scholarly journals Sorting and cultivation of Faecalibacterium prausnitzii from fecal samples using flow cytometry in anaerobic conditions

2020 ◽  
Author(s):  
Samuel Bellais ◽  
Mélanie Nehlich ◽  
Aurore Duquenoy ◽  
Maryne Ania ◽  
Ger van den Engh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Polyclonal Antibodies ◽  
Fecal Microbiota ◽  
Anaerobic Conditions ◽  
Specific Staining ◽  
Fecal Samples ◽  
Promising Tool ◽  
Faecalibacterium Prausnitzii ◽  
Efficient Detection

AbstractBackgroundThere is a growing interest in using gut commensal bacteria as ‘next generation’ probiotics. However, this approach is still hampered by the fact that there are few or no strains available for specific species that are difficult to cultivate. Our objective was therefore to adapt flow cytometry and cell sorting to be able to detect, separate, isolate and cultivate new strains of Extremely Oxygen Sensitive (EOS) species from fecal material, focusing on Faecalibacterium prausnitzii as a proof-of-concept.ResultsA BD Influx® cell sorter was equipped with a glovebox that covers the sorting area. This box is flushed with nitrogen to deplete oxygen in the enclosure. Several non-specific staining methods including Wheat Germ Agglutinin (WGA), Vancomycin BODIPY™ and LIVE/DEAD BacLight were evaluated with three different strains of the EOS species F. prausnitzii. In parallel, we generated polyclonal antibodies directed against this species by immunizing rabbits with heat-inactivated bacteria. Anaerobic conditions were maintained during the full process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii reference strains. In addition, staining solutions did not severely impact bacterial viability while allowing discrimination between groups of strains. Efficient detection was achieved using polyclonal antibodies directed against heat-fixed bacteria. Finally, we were able to detect, isolate and cultivate a variety of F. prausnitzii strains from healthy volunteer’s fecal samples using WGA staining and antibodies. These strains present markedly different phenotypes, thus confirming the heterogeneity of the species.ConclusionsCell-sorting in anaerobic conditions is a promising tool for the study of fecal microbiota. It gives the opportunity to quickly analyze microbial populations and to sort strains of interest using specific antibodies, thus opening new avenues for targeted culturomics experiments.

scholarly journals Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.

1977 ◽  
Vol 25 (7) ◽  
pp. 573-579 ◽  
Author(s):  
R H Jensen
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Deoxyribonucleic Acid ◽  
Emission Spectra ◽  
Hoechst 33258 ◽  
Chromomycin A3 ◽  
Fluorescence Excitation ◽  
Specific Staining ◽  
Cervical Cells ◽  
Two Parameter

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.

scholarly journals Fecal Microbiota Composition and Frailty

2005 ◽  
Vol 71 (10) ◽  
pp. 6438-6442 ◽  
Author(s):  
Sandra P. van Tongeren ◽  
Joris P. J. Slaets ◽  
H. J. M. Harmsen ◽  
Gjalt W. Welling
Keyword(s):  
The Elderly ◽  
Fecal Microbiota ◽  
Microbiota Composition ◽  
Fecal Samples ◽  
Faecalibacterium Prausnitzii ◽  
The Relationship

ABSTRACT The relationship between fecal microbiota composition and frailty in the elderly was studied. Fecal samples from volunteers with high frailty scores showed a significant reduction in the number of lactobacilli (26-fold). At much higher population levels, both the Bacteroides/Prevotella (threefold) and the Faecalibacterium prausnitzii (fourfold) groups showed a significant reduction in percentage of total number of hybridizable bacteria in the elderly with high frailty scores. In contrast to this, the number of Enterobacteriaceae was significantly higher (sevenfold) in samples from very frail volunteers.

scholarly journals Fecal microbiota dynamics during disease activity and remission in newly diagnosed and established ulcerative colitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lena Öhman ◽  
Anders Lasson ◽  
Anna Strömbeck ◽  
Stefan Isaksson ◽  
Marcus Hesselmar ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gut Microbiota ◽  
Disease Activity ◽  
Temporal Dynamics ◽  
Fecal Microbiota ◽  
Disease Stage ◽  
Dietary Interventions ◽  
Microbial Composition ◽  
Fecal Samples ◽  
Specific Species

AbstractPatients with ulcerative colitis (UC) have an altered gut microbiota composition, but the microbial relationship to disease activity needs to be further elucidated. Therefore, temporal dynamics of the fecal microbial community during remission and flare was determined. Fecal samples were collected at 2–6 time-points from UC patients during established disease (cohort EST) and at diagnosis (cohort NEW). Sampling range for cohort EST was 3–10 months and for cohort NEW 36 months. Relapses were monitored for an additional three years for cohort EST. Microbial composition was assessed by Genetic Analysis GA-map Dysbiosis Test, targeting ≥ 300 bacteria. Eighteen patients in cohort EST (8 with maintained remission and 10 experiencing a flare), provided 71 fecal samples. In cohort NEW, 13 patients provided 49 fecal samples. The microbial composition showed no clustering related to disease activity in any cohort. Microbial dissimilarity was higher between than within patients for both cohorts, irrespective of presence of a flare. Microbial stability within patients was constant over time with no major shift in overall composition nor modification in the abundance of any specific species. Microbial composition was not affected by intensified medical treatment or linked to future disease course. Thus in UC, the gut microbiota is highly stable irrespective of disease stage, disease activity or treatment escalation. This suggests that prolonged dietary interventions or repeated fecal transplantations are needed to be able to induce permanent alterations of the gut microbiota.

scholarly journals Comparison of the Fecal Microbiota of Horses with Intestinal Disease and Their Healthy Counterparts

2021 ◽  
Vol 8 (6) ◽  
pp. 113
Author(s):  
Taemook Park ◽  
Heetae Cheong ◽  
Jungho Yoon ◽  
Ahram Kim ◽  
Youngmin Yun ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Metabolic Disorders ◽  
Bacterial Population ◽  
Fecal Microbiota ◽  
Intestinal Disease ◽  
Fecal Samples ◽  
Gut Dysbiosis ◽  
Rrna Sequencing ◽  
Small Intestinal

(1) Background: The intestinal microbiota plays an essential role in maintaining the host’s health. Dysbiosis of the equine hindgut microbiota can alter the fermentation patterns and cause metabolic disorders. (2) Methods: This study compared the fecal microbiota composition of horses with intestinal disease and their healthy counterparts living in Korea using 16S rRNA sequencing from fecal samples. A total of 52 fecal samples were collected and divided into three groups: horses with large intestinal disease (n = 20), horses with small intestinal disease (n = 8), and healthy horses (n = 24). (3) Results: Horses with intestinal diseases had fewer species and a less diverse bacterial population than healthy horses. Lactic acid bacteria, Lachnospiraceae, and Lactobacillaceae were overgrown in horses with large intestinal colic. The Firmicutes to Bacteroidetes ratio (F/B), which is a relevant marker of gut dysbiosis, was 1.94, 2.37, and 1.74 for horses with large intestinal colic, small intestinal colic, and healthy horses, respectively. (4) Conclusions: The overgrowth of two lactic acid bacteria families, Lachnospiraceae and Lactobacillaceae, led to a decrease in hindgut pH that interfered with normal fermentation, which might cause large intestinal colic. The overgrowth of Streptococcus also led to a decrease in pH in the hindgut, which suppressed the proliferation of the methanogen and reduced methanogenesis in horses with small intestinal colic.

scholarly journals Composition of “gold juice” using an ancient method based on intestinal microecology

2020 ◽  
Vol 48 (9) ◽  
pp. 030006052093128
Author(s):  
Qiuwei Li ◽  
Liying Guo ◽  
Li Wang ◽  
Jing Miao ◽  
Huantian Cui ◽  
...  
Keyword(s):  
Chinese Medicine ◽  
Traditional Chinese Medicine ◽  
Fecal Microbiota Transplantation ◽  
Antibiotic Use ◽  
Fecal Microbiota ◽  
Healthy Children ◽  
Fecal Samples ◽  
16S Rdna Sequence Analysis ◽  
History Of ◽  
Chinese Medicine Treatment

Objective To identify potentially effective bacterial components of gold juice, a traditional Chinese medicine treatment used for fecal microbiota transplantation. Methods Fecal samples were collected from five healthy children (two boys and three girls; mean age, 7.52 ± 2.31 years). The children had no history of antibiotic use or intestinal microecological preparation in the preceding 3 months. Fresh fecal samples were collected from children to prepare gold juice in mid-to-late November, in accordance with traditional Chinese medicine methods, then used within 7 days. Finally, 16S rDNA sequence analysis was used to identify potentially effective bacterial components of gold juice. QIIME software was used for comparisons of microbial species among gold juice, diluent, filtrate, and loess samples. Results Microflora of gold juice exhibited considerable changes following “ancient method” processing. Microbial components significantly differed between gold juice and filtrate samples. The gold juice analyzed in our study consisted of microbes that synthesize carbohydrates and amino acids by degrading substances, whereas the filtrate contained probiotic flora, Bacteroides, and Prevotella 9. Conclusions This study of microbial components in gold juice and filtrate provided evidence regarding effective bacterial components in gold juice, which may aid in clinical decisions concerning fecal microbiota transplantation.

scholarly journals Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

2002 ◽  
Vol 68 (11) ◽  
pp. 5209-5216 ◽  
Author(s):  
Kaouther Ben Amor ◽  
Pieter Breeuwer ◽  
Patrick Verbaarschot ◽  
Frank M. Rombouts ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Salt Stress ◽  
Bile Salt ◽  
Cell Sorting ◽  
Physiological Activity ◽  
Membrane Integrity ◽  
Multiparameter Flow Cytometry ◽  
Bifidobacterium Adolescentis ◽  
Multiparametric Flow Cytometry ◽  
Plate Counts

ABSTRACT Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

Flow cytometry and cell sorting for yeast viability assessment and cell selection

Yeast ◽  
1998 ◽  
Vol 14 (2) ◽  
pp. 147-160 ◽  
Author(s):  
Daniel Deere ◽  
Jian Shen ◽  
Graham Vesey ◽  
Philip Bell ◽  
Peter Bissinger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Cell Selection ◽  
Viability Assessment ◽  
Yeast Viability
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