Stefan Roos

A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components

A gene from Lactobacillus reuteri 1063 encoding a cell-surface protein, designated Mub, that adheres to mucus components in vitro has been cloned and sequenced. The deduced amino acid sequence of Mub (358 kDa) shows the presence of 14 approximately 200 aa repeats and features typical for other cell-surface proteins of Gram-positive bacteria. Fusion proteins consisting of different repeats of Mub and the maltose-binding protein (MBP) were produced. These proteins adhered to pig mucus components, with molecular masses ranging from <0.1 to >2 MDa, to pig gastric mucin and to hen intestinal mucus. The binding of Mub to mucus components occurred in the pH range 3-7.4, with maximum binding at pH 4-5 and could be partly inhibited by the glycoprotein fetuin. Affinity-purified antibodies against recombinant Mub were used in immunofluorescence microscopy to demonstrate the presence of Mub on the cell surface of strain 1063. By using the antibodies in a Western blot analysis, Mub could also be detected in the growth medium. The results implicate Mub as a cell-surface protein that is involved in Lactobacillus interactions with mucin and in colonization of the digestive tract.

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.

Lactobacillus reuteri DSM 17938 in Infantile Colic: A Randomized, Double-Blind, Placebo-Controlled Trial

OBJECTIVE: To test the efficacy of Lactobacillus reuteri on infantile colic and to evaluate its relationship to the gut microbiota. STUDY DESIGN: Fifty exclusively breastfed colicky infants, diagnosed according to modified Wessels criteria, were randomly assigned to receive either L reuteri DSM 17 938 (108 colony-forming units) or placebo daily for 21 days. Parental questionnaires monitored daily crying time and adverse effects. Stool samples were collected for microbiologic analysis. RESULTS: Forty-six infants (L reuteri group: 25; placebo group: 21) completed the trial. Daily crying times in minutes/day (median [interquartile range]) were 370 (120) vs 300 (150) (P = .127) on day 0 and 35.0 (85) vs 90.0 (148) (P = .022) on day 21, in the L reuteri and placebo groups, respectively. Responders (50% reduction in crying time from baseline) were significantly higher in the L reuteri group versus placebo group on days 7 (20 vs 8; P = .006), 14 (24 vs 13; P = .007), and 21 (24 vs 15; P = .036). During the study, there was a significant increase in fecal lactobacilli (P = .002) and a reduction in fecal Escherichia coli and ammonia in the L reuteri group only (P = .001). There were no differences in weight gain, stooling frequency, or incidence of constipation or regurgitation between groups, and no adverse events related to the supplementation were observed. CONCLUSION: L. reuteri DSM 17 938 at a dose of 108 colony-forming units per day in early breastfed infants improved symptoms of infantile colic and was well tolerated and safe. Gut microbiota changes induced by the probiotic could be involved in the observed clinical improvement.

Importance and regulation of the colonic mucus barrier in a mouse model of colitis

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.

Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm

Vertebrates engage in symbiotic associations with vast and complex microbial communities that colonize their gastrointestinal tracts. Recent advances have provided mechanistic insight into the important contributions of the gut microbiome to vertebrate biology, but questions remain about the evolutionary processes that have shaped symbiotic interactions in the gut and the consequences that arise for both the microbes and the host. Here we discuss the biological principles that underlie microbial symbiosis in the vertebrate gut and the potential of the development of mutualism. We then review phylogenetic and experimental studies on the vertebrate symbiont Lactobacillus reuteri that have provided novel insight into the ecological and evolutionary strategy of a gut microbe and its relationship with the host. We argue that a mechanistic understanding of the microbial symbiosis in the vertebrate gut and its evolution will be important to determine how this relationship can go awry, and it may reveal possibilities by which the gut microbiome can be manipulated to support health.

Gastroprotective and blood pressure lowering effects of dietary nitrate are abolished by an antiseptic mouthwash.

Recently, it has been suggested that the supposedly inert nitrite anion is reduced in vivo to form bioactive nitric oxide with physiological and therapeutic implications in the gastrointestinal and cardiovascular systems. Intake of nitrate-rich food such as vegetables results in increased levels of circulating nitrite in a process suggested to involve nitrate-reducing bacteria in the oral cavity. Here we investigated the importance of the oral microflora and dietary nitrate in regulation of gastric mucosal defense and blood pressure. Rats were treated twice daily with a commercial antiseptic mouthwash while they were given nitrate-supplemented drinking water. The mouthwash greatly reduced the number of nitrate-reducing oral bacteria and as a consequence, nitrate-induced increases in gastric NO and circulating nitrite levels were markedly reduced. With the mouthwash the observed nitrate-induced increase in gastric mucus thickness was attenuated and the gastroprotective effect against an ulcerogenic compound was lost. Furthermore, the decrease in systemic blood pressure seen during nitrate supplementation was now absent. These results suggest that oral symbiotic bacteria modulate gastrointestinal and cardiovascular function via bioactivation of salivary nitrate. Excessive use of antiseptic mouthwashes may attenuate the bioactivity of dietary nitrate.

Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution

The vertebrate digestive tract, including that of humans, is the habitat to trillions of bacteria that are of significant importance to host biology and health. Although these communities are often postulated to have coevolved with their hosts, evidence is lacking, yet critical for our understanding of microbial symbiosis in vertebrates. To gain insight into the evolution of a gut symbiont, we have characterized the population genetic structure and phylogeny of Lactobacillus reuteri strains isolated from six different host species (human, mouse, rat, pig, chicken and turkey) using Amplified-Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Analysis (MLSA). The results revealed considerable genetic heterogeneity within the L. reuteri population and distinct monophyletic clades reflecting host origin but not provenance. The evolutionary patterns detected indicate a long-term association of L. reuteri lineages with particular vertebrate species and host-driven diversification. Results from a competition experiment in a gnotobiotic mouse model revealed that rodent isolates showed elevated ecological performance, indicating that evolution of L. reuteri lineages was adaptive. These findings provide evidence that some vertebrate gut microbes are not promiscuous, but have diversified into host-adapted lineages by a long-term evolutionary process, allowing the development of a highly specialized symbiosis.

Removal of Antibiotic Resistance Gene-Carrying Plasmids from Lactobacillus reuteri ATCC 55730 and Characterization of the Resulting Daughter Strain, L. reuteri DSM 17938

ABSTRACT The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of β-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known β-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The β-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.

Lactobacillus mucosae sp. nov., a new species with in vitro mucus-binding activity isolated from pig intestine.

A new Lactobacillus species from pig small intestine has been identified. In an attempt to isolate Lactobacillus reuteri strains carrying the putative colonization-factor gene (mub, for mucus binding) a mub-derived gene probe was used to screen pig intestinal material. A number of isolates were obtained and primary characterization showed that they were Gram-positive, catalase-negative, non-spore-forming, non-motile rods. Growth occurred at 45 degrees C but not at 15 degrees C and the DNA G+C content was 46 mol%. Cell wall analysis together with DNA-DNA hybridization and analysis of the 16S rRNA sequence revealed that the new isolates represent a previously undescribed Lactobacillus species closely related to L. reuteri, Lactobacillus fermentum and Lactobacillus pontis. The name Lactobacillus mucosae is proposed for this species and the type strain is S32T.

Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri.

Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867(i)), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins, although present in many L. reuteri isolates, show a high genetic heterogeneity among strains.