Lactobacillus bulgaricus Prevents Intestinal Epithelial Cell Injury Caused by Enterobacter sakazakii-Induced Nitric Oxide both In Vitro and in the Newborn Rat Model of Necrotizing Enterocolitis

Enterobacter sakazakii is an emerging pathogen that has been associated with outbreaks of necrotizing enterocolitis (NEC) as well as infant sepsis and meningitis. Our previous studies demonstrated that E. sakazakii induces NEC in a newborn rat model by inducing enterocyte apoptosis. However, the mechanisms responsible for enterocyte apoptosis are not known. Here we demonstrate that E. sakazakii induces significant production of nitric oxide (NO) in rat intestinal epithelial cells (IEC-6) upon infection. The elevated production of NO, which is due to increased expression of inducible NO synthase, is responsible for apoptosis of IEC-6 cells. Notably, pretreatment of IEC-6 cells with Lactobacillus bulgaricus (ATCC 12278) attenuated the upregulation of NO production and thereby protected the cells from E. sakazakii-induced apoptosis. Furthermore, pretreatment with L. bulgaricus promoted the integrity of enterocytes both in vitro and in the infant rat model of NEC, even after challenge with E. sakazakii. Infection of IEC-6 cells with E. sakazakii upregulated several genes related to apoptosis, cytokine production, and various signaling pathways, as demonstrated by rat gene array analysis, and this upregulation was subdued by pretreatment with L. bulgaricus. In agreement with these data, L. bulgaricus pretreatment protected newborn rats infected with E. sakazakii from developing NEC, resulting in improved survival.Necrotizing enterocolitis (NEC) is a worldwide problem in very-low-birth-weight infants, with a highly variable incidence, affecting 2.6% to 28% of these infants. The precise etiology of NEC is unknown but is widely considered multifactorial. Three major factors have been proposed, including the presence of a pathogenic organism, the challenge of enteral feeding, and altered enteric mucosal integrity. Although mortality rates among infants with NEC may have decreased as a result of improved supportive and surgical care, effective preventive strategies are lacking. The initial management of infants who are suspected of having NEC relies upon aggressive fluid resuscitation and the prompt initiation of broad-spectrum antibiotics (30). Thus, it would be of extreme value to develop a preventive or therapeutic strategy in the management of this disease. Prevention offers benefits over reactive intervention because despite successful treatment, once infants are affected with NEC, they continue to be at risk for multiple morbidities, including short gut syndrome, stricture formation, and even poor neurologic outcomes.Several pathogens have been associated with NEC. However, thus far, none fulfills Koch''s postulates (17). Enterobacter sakazakii is a bacterium that has been implicated in outbreaks of sepsis, meningitis, and NEC in newborns. Such outbreaks have been attributed to E. sakazakii contamination of powdered human infant formula (43). A taxonomic reclassification of E. sakazakii to consist of five species within a new genus, Cronobacter, was recently proposed (20). However, due to the familiarity of the name Enterobacter sakazakii in the literature, we continue to use the same in this study. Additionally, E. sakazakii infection confers a high mortality rate in affected infants (40). Our recent studies have shown that oral feeding of E. sakazakii induces NEC in an experimental animal model (16). We further demonstrated that E. sakazakii binding to rat intestinal epithelial cells (IEC-6) in vitro and in vivo causes enterocyte apoptosis. However, the mechanisms involved in E. sakazakii-induced apoptosis of IEC-6 cells have not yet been identified.Although E. sakazakii may be an important pathogenic trigger in the development of NEC, it has been recognized that there are both quantitative and qualitative changes in the fecal flora before the onset of NEC. A decline in the variety of species and a shift to a predominance of members of the Enterobacteriaceae before the onset of NEC were identified. Gewolb et al. reported that Bifidobacterium and Lactobacillus are found in the stools of <5% of extremely-low-birth-weight infants within the first month of life (11). These data suggest that low levels of colonization of Bifidobacterium and Lactobacillus in low-birth-weight infants may serve as a predisposing factor in microbial infection. Recently, there have been several randomized prospective human clinical trials that have shown a decreased incidence of NEC after prophylactic oral administration of certain probiotic species (2, 4, 15, 29). Although the results for humans are promising, very little is understood regarding the mechanisms by which probiotics may alter disease susceptibility, and an appreciation of the role of specific probiotics is absent. However, there are several theoretical mechanisms through which probiotics may protect against the development of NEC, including blocking enterocyte-binding sites used by pathogenic species through modulation of the intestinal immune system or creating a locally hostile environment (8).NO is a short-lived, highly reactive molecule that plays a key role in the pathogenesis of intestinal barrier failure in NEC (3). NO is produced by three isoforms of NO synthase (NOS). Two of the isoforms, endothelial NOS (eNOS) and neuronal NOS, are expressed at constitutively low levels. The third isoform, inducible NOS (iNOS), is not expressed under normal conditions but is dramatically increased during inflammation, resulting in high levels of NO production (34). Elevated levels of iNOS have been demonstrated in infants with NEC as well as in adults with inflammatory bowel disorders (14, 37). The role of iNOS in E. sakazakii-induced NEC is unknown, as is the role of probiotics in iNOS induction. In this study, we report the following two important phenomena: (i) E. sakazakii stimulates the production of NO, leading to apoptosis of IEC-6 cells; and (ii) oral feeding of Lactobacillus bulgaricus prior to infection with E. sakazakii significantly reduces mortality rates in an experimental animal model of NEC.