ABSTRACT
The assessment of the physiological state of the bacteria in drinking water is a critical issue, especially with respect to the presence of pathogenic bacteria. Though molecular methods can provide insight into the taxonomic composition of the drinking water microflora, the question if a bacterial species is alive or dead still needs to be addressed. To distinguish live and dead bacteria at the taxonomic level, we combined three methods: i) a staining procedure indicating membrane-injured cells (using SYTO9 and Propidium Iodide) that is considered to distinguish between live and dead cells, ii) Fluorescence Activated Cell Sorting (FACS) of the membrane injured and intact bacteria, and iii) molecular analyses of the RNA extracted from the bacteria before and after sorting to analyse the bacterial community at the species level. By staining and FACS analysis the drinking water bacteria could be separated according to their different membrane integrities, and RNA could be extracted from the live and dead sorted bacterial fractions. 16S rRNA based fingerprints revealed a diverse bacterial community in the drinking water samples with the majority being represented by 31 identified phylotypes. Most of the phylotypes referenced belonged to the phyla Proteobacteria (Alpha-, Beta-, Gamma-), Cyanobacteria and Bacteroidetes, and were mostly related to freshwater bacteria. 90% of the total phylotypes could be recovered after FAC-Sorting; 32% of the phylotypes occurred only in the "live" sorted fraction, 21% only in the "dead" sorted fraction, and 46% occurred in both fractions.
Subject(s)
Bacteria/isolation & purification , Water Microbiology/standards , Water Supply/standards , Bacteria/classification , Bacteria/drug effects , Bacteria/genetics , Bacteroides/classification , Bacteroides/genetics , Bacteroides/isolation & purification , Cell Count , Cyanobacteria/classification , Cyanobacteria/genetics , Cyanobacteria/isolation & purification , Flow Cytometry , Fresh Water/microbiology , Phylogeny , Propidium/pharmacology , Proteobacteria/classification , Proteobacteria/genetics , Proteobacteria/isolation & purification , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/isolation & purificationABSTRACT
BACKGROUND: Regulatory T cells (T(regs)) have potential anti-inflammatory effects and are likely to be important in the pathogenesis of chronic inflammatory bowel disease (IBD). However, the induction and expansion of T(regs) at sites of mucosal inflammation are not yet fully understood and may involve antigen presentation by local dendritic cells (DCs) and/or intestinal epithelial cells (IECs). METHODS: To determine the unique ways in which the gut induces or expands T(regs), a transgenic mouse model that is based on the specific expression of a model autoantigen (influenza haemagglutinin (HA)) in the intestinal epithelium (VILLIN-HA) was used. Gut-associated DCs and IECs isolated from these mice were phenotypically and functionally characterised for the potential to interact with HA-specific T(regs) in vitro and in vivo. RESULTS: Intestinal self-antigen expression leads to peripheral expansion of antigen-specific CD4(+)Foxp3(+) T(regs). Although gut-associated DCs can induce antigen-specific CD4(+)Foxp3(+) T cell proliferation, in vivo depletion of DCs did not preclude proliferation of these cells. Interestingly, antigen presentation by primary IECs is sufficient to expand antigen-specific CD4(+)Foxp3(+) T(regs) efficiently. This is dependent on major histocompatibility complex class II, but, in contrast to DCs, is unlikely to require transforming growth factor beta and retinoic acid. CONCLUSION: This study provides experimental evidence for a new concept in mucosal immunity: in contrast to current thinking, expansion of T(regs) can be achieved independently of local DCs through antigen-specific IEC-T cell interactions.
