Extensive DNA sequence conservation throughout the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex includes the four species M. tuberculosis, M. bovis, M. africanum, and M. microti. We sequenced 13 M. tuberculosis complex strains in the 16S-to-23S rDNA internal transcribed spacer (ITS). The ITS has a high rate of nucleotide substitution. Previous reports found three nucleotide substitutions in the ITS between two M. tuberculosis complex strains. In contrast, we found the same ITS sequence in all 13 M. tuberculosis complex strains (including all four species and M. bovis BCG). This finding confirms the conservation of 16S rDNA sequence and the high DNA-DNA relatedness found in previous studies. By the usual criteria, the four species of the M. tuberculosis complex would be considered a single species. In a phylogenetic analysis based on the ITS sequence, the four species of the M. tuberculosis complex were distinct from nontuberculous mycobacteria. The ITS contains at least seven potential sites for oligonucleotide probes with specificity for the M. tuberculosis complex.     

Identification of clinically relevant viridans group streptococci by sequence analysis of the 16S-23S ribosomal DNA spacer region

The feasibility of sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (ITS) for the identification of clinically relevant viridans group streptococci (VS) was evaluated. The ITS regions of 29 reference strains (11 species) of VS were amplified by PCR and sequenced. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The ITS lengths (246 to 391 bp) and sequences were highly conserved among strains within a species. The intraspecies similarity scores for the ITS sequences ranged from 0.98 to 1.0, except for the score for S. gordonii strains. The interspecies similarity scores for the ITS sequences varied from 0.31 to 0.93. Phylogenetic analysis of the ITS regions revealed that evolution of the regions of some species of VS is not parallel to that of the 16S rRNA genes. One hundred six clinical isolates of VS were identified by the Rapid ID 32 STREP system (bioMérieux Vitek, Marcy l'Etoile, France) and by ITS sequencing, and the level of disagreement between the two methods was 18% (19 isolates). Most isolates producing discrepant results could be unambiguously assigned to a specific species by their ITS sequences. The accuracy of using ITS sequencing for identification of VS was verified by 16S rDNA sequencing for all strains except strains of S. oralis and S. mitis, which were difficult to differentiate by their 16S rDNA sequences. In conclusion, identification of species of VS by ITS sequencing is reliable and could be used as an alternative accurate method for identification of VS.     

Comparison of two reverse hybridization methods for mycobacterial identification in clinical practice

AIM OF THE STUDY: To evaluate the performance of two commercial methods for identification of Mycobacterium species: InnoLiPA Mycobacteria first version (Innogenetics) versus Genotype MTBC and Genotype Mycobacterium (HAIN) on, respectively, 2123 and 2164 distinct isolates. MATERIALS AND METHODS: Both techniques are based on the reverse hybridization of PCR products to their complementary probes immobilized on membrane strips. The InnoLiPA assay targets the 16S-23S rRNA spacer region. The HAIN test is composed of two kits: Genotype MTBC, for identification of tuberculosis complex mycobacteria, is based on gyrB DNA sequence polymorphism. Genotype Mycobacterium kit targets the 23S rDNA for identification of mycobacteria other than tuberculosis (MOTT) and tuberculosis complex mycobacteria. Both assays identify complex tuberculosis mycobacteria and respectively, eight and 12 species of MOTT. Moreover, the Genotype MTBC allows species differentiation within the M. tuberculosis complex. RESULTS: Eighty-eight percent and 95% of mycobacteria were identified by InnoLiPA and HAIN, respectively. Hybridization remained negative for 11% of isolates with InnoLiPA and 4% with HAIN. An identification of MOTT was obtained by conventional identification in all cases after the use of InnoLiPA. MOTT and one M. tuberculosis was obtained after HAIN procedure. Unidentified species were complementary to a specific probe in 5% of the cases with InnoLiPA and 17% with HAIN. CONCLUSION: HAIN identifies more mycobacteria species than does InnoLiPA and allows identification in the M. tuberculosis complex. However, failure in identification occurs only with MOTT with InnoLiPA when one M. tuberculosis was found among mycobacteria non identified with HAIN.     

The homology analysis of internal transcribed spacer sequence of ribosomal DNA in common dermatophytes

In order to analyze the sequences of the internal transcribed spacer (ITS) including the 5.8 S ribosomal DNA (rDNA) of common dermatophytes, so as to obtain a rapid and accurate method to identify the species of dermatophytes and to establish the phylogenetic tree of these species to understand their relationship,16 strains of dermatophytes were collected and preliminarily identified by morphological characteristics. General primers for fungi ITS1 and ITS4 were used to amplify the ITS rDNA of each strains with PCR. The PCR products after purification were sequenced directly and were analyzed through internet. In the results,11 strains were identified by means of morphological features, among which 5 strains were Trichophyton,5 strains were Microsporum and 1 was Epidermaphyton, which was consistent with the results by molecular biology. In the 5 unidentifiable strains, 1 strain was proved to be Chrysosporium by molecular biology. These strains studied could be divided into 3 different classes as indicated in the analysis of the phylogenetic tree of the sequences in ITS, which were quite different from those of morphological classification. It is evident from the above observations that the molecular method of analysis on the ITS sequences is a rapid, highly sensitive and accurate approach for the detection of dematophyte species, however, it still exhibits some limitations needing the supplementation with morphological identification.     

Differentiation of Phylogenetically Related Slowly Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences

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.     

Molecular characterization of environmental mycobacterium strains by PCR-restriction fragment length polymorphism of hsp65 and by sequencing of hsp65, and of 16S and ITS1 rDNA

Fifteen mycobacterial strains from the environment, not clearly identifiable by biochemical properties, were analyzed with molecular markers: PCR-restriction enzyme analysis of hsp65 and sequencing of hsp65, and of the internal transcribed spacer 1 (ITS1) and 16S rDNA. The 16S rDNA sequencing closely related the strains to a slow-growing mycobacterial group including Mycobacterium simiae, Mycobacterium lentiflavum, Mycobacterium genavense, Mycobacterium triplex and Mycobacterium heidelbergense. A stretch of T bases at the level of 16S rDNA enabled the separation of M. simiae and M. lentifiavum from M. genavense, M. triplex and M. heidelbergense; hence the attribution of some environmental strains to the first or second group. But the distances between the two clades were very short and the relative positions of environmental strains and of reference strains were not resolved in terms of node robustness (low bootstrap values) in the distance tree. However, the hsp65 restriction profiles suggested assigning six strains to the M. lentiflavum species, although these strains had been found closely related to M. genavense and M. triplex from 16S rDNA nucleotide signatures. The clustering of environmental strains into the same three clusters was deduced from analysis of three sequence data (hsp65, and ITS1 and 16S rDNA), but the taxonomic affiliation of environmental strains to reference strains remained tentative. Among environmental strains and reference strains, the distances found from hsp65 sequences had the same amplitude as those found between different strains of Mycobacterium gordonae. From ITS1 rDNA sequences, the distances found between the strains of the Mycobacterium avium complex also had the same amplitude as those found between environmental strains and reference strains. From our results, it appears that the environmental strains and the reference strains could constitute a complex of subspecies or closely related species. Their taxonomic status must be confirmed by DNA/DNA hybridization experiments.     

Molecular identification and strain typing of dermatophyte fungi.

The rDNA spacer regions provide easily accessible, polymorphic genetic loci for both species and strain identification of dermatophyte fungi. Nucleotide substitutions and length polymorphisms in the internal transcribed spacers (ITS) can be indexed by sequencing or by PCR restriction endonuclease analysis, and provide a rapid and accurate means of identifying dermatophyte taxa. Multiple sets of tandem repeats that vary in copy number both within and between strains produce length heterogeneity in the nontranscribed spacer (NTS) region. Amplification of these repeats using specific PCR, or their detection by Southern hybridisation with a generic ribosomal DNA probe, provides a sensitive and discriminatory technique for strain identification in T. rubrum and other dermatophyte fungi.     

Detection and identification of mycobacteria by amplification of the internal transcribed spacer regions with genus- and species-specific PCR primers

We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from the American Type Culture Collection; 205 clinical isolates of mycobacteria, including 118 Mycobacterium tuberculosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragments of approximately 270 to 400 bp in all mycobacteria. PCR with the M. tuberculosis complex-specific primers amplified an approximately 120-bp fragment only for the M. tuberculosis complex. Multiplex PCR with the panmycobacterial primers and the M. tuberculosis complex-specific primers amplified two fragments that were specific for all mycobacteria and the M. tuberculosis complex, respectively. PCR with M. avium complex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, and M. szulgai-specific primers amplified specific fragments only for the respective target organisms. These novel primers can be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early and accurate diagnosis of mycobacteriosis.     

Differentiation of clinical Mycobacterium tuberculosis complex isolates by gyrB DNA sequence polymorphism analysis

The discriminatory power of gyrB DNA sequence polymorphisms for differentiation of the species of the Mycobacterium tuberculosis complex (MTBC) was evaluated by sequencing and restriction fragment length polymorphism (RFLP) analysis of a 1,020-bp fragment amplified from clinical isolates of M. tuberculosis, Mycobacterium bovis (pyrazinamide [PZA] resistant as well as PZA susceptible), Mycobacterium africanum subtypes I and II, and Mycobacterium microti types vole and llama. We found sequence polymorphisms in four regions described previously and at one additional position. These differences in the gyrB sequences allow an accurate discrimination of M. bovis, M. microti, and M. africanum subtype I. The PZA-susceptible subtypes of M. bovis shared the M. bovis-specific substitution at position 756 with the PZA-resistant strains, but can be unambiguously differentiated by a characteristic substitution at position 1311. As a drawback, M. tuberculosis and M. africanum subtype II showed an identical gyrB sequence that facilitates discrimination from the other species, but not from each other. A PCR-RFLP technique applying three restriction enzymes could be shown to be a rapid and easy-to-perform tool for the differentiation of the members of the MTBC. Based on these results, we present a clear diagnostic algorithm for the differentiation of species of the MTBC.     

Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene (rpoB)

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.     

Malassezia furfur in infantile seborrheic dermatitis

Our objective was to study both incidence and various strains of Malassezia in infantile seborrheic dermatitis (ISD). Sixty infants between 2 weeks and 2 years old with clinical diagnosis of ISD at the Department of Pediatrics, King Chulalongkorn Memorial Hospital from May 2002 to April 2003 were recruited. Malassezia spp. were isolated from cultured skin samples of the patients, genomic DNA was extracted and the ITS1 rDNA region was amplified. The PCR product was examined by agarose gel electrophoresis and DNA sequences were determined. The ITS1 sequences were also subjected to phylogenetic analysis and species identification. ISD is most commonly found in infants below the age of 2 months (64%), followed by those between 2 and 4 months (28%) old. Cultures yielded yeast-like colonies in 15 specimens. PCR yielded 200-bp products (Candida) in 3 patients and 300-bp products (Malassezia furfur) in 12 patients (18%). Sugar fermentation using API 20C aux performed on the three 200-bp PCR products yielded Candida species. M. furfur was the only Malassezia recovered from skin scrapings of children with ISD.     

Identification of medically important Candida and non-Candida yeast species by an oligonucleotide array

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.     

Species distribution of the Mycobacterium tuberculosis complex in clinical isolates from 2007 to 2010 in Turkey: a prospective study

The Mycobacterium tuberculosis complex (MTBC) consists of a group of closely related species that differ in their epidemiological profiles, host ranges, pathogenicities, geographic distributions, and drug resistances. Identification of members in the MTBC is essential for monitoring the epidemiology of tuberculosis (TB) and implementing appropriate public health control measures. In this study, 188 consecutive MTBC clinical isolates from 2007 to 2010 were evaluated to determine the prevalence of MTBC species in Turkey. PCR and restriction fragment length polymorphism analysis (PCR-RFLP) of the gyrB gene were used, and results for species other than M. tuberculosis were confirmed using the GenoType MTBC assay (Hain Lifescience, Nehren, Germany). Most of the strains were found to be M. tuberculosis (94.1%). The prevalences of M. bovis and M. caprae were 4.3% and 1.6%, respectively. Only one M. bovis BCG strain was identified. Overall, the frequency of bovine tuberculosis in humans was 5.3%. We had assumed that bovine TB infection was under control in animal herds, but primary M. bovis infections in humans caused by transmission from infected animals are still an issue in Turkey. Our results indicate that the frequent identification of M. bovis in routine mycobacteriological laboratory work has further importance due to the well-known resistance of this species to pyrazinamide.     

Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses

In this study, 18S rDNA and ITS-5.8S rDNA regions of four Shiraia bambusicola isolates collected from different species of bamboos were amplified by PCR with universal primer pairs NS1/NS8 and ITS5/ITS4, respectively, and sequenced. Phylogenetic analyses were conducted on three selected datasets of rDNA sequences. Maximum parsimony, distance and maximum likelihood criteria were used to infer trees. Morphological characteristics were also observed. The positioning of Shiraia in the order Pleosporales was well supported by bootstrap, which agreed with the placement by Amano (1980) according to their morphology. We did not find significant inter-hostal differences among these four isolates from different species of bamboos. From the results of analyses and comparison of their rDNA sequences, we conclude that Shiraia should be classified into Pleosporales as Amano (1980) proposed and suggest that it might be positioned in the family Phaeosphaeriaceae.     

Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions

Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods.     

Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences Identify Medically Important Yeasts

Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.     

Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic .Eimeria species of the chicken.

We describe a polymerase chain reaction (PCR)-based assay for the detection, identification and differentiation of pathogenic species of .Eimeria in poultry. The internal transcribed spacer 1 (ITS1) regions of ribosomal DNA (rDNA) from .Eimeria acervulina, E. brunetti, E. necatrix and .E. tenella were sequenced and regions of unique sequences identified. Four pairs of oligonucleotide primers, each designed to amplify the ITS1 region of a single .Eimeria species, were synthesised for use in the PCR assay. In tests on purified genomic DNA from all seven species of .Eimeria that infect the chicken, each of the four primer pairs amplified the ITS1 region from only their respective target species. The robustness of the approach was further demonstrated by the amplification of specific DNA fragments from tissues of experimentally infected animals and from oocysts recovered from field samples. We conclude that the ITS1 regions of .Eimeria species contain sufficient inter-specific sequence variation to enable the selection of primers that can be applied in PCR analyses to detect and differentiate between species. In future work they may provide excellent markers for epidemiological studies.     

Detection and identification of Mycobacterium species isolates by DNA microarray

Rapid identification of Mycobacterium species isolates is necessary for the effective management of tuberculosis. Recently, analysis of DNA gyrase B subunit (gyrB) genes has been identified as a suitable means for the identification of bacterial species. We describe a microarray assay based on gyrB gene sequences that can be used for the identification of Mycobacteria species. Primers specific for a gyrB gene region common to all mycobacteria were synthesized and used for PCR amplification of DNA purified from clinical samples. A set of oligonucleotide probes for specific gyrB gene regions was developed for the identification of 14 Mycobacterium species. Each probe was spotted onto a silylated glass slide with an arrayer and used for hybridization with fluorescently labeled RNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for each species of mycobacteria and could differentiate closely related bacterial species. Moreover, the results corresponded well with those obtained by the conventional culture method for the detection of mycobacteria. We conclude that a gyrB-based microarray can rapidly detect and identify closely related mycobacterial species and may be useful in the diagnosis and effective management of tuberculosis.     

Species identification and strain typing of Malassezia species stock strains and clinical isolates based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions

This study demonstrated the application of internal transcribed spacer 1 (ITS1) ribosomal DNA sequences to the species identification and strain typing of 28 standard strains and 46 clinical isolates of the genus Malassezia. The size of ITS1 regions ranged from 162 to 266 bp. Members of the genus Malassezia (M. pachydermatis, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae) were classified into seven ITS1-homologous groups and 22 ITS1-identical, individual groups. The 46 clinical isolates of lipophilic Malassezia spp. were identified as belonging to just three ITS1-homologous groups, i.e., M. furfur (19 strains: 11 from pityriasis versicolor, 4 from seborrhoeic dermatitis and 4 from atopic dermatitis). M. sympodialis (22 strains: 7 from pityriasis versicolor, 3 from seborrhoeic dermatitis, 1 from atopic dermatitis and 11 from healthy controls) and M. slooffiae (five strains: three from chronic otitis media and two from healthy controls).