Evaluation of 16s rRNA and cellular fatty acid profiles as markers of human intestinal bacterial growth in the chemostat

Abstract

Chemostats were used to study the effects of carbon and nitrogen limitation and specific growth rate on 16S rRNA synthesis and cellular fatty acid (CFA) profiles in four human intestinal bacteria (Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Clostridium bifermentans and Cl. difficile). Cellular fatty acid synthesis varied with dilution rate and nutrient availability in different species, but these cellular constituents were relatively stable phenotypic characteristics in Bact. thetaiotaomicron, where branched chain and hydroxy CFA were good taxonomic markers. Conversely, CFA in the Gram‐positive bacteria varied markedly with changes in growth environment. For example, in chemostats, cyclopropane CFA were only synthesized in Cl. bifermentans and Cl. difficile under N‐limited conditions. Similarly, Dimethyl acetal (DMA) fatty acids in Bif. adolescentis were primarily produced during N‐limited growth, and this was inversely related to dilution rate. At low growth rates, 16S rRNA concentrations (µg rRNA per ml culture) correlated with viable bacterial counts, but were more closely related to specific growth rate when expressed as a function of cell mass (µg rRNA per mg dry weight bacteria). However, this did not reveal differences in bacterial population size and rRNA concentration in C‐limited cultures. Thus, at low dilution rates, C limitation strongly reduced rRNA synthesis in Cl. bifermentans, despite viable cell counts being similar to those in N‐limited cultures. These results indicate that, while 16S rRNA is a useful indicator of microbial activity, cell growth rate does not necessarily relate to rRNA concentration under all nutritional conditions. Consequently, bowel habit and diet will affect both CFA and rRNA content in bacteria isolated from intestinal samples, and this should be taken into consideration when interpreting such data measurements.

Characterisation of intestinal bacteria in infant stools using real-time PCR and northern hybridisation analyses

“…To our knowledge, this investigation is the first to demonstrate that SRB can be detected in the stools of very young infants (0–6 months of age). With respect to DNA quantitation, desulfovibrio populations were found to be significantly lower in the stools of this group than in elder infants, although this was not consistent with the RNA analysis, which also gives an indication of metabolic activity [44]. This emphasises an important difference between the two techniques: with real‐time PCR, a specific measure of cell number is achievable, while northern hybridisations give a value that is a percentage of total bacterial 16S rRNA in a sample.…”

Assessment of lactation stage and breed effect on sheep milk fatty acid profile and lipid quality indices

“…Vlaeminck et al (2006) reported that odd-and branched-chain fatty acids in milk fat are largely derived from bacteria leaving the rumen. These fatty acids can be found in animal tissues, especially those of ruminants, and their content can often be used as a taxonomic marker (Hopkins and Macfarlane 2000).…”

Effect of short-chain carbohydrates on human intestinal bifidobacteria and Escherichia coli in vitro

“…Identi®cation according to this method is based upon comparison with a library of FAME pro®les for bacteria grown in identical conditions. The technique is used routinely in this laboratory for the identi®cation of EFFECT OF CARBOHYDRATES ON BIFIDOBACTERIA bacteria at the genus and often the species level [31,32].…”

“…Probe groups targeted were total bacterial rRNA, a Bi®dobacterium genus probe, and a probe for the enterobacterial group. Synthetic HPLC-puri®ed oligonucleotide probes were 59 end-labelled with 32 P by means of polynucleotide kinase (Gibco BRL) and [ã-32 P] ATP (ICN, The Netherlands) with a speci®c activity of .5000 Ci/ mmol and a concentration of 10 mCi/ml, as described previously [16].…”