Antibacterial and anti-inflammatory effects of Lactobacillus reuteri in its biofilm state contribute to its beneficial effects in a rat model of experimental necrotizing enterocolitis.

INTRODUCTION: Necrotizing enterocolitis (NEC) remains a significant surgical emergency in neonates. We have demonstrated the efficacy of Lactobacillus reuteri (Lr) in protecting against experimental NEC when administered as a biofilm by incubation with maltose loaded dextranomer microspheres. Lr possesses antimicrobial and anti-inflammatory properties. We developed mutant strains of Lr to examine the importance of its antimicrobial and anti-inflammatory properties in protecting the intestines from NEC. METHODS: Premature rat pups were exposed to hypoxia/hypothermia/hypertonic feeds to induce NEC. To examine the importance of antimicrobial reuterin and anti-inflammatory histamine, pups received either native or mutant forms of Lr, in either its planktonic or biofilm states, prior to induction of NEC. Intestinal histology was examined upon sacrifice. RESULTS: Compared to no treatment, administration of a single dose of Lr in its biofilm state significantly decreased the incidence of NEC (67% vs. 18%, p < 0.0001), whereas Lr in its planktonic state had no significant effect. Administration of reuterin-deficient or histamine-deficient forms of Lr, in either planktonic or biofilm states, resulted in significant loss of efficacy. CONCLUSION: Antimicrobial and anti-inflammatory effects of Lr contribute to its beneficial effects against NEC. This suggests that both infectious and inflammatory components contribute to the etiology of NEC.

A novel probiotic therapeutic in a murine model of Clostridioides difficile colitis.

For prophylactic therapy, mice received an oral antibiotic cocktail followed by clindamycin injection, followed by probiotic administration (planktonic vs. biofilm state), followed by C. difficile oral gavage. For treatment therapy, mice received antibiotics and C. difficile first, followed by probiotic administration. Clinical sickness scores (CSS) and intestinal histologic injury scores (HIS) were assigned.In the Prophylactic Therapy model, CSS: 67% of untreated mice exposed to C. difficile demonstrated CSS ≥ 6, which is consistent with C. difficile infection (p< .001 compared to unexposed mice). In mice treated with planktonic Lr, 55% had a CSS ≥ 6, but only 19% of mice treated with Lr in its biofilm state had CSS ≥ 6 (p< .001). Mice receiving Lr + DM-Maltose lost the least amount of weight compared to mice receiving saline (p = .004676) or to mice receiving Lr (p= .003185). HIS: 77% of untreated mice exposed to C. difficile had HIS scores ≥4, which is consistent with C. difficile infection. In mice treated with planktonic Lr, 62% had HIS ≥4, but only 19% of mice treated with Lr in its biofilm state had HIS ≥4. (p< .001). Additionally, mice treated with Lr in its biofilm state had better survival compared to untreated mice and to mice treated with planktonic Lr (p ≤ 0.05). Similar findings for weight loss, CSS, HIS and survival were obtained for Treatment Therapy.A single dose of Lactobacillus reuteri in its biofilm state reduces the severity and incidence of experimental C. difficile infection when administered as both prophylactic and treatment therapy.

Development of a Standardized Scoring System to Assess a Murine Model of Clostridium difficile Colitis.

Background: Clostridium difficile infection is the most common cause of antimicrobial-associated diarrhea. Our aim was to introduce a novel and efficient clinical sickness score (CSS), and to define a detailed histologic injury score (HIS) in a murine model of C. difficile colitis. Methods: Mice received an antibiotic cocktail (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) for 96 h. After 48 h, mice received an intraperitoneal injection of clindamycin, followed by oral C. difficile (1.5 × 107 CFU). Signs of sickness were scored using a novel CSS (range 0-12) with scores ≥6 consistent with C. difficile colitis. Intestinal tissue was analyzed utilizing an adapted HIS (range 0-9) with scores ≥4 consistent with C. difficile colitis. Stool was analyzed for C. difficile, and survival evaluated. Results: No control mice showed signs of sickness, whereas 23% of mice receiving antibiotics alone and 65% of mice exposed to antibiotics and subsequently C. difficile demonstrated signs of sickness (p = 0.0134). No control mice had histologic injury, whereas 8% of mice receiving antibiotics alone and 75% of mice exposed to antibiotics followed by C. difficile had evidence of histologic injury (p = 0.0001). Mice exposed to C. difficile lost more weight, although not significant (p = 0.070). Mice that received C. difficile had decreased survival compared to control mice and mice receiving antibiotics only (p = 0.03). Conclusions: We have developed a novel clinical scoring system, and detailed histological grading system, that enables the objective evaluation of a murine C. difficile colitis model. This model allows the study of this disease in a host that demonstrates clinical and histologic signs comparable to human C. difficile infection. This will allow for improved study of therapeutics for this disease in the future.

Treatment of experimental necrotizing enterocolitis with stem cell-derived exosomes.

PURPOSE: Necrotizing enterocolitis (NEC) remains a devastating disease in premature infants. We previously showed that four stem cell (SC) types equivalently improve experimental NEC. Exosomes are intercellular nanovesicles containing RNA, miRNA, DNA, and protein. Because SC therapy faces challenges, our aim was to determine if the beneficial effects of SC are achievable with cell-free exosomes. METHODS: Exosomes from four SC types were compared: (1) amniotic fluid-derived mesenchymal SC (AF-MSC); (2) bone marrow-derived MSC (BM-MSC); (3) amniotic fluid-derived neural SC (AF-NSC); and (4) neonatal enteric NSC (E-NSC). Rat pups exposed to NEC received a varying concentration of a single type of exosome with control pups receiving PBS only. Intestinal damage was graded histologically. RESULTS: The incidence of NEC was 0% in unstressed pups and 60.7% in control pups subjected to NEC. Exosomes (4.0×108) reduced NEC incidence to: AF-MSC 25.0%; BM-MSC 23.1%; AF-NSC 11.1%; E-NSC 27.3%. When administered at a concentration of at least 4.0×108, all groups demonstrated a significant reduction in NEC compared to untreated pups. At this minimum concentration, there was no difference in treatment efficacy between exosomes and the SC from which they were derived. CONCLUSION: Stem cell-derived exosomes reduce the incidence and severity of experimental NEC as effectively as the stem cells from which they are derived, supporting the potential for novel cell-free exosome therapy for NEC. TYPE OF STUDY: Basic science.