Quantification of Bacteria Adherent to Gastrointestinal Mucosa by Real-Time PCR

Abstract

The use of real-time quantitative PCR (5 nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides vulgatus. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5 nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to E. coli and B. vulgatus and with eight other gut microflora species. Mucosal samples spiked with known amounts of E. coli or B. vulgatus DNA showed no PCR inhibition. We conclude that the 5 nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.The human intestinal tract harbors a large and complex community of microbes which is involved in maintaining human health by preventing colonization by pathogens and by producing nutrients. At least 400 bacterial species are thought to be present in the human gastrointestinal tract (4, 9, 14, 19). Microorganisms are not randomly distributed throughout the intestine. The flora of the stomach and proximal small intestine differs significantly from that of the terminal ileum and colon. The numerically predominant species of bacteria are obligate anaerobes and are represented by both gram-positive and gram-negative genera. The most frequently identified anaerobic microorganisms are Bacteroides spp., Bifidobacterium spp., Eubacterium spp., Peptostreptococcus spp., and Fusobacterium spp. (17). The total number of bacterial cells, as determined by fecal culture methods, increases gradually from the small intestine to the large bowel and reaches 10 11 to 10 12 cells/ml of contents. Conventional bacteriological methods like microscopy, culture, and identification are used for the analysis and/or quantification of the intestinal microflora (7,16,20). Limitations of conventional methods are their low sensitivities (5), their inability to detect noncultivatable bacteria and unknown species, their time-consuming aspects, and their low levels of reproducibility due to the multitude of species to be identified and quantified. In addition, the large differences in growth rates and growth requirements of the different species present in the human gut indicate that quantification by culture is bound to be inaccurate. To overcome the problems of culture, techniques based on 16S ribosomal DNA (rDNA) genes were developed (2, 22). These include fluorescent in situ hybridization (8,11,12,13,21), denaturing gradient gel electrophoresis (15, 18), and temperature gradient gel electrophoresis (6, 23). These techniques have low sensitivities and are laborious and technically demanding. We evaluated the suitability of a rea...

Comparison of Real-Time PCR and Culture for Detection of Porphyromonas gingivalis in Subgingival Plaque Samples

“…To check the efficacy of DNA isolation from subgingival plaque samples, a known amount of E. coli (K-12) DNA (equivalent to 50 CFU/reaction mixture) was added to each plaque sample before DNA isolation. After isolation, the DNA was analyzed by real-time PCR with an E. coli-specific primer-probe combination (8). The fluorescent signal was compared to the signals on a standard curve generated with E. coli DNA.…”

GntP Is the Escherichia coli Fructuronic Acid Transporter and Belongs to the UxuR Regulon

“…Taqman probes and primers were designed by using Primer Express software provided with the ABI Prism 7000 sequence detector (Applied Biosystems). Transcript levels were normalized to 16S rRNA as described previously (9), and the values were expressed relative to cells grown on glucose.…”

Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures

“…In a study carried out on the quantification of Campylobacter lanienae in bovine feces by realtime PCR, the limit of reliable quantification was one to eight genomes (Inglis and Kalischuk, 2004). The use of real-time PCR as a tool to study the gastrointestinal microbiota that adheres to the colonic mucosa was also evaluated (Huijsdens et al, 2002). The assay, employing probes and primers targeting the 16S rRNA gene, proved to be very sensitive as levels as low as 1 CFU E. coli and 9 CFU Bacteroides vulgatus could be detected.…”

“…Limitations associated with conventional culturing methods include low sensitivities (Dutta et al, 2001), inability to detect unculturable bacteria and unknown species, slow turnabout time, and poor reproducibility (Huijsdens et al, 2002). In addition, as large differences have been noted in the growth rates and growth requirements of different species, quantification by culture is highly likely to be inaccurate (Huijsdens et al, 2002). Furthermore, because of the ill-defined nature of the accompanying microbiota in NTCs, enumeration of specific species in such populations often proves problematic (O'Sullivan, 2000).…”