Abstract
In this study, we have used different fluorescent dyes and techniques to characterize the heterogeneity and changes of the physiological states encountered by the marine bacterium Deleya aquamarina during a 92-day starvation survival experiment at 20 and 5(deg)C. Changes of physiological states were investigated on a single-cell basis by flow cytometry and epifluorescence microscopy in conjunction with fluorescent dyes specific for various cellular functions and constituents. Heterogeneities within populations with regard to functions (respiration, substrate responsiveness, enzymatic activity, and cytoplasmic membrane permeability), constituent (DNA), and cell volume (light scatter) were compared to the evolution of viable plate counts (CFU). At 20(deg)C, CFU changes were divided into three stages corresponding to stability up to day 13 followed by a rapid drop between days 13 and 42 and then by stabilization at a level of 10 to 20% during the remaining survival period. Most of the cellular fractions showing a metabolic activity were close to the evolution of the culturable cells, suggesting the absence of viable but nonculturable cells. On the other hand, cells with selective cytoplasmic membrane permeability but without any metabolic activity were observed, and this stage was followed by DNA alteration occurring at different rates after the loss of membrane cytoplasmic permeability. We observed a greater maintenance of culturability, physiological functions, DNA, and cellular volume at the lower temperature. These results have different ecological implications from both methodological and conceptual viewpoints.
Full Text
The Full Text of this article is available as a PDF (224.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amann R. I., Ludwig W., Schleifer K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995 Mar;59(1):143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Amy P. S., Morita R. Y. Starvation-survival patterns of sixteen freshly isolated open-ocean bacteria. Appl Environ Microbiol. 1983 Mar;45(3):1109–1115. doi: 10.1128/aem.45.3.1109-1115.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Amy P. S., Pauling C., Morita R. Y. Starvation-survival processes of a marine Vibrio. Appl Environ Microbiol. 1983 Mar;45(3):1041–1048. doi: 10.1128/aem.45.3.1041-1048.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barrett T. D., Hayes E. S., Walker M. J. Lack of selectivity for ventricular and ischaemic tissue limits the antiarrhythmic actions of lidocaine, quinidine and flecainide against ischaemia-induced arrhythmias. Eur J Pharmacol. 1995 Oct 24;285(3):229–238. doi: 10.1016/0014-2999(95)00406-b. [DOI] [PubMed] [Google Scholar]
- Bertone S., Giacomini M., Ruggiero C., Piccarolo C., Calegari L. Automated systems for identification of heterotrophic marine bacteria on the basis of their Fatty Acid composition. Appl Environ Microbiol. 1996 Jun;62(6):2122–2132. doi: 10.1128/aem.62.6.2122-2132.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biosca E. G., Amaro C., Marco-Noales E., Oliver J. D. Effect of low temperature on starvation-survival of the eel pathogen Vibrio vulnificus biotype 2. Appl Environ Microbiol. 1996 Feb;62(2):450–455. doi: 10.1128/aem.62.2.450-455.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dahle A. B., Laake M. Diversity dynamics of marine bacteria studied by immunofluorescent staining on membrane filters. Appl Environ Microbiol. 1982 Jan;43(1):169–176. doi: 10.1128/aem.43.1.169-176.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dobson S. J., McMeekin T. A., Franzmann P. D. Phylogenetic relationships between some members of the genera Deleya, Halomonas, and Halovibrio. Int J Syst Bacteriol. 1993 Oct;43(4):665–673. doi: 10.1099/00207713-43-4-665. [DOI] [PubMed] [Google Scholar]
- Duncan S., Glover L. A., Killham K., Prosser J. I. Luminescence-based detection of activity of starved and viable but nonculturable bacteria. Appl Environ Microbiol. 1994 Apr;60(4):1308–1316. doi: 10.1128/aem.60.4.1308-1316.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eguchi M., Nishikawa T., Macdonald K., Cavicchioli R., Gottschal J. C., Kjelleberg S. Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas sp. Strain RB2256. Appl Environ Microbiol. 1996 Apr;62(4):1287–1294. doi: 10.1128/aem.62.4.1287-1294.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuhrman J. A., McCallum K., Davis A. A. Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans. Appl Environ Microbiol. 1993 May;59(5):1294–1302. doi: 10.1128/aem.59.5.1294-1302.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Giovannoni S. J., DeLong E. F., Olsen G. J., Pace N. R. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol. 1988 Feb;170(2):720–726. doi: 10.1128/jb.170.2.720-726.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huq A., Colwell R. R., Rahman R., Ali A., Chowdhury M. A., Parveen S., Sack D. A., Russek-Cohen E. Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods. Appl Environ Microbiol. 1990 Aug;56(8):2370–2373. doi: 10.1128/aem.56.8.2370-2373.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiang X., Chai T. J. Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells. Appl Environ Microbiol. 1996 Apr;62(4):1300–1305. doi: 10.1128/aem.62.4.1300-1305.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Josephson K. L., Gerba C. P., Pepper I. L. Polymerase chain reaction detection of nonviable bacterial pathogens. Appl Environ Microbiol. 1993 Oct;59(10):3513–3515. doi: 10.1128/aem.59.10.3513-3515.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kogure K., Simidu U., Taga N. A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol. 1979 Mar;25(3):415–420. doi: 10.1139/m79-063. [DOI] [PubMed] [Google Scholar]
- Kurath G., Morita R. Y. Starvation-Survival Physiological Studies of a Marine Pseudomonas sp. Appl Environ Microbiol. 1983 Apr;45(4):1206–1211. doi: 10.1128/aem.45.4.1206-1211.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Langsrud S., Sundheim G. Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound. J Appl Bacteriol. 1996 Oct;81(4):411–418. doi: 10.1111/j.1365-2672.1996.tb03527.x. [DOI] [PubMed] [Google Scholar]
- Lebaron P., Joux F. Flow cytometric analysis of the cellular DNA content of Salmonella typhimurium and Alteromonas haloplanktis during starvation and recovery in seawater. Appl Environ Microbiol. 1994 Dec;60(12):4345–4350. doi: 10.1128/aem.60.12.4345-4350.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee K., Ruby E. G. Symbiotic Role of the Viable but Nonculturable State of Vibrio fischeri in Hawaiian Coastal Seawater. Appl Environ Microbiol. 1995 Jan;61(1):278–283. doi: 10.1128/aem.61.1.278-283.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McFeters G. A., Yu F. P., Pyle B. H., Stewart P. S. Physiological assessment of bacteria using fluorochromes. J Microbiol Methods. 1995 Jan;21(1):1–13. doi: 10.1016/0167-7012(94)00027-5. [DOI] [PubMed] [Google Scholar]
- Moyer C. L., Morita R. Y. Effect of growth rate and starvation-survival on the viability and stability of a psychrophilic marine bacterium. Appl Environ Microbiol. 1989 May;55(5):1122–1127. doi: 10.1128/aem.55.5.1122-1127.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nelson SM, Attwell RW, Dawson MM, Smith CA. The Effect of Temperature on Viability of Carbon- and Nitrogen-Starved Escherichia coli. Microb Ecol. 1996 Jul;32(1):11–21. doi: 10.1007/BF00170103. [DOI] [PubMed] [Google Scholar]
- Nilsson L., Oliver J. D., Kjelleberg S. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. J Bacteriol. 1991 Aug;173(16):5054–5059. doi: 10.1128/jb.173.16.5054-5059.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Porter J., Deere D., Pickup R., Edwards C. Fluorescent probes and flow cytometry: new insights into environmental bacteriology. Cytometry. 1996 Feb 1;23(2):91–96. doi: 10.1002/(SICI)1097-0320(19960201)23:2<91::AID-CYTO1>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
- Porter J., Diaper J., Edwards C., Pickup R. Direct measurements of natural planktonic bacterial community viability by flow cytometry. Appl Environ Microbiol. 1995 Jul;61(7):2783–2786. doi: 10.1128/aem.61.7.2783-2786.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Porter J., Edwards C., Pickup R. W. Rapid assessment of physiological status in Escherichia coli using fluorescent probes. J Appl Bacteriol. 1995 Oct;79(4):399–408. doi: 10.1111/j.1365-2672.1995.tb03154.x. [DOI] [PubMed] [Google Scholar]
- Pyle B. H., Broadaway S. C., McFeters G. A. Factors affecting the determination of respiratory activity on the basis of cyanoditolyl tetrazolium chloride reduction with membrane filtration. Appl Environ Microbiol. 1995 Dec;61(12):4304–4309. doi: 10.1128/aem.61.12.4304-4309.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Quinn J. P. The modification and evaluation of some cytochemical techniques for the enumeration of metabolically active heterotrophic bacteria in the aquatic environment. J Appl Bacteriol. 1984 Aug;57(1):51–57. doi: 10.1111/j.1365-2672.1984.tb02355.x. [DOI] [PubMed] [Google Scholar]
- Ramsing N. B., Fossing H., Ferdelman T. G., Andersen F., Thamdrup B. Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column. Appl Environ Microbiol. 1996 Apr;62(4):1391–1404. doi: 10.1128/aem.62.4.1391-1404.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ravel J., Knight I. T., Monahan C. E., Hill R. T., Colwell R. R. Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation? Microbiology. 1995 Feb;141(Pt 2):377–383. doi: 10.1099/13500872-141-2-377. [DOI] [PubMed] [Google Scholar]
- Rodriguez G. G., Phipps D., Ishiguro K., Ridgway H. F. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol. 1992 Jun;58(6):1801–1808. doi: 10.1128/aem.58.6.1801-1808.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siegele D. A., Kolter R. Life after log. J Bacteriol. 1992 Jan;174(2):345–348. doi: 10.1128/jb.174.2.345-348.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tabor P. S., Neihof R. A. Direct determination of activities for microorganisms of chesapeake bay populations. Appl Environ Microbiol. 1984 Nov;48(5):1012–1019. doi: 10.1128/aem.48.5.1012-1019.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thom S. M., Horobin R. W., Seidler E., Barer M. R. Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity. J Appl Bacteriol. 1993 Apr;74(4):433–443. doi: 10.1111/j.1365-2672.1993.tb05151.x. [DOI] [PubMed] [Google Scholar]
- Ullrich S., Karrasch B., Hoppe H., Jeskulke K., Mehrens M. Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. Appl Environ Microbiol. 1996 Dec;62(12):4587–4593. doi: 10.1128/aem.62.12.4587-4593.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Votyakova T. V., Kaprelyants A. S., Kell D. B. Influence of Viable Cells on the Resuscitation of Dormant Cells in Micrococcus luteus Cultures Held in an Extended Stationary Phase: the Population Effect. Appl Environ Microbiol. 1994 Sep;60(9):3284–3291. doi: 10.1128/aem.60.9.3284-3291.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ward D. M., Weller R., Bateson M. M. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature. 1990 May 3;345(6270):63–65. doi: 10.1038/345063a0. [DOI] [PubMed] [Google Scholar]
- Weichart D., Oliver J. D., Kjelleberg S. Low temperature induced non-culturability and killing of Vibrio vulnificus. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):205–210. doi: 10.1111/j.1574-6968.1992.tb14041.x. [DOI] [PubMed] [Google Scholar]
- Winding A., Binnerup S. J., Sørensen J. Viability of indigenous soil bacteria assayed by respiratory activity and growth. Appl Environ Microbiol. 1994 Aug;60(8):2869–2875. doi: 10.1128/aem.60.8.2869-2875.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winstanley C., Morgan J. A., Pickup R. W., Saunders J. R. Use of a xylE marker gene to monitor survival of recombinant Pseudomonas putida populations in lake water by culture on nonselective media. Appl Environ Microbiol. 1991 Jul;57(7):1905–1913. doi: 10.1128/aem.57.7.1905-1913.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yarmolinsky M. B. Programmed cell death in bacterial populations. Science. 1995 Feb 10;267(5199):836–837. doi: 10.1126/science.7846528. [DOI] [PubMed] [Google Scholar]