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

Abstract

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

Popular Abstract

As we all may know, fruits and vegetables have numerous health-promoting effects as in minerals, vitamins, and fibre. Living in the busy world make us seeking products that do not require too much preparation and that’s why ready-to-eat (RTE) products are getting popular every day. However, RTE salads and salad mixes may have a microscopic surprise that makes our stomachs to feel uneasy or in the worst case, have more severe consequences. This surprise can be Norovirus, enterohemorrhagic Escherichia coli, or Salmonella. These microbes can be found from water or several other foods, but there are no eliminative steps in salad that remove these harmful visitors. For the consumer who wants to eat RTE salads, the best advice is to check that everything looks fresh. Brown or squished salad leaves may seem harmless, but the salad juice is a very powerful nutrient for bacteria and just simply removing the damaged leaves does not change the situation that the bacteria have had a long time to grow inside the bag. But what can science do?

Competition is all around us, also in our salad. The main reason for foodborne illnesses is, that the harmful bacteria have found their way to the salad and been successful in the growth competition. It has been found out that several bacteria, commonly present in the leafy green vegetables can reduce the growth of Escherichia coli, because they are natural part of the microflora of leafy green vegetables. These bacteria can be called antagonists - organisms that are able to interfere the normal growth of harmful bacteria. In this study we added these antagonists to spinach sprouts through the leaves and the seeds, hoping to see the promising antagonistic effect as reduced E. coli concentration. Unfortunately, working with living organisms is not always easy. Even though the antagonist effects have been present in previous experiments, they could not be seen this time as the introduction of the antagonists into the plants did not go as planned.

Our bodies undergo constant competition as well. Sometimes we get sick as the first defence line fails to eliminate the intruder or it takes too long. Luckily we have also the adaptive immune system, which contains several specialized cells and is ready to activate whenever needed. As we are using the antagonistic bacteria to suppress E. coli, it is important to make sure that these antagonists are not found as harmful intruders by the immune system. Since it is not possible to test these things straight on humans, an animal study was carried out. The mice were pre-treated with antibiotics and harmless E.coli to have equal bacterial composition in their guts. The antagonists were given to the mice in drinking water, and their well-being was monitored daily. In the end, the immune response of mice was studied by observing the levels of activated defensive cells in their organs. The preliminary results indicate that the antagonists do not change the consumption behaviour of mice, or trigger a negative immune response. Several defensive cell levels were lower in the mice who got the antagonists, compared with those who got only E. coli. This may indicate that the antagonist are able to suppress the immune response caused by E. coli.

More work is needed to find an optimal way to introduce the antagonist to the plants, and to study the immune response on deeper levels. However, the results of this study indicate that we may have to be able to fight against harmful visitors of our foods by partnering with the antagonist bacteria, and thereby offer everyday consumers safer ready-to-eat salads to enjoy.

• author: Nurminen, Noora Ulla Annikki
• supervisor: Elisabeth Uhlig
• organization: Food Technology and Nutrition (M.Sc.)
• course: KLGM01 20171
• year: 2017
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: ready-to-eat salad, Antagonist, Escherichia coli, food technology, livsmedelsteknologi
• language: English
• id: 8923910
• date added to LUP: 2017-08-30 11:55:35
• date last changed: 2017-08-30 11:55:35