Low Diversity and Instability of the Sinus Microbiota over Time in Adults with Cystic Fibrosis.

Chronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). Recently developed standard of care guidelines for the management of CF CRS suggest treatment of upper airway disease may ameliorate lower airway disease. We sought to determine whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. We performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. Sinus community diversity was low, and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased interleukin-1ß [IL-1ß]). Increased IL-1ß was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. IMPORTANCE Together with prior sinus microbiota studies of adults with CF chronic rhinosinusitis, our study underscores similarities between sinus and lower respiratory tract microbial community structures in CF. We show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1ß in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1ß. Finally, the widespread use of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Child saliva microbiota and caries: a randomized controlled maternal education trial in rural Uganda.

Undernutrition is a public health challenge in sub-Saharan countries, including Uganda. In a previous randomized controlled trial (RCT) with a nutrition, hygiene and stimulation education intervention among mothers of 6 months' old children, we found less caries in the intervention group when the children were 36 months of age. We now examined the effects of (i) the intervention on the microbiota, (ii) microbiota on caries, and (iii) the intervention and microbiota on caries. The original RCT comprised 511 mother/child pairs whereas in the current study we had access to data from 344/511 (67%) children aged 36 months. The saliva microbiota was determined using 16S rRNA gene sequencing. Carious lesions (a proxy for dental health) were identified using close-up intra-oral photographs of the upper front teeth. Statistical models were used to determine host-microbiota associations. The intervention had a significant effect on the microbiota, e.g. an increase in Streptococcus abundance and decreases in Alloprevotella and Tannerella. Significant associations between the microbiota and dental caries were identified: Positive associations of Capnocytophaga and Tannerella suggest that these taxa may be deleterious to dental health while negative associations of Granulicatella, Fusobacterium, and Abiotrophia suggest taxa potentially beneficial or benign contributors to dental health. Based on taxonomic profiles, the effects of the intervention and microbiota on dental health may be independent of one another. Educational interventions with emphasis on nutrition and oral hygiene may provide a feasible strategy to decrease progression of childhood caries in low-resource settings.

Lung transplantation for idiopathic pulmonary fibrosis enriches for individuals with telomere-mediated disease.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is the most common indication for lung transplantation in North America and variants in telomere-maintenance genes are the most common identifiable cause of IPF. We reasoned that younger IPF patients are more likely to undergo lung transplantation and we hypothesized that lung transplant recipients would be enriched for individuals with telomere-mediated disease due to the earlier onset and more severe disease in these patients. METHODS: Individuals with IPF who underwent lung transplantation or were evaluated in an interstitial lung disease specialty clinic who did not undergo lung transplantation were examined. Genetic evaluation was completed via whole genome sequencing (WGS) of 426 individuals and targeted sequencing for 5 individuals. Rare variants in genes previously associated with IPF were classified using the American College of Medical Genetics guidelines. Telomere length from WGS data was measured using TelSeq software. Patient characteristics were collected via medical record review. RESULTS: Of 431 individuals, 149 underwent lung transplantation for IPF. The median age of diagnosis of transplanted vs non-transplanted individuals was significantly younger (60 years vs 70 years, respectively, p<0.0001). IPF lung transplant recipients (IPF-LTRs) were twice as likely to have telomere-related rare variants compared to non-transplanted individuals (24% vs 12%, respectively, p=0.0013). IPF-LTRs had shorter telomeres than non-transplanted IPF patients (p=0.0028) and >85% had telomeres below the age-adjusted mean. Post-transplant survival and CLAD were similar amongst IPF-LTRs with rare variants in telomere-maintenance genes compared to those without, as well as in those with short telomeres versus longer telomeres. CONCLUSIONS: There is an enrichment for telomere-maintenance gene variants and short telomeres among IPF-LTRs. However, transplant outcomes of survival and CLAD do not differ by gene variants or telomere length within IPF-LTRs. Our findings support individual with telomere-mediated disease should not be excluded from lung transplantation and focusing research efforts on therapies directed toward individuals with short-telomere mediated disease.

"Longitudinal Fecal Microbiome Study of Total Body Irradiated Mice Treated With Radiation Mitigators Identifies Bacterial Associations With Survival".

Total body irradiation (TBI) has been demonstrated to alter the intestinal microbiome, but the effects of successful small molecule ionizing radiation mitigators on the intestinal microbiome are not well-known. Our survival experiments examined the effects of anti-cell death radiation mitigators on and in conjunction with the host's microbiota. Mice received 9.25 Gy TBI and then were administered radiation mitigators 24 hours later. Passed stool were collected pre-irradiation, then on days 1, 3, 5, 7, 10, 14, 21, and 30 post-irradiation for 16S rRNA gene (V4 region) sequencing. The Cox proportional hazards (CPH) model was fit with taxonomic composition (time varying covariates) and treatment as predictors. In the first experiment, mice were administered drugs for "granulocyte stimulation and anti-apoptosis" in four protocol combinations: JP4-039 (anti-apoptosis), granulocyte colony-stimulating factor (G-CSF, granulopoietic precursor cell stimulator), both mitigators, and control. Survival improved relative to control (30.0%) for G-CSF (80%, p-value = 0.025), G-CSF/JP4-039 (70%, p-value = 0.084), but not for JP4-039 (50.0%). In the second experiment, mice were administered mitigation drugs "inhibiting programmed cell death" pathways: JP4-039 (anti-apoptosis), necrostatin-1 (anti-necroptosis), and baicalein (anti-ferroptosis), in eight combinations. The survival of JP4-039/baicalein (60.0%, p-value = 0.010) and JP4-039/baicalein/necrostatin-1 (60.0%, p-value = 0.06) treatment combinations were significantly different from the control (26.7%). The JP4-039/necrostatin-1 (46.7%) and baicalein/necrostatin-1 (40.0%) and singlet treatment combinations (26.7%) were not significantly different from the control. Despite differences between the baseline microbiota compositions of the two experiments, consistent changes in composition after irradiation were found: Lactobacillus decreased post-irradiation, relative to baseline. By day 7, microbiota perturbations had incompletely reversed, and no drug-specific differences were identifiable. The CPH model identified Lactobacillus and members of Ruminococcaceae, including Ruminococcus, as protective and Akkermansia as deleterious. By day 30, the microbiota of surviving mice had not returned to baseline, but the differences between experiments suggest the resultant microbiota composition of the survivors are stochastic or batch specific in nature, rather than a requirement for survival. In conclusion, the study determined that key taxa identified in fecal samples, when applied towards the prediction of TBI survival, improves the survival model relative to treatment information alone.

Dynamics of hepatic steatosis resolution and changes in gut microbiome with weight loss in nonalcoholic fatty liver disease.

BACKGROUND: Weight loss is recommended as the primary treatment for nonalcoholic fatty liver disease (NAFLD). However, the magnitude and velocity of hepatic steatosis resolution with weight loss is unclear, making it difficult to counsel patients seeking weight loss for treatment of NAFLD. The aim of this study was to determine the rate of hepatic steatosis improvement and stool microbiome changes associated with rapid diet-induced weight loss in NAFLD. METHODS: Fourteen NAFLD patients (mean ± standard deviation, body mass index [BMI] 36.4 ± 4 kg/m2) enrolled in a 12-week meal replacement program underwent frequent measurement of Fibroscan-controlled attenuation parameter (CAP). Magnetic resonance imaging (MRI-Dixon method) for hepatic fat quantitation and stool microbiome analysis (16S rRNA gene sequencing) were completed in 11 subjects at baseline and Week 12. RESULTS: At Week 12, mean (95% confidence interval) weight loss was -13.4 (-15.2, -11.5)% and CAP score -26.6 (-35.6, -17.6)% (both Ps < 0.001). CAP scores changed at a rate of -4.9 dB/m/kg (-30.1 dB/m per unit BMI) in Weeks 1-4 and -0.6 dB/m/kg (-2.4 dB/m per unit BMI) in Weeks 8-12. MRI-determined hepatic fat fraction decreased by -74.1% (p < 0.001) at a rate of -0.51%/kg (-3.19% per unit BMI), with complete steatosis resolution in 90% patients. BMI change was associated with decreased stool microbial diversity (coefficient = 0.17; Shannon Index), increased abundance of Prevotella_9 (Bacteroidetes; coefficient = 0.96) and decreased abundance of Phascolarctobacterium (Firmicutes; coefficient = -0.42) (both Ps < 0.05). CONCLUSIONS: Diet-induced intensive weight loss is associated with rapid improvement and complete resolution of hepatic steatosis and decreased stool microbial diversity. These findings highlight the dynamic nature of hepatic fat and may help clinicians to develop evidence-based treatment goals for patients with NAFLD and obesity who undertake weight loss interventions. Further research is warranted to understand the effects of intensive weight loss and gut microbiome changes on long-term NAFLD resolution.

The microbiome of pediatric patients with chronic rhinosinusitis.

BACKGROUND: This study aimed to compare the microbiota of pediatric patients with chronic rhinosinusitis (CRS) who are undergoing adenoidectomy to treat their disease with that of healthy control patients. METHODS: Patients undergoing adenoidectomy-only for obstructive sleep apnea (n = 50) and CRS (n = 37) were recruited. Preoperative 22-item Sino-Nasal Outcome Test (SNOT-22) or Sinus and Nasal Quality of Life Survey (SN-5) were collected. Each patient had samples collected from their nasopharynx (adenoid bed) and nasal cavity (sinus) at the onset of surgery. 16S ribosomal ribonucleic acid (rRNA) gene sequencing was subsequently performed to obtain per sample taxonomic abundances. Statistical analyses included permutational multivariate analysis of variance (PERMANOVA), alpha (within sample) diversity measures, and changes in taxonomic abundance. RESULTS: Moraxella was the most abundant organism. Nasopharyngeal swabs demonstrated higher alpha diversity compared to the nasal cavity. The diversity was not different based on CRS vs obstructive history. There was an increase in diversity with increasing age, and eczema contributed to a greater difference in diversity between the nasopharynx and nasal cavity. Diversity was not affected by adenoid size; however, use of nasal steroids, inhaled steroids, and antihistamines influenced diversity in both the nasopharynx and nasal cavity. Nasopharyngeal samples were higher in relative abundance for Fusobacterium, Prevotella, Porphyromonas, and Campylobacter compared to the nasal cavity. CONCLUSION: The nasopharynx and nasal cavity differed in both microbiota composition and diversity. In contrast, no significant difference in composition or diversity were found in CRS vs control patients. Ecological changes in the nasopharyngeal and sinus site may contribute to the etiology for adenoid hypertrophy in both healthy controls and CRS patients.

Antibiotics in early life alter the murine colonic microbiome and adiposity.

Antibiotics administered in low doses have been widely used as growth promoters in the agricultural industry since the 1950s, yet the mechanisms for this effect are unclear. Because antimicrobial agents of different classes and varying activity are effective across several vertebrate species, we proposed that such subtherapeutic administration alters the population structure of the gut microbiome as well as its metabolic capabilities. We generated a model of adiposity by giving subtherapeutic antibiotic therapy to young mice and evaluated changes in the composition and capabilities of the gut microbiome. Administration of subtherapeutic antibiotic therapy increased adiposity in young mice and increased hormone levels related to metabolism. We observed substantial taxonomic changes in the microbiome, changes in copies of key genes involved in the metabolism of carbohydrates to short-chain fatty acids, increases in colonic short-chain fatty acid levels, and alterations in the regulation of hepatic metabolism of lipids and cholesterol. In this model, we demonstrate the alteration of early-life murine metabolic homeostasis through antibiotic manipulation.

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains.

BACKGROUND: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. RESULTS: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75-0.78 Mbp genomes and UUR serovars were 0.84-0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. CONCLUSIONS: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.

Genome-wide expression profiling in Geobacter sulfurreducens: identification of Fur and RpoS transcription regulatory sites in a relGsu mutant.

Rel(Gsu) is the single Geobacter sulfurreducens homolog of RelA and SpoT proteins found in many organisms. These proteins are involved in the regulation of levels of guanosine 3', 5' bispyrophosphate, ppGpp, a molecule that signals slow growth and stress response under nutrient limitation in bacteria. We used information obtained from genome-wide expression profiling of the rel(Gsu) deletion mutant to identify putative regulatory sites involved in transcription networks modulated by Rel(Gsu) or ppGpp. Differential gene expression in the rel(Gsu) deletion mutant, as compared to the wild type, was available from two growth conditions, steady state chemostat cultures and stationary phase batch cultures. Hierarchical clustering analysis of these two datasets identified several groups of operons that are likely co-regulated. Using a search for conserved motifs in the upstream regions of these co-regulated operons, we identified sequences similar to Fur- and RpoS-regulated sites. These findings suggest that Fur- and RpoS-dependent gene expression in G. sulfurreducens is affected by Rel(Gsu)-mediated signaling.

Role of RelGsu in stress response and Fe(III) reduction in Geobacter sulfurreducens.

Geobacter species are key members of the microbial community in many subsurface environments in which dissimilatory metal reduction is an important process. The genome of Geobacter sulfurreducens contains a gene designated rel(Gsu), which encodes a RelA homolog predicted to catalyze both the synthesis and the degradation of guanosine 3',5'-bispyrophosphate (ppGpp), a regulatory molecule that signals slow growth in response to nutrient limitation in bacteria. To evaluate the physiological role of Rel(Gsu) in G. sulfurreducens, a rel(Gsu) mutant was constructed and characterized, and ppGpp levels were monitored under various conditions in both the wild-type and rel(Gsu) mutant strains. In the wild-type strain, ppGpp and ppGp were produced in response to acetate and nitrogen deprivation, whereas exposure to oxygen resulted in an accumulation of ppGpp alone. Neither ppGpp nor ppGp could be detected in the rel(Gsu) mutant. The rel(Gsu) mutant consistently grew to a higher cell density than the wild type in acetate-fumarate medium and was less tolerant of oxidative stress than the wild type. The capacity for Fe(III) reduction was substantially diminished in the mutant. Microarray and quantitative reverse transcription-PCR analyses indicated that during stationary-phase growth, protein synthesis genes were up-regulated in the rel(Gsu) mutant and genes involved in stress responses and electron transport, including several implicated in Fe(III) reduction, were down-regulated in the mutant. The results are consistent with a role for Rel(Gsu) in regulating growth, stress responses, and Fe(III) reduction in G. sulfurreducens under conditions likely to be prevalent in subsurface environments.