Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial ...community composition. Rapid freezing to -80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants' homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to -80 °C. Refrigeration at 4 °C, storage at ambient temperature, and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to -80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available.
Background Asthma pathophysiology and treatment responsiveness are predicted by inflammatory phenotype. However, the relationship between airway microbiology and asthma phenotype is poorly ...understood. Objective We aimed to characterize the airway microbiota in patients with symptomatic stable asthma and relate composition to airway inflammatory phenotype and other phenotypic characteristics. Methods The microbial composition of induced sputum specimens collected from adult patients screened for a multicenter randomized controlled trial was determined by using 16S rRNA gene sequencing. Inflammatory phenotypes were defined by sputum neutrophil and eosinophil cell proportions. Microbiota were defined by using α- and β-diversity measures, and interphenotype differences were identified by using similarity of percentages, network analysis, and taxon fold change. Phenotypic predictors of airway microbiology were identified by using multivariate linear regression. Results Microbiota composition was determined in 167 participants and classified as eosinophilic (n = 84), neutrophilic (n = 14), paucigranulocytic (n = 60), or mixed neutrophilic-eosinophilic (n = 9) asthma phenotypes. Airway microbiology was significantly less diverse ( P = .022) and more dissimilar ( P = .005) in neutrophilic compared with eosinophilic participants. Sputum neutrophil proportions, but not eosinophil proportions, correlated significantly with these diversity measures (α-diversity: Spearman r = −0.374, P < .001; β-diversity: r = 0.238, P = .002). Interphenotype differences were characterized by a greater frequency of pathogenic taxa at high relative abundance and reduced Streptococcus , Gemella , and Porphyromonas taxa relative abundance in patients with neutrophilic asthma. Multivariate regression confirmed that sputum neutrophil proportion was the strongest predictor of microbiota composition. Conclusions Neutrophilic asthma is associated with airway microbiology that is significantly different from that seen in patients with other inflammatory phenotypes, particularly eosinophilic asthma. Differences in microbiota composition might influence the response to antimicrobial and steroid therapies and the risk of lung infection.
The macrolide antibiotic azithromycin reduces exacerbations in adults with persistent symptomatic asthma. However, owing to the pleotropic properties of macrolides, unintended bacteriological ...consequences such as augmented pathogen colonization or dissemination of antibiotic-resistant organisms can occur, calling into question the long-term safety of azithromycin maintenance therapy.
To assess the effects of azithromycin on the airway microbiota, pathogen abundance, and carriage of antibiotic resistance genes.
16S rRNA sequencing and quantitative PCR were performed to assess the effect of azithromycin on sputum microbiology from participants of the AMAZES (Asthma and Macrolides: The Azithromycin Efficacy and Safety) trial: a 48-week, double-blind, placebo-controlled trial of thrice-weekly 500 mg oral azithromycin in adults with persistent uncontrolled asthma. Pooled-template shotgun metagenomic sequencing, quantitative PCR, and isolate whole-genome sequencing were performed to assess antibiotic resistance.
Paired sputum samples were available from 61 patients (
= 34 placebo,
= 27 azithromycin). Azithromycin did not affect bacterial load (
= 0.37) but did significantly decrease Faith's phylogenetic diversity (
= 0.026) and
load (
< 0.0001). Azithromycin did not significantly affect levels of
,
,
, or
. Of the 89 antibiotic resistance genes detected, five macrolide resistance genes and two tetracycline resistance genes were increased significantly.
In patients with persistent uncontrolled asthma, azithromycin reduced airway
load compared with placebo but did not change total bacterial load. Macrolide resistance increased, reflecting previous studies. These results highlight the need for studies assessing the efficacy of nonantibiotic macrolides as a long-term therapy for patients with persistent uncontrolled asthma.
Exacerbations of asthma cause a substantial global illness burden. Adults with uncontrolled persistent asthma despite maintenance treatment require additional therapy. Since macrolide antibiotics can ...be used to treat persistent asthma, we aimed to assess the efficacy and safety of oral azithromycin as add-on therapy in patients with uncontrolled persistent asthma on medium-to-high dose inhaled corticosteroids plus a long-acting bronchodilator.
We did a randomised, double-blind, placebo controlled parallel group trial to determine whether oral azithromycin decreases the frequency of asthma exacerbations in adults (≥18 years) with symptomatic asthma despite current use of inhaled corticosteroid and long-acting bronchodilator, and who had no hearing impairment or abnormal prolongation of the corrected QT interval. Patients were randomly assigned (1:1) to receive azithromycin 500 mg or placebo three times per week for 48 weeks. Patients were centrally allocated using concealed random allocation from a computer-generated random numbers table with permuted blocks of 4 or 6 and stratification for centre and past smoking. Primary efficacy endpoints were the rate of total (severe and moderate) asthma exacerbations over 48 weeks and asthma quality of life. Data were analysed on an intention-to-treat basis. The trial is registered at the Australian and New Zealand Clinical Trials Registry (ANZCTR), number 12609000197235.
Between June 12, 2009, and Jan 31, 2015, 420 patients were randomly assigned (213 in the azithromycin group and 207 in the placebo group). Azithromycin reduced asthma exacerbations (1·07 per patient-year 95% CI 0·85–1·29) compared with placebo (1·86 per patient-year 1·54–2·18; incidence rate ratio IRR 0·59 95% CI 0·47–0·74; p<0·0001). The proportion of patients experiencing at least one asthma exacerbation was reduced by azithromycin treatment (127 61% patients in the placebo group vs 94 44% patients in the azithromycin group, p<0·0001). Azithromycin significantly improved asthma-related quality of life (adjusted mean difference, 0·36 95% CI 0·21–0·52; p=0·001). Diarrhoea was more common in azithromycin-treated patients (72 34% vs 39 19%; p=0·001).
Adults with persistent symptomatic asthma experience fewer asthma exacerbations and improved quality of life when treated with oral azithromycin for 48 weeks. Azithromycin might be a useful add-on therapy in persistent asthma.
National Health and Medical Research Council of Australia, John Hunter Hospital Charitable Trust.
Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were ...investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.
Monofluoroacetate (MFA) is a potent toxin that blocks ATP production via the Krebs cycle and causes acute toxicity in ruminants consuming MFA-containing plants. The rumen bacterium, Cloacibacillus ...porcorum strain MFA1 belongs to the phylum Synergistota and can produce fluoride and acetate from MFA as the end-products of dehalorespiration. The aim of this study was to identify the genomic basis for the metabolism of MFA by this bacterium.
A draft genome sequence for C. porcorum strain MFA1 was assembled and quantitative transcriptomic analysis was performed thus highlighting a candidate operon encoding four proteins that are responsible for the carbon-fluorine bond cleavage. Comparative genome analysis of this operon was undertaken with three other species of closely related Synergistota bacteria.
Two of the genes in this operon are related to the substrate-binding components of the glycine reductase protein B (GrdB) complex. Glycine shares a similar structure to MFA suggesting a role for these proteins in binding MFA. The remaining two genes in the operon, an antiporter family protein and an oxidoreductase belonging to the radical S-adenosyl methionine superfamily, are hypothesised to transport and activate the GrdB-like protein respectively. Similar operons were identified in a small number of other Synergistota bacteria including type strains of Cloacibacillus porcorum, C. evryensis, and Pyramidobacter piscolens, suggesting lateral transfer of the operon as these genera belong to separate families. We confirmed that all three species can degrade MFA, however, substrate degradation in P. piscolens was notably reduced compared to Cloacibacillus isolates possibly reflecting the loss of the oxidoreductase and antiporter in the P. piscolens operon.
Identification of this unusual anaerobic fluoroacetate metabolism extends the known substrates for dehalorespiration and indicates the potential for substrate plasticity in amino acid-reducing enzymes to include xenobiotics.
Hotel quarantine for international travelers has been used to prevent coronavirus disease spread into Australia. A quarantine hotel–associated community outbreak was detected in South Australia. ...Real-time genomic sequencing enabled rapid confirmation tracking the outbreak to a recently returned traveler and linked 2 cases of infection in travelers at the same facility.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
The emergence of antimicrobial-resistant bacteria represents a considerable threat to human health, particularly for vulnerable populations such as those living in residential ...aged care. However, antimicrobial resistance carriage and modes of transmission remain incompletely understood. The Generating evidence on antimicrobial Resistance in the Aged Care Environment (GRACE) study was established to determine principal risk factors of antimicrobial resistance carriage and transmission in residential aged care facilities (RACFs). This article describes the cohort characteristics, national representation, and planned analyses for this study.
Methods
Between March 2019 and March 2020, 279 participants were recruited from five South Australian RACFs. The median age was 88.6 years, the median period in residence was 681 days, and 71.7% were female. A dementia diagnosis was recorded in 54.5% and more than two thirds had moderate to severe cognitive impairment (68.8%). 61% had received at least one course of antibiotics in the 12 months prior to enrolment.
Results
To investigate the representation of the GRACE cohort to Australians in residential aged care, its characteristics were compared to a subset of the historical cohort of the Registry of Senior Australians (ROSA). This included 142,923 individuals who were permanent residents of RACFs on June 30th, 2017. GRACE and ROSA cohorts were similar in age, sex, and duration of residential care, prevalence of health conditions, and recorded dementia diagnoses. Differences were observed in care requirements and antibiotic exposure (both higher for GRACE participants). GRACE participants had fewer hospital visits compared to the ROSA cohort, and a smaller proportion were prescribed psycholeptic medications.
Conclusions
We have assembled a cohort of aged care residents that is representative of the Australian aged care population, and which provides a basis for future analyses. Metagenomic data isolated from participants and built environments will be used to determine microbiome and resistome characteristics of an individual and the facility. Individual and facility risk exposures will be aligned with metagenomic data to identify principal determinants for antimicrobial resistance carriage. Ultimately, this analysis will inform measures aimed at reducing the emergence and spread of antimicrobial resistant pathogens in this high-risk population.
Higher density of Neisseria meningitidis carriage may be associated with transmission of the meningococcus. Our aim was to establish the impact of 4CMenB vaccine on N. meningitidis carriage density.
...We compared 4CMenB vaccine to control among 913 South Australian students aged approximately 15-18 years in a cluster randomized trial who had N. meningitidis carriage at 12 months. Oropharyngeal swabs were collected at baseline and 12 months later to detect N. meningitidis carriage. Colony forming units per millilitre (CFU/ml) were estimated by generating a standard curve that plotted qPCR cycle threshold values against log-normalized CFU.
Among the 913 students with N. meningitidis carriage at 12 months, there was no difference in mean carriage density between the vaccinated (n=434, 3.80 log CFU/ml SD 1.29) and control group (n=479, 3.73 log CFU/ml SD 1.30; p=0.51). Higher N. meningitidis carriage density at baseline was associated with an increase in the odds of persistent carriage at 12 months (n=504, odds ratio per 1.0 log CFU/ml increase in density = 1.36 95% CI, 1.17, 1.58, p<0.001). Students with baseline carriage who were vaccinated had decreased persistent N. meningitidis carriage at 12 months compared to unvaccinated students (82/186 31% vs 105/186 43%, odds ratio 0.60 95% CI, 0.40, 0.90, p=0.01).
4CMenB vaccine did not reduce carriage density of N. meningitidis 12 months post vaccination, despite increased carriage clearance. Higher carriage density is likely to enable transmission through prolonged periods of population exposure.
Clinicaltrials.gov NCT03089086.
Objectives:
To examine if COVID-19 containment strategies were associated with reduced pharyngeal carriage of meningococci in adolescents. Also, to observe if carriage prevalence of meningococcal A, ...C, W and Y differed in meningococcal conjugate ACWY vaccinated and unvaccinated adolescents.
Design:
Repeat cross-sectional study of pharyngeal carriage.
Setting:
In 2020, recruitment commenced from February to March (pre-COVID-19) and recommenced from August to September (during COVID-19 measures) in South Australia.
Participants:
Eligible participants were between 17 and 25 years of age and completed secondary school in South Australia in 2019.
Results:
A total of 1338 school leavers were enrolled in 2020, with a mean age of 18.6 years (standard deviation 0.6). Pharyngeal carriage of disease-associated meningococci was higher during the COVID-19 period compared with the pre-COVID-19 period (41/600 6.83% vs. 27/738 3.66%; adjusted odds ratio aOR, 2.03; 95% CI: 1.22–3.39;
P
= 0.01). Nongroupable carriage decreased during COVID period (1.67% vs. 3.79%; aOR, 0.45; 95% CI: 0.22–0.95). Pharyngeal carriage of groups A, C, W and Y was similar among school leavers vaccinated with meningococcal conjugate ACWY (7/257 2.72%) compared with those unvaccinated (29/1081 2.68%; aOR, 0.86; 95% CI: 0.37–2.02;
P
= 0.73). Clonal complex 41/44 predominated in both periods.
Conclusions:
Meningococcal carriage prevalence was not impacted by public health strategies to reduce severe acute respiratory syndrome coronavirus 2 transmission and is unlikely to be the mechanism for lower meningococcal disease incidence. As international travel resumes and influenza recirculates, clinicians must remain vigilant for signs and symptoms of meningococcal disease. Vaccinating people at the highest risk of invasive meningococcal disease remains crucial despite containment strategies.
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