Molecular Characterization of Spinach (Spinacia Oleracea) Microbial Community Structure and its Interaction With Escherichia coli O157:H7 in Modified Atmosphere Conditions

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.