Bacteroides fragilis Strain ZY-312 Defense against Cronobacter sakazakii-Induced Necrotizing Enterocolitis <i>In Vitro</i> and in a Neonatal Rat Model

Abstract

<i>Cronobacter sakazakii</i> is an important pathogen associated with the development of necrotizing enterocolitis (NEC), infant sepsis, and meningitis. Several randomized prospective clinical trials demonstrated that oral probiotics could decrease the incidence of NEC. Previously, we isolated and characterized a novel probiotic, <i>Bacteroides fragilis</i> strain ZY-312. However, it remains unclear how ZY-312 protects the host from the effects of <i>C. sakazakii</i> infection. To understand the underlying mechanisms triggering the probiotic effects, we tested the hypothesis that there was cross talk between probiotics/probiotics-modulated microbiota and the local immune system, governed by the permeability of the intestinal mucosa, using <i>in vitro</i> and <i>in vivo</i> models for the intestinal permeability. The probiotic effects of ZY-312 on intestinal epithelial cells were first examined, and the results revealed that ZY-312 inhibited <i>C. sakazakii</i> invasion, <i>C. sakazakii</i>-induced dual cell death (pyroptosis and apoptosis), and epithelial barrier dysfunction <i>in vitro</i> and <i>in vivo</i> The presence of ZY-312 also resulted in decreased expression of an inflammasome (NOD-like receptor family member pyrin domain-containing protein 3 [NLRP3]), caspase-3, and serine protease caspase-1 in a neonatal rat model. Furthermore, ZY-312 significantly modulated the compositions of the intestinal bacterial communities and decreased the relative abundances of <i>Proteobacteria</i> and <i>Gammaproteobacteria</i> but increased the relative abundances of <i>Bacteroides</i> and <i>Bacillus</i> in neonatal rats. In conclusion, our findings have shown for the first time that the probiotic <i>B. fragilis</i> ZY-312 suppresses <i>C. sakazakii</i>-induced NEC by modulating the proinflammatory response and dual cell death (apoptosis and pyroptosis).<b>IMPORTANCE</b> <i>Cronobacter sakazakii</i> is an opportunistic pathogenic bacterium that can cause necrotizing enterocolitis (NEC). However, the mechanism of pathogenicity of <i>C. sakazakii</i> is largely unknown. Here we have now demonstrated that apoptotic and pyroptotic stimuli are effectors of <i>C. sakazakii</i>-induced NEC. Previously, we isolated a novel probiotic strain candidate from fecal samples from healthy infants and characterized it as <i>Bacteroides fragilis</i> strain ZY-312. Functional characterization reveals that ZY-312 inhibited <i>C. sakazakii</i> invasion, restoring epithelial barrier dysfunction, decreasing the expression of inflammatory cytokines, and reducing dual cell death (pyroptosis and apoptosis). Furthermore, the presence of ZY-132 was sufficient to hinder the adverse reaction seen with <i>C. sakazakii</i> in a <i>C. sakazakii</i>-induced NEC model. Taking the results together, our study demonstrated the utility of ZY-312 as a promising probiotic agent for the prevention of NEC.