Abstract
The increasing interest in the effects of the gut microbiota on host health has stimulated the investigation of the composition of this microbial community and the factors affecting these microorganisms. This review discusses the recent advances and progress applications in the use of the fluorescent in situ hybridization (FISH) coupled to flow cytometry (FC) technique (FISH-FC) in studies evaluating the gut microbiota published in the last 10 years, with particular emphasis on the effects of foods and dietary interventions. These studies have shown that FISH-FC technique is capable of detecting and quantifying several groups of bacteria found as part of the gut microbiota. FISH-FC can be considered an effective, versatile, and rapid technique to evaluate alterations in gut microbiota composition caused by different foods as assessed in studies in vitro, in vivo, and in clinical trials. Some specific probes have been most used to represent the general gut microbiota, such as those specific to Lactobacillus spp./Enterococcus spp., Bacteroidaceae/Prevotellaceae, Clostridium histolyticum, and Bifidobacterium spp. FISH-FC technique could have an important opportunity for application in studies with next-generation probiotics belonging to the gut microbiota. Optimizations of FISH-FC protocols could allow more discoveries about the gut microbiota, including the development of new probes targeting microorganisms still not explored, the analysis of individual portions of the intestine, and the proposition of novel quantitative approaches.
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References
Aistleitner K, Jeske R, Wölfel R, Wießner A, Kikhney J, Moter A, Stoecker K (2018) Detection of Coxiella burnetii in heart valve sections by fluorescence in situ hybridization. J Med Microbiol 67(4):537–542. https://doi.org/10.1099/jmm.0.000704
Albuquerque TMR, Magnani M, Lima MDS, Castellano LRC, de Souza EL (2021) Effects of digested flours from four different sweet potato (Ipomoea batatas L.) root varieties on the composition and metabolic activity of human colonic microbiota in vitro. J Food Sci 86(8):3707–3719. https://doi.org/10.1111/1750-3841.15852
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl D (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56(6):1919–1925. https://doi.org/10.1128/aem.56.6.1919-1925.1990
Arrigucci R, Bushkin Y, Radford F, Lakehal K, Vir P, Pine R, Gennaro ML (2017) FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Prot 12(6):1245–1260. https://doi.org/10.1038/nprot.2017.039
Ashaolu TJ (2020) Immune boosting functional foods and their mechanisms: a critical evaluation of probiotics and prebiotics. Biomed Pharmacother 130:110625. https://doi.org/10.1016/j.biopha.2020.110625
Conterno L, Martinelli F, Tamburini M, Fava F, Mancini A, Sordo M, Tuohy K (2019) Measuring the impact of olive pomace enriched biscuits on the gut microbiota and its metabolic activity in mildly hypercholesterolaemic subjects. Eur J Nutr 58(1):63–81. https://doi.org/10.1007/s00394-017-1572-2
De Filippis F, Esposito A, Ercolini D (2022) Outlook on next-generation probiotics from the human gut. Cell Mol Life Sci 79(2):76. https://doi.org/10.1007/s00018-021-04080-6
D’hoe K, Conterno L, Fava F, Falony G, Vieira-Silva S, Vermeiren J, Raes J, (2018) Prebiotic wheat bran fractions induce specific microbiota changes. Front Microbiol 9:31. https://doi.org/10.3389/fmicb.2018.00031
Dinoto A, Fukiya S, Yokota A (2006) FISH-flow cytometry: a high-throughput molecular ecological analysis of intestinal microbiota. Jpn J Lact Acid Bact 17(2):110–117. https://doi.org/10.4109/jslab.17.110
Estruel-Amades S, Massot-Cladera M, Pérez-Cano FJ, Franch À, Castell M, Camps-Bossacoma M (2019) Hesperidin effects on gut microbiota and gut-associated lymphoid tissue in healthy rats. Nutrients 11(2):324. https://doi.org/10.3390/nu11020324
Ferrario M, Guerrero S (2017) Impact of a combined processing technology involving ultrasound and pulsed light on structural and physiological changes of Saccharomyces cerevisiae KE 162 in apple juice. Food Microbiol 65:83–94. https://doi.org/10.1016/j.fm.2017.01.012
Gibson GR, Hutkins R, Sanders ME, Prescott SL, Reimer RA, Salminen SJ, Reid G (2017) Expert consensus document: the International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol 14(8):491–502. https://doi.org/10.1038/nrgastro.2017.75
Gómez-Gallego C, Collado MC, Ilo T, Jaakkola UM, Bernal MJ, Periago MJ, Frias R (2012) Infant formula supplemented with polyamines alters the intestinal microbiota in neonatal BALB/cOlaHsd mice. J Nutr Biochem 23(11):1508–1513. https://doi.org/10.1016/j.jnutbio.2011.10.003
Gong J, Yang C (2012) Advances in the methods for studying gut microbiota and their relevance to the research of dietary fiber functions. Food Res Int 48(2):916–929. https://doi.org/10.1016/j.foodres.2011.12.027
Grimaldi R, Cela D, Swann JR, Vulevic J, Gibson GR, Tzortzis G, Costabile A (2017) In vitro fermentation of B-GOS: impact on faecal bacterial populations and metabolic activity in autistic and non-autistic children. FEMS Microbiol Ecol 93(2):fiw233. https://doi.org/10.1093/femsec/fiw233
Grześkowiak Ł, Grönlund MM, Beckmann C, Salminen S, von Berg A, Isolauri E (2012) The impact of perinatal probiotic intervention on gut microbiota: double-blind placebo-controlled trials in Finland and Germany. Anaerobe 18(1):7–13. https://doi.org/10.1016/j.anaerobe.2011.09.006
Grzeskowiak L, Collado MC, Mangani C, Maleta K, Laitinen K, Ashorn P, Salminen S (2012) Distinct gut microbiota in southeastern African and northern European infants. J Ped Gastroenterol Nutr 54(6):812–816. https://doi.org/10.1097/MPG.0b013e318249039c
Habtewold T, Duchateau L, Christophides GK (2016) Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes. Parasit Vectors 9(1):1–10. https://doi.org/10.1186/s13071-016-1438-0
Harmsen HJ, Raangs GC, He T, Degener JE, Welling GW (2002) Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl Environ Microbiol 68(6):2982–2990. https://doi.org/10.1128/AEM.68.6.2982-2990.2002
Hold GL, Schwierts A, Aminov RI, Blaut M, Flint HJ (2003) Oligonucleotide probes that detect quantitatively significant groups of butyrate-producing bacteria in human feces. Appl Environ Microbiol 69(7):4320–4324. https://doi.org/10.1128/AEM.69.7.4320-4324.2003
Kempf VA, Trebesius K, Autenrieth IB (2000) Fluorescent in situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 38(2):830–838. https://doi.org/10.1128/JCM.38.2.830-838.2000
Kumari R, Ahuja V, Paul J (2013) Fluctuations in butyrate-producing bacteria in ulcerative colitis patients of North India. World J Gastroenterol 19(22):3404. https://doi.org/10.3748/wjg.v19.i22.3404
Lahtinen SJ, Forssten S, Aakko J, Granlund L, Rautonen N, Salminen S, Ouwehand AC (2012) Probiotic cheese containing Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus NCFM® modifies subpopulations of fecal lactobacilli and Clostridium difficile in the elderly. Age 34(1):133–143. https://doi.org/10.1007/s11357-011-9208-6
Lennon J, Jones ST (2011) Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat Rev Microbiol 9:119–130. https://doi.org/10.1038/nrmicro2504
Lin L, Song J, Du Y, Wu Q, Gao J, Song Y, Wang W (2020) Quantification of bacterial metabolic activities in the gut by d-amino acid-based in vivo labeling. Angewandte Chemie International Edition 59(29):11923–11926. https://doi.org/10.1002/anie.202004703
Liu L, Poveda C, Jenkins PE, Walton GE (2021) An in vitro approach to studying the microbial community and impact of pre and probiotics under anorexia nervosa related dietary restrictions. Nutrients 13(12):4447. https://doi.org/10.3390/nu13124447
Loo YT, Howell K, Chan M, Zhang P, Ng K (2020) Modulation of the human gut microbiota by phenolics and phenolic fiber-rich foods. Compr Rev Food Sci Food Saf 19(4):1268–1298. https://doi.org/10.1111/1541-4337.12563
Martín-Peláez S, Camps-Bossacoma M, Massot-Cladera M, Rigo-Adrover M, Franch À, Pérez-Cano FJ, Castell M (2017) Effect of cocoa’s theobromine on intestinal microbiota of rats. Mol Nutr Food Res 61(10):1700238. https://doi.org/10.1002/mnfr.201700238
Martín-Peláez S, Mosele JI, Pizarro N, Farràs M, De la Torre R, Subirana I, Fitó M (2017) Effect of virgin olive oil and thyme phenolic compounds on blood lipid profile: implications of human gut microbiota. Eur J Nutr 56(1):119–131. https://doi.org/10.1007/s00394-015-1063-2
Massot-Cladera M, Pérez-Berezo T, Franch A, Castell M, Pérez-Cano FJ (2012) Cocoa modulatory effect on rat faecal microbiota and colonic crosstalk. Arch Biochem Biophys 527(2):105–112. https://doi.org/10.1016/j.abb.2012.05.015
Massot-Cladera M, Abril-Gil M, Torres S, Franch A, Castell M, Pérez-Cano FJ (2014) Impact of cocoa polyphenol extracts on the immune system and microbiota in two strains of young rats. Brit J Nutr 112(12):1944–1954. https://doi.org/10.1017/S0007114514003080
McKinnon KM (2018) Flow cytometry: an overview. Curr Prot Immunol 120(1):5–1. https://doi.org/10.1002/cpim.40
Medeiros VPB, de Souza EL, de Albuquerque TMR, da Costa Sassi CF, dos Santos Lima M, Sivieri K, Magnani M (2021) Freshwater microalgae biomasses exert a prebiotic effect on human colonic microbiota. Algal Res 60:102547. https://doi.org/10.1016/j.algal.2021.102547
Menezes FNDD, de Melo FHC, Vieira ARS, Almeida ETC, Lima MS, Aquino JS, de Souza EL (2020) Acerola (Malpighia glabra L.) and guava (Psidium guayaba L.) industrial processing by-products stimulate probiotic Lactobacillus and Bifidobacterium growth and induce beneficial changes in colonic microbiota. J Appl Microbiol 130:323–1336. https://doi.org/10.1111/jam.14824
Menezes FNDD, da Cruz Almeida ET, da Silva Vieira AR, Aquino JS, dos Santos Lima M, Magnani M, de Souza EL (2021) Impact of cashew (Anacardium occidentale L.) by-product on composition and metabolic activity of human colonic microbiota in vitro indicates prebiotic properties. Curr Microbiol 78(6):2264–2274
Mota de Carvalho NM, Walton GE, Poveda CG, Silva SN, Amorim M, Madureira AR, Jauregi P (2019) Study of in vitro digestion of Tenebrio molitor flour for evaluation of its impact on the human gut microbiota. J Funct Foods 59:101–109. https://doi.org/10.1016/j.jff.2019.05.024
Moter A, Göbel UB (2000) Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J Microbiol Meth 41(2):85–112. https://doi.org/10.1016/S0167-7012(00)00152-4
Moter A, Leist G, Rudolph R, Schrank K, Choi BK, Wagner M, Göbel UB (1998) Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions. Microbiology 144(9):2459–2467. https://doi.org/10.1099/00221287-144-9-2459
Munukka E, Wiklund P, Pekkala S, Völgyi E, Xu L, Cheng S, Cheng S (2012) Women with and without metabolic disorder differ in their gut microbiota composition. Obesity 20(5):1082–1087. https://doi.org/10.1038/oby.2012.8
Munukka E, Pekkala S, Wiklund P, Rasool O, Borra R, Kong L, Cheng S (2014) Gut-adipose tissue axis in hepatic fat accumulation in humans. J Hepatol 61(1):132–138. https://doi.org/10.1016/j.jhep.2014.02.020
Namsolleck P, Thiel R, Lawson P, Holmstrøm K, Rajilic M, Vaughan EE, Blaut M (2004) Molecular methods for the analysis of gut microbiota. Microb Ecol Health Dis 16(2–3):71–85. https://doi.org/10.1080/08910600410032367
Newland GA, Gibson GR, Jackson FL, Wijeyesekera A (2021) Assessment of stool collection and storage conditions for in vitro human gut model studies. J Microbiol Methods 185:106230. https://doi.org/10.1016/j.mimet.2021.106230
Novakova J, Džunková M, Musilova S, Vlkova E, Kokoska L, Moya A, D’Auria G (2014) Selective growth-inhibitory effect of 8-hydroxyquinoline towards Clostridium difficile and Bifidobacterium longum subsp. longum in co-culture analysed by flow cytometry. J Med Microbiol 63(12):1663–1669. https://doi.org/10.1099/jmm.0.080796-0
Owolabi IO, Dat-arun P, Yupanqui CT, Wichienchot S (2020) Gut microbiota metabolism of functional carbohydrates and phenolic compounds from soaked and germinated purple rice. J Funct Foods 66:103787. https://doi.org/10.1016/j.jff.2020.103787
Pärtty A, Luoto R, Kalliomäki M, Salminen S, Isolauri E (2013) Effects of early prebiotic and probiotic supplementation on development of gut microbiota and fussing and crying in preterm infants: a randomized, double-blind, placebo-controlled trial. J Pediatr 163(5):1272–1277. https://doi.org/10.1016/j.jpeds.2013.05.035
Qiao Y, Sun J, Xia S, Tang X, Shi Y, Le G (2014) Effects of resveratrol on gut microbiota and fat storage in a mouse model with high-fat-induced obesity. Food Funct 5(6):1241–1249. https://doi.org/10.1039/c3fo60630a
Robben C, Fister S, Witte AK, Schoder D, Rossmanith P, Mester P (2018) Induction of the viable but non-culturable state in bacterial pathogens by household cleaners and inorganic salts. Sci Reports 8:1–9. https://doi.org/10.1038/s41598-018-33595-5
Rodrigues D, Walton G, Sousa S, Rocha-Santos TA, Duarte AC, Freitas AC, Gomes AM (2016) In vitro fermentation and prebiotic potential of selected extracts from seaweeds and mushrooms. LWT Food Sci Technol 73:131–139. https://doi.org/10.1016/j.lwt.2016.06.004
Rubbens P, Props R, Kerckhof FM, Boon N, Waegeman W (2021) Cytometric fingerprints of gut microbiota predict Crohn’s disease state. ISME J 15(1):354–358. https://doi.org/10.1038/s41396-020-00762-4
Schnell SA, Staines WA, Wessendorf MW (1999) Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47(6):719–730. https://doi.org/10.1177/002215549904700601
Thiel R, Blaut M (2005) An improved method for the automated enumeration of fluorescently labelled bacteria in human faeces. J Microbiol Methods 61(3):369–379. https://doi.org/10.1016/j.mimet.2004.12.014
Tong Y, Liao Z, Yang Q, Chen X, Zeng D, Yang C, Lv M (2021) Effect of a prawn (Macrobrachium rosenbergii)-plant eco-symbiotic culture system (PECS) on intestinal microbiota, organic acids, and ammonia. Aquac Rep 20:100647. https://doi.org/10.1016/j.aqrep.2021.100647
Wang H, Tang X, Cheserek MJ, Shi Y, Le G (2015) Obesity prevention of synthetic polysaccharides in high-fat diet fed C57BL/6 mice. J Funct Foods 17:563–574. https://doi.org/10.1016/j.jff.2015.06.012
Wang X, Gibson GR, Sailer M, Theis S, Rastall RA (2020) Prebiotics inhibit proteolysis by gut bacteria in a host diet-dependent manner: a three-stage continuous in vitro gut model experiment. Appl Environ Microbiol 86(10):e02730-e2819. https://doi.org/10.1128/AEM.02730-19
Wilkinson MG (2016) Flow cytometry in food microbiology: challenges, opportunities and progress to date. Lab Tech 417:722–728
Yap GC, Loo EXL, Aw M, Lu Q, Shek LPC, Lee BW (2014) Molecular analysis of infant fecal microbiota in an Asian at-risk cohort–correlates with infant and childhood eczema. BMC Res Notes 7(1):1–6. https://doi.org/10.1186/1756-0500-7-166
Acknowledgements
Authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil––Finance code 001) for the PhD and MSc. scholarships awarded to K.B. Sampaio and D.S. Nascimento, respectively.
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This study was partially funded by CAPES (Brazil) - Finance code 001.
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KBS and ELS did conceptualization. KBS, DNS, and ELS performed data curation, visualization, writing––original draft and formal analysis. EFG and ELS administrated the project and were involved in writing––review and editing; KBS, DNS, EFG, ELS contributed to resources. ELS did supervision.
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Sampaio, K.B., dos Santos Nascimento, D., Garcia, E.F. et al. An outlook on fluorescent in situ hybridization coupled to flow cytometry as a versatile technique to evaluate the effects of foods and dietary interventions on gut microbiota. Arch Microbiol 204, 469 (2022). https://doi.org/10.1007/s00203-022-03090-7
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DOI: https://doi.org/10.1007/s00203-022-03090-7