DETERMINATION OF DNA CONTENT IN INDIVIDUAL YERSINIA PESTIS CELLS BY USING FLOW CYTOFLUORIMETRY METHOD: COMPARATIVE ANALYSIS OF INHOMOGENEITY IN CULTURES OF STRAINS WITH VARIOUS BIOLOGICAL PROPERTIES


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Aim. Comparative analysis of Yersinia pestis strains with various biological properties by DNA content in individual cells. Materials and methods. Virulent strain 231, avirulent strain KM 260 (12) [231], that is its isogenic (no-plasmid) derivative, and vaccine strain EV NIIEG were used. 48-hour agar cultures of the studied strains reproduced at 28°C and their subcultures obtained by cultivation of the initial cultures by aeration on liquid nutrient medium from 37°C were prepared. DNA of the fixed bacteria was dyed by a mixture of ethidium bromide and mitramycin, and then the bacteria were studied by using flow cytofluorimeter for the determination of rates of cells with relatively low or high DNA content in the studied bacterial populations. The degree of inhomogeneity of a bacterial population was evaluated by DNA histogram variation coefficient value. Results. In 6 hours of growth at 37°C in optically non-dense bacterial cultures a high degree of DNA content per cell inhomogeneity was established that is related to the activation of DNA replication process in bacteria. In 48 hours of growth this inhomogeneity completely disappeared in the virulent strain cultures and remained in the avirulent strain cultures of the plague pathogen. Based on the studied parameters the vaccine strain held an intermediate position. Conclusion. Further studies of the plague culture DNA content per cell inhomogeneity may become a base for the operative strain differentiation based on pathogenicity level (hazard) for humans, and therefore the requirements for the management of safe working conditions with this microorganism.

About the authors

A. L Kravtsov

Russian Research Institute for Plague Control «Microbe», Saratov, Russia

M. N Lyapin

Russian Research Institute for Plague Control «Microbe», Saratov, Russia

T. P Shmelkova

Russian Research Institute for Plague Control «Microbe», Saratov, Russia

E. M Golovko

Russian Research Institute for Plague Control «Microbe», Saratov, Russia

T. A Malyukova

Russian Research Institute for Plague Control «Microbe», Saratov, Russia

T. A Kostyukova

Russian Research Institute for Plague Control «Microbe», Saratov, Russia

I. N Ezhov

Russian Research Institute for Plague Control «Microbe», Saratov, Russia

References

  1. Карташова А.Л. Роль Са2+-зависимости при 37°C в усилении вирулентности Y.pestis и пролиферации клеток злокачественной опухоли. Факты и предположения. Занимательные очерки о деятельности и деятелях противочумной системы России и Советского Союза. М., Информатика, 1998, 8: 208-236.
  2. Кравцов А.Л. Микрофлуориметрическое исследование в потоке гетерогенных популяций бактерий Yersinia pestis и клеток иммунной системы инфицированных чумой лабораторных животных. Автореф. дисс. д-ра биол. наук. Саратов, 1997.
  3. Монцевичюте-Эрингене Е.В. Упрощенные математико-статистические методы в медицинской исследовательской работе. Журн. патол. физиол. экспер. терапии. 1964, 4: 71-78.
  4. Шмелькова Т.П. Взаимодействие чумного микроба и его антигенов с клетками крови человека in vitro. Автореф. дисс. канд. биол. наук. Саратов, 2008.
  5. Шмелькова Т.П., Кравцов А.Л., Щуковская Т.Н., Ляпин М.Н. и др. Влияние биологических свойств чумного микроба на развитие апоптоза лейкоцитов крови человека в системе in vitro. Пробл. особо опасн. инф. 2007, 93 (1): 85-89.
  6. Allman R., Manchee R., Lloyd D. Flow cytometric analysis of heterogenous bacterial populations. In: Flow cytometry in microbiology. D. Lloyd (ed.). Springer-Verlag, 1993, 3: 27-47.
  7. Bubeck S.S., Cantwell A.M., Dube P.H. Delayed inflammatory response to primary pneumonic plague occurs in both outbred and inbred mice. Infect. Immun. 2007, 75 (2): 697-705.
  8. Davey H.M., Winson M.K. Using flow cytometry to quantify microbial heterogeneity. Curr. Issues Mol. Biol. 2003, 5: 9-15.
  9. Maclntyre S., Knight S.D., Fook L. Structure, assembly and applications of the polymeric F1 antigen of Yersinia pestis. In: Yersinia molecular and cellular biology. E.Carniel, B.J. Hinnebusch (ed.). 2004, 18: 363-407.
  10. Marrone D. Flow cytometry: a multipurpose technology for a wide spectrum of global biosecurity applications. J. Association Lab. Automation. 2009, 14 (3):148-156.
  11. Raybourne R.B., Bunning V.K. Bacterial-host interactions at the cellular level: fluorescent labeling of bacteria and analysis of short-term bacterium-phagocyte interaction by flow cytometry. Infect. Immun. 1994, 62 (2): 665-672.
  12. Spinner J.L., Cundiff J.A., Kobayashi S.D. Yersinia pestis type III secretion system-dependent inhibition of human polymorphonuclear leukocyte function. Ibid. 2008, 76 (8): 3754-3760.
  13. Steen H.B., Boye E. Escherichia coli growth studied by dual-parameter flow cytophotometry. J. Bacteriology. 1981, 145 (2): 1091-1094.
  14. Thorsen B.T., Enger Q., Norland S. et al. Long-term starvation survival of Yersinia ruckeri at different salinities studied by microbiological and flow cytometric methods. Appl. Environ. Microbiol. 1992, 58 (5): 1624-1628.
  15. Tyndall R.L., Hand R.E., Mann R.C. et al. Application of flow cytometry to detection and characterization of Legionella spp. Ibid. 1985, 49 (4): 852-857.
  16. Van Amersfoort E.S., Van Berkel T.J., Kuiper J. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin. Microbiol. Rev. 2003, 16 (3): 379-414.

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Copyright (c) 2011 Kravtsov A.L., Lyapin M.N., Shmelkova T.P., Golovko E.M., Malyukova T.A., Kostyukova T.A., Ezhov I.N.

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