Additional file 2 of Quantitative sequencing clarifies the role of disruptor taxa, oral microbiota, and strict anaerobes in the human small-intestine microbiome
Creators: name: Barlow, Jacob T.
name: Leite, Gabriela
name: Romano, Anna E.
name: Sedighi, Rashin
name: Chang, Christine
name: Celly, Shreya
name: Rezaie, Ali
name: Mathur, Ruchi
name: Pimentel, Mark
name: Ismagilov, Rustem F.
Publisher: name: figshare
Year: 2021
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Ecology
subject: Science Policy
subject: 69999 Biological Sciences not elsewhere classified
subject: 110309 Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Total microbial load breakdown by age (A) and gender (B). Figure S2. Distribution of total microbial load from subpopulations of patients: taking probiotics (N=49), active smokers (N=16), taking antibiotics in the past 6 months (N=100), or taking proton pump inhibitors (PPI, N=106). Figure S3. (A) Scatterplot comparing aerobic culture load from MacConkey plates to total load from 16S quantitative sequencing of only the subset of bacteria that are known to grow on MacConkey plates (Escherichia-Shigella, Enterobacteriaceae, Enterococcus, and Aeromonas)1. (B) Scatterplot comparing anaerobic culture load, from blood agar plates, to total load from sequencing of prevalent bacteria that are expected to grow on blood agar plates (Prevotella, Streptococcus, Fusobacterium, Escherichia-Shigella)2. Red dashed line indicates limit of detection of quantitative sequencing method. N = 244. (Six patients in the study were lacking culture data). Figure S4. (A) Cycle threshold (Cq) values yielded by qPCR with Klebsiella-specific primers. Duodenum aspirate samples were classified via quantitative sequencing as containing Enterobacteriaceae (“Entero +”, N=22) or not containing Enterobacteriaceae (“Entero –”, N=8). (B) Total loads of Enterobacteriaceae (copies/mL) in duodenum aspirates as a factor of the approximate Klebsiella load (copies/mL). Enterobacteriaceae measurements are calculated based on 16S rRNA gene copies (8 copies/genome) and Klebsiella measurements are calculated based on the citrate synthase gene (gltA, 1 copy/genome). Figure S5. Receiver operating characteristic (ROC) curve using absolute loads of seven disruptor taxa (Enterobacteriaceae, Escherichia-Shigella, Clostridium sensu stricto 1, Enterococcus, Romboutsia, Aeromonas, Bacteroides) identified in the sequencing data for SIBO classification. SIBO classification was made based on gold-standard aerobic culture results, ≥103 CFU/mL of duodenal aspirate. Data points are connected by a line between each consecutive point. Figure S6. IL8 levels in samples with and without a Clostridium which, based on the V4 region of the 16S rRNA gene, was classified as C. perfringens. Figure S7. Relationship between Lactobacillus load and bloating symptoms in samples containing additional (non-Lactobacillus) disruptor taxa. Figure S8. Violin plots with data points overlaid for patient-reported symptom scores. Binary threshold for determining whether severe symptoms exist was set at the median score reported of each symptom, shown by the red-dashed lines. Figure S9. Disruptor taxa load separated by patient age: 18-39 (N=40), 40-49 (N=31), 50-59 (N=58), 60-69 (N=67), 70-83 (N=54). Figure S10. Relationship between absolute abundance (greater than 105 copies/mL) and relative abundance of disruptor loads (Spearman, P=0.09, not significant). Figure S11. Comparison of total microbial load between human duodenum, mouse duodenum, and mouse duodenum where the mice had coprophagy prevented via tail cup. Mouse data from Bogatyrev et al. 20203. Reported P-values are from Kruskal-Wallis test. Table S1. Summary statistics for the patient cohort used in this study. All patients are from the REIMAGINE study4. Table S2. P-values from significance tests (Kruskal-Wallis) comparing total microbial load between selected subgroups of individuals. Significance is indicated with an asterisk. Table S3. Comparison between prevalence and relative abundance of all taxa in paired saliva and duodenum samples (N=21 participants). Table S4. Two groups of taxa (light blue and dark blue) that have stronger co-correlations with another taxon than with total load. Significance values for all correlations and co-correlations were P &lt; 0.001.