D. H. Klerk1,2, Z. Raouf1, D. J. Scheese1, C. Tragesser1, J. W. Duess1, S. Williams-McLeod1, K. Tsuboi1, M. G. El Baassiri1, W. B. Fulton1, H. Moore1, M. Wang1, T. Prindle1, S. Wang1, C. P. Sodhi1, D. J. Hackam1 1Johns Hopkins University School Of Medicine, Pediatric Surgery, Baltimore, MD, USA 2University Medical Center Groningen, Beatrix Children’s Hospital, Neonatology, Groningen, GRONINGEN, Netherlands
Introduction: The administration of probiotics to premature infants is associated with a significant decrease in necrotizing enterocolitis (NEC) incidence through mechanisms that are largely unknown. We have previously shown that newborn mice display impaired intestinal motility, consistent with that seen in the premature human, due to a lack of enteric glia. We have also shown that impaired intestinal motility is an early trigger for the development of NEC, suggesting that factors that can restore motility may serve to reduce the incidence of NEC. We now hypothesize that colonization of the premature intestine with a multi-strain probiotic will promotegastrointestinal motility and protect against NEC in mice.
Methods: Newborn C57BL/6 mice were fed a multi-strain probiotic from postnatal day 3 to 11, consisting of Bifidobacterium infantis, Bifidobacterium lactis and Streptococcus thermophilus. Colonization was tested by 16s qRT-PCR using strain-specific primers on bacterial DNA isolated from stool. To study the anti-inflammatory roles of probiotics, mice were exposed to lipopolysaccharides (LPS, 5mg/kg, i.p., 6h). To study the role of probiotics in NEC, mice were exposed to an experimental model of NEC by NEC-formula feeding and intermittent hypoxia (5% O2, 10min exposure, twice daily) for 4 days. To evaluate gastrointestinal motility, mice were fed FITC-dextran (70Kda, 10mg/ml, 50ul/mouse), 30-min prior to sacrifice. The small intestine was divided into 1cm sections and FITC fluorescence was measured for each section. The small intestinal transit time was derived from the position of the geometric (Geom) center of FITC-dextran.
Results: Probiotic colonization was confirmed in probiotic-fed mice and not in control mice, confirming the experimental approach. LPS administration induced an increased pro-inflammatory response, which was not attenuated by probiotic administration (Tnf, Probiotics/LPS 10.4 vs. non-probiotics/LPS 8.3, ns), and dysmotility in both probiotic-fed and non-probiotic mice (Geom Center, Probiotics/LPS 4.2 vs. non-probiotics/LPS 4.8, ns). Strikingly, the induction of NEC by formula feeding, hypoxia, NEC bacteria and 0.5% DSS was associated with markedly reduced intestinal motility that was significantly improved in probiotic colonized mice (Geom Center, Probiotics/NEC 4.4 vs. non-probiotics/NEC 2.9, *p<0.05). Importantly, probiotic administration significantly improved clinical features of NEC development (pneumatosis, discoloration of the bowel, and mortality rate (Probiotics/NEC 2 vs. non-probiotics/NEC 5, *p<0.05).
Conclusion: Previous studies show that gastrointestinal dysmotility develops in NEC before the onset of systemic disease. These findings suggest that probiotic administration may prevent NEC in part by restoring gastrointestinal motility.