LONG-TERM SURVIVAL OF AND PLASMID STABILITY IN PSEUDOMONAS AND KLEBSIELLA SPECIES AND APPEARANCE OF NONCULTURABLE CELLS IN AGRICULTURAL DRAINAGE WATER

One year after introduction into agricultural drainage water Pseudomonas fluorescens R2f (RP4), Pseudomonas putida CYM318 (pRK2501), and Klebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air. In contrast to K. aerogenes NCTC418 (pBR322), both Pseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with32P-labelled plasmid RP4 DNA was found in several drainage water samples that originally received P. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 and K. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts. © 1990 Springer-Verlag New York Inc.