Annals of Microbiology

, Volume 69, Issue 1, pp 61–72 | Cite as

In-vitro characterization of potentially probiotic Lactobacillus strains isolated from human microbiota: interaction with pathogenic bacteria and the enteric cell line HT29

  • Yosra Gharbi
  • Imene Fhoula
  • Patricia Ruas-Madiedo
  • Najjari Afef
  • Abdellatif Boudabous
  • Miguel Gueimonde
  • Hadda-Imene OuzariEmail author
Original Article


Among the various tests commonly used for selecting probiotic microorganisms, the tolerance to gastrointestinal transit conditions remains being commonly used to evaluate the probiotic potential of the strains. Besides, the adhesion to epithelial cells and the competition with pathogens constitute significant traits for evaluating the colonization ability and functional performance of candidate strains. In this study, a total of 13 lactic acid bacteria strains isolated from human feces were first identified by biochemical tests and 16S rRNA gene sequencing, and then characterized in vitro for their tolerance to gastrointestinal conditions, hemolytic activity, and antibiotics sensibility. The isolates were identified as Lactobacillus fermentum (06), Lactobacillus rhamnosus (04), Lactobacillus plantarum (02), and Lactobacillus salivarius (01). The adhesion to epithelial cells HT29 was shown to be a strain-dependent character. L. fermentum 88 and L. plantarum 9, being the ones showing higher adhesion values. They were further characterized by determining their antimicrobial activity, hydrophobicity, co-aggregation, antioxidant activity, as well as the ability to inhibit the adhesion of pathogens to the human epithelial cell line HT29. Moreover, these two strains were able to reduce the adhesion of Escherichia coli to HT29 cells, although they failed for inhibiting the adhesion of other pathogens such as Cronobacter sakazaki or Salmonella enterica. These results point out the importance of considering the ecological fitness of the strains in selecting probiotic bacteria and the potential of some of the analyzed strains for the development of food products.


Lactobacillus Feces Probiotic HT29 Adhesion Inhibition of pathogens 


Funding information

This work was funded by the Tunisian Ministry of Higher Education and Scientific research (laboratory project LR03ES03) and the Department of Microbiology and Biochemistry of Dairy products, IPLA-CSIC, Spain.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Arboleya S, Ruas-Madiedo P, Margolles A, Solis G, Salminen S, de los Reyes-Gavilán C, Guiemonde M (2011) Characterization and in vitro properties of potentially probiotic Bifidobacterium strains isolated from breast-milk. Int J Food Microbiol 149:28–35CrossRefGoogle Scholar
  2. Bernardeau M, Vernous JP, Henri-Dubernet S, Guéguen M (2008) Safety assessment of dairy microorganisms: the Lactobacillus genus. Int J Food Microbiol 126:278–285CrossRefGoogle Scholar
  3. Bird (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16:6–21CrossRefGoogle Scholar
  4. Castagliuolo I, Galeazzi F, Ferrari S, Elli M, Brun P, Cavaggioni A, Tormen D, Sturniolo GC, Morelli L, Palu G (2005) Beneficial effect of auto-aggregating Lactobacillus crispatus on experimentally induced colitis in mice. FEMS Immunol Med Microbiol 43:197–204CrossRefGoogle Scholar
  5. Cesena C, Morelli L, Alander M, Siljander T, Tuomola E, Salminen S, Mattila-Sandholm T, Vilpponen-Salmela T, von Wright A (2001) Lactobacillus crispatus and its non-aggregating mutant in human colonization trials. J Dairy Sci 84:1001–1010CrossRefGoogle Scholar
  6. Clinical and Laboratory Standards Institute CLSI (2010) Performance standards for antimicrobial-susceptibility testing. 20th informational supplement. M 100-S20, Wayne, Pannsylvannia 30 (1)Google Scholar
  7. Collado MC, Gonzalez A, Gonzulez R, Hermandez M, Sanz X, Salimen S (2005) Adhesion of selected Bifidobacterium strains to human intestina mucus and the role of adhesion in enteropathogen exclusion. J Food Prot 68:2672–2678CrossRefGoogle Scholar
  8. Collado MC, Jalonen L, Meriluoto J, Salminen S (2006) Protection mechanism of probiotic combination against human pathogens: in vitro adhesion to human intestinal mucus. Asia Pac J Clin Nutr 15:570–575PubMedGoogle Scholar
  9. Collado MC, Meriluoto J, Salminen S (2007) In vitro analysis of probiotic strain combinations to inhibit pathogen adhesion to human intestinal mucus. Food Res Int 40:629–636CrossRefGoogle Scholar
  10. Daffonchio D, Borin S, Frova G, Manachini PL, Sorlini C (1998) PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis. Int J Syst Bacteriol 48(1):107–116CrossRefGoogle Scholar
  11. De Keersmaecker S, Verhoeven TL, Desair J, Marchal K, Vandleyden J, Nagy I (2006) Strong antimicrobial activity of Lactobacillus rhamnosus GG against Salmonella typhimurium is due to accumulation of lactic acid. FEMS Microbiol Lett 259:89–96CrossRefGoogle Scholar
  12. Dunne C, O’Mahony L, Murphy L, Thornton G, Morrissey D, O'Halloran S, Feeney M, Flynn S, Fitzgerald G, Daly C, Kiely B, O'Sullivan GC, Shanahan F, Collins JK (2001) In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am J Clin Nutr 73:386–392CrossRefGoogle Scholar
  13. FAO/WHO (2006) Probiotic in foods: health and nutritional properties and guidelines for evaluation, in FAO Food and Nutrition, pp 85Google Scholar
  14. Fujiwara S, Hashiba H, Hirota T, Forstner JF (2001) Inhibition of the binding of enterotoxigenic Escherichia coli Pb176 to human intestinal epithelial cell line HCT-8 by an extracellular protein fraction containing BIF of Bifidobacterium longum SBT2928: suggestive evidence of blocking of the binding receptor gangliotetraosylceramide on the cell surface. Int J Food Microbiol 67:97–106CrossRefGoogle Scholar
  15. Gilliand SE (1989) Acidophilus milk products a review of potential benefits to consumers. J Dairy Sci 72:2483–2494CrossRefGoogle Scholar
  16. Gobin I (2011) The protective role of lactic acid bacteria to systemic Salmonella enterica serotype typhimurium infection in mice. MSc thesis. Faculty of Food Technology and Biotehnology. University of ZagrebGoogle Scholar
  17. Gómez Zavaglia A, Kociubinski G, Pérez P, Disalvo E, De Antoni G (2002) Effect of bile on the lipid composition and surface properties of bifidobacteria. J Appl Microbiol 93:794–799CrossRefGoogle Scholar
  18. Granato D, Perotti F, Masserey I, Rouvet M, Golliard M, Servin A, Brassart D (1999) Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johsonii LaI to human enterocyte-like Caco-2 cells. Appl Environ Microbiol 65:1071–1077PubMedPubMedCentralGoogle Scholar
  19. Gueimonde M, Noriega L, Margolles A, de los Reyes-Gavilán CG, Salminen S (2005) Ability of Bifidobacterium strains with acquired resistance to bile to adhere to human intestinal mucus. Int J Food Microbiol 101:341–346CrossRefGoogle Scholar
  20. Gueimonde M, Jalonen L, He F, Hiramatsu M, Salminen S (2006) Adhesion and competitive inhibition and displacement of human enteropathogens by selected lactobacilli. Food Res Int 39:467–471CrossRefGoogle Scholar
  21. Gueimonde M, Sánchez B, de los Reyes-Gavilán CG, Margolles A (2013) Antibiotic resistance in probiotic bacteria. Front Microbiol 4:202Google Scholar
  22. Hammes WP, Vogel RF (1995) The genus Lactobacillus. In: Wood BJB, Holzapfel WH (eds) The genera of lactic acid bacteria. Blackie Academic and Professional, London, pp 19–54CrossRefGoogle Scholar
  23. Jacobsen CN, Rosenfeldt Nielsen V, Hayford AE, Møller PL, Michaelsen KF, Pærregaard A, Sandström B, de Tve M, Jakobsen M (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 65:4949–4956PubMedPubMedCentralGoogle Scholar
  24. Jankovic (2012) Aggregation ability of potential probiotic Lactobacillus plantarum strains. IJSER 6:19–24Google Scholar
  25. Kailasapathy K, Chin J (2000) Survival and therapeutic potential of probiotic organism with reference to Lactobacillus acidophilus and Bifidobacterium ssp. Immunol Cell Biol 78:80–88CrossRefGoogle Scholar
  26. Klare I, Konstabel C, Werner G, Huys G, Vankerckhoven V, Klare I, Konstabel C, Werner G, Huys G, Kahlmeter G, Hildebrandt B, Muller-Bertling S, Witte W, Goossens H (2007) Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use. J Antimicrob Chemother 59:900–912CrossRefGoogle Scholar
  27. Klayraung S, viernstein H, Sirithunyalug J, Okonogi S (2008) Probiotic properties of lactobacilli isolated from Thai traditional food. Scientia Pharmaceutica 76(3):485Google Scholar
  28. Klingbery TD, Axelsson L, Naterstad K, Elesser D, Budde BB (2005) Identification of potential probiotic starter, cultures for Scandinavia type fermented sausages. Int J Food Microbiol 105:419–431CrossRefGoogle Scholar
  29. Kochan P, Chmielarczyk A, Szymaniak L, Brykczynski M, Galant K, Zych A, Pakosz K, Giedrys-Kalemba S, Lenouvel E, Heczko PB (2011) Lactobacillus rhamnosus administration causes sepsis in a cardio surgical patient is the time right to revise probiotic safety guidelines? Clin Microbiol Infect 17(10):1589–1592CrossRefGoogle Scholar
  30. Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic S (2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94:981–987CrossRefGoogle Scholar
  31. Kotzamanidis C, Kourelis A (2010) Evaluation of adhesion capacity, cell surface traits and immonumodulatory activity of presumptive probiotic Lactobacillus strains. Int J Food Microbiol 15:154–163CrossRefGoogle Scholar
  32. Krulwich TA, Sachs G, Padan E (2001) Molecular aspects of bacterial pH sensing and homeostasis. Nat Rev Microbiol 9:330–343CrossRefGoogle Scholar
  33. Lema M, Williams L, Rao DR (2001) Reduction of fecal shedding of enterohemorrhagic Escherichia coli O157:H7 in lambs by feeding microbial feed supplement. Small Rumin Res 39:31–39CrossRefGoogle Scholar
  34. Lin MY, Chang FJ (2000) Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356. Dig Dis Sci 45(8):1617–1622CrossRefGoogle Scholar
  35. Lin MY, Yen CL (1999) Antioxidative ability of lactic acid bacteria. J Agric Food Chem 47:1460–1466CrossRefGoogle Scholar
  36. Liong M (2011) Probiotic properties of bifidobacteria and lactobacilli isolated from local dairy products. Ann Microbiol 62:1079–1087Google Scholar
  37. Liong MT, Shah NP (2005) Acid and bile tolerance and cholesterol removal ability of lactobacilli strains. J Dairy Sci 88:55–66CrossRefGoogle Scholar
  38. Mack DR, Michail S, Wet S (1999) Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression. Am J Physiol Cell Physiol 276:941–950Google Scholar
  39. Makras L, Triantafyllou V, Fayol-Messaoudi T, Adriany T, Zoumpopoulou G, Tsakalidou E, Servin A, De Vuyst L (2006) Kinetic analysis of antibacterial activity of probiotic lactobacilli towards Salmonella enteric serovar typhimurium reveals a role for lactic acid and other inhibitory compounds. Res Microbiol 157(3):241–247CrossRefGoogle Scholar
  40. Maldonado NC, Ruiz CS, Otero MC, Sesma F, Nader-Macías (2012) Lactic acid bacteria isolated from young calves e characterization and potential as probiotic. Res Vet Sci 92:342–349CrossRefGoogle Scholar
  41. Mannu L, Communian R, Scintu MF (2000) Mesophilic lactobacilli in Fiore Sardo cheese: PCR-identification and evolution during cheese ripening. Int Dairy J 10:383–389CrossRefGoogle Scholar
  42. Martin R, Jimenez E, Olivares M, Marin ML, Fernanderz L, Xaus J, Rodriguez JM (2006) Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother-child pair. Int J Food Microbiol 122:35–43CrossRefGoogle Scholar
  43. Mary H, Tina AV, Jeeja KJJ, Abiramy MR, Sajina N, JayaSree S (2012) Phytochemical analysis and anticancer activity of essential oil from Myristica fragrans. Int J Curr Pharm Res 2:188–198Google Scholar
  44. Nesser JR, Granato D, Rouvet M, Servin A, Teneberg S, Karlsson KA (2000) Lactobacillus johnsonii La1 shares carbohydrate-binding specificities with several enteropathogenic bacteria. Glycobiology 10:1193–1199CrossRefGoogle Scholar
  45. Owehand AC, Derrien M, de vos W, Tiihonen K, Rautonen N (2005) Prebiotics and other microbial substrates for gut functionality. Curr Opin Biotechnol 16(2):212–217CrossRefGoogle Scholar
  46. Perez PF, Minnaard Y, Disalvo EA, De Antoni GL (1998) Surface properties of bifidobacterial strains of human origin. Appl Environ Microbiol 64:21–26PubMedPubMedCentralGoogle Scholar
  47. Ramos CL, Thorsen L, Schwan RF (2013) Strain-specific probiotics properties of Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus brevis isolates from Brazilian food products. Food Microbiol 36(1):22–29CrossRefGoogle Scholar
  48. Reid G (1999) The scientific basis for probiotics strains of Lactobacillus. Appl Environ Microbiol 65:3763–3766PubMedPubMedCentralGoogle Scholar
  49. Reid G, McGroarty JA, Angotti R, Cook RL (1988) Lactobacillus inhibitor production against Escherichia coli and coaggregation ability with uropathogens. Can J Microbiol 34:344–351CrossRefGoogle Scholar
  50. Ruiz L, Margolles A, Sanchez B (2013) Bile resistance mechanisms in Lactobacillus and Bifidobacterium. Front Microbiol 4:389–396CrossRefGoogle Scholar
  51. Saraniya A, Jeevaratnam K (2014) In vitro probiotic evaluation of phytase producing Lactobacillus species isolated from Uttapam batter and their application in soy milk fermentation. J Food Sci Technol 52(9):5631–5640CrossRefGoogle Scholar
  52. Savage DC (1992) Growth phase cellular hydrophobicity and adhesion in vitro of lactobacilli colonizing the keratinizing gastric epithelium in the mouse. Appl Environ Microbiol 58(6):1992–1995PubMedPubMedCentralGoogle Scholar
  53. Sharpe ME (1979) Identification of lactic acid bacteria. In: Skinner FA, Lovelock DW (eds) Identification methods for microbiologists. Academic press, London, pp 233–259Google Scholar
  54. Shillinger U, Guigas C, Holzapfel WH (2005) In vitro adherence and other properties of lactobacilli used in probiotic yogurt-like products. Int Dairy J 15:1289–1297CrossRefGoogle Scholar
  55. Sommer P, Martin-Rouas C, Mettler E (1999) Influence of the adherent population level on biofilm population, structure and resistance to chlorination. Food Microbiol 16:503–515CrossRefGoogle Scholar
  56. Tagg JR, McGiven AR (1971) Assay system for bacteriocins. Appl Microbiol 21(5):1953–1975Google Scholar
  57. Uugantsetseg E, Batjargal B (2014) Antioxidant activity of probiotic lactic acid bacteria isolated from Mongolian airag. Mongolian J Chem 15:73–78Google Scholar
  58. Valcarce MB, Busalmen JP, Sa’nchez SR (2002) The influence of the surface condition on the adhesion of Pseudomonas fluorescens (ATCC 17552) to copper and aluminium brass. Int Biodeterior Biodegrad 50:61–66CrossRefGoogle Scholar
  59. Velikova PV, Blagoeva GI, Gotcheva VG, Petrova PM (2014) Novel Bulgarian Lactobacillus strains ferment prebiotic carbohydrates. J BioSci Biotech 55–60Google Scholar
  60. Wouters JTM, Ayad EHE, Hugenholtz J, Smit G (2002) Microbes from raw milk for fermented dairy products. Int Dairy J 12:91–109CrossRefGoogle Scholar
  61. Xanthopoulos V, Litopoulou-Tzanetaki E, Tzanetakis N (2000) Characterization of Lactobacillus isolates from infant faeces as dietary adjuncts. Food Microbiol 17(2):205–215Google Scholar
  62. Zhou JS, Pillidge CJ, Gopal PK, Gill HS (2005) Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains. Int J Food Microbiol 98:211–217CrossRefGoogle Scholar
  63. Zivkovic M, Hidalgo-Cantabrana C, Kojic M, Gueimonde M, Golic N, Ruas-Madiedo P (2015) Capability of exopolysaccharide-producing Lactobacillus paraplantarum BGCG11 and its non-producing isogenic strain NB1, to counteract the effect of enteropathogens upon the epithelial cell line HT29-MTX. Food Res Int 74:199–207CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Authors and Affiliations

  1. 1.Faculté des Science de Tunis, LR03ES03 Laboratoire Microorganismes et Biomolécules ActivesUniversité de Tunis El ManarTunisTunisie
  2. 2.Departamento de Microbiología y Bioquímica de Productos LácteosInstituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC)VillaviciosaSpain

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