16S rRNA Gene Copy Numbers Research Articles

Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota.

Fecal bacteria were studied in healthy elderly volunteers (age, 63 to 90 years; n = 35) living in the local community, elderly hospitalized patients (age, 66 to 103; n = 38), and elderly hospitalized patients receiving antibiotic treatment (age, 65 to 100; n = 21). Group- and species-specific primer sets targeting 16S rRNA genes were used to quantitate intestinal bacteria by using DNA extracted from feces and real-time PCR. The principal difference between healthy elderly volunteers and both patient cohorts was a marked reduction in the Bacteroides-Prevotella group following hospitalization. Reductions in bifidobacteria, Desulfovibrio spp., Clostridium clostridiiforme, and Faecalibacterium prausnitzii were also found in the hospitalized patients. However, total 16S rRNA gene copy numbers (per gram of wet weight of feces) were generally lower in the stool samples of the two groups of hospitalized patients compared to the number in the stool samples of elderly volunteers living in the community, so the relative abundance (percentage of the group- and species-specific rRNA gene copies in relation to total bacterial rRNA gene copies) of bifidobacteria, Desulfovibrio spp., C. clostridiiforme, and F. prausnitzii did not change. Antibiotic treatment resulted in further reductions in the numbers of bacteria and their prevalence and, in some patients, complete elimination of certain bacterial communities. Conversely, the numbers of enterobacteria increased in the hospitalized patients who did not receive antibiotics, and due to profound changes in fecal microbiotas during antibiotic treatment, the opportunistic species Enterococcus faecalis proliferated.

Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities

We developed standard curves based on plasmids containing a 16S rRNA gene of a member of one of the three genera Dehalobacter, Desulfitobacterium, and Dehalococcoides. A large difference in amplification efficiency between the standard curves was observed ranging from 1.5 to 2.0. The total eubacterial 16S rRNA gene copy number determined in a sample DNA by using eubacterial primers and the three standard curves led to differences in the estimated copy numbers of a factor up to 73. However, the amplification efficiencies for one specific standard curve were the same independent of the PCR primer pair used. This allowed the determination of the abundance of a population expressed as fractional number, hence, the percentage of genus-specific copy numbers within the total eubacterial 16S rRNA gene copy numbers. Determination of the fractional numbers in DNA mixtures of known composition showed the accuracy of this approach. The average difference in threshold value between two 10-fold dilutions of DNA of pure cultures, mixtures thereof and of environmental samples was −3.45±0.34, corresponding to an average almost optimal amplification efficiency of 1.95. This indicated that the low amplification efficiency of certain standard curves seemed to be mainly a problem of the plasmid DNA used and not of the 16S rRNA gene of the target genera.

Nitrification and nitrifying bacteria in the lower Seine River and estuary (France).

The Achères wastewater treatment plant, located just downstream of Paris, discharges its effluents into the lower Seine River. The effluents contain large numbers of heterotrophic bacteria, organic matter, and ammonium and are a source of nitrifying bacteria. As a result, degradation of organic matter by heterotrophic bacteria and subsequent oxygen depletion occur immediately downstream of the effluent outlet, whereas nitrifying bacteria apparently need to build up a significant biomass before ammonium oxidation significantly depletes the oxygen. We quantified the potential total nitrifying activity and the potential activities of the ammonia- and nitrite-oxidizing communities along the Seine River. In the summer, the maximum nitrifying activity occurs in the upper freshwater estuary, approximately 200 km downstream of Achères. The quantities of nitrifying bacteria, based on amoA gene copy numbers, and of Nitrobacter organisms, based on 16S rRNA gene copy numbers, were correlated with the potential nitrifying activities. The species composition of ammonia-oxidizing bacteria was investigated at two sites: the Triel station just downstream from Achères (km 84) and the Seine freshwater estuary at the Duclair station (km 278). By means of PCR primers targeting the amoA gene, a gene library was created. Phylogenetic analysis revealed that the majority of the analyzed clones at both sites were affiliated with the genus NITROSOMONAS: The Nitrosomonas oligotropha- and Nitrosomonas urea-related clones represented nearly 81% of the community of ammonia-oxidizing bacteria at Triel and 60% at Duclair. Two other ammonia-oxidizing clusters of the beta subclass of the Proteobacteria, i.e., Nitrosomonas europaea- and Nitrosospira-like bacteria, were found in smaller numbers. The major change in the ammonia-oxidizing community between the two stations along the Seine River-upper estuary continuum was the replacement of the N. oligotropha- and N. urea-related bacteria by the Nitrosospira-affiliated bacteria. Although the diversities of the ammonia oxidizers appear to be similar for the two sites, only half of the restriction patterns are common to both sites, which could be explained by the differences in ammonium concentrations, which are much lower in the upper estuary than in the river at the effluent outlet. These results imply a significant immigration and/or selection of the ammonia-oxidizing bacterial population along the continuum of the Seine River from Paris to the estuary.

Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge

A culture-independent molecular technique using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes was applied to the characterization of bacterial communities of activated sludge taken from different municipal sewage treatment plants. 16S rDNA fragments from the bulk DNA of sludge were amplified by PCR with a Cy5-labeled forward primer corresponding to nucleotide positions 8 to 27 and a reverse primer complementary to positions 907 to 926 in the Escherichia coli numbering system. The 16S rDNAs thus obtained were digested with tetrameric restriction enzymes and analyzed using a Pharmacia automated DNA sequencer. A preliminary study on a model DNA mixture prepared from different bacterial species showed that the fluorescence intensity of terminal fragments (T-RFs) of 16S rDNAs amplified and detected was directly proportional to the 16S rRNA gene copy number rather than the amount of genomic DNA of each species present. 16S rDNA fragments amplified from the sludges and digested with HhaI usually generated at least 60 T-RFs, among which T-RFs of around 208 bp were the most abundant regardless of the time or area sampled. Southern blot hybridization with oligonucleotide probes specific to the 5′ terminal regions of the 16S rDNA of different phylogenetic groups indicated that the T-RFs of around 208 bp were derived from members of the β subclass of the class Proteobacteria. Hybridization with a probe specific to the class Actinobacteria failed to detect any appreciable signal. These results did not agree fully with those obtained by quinone profiling. The usefulness and limitations of the T-RFLP method for monitoring bacterial population dynamics in activated sludge were discussed.

Terminal Restriction Pattern Analysis of 16S rRNA Genes for the Characterization of Bacterial Communities of Activated Sludge.

A culture-independent molecular technique using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes was applied to the characterization of bacterial communities of activated sludge taken from different municipal sewage treatment plants. 16S rDNA fragments from the bulk DNA of sludge were amplified by PCR with a Cy5-labeled forward primer corresponding to nucleotide positions 8 to 27 and a reverse primer complementary to positions 907 to 926 in the Escherichia coli numbering system. The 16S rDNAs thus obtained were digested with tetrameric restriction enzymes and analyzed using a Pharmacia automated DNA sequencer. A preliminary study on a model DNA mixture prepared from different bacterial species showed that the fluorescence intensity of terminal fragments (T-RFs) of 16S rDNAs amplified and detected was directly proportional to the 16S rRNA gene copy number rather than the amount of genomic DNA of each species present. 16S rDNA fragments amplified from the sludges and digested with HhaI usually generated at least 60 T-RFs, among which T-RFs of around 208 bp were the most abundant regardless of the time or area sampled. Southern blot hybridization with oligonucleotide probes specific to the 5′ terminal regions of the 16S rDNA of different phylogenetic groups indicated that the T-RFs of around 208 bp were derived from members of the β subclass of the class Proteobacteria. Hybridization with a probe specific to the class Actinobacteria failed to detect any appreciable signal. These results did not agree fully with those obtained by quinone profiling. The usefulness and limitations of the T-RFLP method for monitoring bacterial population dynamics in activated sludge were discussed.

Marker enzyme activities for plasma membrane and intracellular organelles in normal and dysmature foals : C.M. Cottrill, C.A. Seymour a, J. Ousey, A. McGladdery and P.D. Rossdale, Beaufort Cottage Stables, Newmarket, Suffolk and aDept. Medicine, Addenbrooke's Hospital, Cambridge, U.K.

The effects of dietary supplementation with two levels (45 and 90 mg/kg diet, diets PTS-O-45 and PTS-O-90, respectively) of the garlic (Allium sativum)-derived product PTS-O (propyl propane thiosulfonate) on the intestinal microbiota composition and digestibility of nutrients were studied in growing broiler chickens. Lower (P<0.05) lactobacilli log10 16S rRNA gene copy numbers with respect to controls were determined in the crop contents of birds fed on both PTS-O supplemented diets. In the ileal contents, PTS-O-45 feeding resulted in lower (P<0.05) Clostridium coccoides/Eubacterium rectale and Clostridium leptum log10 number of copies. Feeding the PTS-O-90 diet resulted in higher (P<0.05) bacteroides and total bacteria in the ileal contents. Lower (P<0.05) bacteroides log10 number of copies were determined in the caecal contents of birds fed on PTS-O-90, while C. coccoides/E. rectale number of copies of chickens fed on PTS-O-45 was also lower (P<0.05) than controls. Energy, fat, ADF and NDF fecal digestibilities were greater (P<0.05) than controls in chickens fed on diets containing PTS-O. N and NSP fecal digestibilities were greater (P<0.05) than controls in chickens fed diets containing PTS-O-90. No significant effect was found in the enzymatic activities (sucrase, maltase, isomaltase, aminopeptidase, and alkaline phosphatase) of ileal mucosal samples between controls and PTS-O fed birds. This investigation confirmed previous data, and showed that PTS-O modulated intestinal microbiota composition and improved nutrients digestibility in growing broilers.