This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ben-Amor, K. Articles by de Vos, W. M. Search for Related Content PubMed PubMed Citation Articles by Ben-Amor, K. Articles by de Vos, W. M. Agricola Articles by Ben-Amor, K. Articles by de Vos, W. M.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2005, p. 4679-4689, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4679-4689.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Diversity of Viable, Injured, and Dead Fecal Bacteria Assessed by Fluorescence-Activated Cell Sorting and 16S rRNA Gene Analysis

Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands,¹ Laboratory of Food Microbiology, Wageningen University, Wageningen, The Netherlands,² Wageningen Center for Food Sciences, Wageningen, The Netherlands³

Received 1 December 2004/ Accepted 28 February 2005

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.

This article has been cited by other articles:

• Jung, W. K., Koo, H. C., Kim, K. W., Shin, S., Kim, S. H., Park, Y. H. (2008). Antibacterial Activity and Mechanism of Action of the Silver Ion in Staphylococcus aureus and Escherichia coli. Appl. Environ. Microbiol. 74: 2171-2178 [Abstract] [Full Text]  
• Nocker, A., Sossa-Fernandez, P., Burr, M. D., Camper, A. K. (2007). Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology. Appl. Environ. Microbiol. 73: 5111-5117 [Abstract] [Full Text]  
• Dinoto, A., Marques, T. M., Sakamoto, K., Fukiya, S., Watanabe, J., Ito, S., Yokota, A. (2006). Population Dynamics of Bifidobacterium Species in Human Feces during Raffinose Administration Monitored by Fluorescence In Situ Hybridization-Flow Cytometry. Appl. Environ. Microbiol. 72: 7739-7747 [Abstract] [Full Text]  
• Shen, J., Zhang, B., Wei, G., Pang, X., Wei, H., Li, M., Zhang, Y., Jia, W., Zhao, L. (2006). Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis. Appl. Environ. Microbiol. 72: 5232-5238 [Abstract] [Full Text]