Assessment Of Microbiota Vitality And Metabolic Activity

The aforementioned molecular techniques have greatly contributed to our fundamental understanding of the biodiversity, establishment, succession and structure of the intestinal microbiota; yet little is known about the in situ association between the microbial diversity and the metabolic activity of a phylogenetic affiliated group. A further challenge is to determine the physiological activity of the detected cells. This includes those cells that are naturally present within the ecosystem as well as the ingested members from fermented or functional foods. Moreover, the use of specific food-grade lactic acid bacteria as vectors for therapeutic delivery of molecules with targeted activity in the host is being investigated (111,112). These bacteria appear capable of surviving and of being physiologically active at the mucosal surfaces in animal models. Biological containment systems are being developed for these genetically modified lactic acid bacteria to limit their activity to the host and allow their use in human healthcare (113).

In Situ Activity

Quantitative hybridization with fluorescent rRNA probes (as in FISH) is a useful indicator of activity as there is a correlation between the growth rate, which is coupled to efficient protein synthesis, and the number of ribosomes. The FISH technique has been used to estimate growth rates of Escherichia coli cells colonizing the intestinal tract of mice (114). In situ activity of pure cultures of the human commensal Lactobacillus plantarum strain has been measured by correlating the rRNA, as determined by fluorescence intensity, with the cell growth rate (72). However, at very high cell densities, a typical property of L. plantarum at late stages of growth, changes in the cell envelope appeared to prevent effective entry of the probe into the cells. Permeabilization issues may confound application of this technique to certain microbes in complex environments like the intestine. Furthermore, recent data suggest that cellular ribosome content is not always an indicator of physiological activity. Apparently some bacterial cells might be highly active but possess a low ribosome content (115), while other bacterial types possess high RNA even after extended starvation periods (116).

During the last years several innovative methods have been developed to resolve the linkage between taxonomic identity, activity and function in microbial communities. One of these techniques involves microautoradiography (MAR), which when combined with FISH (MAR-FISH), determines the uptake of specific radiochemicals by individual cells (117,118). MAR-FISH allows monitoring of the radio-labeled substrate uptake patterns of the probe-identified organisms under different environmental conditions (117,119). This method has been applied with high throughput DNA microarray analysis to study the complex activated sludge ecosystem (120).

Linking Taxomony to Function

Another recently developed molecular technique coupled with substrate labeling is stable isotope probing (SIP) (121,122). In SIP, either lipid biomarkers (123), DNA (121) orRNA (124) are extracted from microbial communities incubated with 13C-labeled substrates. If cells grow on the added compounds, their pool of macromolecules will be isotopically enriched (heavy) compared to those of inactive organisms. For DNA- or RNA-SIP, identification of the metabolically active organisms (heavy) is achieved by separation of community DNA/RNA according to their buoyant density by means of equilibrium density-gradient centrifugation, followed by PCR-amplification of 16S rRNA genes in the isotopically heavy DNA/RNA pool, cloning and sequencing. The use of RNA was proposed as a more responsive biomarker as its turnover is much higher than that of DNA (124). Phospholipid fatty acids are also used as biomarker for 13C enrichments, but their resolution for diversity analysis is less powerful than for sequence analysis.

Reporters to Monitor Gene Expression

Molecular reporter systems may also be used to monitor activity of specific genes of a microbe of the complex intestinal ecosystem. Generally this involves fusing the reporter gene to the promoter of the bacterial gene of interest, such as stress- and starvation-induced genes and other growth physiology-related genes. It is noteworthy that this approach involves a genetically modified microbe, and consequently, its application is limited to animal studies. The adaptation of ingested lactic acid bacteria has received particular attention in terms of how they adapt their metabolism in order to survive and colonize within the gastrointestinal niches.

The fusion of bacterial promoters from Lactococcus lactis with genes of the reporter protein luciferase (luxA-luxB genes of Vibrio harveyi) was developed to investigate gene expression of this food-grade bacteria in the mouse intestinal tract (125). L. lactis strains marked with reporter genes for luciferase and the green fluorescent protein (GFP; from Aequorea victoria) were studied for their metabolic activity and survival by assessment of lysis, respectively, which revealed differential expression depending on the intestinal conditions and mode of administration (126). Following consumption by rats and analysis of the strains in the different regions of the intestinal tract, the lactococci were demonstrated to survive gastric transit quite well but the majority lost activity and underwent lysis in the duodenum. The luciferase gene reporter system has also been applied to a probiotic Lactobacillus casei strain that is added to fermented dairy products. The luciferase-harboring L. casei derivative was consumed in milk by mice harboring human microbiota. Luciferase activity was undetectable in the stomach to jejunum, but detected when the cells reached the ileum, and the activity remained at a maximum level in the cecum, confirming reinitiation of protein synthesis in the ileal and cecal compartments (127,128).

Several variants of the GFP have been developed such as GFPs with alternative emission wavelengths, or with reduced stability to monitor shifts in gene expression (129,130).

Flow Cytometry-Based Approaches

FCM in combination with a variety of fluorescent physiological probes and cell sorting analysis is invaluable for measuring viability of cells in environmental samples (80,87,131,132). Ability to grow in medium is the current standard to assess viability, but it is recognized that some cells enter a non-culturable state although still exhibit metabolic activity. The criteria by which viability is evaluated by the FCM include membrane permeability or integrity, enzyme activity, and/or maintenance of a membrane-potential (Fig. 2). One of the most widely used dyes for assessment of viability is carboxy-fluorescein diacetate, a non-fluorescent precursor that diffuses across the cell membrane, but is retained only by viable cells with intact membranes which convert it into a membrane-impermeant fluorescent dye by non-specific esterases of active cells. Another probe is PI, a nucleic acid dye, which is excluded by viable cells with intact membranes, but enters cells with damaged membranes and binds to their DNA or RNA. Simultaneous staining of fecal Bifidobacterium species with these two probes was used to assess their viability during bile salt stress (133). Subsequent detection with the FCM and cell sorting revealed three populations representing viable, injured and dead cells, whereby a significant portion (40%) of the injured cells could be cultured. This approach highlights the importance of multi-parametric FCM as a powerful technique to monitor physiological heterogeneity within stressed populations at the single cell level.

FCM also allows monitoring of bacterial heterogeneity at the single cell level and provides a mean to sort sub-populations of interest for further molecular analysis (15). Recently, the viability of fecal microbiota in fecal samples was assessed by combining a viability assay with flow sorting, and subsequent analysis by PCR-DGGE and identification by cloning and 16S rRNA sequencing (80). The fecal cells of four adults were initially discriminated with physiological probes PI and SYTO BC into viable, injured and dead cells. This revealed that only approximately half of the microbial community in fecal samples is viable, while the remainder was injured or dead (about a quarter each of the total community). This is in agreement with a previous analysis of proportions of dead bacteria in 10 persons which ranged from 17% to 34%, as assessed by PI only (134). The 16S rRNA analysis indicated which bacterial groups comprised live, dead or injured populations, for example many butyrate-producers were in the live fractions, while many clones from Bacteroides were found in the dead fractions (80). Specific PCR-DGGE and 16S rRNA analysis of the bifidobacteria! and lactobacilli populations showed sequences with low similarity to the characterized species suggested the potential of as yet uncultured novel species in humans (80,135). This interesting combination of technologies provided ecological information on the in situ diversity and activity of the fecal microbes.

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