Effects and safety of selected antagonistic bacteria against Escherichia coli in leafy green vegetables

Sammanfattning: We live in a busy world, where the demand of ready-to-eat (RTE) salad products has increased rapidly in a relatively short time. Despite of being an easy and fast way to add more vegetables to the daily diet, RTE salads have a serious underlying risk and the amount of vegetable-derived foodborne disease outbreaks has been increasing. The aim of the present study is to protect plants against contamination with human pathogens by inoculating spinach (Spinacia oleracea) seeds with antagonists, inherent on leafy green vegetables. Experiments were carried on to observe the effects of selected antagonistic bacteria against non-pathogenic Escherichia coli CCUG29300T contamination on live spinach sprouts, and to test the safety by studying the immune response in mice. In addition, bacterial isolates from RTE rocket salad (Eruca sativa) were identified by 16S rRNA gene sequencing. The putative results show that bagged rocket salad contains bacteria from several different families and genera, the most abundant being Pseudomonas, commonly found from leafy green vegetables and Aeromonas that are omnipresent in soil, water and vegetation. Surprising findings were Rheinheimera, Chryseobacterium, and Shewanella that are more commonly associated with a marine environment. Unfortunately, the antagonistic effect of selected bacteria could not be seen in this study, as no statistically significant differences between treated samples and negative control were found (p>0.05). The seed pellet may work as a physical barrier hindering the inoculation, or has antimicrobial effects that increases the death rate of two antagonists. FACS analysis of Peyer’s patches and mesenteric lymph nodes of mice indicate that the tested antagonists may be able to suppress non-pathogenic E. coli induced inflammation in mice. In Peyer’s patches, the percentage of gated CCR9+CD8+CD69+ cells was significantly lower in the groups A (15.9 (6.2–23.2), p=0.037) and E (17.1 (7.7–25.3), p=0.029) compared with the control group K (30.3 (20.8–72.1)). These percentages were on the similar level with the untreated group N (13.7 (10.3–17.6)). The percentage of TLR2+TLR4+ activated macrophages in the antagonist groups varied between E 22.7 (21.7–35.7) and B 28.9 (18.6–39.1). All results were significantly lower compared with the group K 62.7 (57.2 – 67.9). There were no significant differences in water or feed intakes between groups, which indicates that antagonist treatment do not make mice feel sick. This study supports the current knowledge of leafy green vegetables harbouring a very diverse microflora with genera with potential human pathogens. Despite of poor results in antagonistic effect experiment, preliminary results indicate that selected antagonists do not trigger a negative immune response in mice