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.

Antifungal Activity of Select Essential Oils against Candida auris and Their Interactions with Antifungal Drugs.

Candida auris is an emerging fungal pathogen that commonly causes nosocomial blood infections in the immunocompromised. Several factors make this pathogen a global threat, including its misidentification as closely related species, its ability to survive for weeks on fomites, and its resistance to commonly prescribed antifungal drugs, sometimes to all three classes of systemic antifungal drugs. These factors demonstrate a need for the development of novel therapeutic approaches to combat this pathogen. In the present study, the antifungal activities of 21 essential oils were tested against C. auris. Several essential oils were observed to inhibit the growth and kill C. auris, Candida lusitaniae, and Saccharomyces cerevisiae when in direct contact and at concentrations considered safe for topical use. The most effective essential oils were those extracted from lemongrass, clove bud, and cinnamon bark. These essential oils also elicited antifungal activity in gaseous form. The efficacies of formulations comprised of these three essential oils in combination with fluconazole, amphotericin B, flucytosine, and micafungin were explored. While synergism was neither observed with cinnamon bark oil nor any of the antifungal drugs, lemongrass oil displayed synergistic, additive, and indifferent interactions with select drugs. Formulations of clove bud oil with amphotericin B resulted in antagonistic interactions but displayed synergistic interactions with fluconazole and flucytosine. These essential oils and their combinations with antifungal drugs may provide useful options for surface disinfection, skin sanitization, and possibly even the treatment of Candida infections.

ROADWAY-ASSOCIATED CULVERTS MAY SERVE AS A TRANSMISSION CORRIDOR FOR PSEUDOGYMNOASCUS DESTRUCTANS AND WHITE-NOSE SYNDROME IN THE COASTAL PLAINS AND COASTAL REGION OF GEORGIA, USA.

White-nose syndrome (WNS) is a disease among hibernating North American bats caused by the psychrophilic fungus Pseudogymnoascus destructans. Since its discovery in New York state, US, in 2006, and as of 2020, WNS has rapidly spread to 34 American states and seven Canadian provinces, causing precipitous declines of native bat populations across North America. The rapid spread of this fungal pathogen has been facilitated by the social behavior of bats, as well as the ability of subterranean hibernacula to support a favorable environment for P. destructans, and is probably exacerbated by anthropogenic transmission events. Although many bat species roost in natural cave environments, bats also selectively use diverse structures for hibernacula. Certain areas of the US lack caves, forcing bats to select different winter roosting environments. Bats have been observed using roadway-associated structures, such as bridges and culverts, for roosting, especially in regions that lack natural cave environments. However, the potential for P. destructans transmission in such roadway-associated structures requires further investigation. Understanding potential pathogen transmission in these widely used anthropogenic structures is crucial to disease management and preventing further declines of imperiled bat populations. Our study investigated these structures as potential pathogen transmission corridors by surveying the use of these structures by Perimyotis subflavus and other susceptible bat populations and by measuring their temperature. The results suggest the environments of roadway-associated culverts are thermally conducive to the proliferation of P. destructans-even in regions with mild winters-and the development of WNS in susceptible bat populations. It is apparent these roadway-associated structures have the potential to spread P. destructans and exacerbate the effect of WNS on susceptible bat populations.

SARS-CoV-2 and Coronavirus Disease Mitigation: Treatment Options, Vaccinations and Variants.

COVID-19 is caused by a novel coronavirus (2019-nCoV), which was declared as a pandemic after it emerged in China 2019. A vast international effort has been conducted to prevent and treat COVID-19 due to its high transmissibility and severe morbidity and mortality rates, particularly in individuals with chronic co-morbidities. In addition, polymorphic variants increased the need for proper vaccination to overcome the infectivity of new variants that are emerging across the globe. Many treatment options have been proposed and more than 25 vaccines are in various stages of development; however, the infection peaks are oscillating periodically, which raises a significant question about the effectiveness of the prevention measures and the persistence of this pandemic disease. In this review, we are exploring the most recent knowledge and advances in the treatment and vaccination options as well as the new emerging variants of 2019-nCoV and the possible mitigation of one of the most aggressive pandemics in the last centuries.

Validation of methylene blue viability staining with the emerging pathogen Candida auris.

Methylene blue viability staining has been traditionally used to assess viability of Saccharomyces cerevisiae in brewing and wine making. Here, this method was tested and validated with the emerging fungal pathogen Candida auris to determine if this species would also deferentially stain, which could provide utility in assessing microbial control and disinfectant efficacy.

Homologous G776G Variant of Transcobalamin-II Gene is Linked to Vitamin B12 Deficiency.

Vitamin B12 (Cobalamin) deficiency, due to improper internalization of cobalamin, is a metabolic disorder prevalent in impoverished and elderly populations and is associated with megaloblastic anemia and dementia. It has been suggested that mutations in transcobalamin II (TCN2) or gastric intrinsic factor (GIF) proteins can alter their binding efficiency to cobalamin or reduce the ability of their receptors to internalize them. In this case-control study, the correlation between vitamin B12 deficiency and alternative alleles of TCN2 and GIF was investigated in a Jordanian population. One hundred individuals with vitamin B12 deficiency (B12 < 200 mg/mL) were enrolled in our study to evaluate the TCN2 and GIF polymorphisms. The control group (B12 > 200 mg/mL) included 100 individuals. Our results indicated a significant association between the homologous variant of the TCN2 gene (G776G) and vitamin B12 deficiency, and an intermediate phenotype in heterozygous individuals (p < 0.001, OR = 5.6, 95% CI = 2.95 to 10.63). The GIF gene, however, showed no correlation between the A68G variant and vitamin B12 deficiency (p = 0.2). This study expounds the association of TCN2 polymorphism with cobalamin levels in a Jordanian population and highlights the necessity of further studies to elucidate the molecular basis and impact of TCN2 and GIF genes polymorphisms on vitamin B12 deficiency and associated disorders.

Lipolytic Activity and the Utilization of Fatty Acids Associated with Bat Sebum by Pseudogymnoascus destructans.

Pseudogymnoascus destructans is the causative agent of a fungal infection of bats known as white-nose syndrome (WNS). Since its discovery in 2006, it has been responsible for precipitous declines of several species of cave-dwelling North American bats. While numerous advancements in the understanding of the disease processes underlying WNS have been made in recent years, there are still many aspects of WNS, particularly with respect to pathogen virulence, that remain unknown. In this preliminary investigation, we sought to further elucidate the disease cycle by concentrating on the pathogen, with specific focus on its ability to utilize lipids that compose bat wing sebum and are found in wing membranes, as a substrate for energy and growth. In vitro growth experiments were conducted with the three most common fatty acids that comprise bat sebum: oleic, palmitic, and stearic acids. None of the fatty acids were observed to contribute a significant difference in mean growth from controls grown on SDA, although morphological differences were observed in several instances. Additionally, as an accompaniment to the growth experiments, bat wing explants from Perimyotis subflavus and Eptesicus fuscus were fluorescently stained to visualize the difference in distribution of 16- and 18-carbon chain fatty acids in the wing membrane. Which substrates contribute to the growth of P. destructans is important to understanding the progressive impact P. destructans has on bat health through the course of the disease cycle.

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.

Detection of antibiotic-producing Actinobacteria in the sediment and water of Ma'in thermal springs (Jordan).

INTRODUCTION: Detection of new Actinobacteria is significant to discover new antibiotics because development of new antibiotics is connected to the characterization of novel bacterial taxa. This study has focused on the identification and isolation of antibiotic-producing Actinobacteria from the sediment and the water of Ma'in thermal springs (48-59°C) situated in the center area of Jordan. METHODS: Samples of sediment and water were transferred to glucose yeast malt agar medium and Actinobacteria were cultivated, isolated and identified according to scanning electron microscopy and 16S rRNA gene analysis. Antibacterial activities of the isolates were then tested against different test bacteria by agar well diffusion method. RESULTS: Three different species of Actinobacteria were isolated (M1-1, M2-2, M3-2) from sediment samples. Based on 16S rRNA gene analysis, isolate M1-1 was found to have only 90% identity percentage with Nocardiopsis sp., however, isolates M2-2 and M3-2 were found to be closely related Streptomyces sp. (97%) and Nocardioides luteus (99%), respectively. The antibacterial activity showed that strain M1-1 is active against P. aeruginosa ATCC 2785 (inhibition zone, 9 mm). Strain M2-2 was found to be active against S. aureus ATCC 29213 (12 mm), B. cereus ATCC 11778 (11 mm), and E. coli ATCC 25922 (9 mm). In respect to strain M3-2, it was found to be active against S. aureus ATCC 29213 (14 mm) and B. cereus ATCC 11778 (9 mm). There were no actinobacterial isolates obtained from water samples despite their significant diversity revealed by our previous metagenomic analysis, which showed the presence of 13 different species dominated by Arthrobacter (an Actinobacterium belonging to family Actinomycetales). CONCLUSION: There were 17 different Actinobacteria that could be detected in Ma'in thermal springs (13 unculturable species and 3 culturable species). The culturable Actinobacteria were found to have some antimicrobial activity. Further chemical analysis of the bioactive compounds is recommended.

Exploring the microbial diversity in Jordanian hot springs by comparative metagenomic analysis.

A culture-independent approach was utilized in this study to reveal the microbial diversity in Jordanian hot springs represented by Ma'in and Afra hot springs. Water samples from Ma'in and Afra hot springs were collected in June 2015. The in situ temperature of water samples range was 38-59°C and the pH range was 7.4-8.4. The metagenome was extracted and analyzed using the next generation technology (bTEFAP® ). A total of 314,310 sequences were parsed and 288,452 were then clustered. The sequences were predominated by bacteria (>84%) and the relative abundance of archaea in each sample was <1%. Eukaryotic microorganisms were detected but with varying abundances (0.6%-15%). Because most of the detected sequences were found to belong to the domain of bacteria (196,936 sequences out 288,452), the bacterial sequences were utilized for further microbial analyses. With respect to alpha and beta diversity, samples were rarefied to 30,000 sequences and bootstrapped at 10,000 sequences. The Shannon-Wiener Index curve plot reaches a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. By examining the relative abundance of phyla detected in each sample, it appears that the biota of both Jordanian hot springs sampled are compositionally similar, with over 50% of the microbial community of each sample being comprised of the phylum Proteobacteria. The second most abundant phylum was the phylum Bacteroidetes which represents more than 13% in each sample. The phylum Firmicutes was also detected with a significant abundance. However, lower abundance of Deinococcus, Verrucomicrobia, Planctomycetes, and Chloroflexi was detected. A principal coordinate analysis plot was generated based upon the weighted UniFrac distance matrix. By utilizing Monte Carlo simulations, we were able to determine that there were no significant differences in the microbial diversity between each sample.

Microbial community analysis of the hypersaline water of the Dead Sea using high-throughput amplicon sequencing.

Amplicon sequencing using next-generation technology (bTEFAP® ) has been utilized in describing the diversity of Dead Sea microbiota. The investigated area is a well-known salt lake in the western part of Jordan found in the lowest geographical location in the world (more than 420 m below sea level) and characterized by extreme salinity (approximately, 34%) in addition to other extreme conditions (low pH, unique ionic composition different from sea water). DNA was extracted from Dead Sea water. A total of 314,310 small subunit RNA (SSU rRNA) sequences were parsed, and 288,452 sequences were then clustered. For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon-Wiener index curve plot reached a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. Archaea was found to be dominating the sequences (52%), whereas Bacteria constitute 45% of the sequences. Altogether, prokaryotic sequences (which constitute 97% of all sequences) were found to predominate. The findings expand on previous studies by using high-throughput amplicon sequencing to describe the microbial community in an environment which in recent years has been shown to hide some interesting diversity.

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.

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.

Chloramination of organophosphorus pesticides found in drinking water sources.

The degradation of commonly detected organophosphorus (OP) pesticides, in drinking water sources, was investigated under simulated chloramination conditions. Due to monochloramine autodecomposition, it is difficult to observe the direct reaction of monochloramine with each OP pesticide. Therefore, a model was developed to examine the reaction of monochloramine (NH(2)Cl) and dichloramine (NHCl(2)) with chlorpyrifos (CP), diazinon (DZ), and malathion (MA). Monochloramine was found not to be very reactive with each OP pesticides, (k)NH(2)Cl,OP = 11-21 M(-1)h(-1). While, dichloramine (NHCl(2)) was found to be 2 orders of magnitude more reactive with each of the OP pesticides than monochloramine, (k)NHCl(2),OP = 2000-2900 M(-1)h(-1), which is still three orders of magnitude less than the hypochlorous acid reaction rate coefficient with each OP pesticide. For each pesticide, the reactivity of the three chlorinated oxidants was then found to correlate with half-wave potentials (E(1/2)) of each oxidant. With reaction rate coefficients for the three chlorinated oxidations as well as neutral and alkaline hydrolysis rate coefficients for the pesticides, the model was used to determine the dominant reaction pathways as a function of pH. At pH 6.5, OP pesticide transformation was mostly due to the reaction of hypochlorous acid and dichloramine. Above pH 8, alkaline hydrolysis or the direct reaction with monochloramine was the primary degradation pathway responsible for the transformation of OP pesticides. This demonstrates the ability of models to be used as tools to elucidate degradation pathways and parameterize critical reaction parameters when used with select yet comprehensive data sets.