Marc Y. Menetrez

Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by Escherichia coli O157:H7

ABSTRACT A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (1010, 109, and 108 PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 109 PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.

Evaluation of radon emanation from soil with varying moisture content in a soil chamber

The EPA chamber (2 × 2 × 4 m long) was constructed to study convective and diffusive soil gas movement under known conditions of 226Ra and222 Rn concentration, moisture, density, soil constituent, and physical response to pressure variation. The radon emanation rates of soil are known to depend strongly on the moisture content of the soil. Because the moisture content varies greatly with depth in the EPAs soil chamber (from saturated at the bottom to nearly dry at the top), it is not possible to fully understand the radon distribution within the chamber without knowing the emanation rate as a function of moisture. Soil radon concentrations vary in the chamber from 7.4 kBq m−3, near the soil surface, to 86.2 kBq m−3, at the chamber bottom. This paper describes measurements of the emanation coefficient and diffusion of radon in soil contained in the chamber, using a wide range of moisture contents. In addition, equal amounts of well-mixed oven-dried soil were placed in 20 L aluminized gas-sampling bags, and, after approximately 1 month of in-growth, radon samples were taken, after which water was added, and another period of in-growth and sampling followed. The emanation coefficients and radon concentrations in the gas bag experiment were observed to increase with increasing moisture content and then decrease before reaching saturated conditions. The emanation and diffusion effects on the radon concentration soil gradient were identified for this sandy soil having approximately 200 Bq kg−1 radium and a soil density of 1682 kg m−3.

Determining Fungi rRNA Copy Number by PCR

The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment.

Growth Response of Stachybotrys chartarum to Moisture Variation on Common Building Materials

The mould Stachybotrys chartarum has been found to be associated with idiopathic pulmonary haemorrhage in infants and indoor exposure has also been linked to other pulmonary diseases, including allergies and asthma. S. chartarum has been studied both for toxin production and its occurrence in water-damaged buildings. Growth of S. chartarum on building materials such as gypsum wallboard has been frequently documented. Given that there may be a high frequency of occurrence and so the risk of exposure, environmental factors leading to the growth of S. chartarum have been studied. Samples of commonly used building materials were sterilised, inoculated with S. chartarum and exposed to controlled levels of relative humidity and wetting. A quantitative analysis of viable S. chartarum was performed on the building materials during a 7-month period. The results indicate that for environments with a relative humidity below total saturation, wetting was necessary for visible growth to occur. Conversely, high levels of relative humidity without wetting did not initiate growth. Porous materials, after becoming sufficiently wet and measuring saturation on a moisture meter, exhibited mould growth in every experiment conducted.

The Measurement of Ambient Bioaerosol Exposure

Monitoring of ambient bioaerosol concentrations through the characterization of outdoor particulate matter (PM) has only been performed on a limited basis in North Carolina (NC) and was the goal of this research. Ambient samples of PM 2.5 (fine) and PM 10−2.5 (coarse) were collected for a six-month period and analyzed for mold, endotoxins and protein. PM 2.5 and PM 10−2.5 concentrations of these bioaerosols were reported as a function of PM mass, as well as volume of air sampled. The mass of PM 2.5 was almost twice that of the PM 10−2.5 ; however, the protein and endotoxin masses were greater in the coarse than the fine PM indicating an enrichment in the coarse PM. The protein and mold results demonstrated a seasonal pattern, both being higher in the summer than in the winter. Except for an occasional excursion, the endotoxin data remained fairly constant throughout the six months of the study.

A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi.

Because of the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces, it is necessary to accurately reflect the organisms responsible for these maladies and to identify them in precise and timely manner. To this end, we have developed a method that is cost effective, easy to perform, and accurate. We performed a simple polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis on multiple members of species known to negatively influence the indoor environment. The genera analyzed were Stachybotrys, Penicillium, Aspergillus, and Cladosporium. Each organism underwent PCR with universal primers that amplified ribosomal sequences generating products from 550 to 600 bp followed by enzymatic digestion with EcoRI, HaeIII, MspI, and HinfI. Our results show that using this combination of restriction enzymes enables the identification of these fungal organisms at the species level.

Testing Antimicrobial Paint Efficacy on Gypsum Wallboard Contaminated with Stachybotrys chartarum

The goal of this research was to reduce occupant exposure to indoor mold through the efficacy testing of antimicrobial paints. An accepted method for handling Stachybotrys chartarum-contaminated gypsum wallboard (GWB) is removal and replacement. This practice is also recommended for water-damaged or mold-contaminated GWB but is not always followed completely. The efficacy of antimicrobial paints to eliminate or control mold regrowth on surfaces can be tested easily on nonporous surfaces. The testing of antimicrobial efficacy on porous surfaces found in the indoor environment, such as gypsum wallboard, can be more complicated and prone to incorrect conclusions regarding residual organisms. The mold S. chartarum has been studied for toxin production and its occurrence in water-damaged buildings. Research to control its growth using seven different antimicrobial paints and two commonly used paints on contaminated, common gypsum wallboard was performed in laboratory testing at high relative humidity. The results indicate differences in antimicrobial efficacy for the period of testing, and that proper cleaning and resurfacing of GWB with an antimicrobial paint can be an option in those unique circumstances when removal may not be possible.

Investigation of the Potential Antimicrobial Efficacy of Sealants Used in HVAC Systems

ABSTRACT Recent experiments confirm field experience that duct cleaning alone may not provide adequate protection from regrowth of fungal contamination on fiberglass duct liner (FGDL). Current recommendations for remediation of fungally contaminated fiberglass duct materials specify complete removal of the materials. But removal of contaminated materials can be extremely expensive. Therefore, a common practice in the duct-cleaning industry is the postcleaning use of antimicrobial surface coatings with the implication that they may contain or limit regrowth. Little information is available on the efficacy of these treatments. This paper describes a study to evaluate whether three commercially available antimicrobial coatings, placed on a cleaned surface that 1 year previously had been actively growing microorganisms, would be able to prevent regrowth. The three coatings contained different active antimicrobial compounds. All three of the coatings were designed for use on heating, ventilation, and air conditioning (HVAC) system components or interior surfaces of lined and unlined duct systems. Coating I was a polyacrylate copolymer containing zinc oxide and borates. Coating II was an acrylic coating containing decabromodiphenyl oxide and antimony trioxide. Coating III was an acrylic primer containing a phosphated quaternary amine complex. The study included field and laboratory assessments. The three treatments were evaluated in an uncontrolled field setting in an actual duct system. The laboratory study broadened the field study to include a range of humidities under controlled conditions. Both static and dynamic chamber laboratory experiments were performed. The results showed that two of the three antimicrobial coatings limited the regrowth of fungal contamination, at least in the short term (the 3-month time span of the study); the third did not. Before use in the field, testing of the efficacy of antimicrobial coatings under realistic use conditions is recommended because antimicrobials have different baseline activities and interact differently with the substrate that contains them and their local environment.

Analysis of Fungal Spore Mycotoxin and the Relationship Between Spore Surface Area and Mycotoxin Content Utilizing a Protein Translation Inhibition Assay

Due to mounting evidence suggesting that biological contamination in the built environment may cause a myr- iad of adverse health effects, research aimed at understanding the potential exposure to fungal organisms and their me- tabolites is of utmost importance. To this end we utilized a protein translation inhibition assay to determine the relative amounts of mycotoxin present in various fungal spore preparations. Basing our results on the transformation of firefly lu- ciferase in a rabbit reticulocyte system our initial data showed that spores from different fungal genera contained varying amounts of mycotoxins. However, by calculating the surface area of the spores and then normalizing the assay by keeping surface areas equivalent we determined that there is a direct relationship between spore size and mycotoxin content. This is an important finding because simply knowing the numbers of spores is clearly not sufficient; one needs to know the specific species present to formulate an effective risk assessment and remediation regimen. This work illuminates the po- tential inhalation exposure to trichothecene mycotoxins that are suspended in the air of the indoor environment. Currently many methods of fungal analysis in the indoor environment are simply based on spore counts. Our analysis clearly dem- onstrates that it is equally important to know the species of organisms present to accurately determine potential exposure to mycotoxins, thereby enabling effective risk management decisions to be made.