Journal: Nature medicine

Article Title: Viable bacterial colonization is highly limited in the human intestine in utero

doi: 10.1038/s41591-020-0761-3

Figure Lengend Snippet: 16S rRNA V4 profiling of a subset (n=10) of banked fetal meconium samples using different library preparation methods: gel extraction and 30 or 35 cycles of amplification, or 30 cycles combined with DASH performed on individual samples (Individual DASH) or on the library pool (Pooled DASH). a. Expansion in Enterobacteriaceae family is detected in 35-cycle amplification method, while small expansion of Pseudomonadaceae is detected post-DASH. Principal coordinates analysis of Bray Curtis distances of libraries using b. 30 cycles of amplification or c. 30 and 35 cycles of amplification, latter to provide an outgroup known to skew bacterial composition. Ellipses indicate 95% confidence intervals. All p-values were calculated using two-sided Satterthwaite’s method on Linear Mixed Effects (LME) modeling to correct for n=10 paired samples that underwent multiple library preparation methods.

Article Snippet: Briefly, 54 sgRNAs targeting the human mtDNA were transcribed from pooled sgRNA templates using custom T7 RNA polymerase generously provided by the DeRisi laboratory at UCSF. sgRNAs were purified and concentrated using a column-based RNA purification kit with DNAse treatment (Zymo) and incubated with purified Cas9 (Berkeley Macrolab) for 10 minutes at 37°C. sgRNA-loaded Cas9 was incubated with either meconium genomic DNA (gDNA) or pooled library of 16S rRNA V4 amplicon (see below) for 2 hours at 37°C.

Techniques: Gel Extraction, Amplification

Journal: Nature medicine

Article Title: Viable bacterial colonization is highly limited in the human intestine in utero

doi: 10.1038/s41591-020-0761-3

Figure Lengend Snippet: PCoA of Bray Curtis distances of 16S rRNA profiles colored by a. meconium dominated by Lactobacillus OTU12 (LM, n=6), Micrococcaceae OTU10 (MM, n=9), or other taxa (OM, n=25) or b. the proportion of PLZF + CD161 + T cells among live, TCRβ + , Vα7.2 − , CD4 + cells in intestinal lamina propria (LP) paired with LM (n=5), MM (n=5) or OM (n=12) samples indicated by ellipses at 95% confidence. c. Proportion of PLZF + CD161 + T cells of live, CD4 + TCRβ + Vα7.2 − cells in LP among samples associated with meconium dominated by OTU10 (MM, n=5) or other OTUs (n=17). d. Principal components (PC) analysis of Euclidean distances of top 10000 variable genes (by coefficient of variation) in OTU10-dominated meconium associated epithelium (OTU10, MM-E, n=7) or other OTU-dominated meconium associated epithelium (Other, n=6) as determined by RNA sequencing. e. Venn diagram, f. heatmap with labeled immune pathway transcripts, and g. volcano plot of top differentially expressed genes between MM-E (n=7, log2 fold change >1, FDR < 0.05) and other OTU-dominated meconium associated epithelium (n=6, log2 fold change<1, FDR < 0.05). h. Normalized enrichment scores of gene set enrichment analysis of transcripts associated with epithelial cell states. For a-g, n indicates biologically independent fetal samples. PERMANOVA test for significance for a-b, d. Two-sided Wilcoxon rank sum test was used for c. DESEQ2 was used to calculate significant genes using a two-sided false discovery rate and log2 fold change. Each dot represents one independent biological replicate in a-d and one transcript in g. Boxplot indicates the median (center), the 25th and 75th percentiles, and the smallest and largest values within 1.5× the interquartile range (whiskers).

Article Snippet: Briefly, 54 sgRNAs targeting the human mtDNA were transcribed from pooled sgRNA templates using custom T7 RNA polymerase generously provided by the DeRisi laboratory at UCSF. sgRNAs were purified and concentrated using a column-based RNA purification kit with DNAse treatment (Zymo) and incubated with purified Cas9 (Berkeley Macrolab) for 10 minutes at 37°C. sgRNA-loaded Cas9 was incubated with either meconium genomic DNA (gDNA) or pooled library of 16S rRNA V4 amplicon (see below) for 2 hours at 37°C.

Techniques: RNA Sequencing Assay, Labeling

Journal: Nature medicine

Article Title: Viable bacterial colonization is highly limited in the human intestine in utero

doi: 10.1038/s41591-020-0761-3

Figure Lengend Snippet: a. Phylogenetic tree of 16S V4 rRNA gene sequences from Lactobacillus -enriched meconium (LM; green), Micrococcaceae -enriched meconium (MM; blue), or procedural swab (red) enriched OTUs (circles) and primary isolate (square) from fetal meconium (Micro36 and reference strain for Micrococcus luteus (MicroRef1). b. Effects of 10 −5 M progesterone (P4) and 10 −6 M β-Estradiol (E2) on the growth of Micro36 compared to ethanol vehicle control in carbon-limiting media (mineral salt media, left) or carbon-rich media (brain heart infusion; right) at 37 °C. Representative growth curves of three independent experiments measured by optical density at 600nm (OD600), error bars denote standard error of the mean (SEM) from center mean between three technical experiments. For carbon-rich media conditions, integral of logistic regression model fitting was used to calculate area under the curve (auc) and difference relative to vehicle control is reported as Δauc. Intracellular survival of c. Micro36, MicroRef1, MicroRef2 in primary human antigen presenting cells isolated from the fetal intestine. Representative data of three biologically independent fetal specimens, error bars indicate SEM from center mean of three cell culture replicates. ANOVA of a generalized linear model of log(CFU+1) against MicroRef1 for each timepoint was used to calculate significance. d. Whole genome average nucleotide identity (ANI) of all available genomes in Micrococcus and Micro36 isolate. When available strain origin is represented, hierarchical clustering was performed on average nucleotide identity, asterisk (*) indicates a reference or a representative genome for the taxon. e . Phylogenetic tree of conserved single-copy genes across all publicly available genomes within Micrococcus and fetal meconium isolate Micro36 with E. coli K12 MG1655 outgroup. For a and e, branch lengths are scaled to the mean number of nucleotide substitutions per site and bootstrap values are represented for relevant nodes.

Article Snippet: Briefly, 54 sgRNAs targeting the human mtDNA were transcribed from pooled sgRNA templates using custom T7 RNA polymerase generously provided by the DeRisi laboratory at UCSF. sgRNAs were purified and concentrated using a column-based RNA purification kit with DNAse treatment (Zymo) and incubated with purified Cas9 (Berkeley Macrolab) for 10 minutes at 37°C. sgRNA-loaded Cas9 was incubated with either meconium genomic DNA (gDNA) or pooled library of 16S rRNA V4 amplicon (see below) for 2 hours at 37°C.

Techniques: Isolation, Cell Culture

Journal: Nature medicine

Article Title: Viable bacterial colonization is highly limited in the human intestine in utero

doi: 10.1038/s41591-020-0761-3

Figure Lengend Snippet: Alignment of 16S V4 rRNA gene sequences of Micro36 to OTU10. Percentage indicates identity to representative OTU sequence.

Article Snippet: Briefly, 54 sgRNAs targeting the human mtDNA were transcribed from pooled sgRNA templates using custom T7 RNA polymerase generously provided by the DeRisi laboratory at UCSF. sgRNAs were purified and concentrated using a column-based RNA purification kit with DNAse treatment (Zymo) and incubated with purified Cas9 (Berkeley Macrolab) for 10 minutes at 37°C. sgRNA-loaded Cas9 was incubated with either meconium genomic DNA (gDNA) or pooled library of 16S rRNA V4 amplicon (see below) for 2 hours at 37°C.

Techniques: Sequencing

Journal: Nature medicine

Article Title: Viable bacterial colonization is highly limited in the human intestine in utero

doi: 10.1038/s41591-020-0761-3

Figure Lengend Snippet: a. Percent identity of samples to 16S rRNA gene of Micro36 in three independent infant stool cohorts. Each symbol represents a sample with a positive hit (>97% sequence identity); symbol shape indicates cohort. Relative abundance of Micrococcus luteus in metagenomic sequencing cohorts across b. body sites at delivery in mother and infant within four months after birth, or c. in maternal stool around delivery and infant stool within the first three months of life. Metagenomic sequences obtained from two independent studies were classified using a custom kraken2 database including the fetal M. luteus Micro 36 genome. Correlation of gestational age with d. total number of OTUs or e. Micrococcaceae OTU10 count in mid-section meconium samples (n= 35 biologically independent fetal specimens) or f. among Micrococaceae meconium (MM, n=9 biologically independent fetal specimens). Pearson’s product-moment correlation coefficient and a one-sided t-distribution p-value is reported for d-f.

Article Snippet: Briefly, 54 sgRNAs targeting the human mtDNA were transcribed from pooled sgRNA templates using custom T7 RNA polymerase generously provided by the DeRisi laboratory at UCSF. sgRNAs were purified and concentrated using a column-based RNA purification kit with DNAse treatment (Zymo) and incubated with purified Cas9 (Berkeley Macrolab) for 10 minutes at 37°C. sgRNA-loaded Cas9 was incubated with either meconium genomic DNA (gDNA) or pooled library of 16S rRNA V4 amplicon (see below) for 2 hours at 37°C.

Techniques: Sequencing

Journal: Applied and Environmental Microbiology

Article Title: Contrasting Microbiome Dynamics of Putative Denitrifying Bacteria in Two Octocoral Species Exposed to Dissolved Organic Carbon (DOC) and Warming

doi: 10.1128/AEM.01886-21

Figure Lengend Snippet: Community compositions and relative abundances of putative denitrifiers in corals X. umbellata and P. flava over the course of the experiment. (a) Relative proportions of denitrifier genera of corals X. umbellata and P. flava inferred by nirS in-silico PCR in relation to the total bacterial community from 16S rRNA gene sequencing. (b) Relative fold changes in copy numbers of nirS gene referenced to 16S rRNA gene and in relation to the day 0 control samples ( n = 3) of corals X. umbellata and P. flava . Values are means ± SD, and the asterisk indicates statistically significant differences (* P < 0.05).

Article Snippet: The 16S rRNA gene amplicon sequencing was conducted at LGC genomics (Berlin, Germany).

Techniques: In Silico, Sequencing

Journal: SpringerPlus

Article Title: Keratinolytic activities of alkaliphilic Bacillus sp. MBRL 575 from a novel habitat, limestone deposit site in Manipur, India

doi: 10.1186/s40064-016-2239-9

Figure Lengend Snippet: Neighbour-joining tree based on 16S rRNA gene sequences, showing the relationships between strain MBRL 575 and other type strains of Bacillus species. Escherichia coli strain SM 25(KF768068.1) was used as the outgroup. Numbers at nodes are levels of bootstrap support (%) for branch points (1000 resamplings). Bar 0.02 substitutions per nucleotide position

Article Snippet: The amplified 16S rRNA gene product was outsourced for sequencing at Xcelris Labs Ltd, India.

Techniques:

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Microbiota composition of suspensions used for human fecal microbiota transplantation. Human microbiota-associated mice were generated by human fecal microbiota transplantation of secondary abiotic mice by gavage on three consecutive days (as described in methods). (A) Microbiota composition of suspensions used for gavage was quantitatively assessed by culture of enterobacteria (EB), enterococci (EC), Gram-positive rods (GPR), Bacteroides/Prevotella spp. (B/P), Clostridium/Eubacterium spp. (C/E), and total bacterial loads (TL) and expressed as colony forming units (CFU) per ml. (B) Microbiota survey was supplemented with quantitative molecular analysis of enterobacteria (EB), enterococci (EC), lactobacilli (LB), bifidobacteria (Bif), Bacteroides/Prevotella spp. (B/P), Clostridium coccoides group (Clocc), Clostridium leptum group (Clept), Mouse Intestinal Bacteroides (MIB), and total eubacterial loads (TL) by qRT-PCR (expressed as 16S rRNA gene numbers per ng DNA). Medians are shown

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Transplantation Assay, Generated, Quantitative RT-PCR

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal total eubacterial gene numbers in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Individual total eubacterial loads were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians are shown. Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of enterobacteria in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Enterobacteria were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians and significance levels ( p values) determined by Mann–Whitney U test are shown. Significant differences between offspring and mothers at a defined time point (as determined by Mann–Whitney U test) are indicated by asterisks ( * p < 0.05). Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of enterococci in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Enterococci were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians and significance levels ( p values) determined by Mann–Whitney U test are shown. Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of lactobacilli in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Lactobacilli were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians and significance levels ( p values) determined by Mann–Whitney U test are shown. Significant differences between offspring and mothers at a defined time point (as determined by Mann–Whitney U test) are indicated by asterisks ( * p < 0.05; ** p < 0.01). Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of bifidobacteria in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Bifidobacteria were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians and significance levels ( p values) determined by Mann–Whitney U test are shown. Significant differences between offspring and mothers at a defined time point (as determined by Mann–Whitney U test) are indicated by asterisks ( *** p < 0.001). Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of Bacteroides/Prevotella species in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Bacteroides/Prevotella spp. were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians are shown. Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of Mouse Intestinal Bacteroides in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Mouse Intestinal Bacteroides were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians are shown. Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of Clostridium leptum group in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Clostridium leptum were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians are shown. Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR

Journal: European Journal of Microbiology & Immunology

Article Title: Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

doi: 10.1556/1886.2017.00002

Figure Lengend Snippet: Fecal gene numbers of Clostridium coccoides group in offspring of human microbiota-associated mice. Human microbiota-associated (hma) mice were generated by human fecal microbiota transplantation of secondary abiotic mice as described in methods. Clostridium coccoides were quantitatively assessed in fecal samples derived from (A) offspring (open circles; n = 21) of hma mice and (B) their mothers (closed circles; n = 5) at defined time points by qRT-PCR and expressed as 16S rRNA gene numbers per ng DNA. Medians and significance levels ( p values) determined by Mann–Whitney U test are shown. Significant differences between offspring and mothers at a defined time point (as determined by Mann–Whitney U test) are indicated by asterisks ( ** p < 0.01). Data were pooled from five litters

Article Snippet: Then, main bacterial groups abundant in the murine and human intestinal microbiota were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) with species-, genera-, or groupspecific 16S rRNA gene primers (Tib MolBiol, Germany) as described previously [ , , ], and numbers of 16S rRNA gene copies per ng DNA of each sample were determined.

Techniques: Generated, Transplantation Assay, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY