The Relationship Between Mucosal Microbiota, Colitis, and Systemic Inflammation in Chronic Granulomatous Disorder

PURPOSE: Chronic granulomatous disorder (CGD) is a primary immunodeficiency which is frequently complicated by inflammatory colitis and is associated with systemic inflammation. Herein, we aimed to investigate the role of the microbiome in the pathogenesis of colitis and systemic inflammation. METHODS: We performed 16S rDNA sequencing on mucosal biopsy samples from each segment of 10 CGD patients’ colons and conducted compositional and functional pathway prediction analyses. RESULTS: The microbiota in samples from colitis patients demonstrated reduced taxonomic alpha-diversity compared to unaffected patients, even in apparently normal bowel segments. Functional pathway richness was similar between the colitic and non-colitic mucosa, although metabolic pathways involved in butyrate biosynthesis or utilization were enriched in patients with colitis and correlated positively with fecal calprotectin levels. One patient with very severe colitis was dominated by Enterococcus spp., while among other patients Bacteroides spp. abundance correlated with colitis severity measured by fecal calprotectin and an endoscopic severity score. In contrast, Blautia abundance is associated with low severity scores and mucosal health. Several taxa and functional pathways correlated with concentrations of inflammatory cytokines in blood but not with colitis severity. Notably, dividing patients into “high” and “low” systemic inflammation groups demonstrated clearer separation than on the basis of colitis status in beta-diversity analyses. CONCLUSION: The microbiome is abnormal in CGD-associated colitis and altered functional characteristics probably contribute to pathogenesis. Furthermore, the relationship between the mucosal microbiome and systemic inflammation, independent of colitis status, implies that the microbiome in CGD can influence the inflammatory phenotype of the condition

Oral Microcosm Biofilms Grown under Conditions Progressing from Peri-Implant Health, Peri-Implant Mucositis, and Peri-Implantitis

Peri-implantitis is a disease influenced by dysbiotic microbial communities that play a role in the short- and long-term outcomes of its clinical treatment. The ecological triggers that establish the progression from peri-implant mucositis to peri-implantitis remain unknown. This investigation describes the development of a novel in vitro microcosm biofilm model. Biofilms were grown over 30 days over machined titanium discs in a constant depth film fermentor (CDFF), which was inoculated (I) with pooled human saliva. Following longitudinal biofilm sampling across peri-implant health (PH), peri-implant mucositis (PM), and peri-implantitis (PI) conditions, the characterisation of the biofilms was performed. The biofilm analyses included imaging by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), selective and non-selective culture media of viable biofilms, and 16S rRNA gene amplification and sequencing. Bacterial qualitative shifts were observed by CLSM and SEM across conditions, which were defined by characteristic phenotypes. A total of 9 phyla, 83 genera, and 156 species were identified throughout the experiment. The phyla Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria showed the highest prevalence in PI conditions. This novel in vitro microcosm model provides a high-throughput alternative for growing microcosm biofilms resembling an in vitro progression from PH-PM-PI conditions

A Multi-scale Biophysical Approach to Develop Structure-Property Relationships in Oral Biofilms

Over the last 5-10 years, optical coherence tomography (OCT) and atomic force microscopy (AFM) have been individually applied to monitor the morphological and mechanical properties of various single-species biofilms respectively. This investigation looked to combine OCT and AFM as a multi-scale approach to understand the role sucrose concentration and age play in the morphological and mechanical properties of oral, microcosm biofilms, in-vitro. Biofilms with low (0.1% w/v) and high (5% w/v) sucrose concentrations were grown on hydroxyapatite (HAP) discs from pooled human saliva and incubated for 3 and 5 days. Distinct mesoscale features of biofilms such as regions of low and high extracellular polymeric substances (EPS) were identified through observations made by OCT. Mechanical analysis revealed increasing sucrose concentration decreased Young's modulus and increased cantilever adhesion (p < 0.0001), relative to the biofilm. Increasing age was found to decrease adhesion only (p < 0.0001). This was due to mechanical interactions between the indenter and the biofilm increasing as a function of increased EPS content, due to increasing sucrose. An expected decrease in EPS cantilever contact decreased adhesion due to bacteria proliferation with biofilm age. The application OCT and AFM revealed new structure-property relationships in oral biofilms, unattainable if the techniques were used independently

Antibiotic resistance, bacterial transmission and improved prediction of bacterial infection in patients with antibody deficiency

Background Antibody-deficient patients are at high risk of respiratory tract infections. Many therefore receive antibiotic prophylaxis and have access to antibiotics for self-administration in the event of breakthrough infections, which may increase antimicrobial resistance (AMR). Objectives To understand AMR in the respiratory tract of patients with antibody deficiency. Methods Sputum samples were collected from antibody-deficient patients in a cross-sectional and prospective study; bacteriology culture, 16S rRNA profiling and PCR detecting macrolide resistance genes were performed. Bacterial isolates were identified using MALDI-TOF, antimicrobial susceptibility was determined by disc diffusion and WGS of selected isolates was done using Illumina NextSeq with analysis for resistome and potential cross-transmission. Neutrophil elastase was measured by a ProteaseTag immunoassay. Results Three hundred and forty-three bacterial isolates from sputum of 43 patients were tested. Macrolide and tetracycline resistance were common (82% and 35% of isolates). erm(B) and mef(A) were the most frequent determinants of macrolide resistance. WGS revealed viridans streptococci as the source of AMR genes, of which 23% also carried conjugative plasmids linked with AMR genes and other mobile genetic elements. Phylogenetic analysis of Haemophilus influenzae isolates suggested possible transmission between patients attending clinic. In the prospective study, a negative correlation between sputum neutrophil elastase concentration and Shannon entropy α-diversity (Spearman’s ρ = −0.306, P = 0.005) and a positive relationship with Berger–Parker dominance index (ρ = 0.502, P < 0.001) were found. Similar relationships were noted for the change in elastase concentration between consecutive samples, increases in elastase associating with reduced α-diversity. Conclusions Measures to limit antibiotic usage and spread of AMR should be implemented in immunodeficiency clinics. Sputum neutrophil elastase may be a useful marker to guide use of antibiotics for respiratory infection

Patterns of subgingival microbiota in different periodontal phenotypes

Objectives: To compare the subgingival microbiota of patients with aggressive (AgP) or chronic periodontitis (CP) to healthy (H), non-periodontitis patients as well as to explore their relevant associations to different host genetic variants. Methods: Following clinical examination, blood and subgingival plaque sampling of 471 study participants (125 AgP, 121 CP, 225 H), subgingival community analysis was performed by next generation sequencing of the 16S rRNA. Microbial data from 266 participants (75 AgP, 95 CP, 98 H) were available for analysis. SNPs in the IL6, IL6R and FTO gene were selected for genetic marker analyses. Results: Combined periodontitis patients (AgP + CP), particularly those classified with AgP, exhibited lower alpha- and beta- diversity. Several genera (including Peptostreptococcaceae, Filifactor, Desulfobulbus, Tannerella and Lachnospiracee) and species were over-abundant in combined periodontitis vs. healthy individuals, while other genera such as Prevotella or Dialister were found to be more abundant in healthy cases. The only genus with difference in abundance between AgP and CP was Granulicatella. No associations between IL6, IL6RA and FTO genetic variants and microbial findings were detected. Conclusion: This study suggests that limited microbial differences existed between AgP and CP and challenges the current notion that periodontitis is associated with increased subgingival microbial diversity compared with periodontal health. Clinical significance: The findings of this study cast some doubts on the notion that the dysbiosis characteristic of periodontal disease is expressed as increased microbial diversity

Dependency of hydration and growth conditions on the mechanical properties of oral biofilms

Within the oral cavity, dental biofilms experience dynamic environments, in part due to changes in dietary content, frequency of intake and health conditions. This can impact bacterial diversity and morpho-mechanical properties. While phenotypic properties of oral biofilms are closely related to their composition, these can readily change according to dynamic variations in the growth environment and nutrient availability. Understanding the interlink between phenotypic properties, variable growth conditions, and community characterization is an essential requirement to develop structure–property relationships in oral-biofilms. In this study, the impact of two distinct growth media types with increasing richness on the properties of oral biofilms was assessed through a new combination of in-vitro time-lapse biophysical methods with microbiological assays. Oral biofilms grown in the enriched media composition presented a decrease in their pH, an increase in soluble EPS production, and a severe reduction in bacterial diversity. Additionally, enriched media conditions presented an increase in biofilm volumetric changes (upon hydration) as well as a reduction in elastic modulus upon indentation. With hydration time considered a major factor contributing to changes in biofilm mechanical properties, we have shown that it is less associated than media richness. Future investigations can now use this time-lapse approach, with a clearer focus on the extracellular matrix of oral biofilms dictating their morpho-mechanical properties

Airway inflammation and dysbiosis in antibody deficiency despite the presence of IgG

Background: Patients with antibody deficiency suffer chronic respiratory symptoms, recurrent exacerbations, and progressive airways disease despite systemic replacement of IgG. Little is known about the respiratory tract biology of these patients. Objective: We sought to measure immunoglobulin levels, inflammatory cytokines, and mediators of tissue damage in serum and sputum from patients with antibody deficiency and healthy controls; to analyze the respiratory microbiome in the same cohorts. Methods: We obtained paired sputum and serum samples from 31 immunocompetent subjects and 67 antibody-deficient patients, the latter divided on computed tomography scan appearance into “abnormal airways” (bronchiectasis or airway thickening) or “normal airways.” We measured inflammatory cytokines, immunoglobulin levels, neutrophil elastase, matrix-metalloproteinase-9, urea, albumin, and total protein levels using standard assays. We used V3-V4 region 16S sequencing for microbiome analysis. Results: Immunodeficient patients had markedly reduced IgA in sputum but higher concentrations of IgG compared with healthy controls. Inflammatory cytokines and tissue damage markers were higher in immunodeficient patients, who also exhibited dysbiosis with overrepresentation of pathogenic taxa and significantly reduced alpha diversity compared with immunocompetent individuals. These differences were seen regardless of airway morphology. Sputum matrix-metalloproteinase-9 and elastase correlated inversely with alpha diversity in the antibody-deficient group, as did sputum IgG, which correlated positively with several inflammatory markers, even after correction for albumin levels. Conclusions: Patients with antibody deficiency, even with normal lung imaging, exhibit inflammation and dysbiosis in their airways despite higher levels of IgG compared with healthy controls