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PubMed2003

Utilization of 16S ribosomal DNA sequencing for diagnosis of septicemia due to Neisseria elongata subsp. glycolytica in a neutropenic patient.

Septicemia due to Neisseria elongata subsp. glycolytica occurs infrequently. We report a case of septicemia in a patient undergoing antimitotic chemotherapy. Gram-negative coccobacilli were isolated from blood cultures. The identity of the isolate by phenotypic methods was uncertain. In contrast, identity was confirmed by 16S ribosomal DNA sequencing, which appeared to be very useful for correct identification.

Aged↗
PubMed1997

Molecular characterization of Mycobacterium avium complex isolates giving discordant results in AccuProbe tests by PCR-restriction enzyme analysis, 16S rRNA gene sequencing, and DT1-DT6 PCR.

Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare isolates and shows the utility of in-house DT1 PCR to detect this group of isolates, which would otherwise have been missed by the AccuProbe system in a routine clinical microbiology laboratory.

Base Sequence↗
PubMed2001

Characterization of halobenzoate-degrading, denitrifying Azoarcus and Thauera isolates and description of Thauera chlorobenzoica sp. nov.

The taxonomic relationships of Azoarcus and Thauera isolates in the beta-subclass of the Proteobacteria capable of degrading fluoro-, chloro- or bromobenzoate under denitrifying conditions were analysed. A detailed classification of these strains was performed using a polyphasic approach, which included studies on morphology, phenotypic characterization, fatty acid analysis, 16S rRNA gene sequence analysis, 16S rRNA gene mapping (ribotyping) and DNA-DNA hybridization. The analyses of fatty acids and 16S rRNA gene sequencing differentiated strains 2FB2, 2FB6 and 4FB10 as new members of the genus Azoarcus and strains 4FB1, 4FB2, 3CB2, 3CB3 and 3BB1 as new members of the genus Thauera. DNA-DNA hybridization experiments established that strains 2FB2, 2FB6 and 4FB10 belong to the species Azoarcus tolulyticus. Strains 3CB2 and 3CB3 were assigned to the species Thauera aromatica on the basis of DNA-DNA hybridization and ribotyping experiments. Strains 4FB1, 4FB2 and 3BB1 showed close relatedness with strain 3CB-1T, previously described as T. aromatica genomovar chlorobenzoica. This group of strains is clearly differentiated from the species T. aromatica on the basis of 16S rRNA sequence analysis, DNA homology and ribotyping analysis. Strains 3CB-1T, 4FB1, 4FB2 and 3BB1 are proposed as members of the new species Thauera chlorobenzoica sp. nov., strain 3CB-1T (= ATCC 700723T) being the type strain.

Azoarcus↗
PubMed1996

Reassessment of the phylogenetic position of the bacterium associated with Whipple's disease and determination of the 16S-23S ribosomal intergenic spacer sequence.

Whipple's disease is a rare chronic illness associated with an unculturable bacterium that is constantly present in affected tissues. This bacterium was previously characterized at the molecular level by PCR and sequencing of the 16S rRNA gene. On the basis of 1,321 nucleotides of the sequence of its gene coding for 16S rRNA (16S rDNA), a phylogenetic relationship to the actinomycetes was established. In this study, we determined an almost complete 16S rDNA sequence (1,495 nucleotides), the 16S-23S ribosomal intergenic spacer sequence, and 200 nucleotides of the 23S rRNA gene. The 16S rDNA sequence was compared with the large number of actinomycete sequences that have been added to the database since the original study. Phylogenetic analysis revealed a branching position as the deepest branch of the cluster comprising the actinomycetes with group B peptidoglycan between this group and the family Cellulomonadaceae. This provides additional information on the phylogenetic position of this bacterium and some clues as to its characteristics. The spacer region between the 16S and 23S rRNA genes is 294 nucleotides long and does not contain tRNA genes. As has been shown in other instances, the increased variability of the ribosomal intergenic spacer compared with the 16S rRNA gene makes it a potential target for use in the differentiation of strains of the bacterium associated with Whipple's disease.

Actinomycetales↗
PubMed2004

[PCR with subsequent sequencing of the 16S gene rRNA in species identification of mycobacteria of the non-tuberculosis complex].

One hundred of mycobacterium cultures were assayed by the method of PCR with subsequent sequencing of the 16S rRNA region. The below mycobacterium species were identified: M. tuberculosis complex (n = 55), M. avium (n = 17), M. intracellulare (n = 4), M. scrofaleceum (n = 2), M. kansasii - M. gastri (n = 3), M. gordonae (n = 3), M. ulcerans - M. marinum (n = 1), M. smegmatis (m = 2), M. fortuitum (n = 11), M. peregrinum (n = 1) and M. chelonae - M. abscessus (n = 1). The method enabled the differentiation of species M. avium from M. intracellulare and M. peregrinum from M. fortuitum, which could not be differentiated by using the classic biochemical and bacteriological methods. Genetic heterogeneity of the mycobacterium strains of M. avium, M. fortuitum and M. gordonae was also established by PCR plus sequencing of the 16S rRNA region.

Base Sequence↗
PubMed1996

A phylogenetic study of Rubrobacter radiotolerans by sequence analysis of the 16S ribosomal RNA gene.

Partial sequence of the 16S ribosomal RNA gene of an extremely high radiotolerant bacterium, Rubrobacter radiotolerans (reclassified from Arthrobacter radiotolerans by chemical characteristics) was determined by PCR-amplification from a small amount of heat-lysed biomass, followed by direct sequencing of the PCR product. The sequence was aligned with seven species of Arthrobacter and also with representatives of various other bacterial groups. R. radiotolerans was confirmed to be out of the Arthrobacter group justifying the reclassification. Moreover, it has an individual position among the selected representatives being closer to the eubacterial groups.

Arthrobacter↗
PubMed2004

A method for comparing multiple bacterial community structures from 16S rDNA clone library sequences.

Culture-independent approaches, based on 16S rDNA sequences, are extensively used in modern microbial ecology. Sequencing of the clone library generated from environmental DNA has advantages over fingerprint-based methods, such as denaturing gradient gel electrophoresis, as it provides precise identification and quantification of the phylotypes present in samples. However, to date, no method exists for comparing multiple bacterial community structures using clone library sequences. In this study, an automated method to achieve this has been developed, by applying pair wise alignment, hierarchical clustering and principle component analysis. The method has been demonstrated to be successful in comparing samples from various environments. The program, named CommCluster, was written in JAVA, and is now freely available, at http://chunlab.snu.ac.kr/commcluster/.

Algorithms↗
PubMed2000

Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys.

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.

Ammonia↗
PubMed2004

Systematic 16S rRNA gene sequencing of atypical clinical isolates identified 27 new bacterial species associated with humans.

Clinical microorganisms isolated during a 5-year study in our hospital that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis. Each isolate yielded a > or =1,400-bp sequence containing <5 ambiguities which was compared with the GenBank 16S rRNA gene library; 1,404 such isolates were tested, and 120 were considered unique (27 isolates) or rare (< or =10 cases reported in the literature) human pathogens. Eleven new species, "Actinobaculum massiliae," "Candidatus Actinobaculum timonae," Paenibacillus sanguinis, "Candidatus Bacteroides massiliae," Chryseobacterium massiliae, "Candidatus Chryseobacterium timonae," Paenibacillus massiliensis, "Candidatus Peptostreptococcus massiliae," "Candidatus Prevotella massiliensis," Rhodobacter massiliensis, and "Candidatus Veillonella atypica" were identified. Sixteen species were obtained from humans for the first time. Our results show the important role that 16S rRNA gene sequence-based bacterial identification currently plays in recognizing unusual and emerging bacterial diseases.

Adolescent↗
PubMed1998

Note: identification of Lactobacillus delbrueckii subspecies bulgaricus and subspecies lactis dairy isolates by amplified rDNA restriction analysis.

Amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) was used to identify Lactobacillus delbrueckii dairy isolates and to assign them to its three subspecies (delbrueckii, lactis and bulgaricus). PCR-ARDRA allowed confirmation of predictions from the DNA database regarding the restriction mapping of the 16S sequence to be verified in practice. PCR-ARDRA was shown to be a reliable and rapid method for identifying Lact. delbrueckii isolates at the subspecies level and for differentiating this species from Lact. helveticus and Lact. acidophilus.

Blotting, Southern↗
PubMed1997

Phylogenetic position of Riemerella anatipestifer based on 16S rRNA gene sequences.

Riemerella anatipestifer, the causative agent of septicemia anserum exsudativa (also called new duckling disease), belongs to the family Flavobacteriaceae of gram-negative bacteria. We determined the DNA sequences of the rrs genes encoding the 16S rRNAs of four R. anatipestifer strains by directly sequencing PCR-amplified rrs genes. A sequence similarity analysis confirmed the phylogenetic position of R. anatipestifer in the family Flavobacteriaceae in rRNA superfamily V and allowed fine mapping of R. anatipestifer on a separate rRNA branch comprising the most closely related species, Bergeyella zoohelcum, as well as Chryseobacterium balustinum, Chryseobacterium indologenes, and Chryseobacterium gleum. The sequences of the rrs genes of the four R. anatipestifer strains varied between 0.5 and 1.0%, but all of the strains occupied the same position on the phylogenetic tree. In general, differences in rrs genes were observed among R. anatipestifer strains, even within a given serotype, as shown by restriction fragment length polymorphism of PCR-amplified rrs genes.

Animals↗
PubMed1999

Identification of the cause of a brain abscess by direct 16S ribosomal DNA sequencing.

We report the case of a young man who apparently suffered successive episodes of meningitis and cerebral abscess over a 1-month period, both of which were diagnosed by two different molecular approaches; PCR for Neisseria meningitidis IS1106 from CSF and 16S rRNA gene sequencing on a specimen of brain pus. In each case, cultures were negative due to prior antibiotic therapy.

Adult↗
PubMed1997

16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia.

The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage-within the gamma-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species.

Base Sequence↗
PubMed2003

Bacterial diversity and sulfur cycling in a mesophilic sulfide-rich spring.

An artesian sulfide- and sulfur-rich spring in southwestern Oklahoma is shown to sustain an extremely rich and diverse microbial community. Laboratory incubations and autoradiography studies indicated that active sulfur cycling is occurring in the abundant microbial mats at Zodletone spring. Anoxygenic phototrophic bacteria oxidize sulfide to sulfate, which is reduced by sulfate-reducing bacterial populations. The microbial community at Zodletone spring was analyzed by cloning and sequencing 16S rRNA genes. A large fraction (83%) of the microbial mat clones belong to sulfur- and sulfate-reducing lineages within delta-Proteobacteria, purple sulfur gamma-Proteobacteria, epsilon -Proteobacteria, Chloroflexi, and filamentous Cyanobacteria of the order Oscillatoria as well as a novel group within gamma-Proteobacteria. The 16S clone library constructed from hydrocarbon-exposed sediments at the source of the spring had a higher diversity than the mat clone library (Shannon-Weiner index of 3.84 compared to 2.95 for the mat), with a higher percentage of clones belonging to nonphototrophic lineages (e.g., Cytophaga, Spirochaetes, Planctomycetes, Firmicutes, and Verrucomicrobiae). Many of these clones were closely related to clones retrieved from hydrocarbon-contaminated environments and anaerobic hydrocarbon-degrading enrichments. In addition, 18 of the source clones did not cluster with any of the previously described microbial divisions. These 18 clones, together with previously published or database-deposited related sequences retrieved from a wide variety of environments, could be clustered into at least four novel candidate divisions. The sulfate-reducing community at Zodletone spring was characterized by cloning and sequencing a 1.9-kb fragment of the dissimilatory sulfite reductase (DSR) gene. DSR clones belonged to the Desulfococcus-Desulfosarcina-Desulfonema group, Desulfobacter group, and Desulfovibrio group as well as to a deeply branched group in the DSR tree with no representatives from cultures. Overall, this work expands the division-level diversity of the bacterial domain and highlights the complexity of microbial communities involved in sulfur cycling in mesophilic microbial mats.

Bacteria↗
PubMed1995

The genus Methylophaga, a new line of descent within phylogenetic branch gamma of Proteobacteria.

The genus Methylophaga with two species, M. marina and M. thalassica, comprises halophilic methylotrophic bacteria. These organisms utilise C1 compounds through the ribulose monophosphate pathway and are unable to grow on methane. Nearly complete 16S rRNA sequences were obtained for both Methylophaga species by directly sequencing the amplified 16S rRNA gene. These sequences were compared with published 16S rRNA sequences of methylotrophic strains and a large number of marine bacterial strains including several members of the alpha, beta and gamma subclasses of Proteobacteria. Phylogenetic trees were inferred using both parsimony and distance matrix methods. Each topology was analysed by bootstrap. The genus Methylophaga was found to be clearly separated from other methylotrophic bacteria and formed a distinct branch within the gamma subclass of Proteobacteria.

Base Sequence↗
PubMed2005

Diversity of bat-associated Leptospira in the Peruvian Amazon inferred by bayesian phylogenetic analysis of 16S ribosomal DNA sequences.

The role of bats as potential sources of transmission to humans or as maintenance hosts of leptospires is poorly understood. We quantified the prevalence of leptospiral colonization in bats in the Peruvian Amazon in the vicinity of Iquitos, an area of high biologic diversity. Of 589 analyzed bats, culture (3 of 589) and molecular evidence (20 of 589) of leptospiral colonization was found in the kidneys, yielding an overall colonization rate of 3.4%. Infection rates differed with habitat and location, and among different bat species. Bayesian analysis was used to infer phylogenic relationships of leptospiral 16S ribosomal DNA sequences. Tree topologies were consistent with groupings based on DNA-DNA hybridization studies. A diverse group of leptospires was found in peri-Iquitos bat populations including Leptospira interrogans (5 clones), L. kirschneri (1), L. borgpetersenii (4), L. fainei (1), and two previously undescribed leptospiral species (8). Although L. kirschenri and L. interrogans have been previously isolated from bats, this report is the first to describe L. borgpetersenii and L. fainei infection of bats. A wild animal reservoir of L. fainei has not been previously described. The detection in bats of the L. interrogans serovar Icterohemorrhagiae, a leptospire typically maintained by peridomestic rats, suggests a rodent-bat infection cycle. Bats in Iquitos maintain a genetically diverse group of leptospires. These results provide a solid basis for pursuing molecular epidemiologic studies of bat-associated Leptospira, a potentially new epidemiologic reservoir of transmission of leptospirosis to humans.

Animals↗