Detection of Biomarkers of Potential Pathogens in Varied Matrices

Methods that use signature biomarkers have become increasingly important in the field of environmental microbiology. Signature lipid biomarker (SLB) analysis is a method of analysis for the quantitative definition of viable biomass, community composition, and nutritional status of microbiota isolated from a wide variety of environmental matrices, including air, soil and water. Biomarkers examined in this thesis were glyco- and phospholipids, and dipicolinic acid. Organisms tested were Cryptosporidium parvum, C. baileyi, C. muris, Eimeria tenella, E. acervulina, E. maxima, Escherichia coli, and Bacillus subtilis.

Three species of Cryptosporidium were examined using signature lipid biomarker analysis. This analysis was successfully applied to the species-specific identification of C. parvum, C. baileyi and C. muris. The glycolipid fraction from the SLB analysis was also examined for C. parvum , as the possibility of a unique biomarker (10-hydroxy stearic acid) was present in research done by others. This compound was shown to be an artifact of sample preparation.

SLB analysis was also used to examine the phospholipids of Eimeria tenella, E. acervulina, and E. maxima. This analysis clearly differentiated these three species from each other.

SLB analysis was also used to examine chlorine-injured Escherichia coli . The experiments presented herein ensure the validity of oxirane fatty acids as indicators of sterilization efficiency. The detection of epoxidated fatty acids firmly establishes that the cells have been in contact with hypochlorite, thus providing a chemical marker for hypochlorite exposure and sterility.

For the examination of dipicolinic acid from endospores, a new extraction and methylation technique was devised and GC/MS analysis was employed for the detection of dimethyl dipicolinate. Preliminary studies also evaluated spectrofluorometric analysis using terbium chloride. Bacillus subtilis spores were detected using the new in situ SFE extraction and methylation technique and GC/MS with a lower limit of quantification of 3 x 104 spores in a sand mixture, and the lower limit of detection was observed to be 3 x 103 spores.