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PubMed2006

Genotypic characteristics of the rrn operon and genome of indigenous soybean Bradyrhizobia in cropping zones of China.

Four genetic assays, 16S rRNA restriction fragment length polymorphism (RFLP), 16S rRNA sequencing, 16S-23S rRNA intergenetic spacer (IGS) RFLP, and amplified fragment length polymorphism (AFLP), were conducted to determine the genotypic characteristics of 44 indigenous strains of Bradyrhizobium from soybean (Glycine max L.) cropping zones of China. The results generated from different assays showed that soybean bradyrhizobial isolates comprised four genomic groups. Group I was composed of strains mainly isolated from the North and Northeast plains of China. All four assays confirmed this group as phylogenetically divergent from all the reference strains. Strains of the group may represent a new species. Strains in Group II isolated from a variety of geographic regions were ascribed to B. liaoningense. Strains in Group III, mainly isolated from Central and East China, were closely related to the reference strains of B. japonicum. Strains in Group IV belonged to B. elkanii.

Bradyrhizobium↗
PubMed1997

[PCR detection of Mycobacterium tuberculosis lacking IS 6110].

We have evaluated the frequency of M. tuberculosis strains which lack IS 6110 among 102 sputa isolated from Moroccan patients. A pair of primers was designed to amplify a 201bp DNA fragment of IS 6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by southern blot hybridization with a 32P-labelled probe (PMTO2). To detect the presence of amplification inhibitors, an internal control DNA was added in each negative PCR result. Among 102 samples, 6 sputa were negative by PCR-IS 6110 but culture positive. The test of detection of M. tuberculosis for 2/6 sputa by PCR Amplicor amplifying 584 pb of rRNA 16s sequence was positive. RFLP analysis of these 2 strains revealed no bands hybridizing IS 6110 but PCR-Mt 308 was positive. These results confirmed that these M. tuberculosis strains are lacking IS 6110.

Adult↗
PubMed2004

T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages.

The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.

Base Sequence↗
PubMed1998

Whole cell protein and partial 16S rRNA gene sequence analysis suggest the existence of a second Rothia species.

OBJECTIVE: To subject ten clinical isolates grouped together based on their biochemical and microbiological profile to further investigations aimed at correct species identification. METHODS: The 16S rRNA gene was partially sequenced using nested amplification. Whole cell protein analysis (SDS-PAGE) and cluster analysis were performed on the 10 strains and also for comparison on 31 reference strains. The API Coryne biochemical kit as well as API 20 Strep were used for analysis of the phenotypic diversity of the strains by use of computerized numerical identification procedures. Antibiotic susceptibility testing was performed using a standardized disk diffusion test. RESULTS: The 265--556-bp-long 16S rRNA gene sequences of all 10 strains showed highest similarity to Rothia dentocariosa. Three strains showed complete identity between the sequences obtained and the sequence of the type strain of Rothia dentocariosa 16S rRNA gene (M59055), and the other seven ranged between 99.7% and 98.3% similarity. Detailed analysis of the sequences revealed a clustering of the strains into two groups. One group consisted of four isolates with the highest degrees of similarity with the reference strain (type I), while the members of another group (type II) showed differences in their nucleotide sequence at four distinct positions in the variable V7 region. T was replaced by C at position 597, C by T at position 608, T by C at position 610, and G by A at position 684 (position numbers according to reference sequence M59055, EMBL/GenBank). Whole cell protein analysis (SDS-PAGE) and cluster analysis also segregated the 10 Rothia dentocariosa strains into two different clusters, with one cluster containing all four strains belonging to 16S rRNA gene type I, and a second cluster containing all six strains belonging to 16S rRNA gene type II. CONCLUSIONS: Partial sequence data of the 16S rRNA gene as well as whole cell protein analysis showed a subdivision of the Rothia species into two groups, genomovar I (Rothia dentocariosa sensu stricto) and genomovar II, a possible new Rothia species.

Journal Article↗
PubMed2025

Evaluation of the gastric microbiota based on body mass index using 16S rRNA gene sequencing.

INTRODUCTION: Obesity is a multifactorial condition influenced by various factors, including the gut microbiota. However, the relationship between the gastric microbiota and obesity remains poorly understood. This study aimed to investigate the composition of gastric microbiota, excluding Helicobacter pylori, in relation to body mass index (BMI) and metabolic indicators. METHODS: Thirty participants undergoing health checkups were classified into three groups-normal weight (BMI 18.5-22.9), overweight (BMI 23.0-24.9), and obese (BMI ≥25.0)-with ten individuals per group. Those with H. pylori infection, atrophic gastritis, or intestinal metaplasia were excluded. Gastric microbiota from four antral biopsies per subject were analyzed using 16S rRNA sequencing and functional profiling by metagenomic prediction. RESULTS AND DISCUSSION: Alpha diversity (Gini-Simpson index) was significantly lower in the combined overweight/obese group than that in the normal group (P=0.049). Beta diversity analysis revealed clear group separation (Bray-Curtis, P=0.005; unweighted UniFrac, P=0.004). Significant species differences between the groups were observed; specifically, the abundances of Muribaculum gordoncarteri, Turicibacter bilis, and Duncaniella dubosii, were significantly reduced in the overweight/obese group. Functional predictions showed differential enrichment of pathways related to fatty acid, amino acid, vitamin, and carbohydrate metabolism across BMI categories. These findings suggest that alterations in the gastric microbiota may be linked to obesity and metabolic dysregulation.

Humans↗
PubMedDate not supplied

[The lactic acid enterococci Enterococcus faecium and Enterococcus durans: nucleotide sequence diversity in 16S rRNA genes].

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.

Armenia↗
PubMed1994

Comparative analysis of the 16S rRNA gene sequence of the putative agent of proliferative ileitis of hamsters.

Proliferative ileitis of hamsters is consistently associated with the presence of intracellular bacteria in affected ileal epithelial cells. The 16S rRNA gene sequence of the putative etiologic agent of proliferative ileitis was determined by using cell culture-maintained organisms. The highest level of relatedness (98.4%) was observed with a newly described obligately intracellular bacterium obtained from porcine intestines, and the level of homology with Desulfovibrio desulfuricans was 87.5%.

Animals↗
PubMed2004

Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing.

Owing to the difficulties in identifying anaerobic, non-sporulating, Gram-positive bacilli in clinical microbiology laboratories, the epidemiology and clinical spectrum of disease of many of these bacteria have been poorly understood. The application of 16S rRNA gene sequencing in characterizing bacteraemia due to anaerobic, non-sporulating Gram-positive bacilli during a 4-year period is described. The first case of Olsenella uli bacteraemia, in a patient with acute cholangitis, is also reported. Among 165 blood culture isolates of anaerobic, Gram-positive bacilli, 75 were identified as Propionibacterium acnes by phenotypic tests and 21 as members of other anaerobic, non-sporulating Gram-positive bacilli by 16S rRNA gene sequencing. Of these 96 isolates, 16 (17 %) were associated with cases of clinically significant bacteraemia, among which 10 (63 %) were caused by Eggerthella, four (25 %) by Lactobacillus and one (6 %) by each of Eubacterium tenue and O. uli. Five of the 10 Eggerthella isolates were Eggerthella lenta, whereas the other five belonged to two novel Eggerthella species, with Eggerthella hongkongensis being almost as prevalent as Eggerthella lenta. Underlying disease in the gastrointestinal tract, isolation of Eggerthella and Lactobacillus, and monomicrobial bacteraemia were associated with clinically significant bacteraemia, whereas isolation of P. acnes and polymicrobial bacteraemia were associated with pseudobacteraemia. Most patients with clinically significant bacteraemia had underlying diseases, with diseases in the gastrointestinal tract being most common. The overall mortality rate was 31 %. Immunocompromised patients with clinically significant bacteraemia due to anaerobic, non-sporulating, Gram-positive bacilli other than P. acnes should be treated with appropriate antibiotics. The unexpected frequency of isolation of Eggerthella from blood cultures and its association with clinically significant disease suggest that this genus is probably of high pathogenicity. Further studies to look for specific virulence factors are warranted.

Adult↗
PubMed1994

Rapid identification of Aeromonas species using 16S rDNA targeted oligonucleotide primers: a molecular approach based on screening of environmental isolates.

Published 16S rDNA sequencing data for Aeromonas species were analysed and the validity of signature sequences derived from our investigations of these sequences was examined by sequencing the corresponding 16S rDNA regions of 67 environmental isolates from sewage effluents and receiving waters around Sydney and one clinical isolate, all previously classified as Aeromonas species. Species-specific probes for Aer. hydrophila and Aer. veronii were designed and tested in PCR assays and clearly discriminated these species from the other Aeromonas isolates as identified by 16S rDNA sequences.

Aeromonas↗
PubMed1992

Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations.

The phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.

Aeromonas↗
PubMed2001

Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt.

Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively. Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells. This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits. We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments. The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.

Cloning, Molecular↗
PubMed2003

Detection and characterization of Pasteuria 16S rRNA gene sequences from nematodes and soils.

Various bacterial species in the genus Pasteuria have great potential as biocontrol agents against plant-parasitic nematodes, although study of this important genus is hampered by the current inability to cultivate Pasteuria species outside their host. To aid in the study of this genus, an extensive 16S rRNA gene sequence phylogeny was constructed and this information was used to develop cultivation-independent methods for detection of Pasteuria in soils and nematodes. Thirty new clones of Pasteuria 16S rRNA genes were obtained directly from nematodes and soil samples. These were sequenced and used to construct an extensive phylogeny of this genus. These sequences were divided into two deeply branching clades within the low-G + C, Gram-positive division; some sequences appear to represent novel species within the genus Pasteuria. In addition, a surprising degree of 16S rRNA gene sequence diversity was observed within what had previously been designated a single strain of Pasteuria penetrans (P-20). PCR primers specific to Pasteuria 16S rRNA for detection of Pasteuria in soils were also designed and evaluated. Detection limits for soil DNA were 100-10,000 Pasteuria endospores (g soil)(-1).

Animals↗
PubMed2005

Prevotella multiformis sp. nov., isolated from human subgingival plaque.

Four bacterial strains isolated from the human oral cavity, PPPA19, PPPA21(T), PPPA28 and PPPA30, were characterized by determining phenotypic and biochemical features, cellular fatty acid profiles and the phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that each of the isolates was a member of the genus Prevotella. These strains were related to Prevotella denticola with about 95 % similarity. The strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. However, the cells of these strains were often cocci (coccobacilli), depending on the cultivation time. Colonies of different sizes were detected on Eggerth Gagnon agar plates for these strains. The cells forming large colonies were cocci, whereas those forming small colonies were cocci and rods. However, 16S rRNA gene sequence comparison of colonies of different sizes revealed that only a single organism was present. Although these strains had phenotypic characteristics that were similar to those of P. denticola JCM 8528, they could be differentiated from P. denticola JCM 8528 by aesculin hydrolysis and d-cellobiose fermentation in API 20A tests. DNA-DNA hybridization experiments revealed the genomic distinction of these four strains with respect to P. denticola JCM 8528. On the basis of these data, a novel Prevotella species, Prevotella multiformis sp. nov., is proposed, with PPPA21(T) (=JCM 12541(T)=DSM 16608(T)) as the type strain.

Bacteroidaceae Infections↗
PubMed1993

Direct sequencing of the polymerase chain reaction-amplified 16S rRNA gene of Flavobacterium gondwanense sp. nov. and Flavobacterium salegens sp. nov., two new species from a hypersaline Antarctic lake.

Phenotypic data and phospholipid ester-linked fatty acid profiles indicate that pigmented bacterial strains isolated from a hypersaline Antarctic lake are members of the "flavobacterium-bacteroides" phylum and may represent new taxa. Nearly complete 16S rRNA sequences were obtained for representative strains by directly sequencing the polymerase chain reaction-amplified 16S rRNA gene. Sequence signatures confirmed that these organisms were members of the flavobacterium-bacteroides phylum. A phylogenetic analysis, in which the sequences of the Antarctic strains were compared with a large number of sequences available for members of the flavobacterium-bacteroides phylum, showed that the Antarctic strains were phylogenetically distinct. The new species cluster with a group of organisms that contains the type species of the genus Flavobacterium, Flavobacterium aquatile. Two new species are described, for which the names Flavobacterium gondwanense and Flavobacterium salegens are proposed; strains ACAM 44 (= DSM 5423) and ACAM 48 (= DSM 5424) are the type strains of F. gondwanense and F. salegens, respectively.

Antarctic Regions↗
PubMed2003

Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory.

An evaluation of the MicroSeq 500 microbial identification system by nucleic acid sequencing and the Mayo Clinic experience with its integration into a routine clinical laboratory setting are described. Evaluation of the MicroSeq 500 microbial identification system was accomplished with 59 American Type Culture Collection (ATCC) strains and 328 clinical isolates of mycobacteria identified by conventional and 16S ribosomal DNA sequencing by using the MicroSeq 500 microbial identification system. Nucleic acid sequencing identified 58 of 59 (98.3%) ATCC strains to the species level or to the correct group or complex level. The identification results for 219 of 243 clinical isolates (90.1%) with a distance score of <1% were concordant with the identifications made by phenotypic methods. The remaining 85 isolates had distance scores of >1%; 35 (41.1%) were identified to the appropriate species level or group or complex level; 13 (15.3%) were identified to the species level. All 85 isolates were determined to be mycobacterial species, either novel species or species that exhibited significant genotypic divergence from an organism in the database with the closest match. Integration of nucleic acid sequencing into the routine mycobacteriology laboratory and use of the MicroSeq 500 microbial identification system and Mayo Clinic databases containing additional genotypes of common species and added species significantly reduced the number of organisms that could not be identified by phenotypic methods. The turnaround time was shortened to 24 h, and results were reported much earlier. A limited number of species could not be differentiated from one another by 16S ribosomal DNA sequencing; however, the method provides for the identification of unusual species and more accurate identifications and offers the promise of being the most accurate method available.

Bacterial Typing Techniques↗
PubMed2003

Granulicatella adiacens and Abiotrophia defectiva bacteraemia characterized by 16S rRNA gene sequencing.

Traditionally, the identification, epidemiology and spectrum of clinical diseases caused by Granulicatella adiacens and Abiotrophia defectiva are dependent upon their phenotypic characterization. During a 6-year period (July 1995-June 2001), seven and two alpha-haemolytic streptococci were identified as G. adiacens and A. defectiva, respectively, by 16S rRNA gene sequencing. Three patients with haematological malignancies and neutropenic fever had primary bacteraemia. Three patients with valvular problems or congenital heart disease had infective endocarditis. A patient with ischemic heart disease and cerebrovascular accident had infected aortic atheroma with dissection. A patient with recurrent pyogenic cholangitis had acute cholangitis and a patient with polypoid cystitis and benign prostatic hypertrophy had acute prostatitis. Four of the nine patients died, including all three with G. adiacens infective endocarditis or infected atheroma. For the seven G. adiacens isolates, the API 20 STREP system successfully identified one and five isolates as G. adiacens with >95 % and 80-90 % confidence, respectively, whereas the Vitek System (GPI) and ATB Expression system (ID32 STREP) successfully identified none and one isolate as G. adiacens. Of the two A. defectiva isolates, none of the three systems successfully identified either of them as A. defectiva. 16S rRNA gene sequencing is the technique of choice for identifying G. adiacens and A. defectiva, and early surgical intervention should be considered when G. adiacens endocarditis is diagnosed.

Adolescent↗
PubMed2005

Prevotella multisaccharivorax sp. nov., isolated from human subgingival plaque.

Six bacterial strains isolated from the human oral cavity, PPPA16, PPPA20(T), PPPA24, PPPA31, EPPA6 and EPPA7, were characterized by determining phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that the isolates represented the same species of the genus Prevotella. The strains were related to Prevotella dentalis with about 89% similarity. In addition, the isolates were related to Prevotella sp. oral clone IDR-CEC-0032, which is a representative of the numerically dominant cluster VI in carious dentine lesions [Nadkarni et al. (2004). J Clin Microbiol 42, 5238-5244], with about 99% similarity. The strains were obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rods. The isolates could be differentiated from other Prevotella species by D-mannitol, D-melezitose, D-sorbitol and D-trehalose fermentation in API 20A tests. The cellular fatty acid composition of strains PPPA16, PPPA20(T), PPPA24, PPPA31, EPPA6 and EPPA7 was significantly different from that of other Prevotella species. Compared with other Prevotella species, only these six strains contained dimethyl acetals. The major menaquinones of the clinical isolates were MK-12 and MK-13, whereas the major menaquinones of other Prevotella species were MK-10 and MK-11. On the basis of these data, a novel Prevotella species, Prevotella multisaccharivorax sp. nov., is proposed, with PPPA20(T) (=JCM 12954(T)=DSM 17128(T)) as the type strain.

Bacterial Typing Techniques↗
PubMed2006

Bacteroides intestinalis sp. nov., isolated from human faeces.

During studies of the microbiota of human faeces, five strains of Gram-negative anaerobic rods were isolated following growth in a polyamine-deficient medium. These strains belonged to the genus Bacteroides on the basis of 16S rRNA gene sequence data. 16S rRNA gene sequence similarity between one of the strains, 341T, and recognized species within the genus Bacteroides was <95 %. The DNA G+C content (44 mol%) and major fatty acid composition (anteiso-C(15 : 0), 32.0 %) supported the affiliation of strain 341(T) to the genus Bacteroides. Biochemical tests and DNA-DNA hybridization analysis demonstrated that strain 341T was distinct from Bacteroides uniformis and Bacteroides helcogenes, to which it was related most closely. On the basis of these data, a novel Bacteroides species, Bacteroides intestinalis sp. nov., is proposed with strain 341T (= JCM 13265T = DSM 17393T) as the type strain.

Bacteroides↗