Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology.     

Unexpected stability of Bacteroidetes and Firmicutes communities in laboratory biogas reactors fed with different defined substrates

In the present study, bacterial communities in 200-liter biogas reactors containing liquid manure consecutively fed with casein, starch, and cream were investigated over a period of up to 33 days. A 16S rRNA gene clone library identified Bacteroidetes and Firmicutes as the most abundant bacterial groups in the starting material, at 58.9% and 30.1% of sequences, respectively. The community development of both groups was monitored by real-time PCR and single-strand conformation polymorphism (SSCP) analysis. The Firmicutes and Bacteroidetes communities were unexpectedly stable and hardly influenced by batch-feeding events. The continuous feeding of starch led to community shifts that nevertheless contributed to a stable reactor performance. A longer starving period and a change in the pH value resulted in further community shifts within the Bacteroidetes but did not influence the Firmicutes. Predominant DNA bands from SSCP gels were cloned and sequenced. Sequences related to Peptococcaceae, Cytophagales, and Petrimonas sulfuriphila were found in all samples from all experiments. Real-time PCR demonstrated the abundance of members of the phylum Bacteroidetes and also reflected changes in gene copy numbers in conjunction with a changing pH value and acetate accumulation.     

Pediatric obesity is associated with an altered gut microbiota and discordant shifts in Firmicutes populations

An altered gut microbiota has been linked to obesity in adulthood, although little is known about childhood obesity. The aim of this study was to characterize the composition of the gut microbiota in obese (n = 42) and normal‐weight (n = 36) children aged 6 to 16. Using 16S rRNA gene‐targeted sequencing, we evaluated taxa with differential abundance according to age‐ and sex‐normalized body mass index (BMI z‐score). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Members of the Bacteroidetes were generally better predictors of BMI z‐score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. In accordance with these observations, the main metabolites produced by gut bacteria, short chain fatty acids (SCFAs), were higher in obese children, suggesting elevated substrate utilisation. Multiple taxa were correlated with SCFA levels, reinforcing the tight link between the microbiota, SCFAs and obesity. Our results suggest that gut microbiota dysbiosis and elevated fermentation activity may be involved in the etiology of childhood obesity.     

Sphingolipid distribution changes with age in the human lens

The formation of an internal barrier to the diffusion of small molecules in the lens during middle age is hypothesized to be a key event in the development of age-related nuclear (ARN) cataract. Changes in membrane lipids with age may be responsible. In this study, we investigated the effect of age on the distribution of sphingomyelins, the most abundant lens phospholipids. Human lens sections were initially analyzed by MALDI mass spectrometry imaging. A distinct annular distribution of the dihydrosphingomyelin, DHSM (d18:0/16:0), in the barrier region was observed in 64- and 70-year-old lenses but not in a 23-year-old lens. An increase in the dihydroceramide, DHCer (d18:0/16:0), in the lens nucleus was also observed in the older lenses. These findings were supported by ESI mass spectrometry analysis of lipid extracts from lenses dissected into outer, barrier, and nuclear regions. A subsequent analysis of 18 lenses ages 20–72 years revealed that sphingomyelin levels increased with age in the barrier region until reaching a plateau at approximately 40 years of age. Such changes in lipid composition will have a significant impact on the physical properties of the fiber cell membranes and may be associated with the formation of a barrier.     

The human microbiota associated with overall health

Human body harbors diverse microbes, the main components include bacteria, eukaryotes and viruses. Emerging evidences show that the human microbiota is intrinsically linked with overall health. The development of next-generation sequencing provides an unprecedented opportunity to investigate the complex microbial communities that are associated with the human body. Many factors like host genetics and environmental factors have a major impact on the composition and dynamic changes of human microbiota. The purpose of this paper is to present an overview of the relationship between human health and human microbiota (skin, nasal, throat, oral, vaginal and gut microbiota), then to focus on the factors modulating the composition of the microbiota and the future challenges to manipulate the microbiota for personalized health.     

The active human gut microbiota differs from the total microbiota

The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around 75% of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis.Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration. Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.     

强直性脊柱炎患者咽部菌群变化

目的 探讨强直性脊柱炎患者的咽部菌群变化。方法 筛选入组7例强直性脊柱炎患者和7例健康者咽拭子样本,提取咽部DNA,扩增16S rRNA基因,在Illumina平台测序,对测序结果进行生物信息学分析。结果 从ACE指数、Chao1指数、Shannon指数和Simpson指数综合来看强直性脊柱炎患者的咽部菌群Alpha多样性差异不大。Beta多样性分析显示两组研究对象咽部菌群样本可被区分。强直性脊柱炎患者咽部菌群组成和含量发生显著改变,主要变化包括：拟杆菌门（Bacteroidetes）和放线菌门（Actinobacteria）显著降低。拟杆菌门中普雷沃杆菌属（Prevotella）相关的纲目科属水平都显著降低。放线菌门变化落实到属水平,放线菌属（Actinomyces）显著降低,丙酸杆菌属（Propionibacterium）和棒状杆菌属（Corynebacterium）显著增高。厚壁菌门（Firmicutes）中,芽胞杆菌纲（Bacilli）所属的与链球菌属（Streptococcus）相关的纲目科属水平显著增加,而梭状芽胞杆菌纲（Clostridia）包含的韦荣球菌属（Veillonella）、消化球菌属（Peptococcus）显著下降。此外,变形菌门中出现弧菌属（Vibrio）的增加和弯曲菌属（Campylobacter）的降低等变化。结论 强直性脊柱炎患者（本次研究样本）的咽部菌群出现紊乱,以普雷沃杆菌属、放线菌属、韦荣球菌属、消化球菌属和弯曲菌属等显著降低,丙酸杆菌属、棒状杆菌属、链球菌属和弧菌属等显著增加为主要特征。     

Age-dependent changes in the gut microbiota and serum metabolome correlate with renal function and human aging

Human aging is invariably accompanied by a decline in renal function, a process potentially exacerbated by uremic toxins originating from gut microbes. Based on a registered household Chinese Guangxi longevity cohort (n = 151), we conducted comprehensive profiling of the gut microbiota and serum metabolome of individuals from 22 to 111 years of age and validated the findings in two independent East Asian aging cohorts (Japan aging cohort n = 330, Yunnan aging cohort n = 80), identifying unique age-dependent differences in the microbiota and serum metabolome. We discovered that the influence of the gut microbiota on serum metabolites intensifies with advancing age. Furthermore, mediation analyses unveiled putative causal relationships between the gut microbiota (Escherichia coli, Odoribacter splanchnicus, and Desulfovibrio piger) and serum metabolite markers related to impaired renal function (p-cresol, N-phenylacetylglutamine, 2-oxindole, and 4-aminohippuric acid) and aging. The fecal microbiota transplantation experiment demonstrated that the feces of elderly individuals could influence markers related to impaired renal function in the serum. Our findings reveal novel links between age-dependent alterations in the gut microbiota and serum metabolite markers of impaired renal function, providing novel insights into the effects of microbiota-metabolite interplay on renal function and healthy aging.     

口腔微生物与全身健康研究进展

口腔微生物是定植于人体口腔的微生物集合。众多研究证实,口腔微生物与多种口腔感染性疾病及系统性疾病紧密相关。随着“人类微生物组计划”及其他微生物宏基因组学相关项目的开展,人们对口腔微生物群落的认识不断深入。本文基于最新研究进展,就口腔微生物的组成、演替特点、与口腔和全身系统性疾病的关系及与肠道微生物的交互作用进行综述。     

Tomographic measurements of age changes in the human parotid gland

This study involved 48 subjects of both sexes with ages ranging from 22 years to 90 years. Computerised tomography was used to scan the right and left parotid gland. Gland mean density was calculated in Hounsfield units and regression graphs drawn. A significant fall in gland density was noted with subject age but no differences were noted between male and female subjects. A significant correlation was observed between the mean density of right and left glands in the same individuals. It is postulated that the fall in gland density is related an increase in fibro-fatty tissue within the gland. This study provides evidence that age related changes occur in the human parotid gland from youth to old age. This work supports the earlier findings of Scott's who studied the superficial lobe of the parotid gland histologically and found an increase in adipose tissue in this area of the gland with age.     

Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum

Age-related changes of IgG N-linked oligosaccharides isolated from normal human serum are reported for 403 individuals (male 227 and female 176), varying in age from 0 to 85 years. The IgG N-linked oligosaccharides were released from the protein by digestion with a glycoamidase and reductively aminated with the fluorescent reagent, 2-aminopyridine. The mixture of pyridylaminated oligosaccharides was separated at high resolution by HPLC using a reverse-phase column. From the results of neutral oligosaccharide analysis, agalactosyl glycoform and bisecting GlcNAc-containing glycoform were shown to increase with increasing age. Spearman's correlation coefficients were 0.503 and 0.473, respectively. Thus, in healthy people, an increase of both types of glycoforms correlates weakly with age. In addition, differences were demonstrated between male and female groups in their twenties. The quantity of agalactosyl glycoform was found to be lower in females than in males. No significant differences, however, were observed in the quantity of bisecting GlcNAc-containing glycoforms between males and females. Abbreviations: Gal, D-galactose: GlcNAc, N-acetyl-D-glucosamine; Man, D-mannose; Fc, C-terminal half of the heavy chain dimers of IgG; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; ODS, octadecylsilyl; PA, pyridylamino This revised version was published online in November 2006 with corrections to the Cover Date.     

Sex ratio at reproductive age: changes over the last century in the Italian population

The radical improvement in living conditions experienced in Italy during the last century caused a reduction in male extra-mortality during the prereproductive years. As a consequence, a progressive increase in the sex ratio at the beginning of the reproductive age (15-19 years) occurred, so that in recent times the sex ratio in the young adult population has approached the almost constant value of 1.06 observed at birth. We calculated that the sex composition would be the same in newborns and in young adults in about one generation: obviously, we have to assume that the sex differentials in mortality and migration are constant over time. The 1:1 equilibrium between sexes, which maximizes reproductive success, occurred in the 15-19 age group at the beginning of the century and shifted to the 30-35 age group in the 1990s. We compared the 1993-1995 sex ratios in different age groups in European Union countries and observed that in Italy as well as in other Mediterranean countries the numerical equality between sexes is reached at 30-35 years of age, while in north-central Europe it is reached later, approximately at the end of reproductive life.     

Development of a real‐time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs

Aims: To investigate whether the relative abundance of the Bacteroidetes and Firmicutes divisions in pigs is different between obese and lean animals. Methods and Results: Group‐specific primers were designed to target the 16S rRNA genes of Bacteroidetes and Firmicutes present in the gut. After the validation of their specificity, these primers were used in the real‐time PCR quantification of all Bacteria, Firmicutes division, Bacteroidetes division and Bacteroides spp. in the faecal samples of obese and lean pigs from Banna mini‐pig inbred line. The obese pigs had a ～61% fewer percentage (based on all Bacteria) of Bacteroidetes division (P = 0·033) and a ～56% fewer proportion of Bacteroides spp. (P = 0·047) than the lean pigs. The proportions of both Bacteroidetes and Bacteroides had a negative correlation (P < 0·01) with the body weight. Conclusion: The results suggested that the fat storage might affect the proportion of Bacteroidetes division in the gut. Significance and Impact of the Study: The real‐time PCR assays developed for Firmicutes and Bacteroidetes will be useful for investigating the composition of gut microbiota.     

Assembly of the human intestinal microbiota

Complex microbial ecosystems occupy the skin, mucosa and alimentary tract of all mammals, including humans. Recent advances have highlighted the tremendous diversity of these microbial communities and their importance to host physiology, but questions remain about the ecological processes that establish and maintain the microbiota throughout life. The prevailing view, that the gastrointestinal microbiota of adult humans is a climax community comprised of the superior competitors for a stable set of niches, does not account for all of the experimental data. We argue here that the unique history of each community and intrinsic temporal dynamics also influence the structure of human intestinal communities.     

Optimizing the analysis of human intestinal microbiota with phylogenetic microarray

Phylogenetic microarrays present an attractive strategy to high-throughput interrogation of complex microbial communities. In this work, we present several approaches to optimize the analysis of intestinal microbiota with the recently developed Microbiota Array. First, we determined how 16S rDNA-specific PCR amplification influenced bacterial detection and the consistency of measured abundance values. Bacterial detection improved with an increase in the number of PCR amplification cycles, but 25 cycles were sufficient to achieve the maximum possible detection. A PCR-caused deviation in the measured abundance values was also observed. We also developed two mathematical algorithms that aimed to account for a predicted cross-hybridization of 16S rDNA fragments among different species, and to adjust the measured hybridization signal based on the number of 16S rRNA gene copies per species genome. The 16S rRNA gene copy adjustment indicated that the presence of members of the class Clostridia might be overestimated in some 16S rDNA-based studies. Finally, we show that the examination of total community RNA with phylogenetic microarray can provide estimates of the relative metabolic activity of individual community members. Complementary profiling of genomic DNA and total RNA isolated from the same sample presents an opportunity to assess population structure and activity in the same microbial community.     

肠道细菌微生态与人类疾病关系研究进展

寄居在人类肠道的数以亿计的肠道细菌,已被证实与人类多种疾病有关。就肠道细菌与内分泌系统疾病、消化系统疾病、心血管系统疾病、神经系统疾病、获得性免疫缺陷综合征以及特应性疾病等的关系研究进展进行综述。