Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.

A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli , Enterococcus faecalis , Staphylococcus aureus , Clostridium perfringens , and Pseudomonas aeruginosa , and the threshold cycle value was found to be linear up to an RNA amount of 10 ⁻³ cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10 ⁰ to 10 ⁵ CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4′,6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients ( n = 38) at a level of 10 ³ cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.

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 10 ² to 10 ⁴ cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 10 ⁸ 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 log ₁₀ (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.

Accumulating evidence suggests that the gut microbiome influences the brain functions and psychological state of its host via the gut–brain axis, and gut dysbiosis has been linked to several mental illnesses, including major depressive disorder (MDD). Animal experiments have shown that a depletion of the gut microbiota leads to behavioral changes, and is associated with pathological changes, including abnormal stress response and impaired adult neurogenesis. Short-chain fatty acids such as butyrate are known to contribute to the up-regulation of brain-derived neurotrophic factor (BDNF), and gut dysbiosis causes decreased levels of BDNF, which could affect neuronal development and synaptic plasticity. Increased gut permeability causes an influx of gut microbial components such as lipopolysaccharides, and the resultant systemic inflammation may lead to neuroinflammation in the central nervous system. In light of the fact that gut microbial factors contribute to the initiation and exacerbation of depressive symptoms, this review summarizes the current understanding of the molecular mechanisms involved in MDD onset, and discusses the therapeutic potential of probiotics, including butyrate-producing bacteria, which can mediate the microbiota–gut–brain axis.

The faecal microbiota of 166 healthy Japanese newborns was analysed periodically from day 1 after birth until the age of 3 years by using the reverse transcription-quantitative PCR. Faecal pH and the organic acid concentration were also examined. Colonisation by both facultative anaerobes and strict anaerobes was confirmed in 95% of the meconium tested. Bifidobacterium-predominant microbiota was established subsequently in most of the infants by 3 months after birth. Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium catenulatum group and Bifidobacterium bifidum were the species mainly detected. Intergroup correlation analysis revealed that the bifidobacterial population levels, but not other strict anaerobe groups, were found to be negatively correlated with those of the Enterobacteriaceae from 7 days until 3 months after birth. Faecal pH was maintained at about 6 until 6 months after birth and reached 6.6 at 3 years after birth. The initial concentration of faecal organic acids (19 μM/g of faeces) just after birth increased until 3 years after birth to the level of 111 μM/g of faeces. Early start of feeding formula milk promoted colonisation by obligate anaerobes such as the Clostridium coccoides group, the Clostridium leptum subgroup, Prevotella, and Atopobium cluster during the 3 months after birth. Population levels of the bifidobacteria until 1 month after birth and those of the Bacteroides fragilis group until 6 months after birth were lower in infants delivered by Caesarean section than in those delivered normally. The results suggested that both earlier start of feeding of formula milk and the mode of infant delivery were found to be important in the development of intestinal microbiota in early infancy.

Various benefits of probiotics to the host have been shown in numerous human clinical trials. These organisms have been proposed to act by improving the balance of the gut microbiota and enhancing the production of short-chain fatty acids, as well as by interacting with host cells in the gastrointestinal tract, including immune cells, nerve cells, and endocrine cells. Although the stimulation of host cells by probiotics and subsequent signaling have been explained by in vitro experiments and animal studies, there has been some skepticism as to whether probiotics can actually interact with host cells in the human gastrointestinal tract, where miscellaneous indigenous bacteria coexist. Most recently, it has been shown that the ileal microbiota in humans after consumption of a fermented milk is occupied by probiotics for several hours, indicating that there is adequate opportunity for the ingested strain to stimulate the host cells continuously over a period of time. As the dynamics of ingested probiotics in the human gastrointestinal tract become clearer, further progress in this research area is expected to elucidate their behavior within the tract, as well as the mechanism of their physiological effects on the host.