Direct flow cytometry of anaerobic bacteria in human feces

Waaij, Laurens A. van der; Mesander, G; Limburg, P. C.; Waaij, D. van der

doi:10.1002/cyto.990160312

Cited by 69 publications

(49 citation statements)

References 29 publications

(29 reference statements)

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“…b All counts were the numbers of bacilli in 50 fields after aerobic incubation at 30°C. specific intact single microorganisms within environmental samples, such as lake water (18), soil (17), and activated sludge (20), and noncultured anaerobic bacteria in human fecal flora (19).…”

Section: Vol 40 2002 Notes 4773mentioning

confidence: 99%

Are There Naturally Occurring Pleomorphic Bacteria in the Blood of Healthy Humans?

et al. 2002

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Dark-field microscopy of blood from healthy individuals revealed the existence of pleomorphic microorganisms. These bacteria exhibited limited growth and susceptibility to antibiotics and could be detected by fluorescent in situ hybridization and flow cytometry. They were further characterized by analysis of their 16S rRNA and gyrB genes.In our search for spirochetes involved in Alzheimer's disease (13), we observed pleomorphic bacteria in the blood of healthy human subjects by dark-field microscopy. This was a surprising finding since it is generally acknowledged that the bloodstream in healthy humans is a sterile environment (7) except when there is a breach in the integrity of the tissue membranes (6). However, the concept of the occurrence of bacteria in the blood of healthy humans is now more plausible because of cultivation-independent laboratory approaches. The main techniques employed in such studies include PCR amplification and sequencing of the16S ribosomal DNA (rDNA). These methods have revealed the presence of a wide diversity of microorganisms in the environment, and indeed within the human body (12). In this report we present evidence based on molecular phylogenetic techniques and light and electron microscopy, as well as other conventional microbiological methods, for the existence of a population of bacteria in healthy human blood. In view of the apparent controversial nature of our findings, it was encouraging to note the recent report of Nikkari et al. (14), who detected blood-associated bacterial rDNA sequences by using real-time PCR methods and a probe targeting conserved regions of bacterial 16S rDNA, and an earlier report by Tedeshi et al. (16) on the presence of pleomorphic bacteria as intraerythrocytic parasites in clinically healthy human subjects.For light microscopic examination, blood samples from 25 healthy volunteers were drawn in a Vacutainer tube with no anticoagulants (Becton Dickinson, Franklin Lakes, N.J.); blood was drawn in the conventional manner involving antisepsis of the skin and avoidance of any introduction of external microorganisms by contamination. (Since external contamination was always a possibility, particular care and precaution were exercised at all times to avoid this. The specific procedures, as well as appropriate controls, are specified throughout the text.) A wet mount of the serum from the clotted blood of each sample, fresh or incubated at 30°C for between 5 to 7 days, was examined by dark-field microscopy (Leitz Dialux 20) for pleomorphic bacteria.For PCR amplification, a 0.5-ml aliquot of incubated blood containing pleomorphic bacteria was used for conventional extraction of DNA by the method of Higuchi (11). Three microliters of the extract was used for DNA amplification by the method of Edwards et al. (8) using the forward primer BSF8/20 (5Ј-AGAGTTTGATCCTGGCTCAG-3Ј) and the reverse primer BSR1541/20 (5Ј-AAGGAGGTGATCCAGCCGCA-3Ј). Thirty cycles of PCR were performed, with 1 cycle consisting of the following steps: (i) denaturation (1 min at 94°C), (i...

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Section: Vol 40 2002 Notes 4773mentioning

confidence: 99%

Are There Naturally Occurring Pleomorphic Bacteria in the Blood of Healthy Humans?

et al. 2002

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“…Also, FCM has been used for analysis of bacteria in environmental samples, such as soil, air, and especially water (31,32,37), and in clinical samples, such as blood, urine, and feces (1,31,36). The potential of FCM for the food industry has been recognized as well (19,32,35).…”

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confidence: 99%

Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters

Bunthof

Abee

2002

Appl Environ Microbiol

126

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“…We are confident in our results because flow cytometry is a rapid and reliable method to quantify bacteria in fecal samples (39,40,53), and Gram staining is a technique that is commonly used in clinical bacteriology to differentiate Gram-positive and Gramnegative bacteria (52); it was also the first method used in initial studies of the gut microbiota (16,17). Transmission electron microscopy has been used for bacterial applications since the late 1960s (22).…”

Section: Discussionmentioning

confidence: 68%

“…The parameters were adjusted using diluted samples with or without beads to the following settings: forward scatter (FSC) 250 nm, SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) 174 nm, FSC photomultiplier tube (PMT) 500 nm, Pacific Blue 313 nm (to visualize the 4=,6-diamidino-2-phenylindole [DAPI] stain), fluorescein isothiocyanate (FITC) 426 nm, and allophycocyanin (APC) 500 nm. Flow cytometry (40) was performed in triplicate with 3 different dilutions (from 10 Ϫ4 to 10 Ϫ6 ) for the 10 control samples and in triplicate with the 10 Ϫ4 dilution for the 16 patient samples. To 500 l of each dilution, 500 l of Triton 0.1% in PBS was added to permeabilize the bacterial cell walls.…”

Section: Methodsmentioning

confidence: 99%

Molecular Studies Neglect Apparently Gram-Negative Populations in the Human Gut Microbiota

et al. 2013

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dStudying the relationships between gut microbiota, human health, and diseases is a major challenge that generates contradictory results. Most studies draw conclusions about the gut repertoire using a single biased metagenomics approach. We analyzed 16 different stool samples collected from healthy subjects who were from different areas, had metabolic disorders, were immunocompromised, or were treated with antibiotics at the time of the stool collection. The analyses performed included Gram staining, flow cytometry, transmission electron microscopy (TEM), quantitative real-time PCR (qPCR) of the Bacteroidetes and Firmicutes phyla, and pyrosequencing of the 16S rRNA gene amplicons targeting the V6 region. We quantified 10 10 prokaryotes per gram of feces, which is less than was previously described. The Mann-Whitney test revealed that Gram-negative proportions of the prokaryotes obtained by Gram staining, TEM, and pyrosequencing differed according to the analysis used, with Gram-negative prokaryotes yielding median percentages of 70.6%, 31.0%, and 16.4%, respectively. A comparison of TEM and pyrosequencing analyses highlighted a difference of 14.6% in the identification of Gram-negative prokaryotes, and a Spearman test showed a tendency toward correlation, albeit not significant, in the Gram-negative/Gram-positive prokaryote ratio ( ‫؍‬ 0.3282, P ‫؍‬ 0.2146). In contrast, when comparing the qPCR and pyrosequencing results, a significant correlation was found for the Bacteroidetes/Firmicutes ratio ( ‫؍‬ 0.6057, P ‫؍‬ 0.0130). Our study showed that the entire diversity of the human gut microbiota remains unknown because different techniques generate extremely different results. We found that to assess the overall composition of bacterial communities, multiple techniques must be combined. The biases that exist for each technique may be useful in exploring the major discrepancies in molecular studies.

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Direct flow cytometry of anaerobic bacteria in human feces

Cited by 69 publications

References 29 publications

Are There Naturally Occurring Pleomorphic Bacteria in the Blood of Healthy Humans?

Are There Naturally Occurring Pleomorphic Bacteria in the Blood of Healthy Humans?

Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters

Molecular Studies Neglect Apparently Gram-Negative Populations in the Human Gut Microbiota

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