Abstract
Probiotic bacterial adhesion to the epithelial cell is a composite process and in vivo adhesion studies can be strengthened with the improved in vitro models for preliminary screening of potentially adherent strains. With this rationale, the study aimed is the first report to demonstrate the colonizing efficiency of probiotic Bacillus licheniformis MCC 2514 in comparison to Bifidobacterium breve NCIM 5671on HT-29 cell line. B. licheniformis (54.28 ± 0.99%) and Bif. breve (70.23 ± 0.85%) adhered in a higher percentage on fibronectin and mucin, respectively. However, the adhesion was higher for B. licheniformis when compared to Bif. breve. In adhesion score, B. licheniformis obtained about 138.85 ± 12.32, whereas Bif. breve got the score of 43.05 ± 9.12. The same trend continued in the adhesion percentage study, where B. licheniformis adhered 75.5 ± 5.2%, higher than Bif. breve which adhered 32.66 ± 3.2%. In invasion assay, both the bacteria significantly decreased the colonization of the pathogen Kocuria rhizophila ATCC 9341 about 97.32 ± 0.81% in the competitive assay, 97.87 ± 0.73% in exclusion assay and 82.19 ± 2.51% in displacement assay. The cytotoxicity effects of the test bacterial strains against HT-29 cell line through MTT assay determined no viability loss in the treated cells. Therefore, the data obtained from the in vitro studies showed that both B. licheniformis and Bif. breve had shown significantly good invasion on pathogen and adhesion capacity on HT-29 cell line.
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References
Achi SC, Halami PM (2019) In vitro comparative analysis of probiotic and functional attributes of indigenous isolates of Bifidobacteria. Curr Microbiol 76:304–311. https://doi.org/10.1007/s00284-018-1615-9
Archer AC, Halami PM (2015) Probiotic attributes of Lactobacillus fermentum isolated from human feces and dairy products. Appl Microbiol Biotechnol 99:8113–8123. https://doi.org/10.1007/s00253-015-6679-x
Ayala FR, Bauman C, Bartolini M et al (2017) Transcriptional regulation of adhesive properties of Bacillus subtilis to extracellular matrix proteins through the fibronectin-binding protein YloA. Mol Microbiol 104:804–821. https://doi.org/10.1111/mmi.13666
Azimirad M, Alebouyeh M, Naji T (2017) Inhibition of lipopolysaccharide-induced interleukin 8 in human adenocarcinoma cell line HT-29 by spore probiotics: B. coagulans and B. subtilis (natto). Probiotics Antimicrob Proteins 9:56–63. https://doi.org/10.1007/s12602-016-9234-x
Baccigalupi L, Di A, Parlato M et al (2005) Small surface-associated factors mediate adhesion of a food-isolated strain of Lactobacillus fermentum to Caco-2 cells. Res Microbiol 156:830–836. https://doi.org/10.1016/j.resmic.2005.05.001
Bernet MF, Brassart D, Neeser JR, Servin AL (1994) Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 35:483–489. https://doi.org/10.1136/gut.35.4.483
Bernet MF, Brassart D, Neeser JR, Servin AL (1993) Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions. Appl Environ Microbiol 59:4121–4128. https://doi.org/10.1128/aem.59.12.4121-4128.1993
Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Lett Appl Microbiol 31:438–442. https://doi.org/10.1046/j.1365-2672.2000.00845.x
Duary RK, Rajput YS, Batish VK, Grover S (2011) Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells. Indian J Med Res 134:664–671. https://doi.org/10.4103/09715916.90992
Elshaghabee FMF, Rokana N, Gulhane RD et al (2017) Bacillus as potential probiotics: status, concerns, and future perspectives. Front Microbiol 8:1–15. https://doi.org/10.3389/fmicb.2017.01490
Erdem L, Avelino F, Xicohtencatl-cortes J, Giro JA (2007) Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli. J Bacteriol 189:7426–7435. https://doi.org/10.1128/JB.00464-07
Fao J, Working WHO, Report G, et al (2002) Guidelines for the evaluation of probiotics in food. 1–11
Gagnon M, Berner AZ, Chervet N et al (2013) Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion. J Microbiol Methods 94:274–279. https://doi.org/10.1016/j.mimet.2013.06.027
Hill C, Guarner F, Reid G et al (2014) Expert consensus document: The international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev GastroenterolHepatol 11:506–514. https://doi.org/10.1038/nrgastro.2014.66
Hong HA, Huang JM, Khaneja R et al (2008) The safety of Bacillus subtilis and Bacillus indicus as food probiotics. J Appl Microbiol 105:510–520. https://doi.org/10.1111/j.1365-2672.2008.03773.x
Inturri R, Molinaro A, Di Lorenzo F et al (2017) Chemical and biological properties of the novel exopolysaccharide produced by a probiotic strain of Bifidobacterium longum. CarbohydrPolym 174:1172–1180. https://doi.org/10.1016/j.carbpol.2017.07.039
Ishibashi N, Yamazaki S (2001) Probiotics and safety. Am J ClinNutr 73:465s–470s. https://doi.org/10.1093/ajcn/73.2.465s
Jacobsen CN, Nielsen VR, Hayford AE et al (1999) Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl Environ Microbiol 65:4949–4956. https://doi.org/10.1128/aem.65.11.4949-4956.1999
Johansson MEV, Phillipson M, Petersson J et al (2008) The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci U S A 105:15064–15069. https://doi.org/10.1073/pnas.0803124105
Juge N (2012) Microbial adhesins to gastrointestinal mucus. Trends Microbiol 20:30–39. https://doi.org/10.1016/j.tim.2011.10.001
Kerneis S, Bilge SS, Fourel V et al (1991) Use of purified F1845fimbrial adhesin to study localization and expression of receptors for diffusely adhering Escherichia coli during enterocytic differentiation of human colon carcinoma cell lines HT-29 and Caco-2 in culture. Infect Immun 59:4013–4018. https://doi.org/10.1128/iai.59.11.4013-4018.1991
Kumar RS, Kanmani P, Yuvaraj N et al (2011) Lactobacillus plantarumAS1 binds to cultured human intestinal cell line HT-29 and inhibits cell attachment by enterovirulent bacterium Vibrio parahaemolyticus. Lett Appl Microbiol 53:481–487. https://doi.org/10.1111/j.1472-765X.2011.03136.x
Lee YK, Salminen S (1995) The coming of age of probiotics. Trends Food Sci Technol 6:241–245. https://doi.org/10.1016/S0924-2244(00)89085-8
Lindell G, Gilshenan K, Carlstedt I (2008) Four modes of adhesion are used during Helicobacter pylori binding to human mucins in the oral and gastric niches. Helicobacter 13:81–93. https://doi.org/10.1111/j.1523-5378.2008.00587.x
Magalhães A, Reis CA, Magalhães A, Reis CA (2010) Helicobacter pylori adhesion to gastric epithelial cells is mediated by glycan receptors. Braz J Med Biol Res 43:611–618. https://doi.org/10.1590/S0100-879X2010007500049
Manjulata S, Kurrey NK, Halami PM (2018) In vitro anti-inflammatory activity among probiotic Lactobacillus species isolated from fermented foods. J Funct Foods 47:19–27. https://doi.org/10.1016/j.jff.2018.05.036
Marraffini LA, Dedent AC, Schneewind O (2006) Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Bio Rev 70:192–221. https://doi.org/10.1128/MMBR.70.1.192
Neish AS (2009) Microbes in gastrointestinal health and disease. Gastroenterology 136:65–80. https://doi.org/10.1053/j.gastro.2008.10.080
Ouwehand A, Vesterlund S (2003) Health aspects of probiotics. IDrugs 6:573–580 (PMID:12811680)
Palva A (2005) Surface layers and their applications. FEMS Microbiol Rev 29:511–529. https://doi.org/10.1016/j.femsre.2005.04.003
Sen PJ, Tsai WC, Chou CC (2001) Surface characteristics of Bacillus cereus and its adhesion to stainless steel. Int J Food Microbiol 65:105–111. https://doi.org/10.1016/S0168-1605(00)00517-1
Rani RP, Anandharaj M, Hema S et al (2016) Purification of antilisterial peptide (Subtilosin A) from novel Bacillus tequilensisFR9 and demonstrate their pathogen invasion protection ability using human carcinoma cell line. Front Microbiol 7:1910. https://doi.org/10.3389/fmicb.2016.01910
Romberger DJ (1997) Fibronectin. Int J Biochem Cell Biol 29:939–943. https://doi.org/10.1016/S1357-2725(96)00172-0
Roos S, Jonsson H (2002) A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components. Microbiology 148:433–442. https://doi.org/10.1099/00221287-148-2-433
Sánchez B, Arias S, Chaignepain S et al (2009) Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin. Microbiology 155:1708–1716. https://doi.org/10.1099/mic.0.025288-0
Shobharani P, Halami PM (2014) Cellular fatty acid profile and H + -ATPase activity to assess acid tolerance of Bacillus sp. for potential probiotic functional attributes. Appl Microbiol Biot 98:9045–9058. https://doi.org/10.1007/s00253-014-5981-3
Swain MR, Anandharaj M, Ray RC, Parveen Rani R (2014) Fermented fruits and vegetables of asia: a potential source of probiotics. Biotechnol Res Int 2014:1–19. https://doi.org/10.1155/2014/250424
Van TML, Miller MJ (2011) Lactobacillus adhesion to mucus. Nutrients 3:613–636. https://doi.org/10.3390/nu3050613
Van Klinken BJW, Dekker J, Buller HA, Einerhand AWC (1995) Mucin gene structure and expression: protection vs. adhesion. Am J Physiol - Gastrointest Liver Physiol 269:613–629. https://doi.org/10.1152/ajpgi.1995.269.5.g613
Xi T, Chen YM, Zheng Y et al (2009) In vitro cytotoxicity of bacterial cellulose scaffolds used for tissue-engineered bone. J BioactCompatPolym 24:137–145. https://doi.org/10.1177/0883911509102710
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The authors would like to thank the Director, CSIR-CFTRI, for the continuous support. The authors acknowledge the work carried under DST funded project (sanction no. EEQ/2016/000310), Department of Science and Technology, New Delhi, and RHS acknowledge the grant of ICMR- senior research fellowship, ICMR, New Delhi for the financial support.
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The research work carried under DST funded project (sanction no. EEQ/2016/000310), Department of Science and Technology, New Delhi.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by HSR. HSR wrote the first draft of the manuscript and PMH commented on previous versions of the manuscript. HSR and PMH read and approved the final manuscript.
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Rohith, H.S., Halami, P.M. In vitro validation studies for adhesion factor and adhesion efficiency of probiotic Bacillus licheniformis MCC 2514 and Bifidobacterium breve NCIM 5671 on HT-29 cell lines. Arch Microbiol 203, 2989–2998 (2021). https://doi.org/10.1007/s00203-021-02257-y
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DOI: https://doi.org/10.1007/s00203-021-02257-y