16S Ribosomal RNA Sequencing Research Articles

A Simple Culture Method Enhances the Recovery of Culturable Actinobacteria From Coastal Sediments.

Molecular methods revealed that the majority of microbes in natural environments remains uncultivated. To fully understand the physiological and metabolic characteristics of microbes, however, culturing is still critical for microbial studies. Here, we used bacterial community analysis and four culture media, namely, traditional marine broth 2216 (MB), water extracted matter (WEM), methanol extracted matter (MEM), and starch casein agar (SCA), to investigate the diversity of cultivated bacteria in coastal sediments. A total of 1,036 isolates were obtained in pure culture, and they were classified into five groups, namely, Alphaproteobacteria (52.51%), Gammaproteobacteria (23.26%), Actinobacteria (13.32%), Firmicutes, and Bacteroidetes. Compared to other three media, WEM recovered a high diversity of actinobacteria (42 of 63 genotypes), with Micromonospora and Streptomyces as the most cultivated genera. Amplicon sequencing of the bacterial 16S ribosomal RNA (rRNA) gene V3–V4 fragment revealed eight dominant groups, Alphaproteobacteria (12.81%), Gammaproteobacteria (20.07%), Deltaproteobacteria (12.95%), Chloroflexi (13.09%), Bacteroidetes (8.28%), Actinobacteria (7.34%), Cyanobacteria (6.20%), and Acidobacteria (5.71%). The dominant members affiliated to Actinobacteria belonged to “Candidatus Actinomarinales,” “Candidatus Microtrichales,” and Nitriliruptorales. The cultivated actinobacteria accounted for a small proportion (<5%) compared to the actinobacterial community, which supported that the majority of actinobacteria are still waiting for cultivation. Our study concluded that WEM could be a useful and simple culture medium that enhanced the recovery of culturable actinobacteria from coastal sediments.

Streptococcus pluranimalium meningoencephalitis in a horse

A 3-y-old, female Quarter Horse with a history of acute neurologic signs was found dead and was submitted for postmortem examination. Areas of petechial and ecchymotic hemorrhage were present on cross-sections of the cerebrum, cerebellum, and brainstem. Histologic examination of the brain revealed severe, purulent meningoencephalitis and vasculitis with a myriad of intralesional gram-positive cocci. Streptococcus pluranimalium was identified from formalin-fixed, paraffin-embedded tissue obtained from sites with active lesions by PCR and nucleotide sequencing of bacterial 16S ribosomal RNA. S. pluranimalium should be considered as a cause of meningoencephalitis in a horse.

Intraspecific nucleotide divergence in Saccharomycodes ludwigii, and proposal of Saccharomycodes pseudoludwigii sp. nov, a new apiculate yeast isolated from China.

The six synonyms currently accepted under Saccharomycodes ludwigii were investigated for by phenotypic properties, however, the sequence diversity of the rRNA and protein coding genes have not yet been determined. Nine strains including the type strains of synonyms of S. ludwigii deposited in the CBS yeast collection, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, were analyzed using a multi-locus sequence analysis (MLSA) approach that included sequences of 18S ribosomal RNA (rRNA), the D1/D2 domains of the 26S rRNA, the ITS region (including the 5.8S rRNA) and fragments of genes encoding the largest subunit of the RNA polymerase II (RPB1 and RPB2) and translation elongation factor 1-α (TEF1). Our results showed that the nine strains have identical D1/D2, 18S and RPB2 sequences and similar ITS, RPB1 and TEF1 sequences, which indicated that they are conspecific. In addition, a novel species of Saccharomycodes, S. pseudoludwigii sp. nov. (type CGMCC 2.4526T) that was isolated from fruit and tree bark in China, is proposed. The MycoBank number of this new species is MB 811,650.

Characterization of cerebrospinal fluid (CSF) microbiota from patients with CSF shunt infection and reinfection using high throughput sequencing of 16S ribosomal RNAgenes.

Nearly 20% of patients with cerebrospinal fluid (CSF) shunt infection develop reinfection. It is unclear whether reinfections are caused by an organism previously present or are independent infection events. We used bacterial culture and high throughput sequencing (HTS) of 16S ribosomal RNA (rRNA) genes to identify bacteria present in serial CSF samples obtained from children who failed CSF shunt infection treatment. We hypothesized that organisms that persist in CSF despite treatment would be detected upon reinfection. Serial CSF samples were obtained from 6 patients, 5 with 2 infections and 1 with 3 infections; the study was limited to those for which CSF samples were available from the end of infection and beginning of reinfection. Amplicons of the 16S rRNA gene V4 region were sequenced. Taxonomic assignments of V4 sequences were compared with bacterial species identified in culture. Seven infection dyads averaging 13.5 samples per infection were analyzed. A median of 8 taxa [interquartile range (IQR) 5-10] were observed in the first samples from reinfection using HTS. Conventional culture correlated with high abundance of an organism by HTS in all but 1 infection. In 6 of 7 infection dyads, organisms identified by culture at reinfection were detected by HTS of culture-negative samples at the end of the previous infection. The median Chao-Jaccard abundance-based similarity index for matched infection pairs at end of infection and beginning of reinfection was 0.57 (IQR 0.07-0.87) compared to that for unmatched pairs of 0.40 (IQR 0.10-0.60) [p = 0.46]. HTS results were generally consistent with culture-based methods in CSF shunt infection and reinfection, and may detect organisms missed by culture at the end of infection treatment but detected by culture at reinfection. However, the CSF microbiota did not correlate more closely within patients at the end of infection and beginning of reinfection than between any two unrelated infections. We cannot reject the hypothesis that sequential infections were independent.

Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BackgroundStudies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls.ResultsThe number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups.ConclusionsDifferences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

Metagenomic Analysis Reveals Reduced Beneficial Microorganism Associations in Roots of Foot-Rot-Affected Citrus Trees

Root endophytes have been shown to influence plant growth and plant responses to pathogens. Phytophthora spp. are important pathogens of citrus that cause foot rot and fibrous root decline. We hypothesize that foot rot infection by Phytophthora spp. can result in changes of the endophytic microbial community in citrus roots. Therefore, the endophytic (including bacterial and fungal) microbiome of roots from Phytophthora foot-rot-affected and healthy citrus trees were analyzed by Illumina sequencing of 16S ribosomal RNA and internal transcribed spacer amplicons. Results indicate that the composition and structure of the endophytic bacterial and fungal communities were changed in roots of foot-rot-affected citrus trees. The populations of bacterial genera Asteroleplasma, Pseudomonas, and Streptomyces were decreased in foot-rot-affected trees. Also, the relative abundance of Exophiala pisciphila and Glomeraceae spp. was significantly decreased in citrus roots. Pseudomonas and Streptomyces spp. are considered beneficial bacteria and E. pisciphila and Glomeraceae spp. are dark septate endophytes and arbuscular mycorrhizal fungi, respectively, which are involved in citrus health and growth. This study provides a baseline to continue investigating the interactions between the citrus host, Phytophthora spp., and beneficial microbes.

Infective endocarditis caused by Neisseria mucosa on a prosthetic pulmonary valve with false positive serology for Coxiella burnetii – The first described case

We present a case of infective endocarditis (IE) on a prosthetic pulmonary valve in a 36-year-old patient with tetralogy of Fallot (TOF). The patient underwent valve replacement surgery and active antibiotic treatment against Gram-negative cocci (Piperacillin Tazobactam then Ceftriaxone) for a total duration of 42 days with a favourable outcome. The causative agent was Neisseria mucosa which was identified on the infected valve by sequencing of 16S ribosomal RNA. To our knowledge, this is the first described case of a N. mucosa infective endocarditis on a pulmonary valve. Initially, serologies performed in clinical settings by immunofluorescence for Coxiella burnetii antibodies showed a major increase in phase I IgG titers at 1024 (normal values <16) corresponding with the diagnostic criteria for Q fever endocarditis. However, this diagnosis could not be confirmed by the National Reference Center, making it the first reported case of a false positive serology for C. burnetii during an infection due to Neisseria spp.

Live and ultrasound-inactivated Lacticaseibacillus casei modulate the intestinal microbiota and improve biochemical and cardiovascular parameters in male rats fed a high-fat diet.

This study aimed to evaluate the effects of ingestion of live (9 log CFU mL-1) and ultrasound-inactivated (paraprobiotic, 20 kHz, 40 min) Lacticaseibacillus casei 01 cells for 28 days on healthy parameters (biochemical and cardiovascular) and intestinal microbiota (amplicon sequencing of 16S ribosomal RNA) of rats fed a high-fat diet. Twenty-four male Wistar rats were divided into four groups of six animals: CTL (standard diet), HFD (high-fat diet), HFD-LC (high-fat diet and live L. casei), and HFD-ILC (high-fat diet and inactivated L. casei). The administration of live and ultrasound-inactivated L. casei prevented the increase (p < 0.05) in cholesterol levels (total and LDL) and controlled the insulin resistance in rats fed a high-fat diet. Furthermore, it promoted a modulation of the intestinal microbial composition by increasing (p < 0.05) beneficial bacteria (Lachnospiraceae and Ruminoccocaceae) and decreasing (p < 0.05) harmful bacteria (Clostridiaceae, Enterobacteriaceae, and Helicobacteriacea), attenuating the effects promoted by the HFD ingestion. Only live cells could increase (p < 0.05) the HDL-cholesterol, while only inactivated cells caused attenuation (p < 0.05) of the blood pressure. Results show beneficial effects of live and inactivated L. casei 01 and indicate that ultrasound inactivation produces a paraprobiotic with similar or improved health properties compared to live cells.

Utilizing Cattle Manure Compost Increases Ammonia Monooxygenase A Gene Expression and Ammonia-oxidizing Activity of Both Bacteria and Archaea in Biofiltration Media for Ammonia Deodorization.

Malodorous emissions are a crucial and inevitable issue during the decomposition of biological waste and contain a high concentration of ammonia. Biofiltration technology is a feasible, low-cost, energy-saving method that reduces and eliminates malodors without environmental impact. In the present study, we evaluated the effectiveness of compost from cattle manure and food waste as deodorizing media based on their removal of ammonia and the expression of ammonia-oxidizing genes, and identified the bacterial and archaeal communities in these media. Ammonia was removed by cattle manure compost, but not by food waste compost. The next-generation sequencing of 16S ribosomal RNA obtained from cattle manure compost revealed the presence of ammonia-oxidizing bacteria (AOB), including Cytophagia, Alphaproteobacteria, and Gammaproteobacteria, and ammonia-oxidizing archaea (AOA), such as Thaumarchaeota. In cattle manure compost, the bacterial and archaeal ammonia monooxygenase A (amoA) genes were both up-regulated after exposure to ammonia (fold ratio of 14.2±11.8 after/before), and the bacterial and archaeal communities were more homologous after than before exposure to ammonia, which indicates the adaptation of these communities to ammonia. These results suggest the potential of cattle manure compost as an efficient biological deodorization medium due to the activation of ammonia-oxidizing microbes, such as AOB and AOA, and the up-regulation of their amoA genes.

Environment has a Stronger Effect than Host Plant Genotype in Shaping Spring Brassica napus Seed Microbiomes

Seed are reproductive units that transfer genetic information to the next generation and harbor microbial communities that may interact with a host plant at all stages of its development. Here, we assessed the effect of the environment and plant genotype on the seed microbiome of eight spring Brassica napus lines harvested from four site years in Saskatchewan, Canada: one location each in 2016 and 2017 and two additional locations in 2017. Seed microbiomes were characterized using high-throughput amplicon sequencing of the bacterial 16S ribosomal RNA and fungal internal transcribed spacer regions. Our results revealed that microbial communities were predominantly shaped by the environment, with location explaining 34% of bacterial and 43% of fungal total variance. Meanwhile, genotype had a smaller effect, accounting for only 9% of bacterial and 13% of fungal variance. The seed microbiome of B. napus predominantly contained members of Enterobacteriales and Pseudomonadales bacterial orders as well as Pleosporales and Capnodiales fungal orders. Additionally, common taxa, including Enterobacteriales, Pseudomonadales, Micrococcales, Sphingomonadales, Pleosporales, Capnodiales, Tremellales, Filobasidiales, and Sporidiobolales, were detected across all site years. Our results demonstrate that the environment plays a dominant role in shaping spring B. napus seed microbiomes, with more subtle contributions related to host plant genotype. Information collected in this study may contribute to the development of novel and sustainable breeding and agricultural strategies that consider microorganisms carried by seed.

Comparing DNA Extraction and 16S rRNA Gene Amplification Methods for Plant-Associated Bacterial Communities

Plant-associated microbes play important roles in global ecology and agriculture. The most common method to profile these microbial communities is amplicon sequencing of the bacterial 16S ribosomal RNA (rRNA) gene. Both the DNA extraction and PCR amplification steps of this process are subject to bias, especially because the latter requires some way to exclude DNA from plant organelles, which would otherwise dominate the sample. We compared several common DNA extraction kits and 16S rRNA gene amplification protocols to determine the relative biases of each and to make recommendations for plant microbial researchers. For DNA extraction, we found that, as expected, kits optimized for soil were the best for soil, though each still included a distinct “fingerprint” of its own biases. Plant samples were less clear, with different species having different “best” options. For 16S rRNA gene amplification, we found that using peptide nucleic acid clamps provides the least taxonomic distortion, while chloroplast-discriminating primers are easy and inexpensive but present significant bias in the results. Blocking oligos require a more complex protocol and optimization for each species and showed significant taxonomic distortion; therefore, we do not recommend them for off-the-shelf applications. Further methods development will hopefully result in protocols that are even more reliable and less biased than these. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Suppurative necrotizing bronchopneumonia caused by Nocardia cyriacigeorgica infection in a stranded striped dolphin (Stenella coeruleoalba) in Japan.

On a coastline in Miyazaki Prefecture, Japan, a wild subadult female striped dolphin was found dead. Necropsy revealed poor nutritional status and bilateral pneumonia, which was histologically diagnosed as severe suppurative necrotizing bronchopneumonia. Special staining detected numerous intralesional filamentous, branching bacteria, which was identified as Nocardia cyriacigeorgica by sequencing of 16S ribosomal RNA and gyrB genes. Other main histological findings included lymphoid depletion in the spleen and superficial cervical and pulmonary lymph nodes. Suppurative nocardiosis without a granulomatous reaction is uncommon, and it is assumed its pathogenesis was related to the host’s immune status. This paper discusses the variable inflammatory response to nocardiosis and describes the first case of N. cyriacigeorgica infection in a wild striped dolphin in Japan.

Profile of Nontuberculous Mycobacteria in Patients Suspected of Tuberculosis and Drug-Resistant Tuberculosis.

Objective Infections due to nontuberculous mycobacteria (NTM) is increasing globally and may present as drug-resistant tuberculosis (DRTB). In India, data on NTM prevalence and species diversity is limited. Present study was conducted to detect the prevalence and profile of NTM among patients suspected of DRTB using paraffin slide culture (PSC)and mycobacteria growth indicator tube (MGIT) culture methods for isolation of NTM. Material and Method A total of 2,938 samples suspected of TB/DRTB were cultured on PSC and MGIT960. Species identification of mycobacterial isolate was done by sequencing of 16s ribosomal RNA gene. Result Among 2938 samples, 35 (1.19%) were found positive for NTM by PSC and 9 (0.30%) were found positive by MGIT. The diversity of NTM species was high (13 species). Out of 35 NTM isolates by PSC, maximum 34.29% (12) isolates were found to be Mycobacterium fortuitum , followed by 11.43% (4) Mycobacterium abscessus and Mycobacterium chelonae , and 42.85% (15) were other species viz. 8.57% (3) were Mycobacterium intracellulare and Mycobacterium kansasii , 5.71% (2) were Mycobacterium peregrinum , and 2.85% (1) were Mycobacterium flavescens, Mycobacterium farcinogenes, Mycobacterium moriokanese, Mycobacterium wolinskyi, Mycobacterium simiae, Mycobacterium goodii , and Mycobacterium terrae each. Coinfection of Mycobacterium tuberculosis (MTB) and NTM was found in 60% (21) samples. Conclusion Prevalence of NTM was low among multidrug resistant tuberculosis/TB suspected patients, similar to other studies done in India. PSC was found better than MGIT for the isolation of NTM, though poor separation of NTM and MTB on subculture may have led to false negativity in cases of coinfection. About 13 species were isolated; M. fortuitum was the most common of all. Since coinfection of NTM and TB can also occur, samples of patients suspected of NTM should be cultured on PSC even if positive for MTB.

Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing.

The exploration of vaginal microbiota by using next-generation sequencing (NGS) of 16S ribosomal RNA (rRNA) gene is widely used. Up to now, different hypervariable regions have been selected to study vaginal microbiota by NGS and there is no standard method for analysis. The study aimed to characterize vaginal microbiota from clinical samples using NGS targeting the 16S rRNA gene and to determine the performance of individual and concatenated hypervariable region sequences to generate the taxonomic profiles of the vaginal microbiota. Fifty-one vaginal DNA samples were subjected to 16S rRNA gene NGS based on the Ion Torrent PGM platform with the use of two primer sets spanning seven hypervariable regions of the 16S rRNA gene. Our analysis revealed that the predominant bacterial genera were Lactobacillus, Gardnerella and Atopobium, which accounted for 78%, 14% and 2%, respectively, of sequences from all vaginal bacterial genera. At the species level, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae accounted for 72%, 10% and 6%, respectively, of the bacterial cells present. Analyses using the V3 region generally indicated the highest bacterial diversity followed by the V6-V7 and V4 regions, while the V9 region gave the lowest bacterial resolution. NGS based on the 16S rRNA gene can give comprehensive estimates of the diversity of vaginal bacterial communities. Selection of sequences from appropriate hypervariable regions is necessary to provide reliable information on bacterial community diversity.

Analysis of bacterial communities in sponges and coral inhabiting Red Sea, using barcoded 454 pyrosequencing.

Microbial communities are linked with marine sponge are diverse in their structure and function. Our understanding of the sponge-associated microbial diversity is limited especially from Red Sea in Saudi Arabia where few species of sponges have been studied. Here we used pyrosequencing to study two marine sponges and coral species sampled from Obhur region from Red sea in Jeddah. A total of 168 operational taxonomic units (OTUs) were identified from Haliclona caerulea, Stylissa carteri and Rhytisma fulvum. Taxonomic identification of tag sequences of 16S ribosomal RNA revealed 6 different bacterial phyla and 9 different classes. A proportion of unclassified reads were was also observed in sponges and coral sample. We found diverse bacterial communities associated with two sponges and a coral sample. Diversity and richness estimates based on OUTs revealed that sponge H. caerulea had significantly high bacterial diversity. The identified OTUs showed unique clustering in three sponge samples as revealed by Principal coordinate analysis (PCoA). Proteobacteria (88–95%) was dominant phyla alonwith Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes and Nitrospirae. Seventeen different genera were identified where genus Pseudoalteromonas was dominant in all three samples. This is first study to assess bacterial communities of sponge and coral sample that have never been studied before to unravel their microbial communities using 454-pyrosequencing method.

A Study of the Gut Bacterial Community of Reticulitermes virginicus Exposed to Chitosan Treatment.

Simple SummaryTermite digestion of lignocellulosic materials is aided by their gut microbial community, which includes protists, bacteria, and archaea. They play important roles in termite growth and environmental adaptation. Dark southern subterranean termites (Reticulitermes virginicus) are native to North America and significantly damage wooden structures. Because of their considerable economic impact, a more thorough understanding of the relationship between host termite and microbial community is needed to develop target-specific and environmentally benign wood protection systems. The overall aim of this study was to investigate the potential influence of chitosan, a biodegradable and antimicrobial compound, on termite gut bacteria. A significant effect of chitosan treatment was observed in the relative abundance of two bacteria phyla (Firmicutes and Actinobacteria). The results suggest that chitosan treatment not only affects the structure of the microbial community in the gut, but other treatments also cause shifts in termite gut communities.A thorough understanding of microbial communities in the gut of lower termites is needed to develop target-specific and environmentally benign wood protection systems. In this study, the bacterial community from Reticulitermes virginicus was examined by Illumina sequencing of 16S ribosomal RNA (rRNA) spanning the V3 and V4 regions. Prior to library preparation, the termites were subjected to five treatments over an 18-day period: three groups were fed on wood treated with 0.5% chitosan, 25% acetic acid, or water, the fourth group was taken directly from the original collection log, and the fifth group was starved. Metagenomic sequences were analyzed using QIIME 2 to understand the treatments’ effects on the dynamics of the gut bacteria. Four dominant phyla were detected: Bacteroidetes (34.4% of reads), Firmicutes (20.6%), Elusimicrobia (15.7%), and Proteobacteria (12.9%). A significant effect of chitosan treatment was observed in two phyla; Firmicutes abundance was significantly lower with chitosan treatment when compared to other groups, while Actinobacteria was lower in unexposed and starved termites. The results suggest that chitosan treatment not only affects the structure of the microbial community in the gut, but other treatments such as starving also cause shifts in termite gut communities.

Epstein-Barr virus-immortalized B lymphocytes exacerbate experimental autoimmune encephalomyelitis in xenograft mice.

Multiple sclerosis (MS) is the most common autoimmune disorder affecting the central nervous system. Epstein-Barr virus (EBV) is a causative agent for infectious mononucleosis (IM) that is associated with MS pathogenesis. However, the exact mechanism by which EBV, specifically in IM, increases the risk for MS remains unknown. EBV immortalizes primary B lymphocytes in vitro and causes excessive B lymphocyte proliferation in IM in vivo. In asymptomatic carriers, EBV-infected B lymphocytes still proliferate to certain degrees, the process of which is tightly controlled by the host immune systems. Experimental autoimmune encephalomyelitis (EAE) mimics key features of MS in humans and is a well-established rodent model for human MS. We have found that xenografts of EBV-immortalized B lymphocytes, which partially resemble the hyperproliferation of EBV-infected cells in IM, exacerbate autoimmune responses in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. After remission, an additional challenge with EBV-immortalized cells induces a relapse in EAE. Moreover, xenografts with EBV-immortalized cells tighten the integrity of the blood-brain barrier (BBB) in the thalamus and hypothalamus areas of the mouse brains. Genomic sequences of prokaryotic 16S ribosomal RNA presented in the feces reveal that EBV-immortalized cells significantly change the diversities of microbial populations. Our data collectively suggest that EBV-mediated proliferation of B lymphocytes may be a risk factor for the exacerbation of MS, which are associated with gut microbiome changes and BBB modulations. Furthermore, multiple xenografts of EBV-immortalized cells into C57BL/6 mice could serve as a useful model for human relapsing-remitting MS with predictable severity and timing.

High-Fat Diet Induced Alteration of Mice Microbiota and the Functional Ability to Utilize Fructooligosaccharide for Ethanol Production.

High-fat diet (HFD) leads to enhancement in various parameters of mice like weight, fasting glucose levels, adipose tissue, and also the liver weight in male C57 BL/6 J mice. Additionally, high-fat diet causes severe liver damage with significant increase in the level of aspartate amino transferase (AST) and alanine transaminase (ALT). The variations in microbiota induced by different diet were analyzed by Illumina MiSeq platform with sequencing of 16S ribosomal RNA (rRNA) gene, and QIIME pipeline was used. The population of Proteobacteria was found to be higher in HFD cecum sample as compared to other treatments. Microbiota analysis suggests that phylum Proteobacteria and Firmicutes were found to be higher in high-fat diet groups as compared to mice fed with normal diet (ND). At the genus level, Bacteroides showed higher population in HFD diet. Bacterial strains belonging to Enterobacteriaceae like Escherichia, Klebsiella, and Shigella were also dominant in HFD treatments. Furthermore, we explored the effects of ethanol production in vitro with supplementation of dietary fibers following inoculation of ND and HFD microbiotas. HFD microbiota of cecum and feces showed high level (P < 0.05) of ethanol production with 2% fructooligosaccharide (FOS) as compared to 2% galactomannan. Microbial fermentation also generated short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate. High levels (P < 0.05) of propionate were found after fermentation of FOS with HFD cecum and feces microbiota. The present study highlights the HFD-induced population of phylum Proteobacteria and genus Bacteroides for ethanol production using FOS as a dietary supplement, and these findings may imply on the harmful effect of HFD even at the microbiota level.

Comparison of Bioinformatics Pipelines and Operating Systems for the Analyses of 16S rRNA Gene Amplicon Sequences in Human Fecal Samples.

Amplicon high-throughput sequencing of 16S ribosomal RNA (rRNA) gene is currently the most widely used technique to investigate complex gut microbial communities. Microbial identification might be influenced by several factors, including the choice of bioinformatic pipelines, making comparisons across studies difficult. Here, we compared four commonly used pipelines (QIIME2, Bioconductor, UPARSE and mothur) run on two operating systems (OS) (Linux and Mac), to evaluate the impact of bioinformatic pipeline and OS on the taxonomic classification of 40 human stool samples. We applied the SILVA 132 reference database for all the pipelines. We compared phyla and genera identification and relative abundances across the four pipelines using the Friedman rank sum test. QIIME2 and Bioconductor provided identical outputs on Linux and Mac OS, while UPARSE and mothur reported only minimal differences between OS. Taxa assignments were consistent at both phylum and genus level across all the pipelines. However, a difference in terms of relative abundance was identified for all phyla (p < 0.013) and for the majority of the most abundant genera (p < 0.028), such as Bacteroides (QIIME2: 24.5%, Bioconductor: 24.6%, UPARSE-linux: 23.6%, UPARSE-mac: 20.6%, mothur-linux: 22.2%, mothur-mac: 21.6%, p < 0.001). The use of different bioinformatic pipelines affects the estimation of the relative abundance of gut microbial community, indicating that studies using different pipelines cannot be directly compared. A harmonization procedure is needed to move the field forward.

Comparing Gut Microbiome in Mothers' Own Breast Milk- and Formula-Fed Moderate-Late Preterm Infants.

Gut microbiome plays an important role in adult human health and diseases. However, how nutritional factors shape the initial colonization of gut bacteria in infants, especially in preterm infants, is still not completely known. In this study, we compared the effects of feeding with mothers’ own breast milk (MBM) and formula on the initial composition and gene expression of gut bacteria in moderate–late preterm infants. Fecal samples were collected from ten formula-fed and ten MBM healthy infants born between 32 and 37 weeks’ gestation after they reached full-volume enteral feedings. Total DNAs were extracted from fecal samples for amplicon sequencing of 16S ribosomal RNA (rRNA) gene and total RNA with rRNA depletion for metatranscriptome RNA-Seq 16S rRNA gene amplicon sequencing results showed that the alpha-diversity was similar between the MBM- and formula-fed preterm infants, but the beta-diversity showed a significant difference in composition (p = 0.002). The most abundant taxa were Veillonella (18.4%) and Escherichia/Shigella (15.2%) in MBM infants, whereas the most abundant taxa of formula-fed infants were Streptococcus (18.6%) and Klebsiella (17.4%). The genera Propionibacterium, Streptococcus, and Finegoldia and order Clostridiales had significantly higher relative abundance in the MBM group than the formula group, whereas bacteria under family Enterobacteriaceae, genera Enterococcus and Veillonella, and class Bacilli were more abundant in the formula group. In general, microbiomes from both diet groups exhibited high functional levels of catalytic activity and metabolic processing when analyzed for gene ontology using a comparative metatranscriptome approach. Statistically, the microbial genes in the MBM group had an upregulation in expression related to glycine reductase, periplasmic acid stress response in Enterobacteria, acid resistance mechanisms, and L-fucose utilization. In contrast, the formula-fed group had upregulations in genes associated with methionine and valine degradation functions. Our data suggest that the nutritional source plays a role in shaping the moderate–late preterm gut microbiome as evidenced by the differences in bacterial composition and gene expression profiles in the fecal samples. The MBM group enriched Propionibacterium. Glycine reductase was highly upregulated in the microbiota from MBM along with the upregulated acid stress tolerance genes, suggesting that the intensity of fermentation process was enhanced.