Takahiro Matsuki

Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Human Feces

ABSTRACT 16S rRNA gene-targeted group-specific primers were designed and validated for specific detection and quantification of the Clostridium leptum subgroup and the Atopobium cluster. To monitor the predominant bacteria in human feces by real-time PCR, we used these specific primers together with four sets of group-specific primers for the Clostridium coccoides group, the Bacteroides fragilis group, Bifidobacterium, and Prevotella developed in a previous study (T. Matsuki, K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka, Appl. Environ. Microbiol. 68:5445-5451, 2002). Examination of DNA extracted from the feces of 46 healthy adults showed that the C. coccoides group was present in the greatest numbers (log10 10.3 ± 0.3 cells per g [wet weight] [average ± standard deviation]), followed by the C. leptum subgroup (log10 9.9 ± 0.7 cells per g [wet weight]), the B. fragilis group (log10 9.9 ± 0.3 cells per g [wet weight]), Bifidobacterium (log10 9.4 ± 0.7 cells per g [wet weight]), and the Atopobium cluster (log10 9.3 ± 0.7 cells per g [wet weight]). These five bacterial groups were detected in all 46 volunteers. Prevotella was found in only 46% of the subjects at a level of log10 9.7 ± 0.8 cells per g (wet weight). Examination of changes in the population and the composition of the intestinal flora for six healthy adults over an 8-month period revealed that the composition of the flora of each volunteer remained stable throughout the test period.

Development of 16S rRNA-Gene-Targeted Group-Specific Primers for the Detection and Identification of Predominant Bacteria in Human Feces

ABSTRACT For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

ABSTRACT A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >106 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

ABSTRACT Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA.

Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The methods ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPD analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2)=0.999, P<0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days, 10(9.1+/-0.5) LcS g(-1) (mean+/-S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0+/-0.9) CFU g(-1) was detected by culture; these values were significantly different (P<0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method.

Gastric acid reduction leads to an alteration in lower intestinal microflora

To clarify the alterations in lower intestinal microflora induced by gastric acid reduction, the dynamics of 12 major genera or groups of bacteria comprising the microflora in feces and colonic contents were examined by quantitative real-time PCR in proton pump inhibitor-treated rats and in asymptomatic human subjects with hypochlorhydria. In both rat and human experiments, most genera or groups of intestinal microflora (facultative and obligate anaerobes) proliferated by gastric acid reduction, and marked and significant increases in the Lactobacilli group and Veillonella, oropharyngeal bacteria, were observed. In rats, potent gastric acid inhibition led to a marked and significant increase of intestinal bacteria, including the Bacteroidesfragilis group, while Bifidobacterium, a beneficial bacterial species, remained at a constant level. These results strongly indicate that the gastric acid barrier not only controls the colonization and growth of oropharyngeal bacteria, but also regulates the population and composition of lower intestinal microflora.

Effects of the enteral administration of Bifidobacterium breve on patients undergoing chemotherapy for pediatric malignancies

PurposeProbiotics are expected to be effective in prophylaxis of infection in cancer patient, since infections in neutropenics are mainly caused by endogenous flora through the intestinal mucosa. However, the experience with the use of probiotics in immunocompromised patients is limited, and precise fecal bacteria analysis has not been reported. The aim of the study was to evaluate the effects of the enteral administration of the probiotic, Bifidobacterium breve strain Yakult, on its ability to prevent infection, fecal micro flora, and intestinal environments in cancer patients on chemotherapy.MethodsA placebo-controlled trial was performed at Juntendo University Hospital. Patients with malignancies admitted for chemotherapy (n = 42) were randomized into two groups receiving probiotic or placebo. The effects on infectious complications, natural killer cells, fecal micro flora, fecal organic acid concentrations, and fecal pH were studied.ResultsThe frequency of fever and the use of intravenous antibiotics were lower in the probiotic group than the placebo group. The probiotic administration enhanced the habitation of anaerobes. Disruption of the intestinal microbiota after chemotherapy such as the increase in the population levels of Enterobacteriaceae was observed at more pronounced manner in the placebo group in comparison to the probiotic group. The concentrations of total organic acids were maintained most of the time at the normal level, which constantly maintained the pH below 7.0 only in the probiotic group.ConclusionThese data, although based on a limited number of patients and samples, suggest that administration of B. breve strain Yakult could be an effective approach for achieving clinical benefits in immunocompromised hosts by improving their intestinal environments.