'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora

Germfree mice were associated via direct and indirect contact with a 'normal' microflora by placing 'normal' mice in an isolator with germfree mice. Relative caecal weights, the ratio of secondary to primary bile acids, the presence of filamentous segmented bacteria in the small intestine and faecal beta-aspartylglycine were normal 5 days after direct contact and 15 days after indirect contact. Enterobacteriaceae were demonstrated by the third day after direct contact and the fourth day after indirect contact. Volatile and non-volatile fatty acids in the caecal contents were variable and appeared to be unrelated to the 'normalization' process of germfree mice after association with a microflora.     

贫铀经口慢性染毒对小鼠肠道菌群多样性的影响

目的观察贫铀经口慢性染毒对小鼠肠道菌群多样性的影响。方法通过将不同剂量的贫铀混入饲料中饲养小鼠,以小鼠肝脏和肾脏铀含量作为判断贫铀在动物体内蓄积的指标,建立贫铀经121慢性染毒小鼠模型,观察各组小鼠之间的体重变化,对各组小鼠进行基于细菌16SrRNAV6－V8区的PCR－DGGE分析。结果食入贫铀的各组小鼠肝脏和肾脏铀含量显著高于对照组（P〈0．05）,各组小鼠体重差异无统计学意义（P〉0．05）,各组小鼠V6－V8IXDGGE图谱的丰富度和多样性指数差异均无统计学意义（P〉0．05）。结论贫铀经日慢性染毒对小鼠肠道菌群多样性无显著影响。     

Association of germfree mice with intestinal microfloras obtained from "normal" mice

A cultured microflora obtained from the caecum of a "normal" mouse was given to 4 groups of germfree mice and was supplied 1x, 2x, 3x and 4x respectively at 5-day intervals. Another group received a 10(-7) dilution of the caecal flora while a group associated with an 'SPF' flora served as control. The difference (measured by 8 parameters) between mice supplied with the cultured flora or with a 10(-7) dilution, both given once only, was small. Supplying the flora 3x resulted in more 'normal' mice compared with mice which received the flora once or twice. The caeca of specified-pathogen-free mice contained more bacteria per gram (microscopic bacterial count), less aerobic and anaerobic bacteria per gram (viable counts), while the yield as percentage of the microscopic bacterial count was lower as compared with the group to which a cultured flora was supplied 4 times.     

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.     

Comprehensive postmortem analyses of intestinal microbiota changes and bacterial translocation in human flora associated mice

Background

Postmortem microbiological examinations are performed in forensic and medical pathology for defining uncertain causes of deaths and for screening of deceased tissue donors. Interpretation of bacteriological data, however, is hampered by false-positive results due to agonal spread of microorganisms, postmortem bacterial translocation, and environmental contamination.

Methodology/Principal Findings

We performed a kinetic survey of naturally occurring postmortem gut flora changes in the small and large intestines of conventional and gnotobiotic mice associated with a human microbiota (hfa) applying cultural and molecular methods. Sacrificed mice were kept under ambient conditions for up to 72 hours postmortem. Intestinal microbiota changes were most pronounced in the ileal lumen where enterobacteria and enterococci increased by 3–5 orders of magnitude in conventional and hfa mice. Interestingly, comparable intestinal overgrowth was shown in acute and chronic intestinal inflammation in mice and men. In hfa mice, ileal overgrowth with enterococci and enterobacteria started 3 and 24 hours postmortem, respectively. Strikingly, intestinal bacteria translocated to extra-intestinal compartments such as mesenteric lymphnodes, spleen, liver, kidney, and cardiac blood as early as 5 min after death. Furthermore, intestinal tissue destruction was characterized by increased numbers of apoptotic cells and neutrophils within 3 hours postmortem, whereas counts of proliferative cells as well as T- and B-lymphocytes and regulatory T-cells decreased between 3 and 12 hours postmortem.

Conclusions/Significance

We conclude that kinetics of ileal overgrowth with enterobacteria and enterococci in hfa mice can be used as an indicator for compromized intestinal functionality and for more precisely defining the time point of death under defined ambient conditions. The rapid translocation of intestinal bacteria starting within a few minutes after death will help to distinguish between relevant bacteria and secondary contaminants thus providing important informations for routine applications and future studies in applied microbiology, forensic pathology, and criminal medicine.     

Development of the human infant intestinal microbiota

Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.     

The microbiota in spider mite feces potentially reflects intestinal bacterial communities in the host

Microorganisms provide many physiological functions to herbivorous hosts. Spider mites (genus Tetranychus) are important agricultural pests throughout the world; however, the composition of the spider mite microbial community, especially gut microbiome, remains unclear. Here, we investigated the bacterial community in five spider mite species and their associated feces by deep sequencing of the 16S rRNA gene. The composition of the bacterial community was significantly different among the five prevalent spider mite species, and some bacterial symbionts showed host‐species specificity. Moreover, the abundance of the bacterial community in spider mite feces was significantly higher than that in the corresponding spider mite samples. However, Flavobacterium was detected in all samples, and represent a “core microbiome”. Remarkably, the maternally inherited endosymbiont Wolbachia was detected in both spider mite and feces. Overall, these results offer insight into the complex community of symbionts in spider mites, and give a new direction for future studies.     

Resistance of germfree rats to indomethacin-induced intestinal lesions.

Indomethacin given orally to conventional rats produced in three days a syndrome, often fatal, of intestinal lesions characterized by multiple ulcers and peritonitis. Male germfree rats were found to be resistant to this effect of indomethacin, while female germfree rats developed very mild lesions. Germfree rats became sensitive again to such lesions when monocontaminated with E. coli. In such animals, however, the lesions were less severe than in conventional animals, presumably because more than one microorganism is necessary for the full syndrome to develop. These results suggest that microorganisms are necessary for the development of indomethacin-induced intestinal lesions. Secondary bile acids, absent in germfree animals, may also be necessary. The prostaglandin deficiency caused by indomethacin appears to weaken the resistance of the intestinal mucosa to microorganisms and/or their toxins. The latter may then penetrate the mucosa, damage the cells and produce ulcers and perforations. Since several prostaglandins also protect against indomethacin-induced lesions, the hypothesis is advanced that certain prostaglandins may protect the mucosa ("cytoprotection") by preventing the spread of microorganisms and/or their toxin through the intestinal wall.     

Towards the human intestinal microbiota phylogenetic core

The paradox of a host specificity of the human faecal microbiota otherwise acknowledged as characterized by global functionalities conserved between humans led us to explore the existence of a phylogenetic core. We investigated the presence of a set of bacterial molecular species that would be altogether dominant and prevalent within the faecal microbiota of healthy humans. A total of 10 456 non-chimeric bacterial 16S rRNA sequences were obtained after cloning of PCR-amplified rDNA from 17 human faecal DNA samples. Using alignment or tetranucleotide frequency-based methods, 3180 operational taxonomic units (OTUs) were detected. The 16S rRNA sequences mainly belonged to the phyla Firmicutes (79.4%), Bacteroidetes (16.9%), Actinobacteria (2.5%), Proteobacteria (1%) and Verrumicrobia (0.1%). Interestingly, while most of OTUs appeared individual-specific, 2.1% were present in more than 50% of the samples and accounted for 35.8% of the total sequences. These 66 dominant and prevalent OTUs included members of the genera Faecalibacterium , Ruminococcus , Eubacterium , Dorea , Bacteroides , Alistipes and Bifidobacterium . Furthermore, 24 OTUs had cultured type strains representatives which should be subjected to genome sequence with a high degree of priority. Strikingly, 52 of these 66 OTUs were detected in at least three out of four recently published human faecal microbiota data sets, obtained with very different experimental procedures. A statistical model confirmed these OTUs prevalence. Despite the species richness and a high individual specificity, a limited number of OTUs is shared among individuals and might represent the phylogenetic core of the human intestinal microbiota. Its role in human health deserves further study.     

口服肝素与小鼠肠道菌群的相互作用

口服肝素药物的开发需要系统地理解口服肝素与肠道菌群之间的互作过程。通过荧光体视镜观察荧光素标记的肝素经小鼠口服后在体内的分布情况,利用高效液相色谱法检测肝素在模拟胃肠液中的稳定性和体外培养肠道菌群模拟肠道菌对肝素的降解作用,发现口服肝素主要分布在小鼠胃肠道内,在体外模拟胃肠液条件下肝素结构稳定,但能够被添加肝素的厌氧培养基培养后的肠道菌群降解。为了进一步揭示口服肝素对健康小鼠肠道菌群的影响,利用Illumina MiSeq高通量测序技术测定口服肝素后C57BL/6J小鼠粪便菌群的16S rRNA序列,与口服生理盐水的小鼠粪便菌群进行对比,发现口服肝素的小鼠粪便菌群的生物多样性降低;在门水平上,菌群结构差异不显著;而在属水平上,别样杆菌属Alistipes、副萨特氏菌属Parasutterella和艾克曼菌属Akkermansia相对丰度增高,而嗜胆菌属Bilophila、肠杆菌属Enterorhabdus、瘤胃梭菌属Ruminiclostridium、普雷沃氏菌科Prevotellaceae_UCG_001、瘤胃梭菌属Ruminiclostridium-9、拟杆菌属Bacteroides、Lachnoclostridium、Candidatus_Saccharimonas、Intestinimonas和Dubosiella的相对丰度减少,表明口服肝素能够影响小鼠肠道菌群结构。此外,实验发现口服肝素对小鼠无明显毒副作用,具有较高安全性。研究结果将为开发肝素口服递送策略提供新的思路,为口服肝素类药物的开发提供参考。     

Spatial heterogeneity and co-occurrence patterns of human mucosal-associated intestinal microbiota

Human gut microbiota shows high inter-subject variations, but the actual spatial distribution and co-occurrence patterns of gut mucosa microbiota that occur within a healthy human instestinal tract remain poorly understood. In this study, we illustrated a model of this mucosa bacterial communities'' biogeography, based on the largest data set so far, obtained via 454-pyrosequencing of bacterial 16S rDNAs associated with 77 matched biopsy tissue samples taken from terminal ileum, ileocecal valve, ascending colon, transverse colon, descending colon, sigmoid colon and rectum of 11 healthy adult subjects. Borrowing from macro-ecology, we used both Taylor''s power law analysis and phylogeny-based beta-diversity metrics to uncover a highly heterogeneous distribution pattern of mucosa microbial inhabitants along the length of the intestinal tract. We then developed a spatial dispersion model with an R-squared value greater than 0.950 to map out the gut mucosa-associated flora''s non-linear spatial distribution pattern for 51.60% of the 188 most abundant gut bacterial species. Furthermore, spatial co-occurring network analysis of mucosa microbial inhabitants together with occupancy (that is habitat generalists, specialists and opportunist) analyses implies that ecological relationships (both oppositional and symbiotic) between mucosa microbial inhabitants may be important contributors to the observed spatial heterogeneity of mucosa microbiota along the human intestine and may even potentially be associated with mutual cooperation within and functional stability of the gut ecosystem.     

罗伊乳杆菌对小鼠血清细胞因子含量和肠道菌群的影响

目的 探讨罗伊乳杆菌对小鼠免疫力及肠道菌群的影响。方法 将50只昆明系小鼠按体重随机分成5组：空白对照组、低剂量组、中剂量组、高剂量组和发酵上清液组。空白对照组灌胃等量生理盐水,其余各组分别灌胃罗伊乳杆菌菌液及发酵液上清21 d后,眼球取血收集血清,采用ELISA法检测IL-2、IFN-γ、IgA和IgG含量;采用16S rRNA基因高通量测序分析小鼠肠道菌群变化。结果 与对照组相比,其余各组小鼠血清IL-2、IFN-γ、IgA和IgG的水平均明显升高,粪便菌群丰度有所增高,多样性降低。与对照组相比,低剂量组和中剂量组小鼠粪便菌群结构无显著差异,而高剂量组和发酵上清液组小鼠粪便菌群结构差异显著。结论 罗伊乳杆菌可以增强小鼠免疫力,提高菌群丰度,改变小鼠肠道菌群结构。     

Plasticity of the adult human small intestinal stoma microbiota

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蜂蜜对洛哌丁胺诱导的便秘小鼠肠道微生态的影响

目的 观察蜂蜜对洛哌丁胺诱导的便秘小鼠肠道微生态的影响。方法 雄性Balb/c小鼠30只,随机分成两组：正常对照组（10只）、模型组（20只）,同时模型组再分为自然恢复组（10只）、蜂蜜组（10只）。应用洛哌丁胺制备便秘小鼠模型,分别于造模后、给蜂蜜后第5天对每只小鼠进行称重、采便;之后于第12天称重、采便,处死小鼠,进行小肠推进率、结肠组织血管活性肠肽（VIP）、P物质（SP）、5-羟色胺（5-HT）测定和肠道菌群检测。结果 洛哌丁胺造模后的第5天,模型组小鼠粪便含水量及体质量下降;蜂蜜组小鼠体质量下降,但粪便含水量无明显变化。蜂蜜治疗至第12天时,蜂蜜组小鼠与模型组比SP增加,粪便含水量及小肠推进率增加,蜂蜜组小鼠与模型组比VIP、5-HT变化不明显。此外蜂蜜干预后便秘小鼠肠道中拟杆菌属和厚壁菌属的细菌丰度有所降低,Alistipes obesi的含量增加。结论 蜂蜜对小鼠5-HT分泌水平无明显影响;可以提高便秘小鼠结肠组织中SP含量、增加粪便含水量、加快小肠推进率;调节肠道微生态失衡,对便秘发挥一定的治疗作用。     

Oral administration of lactobacilli from human intestinal tract protects mice against influenza virus infection

Aims: Our study was conducted to evaluate the potent protective effects of oral administration of probiotic Lactobacillus strains against influenza virus (Flu) infection in a mouse model. Method and Results: Lyophilized Lactobacillus rhamnosus GG (LGG) and Lactobacillus gasseri TMC0356 (TMC0356) were orally administered to BALB/c mice for 19 days. The test mice were intranasally infected with Flu A/PR/8/34 (H1N1) on day 14, and any changes in clinical symptoms were monitored. After 6 days of infection, the mice were killed and pulmonary virus titres were determined. The clinical symptom scores of mice administered oral LGG and TMC0356 were significantly ameliorated, compared to those of the control mice (P < 0·01). The pulmonary virus titres of the mice fed LGG and TMC0356 were also significantly decreased compared to those of control mice (P < 0·05). Conclusions: These results indicate that oral administration of lactobacilli, such as LGG and TMC0356, might protect a host animal against Flu infection. Significance and Impact of the Study: These results demonstrate that oral administration of selected lactobacilli might protect host animals from Flu infection by interactions with gut immunity.     

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.