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PubMed2006

Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities.

BACKGROUND: The beta-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. RESULTS: We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. CONCLUSION: The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities.

Colony Count, Microbial↗
PubMed2003

Extremely acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening sequences in their 16S rRNA genes.

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.

Base Sequence↗
PubMed2004

Phylogenetic analysis of Streptomyces spp. isolated from potato scab lesions in Korea on the basis of 16S rRNA gene and 16S-23S rDNA internally transcribed spacer sequences.

The 16S rRNA gene sequences for 34 strains, including 11 isolates, were determined to classify scab-causing Streptomyces spp. and relatives isolated from potato scab lesions collected in Jeju, Korea. The 16S-23S rDNA internally transcribed spacer (ITS) sequences were determined to investigate whether the 16S-23S ITS region is useful for analysing intra- and interspecific relationships in these bacteria. On the basis of phylogenetic analysis of 16S rRNA gene sequences, most of the isolates were classified as Streptomyces scabiei and Streptomyces acidiscabies. Isolate KJO61 was placed in an ambiguous taxonomic position between Streptomyces reticuliscabiei and Streptomyces turgidiscabies. 16S-23S ITS region sequence analysis showed that tRNA genes were not found in this region of Streptomyces spp. The 16S-23S ITS regions of Streptomyces spp. exhibited various lengths and highly variable sequence similarities (35-100%) within strains as well as intra- and interspecies. It was revealed that Streptomyces europaeiscabiei could be clearly differentiated from Streptomyces scabiei. However, it was clarified that ITS regions are not useful in phylogenetic analysis of Streptomyces spp.

Base Sequence↗
PubMed2004

Description of 'Candidatus Helicobacter heilmannii' based on DNA sequence analysis of 16S rRNA and urease genes.

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as 'Helicobacter heilmannii' or 'Gastrospirillum hominis'. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with 'Candidatus Helicobacter suis', thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name 'Candidatus Helicobacter heilmannii' is proposed.

Adult↗
PubMed1999

Phylogenetic characterization of Centipeda periodontii, Selenomonas sputigena and Selenomonas species by 16S rRNA gene sequence analysis.

The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.

Phylogeny↗
PubMed2000

The evolutionary history of the genus Timarcha (Coleoptera, Chrysomelidae) inferred from mitochondrial COII gene and partial 16S rDNA sequences.

The apterous genus Timarcha consists of three subgenera and more than 100 species in its Palearctic distribution, with specialized feeding on few plant families. Fifty-four sequences sampled from 31 taxa of the genus plus three outgroup leaf beetles were studied for their complete cytochrome oxidase II (COII) and a fragment of 16S rDNA mitochondrial genes, representing a total of about 1200 bp. Phylogenetic analyses using maximum-parsimony and distance methods for each gene separately and for the combined data set gave compatible topologies. The subgenus Metallotimarcha consistently appears in a basal position and is well differentiated from the remaining Timarcha, but no clear monophyletic grouping of Timarchostoma and Timarcha s. str. subgenera can be deduced from our analysis. Calibration of the molecular clock has been done using the opening of the Gibraltar Strait after the Messinian salinity crisis (about 5.5 MYA) as the biogeographic event causing disjunction of two particular taxa. Accordingly, the COII evolutionary rate has been estimated to be of 0.76 x 10(-8) substitution/site/year in Timarcha. Relation between phylogeny and host-plant use indicates widening of trophic regime as a derived character in Timarcha.

Animals↗
PubMed2000

Identification of Yersinia pestis as the causative organism of plague in India as determined by 16S rDNA sequencing and RAPD-based genomic fingerprinting.

Eighteen isolates of bacteria obtained from the sputum of pneumonic plague patients and from the liver and spleen of rodents from the plague-affected areas of India during 1994-1995 when analyzed by 16S rDNA analysis clearly demonstrated that all 18 isolates exhibit an average similarity of 98.5% with the genus Yersinia and 99.1% with Yersinia pestis, thus identifying the isolates as Y. pestis. The isolates from the human plague patients were found to be genetically more homogeneous compared to the isolates from the rodents which were more heterogeneous. An epidemiological linkage among the rodents and human patients is also indicated by 16S rDNA analysis, which suggests that only a sub-population of the rodents was probably the source of the infectious pathogen to the humans initiating the outbreak of the epidemic. The results of the randomly amplified DNA polymorphisms (RAPD)-based DNA fingerprinting are in agreement with the above conclusions.

Bacterial Typing Techniques↗
PubMed2006

Low species diversity and high interindividual variability in faeces of preterm infants as revealed by sequences of 16S rRNA genes and PCR-temporal temperature gradient gel electrophoresis profiles.

Little information regarding the composition of the gut microbiota in preterm infants is available. The purpose of this study was to investigate the bacterial diversity in faeces of preterm infants, using analysis of randomly cloned 16S rRNA genes and PCR-TTGE (temporal temperature gradient gel electrophoresis) profiles, to determine whether noncultivated bacteria represented an important part of the community. The 288 clones obtained from faecal samples of 16 preterm infants were classified into 25 molecular species. All but one molecular species had a cultivated representative in public databases: molecular tools did not reveal any unexplored diversity. The mean number of molecular species per infant was 3.25, ranging from one to eight. There was a high interindividual variability. The main groups encountered were the Enterobacteriaceae family and the genera Enterococcus, Streptococcus and Staphylococcus. Seven preterm infants were colonized by anaerobes and only four by bifidobacteria. TTGE profiles were composed of one to nine bands (mean value: 4.3). Furthermore, 51 of 59 clones (86%) comigrated with a band of the corresponding faecal sample. This study will form a comparative framework for other studies, e.g. on the faecal microbiota of preterm infants with different pathologies or the impact of diet on colonization.

Anti-Bacterial Agents↗
PubMed1988

Escherichia coli 3'-terminal 16S rRNA sequence modulated fidelity during translation.

The ribosome is a central component of the protein synthetic apparatus. Although progress has been made in characterizing the functional role of many of the ribosomal proteins, the properties of ribosomal RNA and its role in ribosome structure and function are not well understood. To investigate the working properties of the highly conserved 3'-end of 16S rRNA, a site-specific deletion was made directly within the 16S rRNA molecule. The terminal deletion did not impair in vitro 30S subunit assembly, but the particles produced lost translational fidelity in an in vitro translation system primed with natural mRNA.

Autoradiography↗
PubMed2001

16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory.

For many years, sequencing of the 16S ribosomal RNA (rRNA) gene has served as an important tool for determining phylogenetic relationships between bacteria. The features of this molecular target that make it a useful phylogenetic tool also make it useful for bacterial detection and identification in the clinical laboratory. Sequence analysis of the 16S rRNA gene is a powerful mechanism for identifying new pathogens in patients with suspected bacterial disease, and more recently this technology is being applied in the clinical laboratory for routine identification of bacterial isolates. Several studies have shown that sequence identification is useful for slow-growing, unusual, and fastidious bacteria as well as for bacteria that are poorly differentiated by conventional methods. The technical resources necessary for sequence identification are significant. This method requires reagents and instrumentation for amplification and sequencing, a database of known sequences, and software for sequence editing and database comparison. Commercial reagents are available, and laboratory-developed assays for amplification and sequencing have been reported. Likewise, there are an increasing number of commercial and public databases. Despite the availability of resources, sequence-based identification is still relatively expensive. The cost is significantly reduced only by the introduction of more automated methods. As the cost decreases, this technology is likely to be more widely applied in the clinical setting.

Bacteria↗
PubMed1992

Phylogenetic relationships between the western aster yellows mycoplasmalike organism and other prokaryotes established by 16S rRNA gene sequence.

Restriction fragments containing the 16S rRNA gene of the western aster yellow mycoplasmalike organism (SAY-MLO) were identified in Southern blots probed with cloned fragments of the western X-disease mycoplasmalike organism 16S rRNA gene. Two fragments which contained the entire SAY-MLO 16S rRNA gene and flanking DNA were cloned in M13 and sequenced. The SAY-MLO 16S rRNA gene is approximately 1,535 bp long, has a G+C content of 47 mol%, and has an overall secondary structure similar to that proposed for Escherichia coli. Putative rRNA promoter sequences and sequences involved in processing of the primary rRNA transcript were similar in the SAY-MLO, two Mycoplasma species, and Bacillus subtilis, suggesting that these prokaryotes and the mycoplasmalike organisms may have similar transcriptional and processing enzymes. We identified two tRNA genes, a tRNA(Tyr) (GTA) gene upstream from the 16S rRNA gene and a tRNA(Ile) (GAT) gene in the spacer region between the 16S and 23S rRNA genes. Comparisons of the SAY-MLO 16S rRNA nucleotide sequence with 16S rRNA sequences of other organisms indicated that the SAY-MLO is phylogenetically related most closely to other plant-pathogenic mycoplasmalike organisms, followed by Anaeroplasma species, Acholeplasma species, and some Mycoplasma species.

Bacteria↗
PubMed2004

Prospective study of use of PCR amplification and sequencing of 16S ribosomal DNA from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting.

We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 x 10(2) to 2 x 10(2) CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.

Base Sequence↗
PubMed2000

Identification of non-tuberculous mycobacteria: 16S rRNA gene sequence analysis vs. conventional methods.

In a retrospective study, 45 clinical isolates of non-tuberculous mycobacteria were identified to the species level by biochemical profile, gas liquid chromatography and partial sequence analysis of 16S rRNA, and were found to represent 13 different species. The results of sequence analysis showed 100% identity with conventional tests for 34 isolates (76%) and could identify species such as M. bohemicum which are difficult to characterise with conventional methods. Most of the discrepant results for the remaining 11 isolates resulted in species of the same group of mycobacteria. Based on these findings. we concluded that direct sequence analysis of amplified 16S rRNA gene is a promising rapid and accurate method for species determination of non-tuberculous mycobacteria.

Bacterial Typing Techniques↗