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PubMed2004

The genomic heterogeneity among Mycobacterium terrae complex displayed by sequencing of 16S rRNA and hsp 65 genes.

The species identification within Mycobacterium terrae complex has been known to be very difficult. In this study, the genomic diversity of M. terrae complex with eighteen clinical isolates, which were initially identified as M. terrae complex by phenotypic method, was investigated, including that of three type strains (M. terrae, M. nonchromogenicum, and M. triviale ). 16S rRNA and 65-kDa heat shock protein (hsp 65) gene sequences of mycobacteria were determined and aligned with eleven other references for the comparison using similarity search against the GenBank and Ribosomal Database Project II (RDP) databases. 16S rRNA and hsp 65 genes of M. terrae complex showed genomic heterogeneity. Amongst the eighteen clinical isolates, nine were identified as M. nonchromogenicum, eight as M. terrae, one as M. mucogenicum with the molecular characteristic of rapid growth. M. nonchromogenicum could be subdivided into three subgroups, while M. terrae could be subdivided into two subgroups using a 5 bp criterion (>1% difference). Seven isolates in two subgroups of M. nonchromogenicum were Mycobacterium sp. strain MCRO 6, which was closely related to M. nonchromogenicum. The hsp 65 gene could not differentiate one M. nonchromogenicum from M. avium or one M. terrae from M. intracellulare. The nucleotide sequence analysis of 16S rRNA and hsp 65 genes was shown to be useful in identifying the M. terrae complex, but hsp 65 was less discriminating than 16S rRNA.

Bacterial Proteins↗
PubMed2000

Phylogenetic characterization of several para- and meta-PCB dechlorinating Clostridium species: 16s rDNA sequence analyses.

The genus Clostridium has more than 127 species, grouped according to their morphology and functions. Nine Clostridium species were identified based on their ability to dechlorinate meta- and para-PCB (polychlorinated biphenyl) contaminated sediments. The phylogenetic relatedness of these PCB-degrading Clostridium species was studied using ribosomal RNA genes. The diversity of small-subunit rRNA genes associated with the domain bacteria was examined using defined operational taxonomic units (OTUs) in samples from PCB contaminated sediments from Lake Medinah, New York. The RFLP (restriction fragment length polymorphism) of the OTVs was measured. OTUs B (105 clones), A (33 clones) and C (45 clones) accounted for 75% of all the 16S rDNA clones expressing anaerobic para- and meta-PCB dechlorinating activity. In this report we describe complete 16S rDNA sequences of OTU-A and OTU-B, and partial rDNA sequences of OTUs C-J. The OTU-B and OTU-I form a phylogenetically related cluster, closely affiliated with Clostridium hydroxybenzoicum strains. OTUs A, C, D, G, H and J also belong to the genus Clostridium, but they represent separate species. OTU-E, a close affiliate to Bacteroides forsynthus, is a meta-PCB dechlorinator. The Cl. hydroxybenzoicum strains (OTU-B) are primarily para-PCB dechlorinators and are the most common. Some less prevalent OTUs (- E, -G, -H and -I), are also mostly para-PCB dechlorinators. Other Clostridium species such as Cl. beijerinckii (OTU-A), Cl. intestinalis (OTU-D) and Cl. thermolacticum (OTU-J) are primarily meta-PCB dechlorinators. Cl. paraputrificum (OTU-C) and Cl. cellulosi (OTU-F), were less prevalent in the total consortium, but they could dechlorinate both para- and meta-PCB. Although a few less prevalent Clostridium species can degrade both para- and meta-PCBs, this study confirms that para- and meta-PCB dechlorinating species are generally phylogenetically different.

Base Sequence↗
PubMed2003

A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products.

The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen. The rapid and sensitive detection of L. monocytogenes is important in the food industry as well as in medical diagnosis. In this study, a PCR-based method for the rapid, specific, and sensitive detection of L. monocytogenes in food products was developed. The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L. monocytogenes within the Listeria genus. The primers for the Listeria genus and for L. monocytogenes were used in the same reaction mix for their simultaneous detection. In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control. For the detection of Listeria and L. monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction. The method detects 1 to 5 CFU in a 25-g sample in < or = 24 h. It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.

Base Sequence↗
PubMed1989

Selective Recovery of 16S rRNA Sequences from Natural Microbial Communities in the Form of cDNA.

Cloning of cDNA obtained from 16S rRNA (16S rcDNA) selectively retrieves species-specific sequence information useful for analyzing the composition and structure of natural microbial communities. With this technique we obtained recombinant 16S rcDNA libraries from Escherichia coli and from a model hot-spring cyanobacterial-mat community. The recombinant plasmids contained exclusively 16S rRNA-derived inserts. This selective approach is independent of biasing culture techniques and eliminates the laborious screening required to locate 16S rRNA gene-bearing recombinants in genomic DNA libraries obtained from natural communities.

Journal Article↗
PubMed2000

Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision.

The genetic diversity among strains in a worldwide collection of Ralstonia solanacearum, causal agent of bacterial wilt, was assessed by using three different molecular methods. PCR-RFLP analysis of the hrp gene region was extended from previous studies to include additional strains and showed that five amplicons were produced not only with all R. solanacearum strains but also with strains of the closely related bacteria Pseudomonas syzygii and the blood disease bacterium (BDB). However, the three bacterial taxa could be discriminated by specific restriction profiles. The PCR-RFLP clustering, which agreed with the biovar classification and the geographical origin of strains, was confirmed by AFLP. Moreover, AFLP permitted very fine discrimination between different isolates and was able to differentiate strains that were not distinguishable by PCR-RFLP. AFLP and PCR-RFLP analyses confirmed the results of previous investigations which split the species into two divisions, but revealed a further subdivision. This observation was further supported by 16S rRNA sequence data, which grouped biovar 1 strains originating from the southern part of Africa.

Africa↗
PubMed1994

Cytochrome b and 16S rRNA sequence variation in the Salmo trutta (Salmonidae, Teleostei) species complex.

PCR amplification and direct sequencing of regions of the cytochrome b and 16S rRNA mitochondrial genes was carried out on Italian and Irish populations of the brown trout (Salmo trutta) species complex. These included S. t. marmoratus, S. t. macrostigma, Salmo carpio, and Salmo fibreni collected in Italy, and S. t. trutta morpha fario, from both Italy and Ireland. Samples of Atlantic salmon (Salmo salar) were also examined and used as an outgroup in the phylogenetic analysis. Results based on 592 bp indicated differentiation between fario morphs from Italy and Ireland. Despite the large phenotypic differences within Italian salmonids, very low genetic variation was found. On the basis of the mtDNA sequences studied, S. carpio and S. fibreni, which have been described as good species, are genetically undifferentiated from the other Italian taxa. The finding of a haplotype from Ireland in the Brenta River in Italy stresses the importance of conservation genetics studies aimed at identifying and preserving native genotypes.

Animals↗
PubMed2001

Characterization by 16S rRNA sequence analysis of pseudomonads causing blotch disease of cultivated Agaricus bisporus.

Bacterial blotch of Agaricus bisporus has typically been identified as being caused by either Pseudomonas tolaasii (brown blotch) or Pseudomonas gingeri (ginger blotch). To address the relatedness of pseudomonads able to induce blotch, a pilot study was initiated in which pseudomonads were selectively isolated from mushroom farms throughout New Zealand. Thirty-three pseudomonad isolates were identified as being capable of causing different degrees of discoloration (separable into nine categories) of A. bisporus tissue in a bioassay. These isolates were also identified as unique using repetitive extragenic palindromic PCR and biochemical analysis. Relationships between these 33 blotch-causing organisms (BCO) and a further 22 selected pseudomonad species were inferred by phylogenetic analyses of near-full-length 16S rRNA gene nucleotide sequences. The 33 BCO isolates were observed to be distributed throughout the Pseudomonas fluorescens intrageneric cluster. These results show that in addition to known BCO (P. tolaasii, P. gingeri, and Pseudomonas reactans), a number of diverse pseudomonad species also have the ability to cause blotch diseases with various discolorations. Furthermore, observation of ginger blotch discoloration of A. bisporus being independently caused by many different pseudomonad species impacts on the homogeneity and classification of the previously described P. gingeri.

Agaricus↗
PubMed2003

Bacteria detected by culture and 16S rRNA sequencing in maxillary sinus samples from intensive care unit patients.

OBJECTIVES/HYPOTHESIS: In critically ill patients, the occurrence of artificial ventilation-acquired sinus disease is common. A possible sinus bacterial infection is occult and is combined with diagnostic difficulties. Culture as a method has limited capacity to verify the presence of bacteria and leaves unanswered the question of a possible infective agent because bacteria are difficult to grow when killed or suppressed by current antibiotic therapy. Hitherto unidentified micro-organisms are also possible in the microenvironments of maxillary sinuses. STUDY DESIGN: Prospective case series. METHODS: Twenty maxillary sinus samples (17 aspirates and 3 lavages) from nine critically ill patients with possible infectious disease were investigated by broad-range 16S rRNA polymerase chain reaction followed by sequencing. These results were compared with the previous culture results from gingiva (passage route), maxillary sinus absorption, and mucosa samples. RESULTS: The contaminations were rare (2 of 20) and corresponded well to culture results. One previously undiagnosed bacterium was found. Two aspirates were negative on both culture and polymerase chain reaction, whereas the corresponding maxillary sinus mucosal cultures had been positive. CONCLUSIONS: Applying a low-contamination maxillary sinuses sampling technique makes 16S rRNA sequencing clinically useful. The polymerase chain reaction and culture results were generally comparable. However, by polymerase chain reaction, bacteria were found that were missed by culturing. Some aspirates were free of bacterial remnants on 16S rRNA polymerase chain reaction, but corresponding mucosal cultures were positive. This could indicate that infection is induced within the tissue. It also indicates that infectious agents introduced into the sinuses may have routes other than the ostium. Further clinical use of 16S rRNA sequencing is required to enlarge our knowledge in applied microbiology and paranasal sinus disease.

Bacteria↗
PubMed2006

Identification of dominant bacteria in feces and colonic mucosa from healthy Spanish adults by culturing and by 16S rDNA sequence analysis.

The aim of this work was to examine by culturing the changes in the total and indicator populations of the feces of two individuals over 1 year and to identify the dominant microbial components of a single sample of feces from each donor. Populations and dominant bacteria from a sample of colonic mucosa from a further individual were also assessed. The culture results were then compared to those obtained with the same samples by 16S rDNA cloning and sequencing. High interindividual variation in representative microbial populations of the gastrointestinal tract (GIT) was revealed by both the culture and the culture-independent techniques. Species belonging to Clostridium clusters (XIVa, IV, and XVIII) predominated in both the fecal and the mucosal samples (except in the mucose cultured isolates), members of Clostridium coccoides cluster XIVa being the most numerous microorganisms. Species of gamma-proteobacteria (Escherichia coli and Shigella spp.), bifidobacteria, and actinobacteria appeared in lower numbers than those of clostridia. From the mucosal cultured sample, only facultative anaerobes and bifidobacteria were recovered, suggesting destruction of the anaerobe population during processing. In accordance with this, the microbial diversity revealed by 16S rDNA sequence analysis was greater than that revealed by culturing. Despite large interindividual differences, distinct human communities may have group-associated GIT microbiota characteristics, such as the low number of Bacteroides seen in the subjects in this study.

Adult↗
PubMed1996

Variability in 3' end of 16S rRNA sequence of Mycobacterium ulcerans is related to geographic origin of isolates.

Mycobacterium ulcerans causes extensive ulcers (Buruli ulcers) in the skin of humans. Analysis of the 3'-terminal region of the 16S rRNA gene sequence of 17 strains of M. ulcerans from Africa, the Americas, and Australia revealed three subgroups corresponding to the continent of origin, and some variable phenotypic characteristics. This sequence is useful for the rapid detection of M. ulcerans and discriminates M. marinum and M. shinshuense from M. ulcerans.

Africa↗
PubMed1994

Phylogeny of human intestinal spirochaetes inferred from 16S rDNA sequence comparisons.

The sequence of 1383 nucleotides of the DNA encoding 16S rDNA was determined for strains of human intestinal spirochaetes, comprising an unnamed isolate and "Brachyspira aalborgi" NCTC 11492. A phylogenetic tree was inferred from aligned sequence comparisons between the intestinal spirochaetes, representatives of the Spirochaetales and Escherichia coli. The type strain of Brachyspira aalborgi, though related to the Serpulina spp. at approx. 96.5% sequence similarity was distinct and separated from the unnamed human intestinal isolate, HIS Oman, N26. The latter formed a separated and novel lineage that bisected the Spirochaetales.

DNA, Ribosomal↗
PubMed1989

16S rRNA sequence indicates that plant-pathogenic mycoplasmalike organisms are evolutionarily distinct from animal mycoplasmas.

The plant-pathogenic mycoplasmalike organisms (MLOs) are so named because they lack cell walls. Many features that are essential to a definitive classification remain uncharacterized, because these organisms have resisted attempts at in vitro culturing. To establish the taxonomic position of the MLOs, the DNA region containing the 16S rRNA gene from a representative of the MLOs has been cloned and sequenced. Sequence comparisons indicate that the MLOs are related to Mycoplasma capricolum and that these two bacteria share their phylogenetic origin with Bacillus subtilis. The low G + C content of this gene and features of its deduced secondary structure further support this grouping. However, the presence of a single tRNAIle gene in the spacer between the 16S rRNA and 23S rRNA genes of the MLOs differentiates the MLOs from other representatives of the mycoplasmas, which indicates an early divergence in the evolution of the members of the class Mollicutes. The presence of certain characteristic oligonucleotides in the 16S rRNA sequence indicates that MLOs may be closely related to acholeplasmas.

Animals↗
PubMed2004

Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA clones.

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

Actinobacteria↗
PubMed1999

Prevalence of corynebacterial 16S rRNA sequences in patients with bacterial and "nonbacterial" prostatitis.

The etiology of chronic prostatitis syndromes in men is controversial, particularly when positive cultures for established uropathogens are lacking. Although identification of bacteria in prostatic fluid has relied on cultivation and microscopy, most microorganisms in the environment, including some human pathogens, are resistant to cultivation. We report here on an rRNA-based molecular phylogenetic approach to the identification of bacteria in prostate fluid from prostatitis patients. Positive bacterial signals were seen for 65% of patients with chronic prostatitis overall. Seven of 11 patients with bacterial signals but none of 6 patients without bacterial signals were cured with antibiotic-based therapy. Results indicate the occurrence in the prostate fluid of a wide spectrum of bacterial species representing several genera. Most rRNA genes were closely related to those of species belonging to the genera Corynebacterium, Staphylococcus, Peptostreptococcus, Streptococcus, and Escherichia. Unexpectedly, a wide diversity of Corynebacterium species was found in high proportion compared to the proportions of other bacterial species found. A subset of these 16S rRNA sequences represent those of undescribed species on the basis of their positions in phylogenetic trees. These uncharacterized organisms were not detected in control samples, suggesting that the organisms have a role in the disease or are the consequence of the disease. These studies show that microorganisms associated with prostatitis generally occur as complex microbial communities that differ between patients. The results also indicate that microbial communities distinct from those associated with prostatitis may occur at low levels in normal prostatic fluid.

Bacterial Infections↗
PubMed2000

Identification of Mycobacterium neoaurum isolated from a neutropenic patient with catheter-related bacteremia by 16S rRNA sequencing.

A rapidly growing pigmented mycobacterial strain with an ambiguous biochemical profile was isolated from the blood culture taken through the Hickman catheter of a 9-year-old girl with acute lymphoblastic leukemia. Whole-cell fatty acid analysis showed that the best match profile was that of Mycobacterium aurum, but the similarity index was only 0.217, meaning that there were no good matches between the isolate and the organisms in the database of the Microbial Identification System. The 16S rRNA gene of the mycobacterial strain was amplified, agarose gel purified, and sequenced. There were 44 base differences between the gene sequence of the isolate and that of M. aurum but only one base difference between the sequence of the isolate and that of Mycobacterium neoaurum, showing that the isolate was indeed a strain of M. neoaurum by using this "gold standard." This represents the first case of M. neoaurum infection documented by 16S rRNA sequencing.

Bacteremia↗
PubMed1992

Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria.

Animals↗
PubMed2002

Septicemia due to Pasteurella pneumotropica: 16S rRNA sequencing for diagnosis confirmation.

Bacteremia due to Pasteurella pneumotropica occurs infrequently. We report a case of septicemia in a 72-year-old woman who had no underlying illness. The microorganism was isolated from 10 blood cultures and identified by conventional and molecular methods. This is the first reported case of P. pneumotropica septicemia in an immunocompetent patient. The history of P. pneumotropica diseases in animals and humans and their varied clinical features are reviewed.

Aged↗