Serum metabolic profile characteristics of offspring rats before and after birth caused by prenatal caffeine exposure.

Epidemiological investigations have confirmed that prenatal caffeine intake could increase the incidence rate of intrauterine growth retardation (IUGR) and multiple diseases after birth. Based on liquid chromatography-mass spectrometry, we analyzed serum metabolic profiles of offspring rats before and after birth in IUGR model induced by prenatal caffeine exposure (PCE). We discovered that differential metabolites in PCE fetuses mainly manifested as amino acids and lipid metabolism. In adulthood, PCE offspring showed less and inconsistent types of differential metabolites compared to those in utero, which still exhibited gender differences. The main differential metabolites induced by PCE, including phospholipids, platelet-activating factor, arachidonic acid, bile acid, sphingosine-1-phosphoric acid, indoxyl sulfuric acid, and cortexolone, may participate in the pathological and physiological processes of organ toxicities. This study demonstrated the short- and long-term developmental toxicity and gender differences of caffeine, providing new ideas for exploring the early warning and drug intervention targets of IUGR offspring.

[Specificity comparison of three Feacalibacterium prausnitzii-specific PCR primer pairs].

OBJECTIVE: To compare the specificity of three 16S rRNA gene-based PCR primer pairs (FPR-1/FPR-2, FPR-2F/Fprau645R and Fprau223F/Fprau420R), which are used to specifically detect and quantify the important human gut bacterium Feacalibacterium prausnitzii. METHODS: Clustal X was used to align the sequences of individual primer and the 16S rRNA gene of F. prausnitzii and other bacteria. The number of Faecalibacterium spp. sequences in Ribosomal Database Project (RDP) matched by each primer was obtained by the Probe Match tool. With the full-length 16S rRNA gene clone library constructed by our laboratory which contained 7255 clones from the gut microbiota of 7 Chinese people, the Simulated PCR (SPCR) program was applied to predict the clone number of F. prausnitzii and other gut bacteria matched by every primer pair; PCR amplification was performed with three primer pairs and representative clones to verify the SPCR prediction. Real-time quantitative PCR was performed with three primer pairs, respectively, for fecal samples from 14 healthy individuals. RESULTS: The first base at the 3' end of Fprau645R showed the highest mismatch level for non-F. prausnitzii bacteria. The percentage of the number of Faecalibacterium spp. sequences matched by Fprau645R accounting for that of matched bacteria sequences in the RDP database was 97.6%, which was significantly higher than that of other primers. As SPCR predicted, all three primer pairs can detect about 1171 clones from F. prausnitzii; the clones of non-Faecalibacterium spp. detected by FPR-2F/Fprau645R mainly were Subdoligranulum spp. , but the non-Faecalibacterium spp. clones detected by FPR-1/FPR-2 and Fprau223F/Fprau420R were mainly Subdoligranulum spp., Oscillibacter spp. , Ruminococcus spp. and unclassified Ruminococcaceae etc. The real PCR showed the same results with SPCR. The real-time quantitative PCR showed FPR-1/FPR-2 and Fprau223F/Fprau420R detected more bacteria than FPR-2F/Fprau645R. CONCLUSION: The three primer pairs can detect F. prausnitzii and Subdoligranulum spp., however, the specificity of FPR-2F/Fprau645R is better than FPR-1/FPR-2 and Fprau223F/Fprau420R.

A diverse bacterial community in an anoxic quinoline-degrading bioreactor determined by using pyrosequencing and clone library analysis.

There is a concern of whether the structure and diversity of a microbial community can be effectively revealed by short-length pyrosequencing reads. In this study, we performed a microbial community analysis on a sample from a high-efficiency denitrifying quinoline-degrading bioreactor and compared the results generated by pyrosequencing with those generated by clone library technology. By both technologies, 16S rRNA gene analysis indicated that the bacteria in the sample were closely related to, for example, Proteobacteria, Actinobacteria, and Bacteroidetes. The sequences belonging to Rhodococcus were the most predominant, and Pseudomonas, Sphingomonas, Acidovorax, and Zoogloea were also abundant. Both methods revealed a similar overall bacterial community structure. However, the 622 pyrosequencing reads of the hypervariable V3 region of the 16S rRNA gene revealed much higher bacterial diversity than the 130 sequences from the full-length 16S rRNA gene clone library. The 92 operational taxonomic unit (OTUs) detected using pyrosequencing belonged to 45 families, whereas the 37 OTUs found in the clone library belonged to 25 families. Most sequences obtained from the clone library had equivalents in the pyrosequencing reads. However, 64 OTUs detected by pyrosequencing were not represented in the clone library. Our results demonstrate that pyrosequencing of the V3 region of the 16S rRNA gene is not only a powerful tool for discovering low-abundance bacterial populations but is also reliable for dissecting the bacterial community structure in a wastewater environment.

Structural shifts of gut microbiota as surrogate endpoints for monitoring host health changes induced by carcinogen exposure.

This study monitored structural shifts of gut microbiota of rats developing precancerous mucosal lesions induced by carcinogen 1,2-dimethyl hydrazine (DMH) treatment using PCR-denaturing gradient gel electrophoresis (DGGE) and 454 pyrosequencing on the 16S rRNA gene V3 region. Partial least square discriminant analysis of DGGE fingerprints showed that the gut microbiota structure of treated animals was similar to that of the controls 1 and 3 weeks after DMH treatments, but significantly different 7 weeks after DMH treatments, when a large number of aberrant crypt foci (ACF) developed in their colons. Martens' uncertainty test, followed by anova test (P<0.05) identified Ruminococcus-like and Allobaculum-like bacteria as key variables for discrimination of DMH-treated rats from controls. Real-time PCR confirmed the significant increase of the Ruminococcus obeum and the Allobaculum-like bacteria in DMH-treated rats. UniFrac analysis based on V3 pyrosequencing further validated that the gut microbiota structures of treated and control animals were similar at an early stage, but segregated after ACF formation. Thirteen operational taxonomic units including Ruminococcus-like and Allobaculum-like bacteria were identified as key variables for the discrimination of DMH-treated rats from controls. Dynamic analysis of gut microbiota may become a noninvasive strategy for monitoring host health changes induced by carcinogen exposure.

Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice.

Both genetic variations and diet-disrupted gut microbiota can predispose animals to metabolic syndromes (MS). This study assessed the relative contributions of host genetics and diet in shaping the gut microbiota and modulating MS-relevant phenotypes in mice. Together with its wild-type (Wt) counterpart, the Apoa-I knockout mouse, which has impaired glucose tolerance (IGT) and increased body fat, was fed a high-fat diet (HFD) or normal chow (NC) diet for 25 weeks. DNA fingerprinting and bar-coded pyrosequencing of 16S rRNA genes were used to profile gut microbiota structures and to identify the key population changes relevant to MS development by Partial Least Square Discriminate Analysis. Diet changes explained 57% of the total structural variation in gut microbiota, whereas genetic mutation accounted for no more than 12%. All three groups with IGT had significantly different gut microbiota relative to healthy Wt/NC-fed animals. In all, 65 species-level phylotypes were identified as key members with differential responses to changes in diet, genotype and MS phenotype. Most notably, gut barrier-protecting Bifidobacterium spp. were nearly absent in all animals on HFD, regardless of genotype. Sulphate-reducing, endotoxin-producing bacteria of the family, Desulfovibrionaceae, were enhanced in all animals with IGT, most significantly in the Wt/HFD group, which had the highest calorie intake and the most serious MS phenotypes. Thus, diet has a dominating role in shaping gut microbiota and changes of some key populations may transform the gut microbiota of Wt animals into a pathogen-like entity relevant to development of MS, despite a complete host genome.

Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16S rRNA gene clone library analysis.

Bacteroides spp. represent a prominent bacterial group in human intestinal microbiota with roles in symbiosis and pathogenicity; however, the detailed composition of this group in human feces has yet to be comprehensively characterized. In this study, the molecular diversity of Bacteroides spp. in human fecal microbiota was analyzed from a seven-member, four-generation Chinese family using Bacteroides spp. group-specific 16S rRNA gene clone library analysis. A total of 549 partial 16S rRNA sequences amplified by Bacteroides spp.-specific primers were classified into 52 operational taxonomic units (OTUs) with a 99% sequence identity cut-off. Twenty-three OTUs, representing 83% of all clones, were related to 11 validly described Bacteroides species, dominated by Bacteroides coprocola, B. uniformis, and B. vulgatus. Most of the OTUs did not correspond to known species and represented hitherto uncharacterized bacteria. Relative to 16S rRNA gene universal libraries, the diversity of Bacteroides spp. detected by the group-specific libraries was much higher than previously described. Remarkable inter-individual differences were also observed in the composition of Bacteroides spp. in this family cohort. The comprehensive observation of molecular diversity of Bacteroides spp. provides new insights into potential contributions of various species in this group to human health and disease.