Kazumasa Matsumoto

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.

Establishment of an Analytical System for the Human Fecal Microbiota, Based on Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNA Molecules

ABSTRACT An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 102 to 104 cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 108 cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RT-qPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log10(6.3 ± 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.

Effects of a probiotic fermented milk beverage containing Lactobacillus casei strain Shirota on defecation frequency, intestinal microbiota, and the intestinal environment of healthy individuals with soft stools.

The effects of drinking a fermented milk beverage that contains Lactobacillus casei strain Shirota (LcS) at 40 billion bacterial cells/bottle for 4 weeks (probiotics, 1 bottle/day) on defecation frequency, intestinal microbiota and the intestinal environment of healthy individuals with soft stools were evaluated. Thirty-four healthy adults who had soft stools were randomised into 2 groups, and the effects of a regular 4-week intake of probiotics were evaluated by a placebo-controlled, double-blind, parallel-group comparative design. Defecation frequency significantly decreased after the 4-week intake period compared with before the probiotic treatment. The stool quality significantly improved (hardened) compared to the placebo. Also, the water content of the stools was lower in the probiotic group than in the placebo group. Live LcS was recovered at 6.9 ± 1.3 and 7.2 ± 0.8 log(10) CFU per 1g of stool after 2 and 4 weeks, respectively, of probiotic treatment. The number of bifidobacteria in the stools also increased significantly compared with the level before starting the probiotics. The organic acid levels (total, acetic acid, propionic acid, and butyric acid) significantly increased compared with the level before intake in both the probiotic and placebo groups, but they returned to the original levels after the end of the intake period. These results suggest that probiotic fermented milk beverage has an intestine-conditioning effect by improving the frequency of defecation and stool quality and increasing the intrinsic bifidobacteria in healthy individuals with soft stool.

Isolation of lactate-utilizing butyrate-producing bacteria from human feces and in vivo administration of Anaerostipes caccae strain L2 and galacto-oligosaccharides in a rat model

Lactate-utilizing butyrate-producers were isolated from human feces and identified based on the sequences of 16S rRNA gene. Anaerostipes caccae strain L2, one of the seven human fecal isolates, was administered to rats with galacto-oligosaccharides (GOS) as bifidogenic carbohydrates for stimulating lactate formation in the hindgut. Ingestion of GOS alone increased concentrations of cecal lactate and butyrate compared with control rats (P<0.05). Additional administration of strain L2 on GOS tended to enhance the promoting effect of GOS on cecal butyrate formation (P=0.06) and lowered the mean value of cecal lactate concentration (P=0.32). Consequently, cecal and fecal butyrate concentrations in rats administered with both strain L2 and GOS were significantly higher than those in the control rats (P<0.01 and P<0.05, respectively). Significant changes were observed in the other fermentation acids, such as succinate, acetate, and propionate, depending on the ingestion of strain L2. Administered strain L2 was retrieved from the cecal content of a rat based on randomly amplified polymorphic DNA analysis. The results suggest that synbiotic ingestion of lactate-utilizing butyrate-producers and GOS alters the microbial fermentation and promotes the formation of beneficial fermentation acids, including butyrate, in the gut.

M-RTLV agar, a novel selective medium to distinguish Lactobacillus casei and Lactobacillus paracasei from Lactobacillus rhamnosus

We developed a novel selective medium, modified-rhamnose-2,3,5-triphenyltetrazolium chloride-LBS-vancomycin agar (M-RTLV agar), that utilizes the fermentability of L-rhamnose to distinguish Lactobacillus casei and Lactobacillus paracasei from Lactobacillus rhamnosus. Whereas L. casei and L. paracasei formed red colonies on the M-RTLV agar, L. rhamnosus formed either pink-toned colonies or white colonies with a red spot. An intervention study was conducted to confirm the capability of M-RTLV agar to detect ingested L. casei when recovered from human feces. Subjects consumed one bottle daily of a fermented milk product (Yakult or Yakult Light, which contains L. casei strain Shirota; LcS) for 7 days. Diluents of the fecal samples were cultivated on M-RTLV agar. We were able to enumerate circular medium-sized red colonies, which were morphologically similar to L. casei/L. paracasei but clearly distinguishable from the remaining colonies owing to the color difference. These colonies were then subjected to enzyme-linked immunosorbent assay in order to identify the LcS. The viable counts of LcS were 6.6+/-0.7 log(10) CFU/g feces after intake of Yakult and 6.5+/-0.6 log(10) CFU/g feces after intake of Yakult Light (mean+/-SD).

Effects of the continuous intake of a milk drink containing Lactobacillus casei strain Shirota on abdominal symptoms, fecal microbiota, and metabolites in gastrectomized subjects

Abstract Objective. This article is based on our previously reported results of irregular bowel movement and disturbances of the intestinal microbiota/environment in gastrectomized patients. A placebo-controlled, double-blind comparative study was carried out to evaluate the effects of a fermented milk beverage containing Lactobacillus casei strain Shirota (LcS) in such patients. The major evaluated factors of this article were “bowel movement” and “quality of life.” The secondary evaluated factors were “fecal microbiota” and “enteric environment.” Methods. Of the 190 gastrectomized subjects who participated in our previously reported defecation survey, 134 subjects judged as having abnormal defecation gave consent to participate in this study. These subjects continuously ingested the test beverage containing 40 billion LcS or placebo (one bottle per day, 4 weeks). Results. In the LcS-ingested group, among the 118 subjects who completed the tests, the assessments of the subjects were based on their division into groups based on their symptoms with our scoring system for constipation/diarrhea; although there was no significant ingestion effect in total, in the constipation group, LcS reduced the degree of constipation compared with that in the placebo group. In the diarrhea group, LcS ingestion improved diarrhea compared with that in the preingestion state. Fecal Staphylococcus level was decreased. Conclusions. The results suggest the possibility that the continuous consumption of LcS-fermented milk relieves irregular bowel movement in gastrectomized patients.

Higher enterococcus counts indicate a lower risk of colorectal adenomas: a prospective cohort study

Intestinal bacteria play an important role in human health. This prospective cohort study aimed to investigate the relationship between the abundance of different intestinal bacteria and the risk of developing colorectal cancer (CRC). Fecal samples from CRC patients (n = 157) were collected at the start of the study wherein patients subsequently underwent endoscopy to remove polyps. Gut bacteria were isolated by using specific culture methods and the fecal counts of various bacteria were quantified by reverse-transcription-quantitative-PCR (RT-qPCR) assays. The obtained data were subjected to cohort analysis in relation to the incidence of colorectal adenomas after 4 years of intervention. No relationship was detected between the counts of major intestinal bacteria and the incidence of colorectal adenomas. However, interestingly, a significant negative correlation was noted between colorectal adenoma incidence and the counts of bacteria grown on Columbia blood agar base (COBA) (P = 0.007). The risk ratio of colorectal adenomas was 0.58 (95% CI: 0.35–0.96) in the group with the highest bacterial count compared to the lowest. Bacteria grown on COBA were more abundant in older patients, non-smoking patients, and patients with a lower body mass index. The RT-qPCR results revealed a significantly lower colorectal adenoma incidence in subjects with higher enterococcal count as compared to subjects with a lower count, with a risk ratio of 0.47 (95% CI: 0.30–0.76). Correlation of a higher enterococci count with a lower risk of CRC development suggests that certain Enterococcus strains may have adenoma suppressive effects.

Effect of Lactobacillus casei on Streptococcus bovis in faecal flora

Bacteraemia caused by Streptococcus bovis is often associated with colorectal tumours. Also, experimental studies have been proposed that S bovis acts as a promoter of tumours. We report the case of a man with colon adenoma who had a high proportion of S bovis in his faecal flora. He was treated with a Lactobacillus casei preparation (BLP) and the effect on the faecal flora was examined. L casei reduced the proportion of S bovis (from 43% down to 9%), and the effect continued after the administration of BLP was stopped. Our data indicate that BLP can repress the excessive colonisation of S bovis.