16S rRNA in situ Hybridization Followed by Flow Cytometry for Rapid Identification of Acetic Acid Bacteria Involved in Submerged Industrial Vinegar Production

ABSTRACT Predominant groups of bacteria from a human fecal flora-associated mouse model challenged with amoxicillin-clavulanic acid were quantified with fluorescence in situ hybridization combined with flow cytometry using specific 16S rRNA targeted oligonucleotide probes. This approach provides a useful tool with high throughput to evaluate fecal microflora under antibiotic treatment.

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Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

Applied and Environmental Microbiology ◽

10.1128/aem.68.9.4225-4232.2002 ◽

2002 ◽

Vol 68 (9) ◽

pp. 4225-4232 ◽

Cited By ~ 90

Author(s):

Erwin G. Zoetendal ◽

Kaouther Ben-Amor ◽

Hermie J. M. Harmsen ◽

Frits Schut ◽

Antoon D. L. Akkermans ◽

...

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

16S Rrna ◽

Fluorescent In Situ Hybridization ◽

Specific Effect ◽

Human Intestine ◽

Fecal Samples ◽

Flow Cytometric ◽

Significant Difference

ABSTRACT A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.

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Detection and cell sorting of Pseudonocardia species by fluorescence in situ hybridization and flow cytometry using 16S rRNA-targeted oligonucleotide probes

Applied Microbiology and Biotechnology ◽

10.1007/s00253-018-8801-3 ◽

2018 ◽

Vol 102 (7) ◽

pp. 3375-3386 ◽

Cited By ~ 8

Author(s):

Mengyan Li ◽

Yu Yang ◽

Ya He ◽

Jacques Mathieu ◽

Cong Yu ◽

...

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Cell Sorting ◽

Oligonucleotide Probes

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Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations

Applied and Environmental Microbiology ◽

10.1128/aem.00161-06 ◽

2006 ◽

Vol 72 (6) ◽

pp. 4293-4301 ◽

Cited By ~ 78

Author(s):

Marina G. Kalyuzhnaya ◽

Rebecca Zabinsky ◽

Sarah Bowerman ◽

David R. Baker ◽

Mary E. Lidstrom ◽

...

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Cell Sorting ◽

Type I ◽

Type Ii ◽

Specific Probe ◽

Artificial Mixtures

ABSTRACT A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 104 or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.

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Ecophysiological Interaction between Nitrifying Bacteria and Heterotrophic Bacteria in Autotrophic Nitrifying Biofilms as Determined by Microautoradiography-Fluorescence In Situ Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1641-1650.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1641-1650 ◽

Cited By ~ 233

Author(s):

Tomonori Kindaichi ◽

Tsukasa Ito ◽

Satoshi Okabe

Keyword(s):

Acetic Acid ◽

In Situ Hybridization ◽

Bacterial Community ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Heterotrophic Bacteria ◽

Nitrifying Bacteria ◽

Community Members ◽

Biofilm Community

ABSTRACT Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4 + as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (α-Proteobacteria), 23%; γ-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. α- and γ-Proteobacteria dominated the utilization of [14C]acetic acid and 14C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-d-[1-14C]glucosamine (NAG). The GNSB accounted for 9% of the 14C-amino acid-consuming bacteria and 27% of the [14C]NAG-consuming bacteria but did not utilize [14C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.

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On the Origin of Tetraploid Vernal Grasses (Anthoxanthum) in Europe

Genes ◽

10.3390/genes12070966 ◽

2021 ◽

Vol 12 (7) ◽

pp. 966

Author(s):

Zuzana Chumová ◽

Terezie Mandáková ◽

Pavel Trávníček

Keyword(s):

Flow Cytometry ◽

In Situ Hybridization ◽

Crucial Role ◽

Evolutionary History ◽

Essential Role ◽

Grass Species ◽

Polyploid Species ◽

Anthoxanthum Odoratum ◽

European Populations

Polyploidy has played a crucial role in the evolution of many plant taxa, namely in higher latitudinal zones. Surprisingly, after several decades of an intensive research on polyploids, there are still common polyploid species whose evolutionary history is virtually unknown. Here, we addressed the origin of sweet vernal grass (Anthoxanthum odoratum) using flow cytometry, DNA sequencing, and in situ hybridization-based cytogenetic techniques. An allotetraploid and polytopic origin of the species has been verified. The chromosome study reveals an extensive variation between the European populations. In contrast, an autopolyploid origin of the rarer tetraploid vernal grass species, A. alpinum, has been corroborated. Diploid A. alpinum played an essential role in the polyploidization of both European tetraploids studied.

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