Region Of Ribosomal RNA Gene Research Articles

The Ramazzini Institute 13-week pilot study on glyphosate and Roundup administered at human-equivalent dose to Sprague Dawley rats: effects on the microbiome

BackgroundGlyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including its effects on microbiome. The present pilot study examines whether exposure to GBHs at doses of glyphosate considered to be “safe” (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero, may modify the composition of gut microbiome in Sprague Dawley (SD) rats.MethodsGlyphosate alone and Roundup, a commercial brand of GBHs, were administered in drinking water at doses comparable to the US glyphosate ADI (1.75 mg/kg bw/day) to F0 dams starting from the gestational day (GD) 6 up to postnatal day (PND) 125. Animal feces were collected at multiple time points from both F0 dams and F1 pups. The gut microbiota of 433 fecal samples were profiled at V3-V4 region of 16S ribosomal RNA gene and further taxonomically assigned and assessed for diversity analysis. We tested the effect of exposure on overall microbiome diversity using PERMANOVA and on individual taxa by LEfSe analysis.ResultsMicrobiome profiling revealed that low-dose exposure to Roundup and glyphosate resulted in significant and distinctive changes in overall bacterial composition in F1 pups only. Specifically, at PND31, corresponding to pre-pubertal age in humans, relative abundance for Bacteriodetes (Prevotella) was increased while the Firmicutes (Lactobacillus) was reduced in both Roundup and glyphosate exposed F1 pups compared to controls.ConclusionsThis study provides initial evidence that exposures to commonly used GBHs, at doses considered safe, are capable of modifying the gut microbiota in early development, particularly before the onset of puberty. These findings warrant future studies on potential health effects of GBHs in early development such as childhood.

Detection of Microbial 16S rRNA Gene in the Blood of Patients With Parkinson's Disease.

Emerging evidence suggests that the microbiota present in feces plays a role in Parkinson’s disease (PD). However, the alterations of the microbiome in the blood of PD patients remain unknown. To test this hypothesis, we conducted this case-control study to explore the microbiota compositions in the blood of Chinese PD patients. Microbiota communities in the blood of 45 patients and their healthy spouses were investigated using high-throughput Illumina HiSeq sequencing targeting the V3-V4 region of 16S ribosomal RNA (rRNA) gene. The relationships between the microbiota in the blood and PD clinical characteristics were analyzed. No difference was detected in the structure and richness between PD patients and healthy controls. The following genera were enriched in the blood of PD patients: Isoptericola, Cloacibacterium, Enhydrobacter and Microbacterium; whereas genus Limnobacter was enriched in the healthy controls after adjusting for age, gender, body mass index (BMI) and constipation. Additionally, the findings regarding these genera were validated in another independent group of 58 PD patients and 57 healthy controls using real-time PCR targeting genus-specific 16S rRNA genes. Furthermore, not only the genera Cloacibacterium and Isoptericola (which were identified as enriched in PD patients) but also the genera Paludibacter and Saccharofermentans were positively associated with disease duration. Some specific genera in the blood were related to mood disorders. We believe this is the first report to provide direct evidence to support the hypothesis that the identified microbiota in the blood are associated with PD. Additionally, some microbiota in the blood are closely associated with the clinical characteristics of PD. Elucidating these differences in blood microbiomes will provide a foundation to improve our understanding of the role of microbiota in the pathogenesis of PD.

Identification of the pathogen Podosphaera erigerontis-canadensis causing powdery mildew disease on dandelion (Taraxacum officinale) in US Arkansas state

Powdery mildew was observed on common dandelion (Taraxacum officinale), collected in Fayetteville, AR, USA, and was identified using morphology and DNA sequences of ITS and 28S ribosomal RNA (rRNA) gene regions. Four primer pairs were used in this study: (1) the powdery mildew specific primer pair PMITS1/2 produced a 696 bp DNA fragment including full sequence of 563 bp DNA fragment amplified by ITS1/4 (KU563734); (2) CTW13/TW14 created a 279 bp DNA fragment including partial sequence of 28S rRNA gene (KU563735); (3) the 28S rRNA region primer pair NL1f/TW14 amplified a 899 bp DNA fragment (KX610821); and (4) the primer pair ITS1/4 amplified a 563 bp DNA fragment including partial sequence of 18S ribosomal RNA gene and complete sequence of ITS1, the 5.8S rRNA gene, ITS2, and partial sequence of the 28S rRNA gene. The similarity analysis by Blast and phylogenetic analysis indicated that the powdery mildew pathogen in dandelion collected from Arkansas was Podosphaera erigerontis-canadensis.

Diversity of the midstream urine microbiome in adults with chronic kidney disease

PurposeTo examine the characteristics of the midstream urine microbiome in adults with stage 3–5 non-dialysis-dependent chronic kidney disease (CKD).MethodsPatients with non-dialysis-dependent CKD (estimated glomerular filtration rate [eGFR] < 60 ml/min/1.73 m2) and diuretic use were recruited from outpatient nephrology clinics. Midstream voided urine specimens were collected using the clean-catch method. The bacterial composition was determined by sequencing the hypervariable (V4) region of the bacterial 16S ribosomal RNA gene. Extraction negative controls (no urine) were included to assess the contribution of extraneous DNA from possible sources of contamination. Midstream urine microbiome diversity was assessed with the inverse Simpson, Chao and Shannon indices. The diversity measures were further examined by demographic characteristics and by comorbidities.ResultsThe cohort of 41 women and 36 men with detectable bacterial DNA in their urine samples had a mean age of 71.5 years (standard deviation [SD] 7.9) years (range 60–91 years). The majority were white (68.0%) and a substantial minority were African-American (29.3%) The mean eGFR was 27.2 (SD 13.6) ml/min/1.73 m2. Most men (72.2%) were circumcised and 16.6% reported a remote history of prostate cancer. Many midstream voided urine specimens were dominated (> 50% reads) by the genera Corynebacterium (n = 11), Staphylococcus (n = 9), Streptococcus (n = 7), Lactobacillus (n = 7), Gardnerella (n = 7), Prevotella (n = 4), Escherichia_Shigella (n = 3), and Enterobacteriaceae (n = 2); the rest lacked a dominant genus. The samples had high levels of diversity, as measured by the inverse Simpson [7.24 (95% CI 6.76, 7.81)], Chao [558.24 (95% CI 381.70, 879.35)], and Shannon indices [2.60 (95% CI 2.51, 2.69)]. Diversity measures were generally higher in participants with urgency urinary incontinence and higher estimated glomerular filtration rate (eGFR). After controlling for demographics and diabetes status, microbiome diversity was significantly associated with estimated eGFR (P < 0.05).ConclusionsThe midstream voided urine microbiome of older adults with stage 3–5 non-dialysis-dependent CKD is diverse. Greater microbiome diversity is associated with higher eGFR.

Pumpkin powdery mildew disease severity influences the fungal diversity of the phyllosphere.

Phyllosphere microbiota play a crucial role in plant-environment interactions and their microbial community and function are influenced by biotic and abiotic factors. However, there is little research on how pathogens affect the microbial community of phyllosphere fungi. In this study, we collected 16 pumpkin (Cucurbita moschata) leaf samples which exhibited powdery mildew disease, with a severity ranging from L1 (least severe) to L4 (most severe). The fungal community structure and diversity was examined by Illumina MiSeq sequencing of the internal transcribed spacer (ITS) region of ribosomal RNA genes. The results showed that the fungal communities were dominated by members of the Basidiomycota and Ascomycota. The Podosphaera was the most dominant genus on these infected leaves, which was the key pathogen responsible for the pumpkin powdery mildew. The abundance of Ascomycota and Podosphaera increased as disease severity increased from L1 to L4, and was significantly higher at disease severity L4 (P < 0.05). The richness and diversity of the fungal community increased from L1 to L2, and then declined from L2 to L4, likely due to the biotic pressure (i.e., symbiotic and competitive stresses among microbial species) at disease severity L4. Our results could give new perspectives on the changes of the leaf microbiome at different pumpkin powdery mildew disease severity.

Nematode-associated microbial taxa do not correlate with host phylogeny, geographic region or feeding morphology in marine sediment habitats.

Studies of host-associated microbes are critical for advancing our understanding of ecology and evolution across diverse taxa and ecosystems. Nematode worms are ubiquitous across most habitats on earth, yet little is known about host-associated microbial assemblages within the phylum. Free-living nematodes are globally abundant and diverse in marine sediments, with species exhibiting distinct buccal cavity (mouth) morphologies that are thought to play an important role in feeding ecology and life history strategies. Here, we investigated patterns in marine nematode microbiomes, by characterizing host-associated microbial taxa in 281 worms isolated from a range of habitat types (deep-sea, shallow water, methane seeps, Lophelia coral mounds, kelp holdfasts) across three distinct geographic regions (Arctic, Southern California and Gulf of Mexico). Microbiome profiles were generated from single worms spanning 33 distinct morphological genera, using a two-gene metabarcoding approach to amplify the V4 region of the 16S ribosomal RNA (rRNA) gene targeting bacteria/archaea and the V1-V2 region of the 18S rRNA gene targeting microbial eukaryotes. Contrary to our expectations, nematode microbiome profiles demonstrated no distinct patterns either globally (across depths and ocean basins) or locally (within site); prokaryotic and eukaryotic microbial assemblages did not correlate with nematode feeding morphology, host phylogeny or morphological identity, ocean region or marine habitat type. However, fine-scale analysis of nematode microbiomes revealed a variety of novel ecological interactions, including putative parasites and symbionts, and potential associations with bacterial/archaeal taxa involved in nitrogen and methane cycling. Our results suggest that in marine habitats, free-living nematodes may utilize diverse and generalist foraging strategies that are not correlated with host genotype or feeding morphology. Furthermore, some abiotic factors such as geographic region and habitat type do not appear to play an obvious role in structuring host-microbe associations or feeding preferences.

Alteration of the fecal microbiota in Chinese patients with Parkinson’s disease

Emerging evidences suggest that gut microbiota dysbiosis plays a role in Parkinson’s disease (PD). However, the alterations in fecal microbiome in Chinese PD patients remains unknown. This case-control study was conducted to explore fecal microbiota compositions in Chinese PD patients. Microbiota communities in the feces of 45 patients and their healthy spouses were investigated using high-throughput Illumina Miseq sequencing targeting the V3-V4 region of 16S ribosomal RNA (rRNA) gene. The relationships between fecal microbiota and PD clinical characteristics were analyzed. The structure and richness of the fecal microbiota differed between PD patients and healthy controls. Genera Clostridium IV, Aquabacterium, Holdemania, Sphingomonas, Clostridium XVIII, Butyricicoccus and Anaerotruncus were enriched in the feces of PD patients after adjusting for age, gender, body mass index (BMI), and constipation. Furthermore, genera Escherichia/Shigella were negatively associated with disease duration. Genera Dorea and Phascolarctobacterium were negatively associated with levodopa equivalent doses (LED). Among the non-motor symptoms (NMSs), genera Butyricicoccus and Clostridium XlVb were associated with cognitive impairment. Overall, we confirmed that gut microbiota dysbiosis occurs in Chinese patients with PD. A well-controlled population involved was beneficial for the identification of microbiota associated with diseases. Additionally, the fecal microbiota was closely related to PD clinical characteristics. Elucidating these differences in the fecal microbiome will provide a foundation to improve our understanding the pathogenesis of PD and to support the potentially therapeutic options modifying the gut microbiota.

Occurrence of endoparasites in wild Antillean manatees (Trichechus manatus manatus) in Colombia

The recognized impact of parasites in wildlife populations demands surveillance of endangered species like the Antillean manatees (Trichechus manatus manatus) in Colombia. We conducted a parasitological survey in four rescued sea cows in order to document the parasite diversity of this sirenian in the Caribbean wetland of Colombia and contribute to the molecular characterization of its trematodes. The flukes Chiorchis fabaceus, Nudacotyle undicola and the protozoans Eimeria manatus and E. nodulosa were identified in analysed faecal samples. For C. fabaceus and N. undicola, partial regions of ribosomal RNA genes were amplified and sequenced in order to infer their phylogenetic relations. The current study constitutes a new sirenian host (T. manatus manatus) record for the genus Eimeria and the trematode N. undicola.

Nasopharyngeal Lactobacillus is associated with a reduced risk of childhood wheezing illnesses following acute respiratory syncytial virus infection in infancy

Early life acute respiratory infection (ARI) with respiratory syncytial virus (RSV) has been strongly associated with the development of childhood wheezing illnesses, but the pathways underlying this association are poorly understood. To examine the role of the nasopharyngeal microbiome in the development of childhood wheezing illnesses following RSV ARI in infancy. We conducted a nested cohort study of 118 previously healthy, term infants with confirmed RSV ARI by RT-PCR. We used next-generation sequencing of the V4 region of the 16S ribosomal RNA gene to characterize the nasopharyngeal microbiome during RSV ARI. Our main outcome of interest was 2-year subsequent wheeze. Of the 118 infants, 113 (95.8%) had 2-year outcome data. Of these, 46 (40.7%) had parental report of subsequent wheeze. There was no association between the overall taxonomic composition, diversity, and richness of the nasopharyngeal microbiome during RSV ARI with the development of subsequent wheeze. However, the nasopharyngeal detection and abundance of Lactobacillus was consistently higher in infants who did not develop this outcome. Lactobacillus also ranked first among the different genera in a model distinguishing infants with and without subsequent wheeze. The nasopharyngeal detection and increased abundance of Lactobacillus during RSV ARI in infancy are associated with a reduced risk of childhood wheezing illnesses at age 2years.

Rigidotubus tephroleucus gen. et sp. nov. (Cystostereaceae, Agaricales) evidenced by morphological characters and phylogenetic analyses

A new poroid wood-inhabiting fungal genus, Rigidotubus, is proposed to accommodate R. tephroleucus sp. nov. based on morphological characters and molecular evidence. The genus is characterized by tough and poroid basidiomata, white to grayish white pore surface, white sterile margin, a monomitic hyphal system with clamped generative hyphae, ovoid to ellipsoid basidiospores negative in Melzer’s reagent and Cotton Blue. The phylogenetic analyses inferred from the internal transcribed spacer (ITS) and nuclear large subunit (28S) ribosomal RNA gene regions confirmed that Rigidotubus belongs to Cystostereaceae in Agaricales. A key to the accepted genera of Cystostereaceae is provided.

Molecular detection and characterisation of Babesia and Theileria in Australian hard ticks

Babesia and Theileria are intraerythrocytic protozoans of the phylum Apicomplexa. These species are capable of infecting wild and domestic animals and have historically caused great economic loss in the agricultural industry. In recent years human babesiosis has been deemed an emerging zoonosis in North America, Europe and Asia. The first locally acquired case of babesiosis in Australia, caused by Babesia microti, was reported in March 2012. A number of native Babesia and Theileria species have been identified in Australian marsupials, however their associated tick vectors and threat to human health is unknown. In the present study DNA was extracted from 1154 ticks collected from across Australia. PCR was used to amplify a Babesia and Theileria-specific partial region of the 18S ribosomal RNA gene. Positive samples were sequenced and phylogenetic analysis was performed. Twenty-nine sequences were obtained from ticks belonging to the genera Ixodes, Haemaphysalis and Bothriocroton. The sequences were closely related to Babesia macropus, and Theileria recently identified in marsupials and monotremes. Bayesian inference and maximum likelihood methods showed that Australian Babesia and Theileria species form monophyletic groups.

Cutaneotrichosporon (Trichosporon) debeurmannianum: A Rare Yeast Isolated from Blood and Urine Samples.

Cutaneotrichosporon (Trichosporon) debeurmannianum is a rarely isolated yeast from clinical samples. Nine isolates of this yeast were identified from clinical samples within a period of 3years from June 2012 to May 2015. These isolates were from blood and urine samples sent to a clinical mycology laboratory of a tertiary care hospital in Assam, North East India. Clinically, the patients were diagnosed as septicemia and urinary tract infection. The age of the patients ranged from 2 to 50years. Identification was made by sequencing the ITS region of ribosomal RNA gene. Antifungal susceptibility test by disk diffusion method (CLSI, M44-A) showed all the isolates to be sensitive to fluconazole and voriconazole. Vitek 2 compact commercial yeast identification system misidentified this yeast as Cryptococcus laurentii and low discrimination Cryptococcus laurentii/Trichosporon mucoides. This species was originally named as Trichosporon debeurmannianum. In 2015, this yeast has been included into new genera Cutaneotrichosporon based on an integrated phylogenetic classification of the Tremellomycetes. To the best of our knowledge, this is the first report of identification of this species from blood and urine samples of clinically suspected cases. We are reporting these isolates because of their rarity in clinical samples. The pathogenic potential and epidemiological relevance of this yeast remains to be seen.

Berberine treatment increases Akkermansia in the gut and improves high-fat diet-induced atherosclerosis in Apoe−/− mice

Background and aimsGut microbiota plays a major role in metabolic disorders. Berberine is used to treat obesity, diabetes and atherosclerosis. The mechanism underlying the role of berberine in modulating metabolic disorders is not fully clear because berberine has poor oral bioavailability. Thus, we evaluated whether the antiatherosclerotic effect of berberine is related to alterations in gut microbial structure and if so, whether specific bacterial taxa contribute to the beneficial effects of berberine. MethodsApoe−/− mice were fed either a normal-chow diet or a high-fat diet (HFD). Berberine was administered to mice in drinking water (0.5 g/L) for 14 weeks. Gut microbiota profiles were established by high throughput sequencing of the V3–V4 region of the bacterial 16S ribosomal RNA gene. The effects of berberine on metabolic endotoxemia, tissue inflammation and gut barrier integrity were also investigated. ResultsBerberine treatment significantly reduced atherosclerosis in HFD-fed mice. Akkermansia spp. abundance was markedly increased in HFD-fed mice treated with berberine. Moreover, berberine decreased HFD-induced metabolic endotoxemia and lowered arterial and intestinal expression of proinflammatory cytokines and chemokines. Berberine treatment increased intestinal expression of tight junction proteins and the thickness of the colonic mucus layer, which are related to restoration of gut barrier integrity in HFD-fed mice. ConclusionsModulation of gut microbiota, specifically an increase in the abundance of Akkermansia, may contribute to the antiatherosclerotic and metabolic protective effects of berberine, which is poorly absorbed orally. Our findings therefore support the therapeutic value of gut microbiota manipulation in treating atherosclerosis.

Cortinarius longistipitatus, a new species in subgenus Telamonia, section Cinnabarini, from Pakistan

A new species of Cortinarius, C. longistipitatus, in subgenus Telamonia sect. Cinnabarini, is described and illustrated based on collections from northern Pakistan. The dark brown to blackish brown coloration of the pileus, the extraordinary long, equal to bulbous stipe, the red universal veil remnants on the stipe base and its occurrence in conifer forests make it a remarkably distinct species in the field. The internal transcribed spacer region (ITS) and large subunit (LSU) of the nuclear ribosomal RNA gene was amplified. The evolutionary relationship of the species was inferred by means of Maximum Likelihood phylogenetic analysis which revealed that C. longistipitatus is distinct from other Cortinarius species.

Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications.

High-throughput sequencing metabarcoding studies in marine biosecurity have largely focused on targeting environmental DNA (eDNA). DNA can persist extracellularly in the environment, making discrimination of living organisms difficult. In this study, bilge water samples (i.e., water accumulating on-board a vessel during transit) were collected from 15 small recreational and commercial vessels. eDNA and eRNA molecules were co-extracted and the V4 region of the 18S ribosomal RNA gene targeted for metabarcoding. In total, 62.7% of the Operational Taxonomic Units (OTUs) were identified at least once in the corresponding eDNA and eRNA reads, with 19.5% unique to eDNA and 17.7% to eRNA. There were substantial differences in diversity between molecular compartments; 57% of sequences from eDNA-only OTUs belonged to fungi, likely originating from legacy DNA. In contrast, there was a higher percentage of metazoan (50.2%) and ciliate (31.7%) sequences in the eRNA-only OTUs. Our data suggest that the presence of eRNA-only OTUs could be due to increased cellular activities of some rare taxa that were not identified in the eDNA datasets, unusually high numbers of rRNA transcripts in ciliates, and/or artefacts produced during the reverse transcriptase, PCR and sequencing steps. The proportions of eDNA/eRNA shared and unshared OTUs were highly heterogeneous within individual bilge water samples. Multiple factors including boat type and the activities performed on-board, such as washing of scientific equipment, may play a major role in contributing to this variability. For some marine biosecurity applications analysis, eDNA-only data may be sufficient, however there are an increasing number of instances where distinguishing the living portion of a community is essential. For these circumstances, we suggest only including OTUs that are present in both eDNA and eRNA data. OTUs found only in the eRNA data need to be interpreted with caution until further research provides conclusive evidence for their origin.

Morphological characteristics and molecular data reveal two new species of Dentipellis from China

Two new species of Dentipellis are described from China based on morphological characteristics and molecular data. Dentipellis longiuscula is characterized by annual, resupinate hydnoid basidiomata with moderately long spines, rough basidiospores (5–6 × 3–3.6 µm) and absence of gloeoplerous hyphae and gloeocystidia. Dentipellis tropicalis is characterized by hydnoid basidiomata with numerous gloeocystidia presenting in hymenium, and minutely rough basidiospores (5–5.5 × 2.5–3.2 µm). Phylogenetic analysis based on the internal transcribed spacer (ITS) and nuclear large subunit ribosomal (nLSU) RNA gene regions confirmed the affinity of the new species in Dentipellis and indicated their relationships with other species in the genus.

High-Salt Diet Has a Certain Impact on Protein Digestion and Gut Microbiota: A Sequencing and Proteome Combined Study.

High-salt diet has been considered to cause health problems, but it is still less known how high-salt diet affects gut microbiota, protein digestion, and passage in the digestive tract. In this study, C57BL/6J mice were fed low- or high-salt diets (0.25 vs. 3.15% NaCl) for 8 weeks, and then gut contents and feces were collected. Fecal microbiota was identified by sequencing the V4 region of 16S ribosomal RNA gene. Proteins and digested products of duodenal, jejunal, cecal, and colonic contents were identified by LC-MS-MS. The results indicated that the high-salt diet increased Firmicutes/Bacteroidetes ratio, the abundances of genera Lachnospiraceae and Ruminococcus (P < 0.05), but decreased the abundance of Lactobacillus (P < 0.05). LC-MS-MS revealed a dynamic change of proteins from the diet, host, and gut microbiota alongside the digestive tract. For dietary proteins, high-salt diet seemed not influence its protein digestion and absorption. For host proteins, 20 proteins of lower abundance were identified in the high-salt diet group in duodenal contents, which were involved in digestive enzymes and pancreatic secretion. However, no significant differentially expressed proteins were detected in jejunal, cecal, and colonic contents. For bacterial proteins, proteins secreted by gut microbiota were involved in energy metabolism, sodium transport, and protein folding. Five proteins (cytidylate kinase, trigger factor, 6-phosphogluconate dehydrogenase, transporter, and undecaprenyl-diphosphatase) had a higher abundance in the high-salt diet group than those in the low-salt group, while two proteins (acetylglutamate kinase and PBSX phage manganese-containing catalase) were over-expressed in the low-salt diet group than in the high-salt group. Consequently, high-salt diet may alter the composition of gut microbiota and has a certain impact on protein digestion.

Gut Microbiota-Dependent Trimethylamine-N-oxide and Serum Biomarkers in Patients with T2DM and Advanced CKD.

Trimethylamine-N-oxide (TMAO) is a product of dietary, gut microbiome, and tissues metabolism. Elevated blood TMAO levels are associated with heart attack, stroke and chronic kidney disease (CKD). The purpose of our study was to investigate the gut microbiota associated with trimethylamine (TMA) production, the precursor of TMAO, and the serum levels of TMAO and inflammatory biomarkers associated with type 2 diabetes mellitus (T2DM) and CKD. Twenty adults with T2DM and advanced CKD and 20 healthy adults participated in the study. Analyses included anthropometric and metabolic parameters, characterization of TMA producing gut microbiota, and concentrations of TMAO, lipopolysaccharides (LPS) endotoxin, zonulin (Zo) gut permeability marker, and serum inflammatory and endothelial dysfunction biomarkers. Diversity of the gut microbiota was identified by amplification of V3–V4 regions of the 16S ribosomal RNA genes and DNA sequencing. TMAO was quantified by Mass Spectrometry and serum biomarkers by ELISA. The significance of measurements justified by statistical analysis. The gut microbiome in T2DM-CKD patients exhibited a higher incidence of TMA-producing bacteria than control, p < 0.05. The serum levels of TMAO in T2DM-CKD patients were significantly higher than controls, p < 0.05. TMAO showed a positive correlation with Zo and LPS, inflammatory and endothelial dysfunction biomarkers. A positive correlation was observed between Zo and LPS in T2DM-CKD subjects. An increased abundance of TMA-producing bacteria in the gut microbiota of T2DM-CKD patients together with excessive TMAO and increased gut permeability might impact their risk for cardiovascular disease through elevation of chronic inflammation and endothelial dysfunction.

The Nasopharyngeal Microbiota of Children With Respiratory Infections in Botswana.

Nearly half of child pneumonia deaths occur in sub-Saharan Africa. Microbial communities in the nasopharynx are a reservoir for pneumonia pathogens and remain poorly described in African children. Nasopharyngeal swabs were collected from children with pneumonia (N = 204), children with upper respiratory infection symptoms (N = 55) and healthy children (N = 60) in Botswana between April 2012 and April 2014. We sequenced the V3 region of the bacterial 16S ribosomal RNA gene and used partitioning around medoids to cluster samples into microbiota biotypes. We then used multivariable logistic regression to examine whether microbiota biotypes were associated with pneumonia and upper respiratory infection symptoms. Mean ages of children with pneumonia, children with upper respiratory infection symptoms and healthy children were 8.2, 11.4 and 8.0 months, respectively. Clustering of nasopharyngeal microbiota identified 5 distinct biotypes: Corynebacterium/Dolosigranulum-dominant (23%), Haemophilus-dominant (11%), Moraxella-dominant (24%), Staphylococcus-dominant (13%) and Streptococcus-dominant (28%). The Haemophilus-dominant [odds ratio (OR): 13.55; 95% confidence interval (CI): 2.10-87.26], the Staphylococcus-dominant (OR: 8.27; 95% CI: 2.13-32.14) and the Streptococcus-dominant (OR: 39.97; 95% CI: 6.63-241.00) biotypes were associated with pneumonia. The Moraxella-dominant (OR: 3.71; 95% CI: 1.09-12.64) and Streptococcus-dominant (OR: 12.26; 95% CI: 1.81-83.06) biotypes were associated with upper respiratory infection symptoms. In children with pneumonia, HIV infection was associated with a lower relative abundance of Dolosigranulum (P = 0.03). Pneumonia and upper respiratory infection symptoms are associated with distinct nasopharyngeal microbiota biotypes in African children. A lower abundance of the commensal genus Dolosigranulum may contribute to the higher pneumonia risk of HIV-infected children.

Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease.

Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD. Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified. Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases. The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.