M. Isani1, J. Bowling1, C. Moneme1, K. A. Durairaj1, M. Elizee1, J. Golden1, B. Bell1, L. Illingworth1, G. Anatoly1, H. Ford1 1Children’s Hospital Los Angeles,Los Angeles, CA, USA
Introduction: Necrotizing enterocolitis (NEC) is a devastating disease that affects premature infants. Lactobacillus probiotics have been shown in a number of studies to protect against NEC.However, results of trials remain inconclusive due to the use of variable species and doses. Furthermore, whether the lactobacilli are colonizing the intestine remains unknown.We propose that an efficient probiotic strain is naturally occurring and should not only protect the intestine against NEC, but should be capable of colonizing the GI tract.
Methods: Animal experiments were approved and neonates were obtained from timed- pregnant rats and subjected to formula feeding and hypoxia for 4 days. Rat pups were sacrificed on day 4. To enumerate and isolate the lactobacilli, large intestine was plated on agar. After incubation at 37oC, colonies were classified according to morphology and Gram stain. Species identity was established by 16S rRNA sequencing. Three species were identified: L.reuteri, L.murinus, and L.acidophilus.
Lactobacillus reuteri was re-introduced to newborn rats at 107 and 108 CFU/animal. Dosage for colonization was determined based on titers of L.reuteri in the large intestine. All three species of Lactobacillus were introduced to neonatal rats at the 107 and 108 CFU/animal in the standard rat model. Next, to determine if the Lactobacillus species protect intestine against a known NEC pathogen, Cronobacter muytjensii, we introduced Cronobacter into our NEC model with the first feeding and the lactobacillus species with the second feed. Terminal ileum sections were stained with H&E and examined to determine NEC score. Sections were also immunostained to determine if COX-2 and iNOS levels were changed in the presence of the lactobacillus species and Cronobacter.
Results: Three species of lactobacilli were isolated from neonatal rat intestine: L.reuteri, L.murinus, and L.acidophilus. Upon re-introduction of L.reuteri at various dosages, we achieved colonization at 107 (p=0.006) and 108 (p=0.0146) CFU/animal. All three species were able to colonize the intestine. However, NEC scores were only lower in groups that received L.murinus (p= 0.0484 at 107 CFU/animal n=25 and p=0.0310 at 108 CFU/animal, n=35). Furthermore, L. murinus was able to protect against NEC when challenged by a known NEC pathogen, Cronobacter muytjensii (p=0.0455 at 108 CFU/animal, n=6). Pups treated with Cronobacter had increased expression of both COX-2 and iNOS; in contrast, those treated with L. murinus had decreased COX-2 and iNOS expression in tissue.
Conclusion: L.murinus is a naturally occurring species that is able to colonize the intestine and protect against NEC in the standard rat model. It can also protect against NEC when challenged by known NEC pathogen, Cronobacter muytjensii. L.murinus decreases iNOS and COX-2 expression in tissue and may serve as a preventive and therapeautic treatment against NEC.