Gut Microbiome and Microbiome-Derived Metabolites in Patients with End-Stage Kidney Disease.

The composition of the gut microbiome is altered in patients with chronic kidney disease (CKD). Dysbiosis leads to decreased levels of stool organic acids (OAs) and systemic inflammation, followed by accumulation of uremic toxins (UTs) and the development of end-stage kidney disease (ESKD). We assessed the relationship between the microbiome and UT levels or the development of ESKD by comparing patients undergoing hemodialysis (HD) and those with normal renal function (NRF). This cross-sectional study recruited 41 patients undergoing HD and 38 sex- and age-matched patients with NRF, and gut microbiome, levels of plasma UTs, inflammatory markers, and stool OAs were compared. The indices of beta-diversity differed significantly between patients with NRF and those undergoing HD, and between patients undergoing HD with and without type 2 diabetes. The levels of stool total OA, inflammatory markers, and UTs differed significantly between the patients with NRF and those undergoing HD. The combined main effects of type 2 diabetes and kidney function status were accumulation of indoxyl sulfate and p-cresyl sulfate. The relative abundances of Negativicutes and Megamonas were associated with development of ESKD and with the levels of UTs, even after adjustment for factors associated with the progression of ESKD. The present study indicates that the gut environment differs between patients with NRF and those undergoing HD and between patients undergoing HD with and without type 2 diabetes. Moreover, ESKD patients with diabetes accumulate more UTs derived from the gut microbiome, which might be associated with cardio-renal diseases and poor prognosis.

Characterization of long-chain fatty acid-linked bile acids: a major conjugation form of 3ß-hydroxy bile acids in feces.

Although most bile acids (BAs) in feces are present in noncovalent forms that can be extracted with ethanol, non-negligible amounts of saponifiable BAs are also present. It is a major concern that such saponifiable BAs are routinely omitted from fecal BA measurements. We compared the BA profiles of healthy stools that were obtained with/without alkaline hydrolysis and found that as much as 29.7% (2.1-67.7%) of total BAs were saponifiable. Specifically, alkaline treatment led to significant elevations of isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) concentrations, suggesting that considerable proportions of isoDCA and isoLCA were esterified. Precursor ion scan data from LC/MS suggested the presence of long-chain FA-linked BAs. We chemically synthesized a series of fatty acid 3ß-acyl conjugates of isoDCA and isoLCA as analytical standards and analyzed their fecal profiles from newborns to adults (n = 64) by LC/MS. FA-conjugated isobile acids (FA-isoBAs) were constantly present from 2 years of age to adulthood. C16- and C18-chain FA-isoBA esters were predominantly found regardless of age, but small amounts of acetic acid esters were also found. FA-isoBA concentrations were not correlated to fecal FA concentrations. Interestingly, there were some adults who did not have FA-isoBAs. Gut bacteria involved in the production of FA-isoBAs have not been identified yet. The present study provides insight into the establishment of early gut microbiota and the interactive development of esterified BAs.The contribution of FA-isoBAs to gut physiology and their role in pathophysiologic conditions such as inflammatory bowel disease are currently under investigation.

Stool preparation under anaerobic conditions contributes to retention of obligate anaerobes: potential improvement for fecal microbiota transplantation.

BACKGROUND: Fecal microbiota transplantation (FMT) in patients with ulcerative colitis has shown variable efficacy depending on the protocol used. A previous randomized controlled trial reported that anaerobic preparation of donor stool contributes to improved efficacy. Despite the suggestion that viable obligate anaerobes would be decreased through aerobic handling, there have been only a limited number of reports on how these aerobic or anaerobic procedures affect the composition of viable microbiota in the fecal slurries used for FMT. METHODS: We adopted 16S and 23S rRNA-targeted reverse transcription-quantitative polymerase chain reaction to quantify viable bacteria in fecal slurries. This study utilized specific primers designed to detect obligate anaerobes (including Clostridium coccoides group, C. leptum subgroup, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, and Prevotella) and facultative anaerobes (including total lactobacilli, Enterobacteriaceae, Enterococcus, Streptococcus, and Staphylococcus). We then calculated the ratio change (RC) between before and after mixing, and compared the resulting values between anaerobic-prep and aerobic-prep in samples fixed immediately after blending (RCAn0 vs. RCAe0) and in samples maintained (under anaerobic or aerobic conditions) for 1 h after blending (RCAn1 vs. RCAe1). RESULTS: For most obligate anaerobes, the median RC tended to be less than 1, indicating that the number of obligate anaerobes was decreased by the blending procedure. However, in samples maintained for 1 h after blending, anaerobic-prep counteracted the decrease otherwise seen for the C. coccoides group and B. fragilis groups (P < 0.01 for both). The C. leptum subgroup also tended to show higher RC by anaerobic-prep than by aerobic-prep, although this effect was not statistically significant. Among facultative anaerobes, Enterobacteriaceae, Enterococcus, and Staphylococcus showed median RC values of more than 1, indicating that these organisms survived and even grew after mixing. Moreover, oxygen exposure had no significant influence on the survival of the facultative anaerobes. CONCLUSIONS: The conditions under which the blending procedure was performed affected the proportion of live anaerobes in fecal slurries. The obligate anaerobes tended to be decreased by blending processes, but anaerobic-prep significantly mitigated this effect. Anaerobic-prep may improve the efficacy of FMT by permitting the efficient transfer of obligate anaerobes to patients with ulcerative colitis.

Impact of Micronutrient Status during Pregnancy on Early Nutrition Programming.

BACKGROUND: Nutrition status prior to conception and during pregnancy and infancy seems to have an influence on the disease risk in adulthood (early nutrition/developmental programming). We aimed to review the current knowledge on the role of micronutrients in early nutrition programming and its implications for healthcare. SUMMARY OF FINDINGS: Globally and even in high-income countries where a balanced diet is generally accessible, an inadequate maternal micronutrient status is common. This may induce health problems in the mother and foetus/newborn both immediately and in later life. Pregnant women and those who may become pregnant should aim to achieve a satisfactory micronutrient status from a well-balanced diet, and where necessary from additional supplements. Key Messages: We emphasise the need for a call to action for healthcare providers and policymakers to better educate women of child-bearing age regarding the short- and long-term benefits of an appropriate micronutrient status. The role of micronutrient status in early nutrition programming needs to be emphasized more to address the still limited awareness of the potential long-term health repercussions of suboptimal micronutrient supply during pregnancy.

Gut Microbiota Composition Before and After Use of Proton Pump Inhibitors.

BACKGROUND: Recently, problems associated with proton pump inhibitor (PPI) use have begun to surface. PPIs influence the gut microbiota; therefore, PPI use may increase the risk of enteric infections and cause bacterial translocation. In this study, we investigated fecal microbiota composition, fecal organic acid concentrations and pH, and gut bacteria in the blood of the same patients before and after PPI use. METHODS: Twenty patients with reflux esophagitis based on endoscopic examination received 8 weeks of treatment with PPIs. To analyze fecal microbiota composition and gut bacteria in blood and organic acid concentrations, 16S and 23S rRNA-targeted quantitative RT-PCR and high-performance liquid chromatography were conducted. RESULTS: Lactobacillus species were significantly increased at both 4 and 8 weeks after PPI treatment compared with bacterial counts before treatment (P = 0.011 and P = 0.002, respectively). Among Lactobacillus spp., counts of the L. gasseri subgroup, L. fermentum, the L. reuteri subgroup, and the L. ruminis subgroup were significantly increased at 4 and 8 weeks after treatment compared with counts before treatment. Streptococcus species were also significantly increased at 4 and 8 weeks after PPI treatment compared with counts before treatment (P < 0.01 and P < 0.001, respectively). There was no significant difference in the total organic acid concentrations before and after PPI treatment. Detection rates of bacteria in blood before and after PPI treatment were 22 and 28%, respectively, with no significant differences. CONCLUSIONS: Our quantitative RT-PCR results showed that gut dysbiosis was caused by PPI use, corroborating previous results obtained by metagenomic analysis.

Gut Microbiota Composition in Healthy Japanese Infants and Young Adults Born by C-Section.

Our gut microbiome plays a fundamental role in our health and disease. The microbial colonization of human gut begins immediately at birth and is an indispensable natural process that modulates our physiology and immunity. Recent studies are elegantly revealing how and when these microbes colonize the gut and what elements could potentially influence this natural phenomenon. The vertical mother-to-baby transmission of microbes is a crucial factor for normal development and maturation of newborn's immune, metabolic as well as neurological health. This important and delicate process of gut microbiota development may be impacted by various factors such as birth mode, type of feeding, gestational age at birth, antibiotics exposure in early life, surrounding environment and hygiene settings, and so on. Perturbations in early life gut microbial colonization have been associated with the development of several diseases such as diabetes, obesity, asthma, allergies, celiac disease, neurodevelopmental disorders, and so on. However, it remains unclear whether predisposition to these diseases is due to the lack of acquisition of the mother's (vaginal and perianal) microbes during birth or because of abnormal exposure to unsolicited bacteria. Hence, studies are required to scrutinize the colonization pattern of infant gut microbiome in context to birth mode and also to elucidate how long these differences could persist. In these contexts, we review and discuss some of the findings obtained from recent investigation of the gut microbiota composition in healthy Japanese infants and young adults born vaginally or by C-section.

Gut Microbiota in Health and Disease.

Intestinal regulatory T (Treg) cells are critical to maintaining immune tolerance to dietary antigens and gut microbiota. This paper reviews several papers on this topic that were recently published by Japanese researchers. Specifically, Prof. K. Honda and his group have found that commensal microbiota capable of metabolizing butyrate induces the differentiation of colonic Treg cells. In a separate work, Prof. Y. Yokoyama and his group used a novel, culture-independent analytical method (the Yakult Intestinal Flora-Scan) for detection of bacteria in the bloodstream. Their work revealed that bacteremia in invasive surgery patients was ameliorated by synbiotic supplementation; similar results were reported in pediatric surgical cases by Dr. T. Okazaki and his group. This cutting-edge method may lead to the evolution of an altered disease concept; an example of this change is provided by the description of bacteremia in patients with type 2 diabetes, as reported by Dr. J. Sato and her group. In a similar work, Prof. Y. Yamashiro and his group found that infants born by cesarean (C)-section, who typically have gut dysbiosis, exhibit higher carriage of toxigenic Clostridium perfringens. The finding suggests that C-section-born infants may serve as a potential reservoir of this opportunistic pathogen. Another separate work by the laboratory of Dr. K. Yamashiro has revealed that gut dysbiosis is associated with altered metabolism and systemic inflammation in patients with ischemic stroke. These papers are consistent with a study by Prof. N. Sudo and his group, who have made significant progress in research on interaction among the microbiota, gut, and brain.

The High Incidence of Bacteremia in Children Undergoing Surgery Can Be Prevented by Bifidobacterium Supplementation.

Major surgical procedures can alter intestinal microbiota and disrupt the intestinal barrier function, leaving the patient at risk for infection. Probiotics are defined as live microorganisms that confer a health benefit on the host when administered in adequate amounts. Although the efficacy of administering probiotics perioperatively to adults has been reported, the clinical significance of probiotics in children undergoing surgery is still unclear. This study provides a brief overview of our randomized controlled trial of preoperative probiotic administration to children, and discusses the relationship between probiotics and their effects in the perioperative period, particularly focusing on bacteremia.

Intestinal Microbiota in Pediatric Surgical Cases Administered Bifidobacterium Breve: A Randomized Controlled Trial.

The efficacy of perioperative probiotic administration has been reported in adults. We examined the effects of orally administered Bifidobacterium breve strain Yakult (BBG-01) on outcomes in pediatric surgical cases by assessing intestinal and blood microbiota. BBG-01 was well tolerated without adverse effects, and postoperative infectious complications were significantly decreased. Fecal analysis showed increased Bifidobacterium and decreased Enterobacteriaceae, Clostridium difficile, and Pseudomonas. Concentrations of fecal acetic acid were significantly increased, maintaining fecal pH at <7.0. The incidence of detecting bacteria in blood was significantly reduced. BBG-01 improved the intestinal environment, and may be implicated in suppressing bacterial translocation.

The Effectiveness of Lactobacillus Beverages in Controlling Infections among the Residents of an Aged Care Facility: A Randomized Placebo-Controlled Double-Blind Trial.

BACKGROUNDS/AIMS: To clarify the usefulness of Lactobacillus casei strain Shirota (LcS)-fermented milk in the normalization of bowel movements and improvement of infection control for the elderly residents and staff of facilities for the elderly. METHODS: A randomized placebo-controlled double-blind test was performed among the elderly residents (average age, 85) and staff members (average age, 37) of facilities for the elderly. The participants randomly received either LcS-fermented milk or a placebo beverage once daily for 6 months. Clinical data and enteric conditions were compared between the 2 groups. RESULTS: A significantly lower incidence of fever and improved bowel movements were seen in the LcS-fermented milk group (n = 36) in comparison to the placebo group (n = 36). The numbers of Bifidobacterium and Lactobacillus were significantly higher (p < 0.01), the numbers of destructive bacteria such as Clostridium difficile were significantly lower (p < 0.05), and the fecal acetic acid concentration and total acidity were significantly higher in the LcS group. A significant difference in the intestinal microbiota, fecal acetic acid, and pH was also observed between the LcS and placebo groups among the facility's staff members. CONCLUSIONS: The long-term consumption of LcS-fermented milk may be useful for decreasing the daily risk of infection and improving the quality of life among the residents and staff of facilities for the elderly.

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes.

BACKGROUND: Clostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults. METHODS: The plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221). RESULTS: The qPCR assays were highly specific and sensitive, with a minimum detection limit of 10(3) bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36% infants and 33% adults, with counts ranging widely (10(3)-10(7) bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0±1.5 log10 bacterial cells/g), as compared to that in adults (4.8±1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9±1.9 and 4.8±0.8 log10 bacterial cells/g in infants and adults, respectively. CONCLUSIONS: These data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of α-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.

Intestinal Microbiota Profiles of Healthy Pre-School and School-Age Children and Effects of Probiotic Supplementation.

OBJECTIVES: This study aims to establish the baseline profile of intestinal microbiota in pre-school and school-age Japanese children and to investigate the effects of a probiotic on the microbiota. METHODS: We analyzed the intestinal microbiota and investigated the effects (before, during and after the ingestion period) on intestinal microbiota and the environment of 6 months of daily ingestion of a probiotic (Lactobacillus casei strain Shirota (LcS)-fermented milk). RESULTS: We performed an open trial in 23 children (14 boys, 9 girls; age 7.7 ± 2.4 years (mean ± SD); BMI 19.6 ± 4.6). The composition of intestinal microbiota of healthy pre-school and school-age children resembled that of adults. During probiotic supplementation, the population levels of Bifidobacterium and total Lactobacillus increased significantly, while those of Enterobacteriaceae, Staphylococcus and Clostridium perfringens decreased significantly. A significant increase in fecal concentrations of organic acids and also a decrease in fecal pH were observed during the ingestion period. However, the patterns of fecal microbiota and intestinal environment were found to revert to the baseline levels (i.e. before ingestion) within 6 months following the cessation of probiotic intake. CONCLUSION: Regular intake of an LcS-containing probiotic product may modify the gut microbiota composition and intestinal environment in pre-school and school-age children while maintaining the homeostasis of the microbiota.

The Two-Way Association of Periodontal Infection with Systemic Disorders: An Overview.

Oral cavity that harbors diverse bacterial populations could also act as a site of origin for spread of pathogenic microorganisms to different body sites, particularly in immunocompromised hosts, patients, the elderly, or the underprivileged. A number of recent publications have advocated that patients with periodontal diseases are more susceptible to metabolic endotoxemia, inflammation, obesity, type 2 diabetes, and other related systemic complications, concluding that periodontal diseases could be a potential contributing risk factor for a wide array of clinically important systemic diseases. However, despite a significant increase in the prevalence of periodontal infections and systemic diseases in the past few decades, the fundamental biological mechanisms of connection between these ailments are still not fully explicated. Consequently, the mechanisms by which this bidirectional damage occurs are being explored with a concentric vision to develop strategies that could prevent or control the complications of these ailments. This paper attempts to summarize and hypothesize the diverse mechanisms that hint to a certain connection between the two prevalent chronic situations.

Current information and Asian perspectives on long-chain polyunsaturated fatty acids in pregnancy, lactation, and infancy: systematic review and practice recommendations from an early nutrition academy workshop.

The Early Nutrition Academy supported a systematic review of human studies on the roles of pre- and postnatal long-chain polyunsaturated fatty acids (LC-PUFA) published from 2008 to 2013 and an expert workshop that reviewed the information and developed recommendations, considering particularly Asian populations. An increased supply of n-3 LC-PUFA during pregnancy reduces the risk of preterm birth before 34 weeks of gestation. Pregnant women should achieve an additional supply ≥200 mg docosahexaenic acid (DHA)/day, usually achieving a total intake ≥300 mg DHA/day. Higher intakes (600-800 mg DHA/day) may provide greater protection against early preterm birth. Some studies indicate beneficial effects of pre- and postnatal DHA supply on child neurodevelopment and allergy risk. Breast-feeding is the best choice for infants. Breast-feeding women should get ≥200 mg DHA/day to achieve a human milk DHA content of ∼0.3% fatty acids. Infant formula for term infants should contain DHA and arachidonic acid (AA) to provide 100 mg DHA/day and 140 mg AA/day. A supply of 100 mg DHA/day should continue during the second half of infancy. We do not provide quantitative advice on AA levels in follow-on formula fed after the introduction of complimentary feeding due to a lack of sufficient data and considerable variation in the AA amounts provided by complimentary foods. Reasonable intakes for very-low-birth weight infants are 18-60 mg/kg/day DHA and 18-45 mg/kg/day AA, while higher intakes (55-60 mg/kg/day DHA, ∼1% fatty acids; 35-45 mg/kg/day AA, ∼0.6-0.75%) appear preferable. Research on the requirements and effects of LC-PUFA during pregnancy, lactation, and early childhood should continue. © 2014 S. Karger AG, Basel.

Longitudinal Analysis of One-Carbon Metabolism-Related Metabolites in Maternal and Cord Blood of Japanese Pregnant Women.

One-carbon metabolism (OCM) is a complex and interconnected network that undergoes drastic changes during pregnancy. In this study, we investigated the longitudinal distribution of OCM-related metabolites in maternal and cord blood and explored their relationships. Additionally, we conducted cross-sectional analyses to examine the interrelationships among these metabolites. This study included 146 healthy pregnant women who participated in the Chiba Study of Mother and Child Health. Maternal blood samples were collected during early pregnancy, late pregnancy, and delivery, along with cord blood samples. We analyzed 18 OCM-related metabolites in serum using stable isotope dilution liquid chromatography/tandem mass spectrometry. We found that serum S-adenosylmethionine (SAM) concentrations in maternal blood remained stable throughout pregnancy. Conversely, S-adenosylhomocysteine (SAH) concentrations increased, and the total homocysteine/total cysteine ratio significantly increased with advancing gestational age. The betaine/dimethylglycine ratio was negatively correlated with total homocysteine in maternal blood for all sampling periods, and this correlation strengthened with advances in gestational age. Most OCM-related metabolites measured in this study showed significant positive correlations between maternal blood at delivery and cord blood. These findings suggest that maternal OCM status may impact fetal development and indicate the need for comprehensive and longitudinal evaluations of OCM during pregnancy.

Modulation of expression of Programmed Death-1 by administration of probiotic Dahi in DMH-induced colorectal carcinogenesis in rats.

UNLABELLED: Interaction of probiotic bacteria with the host immune system elicits beneficial immune modulating effects. Although, there are many published studies on interaction of probiotics with immune system focusing on activation of immune system by bacterial cell wall through the engagement of Toll-like receptor family; very few studies have focused on molecules involved in the T-cell activation, and not much work has been executed to study the correlation of probiotics and programmed death-1 in colorectal carcinogenesis in animal models. Hence, the present study was carried out to assess the effect of probiotic Dahi on expression of programmed death (PD-1) in colorectum of 1, 2-dimethylhydrazine treated Wistar rats. METHODS: DMH was injected subcutaneously at the rate of 40 mg/kg body weight per animal twice a week for 2 weeks. A total of 168 male Wistar rats were randomly allocated to seven groups, each group having twenty-four animals. The rats were euthanized at the 8th, 16th and 32nd week of the experiment and examined for the expression of PD-1 in colorectal tissues by immunohistochemical staining. RESULTS: Expression of PD-1 was observed in colorectal tissues of normal and DMH-treated rats. Feeding rats with probiotic Dahi or the treatment with piroxicam decreased the expression of PD-1 in DMH-induced colorectal mucosa, and the combined treatment with probiotic Dahi and piroxicam was significantly more effective in reducing the expression of PD-1. CONCLUSION: PD-1 expressed independent of carcinogen administration in normal colonic mucosa and may play a role in modulation of immune response in DMH-induced colorectal carcinogenesis. The present study suggests that probiotic Dahi can be used as an effective chemopreventive agent in the management of colorectal cancer.

Association of Fatty Acid Desaturase 1 rs174547 Polymorphism with the Composition of Long-Chain Polyunsaturated Fatty Acids in Serum Glycerophospholipids during Pregnancy.

The increase in fetal requirements of long-chain polyunsaturated fatty acids (LCPUFAs) during pregnancy alters maternal fatty acid metabolism, and therefore, fatty acid desaturase (FADS) gene polymorphisms may change blood fatty acid composition or concentration differently during pregnancy. We investigated the relationship between a FADS1 single-nucleotide polymorphism (SNP) and maternal serum LCPUFA levels in Japanese pregnant women during the first and third trimesters and at delivery. Two hundred and fifty-three pregnant women were included, and fatty acid compositions of glycerophospholipids in serum (weight %) and the FADS1 SNP rs174547 (T/C) were analyzed. LCPUFAs, including arachidonic acid (ARA) and docosahexaenoic acid (DHA), significantly decreased from the first to the third trimester of pregnancy. Furthermore, DHA significantly decreased from the third trimester of pregnancy to delivery. At all gestational stages, linoleic acid (LA) and α-linolenic acid were significantly higher with the number of minor FADS1 SNP alleles, whereas γ-linolenic acid and ARA and the ARA/LA ratio were significantly lower. DHA was significantly lower with the number of minor FADS1 SNP alleles only in the third trimester and at delivery, suggesting that genotype effects become more obvious as pregnancy progresses.

Sensitive quantification of Clostridium difficile cells by reverse transcription-quantitative PCR targeting rRNA molecules.

We established a sensitive and accurate quantification system for Clostridium difficile in human intestines, based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR). We newly developed a species-specific primer set for C. difficile targeting 23S rRNA gene sequences. Both the vegetative cells and the spores of C. difficile in human feces were quantified by RT-qPCR, with a lower detection limit of 10(2.4) cells/g of feces. In an analysis of the feces of residents (n = 83; age, 85 ± 8 years) and staff (n = 19; age, 36 ± 10 years) at a care facility for the elderly, C. difficile was detected by RT-qPCR in 43% of the residents (average count, log(10) 4.0 ± 2.0 cells/g of feces) and 16% of the staff (average count, log(10) 2.2 ± 0.1 cells/g of feces); these rates were far higher than those detected by qPCR (residents, 19%; staff, 0%) or selective cultivation (residents, 18%; staff, 5%). Another analysis of healthy adults (n = 63; age, 41 ± 11 years) also revealed the significant carriage rate of C. difficile in the intestines (detection rate, 13%; average count, log(10) 4.9 ± 1.2 cells/g of feces). From these results, it was suggested that rRNA-targeted RT-qPCR should be an effective tool for analyzing population levels of C. difficile in the human intestine.