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September 22, 2019

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient.

The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (=8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.

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September 22, 2019

Novel syntrophic populations dominate an ammonia-tolerant methanogenic microbiome.

Biogas reactors operating with protein-rich substrates have high methane potential and industrial value; however, they are highly susceptible to process failure because of the accumulation of ammonia. High ammonia levels cause a decline in acetate-utilizing methanogens and instead promote the conversion of acetate via a two-step mechanism involving syntrophic acetate oxidation (SAO) to H2 and CO2, followed by hydrogenotrophic methanogenesis. Despite the key role of syntrophic acetate-oxidizing bacteria (SAOB), only a few culturable representatives have been characterized. Here we show that the microbiome of a commercial, ammonia-tolerant biogas reactor harbors a deeply branched, uncultured phylotype (unFirm_1) accounting for approximately 5% of the 16S rRNA gene inventory and sharing 88% 16S rRNA gene identity with its closest characterized relative. Reconstructed genome and quantitative metaproteomic analyses imply unFirm_1’s metabolic dominance and SAO capabilities, whereby the key enzymes required for acetate oxidation are among the most highly detected in the reactor microbiome. While culturable SAOB were identified in genomic analyses of the reactor, their limited proteomic representation suggests that unFirm_1 plays an important role in channeling acetate toward methane. Notably, unFirm_1-like populations were found in other high-ammonia biogas installations, conjecturing a broader importance for this novel clade of SAOB in anaerobic fermentations. IMPORTANCE The microbial production of methane or “biogas” is an attractive renewable energy technology that can recycle organic waste into biofuel. Biogas reactors operating with protein-rich substrates such as household municipal or agricultural wastes have significant industrial and societal value; however, they are highly unstable and frequently collapse due to the accumulation of ammonia. We report the discovery of a novel uncultured phylotype (unFirm_1) that is highly detectable in metaproteomic data generated from an ammonia-tolerant commercial reactor. Importantly, unFirm_1 is proposed to perform a key metabolic step in biogas microbiomes, whereby it syntrophically oxidizes acetate to hydrogen and carbon dioxide, which methanogens then covert to methane. Only very few culturable syntrophic acetate-oxidizing bacteria have been described, and all were detected at low in situ levels compared to unFirm_1. Broader comparisons produced the hypothesis that unFirm_1 is a key mediator toward the successful long-term stable operation of biogas production using protein-rich substrates.

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September 22, 2019

Rapid infectious disease identification by next-generation DNA sequencing.

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

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September 22, 2019

Moving beyond microbiome-wide associations to causal microbe identification.

Microbiome-wide association studies have established that numerous diseases are associated with changes in the microbiota. These studies typically generate a long list of commensals implicated as biomarkers of disease, with no clear relevance to disease pathogenesis. If the field is to move beyond correlations and begin to address causation, an effective system is needed for refining this catalogue of differentially abundant microbes and to allow subsequent mechanistic studies. Here we demonstrate that triangulation of microbe-phenotype relationships is an effective method for reducing the noise inherent in microbiota studies and enabling identification of causal microbes. We found that gnotobiotic mice harbouring different microbial communities exhibited differential survival in a colitis model. Co-housing of these mice generated animals that had hybrid microbiotas and displayed intermediate susceptibility to colitis. Mapping of microbe-phenotype relationships in parental mouse strains and in mice with hybrid microbiotas identified the bacterial family Lachnospiraceae as a correlate for protection from disease. Using directed microbial culture techniques, we discovered Clostridium immunis, a previously unknown bacterial species from this family, that-when administered to colitis-prone mice-protected them against colitis-associated death. To demonstrate the generalizability of our approach, we used it to identify several commensal organisms that induce intestinal expression of an antimicrobial peptide. Thus, we have used microbe-phenotype triangulation to move beyond the standard correlative microbiome study and identify causal microbes for two completely distinct phenotypes. Identification of disease-modulating commensals by microbe-phenotype triangulation may be more broadly applicable to human microbiome studies.

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September 22, 2019

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

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September 22, 2019

Carbohydrate staple food modulates gut microbiota of Mongolians in China.

Gut microbiota is a determining factor in human physiological functions and health. It is commonly accepted that diet has a major influence on the gut microbial community, however, the effects of diet is not fully understood. The typical Mongolian diet is characterized by high and frequent consumption of fermented dairy products and red meat, and low level of carbohydrates. In this study, the gut microbiota profile of 26 Mongolians whom consumed wheat, rice and oat as the sole carbohydrate staple food for a week each consecutively was determined. It was observed that changes in staple carbohydrate rapidly (within a week) altered gut microbial community structure and metabolic pathway of the subjects. Wheat and oat favored bifidobacteria (Bifidobacterium catenulatum, Bifodobacteriumbifidum, Bifidobacterium adolescentis); whereas rice suppressed bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis) and wheat suppresses Lactobaciilus, Ruminococcus and Bacteroides. The study exhibited two gut microbial clustering patterns with the preference of fucosyllactose utilization linking to fucosidase genes (glycoside hydrolase family classifications: GH95 and GH29) encoded by Bifidobacterium, and xylan and arabinoxylan utilization linking to xylanase and arabinoxylanase genes encoded by Bacteroides. There was also a correlation between Lactobacillus ruminis and sialidase, as well as Butyrivibrio crossotus and xylanase/xylosidase. Meanwhile, a strong concordance was found between the gastrointestinal bacterial microbiome and the intestinal virome. Present research will contribute to understanding the impacts of the dietary carbohydrate on human gut microbiome, which will ultimately help understand relationships between dietary factor, microbial populations, and the health of global humans.

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September 22, 2019

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic and real microbiome sequences. In addition to genome binning, we show that our method links plasmids and other mobile genetic elements to their host species in a real microbiome sample. Incorporation of DNA methylation information into shotgun metagenomics analyses will complement existing methods to enable more accurate sequence binning.

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September 22, 2019

Identification of Burkholderia fungorum in the urine of an individual with spinal cord injury and augmentation cystoplasty using 16S sequencing: copathogen or innocent bystander?

People with neuropathic bladder (NB) secondary to spinal cord injury (SCI) are at risk for multiple genitourinary complications, the most frequent of which is urinary tract infection (UTI). Despite the high frequency with which UTI occurs, our understanding of the role of urinary microbes in health and disease is limited. In this paper, we present the first prospective case study integrating symptom reporting, urinalysis, urine cultivation, and 16S ribosomal ribonucleic acid (rRNA) sequencing of the urine microbiome.A 55-year-old male with NB secondary to SCI contributed 12 urine samples over an 8-month period during asymptomatic, symptomatic, and postantibiotic periods. All bacteria identified on culture were present on 16S rRNA sequencing, however, 16S rRNA sequencing revealed the presence of bacteria not isolated on culture. In particular, Burkholderia fungorum was present in three samples during both asymptomatic and symptomatic periods. White blood cells of =5-10/high power field and leukocyte esterase =2 on urinalysis was associated with the presence of symptoms.In this patient, there was a predominance of pathogenic bacteria and a lack of putative probiotic bacteria during both symptomatic and asymptomatic states. Urinalysis-defined inflammatory markers were present to a greater extent during symptomatic periods compared to the asymptomatic state, which may underscore a role for urinalysis or other inflammatory markers in differentiating asymptomatic bacteriuria from UTI in patients with NB. The finding of potentially pathogenic bacteria identified by sequencing but not cultivation, suggests a need for greater understanding of the relationships amongst bacterial species in the bacteriuric neuropathic bladder.

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September 22, 2019

Improved OTU-picking using long-read 16S rRNA gene amplicon sequencing and generic hierarchical clustering

BACKGROUND: High-throughput bacterial 16S rRNA gene sequencing followed by clustering of short sequences into operational taxonomic units (OTUs) is widely used for microbiome profiling. However, clustering of short 16S rRNA gene reads into biologically meaningful OTUs is challenging, in part because nucleotide variation along the 16S rRNA gene is only partially captured by short reads. The recent emergence of long-read platforms, such as single-molecule real-time (SMRT) sequencing from Pacific Biosciences, offers the potential for improved taxonomic and phylogenetic profiling. Here, we evaluate the performance of long- and short-read 16S rRNA gene sequencing using simulated and experimental data, followed by OTU inference using computational pipelines based on heuristic and complete-linkage hierarchical clustering. RESULTS: In simulated data, long-read sequencing was shown to improve OTU quality and decrease variance. We then profiled 40 human gut microbiome samples using a combination of Illumina MiSeq and Blautia-specific SMRT sequencing, further supporting the notion that long reads can identify additional OTUs. We implemented a complete-linkage hierarchical clustering strategy using a flexible computational pipeline, tailored specifically for PacBio circular consensus sequencing (CCS) data that outperforms heuristic methods in most settings: https://github.com/oscar-franzen/oclust/. CONCLUSION: Our data demonstrate that long reads can improve OTU inference; however, the choice of clustering algorithm and associated clustering thresholds has significant impact on performance.

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September 22, 2019

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes.

Pan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences’ (PacBio’s) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data.Analysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity with regard to taxonomic classification. Examination of a 250-plus species mock community demonstrated correct species-level classification of >?90% of taxa, and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes) was the predominant species throughout, but was found at distinct relative abundances by site.Our microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt ) overcomes deficits of standard marker gene-based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.

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September 22, 2019

Analysis of the duodenal microbiotas of weaned piglet fed with epidermal growth factor-expressed Saccharomyces cerevisiae.

The bacterial community of the small intestine is a key factor that has strong influence on the health of gastrointestinal tract (GIT) in mammals during and shortly after weaning. The aim of this study was to analyze the effects of the diets of supplemented with epidermal growth factor (EGF)-expressed Saccharomyces cerevisiae (S. cerevisiae) on the duodenal microbiotas of weaned piglets.Revealed in this study, at day 7, 14 and 21, respectively, the compositional sequencing analysis of the 16S rRNA in the duodenum had no marked difference in microbial diversity from the phylum to species levels between the INVSc1(EV) and other recombinant strains encompassing INVSc1-EE(+), INVSc1-TE(-), and INVSc1-IE(+). Furthermore, the populations of potentially enterobacteria (e.g., Clostridium and Prevotella) and probiotic (e.g., Lactobacilli and Lactococcus) also remained unchanged among recombinant S. cerevisiae groups (P?>?0.05). However, the compositional sequencing analysis of the 16S rRNA in the duodenum revealed significant difference in microbial diversity from phylum to species levels between the control group and recombinant S. cerevisiae groups. In terms of the control group (the lack of S. cerevisiae), these data confirmed that dietary exogenous S. cerevisiae had the feasibility to be used as a supplement for enhancing potentially probiotic (e.g., Lactobacilli and Lactococcus) (P?

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September 22, 2019

Daily HIV pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate-emtricitabine reduced Streptococcus and increased Erysipelotrichaceae in rectal microbiota.

Daily PrEP is highly effective at preventing HIV-1 acquisition, but risks of long-term tenofovir disoproxil fumarate plus emtricitabine (TDF-FTC) include renal decline and bone mineral density decrease in addition to initial gastrointestinal side effects. We investigated the impact of TDF-FTC on the enteric microbiome using rectal swabs collected from healthy MSM before PrEP initiation and after 48 to 72 weeks of adherent PrEP use. The V4 region of the 16S ribosomal RNA gene sequencing showed that Streptococcus was significantly reduced from 12.0% to 1.2% (p?=?0.036) and Erysipelotrichaceae family was significantly increased from 0.79% to 3.3% (p?=?0.028) after 48-72 weeks of daily PrEP. Catenibacterium mitsuokai, Holdemanella biformis and Turicibacter sanguinis were increased within the Erysipelotrichaceae family and Streptococcus agalactiae, Streptococcus oralis, Streptococcus mitis were reduced. These changes were not associated with host factors including PrEP duration, age, race, tenofovir diphosphate blood level, any drug use and drug abuse, suggesting that the observed microbiome shifts were likely induced by daily PrEP use. Long-term PrEP resulted in increases of Catenibacterium mitsuokai and Holdemanella biformis, which have been associated with gut microbiome dysbiosis. Our observations can aid in characterizing PrEP’s side effects, which is likely to improve PrEP adherence, and thus HIV-1 prevention.

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September 22, 2019

Koumiss consumption alleviates symptoms of patients with chronic atrophic gastritis: A possible link To modulation of gut microbiota

Intestinal dysbiosisis closely related to a variety of medical conditions, especially gastrointestinal diseases. The present study aimed to investigate the effects of koumiss on chronic atrophic gastritis (CAG) in an out-patient clinical trial (n = 10; all female subjects aged 41-55; body mass index ranging from 19.5 to 25.8). Each patient consumed three servings of koumiss per day (i.e. 250 ml daily before each of 3 meals) for a 60-day period. The improvement of patients’ symptoms was monitored by comparing the total scores of symptoms before and after the treatment. Meanwhile, the changes in the patients’ fecal microbiota composition and specific blood parameters were determined. After the 60-day koumiss administration, significant symptom improvements were observed, as evidenced by the reduction of the total symptoms score, and changes in blood platelet and cholesterol levels. The changes in patients’ fecal microbiota composition were found. The patients’ fecal microbiota fell into two distinct enterotypes, Bacteroides dorei/ Bacteroides uniformis (BB-enterotype) and Prevotella copri (P-enterotype). Significant less Bacteroides uniformis was found in the BB-enterotype patient group, while significant more butyrate-producing bacteria (e.g. Eubacterium rectale and Faecalibacterium prausnitzii) were found in the P-enterotype patient group, following koumiss administration. After stopping koumiss consumption, the relative abundance of some biomarker taxa returned to the original level, suggesting that the gut microbiota modulatory effect was not permanent and that continuous koumiss administration was required to maintain the therapeutic effect. In conclusion, koumiss consumption could alleviate the symptoms of CAG patients. Our results may help understand the mechanism of koumiss in alleviating CAG disease symptoms, facilitating the development of such products with desired therapeutic functions.

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September 22, 2019

Analysis of gut microbiota – An ever changing landscape.

In the last two decades, the field of metagenomics has greatly expanded due to improvement in sequencing technologies allowing for a more comprehensive characterization of microbial communities. The use of these technologies has led to an unprecedented understanding of human, animal, and environmental microbiomes and have shown that the gut microbiota are comparable to an organ that is intrinsically linked with a variety of diseases. Characterization of microbial communities using next-generation sequencing-by-synthesis approaches have revealed important shifts in microbiota associated with debilitating diseases such as Clostridium difficile infection. But due to limitations in sequence read length, primer biases, and the quality of databases, genus- and species-level classification have been difficult. Third-generation technologies, such as Pacific Biosciences’ single molecule, real-time (SMRT) approach, allow for unbiased, more specific identification of species that are likely clinically relevant. Comparison of Illumina next-generation characterization and SMRT sequencing of samples from patients treated for C. difficile infection revealed similarities in community composition at the phylum and family levels, but SMRT sequencing further allowed for species-level characterization – permitting a better understanding of the microbial ecology of this disease. Thus, as sequencing technologies continue to advance, new species-level insights can be gained in the study of complex and clinically-relevant microbial communities.

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September 22, 2019

Effects of metal and metalloid pollutants on the microbiota composition of feces obtained from twelve commercial pig farms across China.

Understanding the metal and metalloid contamination and microbiota composition of pig feces is an important step required to support the design and implementation of effective pollution control and prevention strategies. A survey was implemented in 12 locations across China to investigate the content of metals and metalloids, and the main composition of the microbial communities of commercially reared pigs during two growth periods, defined as the early (Q group) and the later fattening growth phases (H group). These data showed widespread Al, Mn, Cu, Zn, and Fe pollution in pig feces. The concentration of Zn in the Q group feces was nearly two times higher than the levels measured in the H group. The microbial composition of the Q group exhibited greater richness of operational taxonomic units (OTUs) and fewer bacteria associated with zoonotic diseases compared with the microbial composition of the H group. Spearman rank correlation analysis showed that Cu and northern latitudes had a significant positive effect on the richness of bacterial communities in pig feces. Zn and Cd exhibited the biggest impact on microbial community composition based on canonical correspondence analysis. Functional metagenomic prediction indicated that about 0.8% genes present in the pig feces bacteria community are related to human diseases, and significantly more predicted pathogenic genes were detected in the H group than in the Q group. These results support the need to monitor heavy metal contamination and to control for zoonotic pathogens disseminated from pig feces in Chinese pig farms. Copyright © 2018. Published by Elsevier B.V.

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