多项分类方法鉴定西藏地区藏灵菇中的乳酸球菌

【目的】采用多项分类法对16株分离自藏灵菇中的乳酸球菌进行准确鉴定。【方法】首先应用传统的生理生化试验,之后采用16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)进行了鉴定,最后,通过16S rRNA基因序列分析进行验证。【结果】将16株菌株初步鉴定为3个菌群:片球菌群、乳球菌群和肠球菌群,进一步鉴定为14株耐久肠球菌,1株乳酸片球菌,1株乳酸乳球菌乳酸亚种,16S rRNA基因序列分析验证的结果与前3种试验方法的结果相一致。【结论】试验结果表明传统的生理生化鉴定和16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)相结合的多项分类方法有利于乳酸球菌种间的准确鉴定。     

Hypervariable regions (V1–V9) of the dinoflagellate 18S rRNA using a large dataset for marker considerations

DNA sequencing methods have been used for the molecular taxonomic discrimination of dinoflagellate protists, particularly using partial 18S rRNA sequences. This study evaluated the taxonomic discrimination power of rRNA gene hypervariable regions (V1 to V9) in dinoflagellates from a large dataset. These included 77 dinoflagellate species (9 orders, 17 families, 40 genera). The complete 18S rRNA sequences of the dinoflagellates ranged from 1,787 to 1,813?bp in length, and consisted of eight V regions with a total combined length of 678 to 699?bp. Regions longer than 100?bp were recoded for V2, V4, and V8 regions; high nucleotide divergences were detected in V1, V2, and V4 regions. Statistic tests showed that the divergences of individual V regions were significantly different (t-test, P?<?0.05) compared with the complete 18S rRNA. The V2 region showed the highest score (83.5%) for PI sites. Moreover, intra-genus DNA similarities of the V2 were considerably low (<93%). Neighbor-joining analyses showed that phylogenetic resolution in the V2–V4 region was 1.32-fold higher than that of the complete 18S rRNA. These results demonstrate that V2 has the highest taxonomic resolving power within the 18S rRNA gene of dinoflagellates, suggesting the V2 and adjacent regions (e.g., V1 to V4) may be the best for marker considerations.     

Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates

In the last few decades, molecular typing has become an important tool in taxonomic, phylogenetic and identification studies of numerous groups of bacteria, including the yersiniae. In this study, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequence Analysis (MLSA) were performed to determine the ability of these techniques to be used in taxonomy and identification of Yersinia strains. A total of 60 Yersinia strains were genotyped by ERIC-PCR and PFGE. Moreover, an in silico analysis was carried out for 16S rRNA gene sequencing and MLSA, using 68 and 49 Yersinia strains, respectively. A phylogenetic tree constructed from the ERIC-PCR, 16S rRNA gene sequencing and MLSA data grouped most of the Yersinia species into distinct species-specific clusters. In the PFGE assay these clusters were not observed. On this basis, ERIC-PCR, 16S rRNA gene sequencing and MLSA seem to be valuable techniques for use in taxonomic and identification studies of the genus Yersinia, whereas PFGE does not. Furthermore, ERIC-PCR has the advantage of being a cheaper, easier and faster assay than 16S rRNA gene sequencing or MLSA, and for these reasons can be considerate an alternative tool in taxonomic studies of yersiniae.     

6种区分乳酸乳球菌乳酸亚种和乳酸乳球菌乳脂亚种的分子生物学方法比较

【目的】比较并评价6种分子生物学技术对乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)和乳酸乳球菌乳脂亚种(Lactococcus lactis subsp.cremoris)的区分效果。【方法】采用16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),随机扩增多态性分析技术(RAPD),重复基因外回文序列分析技术(rep-PCR)和限制性酶切片段多态性分析技术(RFLP)对4株Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris参考菌株进行了区分,并对这6种方法的区分效果进行了比较评价。【结果】16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术无法区分Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris,而其余4种技术可以实现区分。【结论】变性梯度凝胶电泳(DGGE),随机扩增多态性分析技术(RAPD)耗时短,操作简单,试验结果准确稳定,更适合Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris的快速准确区分。     

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.     

Epidemiological surveillance of mycoplasmas belonging to the ‘Mycoplasma mycoides’ cluster: is DGGE fingerprinting of 16S rRNA genes suitable?

Aims:  The analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rRNA gene was previously shown to detect and differentiate a large number of human and animal mycoplasmas. In this study, we further assessed the suitability of the technique for epidemiological surveillance of mycoplasmas belonging to the ' Mycoplasma mycoides ' cluster, a phylogenetic group that includes major ruminant pathogens.
Methods and Results:  The V3 region of 16S rRNA genes from approx. 50 field strains was amplified and analysed by DGGE. Detection and identification results were compared with the ones obtained by antigenic testing and sequence analysis.
Conclusions:  The DGGE technique is robust and valuable as a first-line test, but the patterns obtained for strains belonging to the ' M. mycoides ' cluster were too variable within a taxon and in contrast too conserved between taxa to allow an unequivocal identification of isolates without further analysis.
Significance and Impact of the Study:  Issues raised by the quest for a single universal test able to detect and identify any mycoplasma in one clinical sample are thoroughly documented.     

Estimation of 16S rRNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken

The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.     

Multiple copies of 16s rRNA gene affect the restriction patterns and DGGE profile as revealed by analysis of genome database

The use of 16S rRNA gene has been a "golden" method to determine the diversity of microbial communities in environmental samples, phylogenetic relationships of prokaryotes and taxonomic position of newly isolated organisms. However due to the presence of multiple heterogeneous 16S rRNA gene copies in many strains, the interpretation of microbial ecology via 16S rRNA sequences is complicated. Purpose of present paper is to demonstrate the extent to which the multiple heterogeneous 16S rRNA gene copies affect RFLP patterns and DGG E profiles by using the genome database. In present genome database, there are 782 bacterial strains in total whose genomes have been completely sequenced and annotated. Among the total strains, 639 strains (82%) possess multiple 16S rRNA gene copies, 415 strains (53%) whose multiple copies are heterogeneous in sequences as revealed by alignment, 236 strains (30%) whose multiple copies show different restrict patterns by CSP61 + Hinfl, MspI + Rsal or HhaI as analyzed in silico. Polymorphisms of the multiple copies in certain strains were further characterized by G + C% and phylogentic distances based on the sequences of V3 region, which are linked to DGGE patters. Polymorphisms of a few strains were shown as examples. Using artificial communities, it is demonstrated that the presence of multiple heterogeneous 16S rRNA gene copies potentially leads to over-estimation of the diversity of a community. It is suggested that care must be taken when interpreting 16S rRNA-based RFLP and DGGE data and profiling an environmental community.     

Intra-genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria,Radiolaria) assessed by Sanger,MinION and Illumina sequencing

Ribosomal RNA (rRNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large-scale exploration of environmental microbial diversity through metabarcoding approaches has been focused mainly on the V4 and V9 regions of the 18S rRNA gene. The accurate interpretation of such environmental surveys is hampered by technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra-genomic variability). Here we explored the intra-genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra-genomic variability of Nassellaria and Spumellaria is generally low, yet some Spumellaria specimens showed two different copies of the V4 with <97% similarity. Of the different sequencing methods, Illumina showed the highest number of contaminations (i.e. environmental DNA, cross-contamination, tag-jumping), revealed by its high sequencing depth; and MinION showed the highest sequencing rate error (~14%). Yet the long reads produced by MinION (~2900 bp) allowed accurate phylogenetic reconstruction studies. These results highlight the requirement for a careful interpretation of Illumina-based metabarcoding studies, in particular regarding low abundant amplicons, and open future perspectives towards full-length rDNA environmental metabarcoding surveys.     

Multiple copies of 16S rRNA gene affect the restriction patterns and DGGE profile revealed by analysis of genome database

The use of 16S rRNA gene has been a “golden” method to determine the diversity of microbial communities in environmental samples, phylogenetic relationships of prokaryotes and taxonomic position of newly isolated organisms. However due to the presence of multiple heterogeneous 16S rRNA gene copies in many strains, the interpretation of microbial ecology via 16S rRNA sequences is complicated. Purpose of present paper is to demonstrate the extent to which the multiple heterogeneous 16S rRNA gene copies affect RFLP patterns and DGGE profiles by using the genome database. In present genome database, there are 782 bacterial strains in total whose genomes have been completely sequenced and annotated. Among the total strains, 639 strains (82%) possess multiple 16S rRNA gene copies, 415 strains (53%) whose multiple copies are heterogeneous in sequences as revealed by alignment, 236 strains (30%) whose multiple copies show different restrict patterns by CSP6I+HinfI, MspI+RsaI or HhaI as analyzed in silico. Polymorphisms of the multiple copies in certain strains were further characterized by G+C% and phy-logentic distances based on the sequences of V3 region, which are linked to DGGE patters. Polymorphisms of a few strains were shown as examples. Using artificial communities, it is demonstrated that the presence of multiple heterogeneous 16S rRNA gene copies potentially leads to over-estimation of the diversity of a community. It is suggested that care must be taken when interpreting 16S rRNA-based RFLP and DGGE data and profiling an environmental community.     

Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus

A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5' end hypervariable region (259-273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.     

Fecal Microbiota in Healthy Subjects Following Omnivore,Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling

In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.     

Soil Bacterial Diversity Screening Using Single 16S rRNA Gene V Regions Coupled with Multi-Million Read Generating Sequencing Technologies

The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystems

Efficient profiling of eubacterial diversity within complex communities requires that primers are specific for eubacterial 16S rRNA. Specificity of published primers against eubacterial and archaeal 16S rRNA as well as protozoal and fungal 18S rRNA was assessed in silico. The specificity and sensitivity of the V3 and V6–V8 (F968gc and R1401) Denaturing Gradient Gel Electrophoresis (DGGE) primers was subsequently verified using rumen-derived samples. An assessment of the effects of employing touchdown PCR cycling conditions was also made. For DGGE profiling of eubacteria within rumen samples, primers F968gc and R1401 proved the most specific and sensitive providing that touchdown PCR is not used.     

Ecology and dynamics of coagulase-negative cocci isolated from naturally fermented Italian sausages

Coagulase-negative cocci (CNC) ecology in naturally fermented sausages from Friuli Venezia Giulia region, in the North East of Italy, was investigated. A total of 617 CNC strains, isolated from three different plants during the fermentation process, were identified by traditional methods (biochemical tests) and molecular methods based on species specific PCR, PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of the V3 region of the 16S rRNA gene. The identification, by using biochemical tests, was not successful for 130 strains. Moreover, incongruent results were observed comparing the traditional with the molecular identifications. The same species of CNC were found in all three processing plants, but their contribution to the fermentations was different. In two plants Staphylococcus xylosus was the main species involved in fermentation process, while in the third the maturation was carried out equally by three species: S. xylosus, Staphylococcus warneri and Staphylococcus pasteuri.     

Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3–V4 region to investigate whether the RC-PCR reveals more of the ‘unseen’ diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3–V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.     

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.     

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment

High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions – provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results.