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Open AccessArticle

Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry

1
Department of Medicine and Aging Science, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini, 31, 66100 Chieti, Italy
2
Center for Aging Science and Translational Medicine (CeSI-MeT), Via Luigi Polacchi, 11, 66100 Chieti, Italy
3
Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini, 31, 66100 Chieti, Italy
4
Department of Medical, Oral, and Biotechnological Sciences, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini, 31, 66100 Chieti, Italy
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(21), 5307; https://doi.org/10.3390/ijms20215307
Received: 3 September 2019 / Revised: 18 October 2019 / Accepted: 21 October 2019 / Published: 25 October 2019
(This article belongs to the Special Issue Biofilms and Bacterial Virulence)
Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria–host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples. View Full-Text
Keywords: bacterial membrane vesicles; flow cytometry; Lactobacillus reuteri; Helicobacter pylori; biofilm; extracellular DNA (eDNA); extracellular vesicles bacterial membrane vesicles; flow cytometry; Lactobacillus reuteri; Helicobacter pylori; biofilm; extracellular DNA (eDNA); extracellular vesicles
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Puca, V.; Ercolino, E.; Celia, C.; Bologna, G.; Di Marzio, L.; Mincione, G.; Marchisio, M.; Miscia, S.; Muraro, R.; Lanuti, P.; Grande, R. Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry. Int. J. Mol. Sci. 2019, 20, 5307.

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