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.

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.

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.

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.

Laboratory-scale biofiltration of acrylonitrile by Rhodococcus rhodochrous DAP 96622 in a trickling bed bioreactor.

Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 microl/min ACN and 0.8 microl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l(-1) h(-1) ACN and 83.52 mg l(-1) h(-1) TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100-200 mg l(-1) h(-1) and TOL inlet load of approximately 400 mg l(-1) h(-1), with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was approximately 90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.

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.

PilJ localizes to cell poles and is required for type IV pilus extension in Pseudomonas aeruginosa.

Twitching motility allows Pseudomonas aeruginosa to respond to stimuli by extending and retracting its type IV pili (TFP). PilJ is a protein necessary for this surface-associated twitching motility and bears high sequence identity with Escherichia coli methyl-accepting chemotaxis proteins (MCP). Here, we report that whereas wild-type P. aeruginosa PAO1 cells have extended pili at a single pole, pilJ mutant cells have shortened pili often at both poles despite normal levels of pilin accumulation, suggesting that PilJ is required for full TFP assembly/extension. Using yellow fluorescent protein fusions (pilJ-yfp), both plasmid born and in-frame chromosomal constructs, we determined that PilJ localizes to both poles of the cell. Overexpression of pilJ-yfp resulted in the protein accumulating between the poles.

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.

Novel repair of venous aneurysms secondary to arteriovenous dialysis fistulae.

This report describes the surgical management of 12 hemodialysis patients with arteriovenous fistulae in whom non-infected, fusiform venous aneurysms developed that compromised access for dialysis. The venous aneurysmal changes were too extensive to permit excision and primary veno-venous anastomosis. To avoid the use of synthetic interpositional grafts, the venous aneurysms were left in situ and reduced in size to match the diameters of the veins entering and exiting the aneurysms. After decompression, the lumens of the venous aneurysms were reduced by firing staple lines along the longitudinal axes of the venous aneurysms and excision of the aneurysmal tissue anterior to the staple lines. Twenty-eight aneurysms were repaired by this method of reduction aneurysmoplasty, in 15 operations on 12 patients over the past 10 years. There were no wound infections or dehiscences and no bleeding or hematomas. After the operations, the arteriovenous fistulae were used continuously for hemodialysis until the patients died (7 patients for 36 months -/+ 28 SD), were lost to follow-up (1 patient at 30 days postoperatively), until the arteriovenous fistulae thrombosed following revision of the arteriovenous anastomosis (1 patient at 41 months postoperatively), or until the arteriovenous fistulae was ligated to relieve pain in the upper arm (1 patient at 6 months postoperatively). Two patients continue to use their arteriovenous fistulae until and including the time of this report at 10 and 11 months, respectively. Reduction aneurysmoplasty as described in this report offers an effective and low-risk option for the management of venous aneurysms secondary to arteriovenous fistulae in hemodialysis patients.

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 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.

Pseudomonas aeruginosa, Candida albicans, and device-related nosocomial infections: implications, trends, and potential approaches for control.

For many years, device-associated infections and particularly device-associated nosocomial infections have been of considerable concern. Recently, this concern was heightened as a result of increased antibiotic resistance among the common causal agents of nosocomial infections, the appearance of new strains which are intrinsically resistant to the antibiotics of choice, and the emerging understanding of the role biofilms may play in device-associated infections and the development of increased antibiotic resistance. Pseudomonas aeruginosa and Candida albicans are consistently identified as some of the more important agents of nosocomial infections. In light of the recent information regarding device-associated nosocomial infections, understanding the nature of P. aeruginosa and C. albicans infections is increasingly important. These two microorganisms demonstrate: (1) an ability to form biofilms on the majority of devices employed currently, (2) increased resistance/tolerance to antibiotics when associated with biofilms, (3) documented infections noted for virtually all indwelling devices, (4) opportunistic pathogenicity, and (5) persistence in the hospital environment. To these five demonstrated characteristics, two additional areas of interest are emerging: (a) the as yet unclear relationship of these two microorganisms to those species of highly resistant Pseudomonas spp and Candida spp that are of increasing concern with device-related infections, and (b) the recent research showing the dynamic interaction of P. aeruginosa and C. albicans in patients with cystic fibrosis. An understanding of these two opportunistic pathogens in the context of their ecosystems/biofilms also has significant potential for the development of novel and effective approaches for the control and treatment of device-associated infections.