An In Vitro System Mimics the Intestinal Microbiota of Striped Beakfish (<i>Oplegnathus fasciatus</i>) and Inhibits <i>Vibrio alginolyticus</i> by <i>Limosilactobacillus reuteri</i>-Derived Extracellular Vesicles

Abstract

Lactic acid bacteria (LAB) are often used to improve the probiotic content in aquaculture organisms. Although LAB were fed to aquaculture organisms, these LAB were often not detected when analyzing the gut microbiota. While the intestinal environment was indeed improved, this indicates that the effect of LAB on aquaculture organisms may not occur through the bacteria themselves, but rather through substances such as extracellular vesicles involved in these processes. This study will utilize an in vitro cultured intestinal bacteria system to exclude host interference and investigate the potential of L. reuteri-derived EVs for regulating the intestinal microbiota. Extracellular vesicles (EVs) are functional substances secreted by microbes and host cells, and it has been discovered that they participate in the interactions between different microorganisms. Our recent findings indicate that Limosilactobacillus reuteri-derived EVs have the potential to improve the intestinal microbiota of Oplegnathus fasciatus fish and inhibit pathogenic bacteria. Previous research has reported that the host intestinal cells play a regulatory role in the intestinal microbiota. This suggested that to investigate the mechanisms through which L. reuteri-derived EVs regulate the intestinal microbiota, a system that excludes interference from host intestinal cells should be established. In this study, an in vitro cultured intestinal bacteria system, without host factors, was used to simulate the intestinal microbiota of O. fasciatus fish. After adding L. reuteri-derived EVs to the system, the changes in the microbiota were analyzed. The results showed that L. reuteri-derived EVs effectively reduced the abundance of Vibrio spp. In the results of the in vitro experiments, it was also observed that L. reuteri-derived EVs have the ability to inhibit Vibrio alginolyticus. We further sequenced the small RNA contained in L. reuteri-derived EVs and found that these small RNAs can interfere with genes (LysR, pirin, MIpA/OmpV, CatB, and aspartate-semialdehyde dehydrogenase) related to the growth of V. alginolyticus. Taken together, the results indicate that in the absence of host involvement, the small RNAs present in L. reuteri-derived EVs have the function of inhibiting pathogenic bacteria and exhibit the potential to regulate the intestinal microbiota.