Evaluation of the bacterial populations present in Spring Mix salad and their impact on the behavior of Escherichia coli O157:H7

Abstract

The native bacterial populations present in three brands of Spring Mix salads (brands A, B, and C) were analyzed by plating and 16S rRNA gene sequencing during 15 days of storage at 4 °C. While the traditional plating method did not show significant differences among three brands or between before and after storage, 16S rRNA gene sequencing showed that the “brand” factor significantly impacted the diversity and abundance of native bacterial populations. In order to evaluate the interaction between native bacteria and Escherichia coli O157:H7 (ECO157), day-0 and day-15 salad homogenates were inoculated with ECO157 (a final concentration of 5.5 log CFU/ml). Inoculated samples were stored at 4 °C for additional 15 days and the behavior of inoculated ECO157 was monitored by plating samples on CHROMagar™ O157 agar on days 0, 5, 10 and 15. The top four phyla of bacteria present in all three brands of salad were Proteobacteria, Bacterioidetes, Actinobacteria, and Firmicutes. Changes in the relative abundance and diversity of bacterial populations were significantly altered by inoculation of ECO157. The inoculation remarkably decreased the beta diversity (Jaccard distance) of the bacterial populations (P = 0.005). During the additional 15-day storage, culturable ECO157 cells decreased over the entire storage time. However, fluctuations of culturable ECO157 cells were observed on days 5 and 10. In day-0 brand A and day-15 brands A and C, culturable ECO157 cells were significantly higher on day 10 than on day 5. Microbiome analysis results showed that the relative abundance of Planomicrobium, Aeromonas, Carnobacterium, and Fusobacterium were negatively correlated with the decreasing numbers of culturable ECO157 cells from day 5 to day 10 after inoculation and the relative abundance of genera Methylotenera, Yersinia, Shewanella, Dyadobacter, and Brochothrix were positively correlated with the increasing numbers of culturable ECO157 cells from day 5 to day 10. This study provides in-depth information on the microbiome changes in bagged salad and their impact on food safety, which can assist with the risk assessment of produce products of different shelf life and facilitate the development of new intervention strategies for protecting the microbial safety of fresh produce.