The neonatal gut microbiome and global health.

The role of gut microbiome in health, a century-old concept, has been on the center stage of medical research recently. While different body sites, disease conditions, and populations have been targeted, neonatal and early infancy appear to be the most suitable period for such interventions. It is intriguing to note that, unlike traditional use in diarrhea and maintenance of gastrointestinal health, microbiome-mediating therapies have now addressed the most serious medical conditions in young infants such as necrotizing enterocolitis and neonatal sepsis. Unfortunately, almost all new endeavors in this space have been carried out in the Western world leaving behind millions of neonates that can benefit from such manipulations while serving as a large resource for further learning. In this review, an attempt has been made to quantify the global burden of neonatal morbidity and mortality, examples presented on interventions that have failed as a result of drawing from studies conducted in the West, and a case made for manipulating the neonatal gut microbiome to address the biggest killers in early life. A brief comparative analysis has been made to demonstrate the differences in the gut microbiota of North and South and a large clinical trial of synbiotics conducted by our group in a South Asian setting has been presented. Although challenging, the value of conducting such global health research is introduced with an intent to invite medical scientists to engage in well-planned, scientifically robust research endeavors. This can bring about innovation while saving and serving the most vulnerable citizens now and protecting them from the negative health consequences in the later part of their lives, ultimately shaping a resilient and equitable world as pledged by 193 United Nations member countries in 2015.

Preterm infant with necrotizing enterocolitis and arteritis secondary to streptococcus gallolyticus subspecies pasteurianus.

 Streptococcus gallolyticus subspecies pasteurianus is a subtype of Streptococcus bovis (S. bovis) that has become increasingly recognized as a sepsis-causing pathogen in neonates. It is well documented that S. bovis species have a predilection to both cardiac and gastrointestinal tissue, and in adult populations, isolating these organisms in the bloodstream often triggers further evaluation for co-morbid complications such as colon cancer or endocarditis. However, no such guidance currently exists in neonatal literature. We present a case of a preterm infant with S. gallolyticus subsp. pasteurianus bacteremia presenting as necrotizing enterocolitis (NEC) not previously described in the literature. Furthermore, through a complete diagnostic evaluation, including an echocardiogram, our patient was found to have the rare complication of endocarditis.

THE GUT-BRAIN AXIS: IMPLICATIONS FOR NEUROLOGICAL DISORDERS, MENTAL HEALTH, AND IMMUNE FUNCTION.

A gut-brain axis (GBA) has a long history of conceptual development. Intestinal dysbiosis has now been recognized as a key player in the development of adult neurodevelopmental disorders, obesity, and inflammatory bowel disease. Recent developments in metagenomics suggest those nutrition and gut microbiotas (GM) are important regulators of the gut-brain communication pathways that cause neurodevelopmental and psychiatric problems in adulthood. Intestinal dysbiosis and neurodevelopmental disease outcomes in preterm newborns are being linked by recent research. Recent clinical investigations demonstrate that in critical care units, intestinal dysbiosis occurs before late-onset newborn sepsis and necrotizing enterocolitis. Strong epidemiologic data also shows a connection between necrotizing enterocolitis and extremely low birth weight babies' long-term psychomotor impairments and late-onset neonatal sepsis. The GBA theory suggests that intestinal bacteria may indirectly affect preterm newborns' developing brains. In this review, we emphasize the structure and function of the GBA and discuss how immune-microbial dysfunction in the gut affects the transmission of stress signals to the brain. Preterm babies who are exposed to these signals develop neurologic disorders. Understanding neuronal and humoral communication through the GBA may provide insight into therapeutic and nutritional strategies that may enhance the results of very low-birth-weight babies.

New insights into intestinal macrophages in necrotizing enterocolitis: the multi-functional role and promising therapeutic application.

Necrotizing enterocolitis (NEC) is an inflammatory intestinal disease that profoundly affects preterm infants. Currently, the pathogenesis of NEC remains controversial, resulting in limited treatment strategies. The preterm infants are thought to be susceptible to gut inflammatory disorders because of their immature immune system. In early life, intestinal macrophages (IMφs), crucial components of innate immunity, demonstrate functional plasticity and diversity in intestinal development, resistance to pathogens, maintenance of the intestinal barrier, and regulation of gut microbiota. When the stimulations of environmental, dietary, and bacterial factors interrupt the homeostatic processes of IMφs, they will lead to intestinal disease, such as NEC. This review focuses on the IMφs related pathogenesis in NEC, discusses the multi-functional roles and relevant molecular mechanisms of IMφs in preterm infants, and explores promising therapeutic application for NEC.

Construction of a Synthetic Colostrum Substitute and Its Protection of Intestinal Cells against Inflammation in an In Vitro Model of Necrotizing Enterocolitis.

Colostrum provides bioactive components that are essential for the colonization of microbiota in the infant gut, while preventing infectious diseases such as necrotizing enterocolitis. As colostrum is not always available from the mother, particularly for premature infants, effective and safe substitutes are keenly sought after by neonatologists. The benefits of bioactive factors in colostrum are recognized; however, there have been no accounts of human colostrum being studied during digestion of the lipid components or their self-assembly in gastrointestinal environments. Due to the weaker bile pool in infants than adults, evaluating the lipid composition of human colostrum and linking it to structural self-assembly behavior is important in these settings and thus enabling the formulation of substitutes for colostrum. This study is aimed at the rational design of an appropriate lipid component for a colostrum substitute and determining the ability of this formulation to reduce inflammation in intestinal cells. Gas chromatography was utilized to map lipid composition. The self-assembly of lipid components occurring during digestion of colostrum was monitored using small-angle X-ray scattering for comparison with substitute mixtures containing pure triglyceride lipids based on their abundance in colostrum. The digestion profiles of human colostrum and the substitute mixtures were similar. Subtle differences in lipid self-assembly were evident, with the substitute mixtures exhibiting additional non-lamellar phases, which were not seen for human colostrum. The difference is attributable to the distribution of free fatty acids released during digestion. The biological markers of necrotizing enterocolitis were modulated in cells that were treated with bifidobacteria cultured on colostrum substitute mixtures, compared to those treated with infant formula. These findings provide an insight into a colostrum substitute mixture that resembles human colostrum in terms of composition and structural behavior during digestion and potentially reduces some of the characteristics associated with necrotizing enterocolitis.

New insights into the pathogenesis of necrotizing enterocolitis and the dawn of potential therapeutics.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disorder in premature infants that causes significant morbidity and mortality. Research efforts into the pathogenesis of NEC have discovered a pivotal role for the gram-negative bacterial receptor, Toll-like receptor 4 (TLR4), in its development. TLR4 is activated by dysbiotic microbes within the intestinal lumen, which leads to an exaggerated inflammatory response within the developing intestine, resulting in mucosal injury. More recently, studies have identified that the impaired intestinal motility that occurs early in NEC has a causative role in disease development, as strategies to enhance intestinal motility can reverse NEC in preclinical models. There has also been broad appreciation that NEC also contributes to significant neuroinflammation, which we have linked to the effects of gut-derived pro-inflammatory molecules and immune cells which activate microglia in the developing brain, resulting in white matter injury. These findings suggest that the management of the intestinal inflammation may secondarily be neuroprotective. Importantly, despite the significant burden of NEC on premature infants, these and other studies have provided a strong rationale for the development of small molecules with the capability of reducing NEC severity in pre-clinical models, thus guiding the development of specific anti-NEC therapies. This review summarizes the roles of TLR4 signaling in the premature gut in the pathogenesis of NEC, and provides insights into optimal clinical management strategies based upon findings from laboratory studies.

Probiotics and novel probiotic delivery systems.

Necrotizing enterocolitis (NEC) is an infectious and inflammatory intestinal disease that is the most common surgical emergency in the premature patient population. Although the etiology of the disease is multifactorial, intestinal dysbiosis is a hallmark of this disease. Based on this, probiotics may play a therapeutic role in NEC by introducing beneficial bacteria with immunomodulating, antimicrobial, and anti-inflammatory functions into the gastrointestinal tract. Currently, there is no Food and Drug Administration (FDA)-approved probiotic for the prevention and treatment of NEC. All probiotic clinical studies to date have administered the bacteria in their planktonic (free-living) state. This review will discuss established probiotic delivery systems including planktonic probiotics, prebiotics, and synbiotics, as well as novel probiotic delivery systems such as biofilm-based and designer probiotics. We will also shed light on whether or not probiotic efficacy is influenced by administration with breast milk. Finally, we will consider the challenges associated with developing an FDA-approved probiotic for NEC.

High-resolution microbiome analysis reveals exclusionary Klebsiella species competition in preterm infants at risk for necrotizing enterocolitis.

Intestinal colonization with Klebsiella has been linked to necrotizing enterocolitis (NEC), but methods of analysis usually failed to discriminate Klebsiella species or strains. A novel ~ 2500-base amplicon (StrainID) that spans the 16S and 23S rRNA genes was used to generate amplicon sequence variant (ASV) fingerprints for Klebsiella oxytoca and Klebsiella pneumoniae species complexes (KoSC and KpSC, respectively) and co-occurring fecal bacterial strains from 10 preterm infants with NEC and 20 matched controls. Complementary approaches were used to identify cytotoxin-producing isolates of KoSC. Klebsiella species colonized most preterm infants, were more prevalent in NEC subjects versus controls, and replaced Escherichia in NEC subjects. Single KoSC or KpSC ASV fingerprinted strains dominated the gut microbiota, suggesting exclusionary Klebsiella competition for luminal resources. Enterococcus faecalis was co-dominant with KoSC but present infrequently with KpSC. Cytotoxin-producing KoSC members were identified in most NEC subjects and were less frequent in controls. Few Klebsiella strains were shared between subjects. We conclude that inter-species Klebsiella competition, within an environment of KoSC and E. faecalis cooperation, appears to be an important factor for the development of NEC. Preterm infants seem to acquire Klebsiella primarily through routes other than patient-to-patient transmission.

Bifidobacterium: Host-Microbiome Interaction and Mechanism of Action in Preventing Common Gut-Microbiota-Associated Complications in Preterm Infants: A Narrative Review.

The development and health of infants are intertwined with the protective and regulatory functions of different microorganisms in the gut known as the gut microbiota. Preterm infants born with an imbalanced gut microbiota are at substantial risk of several diseases including inflammatory intestinal diseases, necrotizing enterocolitis, late-onset sepsis, neurodevelopmental disorders, and allergies which can potentially persist throughout adulthood. In this review, we have evaluated the role of Bifidobacterium as commonly used probiotics in the development of gut microbiota and prevention of common diseases in preterm infants which is not fully understood yet. The application of Bifidobacterium as a therapeutical approach in the re-programming of the gut microbiota in preterm infants, the mechanisms of host-microbiome interaction, and the mechanism of action of this bacterium have also been investigated, aiming to provide new insights and opportunities in microbiome-targeted interventions in personalized medicine.

Neonatal necrotizing enterocolitis: Clostridium butyricum and Clostridium neonatale fermentation metabolism and enteropathogenicity.

Bacterial colonization in the gut plays a pivotal role in neonatal necrotizing enterocolitis (NEC) development, but the relationship between bacteria and NEC remains unclear. In this study, we aimed to elucidate whether bacterial butyrate end-fermentation metabolites participate in the development of NEC lesions and confirm the enteropathogenicity of Clostridium butyricum and Clostridium neonatale in NEC. First, we produced C.butyricum and C.neonatale strains impaired in butyrate production by genetically inactivating the hbd gene encoding ß-hydroxybutyryl-CoA dehydrogenase that produces end-fermentation metabolites. Second, we evaluated the enteropathogenicty of the hbd-knockout strains in a gnotobiotic quail model of NEC. The analyses showed that animals harboring these strains had significantly fewer and less intense intestinal lesions than those harboring the respective wild-type strains. In the absence of specific biological markers of NEC, the data provide original and new mechanistic insights into the disease pathophysiology, a necessary step for developing potential novel therapies.

Intra-Amniotic Administration-An Emerging Method to Investigate Necrotizing Enterocolitis, In Vivo (Gallus gallus).

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease in premature infants and a leading cause of death in neonates (1-7% in the US). NEC is caused by opportunistic bacteria, which cause gut dysbiosis and inflammation and ultimately result in intestinal necrosis. Previous studies have utilized the rodent and pig models to mimic NEC, whereas the current study uses the in vivo (Gallus gallus) intra-amniotic administration approach to investigate NEC. On incubation day 17, broiler chicken (Gallus gallus) viable embryos were injected intra-amniotically with 1 mL dextran sodium sulfate (DSS) in H2O. Four treatment groups (0.1%, 0.25%, 0.5%, and 0.75% DSS) and two controls (H2O/non-injected controls) were administered. We observed a significant increase in intestinal permeability and negative intestinal morphological changes, specifically, decreased villus surface area and goblet cell diameter in the 0.50% and 0.75% DSS groups. Furthermore, there was a significant increase in pathogenic bacterial (E. coli spp. and Klebsiella spp.) abundances in the 0.75% DSS group compared to the control groups, demonstrating cecal microbiota dysbiosis. These results demonstrate significant physiopathology of NEC and negative bacterial-host interactions within a premature gastrointestinal system. Our present study demonstrates a novel model of NEC through intra-amniotic administration to study the effects of NEC on intestinal functionality, morphology, and gut microbiota in vivo.

The Metabolome and the Gut Microbiota for the Prediction of Necrotizing Enterocolitis and Spontaneous Intestinal Perforation: A Systematic Review.

Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal emergency in preterm neonates. Research on early predictive biomarkers is fundamental. This is a systematic review of studies applying untargeted metabolomics and gut microbiota analysis to evaluate the differences between neonates affected by NEC (Bell's stage II or III), and/or by spontaneous intestinal perforation (SIP) versus healthy controls. Five studies applying metabolomics (43 cases, 95 preterm controls) and 20 applying gut microbiota analysis (254 cases, 651 preterm controls, 22 term controls) were selected. Metabolomic studies utilized NMR spectroscopy or mass spectrometry. An early urinary alanine/histidine ratio >4 showed good sensitivity and predictive value for NEC in one study. Samples collected in proximity to NEC diagnosis demonstrated variable pathways potentially related to NEC. In studies applying untargeted gut microbiota analysis, the sequencing of the V3−V4 or V3 to V5 regions of the 16S rRNA was the most used technique. At phylum level, NEC specimens were characterized by increased relative abundance of Proteobacteria compared to controls. At genus level, pre-NEC samples were characterized by a lack or decreased abundance of Bifidobacterium. Finally, at the species level Bacteroides dorei, Clostridium perfringens and perfringens-like strains dominated early NEC specimens, whereas Clostridium butyricum, neonatale and Propionibacterium acnei those at disease diagnosis. Six studies found a lower Shannon diversity index in cases than controls. A clear separation of cases from controls emerged based on UniFrac metrics in five out of seven studies. Importantly, no studies compared NEC versus SIP. Untargeted metabolomics and gut microbiota analysis are interrelated strategies to investigate NEC pathophysiology and identify potential biomarkers. Expression of quantitative measurements, data sharing via biorepositories and validation studies are fundamental to guarantee consistent comparison of results.

Next-Generation Probiotic Therapy to Protect the Intestines From Injury.

Probiotics are live microorganisms that, when administered in adequate amounts, provide health benefits to the host. Some strains of the probiotic Lactobacillus reuteri (L. reuteri) have both antimicrobial and anti-inflammatory properties that may be exploited for the treatment and prevention of different gastrointestinal diseases, including necrotizing enterocolitis (NEC) and Clostridioides difficile (C. difficile) infection. Our laboratory has developed a new delivery system for L. reuteri in which the probiotic is incubated with biocompatible, semipermeable, porous dextranomer microspheres (DM) that can be loaded with beneficial and diffusible cargo. L. reuteri can be induced to form a biofilm by incubating the bacteria on the surface of these microspheres, which enhances the efficacy of the probiotic. Loading the DM with sucrose or maltose induces L. reuteri to produce more biofilm, further increasing the efficacy of the probiotic. Using a rat model of NEC, L. reuteri administered in its biofilm state significantly increases animal survival, reduces the incidence of NEC, preserves gut barrier function, and decreases intestinal inflammation. In a murine model of Clostridiodes difficile infection, L. reuteri administered in its biofilm state decreases colitis when administered either before or after C. difficile induction, demonstrating both prophylactic and therapeutic efficacy. There are currently no FDA-approved probiotic preparations for human use. An FDA-approved phase I clinical trial of L. reuteri in its biofilm state in healthy adults is currently underway. The results of this trial will be used to support a phase 1 clinical trial in neonates, with the goal of utilizing L. reuteri in its biofilm state to prevent NEC in premature neonates in the future.

Highly Specialized Carbohydrate Metabolism Capability in Bifidobacterium Strains Associated with Intestinal Barrier Maturation in Early Preterm Infants.

"Leaky gut," or high intestinal barrier permeability, is common in preterm newborns. The role of the microbiota in this process remains largely uncharacterized. We employed both short- and long-read sequencing of the 16S rRNA gene and metagenomes to characterize the intestinal microbiome of a longitudinal cohort of 113 preterm infants born between 240/7 and 326/7 weeks of gestation. Enabled by enhanced taxonomic resolution, we found that a significantly increased abundance of Bifidobacterium breve and a diet rich in mother's breastmilk were associated with intestinal barrier maturation during the first week of life. We combined these factors using genome-resolved metagenomics and identified a highly specialized genetic capability of the Bifidobacterium strains to assimilate human milk oligosaccharides and host-derived glycoproteins. Our study proposes mechanistic roles of breastmilk feeding and intestinal microbial colonization in postnatal intestinal barrier maturation; these observations are critical toward advancing therapeutics to prevent and treat hyperpermeable gut-associated conditions, including necrotizing enterocolitis (NEC). IMPORTANCE Despite improvements in neonatal intensive care, necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality. "Leaky gut," or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to NEC. Early detection and intervention to prevent leaky gut in "at-risk" preterm neonates are critical for decreasing the risk of potentially life-threatening complications like NEC. However, the complex interactions between the developing gut microbial community, nutrition, and intestinal barrier function remain largely uncharacterized. In this study, we reveal the critical role of a sufficient breastmilk feeding volume and the specialized carbohydrate metabolism capability of Bifidobacterium in the coordinated postnatal improvement of the intestinal barrier. Determining the clinical and microbial biomarkers that drive the intestinal developmental disparity will inform early detection and novel therapeutic strategies to promote appropriate intestinal barrier maturation and prevent NEC and other adverse health conditions in preterm infants.

Gut microbiome and necrotizing enterocolitis: Understanding the connection to find a cure.

Necrotizing enterocolitis (NEC), a cause of death among premature babies, has defied therapeutics for decades. Bacterial analyses have expanded insights into NEC pathophysiology and roles of the gut microbiome. We discuss the contribution of the gut microbiome and potential therapeutics, notably lactadherin, that may promote gut homeostasis to alleviate NEC.

Hyaluronic Acid 35 kDa Protects against a Hyperosmotic, Formula Feeding Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC), an inflammatory disease of the intestine, is a common gastrointestinal emergency among preterm infants. Intestinal barrier dysfunction, hyperactivation of the premature immune system, and dysbiosis are thought to play major roles in the disease. Human milk (HM) is protective, but the mechanisms underpinning formula feeding as a risk factor in the development of NEC are incompletely understood. Hyaluronic acid 35 kDa (HA35), a bioactive glycosaminoglycan of HM, accelerates intestinal development in murine pups during homeostasis. In addition, HA35 prevents inflammation-induced tissue damage in pups subjected to murine NEC, incorporating Paneth cell dysfunction and dysbiosis. We hypothesized HA35 treatment would reduce histological injury and mortality in a secondary mouse model of NEC incorporating formula feeding. NEC-like injury was induced in 14-day mice by dithizone-induced disruption of Paneth cells and oral gavage of rodent milk substitute. Mortality and histological injury, serum and tissue cytokine levels, stool bacterial sequencing, and bulk RNA-Seq comparisons were analyzed. HA35 significantly reduced the severity of illness in this model, with a trend toward reduced mortality, while RNA-Seq analysis demonstrated HA35 upregulated genes associated with goblet cell function and innate immunity. Activation of these critical protective and reparative mechanisms of the small intestine likely play a role in the reduced pathology and enhanced survival trends of HA-treated pups subjected to intestinal inflammation in this secondary model of NEC, providing potentially interesting translational targets for the human preterm disease.

Longitudinal gut virome analysis identifies specific viral signatures that precede necrotizing enterocolitis onset in preterm infants.

Necrotizing enterocolitis (NEC) is a serious consequence of preterm birth and is often associated with gut bacterial microbiome alterations. However, little is known about the development of the gut virome in preterm infants, or its role in NEC. Here, using metagenomic sequencing, we characterized the DNA gut virome of 9 preterm infants who developed NEC and 14 gestational age-matched preterm infants who did not. Infants were sampled longitudinally before NEC onset over the first 11 weeks of life. We observed substantial interindividual variation in the gut virome between unrelated preterm infants, while intraindividual variation over time was significantly less. We identified viral and bacterial signatures in the gut that preceded NEC onset. Specifically, we observed a convergence towards reduced viral beta diversity over the 10 d before NEC onset, which was driven by specific viral signatures and accompanied by specific viral-bacterial interactions. Our results indicate that bacterial and viral perturbations precede the sudden onset of NEC. These findings suggest that early life virome signatures in preterm infants may be implicated in NEC.

Interpretable prediction of necrotizing enterocolitis from machine learning analysis of premature infant stool microbiota.

BACKGROUND: Necrotizing enterocolitis (NEC) is a common, potentially catastrophic intestinal disease among very low birthweight premature infants. Affecting up to 15% of neonates born weighing less than 1500 g, NEC causes sudden-onset, progressive intestinal inflammation and necrosis, which can lead to significant bowel loss, multi-organ injury, or death. No unifying cause of NEC has been identified, nor is there any reliable biomarker that indicates an individual patient's risk of the disease. Without a way to predict NEC in advance, the current medical strategy involves close clinical monitoring in an effort to treat babies with NEC as quickly as possible before irrecoverable intestinal damage occurs. In this report, we describe a novel machine learning application for generating dynamic, individualized NEC risk scores based on intestinal microbiota data, which can be determined from sequencing bacterial DNA from otherwise discarded infant stool. A central insight that differentiates our work from past efforts was the recognition that disease prediction from stool microbiota represents a specific subtype of machine learning problem known as multiple instance learning (MIL). RESULTS: We used a neural network-based MIL architecture, which we tested on independent datasets from two cohorts encompassing 3595 stool samples from 261 at-risk infants. Our report also introduces a new concept called the "growing bag" analysis, which applies MIL over time, allowing incorporation of past data into each new risk calculation. This approach allowed early, accurate NEC prediction, with a mean sensitivity of 86% and specificity of 90%. True-positive NEC predictions occurred an average of 8 days before disease onset. We also demonstrate that an attention-gated mechanism incorporated into our MIL algorithm permits interpretation of NEC risk, identifying several bacterial taxa that past work has associated with NEC, and potentially pointing the way toward new hypotheses about NEC pathogenesis. Our system is flexible, accepting microbiota data generated from targeted 16S or "shotgun" whole-genome DNA sequencing. It performs well in the setting of common, potentially confounding preterm neonatal clinical events such as perinatal cardiopulmonary depression, antibiotic administration, feeding disruptions, or transitions between breast feeding and formula. CONCLUSIONS: We have developed and validated a robust MIL-based system for NEC prediction from harmlessly collected premature infant stool. While this system was developed for NEC prediction, our MIL approach may also be applicable to other diseases characterized by changes in the human microbiota.

Bifidobacterium longum Subspecies infantis Strain EVC001 Decreases Neonatal Murine Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a disease mainly of preterm infants with a 30-50% mortality rate and long-term morbidities for survivors. Treatment strategies are limited and have not improved in decades, prompting research into prevention strategies, particularly with probiotics. Recent work with the probiotic B. infantis EVC001 suggests that this organism may generate a more appropriate microbiome for preterm infants who generally have inappropriate gut colonization and inflammation, both risk factors for NEC. Experimental NEC involving Paneth cell disruption in combination with bacterial dysbiosis or formula feeding was induced in P14-16 C57Bl/6 mice with or without gavaged B. infantis. Following completion of the model, serum, small intestinal tissue, the cecum, and colon were harvested to examine inflammatory cytokines, injury, and the microbiome, respectively. EVC001 treatment significantly decreased NEC in a bacterial dysbiosis dependent model, but this decrease was model-dependent. In the NEC model dependent on formula feeding, no difference in injury was observed, but trending to significant differences was observed in serum cytokines. EVC001 also improved wound closure at six and twelve hours compared to the sham control in intestinal epithelial monolayers. These findings suggest that B. infantis EVC001 can prevent experimental NEC through anti-inflammatory and epithelial barrier restoration properties.