs and brain. However, when the administration was started 14 days post-infection, only T-2307 significantly reduced the viable counts in both the lungs and the brain at 1 mg/kg/day. Conclusions: T-2307 shows excellent in vitro and in vivo antifungal activities against C. gattii and would be a promising new candidate for the treatment of cryptococcosis.

Efficacy of High-Dose Meropenem (Six Grams per Day) in Treatment of Experimental Murine Pneumonia Induced by Meropenem-Resistant Pseudomonas aeruginosa.

High-dose meropenem (MEPM; 6 g/day) has been approved as a treatment for purulent meningitis; however, little is known regarding its in vivo efficacy in refractory lower respiratory tract infections. The purpose of this study was to evaluate the efficacy of MEPM at 6 g/day in a murine model of severe pneumonia caused by MEPM-resistant Pseudomonas aeruginosa Experimental pneumonia induced by MEPM-resistant P. aeruginosa was treated with normal-dose MEPM (150 mg/kg of body weight, simulating a 3-g/day regimen in humans) or high-dose MEPM (500 mg/kg, simulating a 6-g/day regimen in humans). Mice treated with high-dose MEPM showed significantly restored survival relative to that of untreated mice and tended to show a survival rate higher than that of mice treated with normal-dose MEPM. The viable bacterial counts (of two clinical isolates) in the lungs decreased significantly in mice treated with high-dose MEPM from those for untreated mice (P < 0.001) or mice treated with normal-dose MEPM (P, <0.01 and <0.05). The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was also significantly lower in mice treated with high-dose MEPM than in untreated mice. The free MEPM concentration in the epithelial lining fluid (ELF) exceeded 16 µg/ml for 85 min in mice treated with high-dose MEPM, but not for mice treated with normal-dose MEPM. Our results demonstrate that high-dose MEPM (6 g/day) might provide better protection against pneumonia caused by MEPM-resistant strains of P. aeruginosa than the dose normally administered (less than 3 g/day).

Roles of vacuolar H+-ATPase in the oxidative stress response of Candida glabrata.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular compartments and plays an important role in oxidative stress response (OSR), but its molecular bases are largely unknown. Here, we investigated how V-ATPase is involved in the OSR by using a strain lacking VPH2, which encodes an assembly factor of V-ATPase, in the pathogenic fungus Candida glabrata The loss of Vph2 resulted in increased H2O2 sensitivity and intracellular reactive oxygen species (ROS) level independently of mitochondrial functions. The Δvph2 mutant also displayed growth defects under alkaline conditions accompanied by the accumulation of intracellular ROS and these phenotypes were recovered in the presence of the ROS scavenger N-acetyl-l-cysteine. Both expression and activity levels of mitochondrial manganese superoxide dismutase (Sod2) and catalase (Cta1) were decreased in the Δvph2 mutant. Phenotypic analyses of strains lacking and overexpressing these genes revealed that Sod2 and Cta1 play a predominant role in endogenous and exogenous OSR, respectively. Furthermore, supplementation of copper and iron restored the expression of SOD2 specifically in the Δvph2 mutant, suggesting that the homeostasis of intracellular cupper and iron levels maintained by V-ATPase was important for the Sod2-mediated OSR. This report demonstrates novel roles of V-ATPase in the OSR in C. glabrata.

In vivo efficacy of biapenem with ME1071, a novel metallo-ß-lactamase (MBL) inhibitor, in a murine model mimicking ventilator-associated pneumonia caused by MBL-producing Pseudomonas aeruginosa.

ME1071, a maleic acid derivative, is a novel, specific inhibitor of metallo-ß-lactamases (MBLs). In vitro, ME1071 can potentiate the activity of carbapenems against MBL-producing Pseudomonas aeruginosa. To confirm the clinical efficacy of ME1071 in ventilator-associated pneumonia (VAP) caused by MBL-producing P. aeruginosa, a mouse model that mimics VAP by placement of a plastic tube in the bronchus was used. Biapenem (100 mg/kg) or ME1071 plus biapenem (each 100 mg/kg) was administered intraperitoneally every 12 h beginning at 12 h after inoculation. Survival was evaluated over 7 days. At 30 h post infection, mice were sacrificed and the numbers of viable bacteria in the lungs and bronchoalveolar lavage fluid (BALF) were compared. Histopathological analysis of lung specimens was also performed. The pharmacokinetics of ME1071 was analysed after initial treatment. The ME1071 plus biapenem combination group displayed significantly longer survival compared with the control and biapenem monotherapy groups (P<0.05). Furthermore, the number of viable bacteria in the lungs was significantly lower in the combination group (P<0.05). Histopathological examination of lung specimens indicated that progression of lung inflammation was prevented in the combination group. Furthermore, total cell and neutrophil counts, as well as cytokine levels, in BALF were significantly decreased (P<0.05) in the combination group. The percentage time above the MIC (%T>MIC) for biapenem without ME1071 was 0% in plasma; however, this value was elevated to 10.8% with ME1071. These results suggest that ME1071 is potent and effective for treatment of VAP caused by MBL-producing P. aeruginosa.

Azithromycin attenuates lung inflammation in a mouse model of ventilator-associated pneumonia by multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is one of the main pathogens that cause ventilator-associated pneumonia (VAP) and is associated with a high rate of mortality. Little is known about the efficacy of macrolides against A. baumannii. In order to confirm the efficacy of azithromycin (AZM) against VAP caused by multidrug-resistant A. baumannii (MDRAB), we used a mouse model that mimics VAP by placement of a plastic tube in the bronchus. AZM (10 and 100 mg/kg of body weight) was administered subcutaneously every 24 h beginning at 3 h after inoculation. Phosphate-buffered saline was administered as the control. Survival was evaluated over 7 days. At 48 h postinfection, mice were sacrificed and the numbers of viable bacteria in lungs and bronchoalveolar lavage fluid were compared. Histopathological analysis of lung specimens was also performed. The treatment groups displayed significantly longer survival than the control group (P < 0.05). AZM did not have an antimicrobial effect. Histopathological examination of lung specimens indicated that the progression of lung inflammation was prevented in the AZM-treated groups. Furthermore, total cell and neutrophil counts, as well as cytokine levels, in bronchoalveolar lavage fluid were significantly decreased (P < 0.05) in the AZM-treated groups. AZM may have a role for the treatment of VAP with MDRAB because of its anti-inflammatory effects.

Efficacy of AiiM, an N-acylhomoserine lactonase, against Pseudomonas aeruginosa in a mouse model of acute pneumonia.

Quorum sensing (QS) in Pseudomonas aeruginosa regulates the production of many virulence factors and plays an important role in the pathogenesis of P. aeruginosa infection. N-acyl homoserine lactones (AHL) are major QS signal molecules. Recently, a novel AHL-lactonase enzyme, AiiM, has been identified. The aim of this study was to evaluate the effect of AiiM on the virulence of P. aeruginosa in a mouse model of acute pneumonia. We developed a P. aeruginosa PAO1 strain harboring an AiiM-expressing plasmid. The production of several virulence factors by the AiiM-expressing strain was examined. Mice were intratracheally infected with an AiiM-expressing PAO1 strain. Lung histopathology, bacterial burden, and bronchoalveolar lavage (BAL) fluid were assessed at 24 h postinfection. AiiM expression in PAO1 reduced production of AHL-mediated virulence factors and attenuated cytotoxicity against human lung epithelial cells. In a mouse model of acute pneumonia, AiiM expression reduced lung injury and greatly improved the survival rates. The levels of proinflammatory cytokines and myeloperoxidase activity in BAL fluid were significantly lower in mice infected with AiiM-expressing PAO1. Thus, AiiM can strongly attenuate P. aeruginosa virulence in a mammalian model and is a potential candidate for use as a therapeutic agent against P. aeruginosa infection.

The heme-binding protein Dap1 links iron homeostasis to azole resistance via the P450 protein Erg11 in Candida glabrata.

The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.

Correlation between triazole treatment history and susceptibility in clinically isolated Aspergillus fumigatus.

This is the first report of a detailed relationship between triazole treatment history and triazole MICs for 154 Aspergillus fumigatus clinical isolates. The duration of itraconazole dosage increased as the itraconazole MIC increased, and a positive correlation was observed (r = 0.5700, P < 0.0001). The number of itraconazole-naïve isolates dramatically decreased as the itraconazole MIC increased, particularly for MICs exceeding 2 µg/ml (0.5 µg/ml versus 2 µg/ml, P = 0.03). We also examined the relationship between cumulative itraconazole usage and the MICs of other azoles. A positive correlation existed between itraconazole dosage period and posaconazole MIC (r = 0.5237, P < 0.0001). The number of itraconazole-naïve isolates also decreased as the posaconazole MIC increased, particularly for MICs exceeding 0.5 µg/ml (0.25 µg/ml versus 0.5 µg/ml, P = 0.004). Conversely, the correlation coefficient obtained from the scattergram of itraconazole usage and voriconazole MICs was small (r = -0.2627, P = 0.001). Susceptibility to three triazole agents did not change as the duration of voriconazole exposure changed. In addition, we carried out detailed analysis, including microsatellite genotyping, for isolates obtained from patients infected with azole-resistant A. fumigatus. We confirmed the presence of acquired resistance to itraconazole and posaconazole due to a G54 substitution in the cyp51A gene for a patient with chronic pulmonary aspergillosis after oral itraconazole therapy. We should consider the possible appearance of azole-resistant A. fumigatus if itraconazole is used for extended periods.

Efficacy of clarithromycin against experimentally induced pneumonia caused by clarithromycin-resistant Haemophilus influenzae in mice.

Clarithromycin is a 14-member lactone ring macrolide with potent activity against Haemophilus influenzae, including ampicillin-resistant strains. We evaluated the in vivo efficacy of clarithromycin at 40 mg/day and 100 mg/day for 3 days in the treatment of a murine model of pneumonia using a macrolide-resistant H. influenzae strain, which was also ampicillin resistant. The MIC of clarithromycin was 64 microg/ml. The viable bacterial counts in infected tissues after treatment with 100 mg clarithromycin/kg of body weight were lower than the counts obtained in control and 40-mg/kg clarithromycin-treated mice. The concentrations of macrophage inflammatory protein 2 (MIP-2) and interleukin 1beta (IL-1beta) in bronchoalveolar lavage fluid (BALF) samples from mice treated at both concentrations were lower than in the control group. Pathologically, following infection, clarithromycin-treated mice, particularly at a dose of 100 mg/kg, showed lower numbers of neutrophils in alveolar walls, and inflammatory changes had apparently improved, whereas large aggregates of inflammatory cells were observed within the alveoli of control mice. In addition, we demonstrated that clarithromycin has bacteriological effects against intracellular bacteria at levels below the MIC. Our results indicate that clarithromycin may be useful in vivo for macrolide-resistant H. influenzae, and this phenomenon may be related to the good penetration of clarithromycin into bronchoepithelial cells. We also believe that conventional drug susceptibility tests may not reflect the in vivo effects of clarithromycin.

In vivo efficacy of sitafloxacin in a new murine model of non-typeable Haemophilus influenzae pneumonia by sterile intratracheal tube.

A novel murine model of non-typeable Haemophilus influenzae (NTHi) pneumonia was established. A plastic tube was inserted into the trachea 7 days before bacterial inoculation. Numbers of NTHi recovered from the lungs and trachea were determined for 7 days. Histologically, bronchioles and adjacent alveoli in the intubation group were filled with numerous inflammatory cells. The efficacy of sitafloxacin was compared with ciprofloxacin using the new murine pneumonia model. The data suggest that sitafloxacin displays equivalent efficacy to ciprofloxacin against H. influenzae pneumonia. This new murine NTHi pneumonia model appears useful not only for in vivo evaluation of antibiotics but also for analysis of the pathogenesis of H. influenzae pneumonia.