Lactobacillus-derived extracellular vesicles counteract Aß42-induced abnormal transcriptional changes through the upregulation of MeCP2 and Sirt1 and improve Aß pathology in Tg-APP/PS1 mice.

Mounting evidence suggests that probiotics are beneficial for treating Alzheimer's disease (AD). However, the mechanisms by which specific probiotics modify AD pathophysiology are not clearly understood. In this study, we investigated whether Lactobacillus paracasei-derived extracellular vesicles (Lpc-EV) can directly act on neuronal cells to modify amyloid-beta (Aß)-induced transcriptional changes and Aß pathology in the brains of Tg-APP/PS1 mice. Lpc-EV treatment in HT22 neuronal cells counteracts Aß-induced downregulation of Brain-derived neurotrophic factor (Bdnf), Neurotrophin 3 (Nt3), Nt4/5, and TrkB receptor, and reverses Aß-induced altered expression of diverse nuclear factors, including the downregulation of Methyl-CpG binding protein 2 (Mecp2) and Sirtuin 1 (Sirt1). Systematic siRNA-mediated knockdown experiments indicate that the upregulation of Bdnf, Nt3, Nt4/5, and TrkB by Lpc-EV is mediated via multiple epigenetic factors whose activation converges on Mecp2 and Sirt1. In addition, Lpc-EV reverses Aß-induced downregulation of the Aß-degrading proteases Matrix metalloproteinase 2 (Mmp-2), Mmp-9, and Neprilysin (Nep), whose upregulation is also controlled by MeCP2 and Sirt1. Lpc-EV treatment restores the downregulated expression of Bdnf, Nt4/5, TrkB, Mmp-2, Mmp-9, and Nep; induces the upregulation of MeCP2 and Sirt1 in the hippocampus; alleviates Aß accumulation and neuroinflammatory responses in the brain; and mitigates cognitive decline in Tg-APP/PS1 mice. These results suggest that Lpc-EV cargo contains a neuroactive component that upregulates the expression of neurotrophic factors and Aß-degrading proteases (Mmp-2, Mmp-9, and Nep) through the upregulation of MeCP2 and Sirt1, and ameliorates Aß pathology and cognitive deficits in Tg-APP/PS1 mice.

Identification of gut dysbiosis in axial spondyloarthritis patients and improvement of experimental ankylosing spondyloarthritis by microbiome-derived butyrate with immune-modulating function.

Introduction: Dysbiosis is an environmental factor that affects the induction of axial spondyloarthritis (axSpA) pathogenesis. In the present study, we investigated differences in the gut microbiota of patients with axSpA and revealed an association between specific gut microbiota and their metabolites, and SpA pathogenesis. Method: Using 16S rRNA sequencing data derived from feces samples of 33 axSpA patients and 20 healthy controls (HCs), we examined the compositions of their gut microbiomes. Results: As a result, axSpA patients were found to have decreased α-diversity compared to HCs, indicating that axSpA patients have less diverse microbiomes. In particular, at the species level, Bacteroides and Streptococcus were more abundant in axSpA patients than in HCs, whereas Faecalibacterium (F). prausnitzii, a butyrate-producing bacteria, was more abundant in HCs. Thus, we decided to investigate whether F. prausnitzii was associated with health conditions by inoculating F. prausnitzii (0.1, 1, and 10 µg/mL) or by administrating butyrate (0.5 mM) into CD4+ T cells derived from axSpA patients. The levels of IL-17A and IL-10 in the CD4+ T cell culture media were then measured. We also assessed osteoclast formation by administrating butyrate to the axSpA-derived peripheral blood mononuclear cells. The CD4+ IL-17A+ T cell differentiation, IL-17A levels were decreased, whereas IL-10 was increased by F. prausnitzii inoculation. Butyrate reduced CD4+ IL-17A+ T cell differentiation and osteoclastogenesis. Discussion: We found that CD4+ IL-17A+ T cell polarization was reduced, when F. prausnitzii or butyrate were introduced into curdlan-induced SpA mice or CD4+ T cells of axSpA patient. Consistently, butyrate treatment was associated with the reduction of arthritis scores and inflammation levels in SpA mice. Taken together, we concluded that the reduced abundance of butyrate-producing microbes, particularly F. prausnitzii, may be associated with axSpA pathogenesis.

Dietary Efficacy Evaluation by Applying a Prediction Model Using Clinical Fecal Microbiome Data of Colorectal Disease to a Controlled Animal Model from an Obesity Perspective.

Obesity associated with a Western diet such as a high-fat diet (HFD) is a known risk factor for inflammatory bowel disease (IBD) and colorectal cancer (CRC). In this study, we aimed to develop fecal microbiome data-based deep learning algorithms for the risk assessment of colorectal diseases. The effects of a HFD and a candidate food (Nypa fruticans, NF) on IBD and CRC risk reduction were also evaluated. Fecal microbiome data were obtained from 109 IBD patients, 111 CRC patients, and 395 healthy control (HC) subjects by 16S rDNA amplicon sequencing. IBD and CRC risk assessment prediction models were then constructed by deep learning algorithms. Dietary effects were evaluated based on fecal microbiome data from rats fed on a regular chow diet (RCD), HFD, and HFD plus ethanol extracts or water extracts of NF. There were significant differences in taxa when IBD and CRC were compared with HC. The diagnostic performance (area under curve, AUC) of the deep learning algorithm was 0.84 for IBD and 0.80 for CRC prediction. Based on the rat fecal microbiome data, IBD and CRC risks were increased in HFD-fed rats versus RCD-fed rats. Interestingly, in the HFD-induced obesity model, the IBD and CRC risk scores were significantly lowered by the administration of ethanol extracts of NF, but not by the administration of water extracts of NF. In conclusion, changes in the fecal microbiome of obesity by Western diet could be important risk factors for the development of IBD and CRC. The risk prediction model developed in this study could be used to evaluate dietary efficacy.

Machine-learning algorithms for asthma, COPD, and lung cancer risk assessment using circulating microbial extracellular vesicle data and their application to assess dietary effects.

Although mounting evidence suggests that the microbiome has a tremendous influence on intractable disease, the relationship between circulating microbial extracellular vesicles (EVs) and respiratory disease remains unexplored. Here, we developed predictive diagnostic models for COPD, asthma, and lung cancer by applying machine learning to microbial EV metagenomes isolated from patient serum and coded by their accumulated taxonomic hierarchy. All models demonstrated high predictive strength with mean AUC values ranging from 0.93 to 0.99 with various important features at the genus and phylum levels. Application of the clinical models in mice showed that various foods reduced high-fat diet-associated asthma and lung cancer risk, while COPD was minimally affected. In conclusion, this study offers a novel methodology for respiratory disease prediction and highlights the utility of serum microbial EVs as data-rich features for noninvasive diagnosis.

Establishment of Patient-Derived Gastric Cancer Organoid Model From Tissue Obtained by Endoscopic Biopsies.

Cancer organoids are three-dimensional mini-organ analogues derived from cancer tissues and have been proposed as models capable of simulating the structure and function of human organs and tissues in vitro. We sought to establish gastric cancer patient-derived organoids (PDOs) from tissues obtained by endoscopic biopsies. Gastric cancer-PDOs were successfully established and cultured from cancer tissues with gastric adenocarcinoma by endoscopic biopsies. To confirm that gastric cancer-PDOs were derived from cancer tissue, the consistency of the original cancer tissue was assessed by histopathological examination. As a result, it was confirmed that the shape and internal structure of gastric cancer-PDO were derived from the original gastric cancer cells, and the tumor specificity of gastric cancer-PDO was confirmed through Periodic acid-Schiff (PAS) and polyclonal carcinoembryonic antigen antibody staining. These results demonstrate that gastric cancer-PDO models show the characteristics of primary tumors and have potential for drug screening and providing a personalized medicine platform.

Metagenomic Analysis of Plasma Microbial Extracellular Vesicles in Patients Receiving Mechanical Ventilation: A Pilot Study.

BACKGROUND: Previous studies reported a significant association between pneumonia outcome and the respiratory microbiome. There is increasing interest in the roles of bacterial extracellular vesicles (EVs) in various diseases. We studied the composition and function of microbiota-derived EVs in the plasma of patients receiving mechanical ventilation to evaluate whether they can be used as a diagnostic marker and to predict clinical outcomes. METHODS: Plasma samples (n = 111) from 59 mechanically ventilated patients (41 in the pneumonia group; 24 in the nursing home and hospital-associated infection [NHAI] group) were prospectively collected on days one and seven. After isolating the bacterial EVs from plasma samples, nucleic acid was extracted for 16S rRNA gene pyrosequencing. The samples were evaluated to determine the α and ß diversity, bacterial composition, and predicted functions. RESULTS: Principal coordinates analysis revealed significantly different clustering of microbial EVs between the pneumonia and non-pneumonia groups. The proportions of Lactobacillus, Cutibacterium, and Sphingomonas were significantly different between the pneumonia and non-pneumonia groups. In addition, the abundances of Lactobacillus and Bifidobacterium were significantly higher in the non-NHAI than the NHAI group. In the analysis of ß diversity, the structure of microbial EVs differed significantly different between 28-day survivors and non-survivors (Bray-Curtis distance, p = 0.014). Functional profiling revealed significant differences between the pneumonia and non-pneumonia groups. The longitudinal change in predicted functions of microbial EV genes showed a significant difference between 28-day survivors and non-survivors. CONCLUSIONS: Bacterial microbiota-derived EVs in the plasma have potential as diagnostic and prognostic markers for patients receiving mechanical ventilation. Further large prospective studies are needed to determine the clinical utility of plasma microbiota-EVs in intubated patients.

A new horizon of precision medicine: combination of the microbiome and extracellular vesicles.

Over several decades, the disease pattern of intractable disease has changed from acute infection to chronic disease accompanied by immune and metabolic dysfunction. In addition, scientific evidence has shown that humans are holobionts; of the DNA in humans, 1% is derived from the human genome, and 99% is derived from microbial genomes (the microbiome). Extracellular vesicles (EVs) are lipid bilayer-delimited nanoparticles and key messengers in cell-to-cell communication. Many publications indicate that microbial EVs are both positively and negatively involved in the pathogenesis of various intractable diseases, including inflammatory diseases, metabolic disorders, and cancers. Microbial EVs in feces, blood, and urine show significant differences in their profiles between patients with a particular disease and healthy subjects, demonstrating the potential of microbial EVs as biomarkers for disease diagnosis, especially for assessing disease risk. Furthermore, microbial EV therapy offers a variety of advantages over live biotherapeutics and human cell EV (or exosome) therapy for the treatment of intractable diseases. In summary, microbial EVs are a new tool in medicine, and microbial EV technology might provide us with innovative diagnostic and therapeutic solutions in precision medicine.

Microbial changes in stool, saliva, serum, and urine before and after anti-TNF-α therapy in patients with inflammatory bowel diseases.

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are chronic immune-mediated intestinal inflammatory disorders associated with microbial dysbiosis at multiple sites, particularly the gut. Anti-tumor necrosis factor-α (TNF-α) agents are important treatments for IBD. We investigated whether microbiome changes at multiple sites can predict the effectiveness of such treatment in IBD. Stool, saliva, serum, and urine biosamples were collected from 19 IBD patients before (V1) and 3 months after (V2) anti-TNF-α treatment, and 19 healthy subjects (control). Microbiota analysis was performed using extracellular vesicles (EVs; all four sample types) and next-generation sequencing (NGS; stool and saliva). The stool, using NGS analysis, was the only sample type in which α-diversity differed significantly between the IBD and control groups at V1 and V2. Relative to non-responders, responders to anti-TNF-α treatment had significantly higher levels of Firmicutes (phylum), Clostridia (class), and Ruminococcaceae (family) in V1 stool, and Prevotella in V1 saliva. Non-responders had significantly higher V2 serum and urine levels of Lachnospiraceae than responders. Finally, Acidovorax caeni was detected in all V1 sample types in responders, but was not detected in non-responders. Microbiome changes at multiple sites may predict the effectiveness of anti-TNF-α treatment in IBD, warranting further research.

Dysbiotic change in gastric microbiome and its functional implication in gastric carcinogenesis.

Although there is a growing interest in the role of gastric microbiome on the development of gastric cancer, the exact mechanism is largely unknown. We aimed to investigate the changes of gastric microbiome during gastric carcinogenesis, and to predict the functional potentials of the microbiome involved in the cancer development. The gastric microbiome was analyzed using gastric juice samples from 88 prospectively enrolled patients, who were classified into gastritis, gastric adenoma, or early/advanced gastric cancer group. Differences in microbial diversity and composition were analyzed with 16S rRNA gene profiling, using next-generation sequencing method. Metagenomic biomarkers were selected using logistic regression models, based on relative abundances at genus level. We used Tax4Fun to predict possible functional pathways of gastric microbiome involved in the carcinogenesis. The microbial diversity continuously decreased in its sequential process of gastric carcinogenesis, from gastritis to gastric cancer. The microbial composition was significantly different among the four groups of each disease status, as well as between the cancer group and non-cancer group. Gastritis group was differently enriched with genera Akkermansia and Lachnospiraceae NK4A136 Group, whereas the cancer group was enriched with Lactobacillus and Veillonella. Predictive analysis of the functional capacity of the microbiome suggested enrichment or depletion of several functional pathways related to carcinogenesis in the cancer group. There are significant changes in the diversity and composition of gastric microbiome during the gastric carcinogenesis process. Gastric cancer was characterized with microbial dysbiosis, along with functional changes potentially favoring carcinogenesis.

Extracellular vesicles derived from small intestinal lamina propria reduce antigen-specific immune response.

BACKGROUND/AIMS: Extracellular vesicles (EVs) are secreted from various types of cells and have specific functions related to their origin. EVs are observed in the small intestinal lamina propria (lpEVs), but their function remains unclear. This study aimed to investigate the role of lpEVs. METHODS: LpEVs were isolated from antigen (ovalbumin [OVA])-fed mice (lpEVs/OVA), and administrated to the naïve mice for 5 days before induction of lung inflammation. Afterwards, the mice were sensitized and challenged with OVA to evaluate the role of lpEVs/OVA in the regulation of immune tolerance. RESULTS: The isolated lpEVs/OVA were sphere-shaped, bi-layered vesicles of approximately 50 to 100 nm in size. The vesicles expressed CD81, A33 antigen, and major histocompatibility complex (MHC) class II on the surface. When administrated to naïve mice, the lpEVs/OVA migrated to the spleen. Intraperitoneal lpEVs/OVA administration to naïve mice decreased the immune response against sensitized antigen in a CD4+FoxP3+T cell-dependent manner. CONCLUSION: EVs are actively secreted from small intestinal epithelial cells to deliver information about orally administered antigens to immune cells, which will facilitate the modulation of the immune response by acting as an intercellular communicasome.

Fecal Microbiota and Gut Microbe-Derived Extracellular Vesicles in Colorectal Cancer.

The human microbiota comprises trillions of microbes, and the relationship between cancer and microbiota is very complex. The impact of fecal microbiota alterations on colorectal cancer (CRC) pathogenesis is emerging. This study analyzed changes in the microbial composition in CRC subjects with both fecal microbiota and gut microbe-derived extracellular vesicles (EVs). From August 2017 to August 2018, 70 CRC patients and 158 control subjects were enrolled in the study. Metagenomic profiling of fecal microbiota and gut microbe-derived EVs in stool was performed using 16S ribosomal DNA sequencing. Relative abundance, evenness, and diversity in both the gut microbiota and gut microbe-derived EVs were analyzed. Additionally, microbial composition changes according to the stage and location of CRC were analyzed. Microbial composition was significantly changed in CRC subjects compared to control subjects, with evenness and diversity significantly lower in the fecal microbiota of CRC subjects. Gut microbe-derived EVs of stool demonstrated significant differences in the microbial composition, evenness, and diversity in CRC subjects compared to the control subjects. Additionally, microbial composition, evenness, and diversity significantly changed in late CRC subjects compared to early CRC subjects with both fecal microbiota and gut microbe-derived EVs. Alistipes-derived EVs could be novel biomarkers for diagnosing CRC and predicting CRC stages. Ruminococcus 2-derived EVs significantly decreased in distal CRC subjects than in proximal CRC subjects. Gut microbe-derived EVs in CRC had a distinct microbial composition compared to the controls. Profiling of microbe-derived EVs may offer a novel biomarker for detecting and predicting CRC prognosis.

Bacteria-Derived Extracellular Vesicles in Urine as a Novel Biomarker for Gastric Cancer: Integration of Liquid Biopsy and Metagenome Analysis.

Early detection is crucial for improving the prognosis of gastric cancer, but there are no non-invasive markers for the early diagnosis of gastric cancer in real clinical settings. Recently, bacteria-derived extracellular vesicles (EVs) emerged as new biomarker resources. We aimed to evaluate the microbial composition in gastric cancer using bacteria-derived EVs and to build a diagnostic prediction model for gastric cancer with the metagenome data. Stool, urine, and serum samples were prospectively collected from 453 subjects (gastric cancer, 181; control, 272). EV portions were extracted from the samples for metagenome analysis. Differences in microbial diversity and composition were analyzed with 16S rRNA gene profiling, using the next-generation sequencing method. Biomarkers were selected using logistic regression models based on relative abundances at the genus level. The microbial composition of healthy groups and gastric cancer patient groups was significantly different in all sample types. The compositional differences of various bacteria, based on relative abundances, were identified at the genus level. Among the diagnostic prediction models for gastric cancer, the urine-based model showed the highest performance when compared to that of stool or serum. We suggest that bacteria-derived EVs in urine can be used as novel metagenomic markers for the non-invasive diagnosis of gastric cancer by integrating the liquid biopsy method and metagenome analysis.

Altered Composition of Microbiota in Women with Ovarian Endometrioma: Microbiome Analyses of Extracellular Vesicles in the Peritoneal Fluid.

Human microbiota refers to living microorganisms which colonize our body and crucially contribute to the metabolism of nutrients and various physiologic functions. According to recently accumulated evidence, human microbiota dysbiosis in the genital tract or pelvic cavity could be involved in the pathogenesis and/or pathophysiology of endometriosis. We aimed to investigate whether the composition of microbiome is altered in the peritoneal fluid in women with endometriosis. We recruited 45 women with histological evidence of ovarian endometrioma and 45 surgical controls without endometriosis. Following the isolation of extracellular vesicles from peritoneal fluid samples from women with and without endometriosis, bacterial genomic DNA was sequenced using next-generation sequencing of the 16S rDNA V3-V4 regions. Diversity analysis showed significant differences in the microbial community at phylum, class, order, family, and genus levels between the two groups. The abundance of Acinetobacter, Pseudomonas, Streptococcus, and Enhydrobacter significantly increased while the abundance of Propionibacterium, Actinomyces, and Rothia significantly decreased in the endometriosis group compared with those in the control group (p < 0.05). These findings strongly suggest that microbiome composition is altered in the peritoneal environment in women with endometriosis. Further studies are necessary to verify whether dysbiosis itself can cause establishment and/or progression of endometriosis.

Microbiome Markers of Pancreatic Cancer Based on Bacteria-Derived Extracellular Vesicles Acquired from Blood Samples: A Retrospective Propensity Score Matching Analysis.

Novel biomarkers for early diagnosis of pancreatic cancer (PC) are necessary to improve prognosis. We aimed to discover candidate biomarkers by identifying compositional differences of microbiome between patients with PC (n = 38) and healthy controls (n = 52), using microbial extracellular vesicles (EVs) acquired from blood samples. Composition analysis was performed using 16S rRNA gene analysis and bacteria-derived EVs. Statistically significant differences in microbial compositions were used to construct PC prediction models after propensity score matching analysis to reduce other possible biases. Between-group differences in microbial compositions were identified at the phylum and genus levels. At the phylum level, three species (Verrucomicrobia, Deferribacteres, and Bacteroidetes) were more abundant and one species (Actinobacteria) was less abundant in PC patients. At the genus level, four species (Stenotrophomonas, Sphingomonas, Propionibacterium, and Corynebacterium) were less abundant and six species (Ruminococcaceae UCG-014, Lachnospiraceae NK4A136 group, Akkermansia, Turicibacter, Ruminiclostridium, and Lachnospiraceae UCG-001) were more abundant in PC patients. Using the best combination of these microbiome markers, we constructed a PC prediction model that yielded a high area under the receiver operating characteristic curve (0.966 and 1.000, at the phylum and genus level, respectively). These microbiome markers, which altered microbial compositions, are therefore candidate biomarkers for early diagnosis of PC.

Helicobacter pylori-derived outer membrane vesicles stimulate interleukin 8 secretion through nuclear factor kappa B activation.

BACKGROUND/AIMS: Bacteria-derived outer membrane vesicles (OMVs) are commonly associated with various biological activities and functions. Helicobacter pylori-derived OMVs are thought to contribute to pathogenesis. This study aimed to investigate the effects of H. pylori-derived OMVs. METHODS: H. pylori strains were isolated from patients with gastritis, gastric ulcer, or gastric cancer using endoscopic biopsy. The U-937, AGS, and MKN-45 cell lines were exposed to H. pylori and H. pylori-derived OMVs. The expression of interleukin 8 (IL-8) messenger RNA (mRNA) was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR, and IL-8 secretion was analyzed using enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB) activation was evaluated by Western blotting. RESULTS: H. pylori and H. pylori-derived OMVs induced the expression of IL-8 mRNA and protein. Importantly, the bacteria induced higher IL-8 mRNA and protein expression than the OMVs. IL-8 expression was induced to different levels in response to H. pylori-derived OMVs from hosts with different gastric diseases. Western blotting revealed the increased phosphorylation and reduced degradation of inhibitor of NF-κB alpha in cells exposed to OMVs. CONCLUSION: H. pylori-derived OMVs may aid the development of various gastric diseases by inducing IL-8 production and NF-κB activation.

The Prevalence of Multidrug Resistance of Helicobacter pylori and Its Impact on Eradication in Korea from 2017 to 2019: A Single-Center Study.

Antimicrobial resistance is one of the major factors determining the efficacy of Helicobacter pylori eradication therapy. This study aimed to estimate the recent prevalence of multidrug resistance of H. pylori and its impact on eradication in Korea. A total of 174 patients were prospectively enrolled at Chung-Ang University Hospital from 2017 to 2019. H. pylori strains were isolated from the gastric body and antrum. The minimum inhibitory concentrations of antibiotics were determined by the serial twofold agar dilution method. Eradication results were reviewed and analyzed in connection with antibiotic resistance. The prevalence of H. pylori infection was 51.7% (90/174). The culture success rate was 77.8% (70/90). The resistance rates for clarithromycin, metronidazole, amoxicillin, tetracycline, levofloxacin, and moxifloxacin were 28.6% (20/70), 27.1% (19/70), 20.0% (14/70), 18.6% (13/70), 42.9% (30/70), and 42.9% (30/70), respectively. The multidrug resistance (resistance to two or more classes of antimicrobials) rate was 42.9% (30/70). Dual resistance to clarithromycin and metronidazole was confirmed in 8.6% (6/70). Eradication with a first-line treatment was successful in 75% (36/48), and those who received second-line treatment all achieved successful eradication. The rate of multidrug resistance is increasing, and standard triple therapy (STT) is no longer an acceptable first-line option for H. pylori eradication in Korea.

Brain tumor diagnostic model and dietary effect based on extracellular vesicle microbiome data in serum.

The human microbiome has been recently associated with human health and disease. Brain tumors (BTs) are a particularly difficult condition to directly link to the microbiome, as microorganisms cannot generally cross the blood-brain barrier (BBB). However, some nanosized extracellular vesicles (EVs) released from microorganisms can cross the BBB and enter the brain. Therefore, we conducted metagenomic analysis of microbial EVs in both serum (152 BT patients and 198 healthy controls (HC)) and brain tissue (5 BT patients and 5 HC) samples based on the V3-V4 regions of 16S rDNA. We then developed diagnostic models through logistic regression and machine learning algorithms using serum EV metagenomic data to assess the ability of various dietary supplements to reduce BT risk in vivo. Models incorporating the stepwise method and the linear discriminant analysis effect size (LEfSe) method yielded 12 and 29 significant genera as potential biomarkers, respectively. Models using the selected biomarkers yielded areas under the curves (AUCs) >0.93, and the model using machine learning resulted in an AUC of 0.99. In addition, Dialister and [Eubacterium] rectale were significantly lower in both blood and tissue samples of BT patients than in those of HCs. In vivo tests showed that BT risk was decreased through the addition of sorghum, brown rice oil, and garlic but conversely increased by the addition of bellflower and pear. In conclusion, serum EV metagenomics shows promise as a rich data source for highly accurate detection of BT risk, and several foods have potential for mitigating BT risk.

Discovery of a Novel Mutation in DNA Gyrase and Changes in the Fluoroquinolone Resistance of Helicobacter pylori over a 14-Year Period: A Single Center Study in Korea.

The efficacy of fluoroquinolone-based eradication therapy largely depends on the fluoroquinolone resistance of H. pylori. The aim of this study was to investigate the changes in the primary resistance rate of H. pylori to fluoroquinolone and the mechanism of resistance in Korea. A total of 153 strains and 48 strains of H. pylori were isolated at a tertiary hospital in 2005/2006 and 2017/2018, respectively. The minimum inhibitory concentrations (MICs) of fluoroquinolone were determined by the serial 2-fold agar dilution method. DNA sequences in the quinolone resistance-determining regions of gyrA/gyrB were analyzed in resistant strains. Subsequent natural transformation study was performed to determine the association between gyrase mutation and resistance. The resistance rates increased from 19.0% (29/153) to 43.8% (21/48) both for levofloxacin and moxifloxacin. The MIC values for resistant strains increased from 2-8 µg/mL to 4-16 µg/mL over time. Mutation of gyrA was detected in 93.1% (27/29) and 100% (21/21) among the resistant strains in both periods, respectively. A novel Gly-85 mutation of gyrA was found and confirmed to be associated with fluoroquinolone resistance. Fluoroquinolone resistance rate of H. pylori has markedly increased over time in Korea. The resistance is mostly due to the point mutation of gyrA. Fluoroquinolone-containing regimens should be carefully selected in Korea, considering the increasing fluoroquinolone resistance.

Lactobacillus paracasei-derived extracellular vesicles attenuate the intestinal inflammatory response by augmenting the endoplasmic reticulum stress pathway.

Lactobacillus paracasei is a major probiotic and is well known for its anti-inflammatory properties. Thus, we investigated the effects of L. paracasei-derived extracellular vesicles (LpEVs) on LPS-induced inflammation in HT29 human colorectal cancer cells and dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. ER stress inhibitors (salubrinal or 4-PBA) or CHOP siRNA were utilized to investigate the relationship between LpEV-induced endoplasmic reticulum (ER) stress and the inhibitory effect of LpEVs against LPS-induced inflammation. DSS (2%) was administered to male C57BL/6 mice to induce inflammatory bowel disease, and disease activity was measured by determining colon length, disease activity index, and survival ratio. In in vitro experiments, LpEVs reduced the expression of the LPS-induced pro-inflammatory cytokines IL-1α, IL-1ß, IL-2, and TNFα and increased the expression of the anti-inflammatory cytokines IL-10 and TGFß. LpEVs reduced LPS-induced inflammation in HT29 cells and decreased the activation of inflammation-associated proteins, such as COX-2, iNOS and NFκB, as well as nitric oxide. In in vivo mouse experiments, the oral administration of LpEVs also protected against DSS-induced colitis by reducing weight loss, maintaining colon length, and decreasing the disease activity index (DAI). In addition, LpEVs induced the expression of endoplasmic reticulum (ER) stress-associated proteins, while the inhibition of these proteins blocked the anti-inflammatory effects of LpEVs in LPS-treated HT29 cells, restoring the pro-inflammatory effects of LPS. This study found that LpEVs attenuate LPS-induced inflammation in the intestine through ER stress activation. Our results suggest that LpEVs have a significant effect in maintaining colorectal homeostasis in inflammation-mediated pathogenesis.

The analysis of virulence factors and antibiotic resistance between Helicobacter pylori strains isolated from gastric antrum and body.

BACKGROUND: Individuals can be infected with multiple strains of Helicobacter pylori. However, the differences among co-infecting strains have not been well analyzed yet. This study aimed to investigate whether the virulence factors and antibiotic resistance patterns of H. pylori differ between strains isolated from different locations of the stomach in the same patient. METHODS: H. pylori isolates were obtained from the antrum and body of the stomach. Genetic differences were examined by random amplified polymorphic DNA (RAPD) fingerprinting. Antibiotic resistance was assessed using the agar dilution method. Virulence factors were identified by polymerase chain reaction and DNA sequencing. RESULTS: Among 80 patients, co-infection by two H. pylori strains was detected in 10 patients. Among the 10 pairs of H. pylori strains, differences in antibiotic resistance patterns were detected in 7 pairs (clarithromycin, 1 patient; quinolone, 3 patients; metronidazole, 4 patients) and differences in virulence factors were detected in 5 pairs. The cagA virulence gene was detected in all 10 patients, and 2 patients had H. pylori strains with different EPIYA motifs. Differences in vacA genotypes were detected in 4 patients. CONCLUSIONS: Co-infection by two H. pylori strains was confirmed by RAPD fingerprinting. Frequently, two H. pylori strains obtained from a single host differed in their virulence factors and antibiotic resistance patterns. Co-infection by multiple H. pylori strains could undermine the success of eradication therapy and should be considered when interpreting the results of antimicrobial susceptibility tests.