Human-derived probiotic Lactobacillus reuteri demonstrate antimicrobial activities targeting diverse enteric bacterial pathogens

Lactobacillus reuteri is a commensal-derived anaerobic probiotic that resides in the human gastrointestinal tract. L. reuteri converts glycerol into a potent broad-spectrum antimicrobial compound, reuterin, which inhibits the growth of gram-positive and gram-negative bacteria. In this study, we compared four human-derived L. reuteri isolates (ATCC 55730, ATCC PTA 6475, ATCC PTA 4659 and ATCC PTA 5289) in their ability to produce reuterin and to inhibit the growth of different enteric pathogens in vitro. Reuterin was produced by each of the four L. reuteri strains and assessed for biological activity. The minimum inhibitory concentration (MIC) of reuterin derived from each strain was determined for the following enteric pathogens: enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, Salmonella enterica, Shigella sonnei and Vibrio cholerae. We also analyzed the relative abilities of L. reuteri to inhibit enteric pathogens in a pathogen overlay assay. The magnitude of reuterin production did not directly correlate with the relative ability of L. reuteri to suppress the proliferation of enteric pathogens. Additional antimicrobial factors may be produced by L. reuteri, and multiple factors may act synergistically with reuterin to inhibit enteric pathogens.     

Glycerol induces reuterin production and decreases Escherichia coli population in an in vitro model of colonic fermentation with immobilized human feces

Lactobacillus reuteri ATCC 55730 is a probiotic strain that produces, in the presence of glycerol, reuterin, a broad-spectrum antimicrobial substance. This strain has been shown to prevent intestinal infections in vivo; however, its mechanisms of action, and more specifically whether reuterin production occurs within the intestinal tract, are not known. In this study, the effects of L. reuteri ATCC 55730 on intestinal microbiota and its capacity to secrete reuterin from glycerol in a novel in vitro colonic fermentation model were tested. Two reactors were inoculated with adult immobilized fecal microbiota and the effects of daily addition of L. reuteri into one of the reactors (c.10(8) CFU mL(-1)) without or with glycerol were tested on major bacterial populations and compared with addition of glycerol or reuterin alone. The addition of glycerol alone or with L. reuteri increased numbers of the Lactobacillus-Enterococcus group and decreased Escherichia coli. The addition of reuterin significantly and selectively decreased E. coli without affecting other bacterial populations. The observed decrease in E. coli concentration during the addition of glycerol (in presence or absence of L. reuteri) could be due to in situ reuterin production because 1,3-propanediol, a typical product of glycerol fermentation, was detected during the addition of glycerol.     

Comparative Genome Analysis of Lactobacillus reuteri and Lactobacillus fermentum Reveal a Genomic Island for Reuterin and Cobalamin Production.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.     

Characterization of growth and metabolite production of Lactobacillus reuteri during glucose/glycerol cofermentation in batch and continuous cultures

In anaerobic batch cultures of Lactobacillus reuteri, glucose/glycerol cofermentation resulted in production of reuterin (-hydroxypropionaldehyde) and 1,3 propanediol at the expense of ethanol and lactate. In anaerobic chemostat cultures of L. reuteri, glucose/glycerol cofermentation resulted in an increased ethanol production and a decreased lactate production. Moreover, reuterin and 1,3 propanediol were produced in significant amounts. These results demonstrate that growing L. reuteri cells have the ability to produce reuterin. © Rapid Science Ltd. 1998     

Fasting increases microbiome-based colonization resistance and reduces host inflammatory responses during an enteric bacterial infection

Reducing food intake is a common host response to infection, yet it remains unclear whether fasting is detrimental or beneficial to an infected host. Despite the gastrointestinal tract being the primary site of nutrient uptake and a common route for infection, studies have yet to examine how fasting alters the host’s response to an enteric infection. To test this, mice were fasted before and during oral infection with the invasive bacterium Salmonella enterica serovar Typhimurium. Fasting dramatically interrupted infection and subsequent gastroenteritis by suppressing Salmonella’s SPI-1 virulence program, preventing invasion of the gut epithelium. Virulence suppression depended on the gut microbiota, as Salmonella’s invasion of the epithelium proceeded in fasting gnotobiotic mice. Despite Salmonella’s restored virulence within the intestines of gnotobiotic mice, fasting downregulated pro-inflammatory signaling, greatly reducing intestinal pathology. Our study highlights how food intake controls the complex relationship between host, pathogen and gut microbiota during an enteric infection.     

Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.     

Salmonella-induced mucosal lectin RegIIIβ kills competing gut microbiota

Intestinal inflammation induces alterations of the gut microbiota and promotes overgrowth of the enteric pathogen Salmonella enterica by largely unknown mechanisms. Here, we identified a host factor involved in this process. Specifically, the C-type lectin RegIIIβ is strongly upregulated during mucosal infection and released into the gut lumen. In vitro, RegIIIβ kills diverse commensal gut bacteria but not Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium). Protection of the pathogen was attributable to its specific cell envelope structure. Co-infection experiments with an avirulent S. Typhimurium mutant and a RegIIIβ-sensitive commensal E. coli strain demonstrated that feeding of RegIIIβ was sufficient for suppressing commensals in the absence of all other changes inflicted by mucosal disease. These data suggest that RegIIIβ production by the host can promote S. Typhimurium infection by eliminating inhibitory gut microbiota.     

Lactobacillus reuteri CRL1098 produces cobalamin

We found that Lactobacillus reuteri CRL1098, a lactic acid bacterium isolated from sourdough, is able to produce cobalamin. The sugar-glycerol cofermentation in vitamin B(12)-free medium showed that this strain was able to reduce glycerol through a well-known cobalamin-dependent reaction with the formation of 1,3-propanediol as a final product. The cell extract of L. reuteri corrected the coenzyme B12 requirement of Lactobacillus delbrueckii subsp. lactis ATCC 7830 and allowed the growth of Salmonella enterica serovar Typhimurium (metE cbiB) and Escherichia coli (metE) in minimal medium. Preliminary genetic studies of cobalamin biosynthesis genes from L. reuteri allowed the identification of cob genes which encode the CobA, CbiJ, and CbiK enzymes involved in the cobalamin pathway. The cobamide produced by L. reuteri, isolated in its cyanide form by using reverse-phase high-pressure liquid chromatography, showed a UV-visible spectrum identical to that of standard cyanocobalamin (vitamin B12).     

Salmonella Typhimurium SPI-1 genes promote intestinal but not tonsillar colonization in pigs

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in several animal species. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections of pigs, however, is not well described. The interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants with disruptions in the SPI-1 genes hilA, sipA and sipB with porcine intestinal epithelial cells were characterized in vitro and in a ligated intestinal loop model in pigs. HilA and SipB were essential in the invasion of porcine intestinal epithelial cells in vitro. A sipA mutant was impaired for invasion using a polarized cell line, but fully invasive in a non-polarized cell line. All SPI-1 mutants induced a significant decrease in influx of neutrophils in the porcine intestinal loop model compared with the wild type strain. Pigs were orally inoculated with 10(8) colony forming units of both the wild type Salmonella Typhimurium strain and its isogenic sipB::kan mutant strain. The sipB mutant strain was significantly impaired to invade the intestinal, but not the tonsillar tissue, one day after inoculation and was unable to efficiently colonize the intestines and the GALT, but not the tonsils, 3 days after inoculation. This study shows that SPI-1 plays a crucial role in the invasion and colonization of the porcine gut and in the induction of influx of neutrophils towards the intestinal lumen, but not in the colonization of the tonsils.     

Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits

AIMS: To determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. METHODS AND RESULTS: Twenty-eight of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin with respect to type strain Lactobacillus reuteri CECT 925T were identified as Lact. reuteri. They were able to survive at pH 3 and subsequent exposure to cholic acid or oxgall, and presented bile salt hydrolase and bacteriocin-like activities. CONCLUSIONS: Reuterin production is a frequently found trait among lactobacilli isolated from pig faeces. Selected Lact. reuteri isolates were able to survive at conditions likely to be encountered throughout the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: High yields of reuterin may be obtained from selected isolates of Lact. reuteri. Probiotic characteristics of isolates studied in the present work suggest their application in food and feed.     

Catecholamines and sympathomimetic drugs decrease early Salmonella Typhimurium uptake into porcine Peyer's patches

Peyer's patches of the small intestine serve as inductive sites for mucosal immunity as well as targets for invasive enteropathogens, including Salmonella. Because they are innervated by catecholamine-containing enteric nerves, the hypothesis that the endogenous catecholamines dopamine and norepinephrine or sympathomimetic drugs alter Salmonella Typhimurium uptake into Peyer's patches was tested. Porcine jejunal Peyer's patch explants were mounted in Ussing chambers and inoculated with a porcine field isolate of Salmonella Typhimurium DT104. Salmonella recovery from gentamicin-treated tissues increased significantly between 30 and 90 min of bacterial exposure to the mucosal surface. Addition of the neuronal conduction blocker saxitoxin (0.1 micromol L(-1)) or dopamine (30 micromol L(-1)) to the contraluminal aspect of explants decreased bacterial recovery after 60 min of Salmonella exposure. The effects of dopamine were mimicked by cocaine and methamphetamine (30 micromol L(-1)), which act on catecholaminergic nerve terminals to increase synaptic neurotransmitter concentrations. These results suggest that enteric catecholaminergic nerves modulate Salmonella colonization of Peyer's patches at the earliest stages of infection, in part by altering epithelial uptake of bacteria.     

Flagellin in combination with curli fimbriae elicits an immune response in the gastrointestinal epithelial cell line HT-29

Flagellin is the major cytokine-releasing factor when Salmonella enterica serovar Typhimurium (S. Typhimurium) infects intestinal epithelial cells. In this work it is shown that curli, an adhesive proteinaceous surface component of Enterobacteriaceae involved in biofilm formation of S. Typhimurium and Escherichia coli strains can bind flagellin and thus elicit an immune response by the intestinal epithelial cell line HT-29.     

Evolution of Coenzyme B(12) Synthesis among Enteric Bacteria: Evidence for Loss and Reacquisition of a Multigene Complex

We have examined the distribution of cobalamin (coenzyme B(12)) synthetic ability and cobalamin-dependent metabolism among enteric bacteria. Most species of enteric bacteria tested synthesize cobalamin under both aerobic and anaerobic conditions and ferment glycerol in a cobalamin-dependent fashion. The group of species including Escherichia coli and Salmonella typhimurium cannot ferment glycerol. E. coli strains cannot synthesize cobalamin de novo, and Salmonella spp. synthesize cobalamin only under anaerobic conditions. In addition, the cobalamin synthetic genes of Salmonella spp. (cob) show a regulatory pattern different from that of other enteric taxa tested. We propose that the cobalamin synthetic genes, as well as genes providing cobalamin-dependent diol dehydratase, were lost by a common ancestor of E. coli and Salmonella spp. and were reintroduced as a single fragment into the Salmonella lineage from an exogenous source. Consistent with this hypothesis, the S. typhimurium cob genes do not hybridize with the genomes of other enteric species. The Salmonella cob operon may represent a class of genes characterized by periodic loss and reacquisition by host genomes. This process may be an important aspect of bacterial population genetics and evolution.     

The effect of cell immobilization on the antibacterial activity of Lactobacillus reuteri DPC16 cells during passage through a simulated gastrointestinal tract system

Cell immobilization has the ability to influence the survival and functional characteristics of probiotic bacterial strains in harsh environments. This study investigated the effect of cell immobilization and passage through a simulated gastrointestinal tract (GI) on the antibacterial activity of Lactobacillus reuteri DPC16. Antibacterial activity, reuterin production and diol dehydratase activity were assayed in recovered isolates of L. reuteri that had been immobilized in Ca alginate-skim milk, and incubated in simulated GI fluids. Among all the recovered isolates tested, any that had undergone immobilization followed by immediate recovery of the cells without subsequent incubation in any fluids demonstrated the highest reuterin production, antimicrobial activity and diol dehydratase enzyme activity. L. reuteri DPC16 cells that had been immobilized, incubated in simulated GI fluids, and subsequently recovered from the beads often showed some loss of antimicrobial activity compared to the immobilized cells. The data confirm that the process of immobilization of L. reuteri in Ca alginate-skim milk, rather than the passage through simulated GI fluids, resulted in enhanced antibacterial activity. This is attributed to increased diol dehydratase activity, resulting in increased reuterin production.     

Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella growth in the inflamed gut

Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization.     

鼠伤寒沙门氏菌诱导昆明小鼠肠道感染模型的建立

目的:建立鼠伤寒沙门氏菌诱导昆明小鼠肠道感染模型。方法:先用5 mg/mL链霉素预处理2 d,提高小鼠对鼠伤寒沙门氏菌的敏感性,然后正常饲养1 d,攻毒前禁水禁食4 h,再分别以不同剂量灌胃攻毒2次,间隔24 h。观察小鼠临床症状,并通过组织病理切片、透射电镜和免疫组织化学的方法,分别观察小鼠肠道组织病理变化、小肠上皮细胞超微结构变化及肠道淋巴细胞增殖状况。结果:攻毒后昆明小鼠会出现昏睡、食欲不振、寒颤,甚至死亡的现象,解剖后发现小鼠肠道充血膨胀。组织病理切片显示小鼠肠粘膜受损,小肠绒毛肿胀,排列杂乱,炎性细胞浸润;透射电镜观察超微结构显示小肠上皮细胞线粒体空泡化,嵴和膜发生融合消失,粗面内质网发生扩张;免疫组织化学的方法显示肠道感染后,淋巴结肿大,T淋巴细胞大量增殖。结论:该模型对探索鼠伤寒沙门氏菌引发肠炎的发病机制、病理生理、免疫等方面作用具有重要意义,并为特异性卵黄抗体被动免疫保护效果的后续评价奠定基础。