Kathene C. Johnson-Henry

Probiotics prevent bacterial translocation and improve intestinal barrier function in rats following chronic psychological stress

Background and aim: Chronic psychological stress, including water avoidance stress (WAS), induces intestinal mucosal barrier dysfunction and impairs mucosal defences against luminal bacteria. The aim of this study was to determine the ability of a defined probiotic regimen to prevent WAS induced intestinal pathophysiology. Methods: Male rats were subjected to either WAS or sham stress for one hour per day for 10 consecutive days. Additional animals received seven days of Lactobacillus helveticus and L rhamnosus in the drinking water prior to stress and remained on these probiotics for the duration of the study. Rats were then sacrificed, intestinal segments assessed in Ussing chambers, and mesenteric lymph nodes cultured to determine bacterial translocation. Results: All animals remained healthy for the duration of the study. Chronic WAS induced excess ion secretion (elevated baseline short circuit current) and barrier dysfunction (increased conductance) in both the ileum and colon, associated with increased bacterial adhesion and penetration into surface epithelial cells. Approximately 70% of rats subjected to WAS had bacterial translocation to mesenteric lymph nodes while there was no bacterial translocation in controls. Probiotic pretreatment alone had no effect on intestinal barrier function. However, WAS induced increased ileal short circuit current was reduced with probiotics whereas there was no impact on altered conductance. Pretreatment of animals with probiotics also completely abrogated WAS induced bacterial adhesion and prevented translocation of bacteria to mesenteric lymph nodes. Conclusion: These findings indicate that probiotics can prevent chronic stress induced intestinal abnormalities and, thereby, exert beneficial effects in the intestinal tract.

Lactobacillus rhamnosus Strain GG Prevents Enterohemorrhagic Escherichia coli O157:H7-Induced Changes in Epithelial Barrier Function

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimately attaches to intestinal epithelial monolayers and produces attaching and effacing (A/E) lesions. In addition, EHEC infection causes disruptions of intercellular tight junctions, leading to clinical sequelae that include acute diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. Current therapy remains supportive since antibiotic therapy increases the risk of systemic complications. This study focused on the potential therapeutic effect of an alternative form of therapy, probiotic Lactobacillus rhamnosus strain GG, to attenuate EHEC-induced changes in paracellular permeability in polarized MDCK-I and T84 epithelial cell monolayers. Changes in epithelial cell morphology, electrical resistance, dextran permeability, and distribution and expression of claudin-1 and ZO-1 were assessed using phase-contrast, immunofluorescence, and transmission electron microscopy and macromolecular flux. This study demonstrated that pretreatment of polarized MDCK-I and T84 cells with the probiotic L. rhamnosus GG reduced morphological changes and diminished the number of A/E lesions induced in response to EHEC O157:H7 infection. With probiotic pretreatment there was corresponding attenuation of the EHEC-induced drop in electrical resistance and the increase in barrier permeability assays. In addition, L. rhamnosus GG protected epithelial monolayers against EHEC-induced redistribution of the claudin-1 and ZO-1 tight junction proteins. In contrast to the effects seen with the live probiotic, heat-inactivated L. rhamnosus GG had no effect on EHEC binding and A/E lesion formation or on disruption of the barrier function. Collectively, these findings provide in vitro evidence that treatment with the probiotic L. rhamnosus strain GG could prove to be an effective management treatment for preventing injury of the epithelial cell barrier induced by A/E bacterial enteropathogens.

Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells.

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface‐layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S‐layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp‐2 and T84) cells were treated with S‐layer protein extract alone, infected with E. coli O157:H7, or pretreated with S‐layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S‐layer protein extracts were characterized by phase‐contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre‐treatment of host epithelial cells with S‐layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching‐effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non‐viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.

Probiotics Reduce Enterohemorrhagic Escherichia coli O157:H7- and Enteropathogenic E. coli O127:H6-Induced Changes in Polarized T84 Epithelial Cell Monolayers by Reducing Bacterial Adhesion and Cytoskeletal Rearrangements

ABSTRACT The aim of this study was to determine if probiotics reduce epithelial injury following exposure to Escherichia coli O157:H7 and E. coli O127:H6. The pretreatment of intestinal (T84) cells with lactic acid-producing bacteria reduced the pathogen-induced drop in transepithelial electrical resistance. These findings demonstrate that probiotics prevent epithelial injury induced by attaching-effacing bacteria.

Unraveling Mechanisms of Action of Probiotics

Probiotics are defined as living organisms that, when administered in sufficient numbers, are of benefit to the host. Current evidence indicates that varying probiotic strains mediate their effects by a variety of different effects that are dependent on the dosage employed as well as the route and frequency of delivery. Some probiotics act in the lumen of the gut by elaborating antibacterial molecules such as bacteriocins; others enhance the mucosal barrier by increasing the production of innate immune molecules, including goblet cell-derived mucins and trefoil factors and defensins produced by intestinal Paneth cells; and other probiotics mediate their beneficial effects by promoting adaptive immune responses (secretory immune globulin A, regulatory T cells, interleukin-10). Some probiotics have the capacity to activate receptors in the enteric nervous system, which could be used to promote pain relief in the setting of visceral hyperalgesia. Future development of the effective use of probiotics in treating various gastroenterological disorders in human participants should take advantage of this new knowledge. The creation of novel formulations of probiotics could be directed to effectively target certain mechanisms of actions that are altered in specific disease states.

Probiotics Reduce Bacterial Colonization and Gastric Inflammation in H. pylori-Infected Mice

Probiotics are characterized by their ability to interact with commensal microflora in the gastrointestinal tract to produce beneficial health effects. In vitro studies suggest that Lactobacillus species have the potential to suppress the growth of Helicobacter pylori. The goal of this study was to determine if pretreatment of mice with a commercial mixture of live probiotics (L. rhamnosus, strain R0011, and L. acidophilus, strain R0052) would suppress colonization of H. pylori, strain SS1. Thirty C57BL/6 female mice were divided into four groups: Group A was fed sterile water, group B received probiotics in sterile drinking water, group C was challenged orogastrically with H. pylori, and group D was pretreated with probiotics in drinking water prior to and following challenge with H. pylori. Rectal swabs, stomach homogenates, and luminal contents from ileum and colon were plated onto colistin nalidixic acid plates. Serial dilutions of stomach homogenates were plated onto H. pylori-sensitive agar plates and incubated under microaerophilic conditions. Tissue samples from the stomach were analyzed histologically to determine the degree of H. pylori colonization, mucosal inflammation, and epithelial cell apoptosis. Probiotics in drinking water did not affect the overall well-being of mice. Lactobacillus species were excreted in stools over the entire duration of treatment. Pretreatment with probiotics reduced the number of mice with H. pylori growth from stomach homgenates (100 to 50%; P = 0.02). The percentage of mice with moderate-severe H. pylori-induced inflammation in the gastric antrum was reduced with probiotic pretreatment (71 to 29%; P = 0.14). However, pretreatment with probiotics did not prevent H. pylori-induced apoptosis in the gastric mucosa. This preparation of probiotics provided a safe and novel approach for reducing H. pylori colonization and bacterial-induced inflammation of mice.

Amelioration of the Effects of Citrobacter rodentium Infection in Mice by Pretreatment with Probiotics

BACKGROUND Citrobacter rodentium is a naturally occurring murine pathogen that causes colonic epithelial-cell hyperplasia, disrupts the colonic mucosa, and elicits a predominantly T helper 1 cellular immune response; it thereby serves as a model for the study of mechanisms of disease induced by human attaching-effacing pathogens. We sought to determine whether pretreatment of mice with a mixture of Lactobacillus rhamnosus and L. acidophilus probiotics would attenuate C. rodentium-induced colonic disease in mice. METHODS Mice were administered sterile drinking water, probiotics (10(9) cfu/mL) in sterile drinking water, maltodextrin in sterile drinking water, orogastric C. rodentium (10(7) cfu in 0.1 mL), or maltodextrin in sterile drinking water for 1 week before C. rodentium infection, or they were pretreated with probiotics (10(9) cfu/mL) for 1 week before challenge with C. rodentium. RESULTS Mice that received viable probiotics remained healthy. C. rodentium infection elicited mucosal inflammation, epithelial-cell hyperplasia, apoptosis in the colon, and interferon (IFN)- gamma production by splenocytes. Pretreatment with probiotics decreased levels of all but IFN- gamma production. CONCLUSIONS Pretreatment with probiotics attenuates the effects of C. rodentium infection in mice. Understanding the mechanism of these beneficial effects will aid in determining the efficacy of probiotics in preventing infection with related attaching-effacing enteric pathogens in humans.

Vitamin D Deficiency Promotes Epithelial Barrier Dysfunction and Intestinal Inflammation

BACKGROUND Vitamin D, an important modulator of the immune system, has been shown to protect mucosal barrier homeostasis. This study investigates the effects of vitamin D deficiency on infection-induced changes in intestinal epithelial barrier function in vitro and on Citrobacter rodentium-induced colitis in mice. METHODS Polarized epithelial Caco2-bbe cells were grown in medium with or without vitamin D and challenged with enterohemorrhagic Escherichia coli O157:H7. Barrier function and tight junction protein expression were assessed. Weaned C57BL/6 mice were fed either a vitamin D-sufficient or vitamin D-deficient diet and then infected with C. rodentium. Disease severity was assessed by histological analysis, intestinal permeability assay, measurement of inflammatory cytokine levels, and microbiome analysis. RESULTS 1,25(OH)2D3 altered E. coli O157:H7-induced reductions in transepithelial electrical resistance (P < .01), decreased permeability (P < .05), and preserved barrier integrity. Vitamin D-deficient mice challenged with C. rodentium demonstrated increased colonic hyperplasia and epithelial barrier dysfunction (P < .0001 and P < .05, respectively). Vitamin D deficiency resulted in an altered composition of the fecal microbiome both in the absence and presence of C. rodentium infection. CONCLUSIONS This study demonstrates that vitamin D is an important mediator of intestinal epithelial defenses against infectious agents. Vitamin D deficiency predisposes to more-severe intestinal injury in an infectious model of colitis.

Strain-specific probiotic (Lactobacillus helveticus) inhibition of Campylobacter jejuni invasion of human intestinal epithelial cells

Campylobacter jejuni is the most common bacterial cause of enterocolitis in humans, leading to diarrhoea and chronic extraintestinal diseases. Although probiotics are effective in preventing other enteric infections, beneficial microorganisms have not been extensively studied with C. jejuni. The aim of this study was to delineate the ability of selected probiotic Lactobacillus strains to reduce epithelial cell invasion by C. jejuni. Human colon T84 and embryonic intestine 407 epithelial cells were pretreated with Lactobacillus strains and then infected with two prototypic C. jejuni pathogens. Lactobacillus helveticus, strain R0052 reduced C. jejuni invasion into T84 cells by 35-41%, whereas Lactobacillus rhamnosus R0011 did not reduce pathogen invasion. Lactobacillus helveticus R0052 also decreased invasion of one C. jejuni isolate (strain 11168) into intestine 407 cells by 55%. Lactobacillus helveticus R0052 adhered to both epithelial cell types, which suggest that competitive exclusion could contribute to protection by probiotics. Taken together, these findings indicate that the ability of selected probiotics to prevent C. jejuni-mediated disease pathogenesis depends on the pathogen strain, probiotic strain and the epithelial cell type selected. The data support the concept of probiotic strain selectivity, which is dependent on the setting in which it is being evaluated and tested.

Probiotics are effective for the prevention and treatment of Citrobacter rodentium-induced colitis in mice.

BACKGROUND Probiotics prevent disease induced by Citrobacter rodentium, a murine-specific enteric pathogen. Whether probiotics can be used to interrupt the infectious process following initiation of infection was determined. METHODS C57BL/6 adult and neonatal mice were challenged with C. rodentium, and a probiotic mixture containing Lactobacillus helveticus and Lactobacillus rhamnosus was provided 1 week before bacterial challenge, concurrently with infection, or 3 days and 6 days after infection. Mice were sacrificed 10 days after infection, and disease severity was assessed by histological analysis and in vivo intestinal permeability assay. Inflammatory pathways and the composition of the fecal microbiome were assessed in adult mice. RESULTS Preadministration and coadministration of probiotics ameliorated C. rodentium-induced barrier dysfunction, epithelial hyperplasia, and binding of the pathogen to host colonocytes in adults, with similar findings in neonatal mice. Upregulated tumor necrosis factor α and interferon γ transcripts were suppressed in the pretreated probiotic group, whereas interleukin 17 transcription was suppressed with probiotics given up to 3 days after infection. Probiotics promoted transcription of interleukin 10 and FOXP3, and increased follicular T-regulatory cells in pretreatment mice. C. rodentium infection resulted in an altered fecal microbiome, which was normalized with probiotic intervention. CONCLUSIONS This study provides evidence that probiotics can prevent illness and treat disease in an animal model of infectious colitis.