A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

Background: Discrimination among viable, active, and inactive cells in aqua tic ecosystems is of great importance to understand which species participa te in microbial processes. In this study, a new approach combining flow cyt ometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the div ersity of total cells and active cells. Methods: Total bacteria were determined by SYBR-II staining. Viable bacteri a were determined in water samples from different sites by plate count tech niques and by the direct viable count (DVC) method. Substrate-responsive ce lls (i.e., DVC+ cells) were distinguished from nonresponsive cells (i.e., D VC- cells) by FCM and sorted. The genetic diversity of the sorted cell frac tion was compared with the diversity of the total microbial community and w ith that of the culturable cell fraction by denaturing gradient gel electro phoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragm ents. The same approach was applied to a seawater sample enriched with nutr ients. In this case, actively respiring cells (CTC+) were also enumerated b y FCM, sorted, and analyzed by DGGE. Results: The diversity of viable cells varied depending on the methods (tra ditional culture or DVC) used for viability assessment. Some phylotypes det ected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring c ells. Inversely, some phylotypes found at the community level were not foun d in viable and active cell-sorted fractions. It suggests that diversity de termined at the community level includes nonactive and nonviable cells. Conclusion: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbia l ecology such as the identification of species able to maintain cellular a ctivity under environmental changes or in the presence of toxic compounds a re now possible. Cytometry 43:314-321, 2001. (C) 2001 Wiley-Liss. Inc.