Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under Mixed<i>Eimeria</i>Infection

We surveyed the fungal microbiota in 16 faecal samples from healthy humans with a vegetarian diet. Fungi were identified using molecular cloning, 454 pyrosequencing and a Luminex analyte‐specific reagent (ASR) assay, all targeting the ITS region of the rRNA genes. Fungi were detected in each faecal sample and at least 46 distinct fungal operational taxonomic units (OTUs) were detected, from two phyla — Ascomycota and Basidiomycota. Fusarium was the most abundant genus, followed by Malassezia, Penicillium, Aspergillus and Candida. Commonly detected fungi such as Aspergillus and Penicillium, as well as known dietary fungi Agaricus bisporus and Ophiocordyceps sinensis, are presumed to be transient, allochthonous members due to their abundance in the environment or dietary associations. No single method identified the full diversity of fungi in all samples; pyrosequencing detected more distinct OTUs than the other methods, but failed to detect OTUs in some samples that were detected by cloning and/or ASR assays. ASRs were limited by the commercially available assays, but the potential to design new, optimized assays, coupled with speed and cost, makes the ASR method worthy of further study. Significance and Impact of the Study Fungi play a role in human gut ecology and health. The field lags immensely behind bacterial gut microbiota research, and studies continue to identify new fungi in faecal samples from healthy humans. However, many of these ‘new’ species are incapable of growth in the human GI tract, let alone making a meaningful contribution to the gut microbial community. Fungi actually inhabiting and impacting the gut likely constitute a small set of species, and an optimized, targeted, probe‐based assay may prove to be the most sensible way of quantifying their abundances.

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“…Data processing and analysis followed Hume et al . (). MrDNA clustered denoised sequences into OTUs at 97% similarity.…”

Section: Methodsmentioning

confidence: 97%

Sequence-based methods for detecting and evaluating the human gut mycobiome

Suhr

Banjara

Hallen‐Adams

2016

Letters in Applied Microbiology

102

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“…After multiple initial PCR amplifications, all subsequent pyrosequencing procedures were performed at the Research and Testing Laboratory (Lubbock, USA). Tag-encoded FLX amplicon pyrosequencing (TEFAP) was performed as described [33], [34], [35] using the bacterium-biased primers, Gray28F (5′-TTTGATCNTGGCTCAG) and Gray519r (5′-GTNTTACNGCGGCKGCTG) and the fungus-biased primers, SSUFungiF (5′-TGGAGGGCAAGTCTGGTG) and SSUFungiR (5′-TCGGCATAGTTTATGGTTAAG). The amplified ∼500-bp fragments spanning the V1 to V3 hypervariable regions of the bacterial 16S rRNA genes and the amplified ∼400-bp fragments of the fungal 18S rRNA genes were generated using a one-step PCR with a total of 30 cycles, a mixture of Hot Start and HotStar high fidelity Taq polymerases, and amplicons originating and extending from the 28F for bacterial diversity and 515F for fungal diversity.…”

Section: Methodsmentioning

confidence: 99%

The Effect of Silver Nanoparticles on Seasonal Change in Arctic Tundra Bacterial and Fungal Assemblages

et al. 2014

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The impact of silver nanoparticles (NPs) and microparticles (MPs) on bacterial and fungal assemblages was studied in soils collected from a low arctic site. Two different concentrations (0.066% and 6.6%) of Ag NPs and Ag MPs were tested in microcosms that were exposed to temperatures mimicking a winter to summer transition. Toxicity was monitored by differential respiration, phospholipid fatty acid analysis, polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequencing. Notwithstanding the effect of Ag MPs, nanosilver had an obvious, additional impact on the microbial community, underscoring the importance of particle size in toxicity. This impact was evidenced by levels of differential respiration in 0.066% Ag NP-treated soil that were only half that of control soils, a decrease in signature bacterial fatty acids, and changes in both richness and evenness in bacterial and fungal DNA sequence assemblages. Prominent after Ag NP-treatment were Hypocreales fungi, which increased to 70%, from only 1% of fungal sequences under control conditions. Genera within this Order known for their antioxidant properties (Cordyceps/Isaria) dominated the fungal assemblage after NP addition. In contrast, sequences attributed to the nitrogen-fixing Rhizobiales bacteria appeared vulnerable to Ag NP-mediated toxicity. This combination of physiological, biochemical and molecular studies clearly demonstrate that Ag NPs can severely disrupt the natural seasonal progression of tundra assemblages.

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“…Bacterial tag-encoded FLX amplicon pyrosequencing (bTE-FAP) was done as described previously [6,[19][20][21][22][23], but with the new technique of bacterial tag-encoded FLX titanium amplicon pyrosequencing (bTETAP). This technique is based on bTEFAP but utilizes titanium-containing reagents and titanium-based procedures, with a one-step PCR, a mixture of hot start and hot start high-fidelity Taq polymerases, and amplicons originating from the 28F 5¢GAGTTTGATCNTGGCTCAG to 519R 5¢GTNTTACNGCGGCKGCTG regions of the rRNA of Escherichia coli, and provides results for the V1-V3 regions of the 16S ribosome.…”

Section: Pyrosequencingmentioning

confidence: 99%

“…Following pyrosequencing of the 16S rRNA in the patient specimens, all unsuccessful sequence readings, low-quality sequence ends, and tags were removed, and any non-bacterial ribosome sequences and chimeras were removed from the specimens with the use of software described previously [6,[19][20][21][22]. Sequences shorter than 350 bp according to the bTEFAP method were excluded.…”

Section: Analysis Of Bacterial Diversity Datamentioning

confidence: 99%

Examination with Next-Generation Sequencing Technology of the Bacterial Microbiota in Bronchoalveolar Lavage Samples after Traumatic Injury

Huebinger

Liu

Dowd³

et al. 2013

Surgical Infections

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Background: We examined the microbiota of bronchoalveolar lavage (BAL) samples with next-generation sequencing (NGS) technology to determine whether its results correlate with those of standard culture methods or affect patient outcome or both. Methods: We collected BAL samples in the surgical intensive care unit (SICU) as part of the standard of care for intubated individuals who had a Clinical Pulmonary Infection Score (CPIS) ‡ 6 points. A portion of the BAL fluid was sequenced for the 16S region of ribosomal deoxyribonucleic acid (rDNA) with the Roche 454 FLX Titanium sequencer. Sequences were analyzed through a data-analysis pipeline to identify the appropriate taxonomic designation (*species) of each 16s sequence. The bacterial microbiota of each BAL sample was compared with the bacteria identified in the sample through standard culture methods. Correlations between the taxonomic diversity of the microbiota and clinical outcome were examined through linear regression and Pearson correlation. Results: Bronchoalveolar lavage samples from 12 individuals in the SICU who had a CPIS ‡ 6 points were examined through 454 pyrosequencing. The number of phylotypes (*species) in the samples ranged from 15 to 129. The number of phyla in the BAL samples ranged from 3 to 14. There was little correlation between the bacteria identified by NGS and those identified with standard culture methods. The same predominant bacterial strain was identified by both culture and sequencing in only a single sample. The correlation between patient days on a ventilator and the number of species in BAL samples was significant (r = 0.7435, p = 0.0056; r 2 = 0.5528). Conclusions: Increasing diversity of the bacterial microbiota in BAL samples correlates with the duration of mechanical ventilation. Bacteria identified through standard culture methods were not well correlated with the findings of NGS.

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Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under MixedEimeriaInfection

Cited by 25 publications

References 41 publications

Sequence-based methods for detecting and evaluating the human gut mycobiome

Sequence-based methods for detecting and evaluating the human gut mycobiome

The Effect of Silver Nanoparticles on Seasonal Change in Arctic Tundra Bacterial and Fungal Assemblages

Examination with Next-Generation Sequencing Technology of the Bacterial Microbiota in Bronchoalveolar Lavage Samples after Traumatic Injury

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