Development of a multi-year white-nose syndrome mitigation strategy using antifungal volatile organic compounds.

Pseudogymnoascus destructans is a fungal pathogen responsible for a deadly disease among North American bats known as white-nose syndrome (WNS). Since detection of WNS in the United States in 2006, its rapid spread and high mortality has challenged development of treatment and prevention methods, a significant objective for wildlife management agencies. In an effort to mitigate precipitous declines in bat populations due to WNS, we have developed and implemented a multi-year mitigation strategy at Black Diamond Tunnel (BDT), Georgia, singly known as one of the most substantial winter colony sites for tricolored bats (Perimyotis subflavus), with pre-WNS abundance exceeding 5000 individuals. Our mitigation approach involved in situ treatment of bats at the colony level through aerosol distribution of antifungal volatile organic compounds (VOCs) that demonstrated an in vitro ability to inhibit P. destructans conidia germination and mycelial growth through contact-independent exposure. The VOCs evaluated have been identified from microbes inhabiting naturally-occurring fungistatic soils and endophytic fungi. These VOCs are of low toxicity to mammals and have been observed to elicit antagonism of P. destructans at low gaseous concentrations. Cumulatively, our observations resolved no detrimental impact on bat behavior or health, yet indicated a potential for attenuation of WNS related declines at BDT and demonstrated the feasibility of this novel disease management approach.

Antimicrobial Activity of Essential Oils Against the Fungal Pathogens Ascosphaera apis and Pseudogymnoascus destructans.

Fungal pathogens are a growing worldwide concern. Declines in a number of economically and agriculturally important plant and animal species pose a significant threat to both biodiversity and food security. Although many effective antifungal agents have been identified, their toxicity often precludes their use with food products or sensitive animal species. This has prompted the exploration of natural products as effective treatment compounds. In the present study, several essential oils were tested for their capacity to limit the growth of the fungal pathogens Ascosphaera apis and Pseudogymnoascus destructans, the causative agents of chalkbrood disease among honey bee larvae and white-nose syndrome among bats, respectively. Essential oils of cinnamon bark, citronella, lemongrass, and orange were exposed to A. apis in contact-dependent oil-agar suspensions as well as in contact-independent shared airspaces. Essential oils of cinnamon bark, citronella, and lemongrass were exposed to P. destructans in contact-dependent oil-agar suspensions. All compounds were found to significantly inhibit mycelial growth at low concentrations, suggesting the potential for these natural products to be used for controlling these and other select fungal pathogens.

A preliminary report on the contact-independent antagonism of Pseudogymnoascus destructans by Rhodococcus rhodochrous strain DAP96253.

BACKGROUND: The recently-identified causative agent of White-Nose Syndrome (WNS), Pseudogymnoascus destructans, has been responsible for the mortality of an estimated 5.5 million North American bats since its emergence in 2006. A primary focus of the National Response Plan, established by multiple state, federal and tribal agencies in 2011, was the identification of biological control options for WNS. In an effort to identify potential biological control options for WNS, multiply induced cells of Rhodococcus rhodochrous strain DAP96253 was screened for anti-P. destructans activity. RESULTS: Conidia and mycelial plugs of P. destructans were exposed to induced R. rhodochrous in a closed air-space at 15°C, 7°C and 4°C and were evaluated for contact-independent inhibition of conidia germination and mycelial extension with positive results. Additionally, in situ application methods for induced R. rhodochrous, such as fixed-cell catalyst and fermentation cell-paste in non-growth conditions, were screened with positive results. R. rhodochrous was assayed for ex vivo activity via exposure to bat tissue explants inoculated with P. destructans conidia. Induced R. rhodochrous completely inhibited growth from conidia at 15°C and had a strong fungistatic effect at 4°C. Induced R. rhodochrous inhibited P. destructans growth from conidia when cultured in a shared air-space with bat tissue explants inoculated with P. destructans conidia. CONCLUSION: The identification of inducible biological agents with contact-independent anti- P. destructans activity is a major milestone in the development of viable biological control options for in situ application and provides the first example of contact-independent antagonism of this devastating wildlife pathogen.

Inhibition of Pseudogymnoascus destructans growth from conidia and mycelial extension by bacterially produced volatile organic compounds.

The recently identified causative agent of white-nose syndrome (WNS), Pseudogymnoascus destructans, has been implicated in the mortality of an estimated 5.5 million North American bats since its initial documentation in 2006 (Frick et al. in Science 329:679-682, 2010). In an effort to identify potential biological and chemical control options for WNS, 6 previously described bacterially produced volatile organic compounds (VOCs) were screened for anti-P. destructans activity. The compounds include decanal; 2-ethyl-1-hexanol; nonanal; benzothiazole; benzaldehyde; andN,N-dimethyloctylamine. P. destructans conidia and mycelial plugs were exposed to the VOCs in a closed air space at 15 and 4 °C and then evaluated for growth inhibition. All VOCs inhibited growth from conidia as well as inhibiting radial mycelial extension, with the greatest effect at 4 °C. Studies of the ecology of fungistatic soils and the natural abundance of the fungistatic VOCs present in these environments suggest a synergistic activity of select VOCs may occur. The evaluation of formulations of two or three VOCs at equivalent concentrations was supportive of synergistic activity in several cases. The identification of bacterially produced VOCs with anti-P. destructans activity indicates disease-suppressive and fungistatic soils as a potentially significant reservoir of biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253.

Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202 units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55 °C (45 and 23 %, respectively) than cells grown in the presence of glucose or fructose (19 and 10 %, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55 °C, as cells grown in the presence of glucose retained 40 % NHase activity as opposed to 19 % without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and stability. Cell envelope components such as mycolic acids are also influenced by sugars and inducers such as cobalt and urea. This is information that can be useful when implementing rhodococcal catalysts in industrial applications.

Recurrent Aspergillus contamination in a biomedical research facility: a case study.

Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.

In vitro interactions of Fusarium and Acanthamoeba with drying residues of multipurpose contact lens solutions.

PURPOSE: To examine in vitro effects of evaporation and drying of multipurpose contact lens solutions on survival of Fusarium and Acanthamoeba. METHODS: Conidia of representative Fusarium from the 2004-2006 keratitis outbreak and trophozoites of Acanthamoeba castellanii were inoculated into commercially available multipurpose contact lens care solutions. These solutions were inoculated with 10(2)-10(6) microbial propagules/mL and were evaporated for at least 24 hours. After drying, nutrient media for recovery of surviving organisms were added to the residues formed in the lids of 38 mm polystyrene Petri dishes. General morphologic patterns of the solution residuals and the distribution and morphologies of the microorganisms were recorded with microscopic imaging. RESULTS: Various multipurpose contact lens disinfection solutions formed distinctive dried residual patterns. Both Fusarium and Acanthamoeba at concentrations tested above 10(3) per mL of disinfection solution were recovered from dried films with replicate testing. Mature cysts of Acanthamoeba not evident in the inocula were observed in sparse numbers in all dried solutions except one (Complete Moisture Plus; Advanced Medical Optics) and control salines where precysts and mature cysts were common. Both fusaria and amoeba tended to be observed in discrete regions of the dried residues. CONCLUSIONS: Regions of drying films of multipurpose contact lens disinfection solutions on contact lens cases may induce and harbor dormant-resistant stages of Fusarium and Acanthamoeba. It is hypothesized that the evaporation and drying of multipurpose contact lens disinfection solutions may have been an added risk factor for case contamination among Fusarium and Acanthamoeba keratitis patients. The need for frequent replacement of contact lens cases is enforced.

Relative in vitro rates of attachment and penetration of hydrogel soft contact lenses by haplotypes of fusarium.

PURPOSE: To investigate the relative abilities of different haplotypes of the Fusarium solani (FSSC)-Fusarium oxysporum (FOSC) complexes to attach to and invade hydrogel contact lenses. METHODS: Silicone hydrogel and traditional hydroxyethylmethacrylate soft contact lenses were exposed to conidia [10 ml in phosphate-buffered saline (PBS)] of different haplotypes of fusaria associated with the Fusarium keratitis outbreak of 2004-2006. Select lenses and fungi were examined under conditions of organic enrichment. The lenses were incubated with shaking at ambient temperatures, then examined microscopically for the presence of penetration pegs (PPs). RESULTS: Attachment to and penetration of balafilcon A lenses in PBS within 96 hours were observed with representative isolates of FSSC 1-a, 1-b, and 2-d. Densities and coiled morphology of the PPs were similar. Eight of 8 FOSC failed to attach and form PP in PBS without prior sorption of organics by the lens. Generally, FSSC 1 isolates showed more rapid development of PP. Representatives of all haplotypes, including FSSC 2-c (ATCC 36031, a standard challenge strain), showed at least sparse attachment and penetration of the balafilcon A lens and, to a lesser degree, the lotrafilcon A lens. The development of PP in etafilcon A and galyfilcon A lenses required extended incubations (>21 days) relative to balafilcon A lenses. CONCLUSIONS: Attachment to and penetration of unworn hydrogel soft contact lenses by Fusarium varied with the strain and lens type rather than with the clinical, environmental, or geographic source of the isolates. Without organic enrichment of the lenses, penetration was more rapid and extensive by representatives of FSSC 1. Penetration was slow and less extensive under these conditions with FOSC and FSSC 2-c and 3. Organic enrichment of the lenses typically favored development of PP by the FOSC. Attachment and penetration of lenses occurred sooner and to a greater extent with surface-treated silicone hydrogel lenses than with the hydroxyethylmethacrylate lens.

Fusarium keratitis and contact lens wear: facts and speculations.

Over the past several decades mycotic keratitis has been considered a rare sequel to hydrogel contact lens wear. In 2005--2006 an upswing in the incidence of Fusarium keratitis was associated with a disproportionate use of one multipurpose contact lens solution (MPS, ReNu with MoistureLoc, Bausch & Lomb, Rochester, NY). The MPS, as manufactured and marketed, was sterile and met regulatory guidelines for antimicrobial activity. A multivariant interaction of poor hygienic practices and the contact lens paraphernalia were associated with a mostly selective contamination in or on the lens storage case by members of the F. solani/F. oxysporum species complexes from the environment of the user. A decline of the anti-fusaria properties of the MPS in the lens case appeared related to its dissociation from drying, or dilution and the potential for sorption of antimicrobial solution components (e.g., alexidine) to various hydrogel lenses. These factors and capacities of the fusaria for rapid amplification by microcycle conidiation, production of dormant resistant cells, and potential for attachment and penetration of hydrogel lenses, were linked to the occasional selective fungal survival and growth during storage of the lens in MPS. Lack of a manual rubbing-cleaning step in the MPS disinfection process was considered a risk factor for keratitis.

Differences among strains of the Fusarium oxysporum-F. solani complexes in their penetration of hydrogel contact lenses and subsequent susceptibility to multipurpose contact lens disinfection solutions.

PURPOSE: To examine in vitro conditions for attachment and penetration of silicone hydrogel (SH) lenses by clinical isolates of the Fusarium oxysporum-F. solani complexes and the relative susceptibilities of the fusaria in the lens matrices to multipurpose contact lens solutions (MPSs). METHODS: SH soft contact lenses were soaked in Sabouraud dextrose broth (SAB) for 2 hours and transferred to 3.0 mL of phosphate-buffered saline (PBS). The lenses were inoculated with representative isolates of both complexes and incubated on a shaker at ambient temperature. Lenses were examined daily by light microscopy before and after rinsing and rubbing in MPS. Selected lenses penetrated by fungi were rinsed and rubbed with MPS and held in MPS for 6 hours, transferred to PBS with 0.03% SAB, and examined daily. RESULTS: The degree and rate of lens penetration of contact lenses by isolates of the F. oxysporum-F. solani complexes varied with lens type and the strain. Isolates obtained from patients with Fusarium keratitis produced on and within lenses chlamydospores that seemed similar to those observed in lenses actually worn by patients when they developed Fusarium keratitis. Clinical isolates showed greater capacities than those of a standard test strain to penetrate lenses and to survive exposures to various MPSs. In general, isolates of F. solani were more readily removed from lenses by rubbing than were isolates of F. oxysporum. CONCLUSIONS: The ability of Fusarium spp. to attach to and penetrate SH lenses in vitro varies with the lens type and strain, and this ability may make infectious keratitis more likely. We recommend the incorporation of a rubbing step in the MPS disinfection of hydrogel lenses to reduce the risk of fungal keratitis.

Attachment to and penetration of conventional and silicone hydrogel contact lenses by Fusarium solani and Ulocladium sp. in vitro.

PURPOSE: To analyze the relative capacities of Fusarium solani and Ulocladium sp. to attach to and penetrate silicone hydrogel soft contact lenses. METHODS: Representative silicone hydrogel (SH, siloxy complexes) and conventional [hydroxyethylmethylacrylate (HEMA)] soft contact lenses were exposed to suspensions of F. solani and Ulocladium sp. in vitro (10 conidia/mL in phosphate-buffered saline). The lenses were incubated with shaking at ambient temperatures and examined after rinsing in a multipurpose contact lens solution (MPS) by light and scanning electron microscopy. RESULTS: Isolates of both genera firmly attached to and penetrated both lens types, but Ulocladium sp. did so in greater density and more rapidly than F. solani. The extent of firm attachment and time needed for penetration into the lenses varied with strain and substratum, particularly with the isolates of F. solani. Morphologic characteristics (eg, penetration pegs, microcycle conidiation, and chlamydospores) of F. solani in the SH and HEMA lenses were similar to those observed in several lenses from patients with ReNu with MoistureLoc (RML)-associated Fusarium keratitis. CONCLUSIONS: To our knowledge, this is the first report that F. solani produces coiled penetration pegs in the matrices of SH hydrophilic soft contact lenses similar in morphology to those found in HEMA lenses. F. solani attaches firmly to SH lenses and rarely penetrates the lens matrix, but viable fungal propagules may remain on the lens after vigorous rinsing with MPS. Failure to use a manual cleaning-disinfection procedure may help to explain the increased incidence of Fusarium keratitis associated with contact lens wear.

Growth and survival of Fusarium solani-F. oxysporum complex on stressed multipurpose contact lens care solution films on plastic surfaces in situ and in vitro.

PURPOSE: To analyze factors implicating the association of ReNu with MoistureLoc (ReNu ML) multipurpose contact lens solution (MPS) with the increased incidence of Fusarium keratitis. METHODS: Used contact lens cases with and without contact lenses and MPS containers were collected from patients with confirmed or possible Fusarium keratitis. Direct microscopy including transparent adhesive tape preparations and swab cultures were used to determine fungal colonization. Survival and growth of selected isolates of Fusarium spp. in drying MPS on plastic surfaces were determined by microscopy and recoverable colony counts on enriched agar. RESULTS: Discrete regions of fungal colonization, including occasional microcycle conidiation and chlamydospore formation, were observed on the surfaces of contact lens cases and, less often, on solution containers that had been used by patients with Fusarium keratitis associated with the use of ReNu ML. Isolates provisionally grouped with the F. solani-F. oxysporum complex were inhibited by fresh MPS in original solution containers and contact lens cases, but survived in stressed (drying) films of MPS, particularly ReNu ML. These in vitro test results were similar to the direct in situ observations of the materials from patients. CONCLUSIONS: Selective, rapid growth and survival of cells of the F. solani-F. oxysporum complex on plastic surfaces, particularly of contact lens cases with stressed ReNu ML films, may explain, in part, the recent Fusarium keratitis outbreak.

In vitro effects of Ag+ on planktonic and adhered cells of fluconazole-resistant and susceptible strains of Candida albicans, C. glabrata and C. krusei.

Planktonic and attached cells of strains of Candida albicans, C. glabrata and C. krusei with varied susceptibilities to fluconazole (FCZ) were compared for their relative susceptibilities to Ag+ via cell recovery and flow cytometric analyses. All strains lost membrane permeability and were non-recoverable upon culture after 1h exposure in morpholino-ethanesulfonic acid (MES) buffer fortified with

In vitro deposition of lysozyme on etafilcon A and balafilcon A hydrogel contact lenses: effects on adhesion and survival of Pseudomonas aeruginosa and Staphylococcus aureus.

PURPOSE: To compare lysozyme adsorption and absorption and bacterial adhesion interactions on conventional (etafilcon A) and silicone (balafilcon A) hydrogel contact lenses. METHOD: Lysozyme concentrations and activities associated with the lenses were determined after solvent extraction (trifluoroacetic acid/acetonitrile) and directly on the lenses without extraction with micrococcal- and micro-bicinchoninic acid (BCA) assays. Cells of bacteria with radiolabeled leucine and a cell recovery procedure were used in determinations of bacterial adhesion to lenses. RESULTS: Lysozyme was adsorbed and absorbed to the conventional etafilcon A lens at about a 10-fold greater concentration than to the balafilcon A silicone hydrogel lens. Enzyme activities on the surfaces of both lenses were similar but replenished after saline extraction only with the etafilcon A lens. Lysozyme on the lens surface showed significant lysis of Micrococcus luteus but had a negligible effect on the adhesion and survival of Staphylococcus aureus. Lysozyme did not appear to affect the survival of Pseudomonas aeruginosa on lenses. CONCLUSION: In vitro experiments show that concentrations of active lysozyme on the surface of the etafilcon A lens, unlike the balafilcon A lens which showed negligible absorption, may be sustained from the lens matrix. Lysozyme deposited on hydrogel lenses had marked activity against M. luteus but relatively minor effects on the primary adhesion of P. aeruginosa and S. aureus.

Mold colonization during use of preservative-treated and untreated air filters, including HEPA filters from hospitals and commercial locations over an 8-year period (1996-2003).

High efficiency particulate arrestance (HEPA; 99.97% efficient at 0.3 microm) filters, filters with ASHRAE particulate arrestance rating of 90-95% at 1 mum (90-95% filters), and lower efficiency cellulosic-polyester filters from air conditioning systems in hospitals and commercial buildings were removed from the systems and examined microscopically for mold colonization. Cellulosic-type filters from systems with water entrainment problems typically were colonized, or became colonized upon incubation in moisture chambers. Species of Acremonium, Aspergillus, and Cladosporium were most common. With air filters of all types, treatment of filter media with an antimicrobial preservative tended to reduce or delay colonization. Mold colonization of HEPA and 90-95% filters was observed most often on the load surfaces, but two untreated HEPA filters were permeated with fungi, one with Aspergillus flavus, the other with Cladosporium sp. Air filters in heating, ventilating, and air conditioning (HVAC) systems, particularly those with chronic or periodic exposure to moisture, may serve as point sources for indoor molds.

Biologically active traditional medicinal herbs from Balochistan, Pakistan.

The biological activities of the following four important medicinal plants of Balochistan, Pakistan were checked; Grewia erythraea Schwein f. (Tiliaceae), Hymenocrater sessilifolius Fisch. and C.A. Mey (Lamiaceae), Vincetoxicum stocksii Ali and Khatoon (Asclepiadaceae) and Zygophyllum fabago L. (Zygophyllaceae). The methanolic extracts were fractionated into hexane, ethyl acetate, chloroform, butanol and water. The antifungal and antibacterial activities of these plants were determined against 12 fungal and 12 bacterial strains by agar well diffusion and disk diffusion assays. The extract of Zygophyllum fabago was found to be highly effective against Candida albicans and Escherichia coli. The extract of Vincetoxicum stocksii was also found to be significantly active against Candida albicans, Bacillus subtilis and Bacillus cereus. Extracts of Hymenocrater sessilifolius and Grewia erythraea showed good activity only against Pseudomonas aeruginosa.

Adherence of Candida albicans to silicone induces immediate enhanced tolerance to fluconazole.

Wild-type and efflux pump-deficient cells of Candida albicans adhering to silicone were compared with planktonic cells by flow cytometry for their relative resistance to fluconazole (FCZ). Flow cytometry data on cells carrying a fusion of green fluorescent protein to efflux pump promoters confirmed that enhanced tolerance of attached cells to FCZ was due in part to increased expression of CaMDR1 and CDR1 promoters. Within 2 h of their attachment to silicone, the adherent cells demonstrated levels of FCZ tolerance shown by cells from 24-h biofilms. Following their mechanical detachment, this subset of cells retained a four- to eightfold increase in tolerance compared with the tolerance of planktonic cells for at least two generations. Enhanced efflux pump tolerance to FCZ appeared to be induced within the initial 15 min of attachment in a subset of cells that were firmly attached to the substrata.

Phagocytosis affects biguanide sensitivity of Acanthamoeba spp.

The incidence of Acanthamoeba keratitis, a disease associated with contact lens wear, has been in apparent decline with the advent of multipurpose contact lens solutions. The concentrations of the biguanides chlorhexidine digluconate (CHX) and particularly polyhexamethylene biguanide (PHMB) included in multipurpose solutions (MPSs) are sublethal for amoebae. We evaluated by flow cytometry the effects of these two biguanides on phagocytosis of particles and the survival of trophozoites of Acanthamoeba castellanii and A. polyphaga. Trophozoites of A. castellanii and A. polyphaga (10(6)/ml) were exposed to solutions of 5 and 50 microg of PHMB and CHX per ml in the presence and absence of particles (i.e., heat-killed yeasts and bacteria and latex beads). In addition, trophozoites were exposed to particles treated with these concentrations of the two biguanides. In the absence of particles, trophozoites of A. polyphaga appeared to be more resistant to the biguanides than those of A. castellanii. In the presence of particles, the rates of survival of both species were decreased. In most instances, particles treated with sublethal concentrations of both biguanides that were adsorbed onto the particles reduced the incidence of phagocytosis. Particles present in MPSs in contact lens cases may be involved in the decreased incidence of Acanthamoeba keratitis.