The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality ...microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.
Coccidioidomycosis is a typically respiratory fungal disease that, in the United States, occurs primarily in Arizona and California. In California, most coccidioidomycosis cases occur in the San ...Joaquin Valley, a primarily agricultural region where the disease poses a risk for outdoor workers. We collected 710 soil samples and 265 settled dust samples from nine sites in the San Joaquin Valley and examined how Coccidioides detection varied by month, site, and the presence and abundance of other fungal species. We detected Coccidioides in 89 of 238 (37.4%) rodent burrow soil samples at five undeveloped sites and were unable to detect Coccidioides in any of 472 surface and subsurface soil samples at four agricultural sites. In what is the largest sampling effort undertaken on agricultural land, our results provide no evidence that agricultural soils in the San Joaquin Valley harbor Coccidioides. We found no clear association between Coccidioides and the greater soil fungal community, but we identified 19 fungal indicator species that were significantly associated with Coccidioides detection in burrows. We also did not find a seasonal pattern in Coccidioides detection in the rodent burrow soils we sampled. These findings suggest both the presence of a spore bank and that coccidioidomycosis incidence may be more strongly associated with Coccidioides dispersal than Coccidioides growth. Finally, we were able to detect Coccidioides in only five of our 265 near-surface settled dust samples, one from agricultural land, where Coccidioides was undetected in soils, and four from undeveloped land, where Coccidioides was common in the rodent burrow soils we sampled. Our ability to detect Coccidioides in few settled dust samples indicates that improved methods are likely needed moving forward, though raises questions regarding aerial dispersal in Coccidioides, whose key transmission event likely occurs over short distances in rodent burrows from soil to naïve rodent lungs.
Biological wastewater treatment is among the most important biotechnological applications and, as drivers of the key processes, microorganisms are central to its success. Therefore, the study of ...wastewater microorganisms has obvious applied significance; however, the importance of wastewater treatment reactors as model systems for microbial ecology is often overlooked. Modern molecular techniques, including environmental genomics, have identified unexpected microbial key players for nutrient removal and sludge bulking and/or foaming, and provided many exciting insights into the diversity, functions and niche differentiations of these predominantly uncultivated microorganisms. It is now time for wastewater microbiology to be recognized as a mature and dynamic discipline in its own right, offering much toward a deeper understanding of life in complex microbial communities. Here, we consider selected key findings to illustrate the past and future roles of molecular ecophysiology and genomics in the development of wastewater microbiology as an important subdiscipline of microbial ecology.
The role of Staphylococcus aureus in the pathogenesis of the chronic sinonasal disease chronic rhinosinusitis (CRS), has not been definitively established. Comparative analyses of S. aureus isolates ...from CRS with those from control participants may offer insight into a possible pathogenic link between this organism and CRS. The intra- and inter-subject S. aureus strain-level diversity in the sinuses of patients with and without CRS were compared in this cross-sectional study. In total, 100 patients (CRS = 64, control = 36) were screened for S. aureus carriage. The overall carriage prevalence of S. aureus in this cohort was 24% (CRS n = 13, control n = 11). Cultured S. aureus isolates from 18 participants were strain-typed using spa gene sequencing. The bacterial community composition of the middle meatus was assessed using amplicon sequencing targeting the V3V4 hypervariable region of the bacterial 16S rRNA gene. S. aureus isolates cultured from patients were grown in co-culture with the commensal bacterium Dolosigranulum pigrum and characterised. All participants harboured a single S. aureus strain and no trend in disease-specific strain-level diversity was observed. Bacterial community analyses revealed a significant negative correlation in the relative abundances of S. aureus and D. pigrum sequences, suggesting an antagonistic interaction between these organisms. Co-cultivation experiments with these bacteria, however, did not confirm this interaction in vitro. We saw no significant associations of CRS disease with S. aureus strain types. The functional role that S. aureus occupies in CRS likely depends on other factors such as variations in gene expression and interactions with other members of the sinus bacterial community.
Photoexcited triplet states are promising candidates for hybrid qubit systems, as they can be used as a controlling gate for nuclear spins. But microwave readout schemes do not generally offer the ...sensitivity needed to approach the single-molecule limit or the scope to integrate such systems into devices. Here, we demonstrate the possibility of electrical readout of triplet spins at room temperature through a specific mechanism of magnetoconductance (MC) in polycrystalline pentacene. We show that hole-only pentacene devices exhibit a positive photoinduced MC response that is consistent with a trap-filling mechanism. Spin and magnetic-field-dependent quenching of photogenerated triplets by holes quantitatively explains the MC response we observe. These results are distinct in both sign and proposed mechanism compared to previous reports on polyacene materials and provide clear design rules for future spintronic devices based on this spin-sensing mechanism.
Chronic rhinosinusitis (CRS) is a common and debilitating inflammatory condition of the sinuses, afflicting 5% of the general population. Although antibiotics are frequently prescribed for the ...medical management of CRS, there is surprisingly little evidence to support their efficacy. In this study, we aimed to establish associations between medication usage, the sinus microbiota and patients' clinical outcomes.
Antibiotic prescription patterns for the year before sample collection of 156 CRS patients, 45 disease control patients (mostly requiring septoplasty and inferior turbinate reduction) and 35 healthy control subjects were examined and analyzed together with previously published bacterial 16S rRNA gene amplicon data from our group.
The highest antibiotic usage was observed among the two CRS patient categories. Despite heavy antibiotic usage, CRS patients' clinical outcomes as indicated by patient questionnaires and radiologic scores were similar to those patients that did not receive any antibiotics. The sinus microbiota was dominated by members of the bacterial genera
and
in all three cohorts. Bacterial community dispersion as measured by principal coordinate analysis was significantly higher in CRS patients compared to healthy control subjects, but not disease control patients. Pairwise comparisons within cohorts revealed differences in the relative 16S rRNA gene sequence abundances of the genera
and
between antibiotic users and non-users. However, overall antibiotic effects were minimal and unpredictable.
The unpredictable effects of antibiotic treatment on the sinus microbiota found in this study, together with the lack of differences in patients' symptom scores between cohorts, do not support preoperative antibiotic treatment for CRS patients.
In studies of the human microbiome, faecal samples are frequently used as a non-invasive proxy for the study of the intestinal microbiota. To obtain reliable insights, the need for bacterial DNA of ...high quality and integrity following appropriate faecal sample collection and preservation steps is paramount. In a study of dietary mineral balance in the context of type 2 diabetes (T2D), faecal samples were collected from healthy and T2D individuals throughout a 13-day residential trial. These samples were freeze-dried, then stored mostly at -20°C from the trial date in 2000/2001 until the current research in 2014. Given the relative antiquity of these samples (~14 years), we sought to evaluate DNA quality and comparability to freshly collected human faecal samples. Following the extraction of bacterial DNA, gel electrophoresis indicated that our DNA extracts were more sheared than extracts made from freshly collected faecal samples, but still of sufficiently high molecular weight to support amplicon-based studies. Likewise, spectrophotometric assessment of extracts revealed that they were of high quality and quantity. A subset of bacterial 16S rRNA gene amplicons were sequenced using Illumina MiSeq and compared against publicly available sequence data representing a similar cohort analysed by the American Gut Project (AGP). Notably, our bacterial community profiles were highly consistent with those from the AGP data. Our results suggest that when faecal specimens are stored appropriately, the microbial profiles are preserved and robust to extended storage periods.
A complex mix of inflammatory and microbial associations underscores the chronic inflammatory condition chronic rhinosinusitis (CRS), and the etiology remains poorly understood. Recent work has begun ...to delineate between variants (endotypes) of CRS on the basis of inflammatory biomarkers. This study aimed to assess inflammatory patterns in CRS phenotypes, identify putative endotypes of CRS, and to assess inflammatory associations with the sinonasal microbiota. Ten cytokines and six inflammatory cell types were assessed in mucosal biopsies from 93 CRS subjects and 17 controls via cytometric bead array and immunohistochemical techniques. Putative endotypes were identified via cluster analysis of subjects on the basis of inflammatory markers and comorbidities including polyposis, asthma, and aspirin sensitivity. Finally, previously published bacterial data for this cohort were reanalyzed to evaluate associations with inflammatory markers and CRS subtypes. Inflammatory patterns were highly variable within standard CRS phenotypes. Cluster analysis identified eight subject clusters, with strong delineation on the basis of polyposis and asthma, but also subtle distinctions in inflammatory markers. An association was also identified between depletion of several "health-associated" bacterial taxa, reduced bacterial diversity and increased overall bacterial load, with markers of inflammation and clinical severity. This study contributes to ongoing efforts to define distinct endotypes of CRS on the basis of underlying inflammatory processes, and also offers compelling evidence of a link between bacterial community dysbiosis and inflammation in CRS. Further resolving the heterogeneity of CRS is vital to inform clinical management and personalized treatment approaches.
Some marine sponges harbor dense and phylogenetically complex microbial communities high microbial abundance (HMA) sponges whereas others contain only few and less diverse microorganisms low ...microbial abundance (LMA) sponges. We focused on the phylum Chloroflexi that frequently occurs in sponges to investigate the different associations with three HMA and three LMA sponges from New Zealand. By applying a range of microscopical and molecular techniques a clear dichotomy between HMA and LMA sponges was observed: Chloroflexi bacteria were more abundant and diverse in HMA than in LMA sponges. Moreover, different HMA sponges contain similar Chloroflexi communities whereas LMA sponges harbor different and more variable communities which partly resemble Chloroflexi seawater communities. A comprehensive phylogenetic analysis of our own and publicly available sponge‐derived Chloroflexi 16S rRNA gene sequences (> 780 sequences) revealed the enormous diversity of this phylum within sponges including 29 sponge‐specific and sponge‐coral clusters (SSC/SCC) as well as a ‘supercluster’ consisting of > 250 sponge‐derived and a single nonsponge‐derived 16S rRNA gene sequence. Interestingly, the majority of sequences obtained from HMA sponges, but only a few from LMA sponges, fell into SSC/SCC clusters. This indicates a much more specific association of Chloroflexi bacteria with HMA sponges and suggests an ecologically important role for these prominent bacteria.
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