Alterations in the microbiome are associated with the development of gastric cancer. Our study aimed to identify dysbiotic features in early gastric cancer (EC). The gastric microbiome was assessed in EC (n = 30), advanced gastric cancer (AC) (n = 30), and chronic gastritis (CG) (n = 60). The results demonstrated significant differences in the microbial profile and composition between EC and AC, suggesting alterations associated with gastric cancer progression. Linear discriminant analysis (LDA) effect size (LEfSe) analyses identified 32 bacterial genera that were associated with EC. Functional analyses of the gastric microbiome showed that the production of urease and synthesis of bacterial flagella were weakened in EC, while the glycolysis of fructose and hydrolysis of glycosides were enhanced. A classifier based on a random forest (RF) machine learning algorithm identified a microbial signature that distinguished EC from CG or AC with high accuracy. The correct identification of the signature was further validated in independent cohorts. This signature enriched of bacteria with varied abundance, high degree of bacterial interactions and carcinogenic potentials. Constrained principal coordinate analyses revealed that the presence of Helicobacter pylori and the cagA and vacA virulence genotypes influenced the structure of the gastric microbiome. To determine the impacts of host genetic variations on the gastric microbiome, six previously reported single nucleotide polymorphisms (SNPs) were examined. The minor allele of MUC1 rs4072037 was associated with an increased abundance of Ochrobactrum. The gastric microbiome altered in EC, which might be attributed in part to host genetic variations, H. pylori infection, bacterial virulence and environmental adaptations. The identified microbial signature could serve as biomarkers for clinical assessment of gastric cancer risk in high-risk patients.

The link between microbiota and gastric cancer (GC) has attracted widespread attention. However, the phylogenetic profiles of niche-specific microbiota in the tumor microenvironment is still unclear. Here, mucosa-associated microorganisms from 62 pairs of matched GC tissues and adjacent non-cancerous tissues were characterized by 16S rRNA gene sequencing. Functional profiles of the microbiota were predicted using PICRUSt, and a co-occurrence network was constructed to analyze interactions among gastric microbiota. Results demonstrated that mucosa-associated microbiota from cancerous and non-cancerous tissues established micro-ecological systems that differed in composition, structure, interaction networks, and functions. Microbial richness and diversity were increased in cancerous tissues, with the co-occurrence network exhibiting greater complexity compared with that in non-cancerous tissue. The bacterial taxa enriched in the cancer samples were predominantly represented by oral bacteria (such as Peptostreptococcus, Streptococcus, and Fusobacterium), while lactic acid-producing bacteria (such as Lactococcus lactis and Lactobacillus brevis) were more abundant in adjacent non-tumor tissues. Colonization by Helicobacter pylori, which is a GC risk factor, also impacted the structure of the microbiota. Enhanced bacterial purine metabolism, carbohydrate metabolism and denitrification functions were predicted in the cancer associated microbial communities, which was consistent with the increased energy metabolism and concentration of nitrogen-containing compounds in the tumor microenvironment. Furthermore, the microbial co-occurrence networks in cancerous and non-cancerous tissues of GC patients were described for the first time. And differential taxa and functions between the two groups were identified. Changes in the abundance of certain bacterial taxa, especially oral microbiota, may play a role in the maintenance of the local microenvironment, which is associated with the development or progression of GC.

Helicobacter pylori infection is widespread worldwide, with more than a half of the world population infected. H. pylori antibiotic-resistant strains and non-compliance to therapy are the major causes of H. pylori eradication failure. The search for new therapies based on plant extracts is a scientific interest field. The present study was conducted to evaluate the effect in vitro of extra virgin olive oil (EVOO), hydroxytyrosol (HT), and oleuropein (Olp) against two H. pylori strains and the effect in vivo of the oral administration of EVOO on the gastric mucosa of BALB/c mice infected with this microorganism. The broth microdilution method assayed the antibacterial in vitro activity of EVOO, HT, and Olp against H. pylori strains. For in vivo studies, male BALB/c mice were infected orally with an H. pylori suspension every 72 h. Four groups were used: (1) Control, (2) H. pylori-infected (HP), (3) EVOO, and (4) HP + EVOO. Mice were sacrificed at 7, 15, and 30 days. The stomachs were removed and observed under a microscope. Scoring of the degree of erosion was determined. Samples were processed by histological techniques for light microscopy. Macroscopic analysis showed that the presence of small erosions increased, both in number and size, in the infected group. Animals infected and treated with EVOO exhibited the presence of fewer erosions, which decreased in number as the treatment progressed. The mucosa of the control and EVOO groups showed normal histological characteristics at the three times studied. The mucosa of animals infected with H. pylori showed disruptions of the lining epithelium, damage to gastric glands, and vasodilation. The mucosa of animals infected with H. pylori and treated with EVOO showed morphological characteristics similar to those of normal and EVOO mucosa. For the first time, the current study showed the effect in vitro and in vivo of EVOO and combined administration of HT and Olp against H. pylori using an animal model. Future studies are needed to establish the mechanism of EVOO's action at the gastric mucosa level to propose this product as a natural antimicrobial agent for the treatment of gastric H. pylori infections.

Afadin is a cytoplasmic protein of the adherens junctions, which regulates the formation and stabilization of both the adherens and the tight junctions. Aberrant expression of Afadin has been shown in cancer and its loss has been associated with epithelial-to-mesenchymal transition (EMT). EMT is characterized by the change from an epithelial to a mesenchymal phenotype, with modifications on the expression of adhesion molecules and acquisition of a migratory and invasive cell behavior. While it is known that Helicobacter pylori disrupts the tight and the adherens junctions and induces EMT, the effect of the bacteria on Afadin is still unknown. The aim of this study was to disclose the effect of H. pylori on Afadin and its impact in the induction of an EMT phenotype in gastric cells. Using two different cell lines, we observed that H. pylori infection decreased Afadin protein levels, independently of CagA, T4SS, and VacA virulence factors. H. pylori infection of cell lines recapitulated several EMT features, displacing and downregulating multiple proteins from cell-cell junctions, and increasing the expression of ZEB1, Vimentin, Slug, N-cadherin, and Snail. Silencing of Afadin by RNAi promoted delocalization of junctional proteins from the cell-cell contacts, increased paracellular permeability, and decreased transepithelial electrical resistance, all compatible with impaired junctional integrity. Afadin silencing also led to increased expression of the EMT marker Snail, and to the formation of actin stress fibers, together with increased cell motility and invasion. Finally, and in line with our in vitro data, the gastric mucosa of individuals infected with H. pylori showed decrease/loss of Afadin membrane staining at cell-cell contacts significantly more frequently than uninfected individuals. In conclusion, Afadin is downregulated by H. pylori infection in vitro and in vivo, and its downregulation leads to the emergence of EMT and to the acquisition of an aggressive phenotype in gastric cells, which can contribute to gastric carcinogenesis.

Helicobacter pylori infection changes gastric microbiota profiles. However, it is not clear whether H. pylori eradication can restore the healthy gastric microbiota. Moreover, there has been no study regarding the changes in gastric microbiota with aging. The objective of this study was to investigate the changes in gastric corpus microbiota with age and following H. pylori eradication. Changes in corpus mucosa-associated microbiota were evaluated in 43 individuals with endoscopic follow-up > 1 year, including 8 H. pylori-uninfected and 15 H. pylori-infected subjects with no atrophy/metaplasia by histology and pepsinogen I/II ratio > 4.0; 17 H. pylori-infected subjects with atrophy/metaplasia and pepsinogen I/II ratio < 2.5; and 3 subjects with atrophy/metaplasia, no evidence of active H. pylori infection, negative for anti-H. pylori immunoglobulin G (IgG) antibody testing, and no previous history of H. pylori eradication. Successful H. pylori eradication was achieved in 21 patients. The gastric microbiota was characterized using an Illumina MiSeq platform targeting 16S ribosomal DNA (rDNA). The mean follow-up duration was 57.4 months (range, 12-145 months), and median follow-up visit was 1 (range, 1-3). Relative abundance of Lactobacillales and Streptococcus was increased with atrophy/metaplasia. In H. pylori-uninfected subjects (n = 8), an increase in Proteobacteria (Enhydrobacter, Comamonadaceae, Sphingobium); a decrease in Firmicutes (Streptococcus, Veillonella), Fusobacteria (Fusobacterium), Nocardioidaceae, Rothia, and Prevotella; and a decrease in microbial diversity were observed during the follow-up (p trend < 0.05). In 10 of 21 subjects (47.6%), H. pylori eradication induced restoration of microbial diversity; however, a predominance of Acinetobacter with a decrease in microbial diversity occurred in 11 subjects (52.3%). The presence of atrophy/metaplasia at baseline and higher neutrophil infiltration in the corpus were associated with the restoration of gastric microbiota after successful eradication, whereas a higher relative abundance of Acinetobacter at baseline was associated with the predominance of Acinetobacter after H. pylori eradication (p < 0.05). To conclude, in H. pylori-uninfected stomach, relative abundance of Proteobacteria increases, relative abundance of Firmicutes and Fusobacteria decreases, and microbial diversity decreases with aging. H. pylori eradication does not always restore gastric microbiota; in some individuals, gastric colonization by Acinetobacter species occurs after anti-Helicobacter treatment.

Accumulating evidence suggests that high-fat diet (HFD) induced metabolic disorders are associated with dysbiosis of gut microbiota. However, no study has explored the effect of HFD on the gastric microbiota. This study established the HFD animal model to determine the impact of HFD on the gastric microbiota and its relationship with the alterations of gut microbiota. A total of 40 male C57BL/6 mice were randomly allocated to receive a standard chow diet (CD) or HFD for 12 weeks (12CD group and 12HFD group) and 24 weeks (24CD group and 24HFD group) (n = 10 mice per group). Body weight and length were measured and Lee's index was calculated at different time points. The insulin sensitivity and serum levels of metabolic parameters including blood glucose, insulin and lipid were also evaluated. The gastric mucosa and fecal microbiota of mice were characterized by 16S rRNA gene sequencing. The body weight was much heavier and the Lee's index was higher in 24HFD group than 12HFD. The insulin resistance and serum level of lipid were increased in 24HFD group compared to 12HFD, indicating the aggravation of metabolic disorders as HFD went on. 16S rRNA gene sequencing showed dysbiosis of gastric microbiota with decreased community diversity while no significant alteration in gut microbiota after 12 weeks of HFD. The phyla Firmicutes and Proteobacteria tended to increase whereas Bacteroidetes and Verrucomicrobia decrease in the gastric microbiota of 12HFD mice compared to 12CD. Moreover, a remarkable reduction of bacteria especially Akkermansia muciniphila, which has beneficial effects on host metabolism, was observed firstly in the stomach of 12HFD group and then in the gut of 24HFD group, indicating the earlier alterations of microbiota in stomach than gut after HFD. We also found structural segregation of microbiota in the stomach as well as gut between 12HFD and 24HFD group, which is accompanied by the aggregation of metabolic disorders. These data suggest that HFD affects not only gut microbiota but also gastric microbiota and the disruption of microbial ecosystem in the digestive tract may play a part in the development and progression of metabolic diseases although molecular mechanism requires further investigation.

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin-antitoxin (TA) systems have emerged as important virulence factors in many pathogenic bacteria. Three type II TA systems have previously been identified in the genome of H. pylori 26695: HP0315-HP0316, HP0892-HP0893, and HP0894-HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967-HP0968, which is encoded by the bicistronic operon hp0968-hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although, the hp0968-hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968-hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968-hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

Helicobacter pylori is a pathogen involved in gastric diseases such as ulcers and carcinomas. H. pylori's urease is an important virulence factor produced in large amounts by this bacterium. In previous studies, we have shown that this protein is able to activate several cell types like neutrophils, monocytes, platelets, endothelial cells, and gastric epithelial cells. Angiogenesis is a physiological process implicated in growth, invasion and metastization of tumors. Here, we have analyzed the angiogenic potential of H. pylori urease (HPU) in gastric epithelial cells. No cytotoxicity was observed in AGS, Kato-III, and MKN28 gastric cell lines treated with 300 nM HPU, as evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As we previously reported in neutrophils, treatment with 300 nM HPU also had an anti-apoptotic effect in gastric epithelial cells leading to a 2.2-fold increase in the levels of Bcl-XL after 6 h, and a decrease of 80% in the content of BAD, after 48 h, two mitochondrial proteins involved in regulation of apoptosis. Within 10 min of exposure, HPU is rapidly internalized by gastric epithelial cells. Treatment of the gastric cells with methyl-β-cyclodextrin abolished HPU internalization suggesting a cholesterol-dependent process. HPU induces the expression of pro-angiogenic factors and the decrease of expression of anti-angiogenic factors by AGS cells. The angiogenic activity of HPU was analyzed using in vitro and in vivo models. HPU induced formation of tube-like structures by human umbilical vascular endothelial cells in a 9 h experiment. In the chicken embryo chorioallantoic membrane model, HPU induced intense neo-vascularization after 3 days. In conclusion, our results indicate that besides allowing bacterial colonization of the gastric mucosa, H. pylori's urease triggers processes that initiate pro-angiogenic responses in different cellular models. Thus, this bacterial urease, a major virulence factor, may also play a role in gastric carcinoma development.

A novel bacterium (Rouxiella badensis subsp. acadiensis) isolated from the microbiota of wild blueberry fruit was investigated for its immunomodulation capabilities and intestinal morpho-functional aspects. The whole-genome shotgun sequencing of this bacterium led to its new taxonomy and showed absence of pathogenicity genes. Although the bacterium was used for blueberry-fermentation and enhancing its anti-inflammatory effects on neurodegeneration, diabetes, and cancer, no study has assessed the effect of the bacterium on health. In this study, we used several in vitro and in vivo assays to evaluate the interaction of R. badensis subsp. acadiensis with the intestinal mucosa and its impact on the localized immune response. The strain antibiotic susceptibility has been investigated as well as its tolerance to gastric and intestinal environment and ability to attach to human intestinal epithelial cells (Caco-2 and HT-29). In addition, Balb/c mice were used to explore the immune-modulatory characteristics of the live bacterium at the intestinal level and its impact on the morpho-functional aspects of the intestinal mucosa. In vitro assays revealed the ability of R. badensis subsp. acadiensis to survive the gastric and intestinal simulated conditions and to satisfactorily adhere to the human intestinal epithelial cells. The bacterium was shown to be sensitive to an array of antibiotics. Immuno-modulation studies with mice orally administered with R. badensis subsp. acadiensis showed a higher number of IgA positive cells in the small intestine, a higher concentration of the anti-inflammatory cytokine IL-10 in the intestinal mucosa, as well as an increase in the number of goblet cells. The anti-inflammatory cytokine miR146a was found to be increased in the ileum and brain. Furthermore, it increases the number of goblet cells which contribute to intestinal barrier integrity. Taken together, our findings reflect the ability of the tested bacterium to modulates the intestinal homeostasis and immune response. Detailed safety unpublished studies and genome data support our finding. The strain Rouxiella badensis subsp. acadiensis has been filed in a provisional patent; a U.S. Provisional Application No. 62/916,921 entitled "Probiotics Composition and Methods." Future studies are still needed to validate the potential utilization of this strain as functional food and its potential probiotic effect.

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and causes peptic ulcers and gastric carcinoma. H. pylori strain 26695 has a small genome (1.67 Mb), which codes for few known transcriptional regulators that control bacterial metabolism and virulence. We analyzed by qRT-PCR the expression of 16 transcriptional regulators in H. pylori 26695, including the three sigma factors under different environmental conditions. When bacteria were exposed to acidic pH, urea, nickel, or iron, the sigma factors were differentially expressed with a particularly strong induction of fliA. The regulatory genes hrcA, hup, and crdR were highly induced in the presence of urea, nickel, and iron. In terms of biofilm formation fliA, flgR, hp1021, fur, nikR, and crdR were induced in sessile bacteria. Transcriptional expression levels of rpoD, flgR, hspR, hp1043, and cheY were increased in contact with AGS epithelial cells. Kanamycin, chloramphenicol, and tetracycline increased or decreased expression of regulatory genes, showing that these antibiotics affect the transcription of H. pylori. Our data indicate that environmental cues which may be present in the human stomach modulate H. pylori transcription.

Gastric infection by Helicobacter pylori is considered a risk factor for gastric and duodenal cancer, and extragastric diseases. Previous data have shown that, in a non-enzymatic way, H. pylori urease (HPU) activates neutrophils to produce ROS and also induces platelet aggregation, requiring ADP secretion modulated by the 12-lipoxygenase pathway, a signaling cascade also triggered by the physiological agonist collagen. Here we investigated further the effects on platelets of recombinant versions of the holoenzyme HPU, and of its two subunits (HpUreA and HpUreB). Although HpUreA had no aggregating activity on platelets, it partially inhibited collagen-induced aggregation. HpUreB induced platelet aggregation in the nanomolar range, and also interfered dose-dependently on both collagen- and ADP-induced platelet aggregation. HPU-induced platelet aggregation was inhibited by antibodies against glycoprotein VI (GPVI), the main collagen receptor in platelets. Flow cytometry analysis revealed exposure of P-selectin in HPU-activated platelets. Anti-glycoprotein IIbIIIa (GPIIbIIIa) antibodies increased the binding of FITC-labeled HPU to activated platelets, whereas anti-GPVI did not. Evaluation of post-transcriptional events in HPU-activated platelets revealed modifications in the pre-mRNA processing of pro-inflammatory proteins, with increased levels of mRNAs encoding IL-1β and CD14. We concluded that HPU activates platelets probably through its HpUreB subunit. Activation of platelets by HPU turns these cells into a pro-inflammatory phenotype. Altogether, our data suggest that H. pylori urease, besides allowing bacterial survival within the gastric mucosa, may have an important, and so far overlooked, role in gastric inflammation mediated by urease-activated neutrophils and platelets.

Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS), which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC) of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12Δ CagA deletion mutant, and a P12Δ PAI deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr) phosphopeptides and analyzed by nano-LC-Orbitrap MS. In total, 85 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK family. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin-linked factors, whereas the T4SS primarily modulates JNK and p38 activation.

Helicobacter pylori (H. pylori) is one of the gram-negative bacteria that mainly colonize the stomach mucosa and cause many gastrointestinal diseases, such as gastritis, peptic ulcer, and gastric cancer. Macrophages play a key role in eradicating H. pylori. Recent data have shown that Notch signaling could modulate the activation and bactericidal activities of macrophages. However, the role of Notch signaling in macrophages against H. pylori remains unclear. In the present study, in the co-culture model of macrophages with H. pylori, the inhibition of Notch signaling using γ-secretase decreased the expression of inducible nitric oxide synthase (iNOS) and its product, nitric oxide (NO), and downregulated the secretion of pro-inflammatory cytokine and attenuated phagocytosis and bactericidal activities of macrophages to H. pylori. Furthermore, we identified that Jagged1, one of Notch signaling ligands, was both upregulated in mRNA and protein level in activated macrophages induced by H. pylori. Clinical specimens showed that the number of Jagged1+ macrophages in the stomach mucosa from H. pylori-infected patients was significantly higher than that in healthy control. The overexpression of Jagged1 promoted bactericidal activities of macrophages against H. pylori and siRNA-Jagged1 presented the opposite effect. Besides, the addition of exogenous rJagged1 facilitated the pro-inflammatory mediators of macrophages against H. pylori, but the treatment of anti-Jagged1 neutralizing antibody attenuated it. Taken together, these results suggest that Jagged1 is a promoting molecule for macrophages against H. pylori, which will provide insight for exploring Jagged1 as a novel therapeutic target for the control of H. pylori infection.

Background:Helicobacter pylori colonizes the gastric mucosa of more than half of the world's population. There is increasing evidence H. pylori protects against the development of obesity and childhood asthma/allergies in which the development of these diseases coincide with transient dysbiosis. However, the mechanism underlying the association of H. pylori eradication with human metabolic and immunological disorders is not well-established. In this study, we aimed to investigate the local and systemic effects of H. pylori eradication through untargeted fecal lipidomics and plasma metabolomics approaches by liquid chromatography mass spectrometry (LC-MS). Results: Our study revealed that eradication of H. pylori eradication (i.e., loss of H. pylori and/or H. pylori eradication therapy) changed many global metabolite/lipid features, with the majority being down-regulated. Our findings primarily show that H. pylori eradication affects the host energy and lipid metabolism which may eventually lead to the development of metabolic disorders. Conclusion: These predictive metabolic signatures of metabolic and immunological disorders following H. pylori eradication can provide insights into dynamic local and systemic metabolism related to H. pylori eradication in modulating human health.

Encapsulation of amoxicillin (AMX) for drug delivery against Helicobacter pylori infection and aspirin-induced ulcers in rat's stomachs was performed using docosahexaenoic acid (DHA)-loaded chitosan/alginate (CA) nanoparticles (NPs) developed by ionotropic gelation method. The physicochemical analyses of the composite NPs were performed by scanning electron microscopy, Fourier transform infrared spectroscopy, zeta potential, X-ray diffraction, and atomic force microscopy. The encapsulation efficiency of AMX was increased to 76% by incorporating DHA, which resulted in a reduction in the particle size. The formed CA-DHA-AMX NPs effectively adhered to the bacteria and rat gastric mucosa. Their antibacterial properties were more potent than those of the single AMX and CA-DHA NPs as demonstrated by the in vivo assay. The composite NPs attained higher mucoadhesive potential during food intake than during fasting (p = 0.029). At 10 and 20 mg/kg AMX, the CA-AMX-DHA showed more potent activities against H. pylori than the CA-AMX, CA-DHA, and single AMX. The in vivo study showed that the effective dose of AMX was lower when DHA was included, indicating better drug delivery and stability of the encapsulated AMX. Both mucosal thickening and ulcer index were significantly higher in the groups receiving CA-DHA-AMX than in the groups receiving CA-AMX and single AMX. The presence of DHA declines the pro-inflammatory cytokines including IL-1β, IL-6, and IL-17A. The synergistic effects of AMX and the CA-DHA formulation increased the biocidal activities against H. pylori infection and improved ulcer healing properties.

S-layer proteins (SLPs), which are present in the external layer of certain strains of lactic acid bacteria isolated from the intestinal tract, are known to recognize and bind to specific proteins and glycan structures and contribute to adsorption to the host intestinal mucosa. The binding properties of certain SLPs are considered to exert a competitive inhibitory effect on infection because similar properties are involved in the infection mechanisms of several viruses. However, little is known regarding whether SLPs directly inhibit viral infection. In the present study, we investigated the effect of an SLP of the Lactobacillus crispatus KT-11 strain, a probiotic strain isolated from a healthy human infant, on human rotavirus infection. The impact of KT-11 lithium chloride extract (KT-11 LE), which contains SLP, on the infection of the P[4] genotype human rotavirus strain DS-1 was evaluated by monitoring the amplification of viral protein 6 (VP6) expression in human intestinal epithelial Caco-2 cells by quantitative reverse transcription-polymerase chain reaction assay after infection. KT-11 LE showed a significant suppressive effect on DS-1 infection in a dose-dependent manner with pre-infection treatment, whereas post-infection treatment was not effective. A 45 KDa protein isolated from KT-11 LE was investigated for homology using the BLAST database and was found to be a novel SLP. KT-11 SLP concentrate (KT-11 SLP) significantly inhibited the proliferative process of the DS-1 strain but not that of the P[8] genotype human rotavirus strain Wa. KT-11 SLP exerted significant inhibitory effect on DS-1 infection by pre-infection treatment even after digestion with gastric juice up to 2 h. Our results provided crucial evidence that SLPs from certain Lactobacillus strains can inhibit human rotavirus infection of intestinal epithelial cells.