2014 GABIO/RAMC Conferences

The 13th annual Georgia Bio Innovation Summit was held October 16th in Atlanta, GA. Five students presented posters of their research.

Kyle Gabriel, PhD student
Category: Molecular and Biological Research
Use of Antifungal Volatile Organic Compounds with a Novel Automated Dispersal Device for the Treatment of White-Nose Syndrome in Bats
    Use of volatile organic compounds (VOCs) with a newly-developed dispersal device may present many advantages over traditional methods for the control of fungi and other microorganisms that colonize areas difficult or impossible to directly access. This research focuses primarily on inhibition of Pseudogymnoascus destructans, a newly-discovered ascomycetous fungus and the etiological agent of a deadly epidemic among North American bats, known as White-Nose Syndrome (WNS). As bats have been known to hibernate in areas that are challenging to access, it is believed that contact-independent antagonism may be a viable option for in and ex situ treatment of WNS.
    Several previously-identified Pseudomonas- and Bacillus-produced antifungal VOCs and VOC formulations were investigated and demonstrated their inhibitory effect through contact-independent antagonism (Cornelison et al., 2013). The development of a device was undertaken to provide autonomous dispersal of these antimicrobial compounds, as microscopic vapor droplets (0.5 – 5 µm diameter), to facilitate rapid evaporation in order to accurately attain an effective gaseous concentration.
    Functioning prototypes of the device have demonstrated accurate dispersal of VOCs and VOC formulations. Additionally, a toxicity study with this device and a highly-effective anti-P. destructans VOC formulation on torpid bats (n=37) yielded favorable results. Necropsies, with specific attention to respiratory tissue, indicated no significant detrimental effects compared to controls.
    The identification of biologically-produced inhibitory volatiles expands the pool of potential control agents of P. destructans, and the development of chemical formulations with significant anti-P. destructans activity, in conjunction with a method for accurate and automated dispersal, provides promising chemical control options for management of WNS. In addition to disease management, this device has the potential to serve a much broader range of uses with microbial control. Furthermore, the design allows for the use of a wide variety of essential oils, VOCs, and VOC formulations, and with the addition of an enclosure and batteries, prolonged use in harsh or remote environments is possible.

Benjamin Poodiack, Masters student
Category: Agriculture Biotechnology
Effects of Rhodococcus rhodochrous strain DAP96253 on Bat Hibernacula Fungal Isolates Grown in Shared Air Space
    Pseudogymnoascus destructans has been positively identified as the causative agent of White Nose Syndrome(WNS). The pathogen infects bats during the Winter hibernation season when bats are in torpor, with greatly depressed body temperatures and immune function. In the past several years, government agencies, cave management services and the scientific community have observed the rapid spread of P. destructans across the Eastern United States and Canada.
    The cumulative effect of the pathogens spread is not only deadly to the bat species themselves but the projected ecological and economic impacts will be devastating. Extremely high mortality rates have been observed in specific caves and a report by the U.S. Fish and Wildlife Service counted the total mortality in 2012 to be 5.5 million bats. Unless human intervention takes place to aid the bats survival the extinction of hibernating North American bat populations appears inevitable.
    Accordingly, laboratories all over the United States are scrambling to find a tool to aid in the control of P. destructans and WNS in bats. A very promising approach is bio-control. Bio-controls are biological agents, either living organisms or agents formed by/from biological organisms and used to control the growth and proliferation of pathogenic organisms. Our project focuses on using a bio-control agent, yet unidentified, volatile organic compound (VOC) produced by the bacterium Rhodococcus rhodochrous strain DAP96253.
    The complexity of the bat hibernacula, many in natural karst environments, would raise many challenges to mechanical control methods. Conceptually placing R. rhodochrous near hibernacula would inhibit growth of P. destructans throughout the winter months and reduce bat mortality associated with WNS. In order to apply the antifungal R. rhodochrous as a bio-control agent in the environment, we need to better understand the effect of R. rhodochrous on naturally found cave microflora. In this study, fungal isolates were collected from bat hibernacula in Canada and the eastern United states. These isolates were then exposed to R. rhodochrous and any effects on growth were observed.

Blake Cherney, Masters student
Category: Molecular and Biological Research
Microbial Control of Ophidiomyces ophiodiicola the causative agent of snake fungal disease by induced Rhodococcus rhodochrous DAP 96253
    Since 2006 the National Wildlife Health Center has observed an increasing number of snakes in captivity and in the wild becoming infected with a fungal dermatitis. Commonly known as Snake Fungal Disease (SFD), Ophidiomyces ophiodiicola is the known causative agent for this fungal dermatitis. It can easily harm healthy snakes and has a 100% mortality rate with Illinois massasauga rattlesnakes, when diagnosed. This disease is currently threatening several species of snakes, including the massasauga and timber rattle snakes. SFD has also shown to become a potential threat in the exotic pet trade business and in many zoos. Most snakes in captivity when infected with this disease go through standard antifungal treatments such as intraconazole, chlorohexidine, and many other antifungal applications with poor results.
    Recently, new biocontrol applications have been developed to treat pathogenic fungal infections and have shown positive results in treating bats infected with white nose syndrome (WNS). Current research has shown, using induced cells of Rhodococcus rhodochrous strain DAP 96253, a naturally occurring organism, inhibition in O. ophiodiicola fungal plug assays over 6 days at 30˚C. In the presence of R. rhodochrous with 24 and 48 hours exposures, on slide agar plates have shown to inhibit spore germinations in concentrations of up to 1.0×10⁹ ml^-1 at 30˚C. R. rhodochrous can potentially be utilized as a biocontrol agent to treat and prevent animals from developing fungalinfections. Starting trials with R. rhodochrous and otherpathogenic fungi closely related to O. ophiodiicola, called Chrysosporium anamorph of Nannizziopsis vriesii (CANV), one of the leading causes of fungal dermatitis in other reptiles, will be crucial in showing that induced cells can be an effective biocontrol treatment for many exotic animals in the pet trade.

Ian Sarad, Masters student
Category: Industrial Production
Shake-Flask Optimization of Fermentation Parameters for the Production of Rhodococcus rhodochrous Strain DAP96253: as a Biological Control Agent of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome
    Since its discovery and documentation in 2006, white-nose syndrome (WNS) has resulted in the death of more than 5.7 million North American bats. WNS is characterized by high mortality rates reaching 100% in some hibernacula. As of August 2014, WNS has been discovered in more than half of the 47 bat species and in more than 30 states in the eastern United States. The fungus Pseudogymnoascus destructans is well documented to be the causative agent of WNS and it has been shown to be susceptible to organic volatile compounds produced by bacteria grown on solid media. Solid media production of antifungal volatiles is time consuming and expensive and the current techniques for fermentation are optimized for alternative applications and antifungal specific fermentation runs lack the volatility needed for large scale application in the control of WNS.
    Refining and optimizing the fermentation for antifungal activity can lead to higher, more productive yields of cell mass, decreasing both time and associated cost. A variety of media and culture preparations have been evaluated in a shake-flask model and have shown positive results for antifungal activity. The transition from solid media to liquid media along with the change in regulation of pH have been the critical changes in shake-flask fermentation techniques.
    The antifungal properties of shake-flask induced R. rhodochrous using these methods have been tested against Aspergillus niger as a control. The results are more efficient than any previously used method of antifungal production. These findings will have significant impacts on production efforts associated with the use of this biological control agent for the control of WNS. The opportunity to reduce the number of future bat deaths and limit the spread of White-Nose Syndrome makes this continued research and optimization vitally important.

John Neville, Masters student
Category: Molecular and Biological Research
The influence of induced Rhodocococcus rhodochrous VOCs on enzyme production and function of Pseudogymnoascus destructans, the causative agent of white-nose syndrome in bats
    White Nose Syndrome is a fungal infection of bats, caused by Pseudogymnoascus destructans, that has devastated North American bat populations. Mortality rates in some hibernacula are estimated at 90% to 100%. P. destructans is an allocthonous pathogen that takes advantage of the bat’s immunologically- and thermally-depressed state while in torpor. In addition to colonizing bat’s muzzles, P. destructans infects the bat’s wings, using it as a nutrient source, thereby destroying the delicate tissue crucial to the bats ability to regulate body temperature and water retention during torpor.
    The enzymatic capacity of P. destructans is a direct link to its virulence as a pathogen. Previously, the exposure of P. destructans to induced R. rhodochrous’s VOCs has been shown to inhibit spore germination and stunt mycelial growth. This study aims to determine if this known method of inhibiting P. destructans, through the exposure to induced R. rhodochrous, will affect the virulence factor of enzyme production and function.
    Solid media assays that produce a colorimetric change in the presence of specific enzymes were made. Assays were made for urease, and β-glucosidase. An assay based on the formation of a precipitate was made for Lipase. Mycilial plugs of P. destructans were placed into the center of the assays. The assays were first inoculated with plugs and examined for a positive color change and mycelial growth to ensure the media was not toxic to P. destructans or lacked any nutrients required for growth. After validation of the assays, the process was repeated and the plates were placed in a shared airspace with induced R. rhodochrous. Quantities of 1, 2.5 and 5 grams of induced R. rhodochrous were used in the exposure assays. Control assays were inoculated with plugs and not exposed to R. rhodochrous. The difference between the color change zones around the plugs in the control and exposure assays were measured to determine if enzyme production or function was being inhibited.
    There was no significant difference seen between the controls and the exposure assays. The assays were photographed and measured until the color change covered the entire plate assay. Even when exposed to quantities of induced R. rhodochrous that were 5 times the amount seen in previous studies, known to inhibit the growth of P. destructans, the enzyme production persisted.
    These results point to the initial conclusion that the extra cellular enzyme production of P. destructans is not the target of the VOCs produced by R. rhodochrous. This directs the investigation of how R. rhodochrous’s VOCs inhibit P. destructans growth to another possible direction such as cell signaling. Also, further experiments should investigate if induced R. rhodochrous VOCs target P. destructans metabolic capabilities.

The 3rd bi-annual Recent Advances in Microbial Control (RAMC) conference, was held November 9-12 in San Francisco, CA.

    George Pierce orally presented research surrounding his induction of Rhodococcus rhodochrous DAP 96253 and its delayed fruit-ripening activity.

    Chris Cornelison orally presented research of the antifungal activity of induced Rhodococcus rhodochrous DAP 96253 and the development of treatment methods for fungal wildlife diseases, focusing on white-nose syndrome in North American bats.

    Kyle Gabriel presented a poster detailing effective formulations of antifungal volatile organic compounds (VOCs) he’s developed to inhibit growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome, and a device he’s designed to disperse those VOCs, for the treatment and prevention of the disease.

Kyle Gabriel with his poster, demonstrating the operation of his VOC dispersal device.