Mycobacterium riyadhense sp. nov., a non-tuberculous species identified as Mycobacterium tuberculosis complex by a commercial line-probe assay (original) (raw)
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Journal of Clinical Microbiology, 2006
The aim of this study was to develop a PCR and reverse line blot hybridization (PCR-RLB) macroarray assay based on 16S-23S rRNA gene internal transcribed spacer sequences for the identification and differentiation of 34 mycobacterial species or subspecies. The performance of the PCR-RLB assay was assessed and validated by using 78 reference strains belonging to 55 Mycobacterium species, 219 clinical isolates which had been identified as mycobacteria by high-performance liquid chromatography or gas chromatography, three skin biopsy specimens from patients with suspected leprosy which had been shown to contain acid-fast bacilli, and isolates of 14 nonmycobacterial species. All mycobacteria were amplified in the PCR and hybridized with a genus-specific probe (probe MYC). The 34 species-specific probes designed in this study hybridized only with the corresponding Mycobacterium species. The mycobacterial PCR-RLB assay is an efficient tool for the identification of clinical isolates of mycobacteria; it can reliably identify mixed mycobacterial cultures and M. leprae in skin biopsy specimens.
Journal of Clinical Microbiology, 1998
Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in Mycobacterium kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szulgai. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus Mycobacterium representing 13 species (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii, M. malmoense, M. marinum, M. shimoidei, M. simiae, M. szulgai, M. triplex, M. ulcerans, and M. xenopi). An alignment of these sequences together with additional sequences available in the EMBL database (for M. intracellulare, M. phlei, M. smegmatis, and M. tuberculosis) was established according to primary-and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for M. simiae, M. genavense, and M. triplex, and distinct branches for M. xenopi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgai revealed ITS sequence similarities of 93 and 88%, respectively. M. marinum and M. ulcerans possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification.
Tubercle and Lung Disease, 1996
Objective: To evaluate the possibility of differentiating species of mycobacteria by amplifying 16S-23S ribosomal deoxyribonucleic acid (DNA) spacer and restriction enzyme analysis of the products. Design: DNA of 113 strains of mycobacteria belonging to 18 species of the genus Mycobacterium were amplified by primers PL1 (5'-GAAGTCGTAACAAGG) and PL2 (5'-CAAGGCATCCACCAT). The amplified products as well as their HaeIII-, MspIand BstXI-digested products were visualized after agarose gel electrophoresis. Results: The amplified products of rapid-growing mycobacteria were different from the slow-growing mycobacteria. The restriction profiles of members of M. tuberculosis complex were the same as each other but different from other investigated species. The restriction profiles of some species, such as Mo avium, M. intracellulare and M. gordonae, were unique, while those of the other species had more than one pattern.
RIDOM: Comprehensive and public sequence database for identification of Mycobacterium species
Bmc Infectious Diseases, 2003
Background: Molecular identification of Mycobacterium species has two primary advantages when compared to phenotypic identification: rapid turn-around time and improved accuracy. The information content of the 5' end of the 16S ribosomal RNA gene (16S rDNA) is sufficient for identification of most bacterial species. However, reliable sequence-based identification is hampered by many faulty and some missing sequence entries in publicly accessible databases.
Objective(s) In addition to several molecular methods and in particular 16S rDNA analysis, the application of a more discriminatory genetic marker, i.e., 16S-23S internal transcribed spacer gene sequence has had a great impact on identification and classification of mycobacteria. In the current study we aimed to apply this sequencing power to conclusive identification of some Iranian clinical strains of mycobacteria. Materials and Methods The test strains consisted of nineteen mycobacterial isolates which were initially identified by the use of conventional phenotypic techniques and molecular methods and subjected to further definitive identification using the 16S-23S internal transcribed spacer gene sequencing. Results Out of 19 studied strains, 7 isolates were found to be rapidly growing and 12 isolates as slowly growing mycobacteria. With the exception of one isolate, i.e., the isolate HNTM87, which yielded a distinct ITS sequence incomparable with all previously identified mycobacteria, the remaining isolates produced the sequences similar to the established mycobacteria and were clearly identified and differentiated from closely related taxa. A phylogenetic tree based on maximum parsimony analysis of 16S-23S internal transcribed spacer gene sequences constructed showing the relatedness of Iranian clinical isolates with the closely related type species of mycobacteria. Conclusion This study showed that the 16S-23S internal transcribed spacer gene of the genus Mycobacterium exhibits a high variation which is of value for discriminating closely related taxa and could be used independently or in combination with 16S rDNA sequencing to delineate the true identity of rare mycobacterial species.
International Journal of Systematic and Evolutionary Microbiology, 2012
A thorough phenotypic and genotypic analysis of 150 strains belonging to the Mycobacterium terrae complex resulted in the identification of a number of previously unreported sequevars (sqvs) within the species known to belong to the complex. For the species Mycobacterium arupense , three sqvs were detected in the 16S rRNA gene, six sqvs in the hsp65 gene and 15 sqvs in the rpoB gene; in Mycobacterium senuense two sqvs were present in each of the three genetic regions; in Mycobacterium kumamotonense four, two and nine sqvs were found, respectively, and in M. terrae three, four and six sqvs were found, respectively. The inappropriate inclusion of Mycobacterium triviale within the M. terrae complex was confirmed. The limited utility of biochemical tests and of mycolic acid analyses for the differentiation of the members of M. terrae complex was also confirmed. The survey allowed the recognition of three previously undescribed species that were characterized by unique sequences in the 1...
Simple and rapid identification of different species of Mycobacteria by PCR
Molecular and Cellular Probes, 1999
A simple polymerase chain reaction (PCR) assay for rapid identification of different species of mycobacteria was developed. This PCR is based on the use of conserved sequences to amplify the genome of several mycobacterial species. The amplification patterns obtained were specific and reproducible for the species tested. In particular, we could identifyMycobacterium tuberculosisandMycobacterium bovis(both produced the same pattern),Mycobacterium avium, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium chelonae, Mycobacterium peregrinum, Mycobacterium fortuitum, Mycobacterium gordonaeandMycobacterium smegmatis. Moreover, due to the numerous copies of the target sequences present in the genome, the PCR showed a very high level of sensitivity.
International journal of systematic and evolutionary microbiology, 2009
An rpoB sequence-based evaluation of 100 Mycobacterium avium complex (MAC) clinical isolates led to the identification of five respiratory tract isolates that were potential representatives of three novel MAC species. Distinctive phenotypic features of isolates 62863 and 5356591(T) included a pseudomycelium morphology and both esterase and acid phosphatase activities. These two isolates exhibited sequence similarities of 99.8 % for the 16S rRNA gene, 86.3 and 86.1 % for 16S-23S rRNA gene internal transcribed spacer (ITS-1) sequence, 96.7 and 97.8 % for rpoB and 97.6 and 97.4 % for hsp65, respectively, with the type strain of Mycobacterium chimaera, the most closely related species. Isolates 3256799 and 5351974(T) lacked alpha-mannosidase and beta-glucosidase activities. They exhibited sequence similarities of 99.6 % for the 16S rRNA gene, 90.1 and 90.4 % for ITS-1, 97.8 % for rpoB and 98.0 and 98.1 % for hsp65, respectively, with the type strain of M. chimaera, the most closely rela...
Journal of Medical Microbiology, 2018
Mycobacteria are common causative agents of bacterial infections in many species of freshwater and marine fish. Identification of mycobacteria to the species level based on phenotypic tests is inappropriate and time consuming. Molecular methods such as partial or entire gene sequence determination in mycobacteria have been employed to resolve these problems. The objective of this study was to assess the use of sequence analysis of the mycobacterial 16S-23S internal transcribed spacer (ITS) region for the identification of different aquatic mycobacteria species. Using published primers, ITS sequences of 64 field and reference strains were determined. The identity of all isolates previously identified as M. marinum by restriction fragment length polymorphism (RFLP) DNA profiling was confirmed as M. marinum by sequence analysis. With the exception of five rapidly growing mycobacteria isolates, all other mycobacteria were easily identified by sequencing of the ITS region. Using this spacer region, it was possible to differentiate between slowly growing and rapidly growing mycobacteria, even before sequence analysis, by the size of the PCR product, although species identification could not be made by size alone. Overall, direct sequencing of this genetic element following PCR has been shown to be useful in the identification of aquatic mycobacteria species.
Journal of clinical microbiology, 1997
The use of high-performance liquid chromatography (HPLC) revealed four previously unreported profiles within a group of mycobacteria consisting of 14 clinical isolates. These mycobacteria, whose identification by conventional tests appeared problematic, mostly resembled Mycobacterium avium complex or Mycobacterium simiae. Genetic analysis revealed, within this group, six different nucleic acid sequences in a hypervariable 16S rRNA segment, but all the isolates appeared to be phylogenetically related to M. simiae. Six isolates representing the largest of groups defined by means of genetic sequencing turned out to belong to the newly described species Mycobacterium lentiflavum. Furthermore, three such clusters precisely coincided with three of those defined by HPLC, while the three remaining clusters shared almost identical HPLC profiles. All but one strain (which, although clearly not belonging to the M. avium complex, hybridized with specific commercial DNA probes) showed high-grade...