Lactobacillus rhamnosus L34 Attenuates Gut Translocation-Induced Bacterial Sepsis in Murine Models of Leaky Gut

Abstract

Gastrointestinal (GI) bacterial translocation in sepsis is well known, but the role of Lactobacillus species probiotics is still controversial. We evaluated the therapeutic effects of Lactobacillus rhamnosus L34 in a new sepsis model of oral administration of pathogenic bacteria with GI leakage induced by either an antibiotic cocktail (ATB) and/or dextran sulfate sodium (DSS). GI leakage with ATB, DSS, and DSS plus ATB (DSS+ATB) was demonstrated by fluorescein isothiocyanate (FITC)-dextran translocation to the circulation. The administration of pathogenic bacteria, either Klebsiella pneumoniae or Salmonella enterica serovar Typhimurium, enhanced translocation. Bacteremia was demonstrated within 24 h in 50 to 88% of mice with GI leakage plus the administration of pathogenic bacteria but not with GI leakage induction alone or bacterial gavage alone. Salmonella bacteremia was found in only 16 to 29% and 0% of mice with Salmonella and Klebsiella administrations, respectively. Klebsiella bacteremia was demonstrated in 25 to 33% and 10 to 16% of mice with Klebsiella and Salmonella administrations, respectively. Lactobacillus rhamnosus L34 attenuated GI leakage in these models, as shown by the reductions of FITC-dextran gut translocation, serum interleukin-6 (IL-6) levels, bacteremia, and sepsis mortality. The reduction in the amount of fecal Salmonella bacteria with Lactobacillus treatment was demonstrated. In addition, an anti-inflammatory effect of the conditioned medium from Lactobacillus rhamnosus L34 was also demonstrated by the attenuation of cytokine production in colonic epithelial cells in vitro In conclusion, Lactobacillus rhamnosus L34 attenuated the severity of symptoms in a murine sepsis model induced by GI leakage and the administration of pathogenic bacteria.

Keywords: Lactobacillus rhamnosus L34; antibiotics; dextran sulfate solution; gastrointestinal leakage; murine model.

FIG 1

(A and B) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation (A) and spontaneous elevation of serum (1→3)-β-d-glucan (BG) levels (B) in mice administered normal saline (NSS), an antibiotic cocktail (ATB), a dextran sulfate sodium (DSS) solution, and DSS with ATB (DSS+ATB). (C to F) Time course of gut leakage for each model with oral administration of bacteria (Salmonella enterica serovar Typhimurium or Klebsiella pneumoniae) 2 days before (day −2), on the day of (day 0), and after (days 1 and 5) administration. FITC-dextran was orally administered 3 h before blood collection at each time point. Values are means ± standard errors.

FIG 2

Survival analysis, bacteremia 6 h and 24 h after bacterial gavage, and organisms identified from blood in gut leakage models of ATB, DSS, and DSS+ATB with oral administration of Salmonella enterica serovar Typhimurium (A to G) or Klebsiella pneumoniae (E to H). Values are means ± standard errors. −ve, negative.

FIG 3

(A to C) Survival analysis with control normal saline solution (NSS) (A) and in gut leakage models of ATB (B) and DSS (C) with oral administration Lactobacillus rhamnosus L34 (Lacto) 1 day prior to administration of Klebsiella pneumoniae. (D to I) Bacteremia, serum IL-6 levels, and gut leakage measured by using FITC-dextran 24 h (D to F) and 120 h (G to I) after Klebsiella pneumoniae administration. Values are means ± standard errors.

FIG 4

(A to C) Survival analysis with control NSS (A) and in gut leakage models of ATB (B) and DSS (C) with oral administration of Lactobacillus rhamnosus L34 1 day prior to administration of Salmonella enterica serovar Typhimurium. (D to I) Bacteremia, serum IL-6 levels, and gut leakage measured by FITC-dextran levels 24 h (D to F) and 120 h (G to I) after Klebsiella pneumoniae administration. Values are means ± standard errors.

FIG 5

(A and B) Fecal burdens of Salmonella enterica serovar Typhimurium measured by turbidity in selenite cystine broth, a selective enrichment medium, 24 h (A) and 120 h (B) after Salmonella enterica serovar Typhimurium administration in gut leakage models of ATB and DSS with oral administration of Lactobacillus rhamnosus L34 1 day prior to administration of Salmonella enterica serovar Typhimurium. (C and D) Semiquantitative analysis of enumeration of fecal bacteria by real-time PCR targeting Salmonella enterica serovar Typhimurium. Values are means ± standard errors.

FIG 6

Representative pictures of the turbidity of selenite cystine broth, a selective enrichment medium. Different levels of growth represent fecal burdens of Salmonella enterica serovar Typhimurium in gut leakage models of ATB (A) and DSS (B) with oral administration of Lactobacillus rhamnosus L34 1 day prior to administration of Salmonella enterica serovar Typhimurium. Only pictures 24 h after Salmonella enterica serovar Typhimurium administration are shown.

FIG 7

Cytokines in the supernatants of Caco-2 and HT-29 cells after incubation with Lactobacillus rhamnosus L34-conditioned medium, with or without heat-killed bacteria (Salmonella enterica serovar Typhimurium or Klebsiella pneumoniae). Independent experiments were performed in triplicate.

FIG 8

Salmonella-induced IL-8 production from colonic epithelial cells (Caco-2 and HT-29) after incubation with conditioned medium from L. rhamnosus L34 (LCM) that was treated by heat exposure (A and B), enzyme digestion (C and D), and fractionation (E and F). Independent experiments were performed in triplicate.

FIG 9

Klebsiella-induced IL-8 production from colonic epithelial cells (Caco-2 and HT-29) after incubation with conditioned medium from L. rhamnosus L34 (LCM) that was treated by heat exposure (A and B), enzyme digestion (C and D), and fractionation (E and F). Independent experiments were performed in triplicate.

FIG 10

Induction of phosphorylated NF-κB (p-NF-κB) and TLR4 expression in colonic epithelial cells (Caco-2 and HT-29) after incubation with conditioned medium from L. rhamnosus L34 (LCM) and administration of heat-killed Salmonella (A to D) and Klebsiella (E to H) bacteria. Independent experiments were performed in triplicate.