Protective Effects and Mechanism of a Novel Probiotic Strain Ligilactobacillus salivarius YL20 against Cronobacter sakazakii-Induced Necrotizing Enterocolitis In Vitro and In Vivo

Abstract

Exposure to probiotics in early life contributes to host intestinal development and prevention of necrotizing enterocolitis (NEC). Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen, can cause NEC, bacteremia, and meningitis in neonates, but the research of probiotics against C. sakazakii is limited relative to other enteropathogens. Here, the protective effect and mechanism of a novel probiotic Ligilactobacillus salivarius (L. salivarius) YL20 isolated from breast milk on C. sakazakii-induced intestinal injury were explored by using two in vitro models, including an C. sakazakii-infected intestinal organoid model and intestinal barrier model, as well as an in vivo experimental animal model. Our results revealed that L. salivarius YL20 could promote epithelial cell proliferation in intestinal organoids, rescue budding-impaired organoids, prevent the decrease of mRNA levels of leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), zonula occludens-1 (Zo-1) and Occludin, and reverse C. sakazakii-induced low level of Mucin 2 (MUC2) in intestinal organoids. Additionally, YL20 could inhibit C. sakazakii invasion, increase the expression of ZO-1 and occludin in C. sakazakii-infected HT-29 cells, and reverse TEER decrease and corresponding permeability increase across C. sakazakii-infected Caco-2 monolayers. Furthermore, YL20 administration could alleviate NEC in C. sakazakii-infected neonatal mice by increasing the mice survival ratio, decreasing pathology scores, and downregulating pro-inflammatory cytokines. Meanwhile, YL20 could also enhance intestinal barrier function in vivo by increasing the number of goblet cells, the level of MUC-2 and the expression of ZO-1. Our overall findings demonstrated for the first time the beneficial effects of L. salivarius YL20 against C. sakazakii-induced NEC by improving intestinal stem cell function and enhancing intestinal barrier integrity.