Escherichia coli can contaminate food, such as beef and spinach, causing illnesses ranging from recurring diarrhea to death in susceptible individuals. They may produce a toxin typically associated with another harmful bacterium known as Shigella spp. This toxin is referred to as “Shiga-toxin”, and E. coli that produce this toxin are known as STECs. While E. coli O157 remains the most common STEC in the United States there are over 100 STECs that can be just as harmful. My project, conducted in collaboration with individuals at CSUEB and the USFDA, involves identifying chemical indicators that would help detect different strains of non-O157 E. coli in contaminated foods, and determining if these STECS are vulnerable to a coliphage and a Bdellovibrio sp. invasion. Specifically, nine different STECs were chosen to be screened for their ability to produce catecholate siderophores (phenolates), such as enterobactin, chelating compounds important for some pathogenic bacteria in the acquisition of iron. The same STECs were screened for the presence of acyl homoserine lactones, chemicals involved in bacterial communication, also known as quorum sensing, and expression of virulence factors. Assays were conducted to determine if a coliphage and a Bdellovibrio sp. can infect these STECs. Detection and identification of non-O157 STECS is not easy, which hinders determination of their origin and, subsequently, how to eliminate them. If new tools, such as the use of predators or chemical indicators were developed to facilitate detection of these bacteria, then determination of the source of contamination (e.g., packing plants and fields) may occur, and quicken measures that prevent or, at least, decrease further infection, disease and/or death.

Edible fungi can accumulate certain toxic metals, including cadmium, lead, and copper, in a species dependent manner. Forests that have been treated with biosolids have elevated soil metals, which could lead to additional accumulation in mushrooms growing in treated sites. This can be a possible safety hazard for people who gather wild mushrooms for consumption. My objective was to determine metal concentrations in several species of edible mushrooms and whether these species accumulate metals above background levels in their soil substrate. Collections of edible mushrooms from biosolid treated forest and control sites were analyzed for toxic metal concentrations. Mushroom concentrations of cadmium and silver were significantly higher in biosolids treated forests, while the other metals were not significantly different. The overall results showed that metals uptake was more strongly influenced by species than by biosolids application. Gomphidius subroseus exceeded the European Union regulatory upper limit with Cd and reached it with Cu. Metals in all other mushrooms in this study were below recommended safe annual limits for moderate levels of consumption.

While necessary for many biological functions, essential heavy metals, such as copper and zinc, are highly toxic when elevated, requiring their concentrations to be tightly regulated.  This need to maintain homeostasis has led to a variety of adaptive mechanisms that chemically sequester excess heavy metal ions.  One such mechanism is the biosynthesis of phytochelatins by the enzyme phytochelatin synthase (PCS), found in plants, algae and several fungal species.  Induced in cells of certain organisms exposed to elevated levels of heavy metals, phytochelatins also bind non-essential heavy metals such as cadmium and arsenic, offering protection against anthropogenic toxins.  Despite their demonstrated ability to synthesize phytochelatins, the PCS gene has not been investigated in conifers.  The simplest method of probing genomc DNA (gDNA) for specific genes is the polymerase chain reaction (PCR) method.  However, lack of sequencing data presents a challenge in designing universal primers for conifers.  The objective of this project was to develop degenerate primers capable of amplifying the PCS gene in a selection of conifers native to Washington State.  The degenerate primers were designed from known and putative PCS sequences of non-conifer plant species obtained from a BLAST query.  The sequences were aligned and primers selected from regions of high consensus, while controlling for degeneracy.  The primers were used in PCR trials to probe the gDNA of thirteen species of conifers.  To date, I have amplified and sequenced PCR gene segements of 267 and 133 base-pairs in Pinus ponderosa and Pinus contorta, respectively; adding two species of conifers to the list of PCS genes sequenced, and allowing me to design primers with increased specificity for conifers for use in subsequent PCR trials.  Future studies could develop molecular assays for expression of the PCS gene for use in assessing metals stress at the cellular level for conifers in contaminated sites.