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PubMed1999

A phylogenetic analysis of the genus Catellatospora based on 16S ribosomal DNA sequences, including transfer of Catellatospora matsumotoense to the genus Micromonospora as Micromonospora matsumotoense comb. nov.

Phylogenetic studies based on the 16S ribosomal gene sequences showed that members of the genus Catellatospora revealed phylogenetic heterogeneity within the family Micromonosporaceae as well as a heterogeneous menaquinone composition. Among them, Catellatospora matsumotoense was closely related to members of the genus Micromonospora, indicating that this organism should be excluded from the genus Catellatospora. On the basis of classical taxonomic characteristics and phylogenetic evidence, Catellatospora matsumotoense is proposed to be transferred to the genus Micromonospora as M. matsumotoense comb. nov.

DNA, Ribosomal↗
PubMed2005

A cryptic Bacillus isolate exhibited narrow 16S rRNA gene sequence divergence with Bacillus thuringiensis and showed low maintenance requirements in hyper-osmotic complex substrate cultivations.

A cryptic Bacillus (K90) isolate obtained from soil samples from the Kuwait desert exhibited lower maintenance requirements in complex substrate cultivations than Bacillus thuringiensis. A mathematical model was used to estimate apparent maintenance coefficients (m(c)) and these were found to be 0.336 and 0.041/h for B. thuringiensis and K90, respectively. The results also showed that the values of apparent maintenance coefficients were inversely related to the specific growth rates. Furthermore, 16S rRNA gene sequencing showed that K90 exhibited 99.81% sequence similarity to that of B. mojavensis and 92.9% with B. thuringiensis. It is evident from the dendrogram that the evolution of B. mojavensis (K90) (B. subtilis group), which may have originated after B. licheniformis could have been influenced by prolonged hyper-osmotic conditions, while B. thuringiensis that evolved before B. oleronius exhibited greater sensitivity as implied by the higher maintenance coefficient obtained for the hyper-osmotic cultures. As K90 exhibited low maintenance requirements in hyperosmotic cultures, close phylogenetic relationship with B. thuringiensis, along with the reported property of encapsulation of insecticidal crystal proteins (Cry) in Bacillus strains and endophytic nature of B. mojavensis, strongly suggest that K90 could be a promising surrogate host for the transgenic delivery of "Cry" proteins.

Bacillus↗
PubMed1998

Use of 16S rRNA gene sequencing to identify Lactobacillus casei in septicaemia secondary to a paraprosthetic enteric fistula.

Human infections caused by Lactobacillus spp. are rarely reported in the literature. Underlying conditions are frequently reported, and identification of lactobacilli to the species level remains rare. A case of Lactobacillus casei septicaemia secondary to a vascular graft infection is reported. The 16S rRNA sequencing technique was used to definitively identify Lactobacillus casei.

Aged↗
PubMed2002

Reevaluation of Streptococcus bovis endocarditis cases from 1975 to 1985 by 16S ribosomal DNA sequence analysis.

Studies that detected an association between Streptococcus bovis endocarditis and colon carcinoma have not taken into account the recently identified genetic diversity among organisms historically classified as S. bovis. With near full-length 16S ribosomal DNA sequence analysis, organisms cultured from the blood of endocarditis patients at the Mayo Clinic from 1975 to 1985 and previously identified as S. bovis or streptococcus group D nonenterococci were shown to represent S. bovis biotypes I (11 isolates) and II/2 (1 isolate), S. salivarius (1 isolate), and S. macedonicus (1 isolate). Two of the S. bovis biotype I cases were associated with colon cancer. Whether S. bovis biotype II or other organisms closely related to and historically identified as S. bovis (e.g., S. macedonicus) are associated with malignant (or premalignant) colon lesions in humans remains to be definitively determined.

Aged↗
PubMed2000

Brachyspira aalborgi infection diagnosed by culture and 16S ribosomal DNA sequencing using human colonic biopsy specimens.

In this study we report on the isolation and characterization of the intestinal spirochete Brachyspira aalborgi using human mucosal biopsy specimens taken from the colon of a young adult male with intestinal spirochetosis. A selective medium, containing 400 microg of spectinomycin/ml and 5 microg of polymyxin/ml was used for the isolation procedure. A high degree of similarity, in terms of phenotypic properties and 16S ribosomal DNA sequence, was observed between the isolated strain, named W1, and the type strain, 513A, of B. aalborgi. A similarity of 99.7% in the nucleotide sequence was found between W1 and 513A(T), based on the almost-complete gene. A short segment of the 16S rRNA gene was amplified by PCR using genetic material enriched from paraffin-embedded biopsy specimens, which were taken from the patient on two occasions. The products showed 16S rRNA gene sequences virtually identical to that of strain 513A(T) in the actual region. Immunohistochemistry was performed on the colonic biopsy specimens with a polyclonal antibody raised against an intestinal spirochete isolated in a previous case of human intestinal spirochetosis. The antibody reacted strongly with the spirochete on the luminal epithelium. No immune reaction was seen within or below the surface epithelium. Routine histology did not reveal signs of colitis. Electron microscopy showed spirochetes attached end-on to the colonic mucosal surface. The isolate grew poorly on a commonly used selective medium for intestinal spirochetes, which may explain previous failures to isolate B. aalborgi.

Adult↗
PubMed2001

Campylobacter hominis sp. nov., from the human gastrointestinal tract.

Sequences of 16S rDNA of a novel campylobacter from faeces of healthy humans were previously shown to originate from a new taxon, 'Candidatus Campylobacter hominis', which could not be cultured. Since phylogenetic analysis suggested that anaerobic conditions might be required for growth, an isolation strategy was developed employing initial non-selective membrane filtration onto fastidious anaerobe agar. Campylobacters were then isolated from the resulting mixed microbial flora by a dilution strategy and/or by immunomagnetic separation with genus-specific polyclonal antibody. Isolates were identified by a genus and taxon-specific PCR assay, and 16S rDNA nucleotide sequence analysis was carried out. All isolates exhibited the typical Campylobacter characteristics of being non-fermentative, oxidase-positive, catalase-negative and Gram-negative. Unusually, however, they were straight rods lacking flagella. The 16S rDNA nucleotide sequence analysis, DNA and mol% G+C were consistent with a new Campylobacter species whose nearest phylogenetic neighbours were Campylobacter gracilis and Campylobacter sputorum. The unique species status of the isolates was further confirmed by taxonomic analysis of 47 phenotypic characteristics. The name Campylobacter hominis sp. nov. is proposed for the new species, the type strain of which is NCTC 13146T (= LMG 19568T).

Bacterial Typing Techniques↗
PubMed1999

Phylogenetic differentiation of two closely related Nitrosomonas spp. That inhabit different sediment environments in an oligotrophic freshwater lake.

The population of ammonia-oxidizing bacteria in a temperate oligotrophic freshwater lake was analyzed by recovering 16S ribosomal DNA (rDNA) from lakewater and sediment samples taken throughout a seasonal cycle. Nitrosospira and Nitrosomonas 16S rRNA genes were amplified in a nested PCR, and the identity of the products was confirmed by oligonucleotide hybridization. Nitrosospira DNA was readily identified in all samples, and nitrosomonad DNA of the Nitrosomonas europaea-Nitrosomonas eutropha lineage was also directly detected, but during the summer months only. Phylogenetic delineation with partial (345 bp) 16S rRNA gene sequences of clones obtained from sediments confirmed the fidelity of the amplified nitrosomonad DNA and identified two sequence clusters closely related to either N. europaea or N. eutropha that were equated with the littoral and profundal sediment sites, respectively. Determination of 701-bp sequences for 16S rDNA clones representing each cluster confirmed this delineation. A PCR-restriction fragment length polymorphism (RFLP) system was developed that enabled identification of clones containing N. europaea and N. eutropha 16S rDNA sequences, including subclasses therein. It proved possible to analyze 16S rDNA amplified directly from sediment samples to determine the relative abundance of each species compared with that of the other. N. europaea and N. eutropha are very closely related, and direct evidence for their presence in lake systems is limited. The correlation of each species with a distinct spatial location in sediment is an unusual example of niche adaptation by two genotypically similar bacteria. Their occurrence and relative distribution can now be routinely monitored in relation to environmental variation by the application of PCR-RFLP analysis.

Cloning, Molecular↗
PubMed1983

DNA sequence of the 16S rRNA/23S rRNA intercistronic spacer of two rDNA operons of the archaebacterium Methanococcus vannielii.

The DNA sequence of the spacer (plus flanking) regions separating the 16S rRNA and 23S rRNA genes of two presumptive rDNA operons of the archaebacterium Methanococcus vannielii was determined. The spacers are 156 and 242 base pairs in size and they share a sequence homology of 49 base pairs following the 3' terminus of the 16S rRNA gene and of about 60 base pairs preceding the 5' end of the 23S rRNA gene. The 242 base pair spacer, in addition contains a sequence which can be transcribed into tRNAAla, whereas no tRNA-like secondary structure can be delineated from the 156 base pair spacer region. Almost complete sequence homology was detected between the end of the 16S rRNA gene and the 3' termini of either Escherichia coli or Halobacterium halobium 16S rRNA, whereas the putative 5' terminal 23S rRNA sequence shared partial homology with E. coli 23S rRNA and eukaryotic 5.8S rRNA.

Base Composition↗
PubMed2006

Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections.

The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.

Arthritis, Infectious↗
PubMed1996

Phylogenetic relationship among termite families based on DNA sequence of mitochondrial 16S ribosomal RNA gene.

Termites (Order Isoptera: Class Insecta), are comprised of a complex assemblage of species, with considerable variation in life history, morphology, social behaviour, caste development and ecology. At present, isoptera is divided into seven families, fourteen subfamilies, approximately 270 genera and over 2000 species. Phylogenetic hypotheses currently available for termite families and genera are based on a limited number of morphological characters and lack rigorous cladistic analysis. In this paper we report on phylogenetic relationships among ten termite genera of five families based on a DNA sequence analysis of a portion of the mitochondrial 16S rRNA gene. Parsimony and distance analysis of DNA sequences supported the existing hypothesis that Mastotermitidae is the basal lineage among extant termites. Kalotermitidae was not found to be a sister taxon of Mastotermitidae as existing hypotheses suggest, but was most closely related to Rhinotermitidae and Termitidae. Representatives of Termopsidae were more basal relative to those of Kalotermitidae. The utility of 16S rRNA nucleotide sequence analysis for inferring phylogenetic relationships among termite families, subfamilies and genera is discussed.

Animals↗
PubMed2007

Evidence for chemoautotrophic symbiosis in a Mediterranean cold seep clam (Bivalvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes.

Symbioses between lucinid clams (Bivalvia: Lucinidae) and autotrophic sulphide-oxidizing bacteria have mainly been studied in shallow coastal species, and information regarding deep-sea species is scarce. Here we study the symbiosis of a clam, resembling Lucinoma kazani, which was recently collected in sediment cores from new cold-seep sites in the vicinity of the Nile deep-sea fan, eastern Mediterranean, at depths ranging from 507 to 1691 m. A dominant bacterial phylotype, related to the sulphide-oxidizing symbiont of Lucinoma aequizonata, was identified in gill tissue by comparative 16S rRNA gene sequence analysis. A second phylotype, related to spirochete sequences, was identified twice in a library of 94 clones. Comparative analyses of gene sequences encoding the APS reductase alpha subunit and ribulose-1,5-bisphosphate carboxylase oxygenase support the hypothesis that the dominant symbiont can perform sulphide oxidation and autotrophy. Transmission electron micrographs of gills confirmed the dominance of sulphide-oxidizing bacteria, which display typical vacuoles, and delta(13)C values measured in gill and foot tissue further support the hypothesis for a chemoautotrophic-sourced host carbon nutrition.

Animals↗
PubMed2001

Specific detection and prevalence of Helicobacter heilmannii-like organisms in the human gastric mucosa by fluorescent in situ hybridization and partial 16S ribosomal DNA sequencing.

Gastric infection with Helicobacter heilmannii (previously known as Gastrospirillum hominis) is invariably linked with the presence of chronic gastritis and the risk of developing low-grade mucosa-associated lymphoid tissue lymphoma in humans. In contrast to Helicobacter pylori, various H. heilmannii species colonize the stomachs of domestic animals, which might be a reservoir for transmission to humans (zoonosis). To identify the number and prevalence of different H. heilmanni types in humans, we analyzed 89 gastric biopsy samples histologically identified as H. heilmannii positive by fluorescence in situ hybridization. Of these gastric specimens, 84 (94.4%) contained a single H. heilmannii type. In five samples, however, two different H. heilmannii types were detected. The most prevalent species in monoinfected samples is H. heilmannii type 1, found in 78.5% (66 of 84) of the specimens, followed by a novel H. heilmannii-like organism (HHLO), HHLO type 4, identified in 9.6% (8 of 84) of tissue sections. H. heilmannii type 2 and a further HHLO type not described before, type 3, were found in 8.3% (7 of 84) and 1.2% (1 of 84) of the monoinfected samples, respectively. Additionally, HHLO type 5 with a 16S ribosomal DNA sequence identical to that of Helicobacter salomonis was found with a prevalence of 2.4% (2 of 89). Thirteen of these biopsy samples were also investigated by a PCR approach developed for this study that allows a Helicobacter-specific amplification of a variable portion of the 16S rRNA gene and subsequent sequencing. In total, five different types of HHLOs could be identified within these samples. We conclude that humans can be infected by at least five different HHLO types, which presumably have their origin in animal species like dogs, cats, and pigs.

Biopsy↗
PubMed2002

[Genetic diversity and phylogeny of rhizobia isolated from peanut (Arachis hypogaea)].

Forty three rhizobium strains isolated from peanut (Arachis hypogaea) and 15 reference strains from other genus and species were analyzed by the method of 16S rRNA RFLP, 16S rRNA sequencing and 16S-23S IGS PCR RFLP. The results of the 16S rRNA RFLP shown that 43 strains tested were all ascribed to the genus of Bradyrhizobium phylogenetically. Strains tested were adjacent to the B. japonicum and far from B. elkanii 16S rRNA genotype. The genotypes generated by the 4 restriction endonucleases, Mbo I, Dde I, Hae III and Msp I, were same as the representatives of B. japonicum. The dendrogram generated by 16S rRNA sequence and Neighbor-joining method shown that peanut rhizobia clustered into the subcluster represented by B. japonicum and B. liaoningense, were more close to B. liaoningense genetically, and the sequence difference between them was less than 1%. High sequence similarity was also determined between B. liaoningense and B. japonicum. JZ1, representative strain of peanut rhizobia were systematically far from the B. elkanii, and the sequence divergence about 2%. The results from IGS RFLP analysis indicated that although they were phylogenetically close to B. japonicum and B. elkanii, peanut rhizobia forming an independent group at the similarity of 71% could be further divided into four subgroups, A, B, C and D. Subgroup A consisted of strains from different region, subgroup B was composed of strains from Wuchang, Qianjiang and Jingzhou, subgroup C was mainly composed of strains from Jingzhou and starins of subgroup D mainly from Neijiang. Reference strains from B. japonicum and B. elkanii were independently clustered into the subgroup E at the similarity of 71%. The geographical factor effect on genetic diversity of rhizobia was found.

Arachis↗
PubMed1994

Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis.

Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool.

AIDS-Related Opportunistic Infections↗
PubMed2000

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing.

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Animals↗
PubMed1996

Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria.

We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.

Ammonia↗
PubMed2005

Seasonal change in bacterial flora and biomass in mountain snow from the Tateyama Mountains, Japan, analyzed by 16S rRNA gene sequencing and real-time PCR.

The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4 degrees C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 x 10(5)-fold, 1.5 x 10(5)-fold, and 1.0 x 10(4)-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world.

Bacteria↗