In Vitro Antifungal Susceptibilities of Scopulariopsis Isolates

MICs and minimum fungicidal concentrations of amphotericin B, miconazole, itraconazole, ketoconazole, fluconazole, and flucytosine against 17 isolates of Scopulariopsis spp. were determined by a broth microdilution method. All the isolates were resistant to itraconazole, fluconazole, and flucytosine, and amphotericin B, miconazole, and ketoconazole MICs were low for only a few.     

In Vitro and In Vivo Antifungal Susceptibilities of the Mucoralean Fungus Cunninghamella

We have determined the in vitro activities of amphotericin B (AMB), voriconazole, posaconazole (PSC), itraconazole (ITC), ravuconazole, terbinafine, and caspofungin against five strains of Cunninghamella bertholletiae and four of Cunninghamella echinulata. The best activity was shown by terbinafine against both species (MIC range = 0.3 to 0.6 μg/ml) and PSC against Cunninghamella bertholletiae (MIC = 0.5 μg/ml). We have also evaluated the efficacies of PSC, ITC, and AMB in neutropenic and diabetic murine models of disseminated infection by Cunninghamella bertholletiae. PSC at 40, 60, or 80 mg/kg of body weight/day was as effective as AMB at 0.8 mg/kg/day in prolonging survival and reducing the fungal tissue burden in neutropenic mice. PSC at 80 mg/kg/day was more effective than AMB at 0.8 mg/kg/day in reducing the fungal load in brain and lung of diabetic mice. Histological studies revealed an absence of fungal elements in organs of mice treated with either AMB at 0.8 mg/kg/day or PSC at 60 or 80 mg/kg/day in both models. ITC showed limited efficacy in both models. PSC could be a therapeutic option for the treatment of systemic infections caused by Cunninghamella bertholletiae.The genus Cunninghamella in the order Mucorales encompasses filamentous fungi that are inhabitants of soil and other environments and are common laboratory contaminants. Cunninghamella bertholletiae is the only member of the genus documented to cause human infections (8), although recently the species C. echinulata was also found in clinical samples (3). Infections caused by Cunninghamella are less frequent than those produced by other genera of Mucorales, e.g., Rhizopus and Mucor, but the mortality rate is higher (76%) (5, 19). In general, infections caused by the members of Mucorales are life-threatening and devastating, requiring prompt and aggressive treatment. There are several simultaneous approaches recommended for patient management, including surgical debridement, antifungal therapy, and correction of the underlying predisposing factors, when possible (5, 20).Amphotericin B (AMB) is the drug of choice, although very few data exist on the activity of this drug against some of the less frequent genera of Mucorales, such as Cunninghamella (1, 2). In a recent study in which a large set of clinical isolates of Mucorales was tested, C. bertholletiae demonstrated the highest resistance to AMB in vitro, with only 63% of the isolates tested showing MIC values under the working interpretative breakpoints described by the CLSI (2, 6). In addition, the failure of AMB therapy has been reported in clinical cases caused by Cunninghamella (4, 15, 18, 19). Posaconazole (PSC) appears to be a promising drug for the treatment of zygomycosis, having been successfully used as salvage therapy for patients who are refractory to or intolerant of AMB (10, 23). PSC is safer than AMB and shows good in vitro activity and in vivo efficacy against some zygomycetes (1, 2, 7, 22). There are only two clinical reports on the use of PSC for the management of Cunninghamella infection (9, 16), and there are no data on the use of this drug in animal models of C. bertholletiae infections. The aim of the present study, therefore, was to evaluate the in vitro activities of seven drugs against isolates of both C. bertholletiae and C. echinulata and to assess the in vivo efficacies of AMB, itraconazole (ITC), and PSC in neutropenic and diabetic murine models of disseminated infection by five strains of C. bertholletiae.     

Sordarins: In Vitro Activities of New Antifungal Derivatives against Pathogenic Yeasts,Pneumocystis carinii,and Filamentous Fungi

GM 193663, GM 211676, GM 222712, and GM 237354 are new semisynthetic derivatives of the sordarin class. The in vitro antifungal activities of GM 193663, GM 211676, GM 222712, and GM 237354 against 111 clinical yeast isolates of Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei, and Cryptococcus neoformans were compared. The in vitro activities of some of these compounds against Pneumocystis carinii, 20 isolates each of Aspergillus fumigatus and Aspergillus flavus, and 30 isolates of emerging less-common mold pathogens and dermatophytes were also compared. The MICs of GM 193663, GM 211676, GM 222712, and GM 237354 at which 90% of the isolates were inhibited (MIC₉₀s) were 0.03, 0.03, 0.004, and 0.015 μg/ml, respectively, for C. albicans, including strains with decreased susceptibility to fluconazole; 0.5, 0.5, 0.06, and 0.12 μg/ml, respectively, for C. tropicalis; and 0.004, 0.015, 0.008, and 0.03 μg/ml, respectively, for C. kefyr. GM 222712 and GM 237354 were the most active compounds against C. glabrata, C. parapsilosis, and Cryptococcus neoformans. Against C. glabrata and C. parapsilosis, the MIC₉₀s of GM 222712 and GM 237354 were 0.5 and 4 μg/ml and 1 and 16 μg/ml, respectively. The MIC₉₀s of GM 222712 and GM 237354 against Cryptococcus neoformans were 0.5 and 0.25 μg/ml, respectively. GM 193663, GM 211676, GM 222712, and GM 237354 were extremely active against P. carinii. The efficacies of sordarin derivatives against this organism were determined by measuring the inhibition of the uptake and incorporation of radiolabelled methionine into newly synthesized proteins. All compounds tested showed 50% inhibitory concentrations of <0.008 μg/ml. Against A. flavus and A. fumigatus, the MIC₉₀s of GM 222712 and GM 237354 were 1 and 32 μg/ml and 32 and >64 μg/ml, respectively. In addition, GM 237354 was tested against the most important emerging fungal pathogens which affect immunocompromised patients. Cladosporium carrioni, Pseudallescheria boydii, and the yeast-like fungi Blastoschizomyces capitatus and Geotrichum clavatum were the most susceptible of the fungi to GM 237354, with MICs ranging from ≤0.25 to 2 μg/ml. The MICs of GM 237354 against Trichosporon beigelii and the zygomycetes Absidia corymbifera, Cunninghamella bertholletiae, and Rhizopus arrhizus ranged from ≤0.25 to 8 μg/ml. Against dermatophytes, GM 237354 MICs were ≥2 μg/ml. In summary, we concluded that some sordarin derivatives, such as GM 222712 and GM 237354, showed excellent in vitro activities against a wide range of pathogenic fungi, including Candida spp., Cryptococcus neoformans, P. carinii, and some filamentous fungi and emerging invasive fungal pathogens.During the past two decades, the incidence of infections caused by opportunistic fungal pathogens in immunocompromised patients has increased substantially (1, 11, 30, 31, 36). Candida albicans is the major opportunistic pathogen, although the incidence of fungal infections caused by non-C. albicans species is increasing (37). Pneumocystis carinii remains an important pathogen in AIDS patients and other immunocompromised individuals (17), and invasive pulmonary aspergillosis remains a frequently fatal complication of bone marrow transplantation and of cancer chemotherapy in patients with hematologic neoplasms (23, 26, 29). Although there has been an expansion in the number of antifungal drugs available (8–10), in many cases, treatment of fungal diseases remains unsatisfactory. This situation has led to an ongoing search for fungicidal agents with different modes of action and fewer side effects and which can be administered both orally and parenterally.One of the major challenges to finding a potent yet safe antifungal agent is the similarity between fungal and mammalian cells. Like mammalian cells, fungi are eukaryotic, so they have many of the same structures and metabolic pathways as mammalian cells, making it more difficult to find targets of differential toxicity. Although protein synthesis is a universal process in living cells, it has always been considered as one of the more attractive targets for the development of antimicrobial agents (8, 12, 36). It is known that fungal protein has exploitable differences relative to its mammalian counterpart, e.g., the two soluble protein factors elongation factor 3 (EF-3) (19, 34) which is absent from mammalian cells, EF-2, which is functionally distinct from its mammalian counterpart (5, 6). On the basis of these differences, a target-based screening program was established, with the objective of isolating selective protein synthesis inhibitors of the fungal machinery (2). As part of this screening program, a novel antifungal compound, GR 135402, was isolated from fermentation broth of Graphium putredinis and characterized (20). This new compound is the first natural product described to date which possesses antifungal activity through inhibition of fungal but not mammalian protein synthesis (20). GR 135402 belongs to the sordarin class (13), and although it has some structural similarity to zofimarin (27), sordarin (33), and sordarin derivatives (3, 28), no mode of action was described for the antifungal activity of these compounds. A synthetic chemical program was initiated to improve the biological properties of GR 135402, and four compounds, designated GM 193663, GM 211676, GM 222712, and GM 237354, were selected for evaluation.In this study, we analyze the in vitro antifungal activities of these four new sordarin derivatives against several groups of clinical isolates.(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997 [15, 16].)     

In Vitro Evaluation of Drug Susceptibilities of Babesia divergens Isolates

The susceptibilities of three bovine and two human Babesia divergens isolates to antimicrobial agents were evaluated in vitro by a tritiated hypoxanthine incorporation assay. The MICs at which 50% of isolates are inhibited (MIC₅₀s) for mefloquine (chlorhydrate), chloroquine (sulfate), quinine (chlorhydrate), clindamycin (phosphate), pentamidine (isethionate), phenamidine (isethionate) plus oxomemazine (chlorhydrate), lincomycin (chlorhydrate monohydrate), and imidocarb (dipropionate) were determined. Except for imidocarb, the MIC₅₀s observed for the different isolates were close. Imidocarb and the combination of phenamidine plus oxomemazine exhibited the highest in vitro activity, while antimalarial agents such as mefloquine, choroquine, and quinine were inactive. Other drugs had intermediate activities. The data support further in vitro evaluation of antimicrobial agents active against B. divergens for the improvement of therapeutic strategies.     

Evaluation of the In Vitro Antifungal Activity of Allicin

Allicin was effective in vitro against Candida, Cryptococcus, Trichophyton, Epidermophyton, and Microsporum. The minimal inhibitory concentrations (MICs) of allicin against these organisms were 3.13 to 6.25 μg/ml by the agar dilution method and 1.57 to 6.25 μg/ml by the broth dilution method, using Sabouraud glucose (SG) medium. However decreased activity was demonstrated against Aspergillus. The MIC of allicin against various pathogenic fungi was affected considerably by differences in the experimental conditions, e.g., incubation time, inoculum size, type of medium, and medium pH. The MIC of allicin against Candida, Cryptococcus, and Aspergillus remained constant after more than 3 days of incubation, and that against Dermatophytes remained constant after more than 10 days of incubation. Decreasing the inoculum size increased the susceptibility to allicin. The antifungal activity of allicin was stronger on SG agar medium with a pH of 5.6 than on the same medium with a pH of 6.0 or higher. By microscopical observation, allicin induced morphological abnormalities in hyphae of Trichophyton mentagrophytes Morita. Percent germination of spores of the Morita strain at 24 h in SG agar medium was greatly decreased with an allicin concentration of 3.13 μg/ml, and the lethal dose for the spores was about four times higher than the fungistatic concentration. These results suggest that allicin inhibits both germination of spores and growth of hyphae.     

Population-Based Survey of Filamentous Fungi and Antifungal Resistance in Spain (FILPOP Study)

A population-based survey was conducted to investigate the epidemiology of and antifungal resistance in Spanish clinical strains of filamentous fungi isolated from deep tissue samples, blood cultures, and respiratory samples. The study was conducted in two different periods (October 2010 and May 2011) to analyze seasonal variations. A total of 325 strains were isolated in 29 different hospitals. The average prevalence was 0.0016/1,000 inhabitants. Strains were identified by sequencing of DNA targets and susceptibility testing by the European Committee for Antimicrobial Susceptibility Testing reference procedure. The most frequently isolated genus was Aspergillus, accounting for 86.3% of the isolates, followed by Scedosporium at 4.7%; the order Mucorales at 2.5%; Penicillium at 2.2%, and Fusarium at 1.2%. The most frequent species was Aspergillus fumigatus (48.5%), followed by A. flavus (8.4%), A. terreus (8.1%), A. tubingensis (6.8%), and A. niger (6.5%). Cryptic/sibling Aspergillus species accounted for 12% of the cases. Resistance to amphotericin B was found in 10.8% of the isolates tested, while extended-spectrum triazole resistance ranged from 10 to 12.7%, depending on the azole tested. Antifungal resistance was more common among emerging species such as those of Scedosporium and Mucorales and also among cryptic species of Aspergillus, with 40% of these isolates showing resistance to all of the antifungal compounds tested. Cryptic Aspergillus species seem to be underestimated, and their correct classification could be clinically relevant. The performance of antifungal susceptibility testing of the strains implicated in deep infections and multicentric studies is recommended to evaluate the incidence of these cryptic species in other geographic areas.     

Positions and Numbers of FKS Mutations in Candida albicans Selectively Influence In Vitro and In Vivo Susceptibilities to Echinocandin Treatment

Candidemia is the fourth most common kind of microbial bloodstream infection, with Candida albicans being the most common causative species. Echinocandins are employed as the first-line treatment for invasive candidiasis until the fungal species is determined and confirmed by clinical diagnosis. Echinocandins block the FKS glucan synthases responsible for embedding β-(1,3)-d-glucan in the cell wall. The increasing use of these drugs has led to the emergence of antifungal resistance, and elevated MICs have been associated with single-residue substitutions in specific hot spot regions of FKS1 and FKS2. Here, we show for the first time the caspofungin-mediated in vivo selection of a double mutation within one allele of the FKS1 hot spot 1 in a clinical isolate. We created a set of isogenic mutants and used a hematogenous murine model to evaluate the in vivo outcomes of echinocandin treatment. Heterozygous and homozygous double mutations significantly enhance the in vivo resistance of C. albicans compared with the resistance seen with heterozygous single mutations. The various FKS1 hot spot mutations differ in the degree of their MIC increase, substance-dependent in vivo response, and impact on virulence. Our results demonstrate that echinocandin EUCAST breakpoint definitions correlate with the in vivo response when a standard dosing regimen is used but cannot predict the in vivo response after a dose escalation. Moreover, patients colonized by a C. albicans strain with multiple mutations in FKS1 have a higher risk for therapeutic failure.     

In Vitro Susceptibilities of Candida Bloodstream Isolates to the New Triazole Antifungal Agents BMS-207147, Sch 56592, and Voriconazole

BMS-207147, Sch 56592, and voriconazole are three new investigational triazoles with broad-spectrum antifungal activity. The in vitro activities of these three agents were compared with those of itraconazole and fluconazole against 1,300 bloodstream isolates of Candida species obtained from over 50 different medical centers in the United States. The MICs of all of the antifungal drugs were determined by broth microdilution tests performed according to the National Committee for Clinical Laboratory Standards method using RPMI 1640 as a test medium. BMS-207147, Sch 56592, and voriconazole were all quite active against all Candida sp. isolates (MICs for 90% of the isolates tested [MIC₉₀s], 0.5, 1.0, and 0.5 μg/ml, respectively). Candida albicans was the most susceptible species (MIC₉₀s, 0.03, 0.06, and 0.06 μg/ml, respectively), and C. glabrata was the least susceptible (MIC₉₀s, 4.0, 4.0, and 2.0 μg/ml, respectively). BMS-207147, Sch 56592, and voriconazole were all more active than itraconazole and fluconazole against C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. There existed a clear rank order of in vitro activity of the five azoles examined in this study when they were tested versus C. glabrata: voriconazole > BMS-207147 = Sch 56592 = itraconazole > fluconazole (MIC₉₀s, 2.0, 4.0, 4.0, 4.0, and 64 μg/ml, respectively). For isolates of Candida spp. with decreased susceptibility to both itraconazole and fluconazole, the MICs of BMS-207147, Sch 56592, and voriconazole were also elevated. These results suggest that BMS-207147, Sch 56592, and voriconazole all possess promising antifungal activity and that further in vitro and in vivo investigations are warranted to establish the clinical value of this improved potency.     

In Vitro Susceptibilities of Neisseria gonorrhoeae to Fatty Acids and Monoglycerides

The susceptibility of Neisseria gonorrhoeae to several medium-chain fatty acids and their 1-monoglycerides was tested at a short inactivation time of 1 min. The results indicate that monocaprin, a monoglyceride of capric acid (10 carbon atoms, no double bonds), causes the fastest and most effective killing of all strains of N. gonorrhoeae tested.     

In Vitro and In Vivo Efficacy of Florfenicol for Treatment of Francisella asiatica Infection in Tilapia

Francisella asiatica is a recently described, Gram-negative, facultative intracellular fish pathogen, known to be the causative agent of francisellosis in warm-water fish. Francisellosis outbreaks have increased in frequency among commercial aquaculture operations and have caused severe economic losses in every case reported. The lack of effective treatments for piscine francisellosis led us to investigate the potential efficacy of florfenicol for inhibition of F. asiatica in vitro and as an oral therapeutic agent in vivo. The MIC of florfenicol for F. asiatica, as determined by the broth dilution method, was 2 μg/ml, which indicates its potential efficacy as a therapeutic agent for treatment of francisellosis. The intracellular susceptibility of the bacterium to florfenicol in tilapia head kidney-derived macrophages (THKDM) was also investigated. Addition of florfenicol to the medium at 10 μg/ml was sufficient to significantly reduce bacterial loads in the THKDM in vitro. Cytotoxicity assays done in infected THKDM also demonstrated drug efficacy in vivo, as determined by lactate dehydrogenase (LDH) release. Levels of LDH released from infected THKDM were significantly lower in macrophages treated with florfenicol (P < 0.001) than in untreated cells. In medicated-feed trials, fish were fed 15 mg of florfenicol/kg of fish body weight for 10 days, and the feeding was initiated at either 1, 3, or 6 days postchallenge. Immersion challenges resulted in 30% mean percent survival in nontreated fish, and fish receiving medicated feed administered at 1 and 3 days postinfection showed higher mean percent survival (100% and 86.7%, respectively). A significant decrease (P < 0.001) in bacterial numbers (number of CFU/g of spleen tissue) was observed in treated groups compared to nontreated infected fish at both 1 and 3 days postchallenge. There were no differences in bacterial burden in the spleens between fish treated 6 days postchallenge and untreated controls. In conclusion, if florfenicol is administered during early stages of infection, it has the potential for effectively treating piscine francisellosis, including the capacity for intracellular penetration and bacterial clearance.Members of the genus Francisella are small, pleomorphic, Gram-negative bacteria belonging to the Gammaproteobacteria (3, 7, 28). Many Francisella spp. are facultative intracellular pathogens, capable of replicating in macrophages and other various cell types in humans, rabbits, rodents, nonhuman primates, and fish. The bacteria may also exist as endosymbionts of amoebae and arthropods (1, 2, 7, 26, 31, 34). Francisella asiatica and Francisella noatunensis are two recently described members of the genus that causes piscine francisellosis in a wide variety of fish species (19).During the past 5 years F. asiatica has been implicated as the causative agent of mortality in tilapia (Oreochromis sp.) and other important warm-water species cultured in the United States (including Hawaii), Taiwan, Latin America (particularly Costa Rica), and Japan (13, 14, 15, 17, 19, 20, 29, 33). Due to its increase in incidence, high infectivity rates, and wide range of fish hosts, francisellosis is now considered one of the most important emergent diseases in aquaculture (13, 14, 15, 17, 19, 20, 29, 33). In tilapia the disease can present as an acute syndrome with few nonspecific clinical signs and high mortality rates or as a subacute to chronic syndrome with nonspecific clinical signs like anorexia, exophthalmia, and anemia. The bacterium has a high infectivity rate in tilapia fingerlings. Low numbers (1 to 10 CFU) of the bacterium injected intraperitoneally can cause colonization and significant damage to the head kidney and spleen, with a dose as low as 23 bacteria resulting in mortality (30). Macroscopic and microscopic examination often reveals enlarged internal organs containing widespread multifocal white nodules (13, 14, 15, 17, 19, 20, 29). Moreover, F. asiatica has been found to be resistant to serum killing and can penetrate, replicate, and survive in tilapia head kidney-derived macrophages (THKDM) (31).Very limited data on fish pathogen susceptibility to antibiotics have been published. Only recently have guidelines been published for broth microdilution testing of fish pathogens (8); however, methods for fastidious organisms such as F. asiatica are not included in this publication. Clinical breakpoints are not available for this class of fish pathogens either. Currently, only three antibiotics have been approved by the U.S. Food and Drug Administration for use in U.S. aquaculture: oxytetracycline (Terramycin 200 for fish; Phibro Animal Health, Fairfield, NJ), ormetoprim-sulfadimethoxine (Romet-30 type A medicated article; Pharmaq AS, Oslo, Norway), and florfenicol (Aquaflor type A medicated article; Intervet/Schering-Plough Animal Health, Roseland, NJ). Florfenicol is a fluorinated derivative of thiamphenicol that blocks the peptidyltransferase at the 50S ribosome subunit and acts against a wide variety of both Gram-positive and Gram-negative bacteria (5). As a medicated feed, florfenicol has been used to treat a wide variety of fish diseases in various warm- and cold-water cultured fish species, including Vibrio anguillarum, Aeromonas salmonicida, Streptococcus iniae, Listonella anguillarum, and Edwardsiella ictaluri, among others (9, 11, 23, 24, 27).Due to the emergent nature of francisellosis in fish and the fastidious characteristics of the bacteria, there is currently very little published data regarding antibiotic susceptibility of F. asiatica in vivo or in vitro, and at present there are no known efficacious chemotherapeutics or vaccines available (17, 20, 29, 30, 31, 32). Additionally, antimicrobial therapy in facultative intracellular bacteria is more complex than in extracellular bacteria since the efficacy of the drug depends on its ability to penetrate and accumulate within the cell, cellular metabolism, subcellular disposition, and bioavailability of the drug (25). For F. noatunensis, in vitro data were presented that indicated that strain GM2212T was resistant to trimethoprim-sulfamethoxazole, penicillin, ampicillin, cefuroxime, and erythromycin yet susceptible to ceftazidime, tetracycline, gentamicin, and ciprofloxacin (21). No further research has been published to demonstrate the potential use of any of these drugs in medicated feed for the treatment of francisellosis in fish. Moreover, at this point it is unknown if the antimicrobial susceptibilities of F. noatunensis and F. asiatica are the same.The goal of the present study was to determine the ability of florfenicol-medicated feed to control experimentally induced F. asiatica infection in tilapia. Additionally, we evaluated the capacity of florfenicol to eliminate intracellular F. asiatica from THKDM in vitro.     

Antifungal Activity of Propolis Against Yeasts Isolated From Blood Culture: In Vitro Evaluation

Background: Due to the failure of available antifungal agents in the treatment of candidemia and the toxic activities of these drugs, a lot of researches are being conducted to develop new nontoxic and effective antifungal agents for optimal control of fungal pathogens. The aim of this study is to evaluate the in vitro antifungal activity of propolis against yeasts isolated from the blood cultures of intensive care unit patients. Methods: Seventy‐six strains were included in this study. The in vitro antifungal activity of propolis, fluconazole (FLU), and itraconazole (ITR) was investigated by the microdilution broth methods (CLSI guidelines M27‐A3 for yeast). The propolis sample was collected from Kayseri, Turkey. Results: Of the 76 isolates, 33 were identified as Candida albicans while 37 were C. parapsilosis, three were C. tropicalis, and three were identified as C. glabrata. The geometric mean range for MIC (μg/ml) with regard to all isolates was 0.077 to 3 μg/ml for FLU and ITR, and 0.375 to 0.70 μg/ml for propolis. It was shown that propolis had significant antifungal activity against all Candida strains and the MIC range of propolis was determined as 0185 to 3 μg/ml. Conclusion: This study demonstrated that propolis had significant antifungal activity against yeasts isolated from blood culture compared with FLU and ITR. The propolis MIC in azole‐resistant strains such as C. glabrata was found lower than the FLU MIC.     

Antifungal Activity of Colistin against Mucorales Species In Vitro and in a Murine Model of Rhizopus oryzae Pulmonary Infection

In immunosuppressed hosts, mucormycosis is a life-threatening infection with few treatment options. We studied the activity of colistin (polymyxin E) against Mucorales species in vitro and in a murine model of pulmonary Rhizopus oryzae infection. Colistin exhibited fungicidal activity in vitro against Mucorales spores and mycelia. At the colistin MIC, initial R. oryzae hyphal damage was followed by rapid regrowth; however, regrowth was prevented by combining colistin with a subinhibitory concentration of amphotericin B. Using electron microscopy and FM4-64 staining, we demonstrated that colistin disrupts R. oryzae cytoplasmic and vacuolar membranes, resulting in the leakage of intracellular contents. The prophylactic intranasal treatment of immunosuppressed mice with colistimethate significantly reduced the mortality rate and pulmonary fungal burden resulting from inhalational challenge with R. oryzae spores, whereas intraperitoneal colistimethate treatment had no effect. We conclude that colistin has modest in vitro and in vivo fungicidal activity against Mucorales spp. Further studies are warranted to assess the use of this drug in the prevention and treatment of mucormycosis.During the past decade, pulmonary mucormycosis (PMM) has emerged as an important life-threatening opportunistic infection in severely immunocompromised patients, such as those with prolonged neutropenia and recipients of allogeneic hematopoietic stem cell transplantation (12, 21). In contrast to diabetic patients who develop rhinocerebral mucormycosis, surgical debridement is impractical in patients with PMM, and the correction of the underlying immunosuppression is challenging (12, 21); therefore, antifungal chemotherapy remains the chief therapeutic intervention for many patients with PMM. Unfortunately, Mucorales species are inherently resistant to most clinically available antifungal agents except amphotericin B, which is associated with renal and infusional toxicity, and posaconazole, which is available only as an oral formulation and has variable absorption from the gastrointestinal tract (12, 21). Hence, there is an urgent need for novel drugs with activity against Mucorales spp.Polymyxins are cyclic, positively charged peptide antibiotics produced by the bacterium Bacillus polymyxa (16). Polymyxins bind to and disrupt the anionic outer cell membrane of Gram-negative bacteria and have a neutralizing effect on bacterial lipopolysaccharides (16). Interestingly, polymyxin B also has been shown to disrupt the cytoplasmic and vacuolar membranes of the model yeast Saccharomyces cerevisiae (23), suggesting that its antimicrobial activity extends to eukaryotic organisms. We studied the activity of polymyxin E (colistin) against Mucorales spp. in vitro and in a murine model of PMM.     

In Vitro Interactions between Target of Rapamycin Kinase Inhibitor and Antifungal Agents against Aspergillus Species

In vitro interactions of INK128, a target of rapamycin (TOR) kinase inhibitor, and antifungals, including itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin, against Aspergillus spp. were assessed with the broth microdilution checkerboard technique. Our results suggested synergistic effects between INK128 and all azoles tested, against multiple Aspergillus fumigatus and Aspergillus flavus isolates. However, no synergistic effects were observed when INK128 was combined with amphotericin B or caspofungin. No antagonism was observed for any combination.     

Evaluation of VT-1161 for Treatment of Coccidioidomycosis in Murine Infection Models

Coccidioidomycosis, or valley fever, is a growing health concern endemic to the southwestern United States. Safer, more effective, and more easily administered drugs are needed especially for severe, chronic, or unresponsive infections. The novel fungal CYP51 inhibitor VT-1161 demonstrated in vitro antifungal activity, with MIC₅₀ and MIC₉₀ values of 1 and 2 μg/ml, respectively, against 52 Coccidioides clinical isolates. In the initial animal study, oral doses of 10 and 50 mg/kg VT-1161 significantly reduced fungal burdens and increased survival time in a lethal respiratory model in comparison with treatment with a placebo (P < 0.001). Oral doses of 25 and 50 mg/kg VT-1161 were similarly efficacious in the murine central nervous system (CNS) model compared to placebo treatment (P < 0.001). All comparisons with the positive-control drug, fluconazole at 50 mg/kg per day, demonstrated either statistical equivalence or superiority of VT-1161. VT-1161 treatment also prevented dissemination of infection from the original inoculation site to a greater extent than fluconazole. Many of these in vivo results can be explained by the long half-life of VT-1161 leading to sustained high plasma levels. Thus, the efficacy and pharmacokinetics of VT-1161 are attractive characteristics for long-term treatment of this serious fungal infection.     

In Vitro Antifungal Susceptibilities and Amplified Fragment Length Polymorphism Genotyping of a Worldwide Collection of 350 Clinical,Veterinary, and Environmental Cryptococcus gattii Isolates

The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC₉₀ (0.125 μg/ml) for all C. gattii isolates, followed by a 1 log₂ dilution step increase (MIC₉₀, 0.25 μg/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC₉₀ of 0.5 μg/ml, but fluconazole and flucytosine had relatively high MIC₉₀s of 8 μg/ml.The basidiomycetous yeast Cryptococcus gattii is responsible for life-threatening invasive disease in apparently healthy humans and animals (7, 19). A typical C. gattii infection is acquired through the respiratory tract, from which it can further disseminate to the central nervous system, resulting in fatal meningitis (7, 19, 32). Cryptococcosis caused by the primary pathogenic yeast C. gattii was, until a decade ago, a rarely encountered infection outside tropical and subtropical regions (17, 26, 27). However, this changed due to an unprecedented outbreak that emerged in the temperate climate of Vancouver Island (British Columbia, Canada) that subsequently expanded farther into the Pacific Northwest (1, 8, 10, 16). Its sibling species, Cryptococcus neoformans, differs ecologically and epidemiologically from C. gattii since it occurs on a global scale and is linked with disease occurring in immunocompromised individuals, such as HIV-positive patients and transplant patients who receive immune-suppressive medicines (7, 10, 18, 19, 31).Cryptococcus gattii can be discerned from C. neoformans using a wide range of microbiological and molecular techniques (7, 20). A convenient method is the use of canavanine-glycine-bromothymol blue (CGB) medium, which allows C. gattii but not C. neoformans to grow and which changes the pH indicator in the medium from green-yellowish to blue (18). With the increasing use of molecular techniques, such as PCR fingerprinting, restriction fragment length polymorphism (RFLP) analysis of the PLB1 and URA5 loci, and amplified fragment length polymorphism (AFLP) fingerprint analysis, as well as several multilocus sequence typing (MLST) approaches, it became clear that C. gattii could be divided into five distinct genotypes, named AFLP4/VGI, AFLP5/VGIII, AFLP6/VGII, AFLP7/VGIV, and AFLP10 (the last one of which is a recently observed novel genotype) (2, 6, 7, 13, 16, 20, 21, 23). Until recently, a serotype agglutination assay was widely used to distinguish C. neoformans (serotypes A and D) from C. gattii (serotypes B and C) (7, 27). In general, serotype B strains are found in each of the five C. gattii AFLP genotypes, but it seems that C. gattii serotype C strains are restricted to genotypes AFLP5/VGIII and AFLP7/VGIV (2, 6, 16, 21, 27).In addition, it was found that C. gattii and C. neoformans can form interspecies hybrids, named genotype AFLP8 (C. neoformans var. neoformans AFLP2/VNIII serotype D × C. gattii AFLP4/VGI serotype B) and AFLP9 (C. neoformans var. grubii AFLP1/VNI serotype A × C. gattii AFLP4/VGI serotype B). These interspecies hybrids have, until now, been isolated only from clinical samples, and they might have a higher virulence potential than regular C. gattii or C. neoformans isolates (4, 5; F. Hagen, K. Tintelnot, and T. Boekhout, unpublished data).Treatment of cryptococcosis depends on, besides the immune status of the patient, the severity and localization of the infection (11). Severe cases of cryptococcosis in immunocompetent and -compromised patients are treated according to the guidelines of the Infectious Diseases Society of America, according to which treatment consists of an induction therapy for 2 weeks with a combination of amphotericin B and flucytosine, followed by a 10-week consolidation therapy with fluconazole (11, 24).Cryptococcus neoformans has been extensively studied for its in vitro susceptibility to a wide variety of antifungal compounds, including the new triazoles posaconazole, voriconazole, ravuconazole, and isavuconazole (12, 14, 28, 29, 33). Despite the ongoing C. gattii outbreak, only a few studies using relatively small sets of C. gattii isolates have been performed to investigate their in vitro susceptibilities to amphotericin B, flucytosine, fluconazole, and the new triazole antifungals (12, 15, 28-30). A few studies divided the C. gattii isolates into groups according to their serotype or genotype (15, 29).Therefore, we studied the in vitro susceptibilities of each of the C. gattii genotypes from a large worldwide collection, subdivided by AFLP genotyping, to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and the new experimental broad-spectrum antifungal triazole isavuconazole.     

In Vitro Antifungal Susceptibility of Candida glabrata to Caspofungin and the Presence of FKS Mutations Correlate with Treatment Response in an Immunocompromised Murine Model of Invasive Infection

It has been argued that the in vitro activity of caspofungin (CSP) is not a good predictor of the outcome of echinocandin treatment in vivo. We evaluated the in vitro activity of CSP and the presence of FKS mutations in the hot spot 1 (HS1) region of the FKS1 and FKS2 genes in 17 Candida glabrata strains with a wide range of MICs. The efficacy of CSP against systemic infections from each of the 17 strains was evaluated in a murine model. No HS1 mutations were found in the eight strains showing MICs for CSP of ≤0.5 μg/ml, but they were present in eight of the nine strains with MICs of ≥1 μg/ml, i.e., three in the FKS1 gene and five in the FKS2 gene. CSP was effective for treating mice infected with strains with MICs of ≤0.5 μg/ml, showed variable efficacy in animals challenged with strains with MICs of 1 μg/ml, and did not work in those with strains with MICs of >1 μg/ml. In addition, mutations, including one reported for the first time, were found outside the HS1 region in the FKS2 gene of six strains with different MICs, but their presence did not influence drug efficacy. The in vitro activity of CSP was compared with that of another echinocandin, anidulafungin, suggesting that the MICs of both drugs, as well as mutations in the HS1 regions of the FKS1 and FKS2 genes, are predictive of outcome.     

In Vitro Profiling of Pramiconazole and In Vivo Evaluation in Microsporum canis Dermatitis and Candida albicans Vaginitis Laboratory Models

The triazole antifungal pramiconazole (Stiefel, a GSK company) was compared with itraconazole, miconazole, and terbinafine in vitro and in vivo. Potent in vitro activities against Candida spp. (50% inhibitory concentration [IC₅₀], 0.04 to 1.83 μM) and Microsporum and Trichophyton spp. (IC₅₀, 0.15 to 1.34 μM) were obtained but not, however, against other filamentous molds and zygomycetes. In the M. canis guinea pig model and C. albicans vulvovaginitis rat model, pramiconazole was superior to the reference compounds after oral and topical administration.Although considerable research is invested in finding novel strategies for the treatment of fatal invasive mycoses (6), nonfatal superficial mycoses believed to infect about 25% of the world population should not be overlooked (1). The most widespread dermatomycoses are caused by Trichophyton, Epidermophyton, and Microsporum species. Treatment is oral or topical with the allylamine terbinafine or any of the azoles (4, 13, 14). Yeasts also cause superficial infections of skin and mucous membranes, whereby vulvovaginal candidiasis (VVC) affects at least 75% of all women at least once in their lives (15, 21). Standard therapy involves intravaginal application of clotrimazole or miconazole or oral treatment with fluconazole or itraconazole (15). Although current treatment options may suffice, new antifungals would still be acceptable to improve treatment compliance or reduce adverse effects and drug interactions. The triazole pramiconazole shows good in vitro and clinical activity against dermatophytes and yeasts (12, 16, 17) and Malassezia infections (10, 11, 19). Laboratory data always refer to oral treatment of mice and guinea pigs (16, 17); however, no data on topical application are available. No data have yet been published on pramiconazole in VVC in comparison with reference drugs, although it is in clinical de-velopment for these indications (8, 9). The specific aims of this laboratory study were (i) to perform an in vitro profiling of pramiconazole and (ii) to evaluate oral and topical treatment schemes against Microsporum canis in guinea pigs and Candida albicans VVC in rats.Miconazole (MC), itraconazole (ITC), and terbinafine (TRB) were purchased from Sigma, while pramiconazole (PRC) was provided by Stiefel-GSK. The fungal isolates were obtained from the Scientific Institute of Public Health (IHEM, Brussels, Belgium) and cultivated on Sabouraud dextrose agar (SDA) (Oxoid). For all species, a stock of 5 × 10⁶ CFU/ml was prepared in RPMI-MOPS medium with 10% glycerol and stored in liquid nitrogen for later use in all in vitro tests. Fresh inocula were used for animal infections. The in vitro susceptibility screens were performed as previously described (7). Briefly, 10 μl of prediluted compound solution was spotted onto 96-well plates (U-bottom; Greiner Bio-One) with 64 μM as the highest concentration; 10³ CFU in 200 μl RPMI-MOPS was added to each well. After incubation, growth inhibition was measured after adding 10 μl/well 0.005% (wt/vol) resazurin (Sigma), allowing fluorimetric reading (λ_ex, 550 nm; λ_em, 590 nm) (23). Activity is expressed as IC₅₀, i.e., the concentration that inhibits growth for 50% compared to nontreated controls. Cytotoxicity was simultaneously tested on human lung fibroblasts (MRC-5_SV40) (Invitrogen). Five independent replicates were performed for each observation.The in vitro IC₅₀s for reference drugs were comparable to the ranges in literature (2, 5, 20), and available data on pramiconazole were also confirmed (16, 17) (Table ​(Table1).1). TRB performs marginally better against dermatophytes. Except for Trichophyton quinckeanum, PRC activity remained below 0.5 μM. Against Candida spp., activities remained below 1 μM, except for C. albicans B2630. PRC failed to show activity (IC₅₀, >64 μM) against the other filamentous molds and zygomycetes (data not shown).

TABLE 1.

Cytotoxicity (CC₅₀) and activity (IC₅₀) against four dermatophyte and four Candida species