This dissertation examined the microbial ecology of Listeria monocytogenes in three distinctly different environments: a cheese microbial community; a farm environment; and a soil microbial community. The aim of the first study was to investigate the effects of L. monocytogenes on the composition of the surface microflora on washed rind soft cheese. Two trials with washed rind cheeses that were inoculated with 100cfu cm⁻² of a L. monocytogenes six strain cocktail were conducted. The first trial had to be terminated early (day 28) as contamination of Pseudomonas spp. from the initial brine did not produce the expected characteristics of the cheese during the aging period. For the second trial, cheese samples were aged in the lab for 60 days according to the cheesemakers specifications. Surface cheese rind samples were collected from both control and inoculated cheeses every 7 days. Cheese rind samples were analyzed through the standard BAM method for enumeration of L. monocytogenes and through amplification of the V4 region of 16S rRNA and ITS regions for identification of the surface rind bacterial and fungal communities, respectively. Our data showed that Pseudomonas spp. significantly changed the composition of the microorganisms found on the surface of the rind while L. monocytogenes had little effect. In addition, although the concentration of L. monocytogenes increased to levels of 10⁶ cfu cm⁻² based on the enumeration data, the genetic data was not able to identify it in the flora due to the fact that other genera were found at much higher concentrations, which is a limitation of molecular methods used for identification of pathogens in foods. For the second study the presence and incidence of L. monocytogenes on farms that either produce raw milk cheese or supply the milk for raw milk cheese production was investigated. Five farms were visited and in total 266 samples were collected from barn, environmental, and milk sites. L. monocytogenes prevalence was found to be at 6% from all the farms tested with 10 isolates found in the barn samples, 5 from environmental sites and 1 from milking equipment. Samples were identified to the genus level through a modified BAM method and speciated though multiplex PCR. Included in the pathogenic isolates was a DUP-1042B L. monocytogenes strain that has been implicated in major outbreaks, which emphasizes the adaptability and persistence of highly pathogenic stains in food manufacturing environments. Results from this study continue to support the fact that contaminated silage can be an important reservoir of the pathogen in a dairy farm setting. From our data and field observations we identified that drinking water sources for the animals is also an important reservoir of L. monocytogenes in farm environments. More importantly this study has shown the importance of continuous monitoring of environmental sites for the presence of the pathogen, particularly in silage. Lastly manure amended soils in the northeastern U.S. were tested for the presence and survival of rifampicin resistant Escherichia coli (rE. coli), generic E. coli (gE. coli) and Listeria spp.. Both gE.coli and rE.coli samples were processed using either direct enumeration, MPN or bag enrichment methods. Samples were taken from both tilled and surface dairy solid manure-amended plots. Listeria samples were processed using a modified BAM method. Listeria presence was constant throughout the study. In contrast, rE. coli and gE. coli levels declined with time. The main conclusions of this study were that soil type, location and physical characteristics have a significant role in the survival of bacterial populations of rE. coli, gE. coli and Listeria spp. in soil. Dairy solids application does not seem to have a long term effect on the natural microbial population of soils. Tilling of soils results in increased survival of the bacterial population due to the fact that it increases soil pore size and facilitates moisture entry, which in turn has been shown to increase bacterial survival rates. Data from this research will assist in the creation of preventative measures that lead to the elimination of pathogen reservoirs. It will be further used to verify that a 120 day interval following manure application should be sufficient to ensure food safety of edible crops subsequently planted on these soils.

Molecular biological methods are fast and efficient tool for the identification of microorganisms in real samples. The aim of this diploma thesis was analysis of microflora in control and contaminated brined cheeses. DNA isolated from analyzed samples was used to optimize the PCR course using primers with GC clamp on the distribution of amplicons using DGGE. DGGE products were reamplified after optimization and prepared for DNA sequencing. DNA isolated from analyzed samples was used in real-time PCR with high resolution melt analysis of the amplicons (HRMA). Samples of cheese and bacterial cultures isolated from cheeses were compared by DGGE and HRMA. Comparing the position of the amplicons was found that contaminants may be Bacillus licheniformis, Staphylococcus epidermidis, and Acinetobacter baumanii/ calcoaceticus. Sequence analysis of cheese and pickles amplicons, the presence of Bacillus sp. and other microorganisms spree in five genera were detected. Representatives of the tree genera were cultured. It is considered contamination Bacillus sp., or microorganisms which are not culturable methods used. The method is suitable for the analysis of complex microflora in cheese and pickles after further optimization.