Diagnostic accuracy of a real-time PCR assay for detection of Helicobacter pylori and resistance to clarithromycin and levofloxacin directly from stool.

OBJECTIVE: The non-invasive detection of Helicobacter pylori (H. pylori) and its resistance to clarithromycin and levofloxacin significantly improves the management of infected patients by enabling tailored eradication treatments without the need for endoscopic procedures. This study aimed to assess the effectiveness of real-time PCR (RT-PCR) assays in identifying H. pylori infection and antibiotic resistance in stool and gastric biopsy specimens. PATIENTS AND METHODS: Stool and gastric biopsy samples were collected from patients within three days of post-hospitalization. A total of 115 samples were analyzed for H. pylori infection, and an additional 115 samples were evaluated for resistance to clarithromycin and levofloxacin using an RT-PCR-based molecular test. Statistical analyses were performed using (SPSS 26.0 IBM Corp., Armonk, NY, USA). RESULTS: Among 115 patients (53 males, average age 50.8±13.2 years), H. pylori was detected in 93.1% of stool samples and 93.9% of gastric biopsies. The RT-PCR assay demonstrated a sensitivity of 99.1% and a specificity of 100%, with an overall diagnostic accuracy of 99.1%. Clarithromycin resistance was found in 37.3% of stool and 46.9% of gastric biopsy specimens, with the assay showing 79.6% sensitivity and 98.4% specificity. Levofloxacin resistance was identified in 32.1% of stool samples and 31.3% of gastric biopsies, with 86.3% sensitivity and 91.1% specificity of the molecular test. CONCLUSIONS: The RT-PCR-based detection of H. pylori and its resistance to clarithromycin and levofloxacin in stool samples represents a promising approach to enhance eradication therapy outcomes, potentially improving treatment efficacy. Chictr.org.cn: ChiCTR2300070267.

Helicobacter pylori infection alters gastric microbiota structure and biological functions in patients with gastric ulcer or duodenal ulcer.

BACKGROUND: Helicobacter pylori (H. pylori) infection is closely associated with gastrointestinal diseases. Our preliminary studies have indicated that H. pylori infection had a significant impact on the mucosal microbiome structure in patients with gastric ulcer (GU) or duodenal ulcer (DU). AIM: To investigate the contributions of H. pylori infection and the mucosal microbiome to the pathogenesis and progression of ulcerative diseases. METHODS: Patients with H. pylori infection and either GU or DU, and healthy individuals without H. pylori infection were included. Gastric or duodenal mucosal samples was obtained and subjected to metagenomic sequencing. The compositions of the microbial communities and their metabolic functions in the mucosal tissues were analyzed. RESULTS: Compared with that in the healthy individuals, the gastric mucosal microbiota in the H. pylori-positive patients with GU was dominated by H. pylori, with significantly reduced biodiversity. The intergroup differential functions, which were enriched in the H. pylori-positive GU patients, were all derived from H. pylori, particularly those concerning transfer RNA queuosine-modification and the synthesis of demethylmenaquinones or menaquinones. A significant enrichment of the uibE gene was detected in the synthesis pathway. There was no significant difference in microbial diversity between the H. pylori-positive DU patients and healthy controls. CONCLUSION: H. pylori infection significantly alters the gastric microbiota structure, diversity, and biological functions, which may be important contributing factors for GU.

Helicobacter pylori East Asian type CagA hijacks more SHIP2 by its EPIYA-D motif to potentiate the oncogenicity.

CagA is a significant oncogenic factor injected into host cells by Helicobacter pylori, which is divided into two subtypes: East Asian type (CagAE), characterized by the EPIYA-D motif, and western type (CagAW), harboring the EPIYA-C motif. CagAE has been reported to have higher carcinogenicity than CagAW, although the underlying reason is not fully understood. SHIP2 is an intracellular phosphatase that can be recruited by CagA to perturb the homeostasis of intracellular signaling pathways. In this study, we found that SHIP2 contributes to the higher oncogenicity of CagAE. Co-Immunoprecipitation and Pull-down assays showed that CagAE bind more SHIP2 than CagAW. Immunofluorescence staining showed that a higher amount of SHIP2 recruited by CagAE to the plasma membrane catalyzes the conversion of PI(3,4,5)P3 into PI(3,4)P2. This alteration causes higher activation of Akt signaling, which results in enhanced IL-8 secretion, migration, and invasion of the infected cells. SPR analysis showed that this stronger interaction between CagAE and SHIP2 stems from the higher affinity between the EPIYA-D motif of CagAE and the SH2 domain of SHIP2. Structural analysis revealed the crucial role of the Phe residue at the Y + 5 position in EPIYA-D. After mutating Phe of CagAE into Asp (the corresponding residue in the EPIYA-C motif) or Ala, the activation of downstream Akt signaling was reduced and the malignant transformation of infected cells was alleviated. These findings revealed that CagAE hijacks SHIP2 through its EPIYA-D motif to enhance its carcinogenicity, which provides a better understanding of the higher oncogenic risk of H. pylori CagAE.

Boosting the Anti-Helicobacter Efficacy of Azithromycin through Natural Compounds: Insights From In Vitro, In Vivo, Histopathological, and Molecular Docking Investigations.

BACKGROUND: Antimicrobial-resistant Helicobacter pylori (H. pylori) poses a significant public health concern, especially given the limited therapeutic options for azithromycin-resistant strains. Hence, there is a necessity for new studies to reconsider the use of azithromycin, which has diminished in effectiveness against numerous strains. Thus, we aimed to augment azithromycin's anti-Helicobacter properties by combining it with curcumin in different formulations, including curcumin in clove oil, curcumin nano-gold emulsion, and curcumin nanoemulsion. METHODS: The antimicrobial activities of the investigated compounds, both individually and in combination with other anti-Helicobacter drugs, were evaluated. Their antibiofilm and anti-virulence properties were assessed using both phenotypic and genotypic methods, alongside molecular docking studies. Our findings were further validated through mouse protection assays and histopathological analysis. RESULTS: We observed high anti-Helicobacter activities of curcumin, especially curcumin nanoemulsion. A synergistic effect was detected between curcumin nanoemulsion and azithromycin with fraction inhibitory concentration index (FICI) values <0.5. The curcumin nanoemulsion was the most active anti-biofilm and anti-virulence compound among the examined substances. The biofilm-correlated virulence genes (babA and hopQ) and ureA genes were downregulated (fold change <1) post-treatment with curcumin nanoemulsion. On the protein level, the anti-virulence activities of curcumin nanoemulsion were documented based on molecular docking studies. These findings aligned with histopathological scoring of challenge mice, affirming the superior efficacy of curcumin nanoemulsion/azithromycin combination. CONCLUSION: The anti-Helicobacter activities of all curcumin physical forms pose significant challenges due to their higher  minimum inhibitory concentration (MIC) values exceeding the maximum permissible level. However, using curcumin nanoemulsion at sub-MIC levels could enhance the anti-Helicobacter activity of azithromycin and exhibit anti-virulence properties, thereby improving patient outcomes and addressing resistant pathogens. Therefore, more extensive studies are necessary to assess the safety of incorporating curcumin nanoemulsion into H. pylori treatment.

Antimicrobial Resistance of Helicobacter pylori Isolated From Latin American Children and Adolescents (2008-2023): A Systematic Review.

BACKGROUND: Latin America has a high prevalence of Helicobacter pylori in children that may lead to peptic ulcer disease and eventually gastric cancer in adulthood. Successful eradication is hindered by rising antimicrobial resistance. We summarize H. pylori resistance rates in Latin American children from 2008 to 2023. MATERIAL AND METHODS: Systematic review following PRISMA guidelines and National Heart, Lung, and Blood Institute checklist to assess risk of bias (PROSPERO CRD42024517108) that included original cross-sectional observational studies reporting resistance to commonly used antibiotics in Latin American children and adolescents. We searched in PubMed, LILACS, and SciELO databases. RESULTS: Of 51 studies, 45 were excluded. The quality of the six analyzed studies (297 H. pylori-positive samples) was satisfactory. Phenotypic methods (N = 3) reported higher resistance rates than genotypic studies (N = 3). Clarithromycin resistance ranged from 8.0% to 26.7% (6 studies; 297 samples), metronidazole from 1.9% to 40.2% (4 studies; 211 samples), amoxicillin from 0% to 10.4% (3 studies; 158 samples), tetracycline resistance was not detected (3 studies; 158 samples), and levofloxacin resistance was 2.8% (1 study; 36 samples). CONCLUSION: Scarce Latin American studies on H. pylori resistance, along with methodological heterogeneity, hinder conclusive findings. Clarithromycin and metronidazole (first-line drugs) resistance is worrisome, likely impacting lower eradication rates. Urgent systematic surveillance or individual testing before treatment is necessary to enhance eradication.

Human gastric microbiota analysis of refractory H. pylori infection.

H. pylori infection is gaining increasing attention, but detailed investigations into its impact on gastric microbiota remain limited. We collected gastric mucosa samples from 47 individuals divided into three groups: 1. Group HP: patients with initial positive H. pylori infection (25 cases); 2. Group ck: H. pylori-negative patients (14 cases); 3. Group DiffHP: patients with refractory H. pylori infection (8 cases). The samples were analyzed using 16S rDNA sequencing and functional prediction with PICRUSt. Group HP showed differences in flora distribution and function compared to Group ck, while Group DiffHP overlapped with Group HP. The abundances of Aeromonas piscicola, Shewanella algae, Vibrio plantisponsor, Aeromonas caviae, Serratia marcescens, Vibrio parahaemolyticus, Microbacterium lacticum, and Prevotella nigrescens were significantly reduced in both Group DiffHP and Group HP compared to Group ck. Vibrio shilonii was reduced only in Group DiffHP compared to Group ck, while Clostridium perfringens and Paracoccus marinus were increased only in Group DiffHP. LEfSe analysis revealed that Clostridium perfringens and Paracoccus marinus were enriched, whereas Vibrio shilonii was reduced in Group DiffHP compared to Group ck at the species level. In individuals with refractory H. pylori infection, the gastric microbiota exhibited enrichment in various human diseases, organic systems, and metabolic pathways (amino acid metabolism, carbohydrate metabolism, transcription, replication and repair, cell cycle pathways, and apoptosis). Patients with multiple failed H. pylori eradication exhibited significant changes in the gastric microbiota. An increase in Clostridium perfringens and Paracoccus marinus and a decrease in Vibrio shilonii appears to be characteristic of refractory H. pylori infection.

Intratumoral microbiome is associated with gastric cancer prognosis and therapy efficacy.

The role of the intratumoral microbiome in gastric cancer (GC) has not been comprehensively assessed. Here, we explored the relationship between the microbial community and GC prognosis and therapy efficacy. Several cancer-associated microbial characteristics were identified, including increased α-diversity, differential ß-diversity, and decreased Helicobacter pylori abundance. After adjusting for clinical features, prognostic analysis revealed 2 phyla, 14 genera, and 5 species associated with the overall survival of patients with GC. Additionally, 2 phyla, 14 genera, and 6 species were associated with adjuvant chemotherapy (ACT) efficacy in patients with stage II - III GC. Furthermore, we classified GC microbiome structures into three microbial subtypes (MS1, MS2 and MS3) with distinguishing features. The MS1 subtype exhibited high immune activity and enrichment of microbiota related to immunotherapy and butyric acid-producing, as well as potential benefits in immunotherapy. MS2 featured the highest α-diversity and activation of the TFF pathway, MS3 was characterized by epithelial-mesenchymal transition and was associated with poor prognosis and reduced ACT efficacy. Collectively, the results of this study provide valuable insights into the microbial characteristics associated with GC prognosis and therapy efficacy.

A prominent role of LncRNA H19 in H. pylori CagA induced DNA damage response and cell malignancy.

Helicobacter pylori (H. pylori), together with its CagA, has been implicated in causing DNA damage, cell cycle arrest, apoptosis, and the development of gastric cancer. Although lncRNA H19 is abundantly expressed in gastric cancer and functions as a pro-oncogene, it remains unclear whether lncRNA H19 contributes to the oncogenic process of H. pylori CagA. This study investigates the role of H19 in the DNA damage response and malignancy induced by H. pylori. It was observed that cells infected with CagA+ H. pylori strain (GZ7/cagA) showed significantly higher H19 expression, resulting in increased γH2A.X and p-ATM expression and decreased p53 and Rad51 expression. Faster cell migration and invasion was also observed, which was reversed by H19 knockdown in H. pylori. YWHAZ was identified as an H19 target protein, and its expression was increased in H19 knockdown cells. GZ7/cagA infection responded to the increased YWHAZ expression induced by H19 knockdown. In addition, H19 knockdown stimulated cells to enter the G2-phase and attenuated the effect of GZ7/cagA infection on the cellular S-phase barrier. The results suggest that H. pylori CagA can upregulate H19 expression, participate in the DNA damage response and promote cell migration and invasion, and possibly affect cell cycle arrest via regulation of YWHAZ.

Insights into the molecular mechanism of ParABS system in chromosome partition by HpParA and HpParB.

The ParABS system, composed of ParA (an ATPase), ParB (a DNA binding protein), and parS (a centromere-like DNA), regulates bacterial chromosome partition. The ParB-parS partition complex interacts with the nucleoid-bound ParA to form the nucleoid-adaptor complex (NAC). In Helicobacter pylori, ParA and ParB homologs are encoded as HpSoj and HpSpo0J (HpParA and HpParB), respectively. We determined the crystal structures of the ATP hydrolysis deficient mutant, HpParAD41A, and the HpParAD41A-DNA complex. We assayed the CTPase activity of HpParB and identified two potential DNA binding modes of HpParB regulated by CTP, one is the specific DNA binding by the DNA binding domain and the other is the non-specific DNA binding through the C-terminal domain under the regulation of CTP. We observed an interaction between HpParAD41A and the N-terminus fragment of HpParB (residue 1-10, HpParBN10) and determined the crystal structure of the ternary complex, HpParAD41A-DNA-HpParBN10 complex which mimics the NAC formation. HpParBN10 binds near the HpParAD41A dimer interface and is clamped by flexible loops, L23 and L34, through a specific cation-π interaction between Arg9 of HpParBN10 and Phe52 of HpParAD41A. We propose a molecular mechanism model of the ParABS system providing insight into chromosome partition in bacteria.

Helicobacter pylori CagA Promotes the Formation of Gallstones by Increasing the Permeability of Gallbladder Epithelial Cells.

BACKGROUND: The formation of gallstones is often accompanied by chronic inflammation, and the mechanisms underlying inflammation and stone formation are not fully understood. Our aim is to utilize single-cell transcriptomics, bulk transcriptomics, and microbiome data to explore key pathogenic bacteria that may contribute to chronic inflammation and gallstone formation, as well as their associated mechanisms. METHODS: scRNA-seq data from a gallstone mouse model were extracted from the Gene Expression Omnibus (GEO) database and analyzed using the FindCluster() package for cell clustering analysis. Bulk transcriptomics data from patients with gallstone were also extracted from the GEO database, and intergroup functional differences were assessed using GO and KEGG enrichment analysis. Additionally, 16S rRNA sequencing was performed on gallbladder mucosal samples from asymptomatic patients with gallstone (n = 6) and liver transplant donor gallbladder mucosal samples (n = 6) to identify key bacteria associated with stone formation and chronic inflammation. Animal models were constructed to investigate the mechanisms by which these key pathogenic bacterial genera promote gallstone formation. RESULTS: Analysis of scRNA-seq data from the gallstone mouse model (GSE179524) revealed seven distinct cell clusters, with a significant increase in neutrophil numbers in the gallstone group. Analysis of bulk transcriptomics data from patients with gallstone (GSE202479) identified chronic inflammation in the gallbladder, potentially associated with dysbiosis of the gallbladder microbiota. 16S rRNA sequencing identified Helicobacter pylori as a key bacterium associated with gallbladder chronic inflammation and stone formation. CONCLUSIONS: Dysbiosis of the gallbladder mucosal microbiota is implicated in gallstone disease and leads to chronic inflammation. This study identified H. pylori as a potential key mucosal resident bacterium contributing to gallstone formation and discovered its key pathogenic factor CagA, which causes damage to the gallbladder mucosal barrier. These findings provide important clues for the prevention and treatment of gallstones.

Immunoinformatics-Based Designing of Novel and Potent Multi-Epitope PSA D15 and Cag11 Immunogens for Helicobacter pylori Immunodiagnostic Assay Development.

Helicobacter pylori (H. pylori) strain is the most genetically diverse pathogenic bacterium and now alarming serious human health concern ranging from chronic gastritis to gastric cancer and human death all over the world. Currently, the majority of commercially available diagnostic assays for H. pylori is a challenging task due to the heterogeneity of virulence factors in various geographical regions. In this concern, designing of universal multi-epitope immunogenic biomarker targeted for all H. pylori strains would be crucial to successfully immunodiagnosis assay and vaccine development for H. pylori infection. Hence, the present study aimed to explore the potential immunogenic epitopes of PSA D15 and Cag11 proteins of H. pylori, using immunoinformatics web tools in order to design novel immune-reactive multi-epitope antigens for enhanced immunodiagnosis in humans. Through an in silico immunoinformatics approach, high-ranked B-cell, MHC-I, and MHC-II epitopes of PSA D15 and Cag11 proteins were predicted, screened, and selected. Subsequently, a novel multi-epitope PSA D15 and Cag11 antigens were designed by fused the high-ranked B-cell, MHC-I, and MHC-II epitopes and 50S ribosomal protein L7/L12 adjuvant using linkers. The antigenicity, solubility, physicochemical properties, secondary and tertiary structures, 3D model refinement, and validations were carried. Furthermore, the designed multi-epitope antigens were subjected to codon adaptation and in silico cloning, immune response simulation, and molecular docking with receptor molecules. A novel, stable multi-epitope PSA D15 and Cag11 H. pylori antigens were developed and immune simulation of the designed antigens showed desirable levels of immunological response. Molecular docking of designed antigens with immune receptors (B-cell, MHC-I, MHC-II, and TLR-2/4) revealed robust interactions and stable binding affinity to the receptors. The codon optimized and in silico cloned showed that the designed antigens were successfully expressed (CAI value of 0.95 for PSA D15 and 1.0 for Cag11) after inserted into pET-32ba (+) plasmid of the E. coli K12 strain. In conclusion, this study revealed that the designed multi-epitope antigens have a huge immunological potential candidate biomarker and useful in developing immunodiagnostic assays and vaccines for H. pylori infection.

DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA.

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL-1, 1.5 copies⋅µL-1, and 0.17 copies⋅µL-1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

Outer membrane vesicles from genetically engineered Salmonella enterica serovar Typhimurium presenting Helicobacter pylori antigens UreB and CagA induce protection against Helicobacter pylori infection in mice.

Helicobacter pylori causes globally prevalent infections that are highly related to chronic gastritis and even development of gastric carcinomas. With the increase of antibiotic resistance, scientists have begun to search for better vaccine design strategies to eradicate H. pylori colonization. However, while current strategies prefer to formulate vaccines with a single H. pylori antigen, their potential has not yet been fully realized. Outer membrane vesicles (OMVs) are a potential platform since they could deliver multiple antigens. In this study, we engineered three crucial H. pylori antigen proteins (UreB, CagA, and VacA) onto the surface of OMVs derived from Salmonella enterica serovar Typhimurium (S. Typhimurium) mutant strains using the hemoglobin protease (Hbp) autotransporter system. In various knockout strategies, we found that OMVs isolated from the ΔrfbP ΔfliC ΔfljB ΔompA mutants could cause distinct increases in immunoglobulin G (IgG) and A (IgA) levels and effectively trigger T helper 1- and 17-biased cellular immune responses, which perform a vital role in protecting against H. pylori. Next, OMVs derived from ΔrfbP ΔfliC ΔfljB ΔompA mutants were used as a vector to deliver different combinations of H. pylori antigens. The antibody and cytokine levels and challenge experiments in mice model indicated that co-delivering UreB and CagA could protect against H. pylori and antigen-specific T cell responses. In summary, OMVs derived from the S. Typhimurium ΔrfbP ΔfliC ΔfljB ΔompA mutant strain as the vector while importing H. pylori UreB and CagA as antigenic proteins using the Hbp autotransporter system would greatly benefit controlling H. pylori infection.

Outer membrane vesicles (OMVs), as a novel antigen delivery platform, has been used in vaccine design for various pathogens and even tumors. Salmonella enterica serovar Typhimurium (S. Typhimurium), as a bacterium that is easy to engineer and has both adjuvant efficacy and immune stimulation capacity, has become the preferred bacterial vector for purifying OMVs after Escherichia coli. This study focuses on the design of Helicobacter pylori ;(H. pylori) vaccines, utilizing genetically modified Salmonella OMVs to present several major antigens of H. pylori, including UreB, VacA and CagA. The optimal Salmonella OMV delivery vector and antigen combinations are screened and identified, providing new ideas for the development of H. pylori vaccines and an integrated antigen delivery platform for other difficult to develop vaccines for bacteria, viruses, and even tumors.

Helicobacter pylori infection in infant rhesus macaque monkeys is associated with an altered lung and oral microbiome.

It is estimated that more than half of the world population has been infected with Helicobacter pylori. Most newly acquired H. pylori infections occur in children before 10 years of age. We hypothesized that early life H. pylori infection could influence the composition of the microbiome at mucosal sites distant to the stomach. To test this hypothesis, we utilized the infant rhesus macaque monkey as an animal model of natural H. pylori colonization to determine the impact of infection on the lung and oral microbiome during a window of postnatal development. From a cohort of 4-7 month-old monkeys, gastric biopsy cultures identified 44% of animals infected by H. pylori. 16S ribosomal RNA gene sequencing of lung washes and buccal swabs from animals showed distinct profiles for the lung and oral microbiome, independent of H. pylori infection. In order of relative abundance, the lung microbiome was dominated by the phyla Proteobacteria, Firmicutes, Bacteroidota, Fusobacteriota, Campilobacterota and Actinobacteriota while the oral microbiome was dominated by Proteobacteria, Firmicutes, Bacteroidota, and Fusobacteriota. In comparison to the oral cavity, the lung was composed of more genera and species that significantly differed by H. pylori status, with a total of 6 genera and species that were increased in H. pylori negative infant monkey lungs. Lung, but not plasma IL-8 concentration was also associated with gastric H. pylori load and lung microbial composition. We found the infant rhesus macaque monkey lung harbors a microbiome signature that is distinct from that of the oral cavity during postnatal development. Gastric H. pylori colonization and IL-8 protein were linked to the composition of microbial communities in the lung and oral cavity. Collectively, these findings provide insight into how H. pylori infection might contribute to the gut-lung axis during early childhood and modulate future respiratory health.

Counterclockwise rotation of the flagellum promotes biofilm initiation in Helicobacter pylori.

Motility promotes biofilm initiation during the early steps of this process: microbial surface association and attachment. Motility is controlled in part by chemotaxis signaling, so it seems reasonable that chemotaxis may also affect biofilm formation. There is a gap, however, in our understanding of the interactions between chemotaxis and biofilm formation, partly because most studies analyzed the phenotype of only a single chemotaxis signaling mutant, e.g., cheA. Here, we addressed the role of chemotaxis in biofilm formation using a full set of chemotaxis signaling mutants in Helicobacter pylori, a class I carcinogen that infects more than half the world's population and forms biofilms. Using mutants that lack each chemotaxis signaling protein, we found that chemotaxis signaling affected the biofilm initiation stage, but not mature biofilm formation. Surprisingly, some chemotaxis mutants elevated biofilm initiation, while others inhibited it in a manner that was not tied to chemotaxis ability or ligand input. Instead, the biofilm phenotype correlated with flagellar rotational bias. Specifically, mutants with a counterclockwise bias promoted biofilm initiation, e.g., ∆cheA, ∆cheW, or ∆cheV1; in contrast, those with a clockwise bias inhibited it, e.g., ∆cheZ, ∆chePep, or ∆cheV3. We tested this correlation using a counterclockwise bias-locked flagellum, which induced biofilm formation independent of the chemotaxis system. These CCW flagella, however, were not sufficient to induce biofilm formation, suggesting there are downstream players. Overall, our work highlights the new finding that flagellar rotational direction promotes biofilm initiation, with the chemotaxis signaling system operating as one mechanism to control flagellar rotation. IMPORTANCE: Chemotaxis signaling systems have been reported to contribute to biofilm formation in many bacteria; however, how they regulate biofilm formation remains largely unknown. Chemotaxis systems are composed of many distinct kinds of proteins, but most previous work analyzed the biofilm effect of loss of only a few. Here, we explored chemotaxis' role during biofilm formation in the human-associated pathogenic bacterium Helicobacter pylori. We found that chemotaxis proteins are involved in biofilm initiation in a manner that correlated with how they affected flagellar rotation. Biofilm initiation was high in mutants with counterclockwise (CCW) flagellar bias and low in those with clockwise bias. We supported the idea that a major driver of biofilm formation is flagellar rotational direction using a CCW-locked flagellar mutant, which stays CCW independent of chemotaxis input and showed elevated biofilm initiation. Our data suggest that CCW-rotating flagella, independent of chemotaxis inputs, are a biofilm-promoting signal.

The role of THBS1 and PDGFD in the immune microenvironment of Helicobacter pylori-associated gastric cancer.

BACKGROUND AND STUDY AIMS: Immunotherapy has emerged as a hot topic in cancer treatment in recent years and has also shown potential in the treatment of Helicobacter pylori-associated gastric cancer. However, there is still a need to identify potential immunotherapy targets. MATERIAL AND METHODS: We used the GSE116312 dataset of Helicobacter pylori-associated gastric cancer to identify differentially expressed genes, which were then overlapped with immune genes from the ImmPort database. The identified immune genes were used to classify gastric cancer samples and evaluate the relationship between classification and tumor mutations, as well as immune infiltration. An immune gene-based prognostic model was constructed, and the expression levels of the genes involved in constructing the model were explored in the tumor immune microenvironment. RESULTS: We successfully identified 60 immune genes and classified gastric cancer samples into two subtypes, which showed differences in prognosis, tumor mutations, immune checkpoint expression, and immune cell infiltration. Subsequently, we constructed an immune prognostic model consisting of THBS1 and PDGFD, which showed significant associations with macrophages and fibroblasts. CONCLUSION: We identified abnormal expression of THBS1 and PDGFD in cancer-associated fibroblasts (CAFs) within the tumor immune microenvironment, suggesting their potential as therapeutic targets.

Genetic variations of penicillin-binding protein 1A: insights into the current status of amoxicillin-based regimens for Helicobacter pylori eradication in Malaysia.

Introduction. Resistance towards amoxicillin in Helicobacter pylori causes significant therapeutic impasse in healthcare settings worldwide. In Malaysia, the standard H. pylori treatment regimen includes a 14-day course of high-dose proton-pump inhibitor (rabeprazole, 20 mg) with amoxicillin (1000 mg) dual therapy.Hypothesis/Gap Statement. The high eradication rate with amoxicillin-based treatment could be attributed to the primary resistance rates of amoxicillin being relatively low at 0%, however, a low rate of secondary resistance has been documented in Malaysia recently.Aim. This study aims to investigate the amino acid mutations and related genetic variants in PBP1A of H. pylori, correlating with amoxicillin resistance in the Malaysian population.Methodology. The full-length pbp1A gene was amplified via PCR from 50 genomic DNA extracted from gastric biopsy samples of H. pylori-positive treatment-naïve Malaysian patients. The sequences were then compared with reference H. pylori strain ATCC 26695 for mutation and variant detection. A phylogenetic analysis of 50 sequences along with 43 additional sequences from the NCBI database was performed. These additional sequences included both amoxicillin-resistant strains (n=20) and amoxicillin-sensitive strains (n=23).Results. There was a total of 21 variants of amino acids, with three of them located in or near the PBP-motif (SKN402-404). The percentages of these three variants are as follows: K403X, 2%; S405I, 2% and E406K, 16%. Based on the genetic markers identified, the resistance rate for amoxicillin in our sample remained at 0%. The phylogenetic examination suggested that H. pylori might exhibit unique conserved pbp1A sequences within the Malaysian context.Conclusions. Overall, the molecular analysis of PBP1A supported the therapeutic superiority of amoxicillin-based regimens. Therefore, it is crucial to continue monitoring the amoxicillin resistance background of H. pylori with a larger sample size to ensure the sustained effectiveness of amoxicillin-based treatments in Malaysia.

Comparison of Helicobacter pylori eradication rates between 7 and 14 days of tailored therapy according to clarithromycin resistance test: A randomized, multicenter, non-inferiority study.

BACKGROUND: Recently, a simple tailored therapy based on clarithromycin resistance has been implemented as Helicobacter pylori (H. pylori) eradication therapy. Nonetheless, despite the tailored therapy and frequent adverse events, studies on treatment period are lacking. This study aimed to compare the H. pylori eradication rates of 7-day and 14-day tailored therapy regimens according to clarithromycin resistance. MATERIALS AND METHODS: This multicenter, prospective, randomized, noninferiority trial enrolled H. pylori-positive patients who were randomly assigned to 7-day and 14-day regimen groups, depending on the presence or absence of clarithromycin resistance by 23S rRNA gene point mutations. Standard triple therapy (STT) (20 mg rabeprazole, 1 g amoxicillin, and 500 mg clarithromycin twice daily) or bismuth quadruple therapy (BQT) (20 mg rabeprazole twice daily, 500 mg metronidazole thrice daily, 120 mg bismuth four times daily, and 500 mg tetracycline four times daily) was assigned by clarithromycin resistance. Eradication rates and adverse events were evaluated. RESULTS: A total of 314 and 278 patients were included in the intention-to-treat (ITT) and per-protocol (PP) analyses, respectively; however, 31 patients were lost to follow-up, whereas five patients violated the protocol. Both the 7-day and 14-day regimens showed similar eradication rates in the ITT (7-day vs. 14-day: 78.3% vs. 78.3%, p > 0.99) and PP (87.9% vs. 89.1%, p = 0.851) analyses. Non-inferiority was confirmed (p < 0.025). A subgroup analysis according to clarithromycin resistance (clarithromycin resistance rate: 28.7%) revealed no significant difference in eradication rates between the 7-day and 14-day STT (90.0% vs. 90.1%, p > 0.99) and BQT (82.5% vs. 86.5%, p = 0.757). Furthermore, adverse events did not significantly differ between the two groups. CONCLUSIONS: The 7-day triple and quadruple therapy according to clarithromycin resistance showed similar eradication rates, as compared to the 14-day therapy.

MicroRNAs in Helicobacter pylori-infected gastric cancer: Function and clinical application.

Gastric cancer (GC) is the leading cause of cancer-related death worldwide, and it is associated with a combination of genetic, environmental, and microbial risk factors. Helicobacter pylori (H. pylori) is classified as a type I carcinogen, however, the exact regulatory mechanisms underlying H. pylori-induced GC are incompletely defined. MicroRNAs (miRNAs), one of small non-coding RNAs, negatively regulate gene expression through binding to their target genes. Dysregulation of miRNAs is crucial in human cancer. A noteworthy quantity of aberrant miRNAs induced by H. pylori through complex regulatory networks have been identified. These miRNAs substantially affect genetic instability, cell proliferation, apoptosis, invasion, metastasis, autophagy, chemoresistance, and the tumor microenvironment, leading to GC development and progression. Importantly, some H. pylori-associated miRNAs hold promise as therapeutic tools and biomarkers for GC prevention, diagnosis, and prognosis. Nonetheless, clinical application of miRNAs remains in its infancy with multiple issues, including sensitivity and specificity, stability, reliable delivery systems, and off-target effects. Additional research on the specific molecular mechanisms and more clinical data are still required. This review investigated the biogenesis, regulatory mechanisms, and functions of miRNAs in H. pylori-induced GC, offering novel insights into the potential clinical applications of miRNA-based therapeutics and biomarkers.