scholarly journals High-throughput isolation and sorting of gut microbes reduce biases of traditional cultivation strategies

2019 ◽  
Author(s):  
William J. Watterson ◽  
Melikhan Tanyeri ◽  
Andrea R. Watson ◽  
Candace M. Cham ◽  
Yue Shan ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Fold Increase ◽  
Taxonomic Composition ◽  
Biased Sampling ◽  
Human Gut ◽  
Rare Taxa ◽  
Conventional Culture ◽  
16S Ribosomal Rna Gene ◽  
Slow Growing ◽  
New Strategies

AbstractTraditional cultivation approaches in microbiology are labor-intensive, low-throughput, and often yield biased sampling of taxa due to ecological and evolutionary factors. New strategies are needed to enable ample representation of rare taxa and slow-growers that are outcompeted by fast-growing organisms. We developed a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts droplets based on colony density. We applied our strategy to mouse and human gut microbiomes and used 16S ribosomal RNA gene amplicons to characterize taxonomic composition of cells grown using different media. We found up to 4-fold increase in richness and larger representation of rare taxa among cells grown in droplets compared to conventional culture plates. Automated sorting of droplets for slow-growing colonies further enhanced the relative abundance of rare populations. Our method improves the cultivation and analysis of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.

scholarly journals Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
William J Watterson ◽  
Melikhan Tanyeri ◽  
Andrea R Watson ◽  
Candace M Cham ◽  
Yue Shan ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Fecal Microbiota ◽  
Biased Sampling ◽  
Growth Media ◽  
Taxonomic Richness ◽  
Fecal Microbiota Transplant ◽  
Antibiotic Resistant ◽  
Slow Growing ◽  
New Strategies ◽  
Environmental Microbes

Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and evolutionary factors. New strategies are needed for ample representation of rare taxa and slow-growers that are often outcompeted by fast-growers in cultivation experiments. Here we describe a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts them based on colony density to enhance slow-growing organisms. We applied our strategy to a fecal microbiota transplant (FMT) donor stool using multiple growth media, and found significant increase in taxonomic richness and larger representation of rare and clinically relevant taxa among droplet-grown cells compared to conventional plates. Furthermore, screening the FMT donor stool for antibiotic resistance revealed 21 populations that evaded detection in plate-based assessment of antibiotic resistance. Our method improves cultivation-based surveys of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.

scholarly journals Molecular Approaches to Studying Microbial Communities: Targeting the 16S Ribosomal RNA Gene

2016 ◽  
Vol 38 (3) ◽  
pp. 223-232 ◽  
Author(s):  
Kazumasa FUKUDA ◽  
Midori OGAWA ◽  
Hatsumi TANIGUCHI ◽  
Mitsumasa SAITO
Keyword(s):  
Microbial Communities ◽  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
Approaches To Studying ◽  
Ribosomal Rna Gene ◽  
Molecular Approaches ◽  
16S Ribosomal Rna Gene

scholarly journals Evaluation of 16S, map1 and pCS20 probes for detection of Cowdria and Ehrlichia species

1999 ◽  
Vol 122 (2) ◽  
pp. 323-328 ◽  
Author(s):  
M. T. E. P. ALLSOPP ◽  
C. M. HATTINGH ◽  
S. W. VOGEL ◽  
B. A. ALLSOPP
Keyword(s):  
16S Rrna ◽  
Ribosomal Rna ◽  
Pcr Amplification ◽  
16S Ribosomal Rna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Amblyomma Hebraeum ◽  
Ribosomal Rna Gene ◽  
Group Iii ◽  
16S Ribosomal Rna Gene

A panel of 16S ribosomal RNA gene probes has been developed for the study of the epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five distinct genotypes; one detects any Group III Ehrlichia species other than Cowdria and one detects any Group II Ehrlichia species. These probes have been used on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other cowdria genes (map1 and pCS20) which have previously been used for heartwater epidemiology. This paper describes the first direct comparison of all currently available DNA probes for heartwater-associated organisms.

scholarly journals A Case of Sepsis in a 92-Year-Old Korean Woman Caused byAerococcus urinaeand Identified by Sequencing the 16S Ribosomal RNA Gene

2016 ◽  
Vol 47 (2) ◽  
pp. e15-e17 ◽  
Author(s):  
Min Young Lee ◽  
Myeong Hee Kim ◽  
Woo In Lee ◽  
So Young Kang ◽  
You La Jeon
Keyword(s):  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
Korean Woman ◽  
Ribosomal Rna Gene ◽  
16S Ribosomal Rna Gene

scholarly journals Bacterial diversity in aortic aneurysms determined by 16S ribosomal RNA gene analysis

2006 ◽  
Vol 44 (5) ◽  
pp. 1055-1060 ◽  
Author(s):  
Rafael Marques da Silva ◽  
Dominique A. Caugant ◽  
Emenike R.K. Eribe ◽  
Jørn A. Aas ◽  
Per S. Lingaas ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Ribosomal Rna ◽  
Aortic Aneurysms ◽  
16S Ribosomal Rna ◽  
Gene Analysis ◽  
Ribosomal Rna Gene ◽  
16S Ribosomal Rna Gene

scholarly journals Ileal mucosa-associated microbiota overgrowth associated with pathogenesis of primary biliary cholangitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shogo Kitahata ◽  
Yasunori Yamamoto ◽  
Osamu Yoshida ◽  
Yoshio Tokumoto ◽  
Tomoe Kawamura ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Primary Biliary Cholangitis ◽  
Small Intestinal Mucosa ◽  
Ileal Mucosa ◽  
16S Ribosomal Rna Gene ◽  
Pathological Development ◽  
Small Intestinal ◽  
Chronic Nonsuppurative Destructive Cholangitis ◽  
Independent Association ◽  
The Relationship

AbstractThe small intestinal mucosa-associated microbiota (MAM) can potentially impact the etiology of primary biliary cholangitis (PBC). Herein, we investigate the MAM profile to determine its association with liver pathology in patients with PBC. Thirty-four patients with PBC and 21 healthy controls who underwent colonoscopy at our hospital were enrolled in our study. We performed 16S ribosomal RNA gene sequencing of MAM samples obtained from the mucosa of the terminal ileum and examined the relationship between the abundance of ileal MAM and chronic nonsuppurative destructive cholangitis using liver specimens from patients with PBC. There was a significant reduction in microbial diversity within individuals with PBC (P = 0.039). Dysbiosis of ileal MAM was observed in patients with PBC, with a characteristic overgrowth of Sphingomonadaceae and Pseudomonas. Multivariate analysis showed that the overgrowth of Sphingomonadaceae and Pseudomonas is an independent association factor for PBC (P = 0.0429, P = 0.026). Moreover, the abundance of Sphingomonadaceae was associated with chronic nonsuppurative destructive cholangitis in PBC (P = 0.00981). The overgrowth of Sphingomonadaceae and Pseudomonas in ileal MAM was found in patients with PBC. Sphingomonadaceae may be associated with the pathological development of PBC.

scholarly journals Alterations in the gut microbiota can elicit hypertension in rats

2017 ◽  
Vol 49 (2) ◽  
pp. 96-104 ◽  
Author(s):  
Sareema Adnan ◽  
James W. Nelson ◽  
Nadim J. Ajami ◽  
Venugopal R. Venna ◽  
Joseph F. Petrosino ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Gut Microbiota ◽  
Systolic Blood Pressure ◽  
Ribosomal Rna ◽  
Shr Rats ◽  
Spontaneously Hypertensive ◽  
Gut Dysbiosis ◽  
16S Ribosomal Rna Gene ◽  
Previously Treated ◽  
Normotensive Strain

Gut dysbiosis has been linked to cardiovascular diseases including hypertension. We tested the hypothesis that hypertension could be induced in a normotensive strain of rats or attenuated in a hypertensive strain of rats by exchanging the gut microbiota between the two strains. Cecal contents from spontaneously hypertensive stroke prone rats (SHRSP) were pooled. Similarly, cecal contents from normotensive WKY rats were pooled. Four-week-old recipient WKY and SHR rats, previously treated with antibiotics to reduce the native microbiota, were gavaged with WKY or SHRSP microbiota, resulting in four groups; WKY with WKY microbiota (WKY g-WKY), WKY with SHRSP microbiota (WKY g-SHRSP), SHR with SHRSP microbiota (SHR g-SHRSP), and SHR with WKY microbiota (SHR g-WKY). Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. At 11.5 wk of age systolic blood pressure increased 26 mmHg in WKY g-SHRSP compared with that in WKY g-WKY (182 ± 8 vs. 156 ± 8 mmHg, P = 0.02). Although the SBP in SHR g-WKY tended to decrease compared with SHR g-SHRSP, the differences were not statistically significant. Fecal pellets were collected at 11.5 wk of age for identification of the microbiota by sequencing the 16S ribosomal RNA gene. We observed a significant increase in the Firmicutes:Bacteroidetes ratio in the hypertensive WKY g-SHRSP, as compared with the normotensive WKY g-WKY ( P = 0.042). Relative abundance of multiple taxa correlated with SBP. We conclude that gut dysbiosis can directly affect SBP. Manipulation of the gut microbiota may represent an innovative treatment for hypertension.

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