Adhesion Inhibition Assay for Helicobacter pylori to Mucin by Lactobacillus.

The ability of Lactobacillus to adhere to mucin is a parameter for evaluating the effectiveness of probiotics. In particular, a competitive inhibition assay of pathogenic bacteria using mucin-adherent lactobacilli is useful for identifying Lactobacillus strains capable of preventing mucus from being colonized by pathogens. Here, we describe an adhesion inhibition assay method for Helicobacter pylori to porcine gastric mucin by Limosilactobacillus reuteri.

Genome sequence of Enterococcus gallinarum AH4, a milk oligosaccharide-degrading strain isolated from suckling rats.

We had previously isolated Enterococcus gallinarum AH4, a strain capable of degrading rat milk oligosaccharides. In this study, we determined the whole-genome sequence of AH4. This whole-genome information will expand our understanding of milk oligosaccharide-mediated symbioses between bacteria and host mammals.

Sharing of Moonlighting Proteins Mediates the Symbiotic Relationship among Intestinal Commensals.

The human gastrointestinal tract is inhabited by trillions of symbiotic bacteria that form a complex ecological community and influence human physiology. Symbiotic nutrient sharing and nutrient competition are the most studied relationships in gut commensals, whereas the interactions underlying homeostasis and community maintenance are not fully understood. Here, we provide insights into a new symbiotic relationship wherein the sharing of secreted cytoplasmic proteins, called "moonlighting proteins," between two heterologous bacterial strains (Bifidobacterium longum and Bacteroides thetaiotaomicron) was observed to affect the adhesion of bacteria to mucins. B. longum and B. thetaiotaomicron were cocultured using a membrane-filter system, and in this system the cocultured B. thetaiotaomicron cells showed greater adhesion to mucins compared to that shown by monoculture cells. Proteomic analysis showed the presence of 13 B. longum-derived cytoplasmic proteins on the surface of B. thetaiotaomicron. Moreover, incubation of B. thetaiotaomicron with the recombinant proteins GroEL and elongation factor Tu (EF-Tu)-two well-known mucin-adhesive moonlighting proteins of B. longum-led to an increase in the adhesion of B. thetaiotaomicron to mucins, a result attributed to the localization of these proteins on the B. thetaiotaomicron cell surface. Furthermore, the recombinant EF-Tu and GroEL proteins were observed to bind to the cell surface of several other bacterial species; however, the binding was species dependent. The present findings indicate a symbiotic relationship mediated by the sharing of moonlighting proteins among specific strains of B. longum and B. thetaiotaomicron. IMPORTANCE The adhesion of intestinal bacteria to the mucus layer is an important colonization strategy in the gut environment. Generally, the bacterial adhesion process is a characteristic feature of the individual cell surface-associated adhesion factors secreted by a particular bacterium. In this study, coculture experiments between Bifidobacterium and Bacteroides show that the secreted moonlighting proteins adhere to the cell surface of coexisting bacteria and alter the adhesiveness of the bacteria to mucins. This finding indicates that the moonlighting proteins act as adhesion factors for not only homologous strains but also for coexisting heterologous strains. The presence of a coexisting bacterium in the environment can significantly alter the mucin-adhesive properties of another bacterium. The findings from this study contribute to a better understanding of the colonization properties of gut bacteria through the discovery of a new symbiotic relationship between them.

Efficacy of soft palatal augmentation prosthesis for oral functional rehabilitation in patients with dysarthria and dysphagia: a protocol for a randomised controlled trial.

INTRODUCTION: Palatal augmentation prosthesis (PAP) is used in patients with articulation and swallowing disorders caused by postoperative loss of tongue tissue due to tongue cancer, cerebrovascular disease sequelae and age-related hypofunction. We have previously reported a newly designed soft PAP fabricated using an thermoplastic material that is particularly appropriate for early intervention. However, the effect of soft PAP on oral function improvement remains to be elucidated. The aim of this study is to investigate whether soft PAP can improve dysarthria and dysphagia occurring as cerebrovascular disease sequelae. METHODS AND ANALYSIS: This prospective, randomised, controlled trial will compare the immediate and training effects of rehabilitation using soft PAP with those of rehabilitation without using it. Primary outcomes are the single-word intelligibility test score and pharyngeal transit time (PTT). Secondary outcomes are tongue function (evaluated based on maximum tongue pressure, repetitions of tongue pressure and endurance of tongue pressure), articulation function (evaluated based on speech intelligibility, oral diadochokinesis, Voice-Related Quality of Life (V-RQOL)) and swallowing function (evaluated using Eating Assessment Tool-10). The study results will help determine the efficacy of Soft PAP in improving functional outcomes of word intelligibility and PTT. We hypothesised that early rehabilitation using Soft PAP would more effectively improve articulation and swallowing function compared with conventional rehabilitation without using soft PAP. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Okayama University Certified Review Board. The study findings will be published in an open access, peer-reviewed journal and presented at relevant conferences and research meetings. TRIAL REGISTRATION NUMBER: jRCTs062200054.

O-acetylesterase activity of Bifidobacterium bifidum sialidase facilities the liberation of sialic acid and encourages the proliferation of sialic acid scavenging Bifidobacterium breve.

Bifidobacterium bifidum possesses two extracellular sialidases (SiaBb1 and SiaBb2) that release free sialic acid from mucin sialoglycans, which can be utilized via cross-feeding by Bifidobacterium breve that, otherwise, is prevented from utilizing this nutrient source. Modification of sialic acids with O-acetyl esters is known to protect mucin glycans from degradation by bacterial sialidases. Compared to SiaBb2, SiaBb1 has an additional O-acetylesterase (Est) domain. We aimed to elucidate the role of the SiaBb1 Est domain from B. bifidum in sialic acid cross-feeding within Bifidobacterium. Pre-treatment of mucin secreted from bovine submaxillary glands (BSM) using His6 -tagged-Est and -SiaBb2 released a higher amount of sialic acid compared to the pre-treatment by His6 -SiaBb2. Growth of B. breve increased with an increase in nanE expression when supplemented with both His6 -Est- and His6 -SiaBb2-treated BSM. These results indicate that the esterase activity of the SiaBb1 Est domain enhances the efficiency of SiaBb2 to cleave sialic acid from mucin. This free sialic acid can be utilized by coexisting sialic acid scavenging B. breve via cross-feeding. Here, we provide the molecular mechanism underlying the unique sialoglycan degradation property of B. bifidum which is mediated by the complementary activities of SiaBb1 and SiaBb2 in the context of sialic acid cross-feeding.

Roles of the Cell Surface Architecture of Bacteroides and Bifidobacterium in the Gut Colonization.

There are numerous bacteria reside within the mammalian gastrointestinal tract. Among the intestinal bacteria, Akkermansia, Bacteroides, Bifidobacterium, and Ruminococcus closely interact with the intestinal mucus layer and are, therefore, known as mucosal bacteria. Mucosal bacteria use host or dietary glycans for colonization via adhesion, allowing access to the carbon source that the host's nutrients provide. Cell wall or membrane proteins, polysaccharides, and extracellular vesicles facilitate these mucosal bacteria-host interactions. Recent studies revealed that the physiological properties of Bacteroides and Bifidobacterium significantly change in the presence of co-existing symbiotic bacteria or markedly differ with the spatial distribution in the mucosal niche. These recently discovered strategic colonization processes are important for understanding the survival of bacteria in the gut. In this review, first, we introduce the experimental models used to study host-bacteria interactions, and then, we highlight the latest discoveries on the colonization properties of mucosal bacteria, focusing on the roles of the cell surface architecture regarding Bacteroides and Bifidobacterium.

Milk oligosaccharide-mediated cross-feeding between Enterococcus gallinarum and lactobacilli in the gut microbiota of infant rats.

We investigated bacteria that have a nutritional symbiotic relationship with respect to milk oligosaccharides in gut microbiota of suckling rats, with specific reference to sialyllactose (SL) degrading Enterococcus gallinarum. Our next generation sequencing analysis of the colonic contents of 12-day-old suckling rats revealed that almost half of the bacteria in the microbiota belonged to the Lactobacillaceae family. Major Lactobacillus species in the contents were identified as L. johnsonii, L. murinus, and L. reuteri. We then monitored changes in numbers of the above Lactobacillus species, E. gallinarum, and the bacteria belonging to the family Enterobacteriaceae (i.e., enterobacteria) in the colonic contents of infant rats at 7, 12, 21, 28, and 35 days of age by using real-time PCR assays targeting these bacterial groups. The 7-day-old infant rats had a gut microbiota in which enterobacteria were predominant. Such dominance was replaced by L. johnsonii and the concomitant E. gallinarum markedly increased in those of 12 and 21 days of ages. During this period, the number of enterobacteria declined dramatically, but that of L. reuteri surged dramatically. Our separate in vitro experiment showed that supplementation of culture media with SL promoted the growth of L. johnsonii and E. gallinarum, with marked production of lactic acid. These findings revealed possible milk oligosaccharide-mediated cross-feeding between E. gallinarum and L. johnsonii, with the former degrading SL to release lactose to be utilized by the latter.

Isolation and identification of milk oligosaccharide-degrading bacteria from the intestinal contents of suckling rats.

We report the isolation of bacteria capable of degrading milk oligosaccharides from suckling infant rats. The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted intestinal contents of infant rats were individually incubated in an enrichment medium containing 3'-sialyllactose (3'-SL), followed by the isolation of candidate strains from streaked agar plates and selection of 3'-SL-degrading strains using thin-layer chromatography. Subsequent genomic and phenotypic analyses identified all strains as Enterococcus gallinarum. The strains were capable of degrading both 3'-SL and 6'-SL, which was not observed with the type strain of E. gallinarum used as a reference. Furthermore, a time-course study combining high-performance anion-exchange chromatography with pulsed amperometric detection revealed that the representative strain AH4 degraded 3'-SL completely to yield an equimolar amount of lactose and an approximately one-fourth equimolar amount of sialic acid after 24 hr of anaerobic incubation. These findings point to a possibility that the enterococci degrade rat milk oligosaccharides to "cross-feed" their degradants to other members of concomitant bacteria in the gut of the infant rat.

Extracellular Vesicles Produced by Bifidobacterium longum Export Mucin-Binding Proteins.

Extracellular proteins are important factors in host-microbe interactions; however, the specific factors that enable bifidobacterial adhesion and survival in the gastrointestinal (GI) tract are not fully characterized. Here, we discovered that Bifidobacterium longum NCC2705 cultured in bacterium-free supernatants of human fecal fermentation broth released a myriad of particles into the extracellular environment. The aim of this study was to characterize the physiological properties of these extracellular particles. The particles, approximately 50 to 80 nm in diameter, had high protein and double-stranded DNA contents, suggesting that they were extracellular vesicles (EVs). A proteomic analysis showed that the EVs primarily consisted of cytoplasmic proteins with crucial functions in essential cellular processes. We identified several mucin-binding proteins by performing a biomolecular interaction analysis of phosphoketolase, GroEL, elongation factor Tu (EF-Tu), phosphoglycerate kinase, transaldolase (Tal), and heat shock protein 20 (Hsp20). The recombinant GroEL and Tal proteins showed high binding affinities to mucin. Furthermore, the immobilization of these proteins on microbeads affected the permanence of the microbeads in the murine GI tract. These results suggest that bifidobacterial exposure conditions that mimic the intestine stimulate B. longum EV production. The resulting EVs exported several cytoplasmic proteins that may have promoted B. longum adhesion. This study improved our understanding of the Bifidobacterium colonization strategy in the intestinal microbiome.IMPORTANCEBifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. Morphological observations revealed that extracellular appendages of bifidobacteria in complex microbial communities are important for understanding its adaptations to the GI tract environment. We identified dynamic extracellular vesicle (EV) production by Bifidobacterium longum in bacterium-free fecal fermentation broth that was strongly suggestive of differing bifidobacterial extracellular appendages in the GI tract. In addition, export of the adhesive moonlighting proteins mediated by EVs may promote bifidobacterial colonization. This study provides new insight into the roles of EVs in bifidobacterial colonization processes as these bacteria adapt to the GI environment.

Vibiro vulnificus hemolysin associates with gangliosides.

BACKGROUND: Vibrio vulnificus hemolysin (VVH) is a pore-forming toxin secreted by Vibrio vulnificus. Cellular cholesterol was believed to be the receptor for VVH, because cholesterol could bind to VVH and preincubation with cholesterol inhibited cytotoxicity. It has been reported that specific glycans such as N-acetyl-D-galactosamine and N-acetyl-D-lactosamine bind to VVH, however, it has not been known whether these glycans could inhibit the cytotoxicity of VVH without oligomer formation. Thus, to date, binding mechanisms of VVH to cellular membrane, including specific receptors have not been elucidated. RESULTS: We show here that VVH associates with ganglioside GM1a, Fucosyl-GM1, GD1a, GT1c, and GD1b by glycan array. Among them, GM1a could pulldown VVH. Moreover, the GD1a inhibited the cytotoxicity of VVH without the formation of oligomers. CONCLUSION: This is the first report of a molecule able to inhibit the binding of VVH to target cells without oligomerization of VVH.

Prevention of postoperative pneumonia by perioperative oral care in patients with esophageal cancer undergoing surgery: a multicenter retrospective study of 775 patients.

PURPOSE: Postoperative pneumonia is one of the major complications after esophageal cancer surgery. The risk factors associated with postoperative pneumonia are poor general health, smoking, decreased pulmonary function, diabetes mellitus, surgical stress, old age, postoperative aspiration, and oral hygiene. In this study, we examined the effect of perioperative oral care on reducing postoperative pneumonia since the evidence to-date is not clear. METHODS: A multicenter, retrospective investigation of the relationship between perioperative oral care and incidence of postoperative pneumonia in patients undergoing esophageal cancer surgery was conducted. A total of 775 patients who underwent thoracoscopic esophageal resection at 25 hospitals between 2016 and 2017 were enrolled in the study. Various factors were examined for correlation with development of postoperative pneumonia. RESULTS: Multivariate analysis showed that old age, smoking habit, lower hemoglobin, higher creatinine, postoperative dysphagia, and lack of oral care intervention were independent risk factors for pneumonia. Oral care was more effective in preventing pneumonia in hospitals in which the incidence of postoperative pneumonia was lower than 20%, while it was not effective in hospitals in which the incidence was higher than 20%. CONCLUSION: Results of the study suggest that it is recommended to carry out perioperative oral care in esophageal cancer surgery.

A new approach for analyzing an adhesive bacterial protein in the mouse gastrointestinal tract using optical tissue clearing.

Several bacterial moonlighting proteins act as adhesion factors, which are important for bacterial colonization of the gastrointestinal (GI) tract. However, little is known about the adherence properties of moonlighting proteins in the GI tract. Here, we describe a new approach for visualizing the localization of moonlighting protein-coated fluorescent microbeads in the whole GI tract by using a tissue optical clearing method, using elongation factor Tu (EF-Tu) as an example. As a bacterial cell surface-localized protein mimic, recombinant EF-Tu from Lactobacillus reuteri was immobilized on microbeads. EF-Tu-coating promoted the interaction of the microbeads with a Caco-2 cell monolayer. Next, the microbeads were orally administered to mice. GI whole tissues were cleared in aqueous fructose solutions of increasing concentrations. At 1 h after administration, the microbeads were diffused from the stomach up to the cecum, and after 3 h, they were diffused throughout the intestinal tract. In the lower digestive tract, EF-Tu-beads were significantly more abundant than non-coated control beads, suggesting that EF-Tu plays an important role in the persistence of the microbeads in the GI tract. The new approach will help in evaluating how moonlighting proteins mediate bacterial colonization.

Adhesion of Lactobacillus to Intestinal Mucin.

Adhesion to intestinal mucin is one of the main in vitro tests for the study of probiotic strains. Mucins immobilized on microtiter plates and glass slides are easy and excellent methods used for the quantitative analysis of Lactobacillus adhesion. However, to maintain the performance of the quantitative analysis, these methods must be chosen appropriately according to the nature and characteristics of the strain, such as tolerance to surfactant and the ability to self-aggregate. Here we describe two methodologies used to evaluate adhesion of Lactobacillus to mucin, in addition to the isolation and purification of mucins from porcine colonic tissues.

Two extracellular sialidases from Bifidobacterium bifidum promote the degradation of sialyl-oligosaccharides and support the growth of Bifidobacterium breve.

We investigated the roles of extracellular sialidases (SiaBb1 and SiaBb2) in cross-feeding between sialidase-carrying Bifidobacterium bifidum and sialic acid-utilizing Bifidobacterium breve. Using 6' sialyllactose (6'SL) as a carbon source, the number of wild-type B. bifidum cells increased while that of a siabb2-inactivated strain (Δsiabb2) did not. Coculture of these two strains in the presence of 6'SL resulted in similar increase in cell numbers. Coculture of wild-type B. bifidum, but not the Δsiabb2 strain, with sialic acid-utilizing Bifidobacterium breve, which cannot release sialic acids from carbohydrates, in the presence of 6'SL increased the number of B. breve cells. Moreover, when mucin was used as a carbon source, B. breve growth was increased in cocultures with B. bifidum wild-type and Δsiabb2 strains, suggesting that SiaBb1 may be involved. Additionally, B. breve cell numbers increased during cultivation with recombinant SiaBb1-and SiaBb2-treated mucin as the sole carbon source. These results indicated that B. bifidum SiaBb2 liberated sialic acid from sialyl-human milk oligosaccharides and -mucin glycans, supporting the growth of B. breve through sialic acid cross-feeding. SiaBb1 may assist in the degradation of mucin glycan. Collectively, our results revealed that both the B. bifidum extracellular sialidases promote the utilization of sialylated carbohydrates and supply free sialic acid to other Bifidobacterium strains.

Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation.

Bifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galß] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates.IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains.

Influences of FLU/BU and FLU/MEL on incidence and severity of oral mucositis among allogeneic hematopoietic stem cell transplantation patients.

In the present study, we compared the incidence and severity of oral mucositis among patients undergoing allogeneic hematopoietic stem cell transplantation after fludarabine-based regimens with busulfan 12.8 mg/kg (FB12.8), with busulfan less than or equal to 9.6 mg/kg (FB9.6), and with melphalan 140 mg/m2 (FM). The incidence of oral mucositis after FB12.8 was the highest among these 3 groups. After FM, all of the patients had developed oral mucositis by day 7. The mean disease duration of oral mucositis after FB12.8 was 13.5 days, whereas the mean disease duration after FM was 24.9 days, and was significantly prolonged as compared to that after FB12.8 (p=0.0009). The incidence of severe oral mucositis (grade 3) after FM was significantly higher than that after FB12.8 (p=0.03). As stated above, although the incidence of oral mucositis after FB12.8 was higher than that after FM, oral mucositis after FB12.8 showed improvement relatively quickly without deterioration. In contrast, the higher incidence of severe oral mucositis and the delay in resolution of mucositis after FM were remarkable.

Correction: Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri.

[This corrects the article DOI: 10.1371/journal.pone.0083703.].

Anchorless cell surface proteins function as laminin-binding adhesins in Lactobacillus rhamnosus FSMM22.

Anchorless cell surface proteins (CSPs) were extracted with 1 M lithium chloride solution from Lactobacillus rhamnosus FSMM22. Loss of the anchorless CSPs resulted in a 2-fold decrease in FSMM22 cells bound to a constitutive extracellular matrix glycoprotein, laminin, in vitro. DNA-binding protein HU, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and 30S ribosomal protein S19 (RpsS) were identified by mass spectrometry in the extract as laminin-binding adhesins. Among the four proteins, RpsS was immunohistochemically confirmed to exist on the cell surface. Our findings strongly suggest that anchorless CSPs can enhance bacterial adhesion to the host.

Adhesion Properties of Lactic Acid Bacteria on Intestinal Mucin.

Lactic acid bacteria (LAB) are Gram-positive bacteria that are natural inhabitants of the gastrointestinal (GI) tracts of mammals, including humans. Since Mechnikov first proposed that yogurt could prevent intestinal putrefaction and aging, the beneficial effects of LAB have been widely demonstrated. The region between the duodenum and the terminal of the ileum is the primary region colonized by LAB, particularly the Lactobacillus species, and this region is covered by a mucus layer composed mainly of mucin-type glycoproteins. The mucus layer plays a role in protecting the intestinal epithelial cells against damage, but is also considered to be critical for the adhesion of Lactobacillus in the GI tract. Consequently, the adhesion exhibited by lactobacilli on mucin has attracted attention as one of the critical factors contributing to the persistent beneficial effects of Lactobacillus in a constantly changing intestinal environment. Thus, understanding the interactions between Lactobacillus and mucin is crucial for elucidating the survival strategies of LAB in the GI tract. This review highlights the properties of the interactions between Lactobacillus and mucin, while concomitantly considering the structure of the GI tract from a histochemical perspective.

Non-absorbable apple procyanidins prevent obesity associated with gut microbial and metabolomic changes.

Several studies have suggested that flavan-3-ols/procyanidins are associated with a reduced risk of developing obesity and metabolic syndrome. However, the role of highly polymeric procyanidins (PP), which are major non-absorbable flavonoids, in the biological effects, is not completely understood. Here, we show that 0.5% PP administration for 20 weeks alleviated obesity and regulate expression of genes related to lipid metabolism in C57BL/6J mice fed a high-fat/high-sucrose diet. PP-treatment attenuated weight gain and inflammatory effects including lipopolysaccharide and gut permeability. Additionally, metabolic urine profiling using high-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry demonstrated that PP-treatment decreased the levels of endogenous metabolites associated with insulin resistance. Furthermore, microbial 16S rRNA gene sequencing of the cecum demonstrated that PP administration markedly decreased the Firmicutes/Bacteroidetes ratio and increased eight times the proportion of Akkermansia. These data suggest that PPs influence the gut microbiota and the intestinal metabolome to produce beneficial effects on metabolic homeostasis.