Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Lactobacillus Persisters Formation and Resuscitation

  • Hyein Kim (Department of Animal Science, Jeonbuk National University) ;
  • Sejong Oh (Division of Animal Science, Chonnam National University) ;
  • Sooyeon Song (Department of Animal Science, Jeonbuk National University)
  • Received : 2023.12.27
  • Accepted : 2024.01.16
  • Published : 2024.04.28
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Abstract

Lactobacillus is a commonly used probiotic, and many researchers have focused on its stress response to improve its functionality and survival. However, studies on persister cells, dormant cells that aid bacteria in surviving general stress, have focused on pathogenic bacteria that cause infection, not Lactobacillus. Thus, understanding Lactobacillus persister cells will provide essential clues for understanding how Lactobacillus survives and maintains its function under various environmental conditions. We treated Lactobacillus strains with various antibiotics to determine the conditions required for persister formation using kill curves and transmission electron microscopy. In addition, we observed the resuscitation patterns of persister cells using single-cell analysis. Our results show that Lactobacillus creates a small population of persister cells (0.0001-1% of the bacterial population) in response to beta-lactam antibiotics such as ampicillin and amoxicillin. Moreover, only around 0.5-1% of persister cells are heterogeneously resuscitated by adding fresh media; the characteristics are typical of persister cells. This study provides a method for forming and verifying the persistence of Lactobacillus and demonstrates that antibiotic-induced Lactobacillus persister cells show characteristics of dormancy, sensitivity of antibiotics, same as exponential cells, multi-drug tolerance, and resuscitation, which are characteristics of general persister cells. This study suggests that the mechanisms of formation and resuscitation may vary depending on the characteristics, such as the membrane structure of the bacterial species.

Keywords

Acknowledgement

This work was supported by funds derived from a National Research Foundation of Korea (NRF) grant from the Korean Government (2020R1F1A107239713, RS-2023-00210305) for SYS and (NRF-2021R1A4A1031220) for SO. This paper was supported by research funds for newly appointed professors of Jeonbuk National University in 2020.

References

  1. Zhihong Sun, Hugh MB Harris, Angela McCann, Chenyi Guo, Silvia Argimon, Wenyi Zhang, et al. 2015. Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. Nat. Commun. 6: 8322.
  2. Liu YW, Liong MT, Tsai YC. 2018. New perspectives of Lactobacillus plantarum as a probiotic: the gut-heart-brain axis. J. Microbiol. 56: 601-613.
  3. Pathmakanthan S, Li CK, Cowie J, Hawkey CJ. 2004. Lactobacillus plantarum 299: beneficial in vitro immunomodulation in cells extracted from inflamed human colon. J. Gastroenterol. Hepatol. 19: 166-73.
  4. Vareille-Delarbre M, Miquel S, Garcin S, Bertran T, Balestrino D, Evrard B, et al. 2019. Immunomodulatory effects of Lactobacillus plantarum on inflammatory response induced by Klebsiella pneumoniae. Infect. Immun. 87: 10-1128.
  5. Roberta Prete, Natalia Garcia-Gonzalez, Carla D Di Mattia, Aldo Corsetti, Natalia Battista. 2020. Food-borne Lactiplantibacillus plantarum protect normal intestinal cells against inflammation by modulating reactive oxygen species and IL-23/IL-17 axis. Sci. Rep. 10: 16340.
  6. Kazmierczak-Siedlecka K, Daca A, Folwarski M, Witkowski JM, Bryl E, Makarewicz W. 2020. The role of Lactobacillus plantarum 299v in supporting treatment of selected diseases. Cent. Eur. J. Immunol. 45: 488-493.
  7. Bisutti IL, Hirt K, Stephan D. 2015. Influence of different growth conditions on the survival and the efficacy of freeze-dried Pseudomonas fluorescens strain Pf153. Biocontrol. Sci. Technol. 25: 1269-1284.
  8. Gao X, Kong J, Zhu H, Mao B, Cui S, Zhao J. 2022. Lactobacillus, Bifidobacterium and Lactococcus response to environmental stress: mechanisms and application of cross-protection to improve resistance against freeze-drying. J. Appl. Microbiol. 132: 802-821.
  9. Mbye M, Baig MA, AbuQamar SF, El-Tarabily KA, Obaid RS, Osaili TM, Ayyash MM, et al. 2020. Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses. Compr. Rev. Food Sci. Food Saf. 19: 1110-1124.
  10. Bergenholtz AS, Wessman P, Wuttke A, Hakansson S. 2012. A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying. Cryobiology 64: 152-159.
  11. Chen MJ, Tang HY, Chiang ML. 2017. Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1. Food Microbiol. 66: 20-27.
  12. Li Y, Yan P, Lei Q, Li B, Sun Y, Li S, Xie N, et al. 2019. Metabolic adapt ability shifts of cell membrane fatty acids of Komagataeibacter hansenii HDM1-3 improve acid stress resistance and survival in acidic environments. J. Ind. Microbiol. Biotechnol. 46: 1491-1503.
  13. Leon MJ, Hoffmann T, Sanchez-Porro C, Heider J, Ventosa A, Bremer E. 2018. Compatible solute synthesis and import by the moderate halophile Spiribacter salinus: physiology and genomics. Front. Microbiol. 9: 108.
  14. Al-Naseri A, Bowman JP, Wilson R, Nilsson RE, Britz ML. 2013. Impact of lactose starvation on the physiology of Lactobacillus casei GCRL163 in the presence or absence of tween 80. J. Proteome Res. 12: 5313-5322.
  15. Xu HS, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Colwell RR. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8: 313-323.
  16. Hobby GL, Meyer K, Chaffee E. 1942. Observations on the mechanism of action of penicillin. Proc. Soc. Exp. Biol. Med. 50: 281-285.
  17. Kim JS, Chowdhury N, Yamasaki R, Wood TK. 2018. Viable but non-culturable and persistence describe the same bacterial stress state. Environ. Microbiol. 20: 2038-2048.
  18. Um HY, Kong HG, Lee HJ, Choi HK, Park EJ, Kim ST, et al. 2013. Altered Gene Expression and intracellular changes of the viable but nonculturable state in Ralstonia solanacearum by copper treatment. Plant Pathol. J. 29: 374-85.
  19. Van den Bergh B, Fauvart M, Michiels J. 2017. Formation, physiology, ecology, evolution and clinical importance of bacterial persisters. FEMS Microbiol. Rev. 41: 219-251.
  20. Song S, Wood TK. 2020. ppGpp ribosome dimerization model for bacterial persister formation and resuscitation. Biochem. Biophys. Res. Commun. 523: 281-286.
  21. Tagami K, Nanamiya H, Kazo Y, Maehashi M, Suzuki S, Kawamura F, et al. 2012. Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp(0) mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome. MicrobiologyOpen 1: 115-134.
  22. Yamasaki R, Song S, Benedik MJ, Wood TK. 2020. Persister cells resuscitate using membrane sensors that activate chemotaxis, lower cAMP levels, and revive ribosomes. Iscience 23: 100792.
  23. Fisher RA, Gollan B, Helaine S. 2017. Persistent bacterial infections and persister cells. Nat. Rev. Microbiol. 15: 453-464.
  24. De Man JC, Rogosa D, Sharpe ME. 1960. A medium for the cultivation of lactobacilli. J. Appl. Microbiol. 23: 130-135.
  25. Lima LM, Silva BNMD, Barbosa G, Barreiro EJ. 2020. β-lactam antibiotics: an overview from a medicinal chemistry perspective. Eur. J. Med. Chem. 208: 112829.
  26. Kim JS, Wood TK. 2016. Persistent persister misperceptions. Front. Microbiol. 208: 112829.
  27. Kim JS, Yamasaki R, Song S, Zhang W, Wood TK. 2018. Single cell observations show persister cells wake based on ribosome content. Environ. Microbiol. 20: 2085-2098.
  28. Kim JS, Wood TK. 2017. Tolerant, growing cells from nutrient shifts are not persister cells. MBio 8: 10-1128.
  29. Martinez OV, Gratzner HG, Malinin TI, Ingram M. 1982. The effect of some beta-lactam antibiotics on Escherichia coli studied by flow cytometry. Cytometry 3: 129-133.
  30. Yao Z, Kahne D, Kishony R. 2012. Distinct single-cell morphological dynamics under beta-lactam antibiotics. Mol. Cell 48: 705-712.
  31. Fan X, Bao T, Yi H, Zhang Z, Zhang K, Liu X, Feng Z, et al. 2021. Ribosome profiling and RNA sequencing reveal genome-wide cellular translation and transcription regulation under osmotic stress in Lactobacillus rhamnosus ATCC 53103 Front. Microbiol. 12: 781454.
  32. Anjum N, Maqsood S, Masud T, Ahmad A, Sohail A, Momin A. 2014. Lactobacillus acidophilus: characterization of the species and application in food production. Crit. Rev. Food Sci. Nutr. 54: 1241-1251.
  33. Neville BA, Forde BM, Claesson MJ, Darby T, Coghlan A, Nally K, et al. 2012. Characterization of pro-inflammatory flagellin proteins produced by Lactobacillus ruminis and related motile lactobacilli. PLoS One 7: e40592.
  34. Rohde M. 2019. The Gram-positive bacterial cell wall. Microbiol. Spectr. 7. doi: 10.1128/microbiolspec.GPP3-0044-2018.
  35. Wu C, Zhang J, Wang M, Du G, Chen J. 2012. Lactobacillus casei combats acid stress by maintaining cell membrane functionality. J. Ind. Microbiol. Biotechnol. 39: 1031-1039.