Leafy greens like lettuce and spinach are a common vehicle for foodborne illness in United States. It is unknown if native plant epiphytic bacteria may play a role in the establishment of enteric pathogens on leaf surfaces. The objective of this study was to characterize the bacterial communities of fresh and packaged spinach leaves and to explore interactions with E. coli O157:H7. We assessed the bacterial diversity present on the spinach leaf surfaces and how parameters such as spinach cultivar, field conditions, post-harvest operations and the presence of E. coli O157:H7 affected its diversity. Differences in bacterial population size and species richness were associated with differences in plant topography; flat leaves had smaller bacterial populations than savoy leaves, which correlated with larger number of stomata and trichomes in savoy leaves. During spinach growing season shifts in environmental conditions affected richness and population size of the spinach bacterial community. Decreases in the overall soil and ambient temperature and increased rainfall decreased richness and bacterial population size. Fresh spinach richness and composition assessed by parallel pyrosequencing of 16S rRNA elucidated 600 operational taxonomic units, with 11 different bacterial phyla. During postharvest operations diversity indexes and evenness tended to decrease, likely attributed to storage at low temperature and time of storage (4°C and 10°C), that promoted the dominance of g-Proteobacteria. Bacteria isolated from fresh spinach elicited growth inhibition of E. coli O157:H7 in vitro, which was associated with nutrient competition. In contrast growth enhancement produced by epiphytes was associated to low correlations in carbon source utilization and the ability of E. coli O157:H7 to rapidly utilize carbon resources. In packaged spinach, E. coli O157:H7 altered the composition of the bacterial community and its growth was promoted on packaged spinach when a disinfection and temperature abuse occurred, removal of the epiphytic bacteria resulted in significant increases in numbers of E. coli O157:H7 at 10°C and was associated with increased expression of E. coli O157:H7 virulence and stress response genes. The large diversity present on the surface of spinach leaves significantly impacted the ecology of enteric pathogens like E. coli O157:H7 on the phyllosphere. / Ph. D.

Leafy greens such as spinach have been the object of several recent food-borne pathogen outbreaks. The purpose of this study was to isolate bacteria spinach epiphytic bacteria that inhibit growth of E. coli O157:H7, which we describe as antagonism. The mechanism of antagonism was investigated and we attempted to improve the antagonistic potential in vitro and on spinach leaves when cellobiose, a carbon source utilized by the antagonists but not E. coli O157:H7, was added. There were larger culturable populations of bacteria on the leaves of savoyed cultivars compared to flat. From the isolated colonies, 47 displayed antagonism towards E.coli O157:H7, and were identified as members of 11 different genera and sixteen species. A representative isolate from each species was evaluated for three possible mechanisms of antagonism: acid production, secretion of an inhibitory compounds or secreted protease. The majority (14/16) produced at least a moderate level of acid. Two of these strains, Paenibacillus polymyxa and Pseudomonas espejiana, were found to secrete a non- protease antagonistic compound. These antagonists varied in their reduction of E.coli O157:H7 numbers in vitro, but all significantly reduced numbers in 48 hours of co-culturing in nutrient rich media. Five antagonists resulted in a significant reduction in E.coli O157:H7 populations when co-cultured on spinach leaves. Application of cellobiose did not improve the amount of antagonism in vitro or on the leaf surface after 24 hours. / Master of Science

The oral pathogen Streptococcus mutans is a known etiological agent for dental root decay and coronal caries. It has been hypothesized, but not yet proven, that S. mutans expression of virulence genes in dental plaque may be influenced by its interaction with co-aggregating partners, notably Fusobacterium nucleatum and Actinomyces viscosus. Investigation of the suitability of mixed cultures of S. mutans with F. nucleatum versus S. mutans with A. viscosus proved that A. viscosus was a better target in the present laboratory setting. Furthermore, A. viscosus, a causative agent of mandible osteomyelitis and endocarditis, has been shown to have direct interaction ability with S. mutans. DNA microarray analysis was used in the present study to investigate the influence of co-aggregation with A. viscosus on the expression of S. mutans genes. Microarrays have been used successfully in the analysis of differential gene expression in S. mutans as a function of culture conditions, such as in biofilms versus planktonic states. This technology however, has not yet been applied to the analysis of homologous versus heterologous cultures. The present study was conducted in order to identify potential problems associated with the application of microarray analysis to mixed cultures. The data obtained encourage the further testing of microarrays for the analysis of heterologous cultures of oral bacteria.

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