pyrosequencing of 16S rRNA gene amplicons for characterization of the phylloepiphytic bacteria of fresh and minimally processed spinach stored at refrigeration temperatures

Spinach was chosen as a model to examine alterations to the community structure and population dynamics of phylloepiphytic bacteria associated with packaging and refrigeration of ready to eat fresh produce using pyrosequencing of 16s rRNA gene amplicons. The fresh and packaged spinach phylloepiphytic communities were diverse harboring a wide variety of members. Sequences were assigned to 11 different phyla with the largest number of reads belonging to Proteobacteria followed by Firmicutes, Actinobacteria and Acidobacteria. Other phyla represented in lower relative abundance were Planctomycetes, Verrucomicrobia and TM7 which contained novel genera not previously described in the phyllosphere. Spinach packaging and storage affected the richness, diversity and evenness of the spinach bacterial community. Overall packaging and refrigeration resulted in a decrease in richness and diversity of the spinach phylloepiphytic community. Storage at both temperatures was associated with increases in members of the Enterobacteriaceae and the genera Pseudomonas spp. Refrigeration was associated with increases in the relative abundance of several families known to contain psychrotrophs, while the relative abundance of sequences from mesophilic bacteria decreased. The application of pyrosequencing allowed a broader description of the composition and diversity of fresh spinach and allowed in greater detail to identify the changes in bacterial composition associated with spinach packaging and time of storage.