Effect of probiotic yoghurt on animal-based diet-induced change in gut microbiota: an open, randomised, parallel-group study.

Diet has a significant influence on the intestinal environment. In this study, we assessed changes in the faecal microbiota induced by an animal-based diet and the effect of the ingestion of yoghurt supplemented with a probiotic strain on these changes. In total, 33 subjects were enrolled in an open, randomised, parallel-group study. After a seven-day pre-observation period, the subjects were allocated into three groups (11 subjects in each group). All of the subjects were provided with an animal-based diet for five days, followed by a balanced diet for 14 days. Subjects in the first group ingested dairy in the form of 200 g of yoghurt supplemented with Bifidobacterium longum during both the animal-based and balanced diet periods (YAB group). Subjects in the second group ingested yoghurt only during the balanced diet period (YB group). Subjects who did not ingest yoghurt throughout the intervention were used as the control (CTR) group. Faecal samples were collected before and after the animal-based diet was provided and after the balanced diet was provided, followed by analysis by high-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene. In the YB and CTR groups, the animal-based diet caused a significant increase in the relative abundance of Bilophila, Odoribacter, Dorea and Ruminococcus (belonging to Lachnospiraceae) and a significant decrease in the level of Bifidobacterium after five days of intake. With the exception of Ruminococcus, these changes were not observed in the YAB group. No significant effect was induced by yoghurt supplementation following an animal-based diet (YB group vs CTR group). These results suggest that the intake of yoghurt supplemented with bifidobacteria played a role in maintaining a normal microbiota composition during the ingestion of a meat-based diet. This study protocol was registered in the University Hospital Medical Information Network: UMIN000014164.

Effects of heat-killed Lactobacillus kunkeei YB38 on human intestinal environment and bowel movement: a pilot study.

It is well known that lactic acid bacteria supplementation is beneficial for intestinal conditions such as microbiota; however, the effects of killed-lactic acid bacteria on intestinal conditions are largely unclear. This study aimed to evaluate the effect of heat-killed Lactobacillus kunkeei YB38 (YB38) at a dose of approximately 10 mg/day on human intestinal environment and bowel movement. This single-blind study enrolled 29 female subjects with a low defecation frequency who consumed heat-killed YB38 at four increasing dosage levels: 0 (placebo), 2, 10, and 50 mg. Each dose was consumed daily for two weeks, with a two-week baseline period preceding the dosing-period and a two-week washout period ending the study. Observed levels of Bacteroides fragilis group significantly decreased with intake of heat-killed YB38 at ≥10 mg/day compared with levels during placebo intake (P<0.01). Faecal pH significantly decreased with 10 and 50 mg/day intake (P<0.01 and 0.05, respectively). Acetic acid levels tended to increase in faeces at the 50 mg/day dose (P<0.1). Bowel movement tended to increase in all heat-killed YB38 intake periods (P<0.1). In conclusion, heat-killed YB38 altered human intestinal microbiota at doses of ≥10 mg/day and tended to increase bowel movement at ≥2 mg/day. This is the first study to show the intestinal microbiota-altering effect of L. kunkeei and to report the bowel movement-improving effect of heat-killed lactic acid bacteria.

Quantification of Blautia wexlerae and Blautia luti in human faeces by real-time PCR using specific primers.

The Clostridium coccoides group, including the genus Blautia and other genera, is one of the predominant bacterial groups in the human intestine. We re-examined 266 human faecal clones and 58 isolates in the C. coccoides group isolated by Hayashi et al. (2002) in order to elucidate the detailed distribution of Blautia wexlerae and Blautia luti in human faeces. Subsequently, we designed a primer pair specific for B. wexlerae and B. luti based on the 16S ribosomal RNA (16S rRNA) gene sequence. The number of B. wexlerae and B. luti in faecal samples of 12 healthy Japanese subjects was examined by real-time PCR assay. The number of the C. coccoides group in the 12 faecal samples was also determined using C. coccoides group-specific primers. Re-examination of the human faecal clones and isolates revealed that B. wexlerae and B. luti accounted for 19.5% of the clones and 25.9% of the isolates. B. wexlerae and B. luti were detected in all faecal samples with 5.3±3.2×10(9) cells/g faeces (wet weight, average ± standard deviation) as assessed by real-time PCR. Furthermore, B. wexlerae and B. luti constituted 32.3±12.7% (average ± standard deviation) of the C. coccoides group (1.7±0.8×10(10) cells/g faeces). This demonstrates that B. wexlerae and B. luti were presented in human faeces with a high frequency as the dominant bacteria.

Analysis of the human intestinal microbiota from 92 volunteers after ingestion of identical meals.

The intestinal microbiota composition of 92 volunteers living in Japan was identified following the consumption of 'identical meals' (1,879 kcal/day) for 3 days. When faecal samples were analysed by terminal restriction fragment length polymorphism with several primer-restriction enzyme systems and then clustered, the patterns could be divided into 2 clusters. Contribution tests and partition modelling showed that OTU211 of the 35f-MspI system and OTU237 of the 35f-AluI system were key factors in the distribution of these groups. However, significant differences among these groups in terms of body mass index and age were not observed.

Faecal microbiota profile of Crohn's disease determined by terminal restriction fragment length polymorphism analysis.

BACKGROUND: Terminal restriction fragment length polymorphism (T-RFLP) analyses are powerful tools to assess the diversity of complex microbiota. T-RFLPs permit rapid comparisons of microbiota from many samples. AIM: To perform T-RFLP analyses of faecal microbiota in Crohn's disease (CD) patients to investigate potential alterations in faecal microbial communities and furthermore to analyse the effects of elemental diet on faecal microbiota profiles. METHODS: Thirty-four patients with CD and 30 healthy individuals were enrolled in the study. DNA was extracted from stool samples and 16S rRNA genes were amplified by PCR. PCR products were digested with BslI restriction enzymes and T-RF lengths were determined. RESULTS: Faecal microbial communities were classified into seven clusters. Almost all healthy individuals (28/30) were included in cluster I, II and III, but the majority of CD patients (25/34) could be divided into another four clusters (cluster IV-VII). Prediction of bacteria based on the BslI-digested T-RFLP database showed a significant decrease in Clostridium cluster IV, Clostridium cluster XI and subcluster XIVa in CD patients. In contrast, Bacteroides significantly increased in CD patients. Significant increases in Enterobacteriales were also observed in CD patients. Furthermore, elemental diets modulated faecal bacterial communities in CD patients. CONCLUSIONS: Terminal restriction fragment length polymorphism analyses showed that the diversity of faecal microbiota in patients with CD differed from that of healthy individuals. Furthermore, elemental diets modulated faecal microbiota composition, and this effect may be involved in mechanisms of clinical effects of elemental diet.

Molecular analysis of the root canal microbiota associated with endodontic treatment failures.

INTRODUCTION: The failure of endodontic treatment is usually caused by persistent/secondary intraradicular infections and Enterococcus faecalis has been considered to be the main pathogen involved. Nevertheless, the breadth of bacterial diversity involved with endodontic treatment failures remains to be consistently explored by culture-independent approaches. METHODS: This study determined the intraradicular microbiota of root-canal-treated teeth with post-treatment apical periodontitis using 16S ribosomal RNA gene clone library analysis. RESULTS: Bacteria were present in all cases, confirming the infectious etiology of post-treatment disease. Seventy-four bacterial taxa belonging to six phyla were found in the nine cases investigated. Of these, 55% were identified as as-yet-uncultivated phylotypes, which also made up a significant proportion of the microbiota in many cases. Twenty-five new phylotypes were identified. Most teeth harbored a mixed consortium, with a mean number of 10 taxa per case. Only 11 taxa were found in more than one case, revealing a high interindividual variability in the composition of the microbiota. CONCLUSION: The current findings revealed new candidate endodontic pathogens, including as-yet-uncultivated bacteria and taxa other than E. faecalis, which may participate in the mixed infections associated with post-treatment apical periodontitis.

A novel keratinase from Clostridium sporogenes bv. pennavorans bv. nov., a thermotolerant organism isolated from solfataric muds.

A bacterium which can grow on chicken feathers and which exhibits keratinolytic activity was isolated from solfataric muds. It was classified as belonging to the genus Clostridium and closely related to C. sporogenes. Based on its unique capability to degrade chicken feathers, it was designated as Clostridium sporogenes bv. pennavorans bv. nov. The keratinase purified from the culture supernatant is a monomer of 28.7kDa molecular mass. The enzyme is relatively thermostable and is active over a broad range of temperature and pH. Specific enzymatic assays demonstrate that keratinase can act on a large variety of soluble and insoluble protein substrates.

Fluctuation of fecal microbiota in individuals with Japanese cedar pollinosis during the pollen season and influence of probiotic intake.

BACKGROUND: We have previously reported the results of a randomized, double-blind, placebo-controlled trial that found the intake of yogurt supplemented with a probiotic strain, Bifidobacterium longum BB536, alleviates symptoms and affects blood parameters in individuals with Japanese cedar pollinosis (JCPsis) during the pollen season. OBJECTIVE: In the present study, fecal microbiota were investigated to examine whether any changes occur during the pollen season and whether any influence is exerted by probiotic intake. METHODS: Yogurt either with BB536 (BB536 yogurt) or without BB536 (placebo yogurt) was administered for 14 weeks at 2 x 100 g per day to 40 subjects (17 men, 23 women) with a clinical history of JCPsis. Fecal samples were obtained from 23 subjects (placebo group, n=13; BB536 group, n=10) before and during the intervention (weeks 4, 9 and 13) and fecal microbiota were analyzed using terminal-restriction fragment length polymorphism and real-time polymerase chain reaction (PCR) methods. RESULTS: From the fluctuation patterns of terminal-restriction fragments, the Bacteroides fragilis group and bifidobacteria were among the species that changed most with pollen dispersion. Real-time PCR analyses indicated that the cell numbers of the B fragilis group increased significantly along with pollen dispersion in both BB536 and placebo groups. Cell numbers of bifidobacteria were significantly higher in the BB536 group compared with the placebo group (P < .05 at weeks 4 and 9). The ratio of cell numbers of the B fragilis group to bifidobacteria increased significantly during the pollen season in the placebo group (P < .01 at weeks 9 and 14), but not in the BB536 group. An in vitro study using peripheral blood mononuclear cells from JCPsis subjects indicated that strains of the B fragilis group induced significantly more helper T cell (T(H)) type2 cytokines (interleukin [IL]-6) but fewer T(H)1 cytokines (IL-12 and interferon) compared with those of bifidobacteria. CONCLUSIONS: These results suggest a relationship between fluctuation in intestinal microbiota and pollinosis allergy. Furthermore, intake of BB536 yogurt appears to exert positive ihfluences on the formation of anti-allergic microbiota.

LKM512 yogurt consumption improves the intestinal environment and induces the T-helper type 1 cytokine in adult patients with intractable atopic dermatitis.

BACKGROUND: In atopic dermatitis (AD) patients, the intestinal mucosal barrier function is weakened, permiting frequent invasion by antigens. Polyamines and short-chain fatty acids (SCFA) produced by intestinal bacteria are involved in the promotion of intestinal mucosal barrier functions. AIM: Our aim was to investigate the effect of pro-biotic yogurt containing Bifidobacterium animalis subsp. lactis LKM512 (LKM512 yogurt) on subjective symptoms, intestinal microbiota, intestinal bacterial metabolites (polyamines and SCFA), and T-helper type 1 (Th1)/Th2 balance in intractable AD patients. METHODS: In a double-blind, placebo-controlled, crossover study, LKM512 yogurt was given for 4 weeks to 10 adult AD patients who were diagnosed with moderate AD (four males and six females; average age, 22.1 years). The subjective symptoms were recorded after each intervention. The dynamics of fecal microbiota were analysed by the terminal-restriction fragment length polymorphism method. The effects of LKM512 yogurt on fecal polyamines, SCFA, and serum cytokines were evaluated. RESULTS: Scores of itch and burning tended to improve to a greater extent by LKM512 yogurt consumption than by placebo consumption. LKM512 yogurt (P<0.005) and placebo consumption (P<0.05) significantly increased the serum IFN-gamma concentration by six- and threefold, respectively. Fecal microbiota was altered dynamically by LKM512 yogurt consumption, in particular, the bacterial species and phylotypes of Bifidobacterium, Clostridium cluster IV and subcluster XIVa were increased in number. In addition, fecal spermidine concentration was significantly (P<0.05) increased, while fecal butyrate also tended to be increased by LKM512 yogurt consumption. CONCLUSION: We conclude that LKM512 yogurt consumption may be effective against intractable adult-type AD and this effect depends on the recovery of the intestinal mucosal barrier function and the induction of the Th1-type cytokine by polyamines and SCFA, particularly, butyrate, produced by the altered intestinal microbiota.

Bacterial reduction and persistence after endodontic treatment procedures.

Bacteria that persist after endodontic disinfection procedures may lead to treatment failure. Over 50% of the bacteria found in endodontic infections are as-yet-uncultivated so investigations of bacteria that endure treatment procedures should include techniques that side-step cultivation. This culture-independent study evaluated the bacterial reduction promoted by intracanal disinfection procedures and identified the taxa persisting after treatment. Samples taken from the infected canals of teeth with apical periodontitis before treatment (S1), after instrumentation using NaOCl as irrigant (S2) and after interappointment medication with a calcium hydroxide paste (S3) were subjected to 16S rRNA gene clone library and real-time polymerase chain reaction analyses. The S2 and S3 samples from five of the 15 canals showed negative results. In the other cases, instrumentation and instrumentation/medication promoted a significant reduction (99.67% and 99.85%, respectively) in the number of bacteria when compared to S1 samples. Forty-three distinct bacterial taxa were identified, of which 24 (56%) were as-yet-uncultivated phylotypes. Nineteen of these 43 taxa (including eight as-yet-uncultivated phylotypes) were disclosed in post-treatment samples, with streptococci being the most prevalent taxa. Findings demonstrated that culture-independent methods provide a detailed insight into the effects of intracanal disinfection protocols, helping to define more effective strategies to deal with endodontic bacteria, including as-yet-uncultivated phylotypes.

Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections.

The purpose of the present study was to use terminal restriction fragment length polymorphism analysis and the 16S rRNA gene clone library to investigate the diversity of the microbiota associated with asymptomatic and symptomatic endodontic infections and to compare the bacterial community structure in these two clinical conditions. Samples were taken from asymptomatic endodontic infections associated with chronic periradicular lesions and from symptomatic infections clinically diagnosed as acute abscesses. 16S rRNA genes from DNA isolated from clinical samples were used to construct clone libraries or were subjected to terminal restriction fragment length polymorphism analysis. Sequence analysis of 186 clones revealed 42 taxa; 23 (55%) were uncultivated phylotypes, of which seven were unique to endodontic infections. Clone sequencing and terminal restriction fragment length polymorphism analysis revealed that the most commonly detected taxa were Fusobacterium nucleatum (including terminal restriction fragment types 1 and 2), Peptostreptococcus micros/Peptostreptococcus sp. oral clone AJ062/BS044/FG014, Prevotella species, Dialister species, Mogibacterium species, Lachnospiraceae oral clone 55A-34, Filifactor alocis, Megasphaera sp. oral clone CS025/BS073, and Veillonella sp. oral clone BP1-85/Veillonella dispar/V. parvula. Bacteroides-like sp. oral clone X083/Bacteroidales oral clone MCE7_20 and Dialister sp. oral clone BS016/MCE7_134 were detected only in asymptomatic teeth. On the other hand, F. nucleatum terminal restriction fragment type 2, Prevotella intermedia, Dialister pneumosintes, and some phylotypes were exclusively detected in symptomatic samples. Bacterial profiles of symptomatic endodontic infections generated by terminal restriction fragment length polymorphism analysis were clearly different from those of asymptomatic infections. Overall, the average number of terminal restriction fragments in symptomatic samples was significantly larger than in asymptomatic samples. Molecular analysis of the microbiota associated with symptomatic or asymptomatic endodontic infections indicates that the endodontic bacterial diversity is greater than previously described by culture methods and that the structure of the microbiota differ significantly between asymptomatic and symptomatic infections.

Mucosa-associated bacteria in ulcerative colitis before and after antibiotic combination therapy.

BACKGROUND: We proposed that Fusobacterium varium is one of the causative agents in ulcerative colitis. AIM: To examine the efficacy of antibiotic combination therapy against F. varium and to investigate the mucosa-associated bacteria before and after the therapy using a new molecular approach. METHODS: Twenty patients with ulcerative colitis were randomly assigned into the antibiotic treatment group (amoxicillin, tetracycline and metronidazole for 2 weeks) and no-antibiotics group. Clinical assessment, colonoscopic and histological evaluations were performed at 0 and 3-5 months after the treatment. DNA from mucosal bacteria was isolated from biopsy specimens. We investigated the mucosa-associated bacterial components by terminal restriction fragment length polymorphism with the restriction enzyme HhaI and MspI, and quantified the change in the number of bacteria by real-time polymerase chain reaction. Immunohistochemical detection of F. varium in biopsy specimens was also performed. RESULTS: After the treatment, the clinical assessment, colonoscopic and histological scores improved in the antibiotic group compared with the control group. Three peaks of terminal restriction fragment length polymorphism decreased after treatment only in the antibiotic group. Eubacterium rectale, Dorea formicigenerans, Clostridium clostridioforme and F. varium were included in these peaks. Based on the real-time polymerase chain reaction study, only F. varium was significantly reduced after treatment. In the immunostaining, post-treatment scores in treatment group were significantly lower than that in control group. CONCLUSIONS: Antibiotics combination therapy was effective for ulcerative colitis. The number of mucosa-associated F. varium significantly decreased after the treatment.

Biodiversity of Lactobacillus sanfranciscensis strains isolated from five sourdoughs.

AIMS: Five different sourdoughs were investigated for the composition of lactic acid bacteria (LAB) and the biodiversity of Lactobacillus sanfranciscensis strains. METHODS AND RESULTS: A total of 57 strains were isolated from five sourdoughs. Isolated strains were all identified by the 16S rDNA sequence and species-specific primers for L. sanfranciscensis. Results of identification showed that LAB strains were L. sanfranciscensis, Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus fermentum, Lactobacillus pontis, Lactobacillus casei, Weisella confusa and Pediococcus pentosaceus. A total of 21 strains were identified as L. sanfranciscensis and these isolates were detected in all five sourdoughs. Ribotyping was applied to investigate the relationship between intraspecies diversity of L. sanfranciscensis and sourdough. A total of 22 strains of L. sanfranciscensis including L. sanfranciscensis JCM 5668T were compared by ribotyping. The dendrogram of 21 ribotyping patterns showed four clusters, and L. sanfranciscensis JCM 5668T was independent of the others. The different biotypes of L. sanfranciscensis were present in two sourdoughs compared with other three sourdoughs. CONCLUSIONS: The LAB compositions of five sourdoughs were different and the relationship between intraspecies diversity of L. sanfranciscensis strains and five sourdoughs was shown by ribotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that ribotyping was useful for distinguishing L. sanfranciscensis strains. A further important result is that the intra-species diversity of L. sanfranciscensis strains seems to be related to the sourdough preparation.

Exfoliation of Helicobacter pylori from gastric mucin by glycopolypeptides from buttermilk.

In the human stomach, Helicobacter pylori, an ulcer pathogenic bacterium, colonizes the gastric mucosal layer primarily. The ability of glycopolypeptides (GPP) prepared from buttermilk to exfoliate H. pylori bound to gastric mucin was investigated. The GPP were prepared from buttermilk by digestion with trypsin, papain, pancreatin, bromelain, or pepsin. Helicobacter pylori ATCC 43504T and 43579 adhered more strongly to all of the GPP tested than to whole buttermilk, the soluble fraction of buttermilk, gastric mucin prepared from mouse stomach, or commercial pig gastric mucin. The GPP digested with trypsin, papain, or pancreatin were significantly more adherent. When the GPP concentration was 10 mg/mL, bound H. pylori ATCC 43504T, 43579, and 5 clinical isolates were exfoliated markedly from immobilized porcine gastric mucin following treatment with GPP digested with trypsin or pancreatin. This ability of GPP did not correlate with sialic acid content, indicating that sialic acid content is not important in the exfoliation of this microorganism. Such an ability may depend on the structure or number of sugar chains, or the position of sialic acid. We conclude that GPP promote the exfoliation of H. pylori bound to gastric mucin and prevent the de novo adherence of this microorganism. As such, GPP are a promising food material for preventing H. pylori infection.

Treponema socranskii, Treponema denticola, and Porphyromonas gingivalis are associated with severity of periodontal tissue destruction.

BACKGROUND: The aim of the present study was to identify Treponema socranskii in addition to Treponema denticola and Porphyromonas gingivalis by polymerase chain reaction (PCR), and to clarify the relationship between the presence of these microorganisms and the severity of clinical periodontal parameters. METHODS: Saliva and subgingival plaque collected from 123 subjects (38 aggressive periodontitis patients, 65 chronic periodontitis patients, 20 healthy patients) were subjected to PCR to detect the aforementioned 3 microorganisms. RESULTS: Detection frequencies of T. socranskii, T. denticola, and P. gingivalis in plaque samples from aggressive periodontitis patients (71.1%, 73.7%, 84.2%, respectively) and chronic periodontitis patients (89.2%, 93.8%, 95.3%) were much higher than those from healthy subjects (30%, 5.0%, 10.0%). In aggressive and chronic periodontitis patients, these 3 species of bacteria were detected frequently at sites that showed deep periodontal pockets and severe attachment loss. The percentage of these bacteria-positive sites increased as the gingival index score of chronic periodontitis patients increased. T. socranskii was frequently detected together with T. denticola or P. gingivalis at the same sites, and coexistence of these microorganisms was frequently observed in deep periodontal pockets of aggressive periodontitis patients. CONCLUSIONS: T. socranskii, T. denticola, and P. gingivalis were frequently detected in periodontitis patients by PCR. The prevalence of these 3 microorganisms was correlated with various clinical parameters. Taken together, our findings suggest that T. socranskii, T. denticola, and P. gingivalis are associated with the severity of periodontal tissue destruction.

Differences in composition and mucosal adhesion of bifidobacteria isolated from healthy adults and healthy seniors.

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30-40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people.

Impact of LKM512 yogurt on improvement of intestinal environment of the elderly.

Improvement of the intestinal environment by administration of LKM512 yogurt was examined using polyamine, haptoglobin and mutagenicity as indexes which directly reflect the health condition of the host. The concentration of spermine in feces increased significantly by 3-fold (P<0.05) at week 2 of administration of LKM512 yogurt compared with before administration, and that of putrescine, spermidine, and cadaverine also tended to increase with administration of LKM512 yogurt. The haptoglobin content in feces decreased significantly (P<0.05) at week 2 of administration of LKM512 yogurt, and it showed a negative correlation with the polyamine content, indicating that acute intestinal inflammation was suppressed. Fecal mutagenicity was measured using fecal extract and fecal precipitate. Both preparations showed similar significant decreases (P<0.05) by the administration of LKM512 yogurt, as well as a negative correlation with polyamine content. This result indicated that antimutagenicity due to administration of LKM512 yogurt was not based on binding of the mutagen to the bacterial cell wall. Many reports have suggested that polyamines increased by the administration of LKM512 yogurt led to inhibition of inflammation and antimutagenicity in the intestinal tract.

Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping.

A total of 91 type and reference strains of the Lactobacillus casei group and the L acidophilus group were characterized by the automated ribotyping device Riboprinter microbial characterization system. The L. casei group was divided into five (C1-C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1-A11, B1-B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter system yields rapid, accurate and reproducible genetic information for the identification of many strains.

Rapid detection of human fecal Eubacterium species and related genera by nested PCR method.

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.