Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting

  • Yang, Zhen-quan (College of Food Science and Engineering, Yangzhou University) ;
  • Xue, Yu (College of Food Science and Engineering, Yangzhou University) ;
  • Rao, Sheng-qi (College of Food Science and Engineering, Yangzhou University) ;
  • Zhang, Mi (College of Food Science and Engineering, Yangzhou University) ;
  • Gao, Lu (College of Food Science and Engineering, Yangzhou University) ;
  • Yin, Yong-qi (College of Food Science and Engineering, Yangzhou University) ;
  • Chen, Da-wei (Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University) ;
  • Zhou, Xiao-hui (Department of Pathobiology and Veterinary Science, University of Connecticut) ;
  • Jiao, Xin-an (Jiangsu Key Laboratory of Zoonosis)
  • Received : 2017.05.22
  • Accepted : 2017.09.14
  • Published : 2017.11.28
Download PDF
⟨ Previous Next ⟩

Abstract

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

Keywords

References

  1. Saxelin M, Tykkynen S, Mattila-Sandholm T, de Vos WM. 2005. Probiotic and other functional microbes: from markets to mechanisms. Curr. Opin. Biotechnol. 16: 204-211. https://doi.org/10.1016/j.copbio.2005.02.003
  2. Doron S, Snydman DR, Gorbach SL. 2005. Lactobacillus GG:bacteriology and clinical applications. Gastroenterol. Clin. North Am. 34: 483-498.
  3. Pirarat N, Kobayashi T, Katagiri T, Maita M, Endo M. 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet. Immunol. Immunopathol. 113: 339-347. https://doi.org/10.1016/j.vetimm.2006.06.003
  4. Hojsak I, Snovak N, Abdovic S, Szajewska H, Misak Z, Kolacek S. 2010. Lactobacillus GG in the prevention of gastrointestinal and respiratory tract infections in children who attend day care centers: a randomized, double-blind, placebo-controlled trial. Clin. Nutr. 29: 312-316. https://doi.org/10.1016/j.clnu.2009.09.008
  5. Goldin BR, Gorbach SL, Saxelin M, Barakat S, Gualtieri L, Salminen S. 1991. Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. Dig. Dis. Sci. 37: 121-128.
  6. Tuomola EM, Ouwehand AC, Salminen SJ. 1999. The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus. FEMS Immunol. Med. Microbiol. 26: 137-142. https://doi.org/10.1111/j.1574-695X.1999.tb01381.x
  7. Alander M, Satokari R, Korpela R, Saxelin M, Vilpponen-Salmela T, Mattila-Sandholm T, et al. 1999. Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl. Environ. Microbiol. 65: 351-354.
  8. Kankainen M, Paulin L, Tynkkynen S, von Ossowski I, Reunanen J, Partanen P, et al. 2009. Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein. Proc. Natl. Acad. Sci. USA 106: 17193-17198. https://doi.org/10.1073/pnas.0908876106
  9. Kant R, Rintahaka J, Yu X, Sigvart-Mattila P, Paulin L, Mecklin JP, et al. 2014. A comparative pan-genome perspective of niche-adaptable cell-surface protein phenotypes in Lactobacillus rhamnosus. PLoS One 9: e102762. https://doi.org/10.1371/journal.pone.0102762
  10. Reunanen J, von Ossowski I, Hendrickx APA, Palva A, de Vos WM. 2012. Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG. Appl. Environ. Microbiol. 78: 2337-2344. https://doi.org/10.1128/AEM.07047-11
  11. Markowicz C, Olejnik-Schmidt A, Borkowska M, CSchmidt MT. 2014. SpaCBA sequence instability and its relationship to the adhesion efficiency of Lactobacillus casei group isolates to Caco-2 cells. Acta Biochim. Pol. 61: 341-347.
  12. Lebeer S, Claes I, Tytgat HLP, Verhoeven TLA, Marien E, von Ossowski I, et al. 2012. Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells. Appl. Environ. Microbiol. 78: 185-193. https://doi.org/10.1128/AEM.06192-11
  13. Bullens DM, Vanderleyden J, Lebeer S. 2015. Piliation of Lactobacillus rhamnosus GG promotes adhesion, phagocytosis, and cytokine modulation in macrophages. Appl. Environ. Microbiol. 81: 2050-2062. https://doi.org/10.1128/AEM.03949-14
  14. Douillard FP, Ribbera A, Kant R, Pietila TE, Jarvinen HM, Messing M, et al. 2013. Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG. PLoS Genet. 9: 535-554.
  15. von Ossowski I, Reunanen J, Satokari R, Vesterlund S, Kankainen M, Huhtinen H, et al. 2011. Mucosal adhesion properties of the probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED pilin subunits. Appl. Environ. Microbiol. 76: 2049-2057.
  16. Grzeskowiak L, Isolauri E, Salminen S, Gueimonde M. 2011. Manufacturing process influences properties of probiotic bacteria. Br. J. Nutr. 105: 887-894. https://doi.org/10.1017/S0007114510004496
  17. Belyi YF, Varfolomeeva NA, Tartakovskii IS. 1995. As imple colony-blot method for identification of Listeria in food samples. Med. Microbiol. Immunol. 184: 105-108.
  18. Wieckowska-Szakiel M, Bubert A, Rozalski M, Krajewska U, Rudnicka W, Rozalska B. 2002. Colony-blot assay with antip60 antibodies as a method for quick identification of Listeria in food. Int. J. Food Microbiol. 72: 63-71. https://doi.org/10.1016/S0168-1605(01)00606-7
  19. Duez H, Pelletier C, Cools S, Aissi E, Cayuela C, Gavini F, et al. 2000. Colony immunoblotting method for quantitative detection of a Bifidobacterium animalis probiotic strain in human faeces. J. Appl. Microbiol. 88: 1019-1027. https://doi.org/10.1046/j.1365-2672.2000.01073.x
  20. Moe GR, Zuno-Mitchell P, Lee SS, Lucas AH, Granoff DM. 2001. Functional activity of anti-neisserial surface protein A monoclonal antibodies against strains of Neisseria meningitidis serogroup B. Infect. Immun. 69: 3762-3771. https://doi.org/10.1128/IAI.69.6.3762-3771.2001
  21. Palumbo JD, Borucki MK, Mandrell RE, Gorski L. 2003. Serotyping of Listeria monocytogenes by enzyme-linked immunosorbent assay and identification of mixed-serotype cultures by colony immunoblotting. J. Clin. Microbiol. 41: 564-571. https://doi.org/10.1128/JCM.41.2.564-571.2003
  22. Wei YF, Yang ZQ, Gao L, Rao SQ, Yin YQ, Fang WM, et al. 2016. Expression, antibody production and species specificity of SpaA pilin subunit from Lactobacillus rhamnosus. Microbiol. China 43: 1288-1294.
  23. Rao SQ, Zang XY, Yang ZQ, Gao L, Yin YQ, Fang WM. 2016. Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELPSUMO dual fusion system. Protein Expr. Purif. 118: 113-119. https://doi.org/10.1016/j.pep.2015.11.005
  24. Lei Z, Anand A, Mysore KS, Sumner LW. 2007. Electroelution of intact proteins from SDS-PAGE gels and their subsequent MALDI-TOF MS analysis. Methods Mol. Biol. 355: 353-363.
  25. Chao SH, Tomii Y, Watanabe K, Tsai YC. 2008. Diversity of lactic acid bacteria in fermented brines used to make stinky tofu. Int. J. Food Microbiol. 123: 134-141. https://doi.org/10.1016/j.ijfoodmicro.2007.12.010
  26. Gu R, Yang Z, Cai J, Li Z, Chen S, Luo Z. 2008. Analysis of different Lactobacillus rhamnosus by random amplified polymorphic DNA. Acta Microbiol. Sin. 48: 426-431.
  27. Nueno-Palop C, Narbad A. 2011. Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut. Int. J. Food Microbiol. 145: 390-394. https://doi.org/10.1016/j.ijfoodmicro.2010.12.029
  28. Rahman MM, Kim WS, Kumura H, Shimazaki K. 2008. Autoaggregation and surface hydrophobicity of bifidobacteria. World J. Microbiol. Biotechnol. 24: 1593-1598. https://doi.org/10.1007/s11274-007-9650-x
  29. Caggia C, de Angelis M, Pitino I, Pino A, Randazzo CL. 2015. Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano cheeses. Food Microbiol. 50: 109-117. https://doi.org/10.1016/j.fm.2015.03.010
  30. Douillard FP, Ribbera A, Jarvinen HM, Kant R, Pietila TE, Randazzo C, et al. 2013. Comparative genomic and functional analysis of Lactobacillus casei and Lactobacillus rhamnosus strains marketed as probiotics. Appl. Environ. Microbiol. 79: 1923-1933. https://doi.org/10.1128/AEM.03467-12
  31. Munoz-Provencio D, Rodriguez-Diaz J, Collado MC, Langella P, Bermudez-Humaran LG, Monedero V. 2012. Functional analysis of the Lactobacillus casei BL23 sortases. Appl. Environ. Microbiol. 78: 8684-8693. https://doi.org/10.1128/AEM.02287-12
  32. Toh H, Oshima K, Nakano A, Takahata M, Murakami M, Takaki T, et al. 2013. Genomic adaptation of the Lactobacillus casei group. PLoS One 8: e75073. https://doi.org/10.1371/journal.pone.0075073
  33. Lena MD, Quero GM, Santovito E, Verran J, de Angelis M, Fusco VA. 2015. Selective medium for isolation and accurate enumeration of Lactobacillus casei-group lactobacilli in probiotic milks and dairy products. Int. Dairy J. 47: 27-36. https://doi.org/10.1016/j.idairyj.2015.01.018
  34. Huang CJ, Lee FL. 2011. The dnaK gene as a molecular marker for the classification and discrimination of the Lactobacillus casei group. Antonie Van Leeuwenhoek 99: 319-327. https://doi.org/10.1007/s10482-010-9493-6
  35. Ahrne S, Lonnermark E, Wold AE, Aberg N, Hesselmar B, Saalman R, et al. 2005. Lactobacilli in the intestinal microbiota of Swedish infants. Microb. Infect. 7: 1256-1262. https://doi.org/10.1016/j.micinf.2005.04.011
  36. Kirtzalidou E, Pramateftaki P, Kotsou M, Kyriacou A. 2011. Screening for lactobacilli with probiotic properties in the infant gut microbiota. Anaerobe 17: 440-443. https://doi.org/10.1016/j.anaerobe.2011.05.007
  37. Rohani M, Noohi N, Talebi M, Katouli M, Pourshafie MR. 2015. Highly heterogeneous probiotic Lactobacillus species in healthy Iranians with low functional activities. PLoS One 10: e0144467. https://doi.org/10.1371/journal.pone.0144467
  38. Gu R, Yang Z, Li Z, Chen S, Luo Z. 2008. Probiotic properties of lactic acid bacteria isolated from stool samples of longevous people in regions of Hotan, Xinjiang and Bama, Guangxi, China. Anaerobe 14: 313-317. https://doi.org/10.1016/j.anaerobe.2008.06.001
  39. Salminen MK, Tynkkynen S, Rautelin H, Saxelin M, Vaara M, Ruutu P, et al. 2002. Lactobacillus b acteremia during a r ap id increase in probiotic use of Lactobacillus rhamnosus GG in Finland. Clin. Infect. Dis. 35: 1155-1160. https://doi.org/10.1086/342912
  40. Conway PL. 1996. Selection criteria for probiotic microorganisms. Asia Pac. J. Clin. Nutr. 5: 10-14.
  41. van Tassell ML, Miller MJ. 2011. Lactobacillus adhesion to mucus. Nutrients 3: 613-636. https://doi.org/10.3390/nu3050613
  42. 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
  43. Pan WH, Li PL, Liu Z. 2006. The correlation between surface hydrophobicity and adherence of Bifidobacterium strains from centenarians' faeces. Anaerobe 12: 148-152. https://doi.org/10.1016/j.anaerobe.2006.03.001
  44. Saarela M, Mogensen G, Fonden R, Matto J, Mattila-Sandholm T. 2000. Probiotic bacteria: safety, functional and technological properties. J. Biotechnol. 84: 197-215. https://doi.org/10.1016/S0168-1656(00)00375-8

Cited by

  1. Electrochemical Magnetic Bead-Based Immunosensor for Rapid and Quantitative Detection of Probiotic Lactobacillus rhamnosus in Dairy Products vol.12, pp.5, 2019, https://doi.org/10.1007/s12161-019-01457-z
  2. N, O-codoped hierarchical porous graphitic carbon for electrochemical immunosensing of Lactobacillus rhamnosus GG vol.189, pp.1, 2017, https://doi.org/10.1007/s00604-021-05049-9