Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Genomic Characterization and Safety Assessment of Bifidobacterium breve BS2-PB3 as Functional Food

  • Kristin Talia Marbun (Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan) ;
  • Marcelia Sugata (Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan) ;
  • Jonathan Suciono Purnomo (Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan) ;
  • Dikson (Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan) ;
  • Samuel Owen Mudana (Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan) ;
  • Tan Tjie Jan (Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan) ;
  • Juandy Jo (Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan)
  • Received : 2023.11.21
  • Accepted : 2023.12.14
  • Published : 2024.04.28
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Abstract

Our group had isolated Bifidobacterium breve strain BS2-PB3 from human breast milk. In this study, we sequenced the whole genome of B. breve BS2-PB3, and with a focus on its safety profile, various probiotic characteristics (presence of antibiotic resistance genes, virulence factors, and mobile elements) were then determined through bioinformatic analyses. The antibiotic resistance profile of B. breve BS2-PB3 was also evaluated. The whole genome of B. breve BS2-PB3 consisted of 2,268,931 base pairs with a G-C content of 58.89% and 2,108 coding regions. The average nucleotide identity and whole-genome phylogenetic analyses supported the classification of B. breve BS2-PB3. According to our in silico assessment, B. breve BS2-PB3 possesses antioxidant and immunomodulation properties in addition to various genes related to the probiotic properties of heat, cold, and acid stress, bile tolerance, and adhesion. Antibiotic susceptibility was evaluated using the Kirby-Bauer disk-diffusion test, in which the minimum inhibitory concentrations for selected antibiotics were subsequently tested using the Epsilometer test. B. breve BS2-PB3 only exhibited selected resistance phenotypes, i.e., to mupirocin (minimum inhibitory concentration/MIC >1,024 ㎍/ml), sulfamethoxazole (MIC>1,024 ㎍/ml), and oxacillin (MIC >3 ㎍/ml). The resistance genes against those antibiotics, i.e., ileS, mupB, sul4, mecC and ramA, were detected within its genome as well. While no virulence factor was detected, four insertion sequences were identified within the genome but were located away from the identified antibiotic resistance genes. In conclusion, B. breve BS2-PB3 demonstrated a sufficient safety profile, making it a promising candidate for further development as a potential functional food.

Keywords

Acknowledgement

A portion of the data was presented at the International Conference of Fermented Food, Tangerang, Indonesia in November 2023. This work was supported by the Institute of Research and Community Service of Universitas Pelita Harapan (P-14-FaST/VIII/2022 and P-01-FaST/I/2023).

References

  1. Kandasamy S, Yoo J, Yun J, Lee KH, Kang HB, Kim JE, et al. 2022. Probiogenomic in-silico analysis and safety assessment of Lactiplantibacillus plantarum DJF10 strain isolated from Korean raw milk. Int. J. Mol. Sci. 23: 14494.
  2. FAO/WHO. 2001. Report of a joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Cordoba, Argentina: Food and Agricultural Organization of the United Nations, World Health Organization.
  3. Reuter G. 2001. The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and succession. Curr. Issues Intest. Microbiol. 2: 43-53.
  4. Latif A, Shehzad A, Niazi S, Zahid A, Ashraf W, Iqbal MW, et al. 2023. Probiotics: mechanism of action, health benefits and their application in food industries. Front. Microbiol. 14: 1216674.
  5. Chen J, Chen X, Ho CL. 2021. Recent development of probiotic Bifidobacteria for treating human diseases. Front. Bioeng. Biotechnol. 9: 770248.
  6. Bozzi Cionci N, Baffoni L, Gaggia F, Di Gioia D. 2018. Therapeutic microbiology: the role of Bifidobacterium breve as food supplement for the prevention/treatment of paediatric diseases. Nutrients 10: 1723.
  7. Thibault H, Aubert-Jacquin C, Goulet O. 2004. Effects of long-term consumption of a fermented infant formula (with Bifidobacterium breve c50 and Streptococcus thermophilus 065) on acute diarrhea in healthy infants. J. Pediatr. Gastroenterol. Nutr. 39: 147-152. https://doi.org/10.1002/j.1536-4801.2004.tb00786.x
  8. Wong CB, Iwabuchi N, Xiao J-z. 2019. Exploring the science behind Bifidobacterium breve M-16V in infant health. Nutrients 11: 1724.
  9. Choi IY, Kim J, Kim SH, Ban OH, Yang J, Park MK. 2021. Safety evaluation of Bifidobacterium breve IDCC4401 isolated from infant feces for use as a commercial probiotic. J. Microbiol. Biotechnol. 31: 949-955. https://doi.org/10.4014/jmb.2103.03041
  10. Shori AB. 2022. Application of Bifidobacterium spp. In beverages and dairy food products: an overview of survival during refrigerated storage. Food Sci. Technol. 42: e41520.
  11. Linares DM, Gomez C, Renes E, Fresno J, Tornadijo M, Ross RP, et al. 2017. Lactic acid bacteria and Bifidobacteria with potential to design natural biofunctional health-promoting dairy foods. Front. Microbiol. 8: 846.
  12. Xiao J, Katsumata N, Bernier F, Ohno K, Yamauchi Y, Odamaki T, et al. 2020. Probiotic Bifidobacterium breve in improving cognitive functions of older adults with suspected mild cognitive impairment: a randomized, double-blind, placebo-controlled trial. J. Alzheimers Dis. 77: 139-147. https://doi.org/10.3233/JAD-200488
  13. Minami J, Kondo S, Yanagisawa N, Odamaki T, Xiao JZ, Abe F, et al. 2015. Oral administration of Bifidobacterium breve B-3 modifies metabolic functions in adults with obese tendencies in a randomised controlled trial. J. Nutr. Sci. 4: E17.
  14. Umborowati MA, Damayanti D, Anggraeni S, Endaryanto A, Surono IS, Effendy I, et al. 2022. The role of probiotics in the treatment of adult atopic dermatitis: a meta-analysis of randomized controlled trials. J. Health Popul. Nutr. 41: 37.
  15. Sanders ME, Akkermans LM, Haller D, Hammerman C, Heimbach J, Hormannsperger, G, et al. 2010. Safety assessment of probiotics for human use. Gut Microbes 1: 164-185. https://doi.org/10.4161/gmic.1.3.12127
  16. Gueimonde M, Sanchez B, G de Los Reyes-Gavilan C, Margolles A. 2013. Antibiotic resistance in probiotic bacteria. Front. Microbiol. 4: 202.
  17. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37: 540-546. https://doi.org/10.1038/s41587-019-0072-8
  18. Tanizawa Y, Fujisawa T, Nakamura Y. 2018. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics 34: 1037-1039. https://doi.org/10.1093/bioinformatics/btx713
  19. Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen C, et al. 2023. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 51: W484-W492.
  20. Chen L, Yang J, Yu J, Yao Z, Sun L, Shen Y, et al. 2005. VFDB: a reference database for bacterial virulence factors. Nucleic Acids Res. 33: 325-328. https://doi.org/10.1093/nar/gki177
  21. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. 2006. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res. 34: D32-D36. https://doi.org/10.1093/nar/gkj014
  22. McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, et al. 2013. The comprehensive antibiotic resistance database. Antimicrob. Agents Chemother. 58: 212-220.
  23. Jorgensen JH, Turnidge JD. 2015. Manual of clinical microbiology, pp. 1253-1273. 11th ed. American Society of Microbiology, Washington, DC, USA
  24. Latorre-Perez A, Villalba-Bermell P, Pascual J, Vilanova C. 2020. Assembly methods for nanopore-based metagenomic sequencing: a comparative study. Sci. Rep. 10: 13588.
  25. Pfeiffer F, Grober C, Blank M, Handler K, Beyer M, Schultze JL, et al. 2018. Systematic evaluation of error rates and causes in short samples in next-generation sequencing. Sci. Rep. 8: 10950.
  26. Valdez-Baez, J, da Costa FMR, Pinto Gomide AC, Profeta R, da Silva AL, Sousa TJ, et al. 2022. Comparative genomics and in silico evaluation of genes related to the probiotic potential of Bifidobacterium breve 1101A. Bacteria 1: 161-182. https://doi.org/10.3390/bacteria1030013
  27. Rodrigo-Torres L, Rodriguez E, Peiroten A, Langa S, Medina M, Arahal DR, et al. 2021. Genome sequence of Bifidobacterium breve INIA P734 (CECT 8178), a strain isolated from human breast milk. Microbiol. Resour. Announc. 10: e00871-20.
  28. Parlindungan E, Lugli GA, Ventura M, Van Sinderen D, Mahony J. 2021. Lactic acid bacteria diversity and characterization of probiotic candidates in fermented meats. Foods 10: 1519.
  29. Aakko J, Sanchez B, Gueimonde M, Salminen S. 2014. Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG. J. Appl. Microbiol. 117: 239-248. https://doi.org/10.1111/jam.12520
  30. Schopping M, Zeidan AA, Franzen CJ. 2022. Stress response in bifidobacteria. Microbiol. Mol. Biol. Rev. 86: e0017021
  31. Xu Q, Zhai Z, An H, Yang Y, Yin J, Wang G, et al. 2019. The MarR family regulator BmrR is involved in bile tolerance of Bifidobacterium longum BBMN68 via controlling the expression of an ABC transporter. Appl. Environ. Microbiol. 85: e02453-18.
  32. Ouwehand AC, Tolkko S, Salminen S. 2001. The effect of digestive enzymes on the adhesion of probiotic bacteria in vitro. J. Food Sci. 66: 856-859. https://doi.org/10.1111/j.1365-2621.2001.tb15186.x
  33. Zhang J, Wang S, Zeng Z, Qin Y, Li P. 2019. The complete genome sequence of Bifidobacterium animalis subsp. lactis 01 and its integral components of antioxidant defense system. 3 Biotech. 9: 352.
  34. Lee C, Hyun K, Kim G, Kwon H, Kwon H. 2023. Exploring probiotic effector molecules and their mode of action in gut-immune interactions. FEMS Microbiol. Rev. 47: fuad046.
  35. Clinically and laboratory standards institute. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard CLSI document M07-A9. Wayne, PA, USA.
  36. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). 2012. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J. 10: 2740.
  37. Turroni F, Foroni E, Pizzetti P, Giubellini V, Ribbera A, Merusi P, et al. 2009. Exploring the diversity of the bifidobacterial population in the human intestinal tract. Appl. Environ. Microbiol. 75: 1534-1545. https://doi.org/10.1128/AEM.02216-08
  38. Serafini F, Bottacini F, Viappiani A, Baruffini E, Turroni F, Foroni E, et al. 2011. Insights into physiological and genetic mupirocin susceptibility in bifidobacteria. Appl. Environ. Microbiol. 77: 3141-3146. https://doi.org/10.1128/AEM.02540-10
  39. Taft DH, Liu J, Maldonado-Gomez MX, Akre S, Huda MN, Ahmad S M, et al. 2018. Bifidobacterial dominance of the gut in early life and acquisition of antimicrobial resistance. mSphere 3: e00441-18
  40. Ammor MS, Belen Florez A, Mayo B. 2007. Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria. Food Microbiol. 24: 559-570. https://doi.org/10.1016/j.fm.2006.11.001
  41. Moubareck C, Gavini F, Vaugien L, Butel MJ, Doucet-Populaire F. 2005. Antimicrobial susceptibility of bifidobacteria. J. Antimicrob. Chemother. 5: 38-44. https://doi.org/10.1093/jac/dkh495
  42. Masco L, Van Hoorde K, De Brandt E, Swings J, Huys G. 2006. Antimicrobial susceptibility of Bifidobacterium strains from humans, animals and probiotic products. J. Antimicrob. Chemother. 58: 85-94. https://doi.org/10.1093/jac/dkl197
  43. Kim M J, Ku S, Kim SY, Lee HH, Jin H, Kang S, et al. 2018. Safety evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI. Int. J. Mol. Sci. 19: 1422.
  44. Mancino W, Lugli GA, Van Sinderen D, Ventura M, Turroni F. 2019. Mobilome and resistome reconstruction from genomes belonging to members of the Bifidobacterium genus. Microorganisms 7: 638.
  45. Tsuchida S, Maruyama F, Ogura Y, Toyoda A, Hayashi T, Okuma M, et al. 2017. Genomic characteristics of Bifidobacterium thermacidophilum pig isolates and wild boar isolates reveal the unique presence of a putative mobile genetic element with tetW for pig farm. Front. Microbiol. 8: 1540.
  46. Cho H, Uehara T, Bernhardt TG. 2014. Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery. Cell 159: 1300-1311. https://doi.org/10.1016/j.cell.2014.11.017
  47. Paterson GK, Harrison EM, Holmes MA. 2014. The emergence of mecC methicillin-resistant Staphylococcus aureus. Trends Microbiol. 22: 42-47. https://doi.org/10.1016/j.tim.2013.11.003
  48. Ricci V, Blair JM, Piddock LJ. 2014. RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease. J. Antimicrob. Chemother. 69: 643-650. https://doi.org/10.1093/jac/dkt432
  49. Levy C, Minnis D, Derrick JP. 2008. Dihydropteroate synthase from Streptococcus pneumoniae: structure, ligand recognition and mechanism of sulfonamide resistance. Biochem. J. 412: 379-388. https://doi.org/10.1042/BJ20071598
  50. Razavi M, Marathe NP, Gillings MR, Flach CF, Kristiansson E, Larsson JDG. 2017. Discovery of the fourth mobile sulfonamide resistance gene. Microbiome 5: 160.
  51. Seah C, Alexander DC, Louie L, Simor A, Low DE, Longtin J, et al. 2012. MupB, a new high-level mupirocin resistance mechanism in Staphylococcus aureus. Antimicrob. Agents Chemother. 56: 1916-1920. https://doi.org/10.1128/AAC.05325-11
  52. Hurdle JG, O'Neill AJ, Ingham E, Fishwick C, Chopra I. 2004. Analysis of mupirocin resistance and fitness in Staphylococcus aureus by molecular genetic and structural modeling techniques. Antimicrob. Agents Chemother. 48: 4366-4376. https://doi.org/10.1128/AAC.48.11.4366-4376.2004
  53. Abdulgader SM, Lentswe T, Whitelaw A, Newton-Foot M. 2020. The prevalence and molecular mechanisms of mupirocin resistance in Staphylococcus aureus isolates from a hospital in Cape Town, South Africa. Antimicrob. Resist. Infect. Control 9: 47.
  54. Hetem D J, Bonten MJM. 2013. Clinical relevance of mupirocin resistance in Staphylococcus aureus. J. Hosp. Infect. 85: 249-256. https://doi.org/10.1016/j.jhin.2013.09.006
  55. Charteris WP, Kelly PM, Morelli L, Collins JK. 1998. Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Prot. 61: 1636-1643. https://doi.org/10.4315/0362-028X-61.12.1636
  56. Aditi FY, Rahman SS, Hossain M. 2017. A study on the microbiological status of mineral drinking water. Open Microbiol. J. 11: 31-44. https://doi.org/10.2174/1874285801711010031
  57. Chukiatsiri K, Sasipreeyajan J, Blackall PJ, Yuwatanichsampan S, Chansiripornchai N. 2012. Serovar identification, antimicrobial sensitivity, and virulence of Avibacterium paragallinarum isolated from chickens in Thailand. Avian. Dis. 56: 359-364. https://doi.org/10.1637/9881-080811-Reg.1
  58. Patel JB, Gorwitz RJ, Jernigan JA. 2009. Mupirocin resistance. Clin. Infect. Dis. 49: 935-941. https://doi.org/10.1086/605495
  59. Clinical and Laboratory Standards Institute (CLSI). 2014. Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement, CLSI document M100-S24. Clinical and Laboratory Standards Institute, Wayne, PA, USA.
  60. Rajkumari N, Mathur P, Bhardwaj N, Gupta G, Dahiya R, Behera B, et al. 2014. Resistance pattern of mupirocin in methicillin-resistant Staphylococcus aureus in trauma patients and comparison between disc diffusion and E-test for better detection of resistance in low resource countries. J. Lab. Phyicians 6: 91-95. https://doi.org/10.4103/0974-2727.141505