1887

Microbiology Society logo

Volume 148, Issue 5

Formation and resuscitation of ‘non-culturable’ cells of and in prolonged stationary phase Free

Abstract

After growth of in Sauton’s medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3–4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of ) appeared to be responsible for some of the activity in the culture supernatant. The formation of ‘non-culturable’ cells of the ‘Academia’ strain of was similarly observed following growth in Sauton’s medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4–5 months post-inoculation, of a homogeneous population of ostensibly ‘non-culturable’ cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10 organisms ml, and this value was further increased by one log using supernatant from an actively growing culture. Populations of ‘non-culturable’ cells of were also obtained by the filtration of ‘clumpy’ cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The ‘non-culturable’ cells that accumulated during prolonged stationary phase in both the and the cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with ‘non-culturable’ bacterial suspensions invites the speculation that one, or more, of the cognate Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): ADC, albumen, glucose and NaCl , CTC, 5-cyano-2,3-ditolyl tetrazolium chloride , dormancy , Micrococcus luteus , MPN, most probable number , Nocardiaceae , Rpf , Rpf, resuscitation-promoting factor , SN, supernatant , VBNC and VBNC, viable but non-culturable
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-5-1581
2002-05-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/5/1481581a.html?itemId=/content/journal/micro/10.1099/00221287-148-5-1581&mimeType=html&fmt=ahah

References

  1. Barer M. R. 1997; Viable but non-culturable and dormant bacteria: time to resolve an oxymoron and a misnomer?. J Med Microbiol 46:629–631
     [Google Scholar]
  2. Barer M. R., Harwood C. R. 1999; Bacterial viability and culturability. Adv Microb Physiol 41:93–137
     [Google Scholar]
  3. Barer M. R., Kaprelyants A. S., Weichart D. H., Harwood C. R., Kell D. B. 1998; Microbial stress and culturability: conceptual and operational domains. Microbiology 144:2009–2010 [CrossRef]
     [Google Scholar]
  4. Biketov S., Mukamolova G. V., Potapov V., Gilenkov E., Vostroknutova G., Kell D. B., Young M., Kaprelyants A. S. 2000; Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery. FEMS Immunol Med Microbiol 29:233–240 [CrossRef]
     [Google Scholar]
  5. Connell N. D. 1994; Mycobacterium : isolation, maintenance, transformation, and mutant selection. Methods Cell Biol 45:107–125
     [Google Scholar]
  6. Cunningham A. F., Spreadbury C. L. 1998; Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton α-crystallin homolog. J Bacteriol 180:801–808
     [Google Scholar]
  7. Davey H. M., Kaprelyants A. S., Weichart D. H., Kell D. B. 1999; Approaches to the estimation of microbial viability using flow cytometry. In Current Protocols in Cytometry vol. 11Microbial Cytometry Edited by Robinson J. P. New York: Wiley;
     [Google Scholar]
  8. de Man J. C. 1975; The probability of most probable numbers. Eur J Appl Microbiol 1:67–78 [CrossRef]
     [Google Scholar]
  9. de Wit D., Wootton M., Dhillon J., Mitchison D. A. 1995; The bacterial DNA content of mouse organs in the Cornell model of dormant tuberculosis. Tuber Lung Dis 76:555–562 [CrossRef]
     [Google Scholar]
  10. Desjardin L. E., Hayes L. G., Sohaskey C. D., Wayne L. G., Eisenach K. D. 2001; Microaerophilic induction of the alpha-crystallin chaperone protein homologue ( hspX ) mRNA of Mycobacterium tuberculosis . J Bacteriol 183:5311–5316 [CrossRef]
     [Google Scholar]
  11. Dick T., Lee B. H., Murugasu-Oei B. 1998; Oxygen depletion induced dormancy in Mycobacterium smegmatis . FEMS Microbiol Lett 163:159–164 [CrossRef]
     [Google Scholar]
  12. Flynn J. L., Chan J. 2001; Tuberculosis: latency and reactivation. Infect Immun 69:4195–4201 [CrossRef]
     [Google Scholar]
  13. Gangadharam P. R. J. 1995; Mycobacterial dormancy. Tuber Lung Dis 76:477–479 [CrossRef]
     [Google Scholar]
  14. Hu Y. M., Butcher P. D., Sole K., Mitchison D. A., Coates A. R. M. 1998; Protein synthesis is shut down in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock. FEMS Microbiol Lett 158:139–145 [CrossRef]
     [Google Scholar]
  15. Hu Y., Butcher P. D., Mangan J. A., Rajandream M. A., Coates A. R. 1999; Regulation of hmp gene transcription in Mycobacterium tuberculosis : effects of oxygen limitation and nitrosative and oxidative stress. J Bacteriol 181:3486–3493
     [Google Scholar]
  16. Hutter B., Dick T. 1998; Increased alanine dehydrogenase activity during dormancy in Mycobacterium smegmatis . FEMS Microbiol Lett 167:7–11 [CrossRef]
     [Google Scholar]
  17. Kaprelyants A. S., Kell D. B. 1992; Rapid assessment of bacterial viability and vitality using rhodamine 123 and flow cytometry. J Appl Bacteriol 72:410–422 [CrossRef]
     [Google Scholar]
  18. Kaprelyants A. S., Kell D. B. 1993a; Dormancy in stationary-phase cultures of Micrococcus luteus : flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59:3187–3196
     [Google Scholar]
  19. Kaprelyants A. S., Kell D. B. 1993b; The use of 5-cyano-2,3-ditolyl tetrazolium chloride and flow-cytometry for the visualization of respiratory activity in individual cells of Micrococcus luteus . J Microbiol Methods 17:115–122 [CrossRef]
     [Google Scholar]
  20. Kaprelyants A. S., Kell D. B. 1996; Do bacteria need to communicate with each other for growth?. Trends Microbiol 4:237–242 [CrossRef]
     [Google Scholar]
  21. Kaprelyants A. S., Gottschal J. C., Kell D. B. 1993; Dormancy in non-sporulating bacteria. FEMS Microbiol Rev 104:271–286 [CrossRef]
     [Google Scholar]
  22. Kaprelyants A. S., Mukamolova G. V., Kell D. B. 1994; Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent medium at high dilution. FEMS Microbiol Lett 115:347–352 [CrossRef]
     [Google Scholar]
  23. Kaprelyants A. S., Mukamolova G. V., Davey H. M., Kell D. B. 1996; Quantitative analysis of the physiological heterogeneity within starved cultures of Micrococcus luteus by flow cytometry and cell sorting. Appl Environ Microbiol 62:1311–1316
     [Google Scholar]
  24. Kaprelyants A. S., Mukamolova G. V., Kormer S. S., Weichart D. H., Young M., Kell D. B. 1999; Intercellular signalling and the multiplication of prokaryotes: bacterial cytokines. In Microbial Signalling and CommunicationSociety for General Microbiology Symposium no. 57 pp 33–69 Edited by England R. Hobbs G., Bainton N., Roberts D. McL. Cambridge: Cambridge University Press;
     [Google Scholar]
  25. Keer J., Smeulders M. J., Gray K. M., Williams H. D. 2000; Mutants of Mycobacterium smegmatis impaired in stationary-phase survival. Microbiology 146:2209–2217
     [Google Scholar]
  26. Keer J., Smeulders M. J., Williams H. D. 2001; A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phase. Microbiology 147:473–481
     [Google Scholar]
  27. Kell D. B., Young M. 2000; Bacterial dormancy and culturability: the role of autocrine growth factors. Curr Opin Microbiol 3:238–243 [CrossRef]
     [Google Scholar]
  28. Kell D. B., Kaprelyants A. S., Weichart D. H., Harwood C. L., Barer M. R. 1998; Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Leeuwenhoek 73:169–187 [CrossRef]
     [Google Scholar]
  29. Lim A., Eleuterio M., Hutter B., Murugasu-Oei B., Dick T. 1999; Oxygen depletion-induced dormancy in Mycobacterium bovis BCG. J Bacteriol 181:2252–2256
     [Google Scholar]
  30. McCune R. M., Feldman F. M., Lambert H., McDermott W. 1966; Microbial persistence. I. The capacity of tubercle bacilli to survive sterilization in mouse tissues. J Exp Med 123:224–268
     [Google Scholar]
  31. Mukamolova G. V., Yanopolskaya N. D., Votyakova T. V., Popov V. I., Kaprelyants A. S., Kell D. B. 1995; Biochemical changes accompanying the long-term starvation of Micrococcus luteus cells in spent growth medium. Arch Microbiol 163:373–379
     [Google Scholar]
  32. Mukamolova G. V., Kaprelyants A. S., Young D. I., Young M., Kell D. B. 1998; A bacterial cytokine. Proc Natl Acad Sci U S A 95:8916–8921 [CrossRef]
     [Google Scholar]
  33. Ogloblina L. S., Ravich-Birger E. D. 1958 Catalogue of Strains, issue 2 p 6 Moscow: Tarasevich State Control Institute of Medical and Biological Preparations;
     [Google Scholar]
  34. Osterman I. 1985 Chromatography of Proteins and Nucleic Acids Moscow: Nauka;
     [Google Scholar]
  35. Parrish N. M., Dick J. D., Bishai W. R. 1998; Mechanisms of latency in Mycobacterium tuberculosis . Trends Microbiol 6:107–112 [CrossRef]
     [Google Scholar]
  36. Ray B., Speck M. L. 1973; Freeze-injury in bacteria. CRC Crit Rev Clin Lab Sci 4:161–213 [CrossRef]
     [Google Scholar]
  37. Smeulders M. J., Keer J., Speight R. A., Williams H. D. 1999; Adaptation of Mycobacterium smegmatis to stationary phase. J Bacteriol 181:270–283
     [Google Scholar]
  38. Sun Z., Zhang Y. 1999; Spent culture supernatant of Mycobacterium tuberculosis H37Ra improves viability of aged cultures of this strain and allows small inocula to initiate growth. J Bacteriol 181:7626–7628
     [Google Scholar]
  39. Wayne L. G. 1994; Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis 13:908–914 [CrossRef]
     [Google Scholar]
  40. Wayne L. G., Hayes L. G. 1996; An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through 2 stages of nonreplicating persistence. Infect Immun 64:2062–2069
     [Google Scholar]
  41. Wayne L. G., Sohaskey C. D. 2001; Nonreplicating persistence of Mycobacterium tuberculosis . Annu Rev Microbiol 55:139–163 [CrossRef]
     [Google Scholar]
  42. Wayne L. G., Sramek H. A. 1994; Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis . Antimicrob Agents Chemother 38:2054–2058 [CrossRef]
     [Google Scholar]
  43. Yuan Y., Crane D. D., Barry C. E. III 1996; Stationary phase-associated protein expression in Mycobacterium tuberculosis : function of the mycobacterial alpha-crystallin homolog. J Bacteriol 178:4484–4492
     [Google Scholar]
  44. Zhang Y., Yang Y., Woods A., Cotter R. J., Sun Z. 2001; Resuscitation of dormant Mycobacterium tuberculosis by phospholipids or specific peptides. Biochem Biophys Res Commun 284:542–547 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-5-1581
Loading
Formation and resuscitation of ‘non-culturable’ cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase
Microbiology 148, 1581 (2002); https://doi.org/10.1099/00221287-148-5-1581
/content/journal/micro/10.1099/00221287-148-5-1581
/content/journal/micro/10.1099/00221287-148-5-1581
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error