Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture-based method. Direct counts ranged from 1.9 X 10" to 4.0 X 10" cells/g (wet weight), and plate counts totaled 6.6 X 10(10) to 1.2 X 10(11) CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.     

Identification of bacteria using two degenerate 16S rDNA sequencing primers.

Two degenerate 16S rDNA primers have been designed for broad-range identification of eubacteria by PCR and automated sequencing. Using a simple method, the primers have proven useful in identification of proteobacteria (Campylobacter, Enterobacter, Escherichia, Helicobacter, Klebsiella), gram-positive bacteria (Mycobacterium, Staphylococcus, Streptococcus) and spirochetes (Borrelia) derived from clinical samples. In several cases, the samples could be identified at the species level.     

Identification of a pheA gene associated with Streptococcus mitis by using suppression subtractive hybridization

We performed suppression subtractive hybridization to identify genomic differences between Streptococcus mitis and Streptococcus pneumoniae. Based on the pheA gene, a primer set specific to S. mitis detection was found in 18 out of 103 S. mitis-specific clones. Our findings would be useful for discrimination of S. mitis from other closely related cocci in the oral environment.     

Evolution of Streptococcus pneumoniae and its close commensal relatives

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.     

Determinants of the developing oral flora in normal newborns.

The ability of Streptococcus species to selectively adhere to the oral epithelial cells of newborns was studied in vitro. On day 1 of life, mucosal cells from normal infants demonstrated selective attraction for the natural distribution of streptococci that would soon colonize these surfaces. Streptococcus salivarius and Streptococcus mitis adhered well in vitro to scraped cells from cheek and tongue surfaces. Streptococcus mutans, on the other hand, exhibited feeble or no adherence to cheek or tongue cells. Adherence of Escherichia coli to oral epithelial cells was also studied. The ability of strains of E. coli to adhere to cheek and tongue cells correlated solely with the presence of cell surface substances, probably pili. These observations, made on infants at the critical moment of their developing flora, strengthen the hypothesis that the ability of bacteria to adhere to surfaces is an important determinant of their ecological place in the oral microflora.     

Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil

Four fungal 18S rDNA and internal transcribed spacer (ITS) polymerase chain reaction (PCR) primer pairs were tested for their specificity towards target fungal DNA in soil DNA extracts, and their ability to assess the diversity of fungal communities in a natural grassland soil was compared. Amplified PCR products were cloned, and approximately 50 clones from each library were sequenced. Phylogenetic analysis and database searches indicated that each of the sequenced cloned DNA fragments was of fungal origin for each primer pair, with the exception of the sequences generated using the 18S rDNA primers nu-SSU-0817 and nu-SSU-1196, where 35 of the 50 sequenced clones represented soil invertebrates. Although some of the primers have previously been suggested to be biased towards certain fungal taxonomic groups, the ratio of sequences representing each of the four main fungal phyla, Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, was similar for each of the primer pairs, suggesting that primer bias may be less significant than previously thought. Collector's curves were plotted to estimate the coverage obtained for each of the clone libraries after clustering the sequences into operational taxonomic units at a level of 99% sequence similarity. The curves indicated that good coverage of diversity was achieved, with the exception of the clone library constructed using primers nu-SSU-0817 and nu-SSU-1196, on account of the high number of non-fungal sequences obtained. The work demonstrates the usefulness of 18S rDNA and ITS PCR primers for assessing fungal diversity in environmental samples, and it also highlights some potential limitations of the approach with respect to PCR primer specificity and bias.     

Altamira cave Paleolithic paintings harbor partly unknown bacterial communities

Since it has been reported that microorganisms can affect painting pigments, Paleolithic painting microbiology deserves attention. The present study is the first report on the bacterial colonization of the valuable Paleolithic paintings in the famous Altamira cave (Spain). One sample taken from a painting area in the Polychromes Hall was analyzed culture-independently. This was the first time microbiologists were allowed to take sample material directly from Altamira paintings. Identification methods included PCR amplification of 16S rRNA genes (16S rDNA) and community fingerprinting by denaturing gradient gel electrophoresis (DGGE). The applied approach gave insight into a great bacterial taxonomic diversity, and allowed the detection of unexpected and unknown bacteria with potential effects on the conservation of the painting. Regarding the number of 29 visible DGGE bands in the community fingerprint, the numbers of analyzed clones described about 72% of the phylogenetic diversity present in the sample. Thirty-eight percent of the sequences analyzed were phylogenetically most closely related to cultivated bacteria, while the majority (62%) were most closely related to environmental 16S rDNA clones. Bacteria identified in Altamira were related with sequence similarities between 84.8 and 99.4% to members of the cosmopolitan Proteobacteria (52.3%), to members of the Acidobacterium division (23.8%), Cytophaga/Flexibacter/Bacteroides phylum (9.5%), green non-sulfur bacteria (4.8%), Planctomycetales (4.8%) and Actinobacteria (4.8%). The high number of clones most closely related to environmental 16S rDNA clones showed the broad spectrum of unknown and yet to be cultivated bacteria in Altamira cave.     

Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.     

Rumen Bacterial Community Transition During Adaptation to High-grain Diet

Transitional changes of the ruminal bacterial community structure in cows during the switch from roughage to high-grain diet were monitored by PCR amplification and sequencing of 16S rDNA clone libraries. In total, one hundred fifty 16S rDNA sequences of almost full-length (1.4 kb) were analysed from three libraries prepared from the rumen fluid on day 0, 3, and 28 of switch to high-grain diet. In the first library (day 0, hay diet) of 51clones, 90.2% of sequences were belonging to the low G+C Gram-positive bacteria (LGCGPB) phylum, with the minor inclusion of theCytophaga-Flavobacter-Bacteroides (CFB;3.9%), Proteobacteria (3.9%) and high G+C Gram-positive bacteria (HGCGPB;2.0%) phyla-related sequences. Six LGCGPB sequences were clustered with the well-known cellulolytics of the rumen, Ruminococcus flavefaciens and R. albus. In the second library (day 3 of high-grain diet) of 58 clones, the LGCGPB-related sequences still dominated (72.4%), albeit being represented by other species than in the first library. In particular, this library was enriched by representatives of Selenomonas-Succiniclasticum-Megasphaera group IX (17.2%), lactobacilli- (6.9%) and Butyrivibrio fibrisolvens lineage 3-related (8.6%) sequences. Other phyla were represented by CFB (22.4%) and HGCGPB (3.4%). In the third library (day 28 of high-grain diet) of 41 clones, 95% of sequences fell into the LGCGPB phylum. About half of them (46%) were clustered within theSelenomonas-Succiniclasticum-Megasphaera group in Clostridium cluster IX. No HGCGPB-related sequences were detected and CFB was represented by only a single clone. No Streptococcus bovis -related sequences were detected in any of the three clone libraries.     

Diversity of 16S rDNA and Naphthalene Dioxygenase Genes from Coal-Tar-Waste-Contaminated Aquifer Waters

Microbial diversity in four wells along a groundwater flowpath in a coal-tar-waste-contaminated aquifer was examined using RFLP analysis of both 16S rDNA and naphthalene dioxygenase (NDO) genes. Amplified ribosomal DNA restriction analysis (ARDRA) relied upon eubacteria-specific primers to generate four clone libraries. From each library, 100 clones were randomly picked for analysis. Sixty percent of 400 clones contained unique ARDRA patterns. Diversity indices calculated for each community were high (Shannon-Weaver, H = 3.53 to 3.69). Clones representing ARDRA patterns found in the highest abundance were sequenced (31 total). Sequences related to aerobic bacteria (e.g., Nitrospira, Methylomonas, and Gallionella) predominated among those retrieved from the uncontaminated area of the site, whereas sequences related to facultatively aerobic and anaerobic bacteria (e.g. Azoarcus, Syntrophus, and Desulfotomaculum) predominated among those retrieved from contaminated areas of the site. Using NDO-specific primers and low-stringency PCR conditions, variability in RFLP patterns was only detected in community-derived DNA (3 of 4 wells) and not in 5 newly isolated naphthalene-degrading pure cultures. The ARDRA patterns of the pure culture isolates were not found in the clone libraries. Polymorphisms in community 16S rDNA and NDO genes found in well-water microorganisms reflected distinctive geochemical conditions across the site. Sequences related to sulfate-reducing bacteria were found in groundwater that contained sulfide, while sequences related to Gallionella, Syntrophus, and nitrate-reducing aromatic hydrocarbon-degrading bacteria were found in groundwater that contained ferrous iron, methane, and naphthalene, respectively.     

A molecular phylogenetic survey of sea-ice microbial communities (SIMCO)

16S rDNA clone library analysis was used to identify bacterial biodiversity in a variety of sea-ice microbial communities (SIMCO). DNA was extracted from seven Antarctic sea-ice samples and one Arctic sea-ice sample and 16S rDNA PCR-amplified using universal and Archaea-specific primers. Recombinant 16S rDNA clones were obtained and dereplicated using restriction fragment length polymorphism analysis (RFLP). After RFLP analysis, 100 distinct phylotypes (a unique clone or group of clones with sequence similarity of >0.98) were defined. From the clone libraries 16S rDNA sequences of bacterial and eukaryotic origin were detected, however Archaea were not detected either with universal or Archaea-specific 16S rDNA primer sets. Bacterial phylotypes grouped within the alpha and gamma proteobacteria, the Cytophaga-Flavobacterium-Bacteroides division, the Gram-Positive bacteria and the orders Chlamydiales and Verrucomicrobiales. The majority of bacterial phylotypes were affiliated with heterotrophic taxa and many grouped closely with cultivated genera and species. Eukaryotic clones were affiliated with a variety of autotrophic and heterotrophic nanoplankton and included a large number of chloroplast 16S rDNA genes. The findings of this investigation corroborated culture data indicating bacterial biodiversity increased in SIMCO displaying high levels of primary production, however the bacterial communities within SIMCO were highly heterogeneous at the genus/species-level between different samples. A comparison of Antarctic and Arctic SIMCO revealed certain sea-ice dwelling bacterial genera are common at both poles.     

采用未培养技术对荷斯坦奶牛瘤胃细菌多样性进行初步分析

采用未培养(Culture independent)技术直接从荷斯坦奶牛瘤胃液中提取瘤胃细菌微生物混合DNA(也叫元基因组DNA),利用细菌16SrDNA通用引物27F与1492R,扩增瘤胃混合微生物的16SrDNA,根据16SrDNA序列对瘤胃细菌多样性进行初步分析。通过16SrDNA序列同源性分析,发现有多于一半以上的序列与可培养的菌株的同源性小于90%,属于不可培养的菌株。选用45条测得序列与已知序列构建系统发育树,分析结果表明,它们分属于两大类LGCGPB(the lowG CGram positivebac teria)和CFB(Cytophaga_Flexibacter $CBacteroides group),剩下的克隆尚难确定其分类地位,可能是代表新属和种的序列,这些序列已向GenBank提交并得到序列号(AY986777_AY986791)。     

16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica

The prokaryotic diversity of aerobic and anaerobic bacterial isolates and of bacterial and archaeal 16S rDNA clones was determined for a microbial mat sample from the moated region of Lake Fryxell, McMurdo Dry Valleys, Antarctica. Among the anaerobic bacteria, members of Clostridium estertheticum and some other psychrotolerant strains dominated whereas methanogens and other Archaea were lacking. Isolates highly related to Flavobacterium hibernum, Janthiniobacterium lividum, and Arthrobacter flavus were among the aerobic bacteria most frequently isolated. Assessment of more than 350 partial 16S rDNA clone sequences of libraries generated by Bacteria- and Archaea-specific PCR primers revealed a rich spectrum of bacterial diversity but only two different archaeal clone sequences. Among the Bacteria, representative sequences belonged to the class Proteobacteria, order Verrucomicrobiales, class Actinobacteria, Clostridium/Bacillus subphylum of Gram-positives, and the Cytophaga-Flavobacterium-Bacteroides phylum. The clones formed about 70 higher taxonomy groups (<98% sequence similarity) and 133 potential species, i.e., groups of clones sharing greater than 98% similarity. Only rarely were clone sequences found to be highly related to Lake Fryxell isolates and to strains of described species. Subsequent analysis of ten sequencing batches of 36 individual clones indicated that the diversity might be still higher than had been assessed.     

16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure

A laboratory-scale continuously stirred anaerobic thermophilic batch digester was inoculated with cattle manure. Bacterial and archaeal communities, as well as digester performances, were analysed during reactor start-up for about 20 days. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used for overall detection and for study of the dynamics of microbial populations. Dominant bacteria and archaea 16S rDNAs were sequenced from the sample on day 12. Ten bacteria and 3 archaea OTUs (operational taxonomic units) were identified from the 52 clones sequenced. Sequences corresponding to the dominant bacterial SSCP peak were phylogenetically close to the 16S rDNA sequence of Bacillus thermoterrestris, whereas sequences corresponding to the two dominant archaeal SSCP peaks were phylogenetically close to the 16S rDNA sequence of Methanoculleus thermophilicus and Methanosarcina thermophila.     

Microbial diversity of a mesophilic hydrogen-producing sludge

A hydrogen-producing sludge degraded 99% of glucose at 36 degrees C and pH 5.5, producing a methane-free biogas (comprising 64% hydrogen) and an effluent comprising mostly butyrate, acetate, and ethanol. The yield was 0.26 l H2 g(-1) glucose and the production rate per gram of volatile suspended solids was 4.6 1 H2 day(-1). A 16S rDNA library was constructed from the sludge for microbial species determination. A total of 96 clones were selected for plasmids recovery, screened by denaturing gradient gel electrophoresis, and sequenced for rDNA. Based on the phylogenetic analysis of the rDNA sequences, 64.6% of all the clones were affiliated with three Clostridium species (Clostridiaceae), 18.8% with Enterobacteriaceae, and 3.1% with Streptococcus bovis (Streptococcaceae). The remaining 13.5% belonged to eight operational taxonomic units, the affiliations of which were not identified.     

新疆野生胀果甘草内生细菌多样性的非培养初步分析

摘要：【目的】了解新疆野生甘草内生细菌多样性,为开发新的微生物资源奠定基础。【方法】采用改进的CTAB (十六烷基三甲基溴化铵)法提取新疆野生胀果甘草根部总DNA,利用细菌16S rDNA 基因通用引物对甘草总DNA 进行16S rDNA 基因扩增,构建甘草内生细菌16S rDNA基因文库;挑选具有不同酶切图谱的克隆进行测序、比对并构建16S rDNA 基因系统发育树。【结果】构建的甘草内生细菌16S rDNA基因文库中, 150个克隆分属于32个不同的分类单元,Blast结果表明大部分克隆与已知细菌的16S rDNA基因序列相似性较高,分别归属于变形杆菌门(Proteobacteria)的alpha、gamma亚群,厚壁菌门(Firmicutes),放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes)中的鞘脂菌属(Sphingobium),叶杆菌属(Phyllobacterium),生丝单胞菌属(Hyphomonas),土壤杆菌属(Agrobacterium)等14个属, 其中26%的克隆与已知细菌16S rDNA 基因相似性小于96%,可能代表新的分类单元.【结论】甘草内生细菌多样性丰富且存在尚未被认识的新物种。     

Inhibition of Streptococcus mutans NS adhesion to glass with and without a salivary conditioning film by biosurfactant- releasing Streptococcus mitis strains

The release of biosurfactants by adhering microorganisms as a defense mechanism against other colonizing strains on the same substratum surface has been described previously for probiotic bacteria in the urogenital tract, the intestines, and the oropharynx but not for microorganisms in the oral cavity. Two Streptococcus mitis strains (BA and BMS) released maximal amounts of biosurfactants when they were grown in the presence of sucrose and were harvested in the early stationary phase. The S. mitis biosurfactants reduced the surface tensions of aqueous solutions to about 30 to 40 mJ m(-2). Biochemical and physicochemical analyses revealed that the biosurfactants released were glycolipids. An acid-precipitated fraction was extremely surfactive and was identified as a rhamnolipidlike compound. In a parallel-plate flow chamber, the number of Streptococcus mutans NS cells adhering to glass with and without a salivary conditioning film in the presence of biosurfactant-releasing S. mitis BA and BMS (surface coverage, 1 to 4%) was significantly reduced compared with the number of S. mutans NS cells adhering to glass in the absence of S. mitis. S. mutans NS adhesion in the presence of non-biosurfactant-releasing S. mitis BA and BMS was not reduced at all. In addition, preadsorption of isolated S. mitis biosurfactants to glass drastically reduced the adhesion of S. mutans NS cells and the strength of their bonds to glass, as shown by the increased percentage of S. mutans NS cells detached by the passage of air bubbles through the flow chamber. Preadsorption of the acid-precipitated fraction inhibited S. mutans adhesion up to 80% in a dose-responsive manner. These observations indicate that S. mitis plays a protective role in the oral cavity and protects against colonization of saliva-coated surfaces by cariogenic S. mutans.