Differentiation between Mycobacterium farcinogenes and Mycobacterium senegalense strains based on 16S-23S ribosomal DNA internal transcribed spacer sequences

16S ribosomal DNA (rDNA) and 16S-23S internal transcribed spacer rDNA sequence analyses were performed on Mycobacterium farcinogenes and M. senegalense strains and 26 strains of other rapidly growing mycobacteria to investigate the phylogenetic structure of bovine farcy mycobacteria within the M. fortuitum complex. M. farcinogenes and M. senegalense were indistinguishable in their 5"-end 16S rDNA but showed both considerable interspecies spacer sequence divergence and a high level of intraspecies sequence stability. A rapid detection assay using PCR and hybridization with species-specific probes was developed. The assay was specific among 46 species other than M. farcinogenes and M. senegalense and correctly identified all M. farcinogenes and M. senegalense strains. PCR- and 16S-23S rDNA sequence-based detection will be a valuable approach for diagnosis of the causal agents of African bovine farcy in cattle.     

Differentiation of two biovars of Ureaplasma urealyticum based on the 16S-23S rRNA intergenic spacer region

The 16S-23S rRNA intergenic spacer regions of 14 strains representing the 14 serovars of Ureaplasma urealyticum were amplified by PCR and sequenced for genetic differentiation between the two biovars Parvo and T960. Although the spacer region of the Parvo and T960 biovars comprised 302 nucleotides and lacked spacer tRNA genes, 15 nucleotides were different between the two biovars. The four nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 1, 3, 6, and 14 in the Parvo biovar were found to be identical. Similarly, the 10 nucleotide sequences of the 16S-23S rRNA intergenic spacer region of serovars 2, 4, 5, and 7 to 13 in the T960 biovar were found to be identical. The nucleotide sequence of the T960 biovar contains multiple restriction sites for restriction endonuclease SspI, which allows differentiation of the T960 biovar from the Parvo biovar.     

Efficient discrimination of Mycobacterium tuberculosis strains by 16S-23S spacer region-based random amplified polymorphic DNA analysis.

Amplification of the region separating the genes coding for 16S and 23S rRNA was performed with 15 Mycobacterium tuberculosis isolates and the type strain, ATCC 27294. Reproducible amplification patterns were obtained. PCR products were then used as target DNA for random amplified polymorphic DNA (RAPD) analysis. The discriminatory power was higher than when whole genomic DNA was used as a RAPD template. 16S-23S spacer region-based RAPD analysis was a simple and efficient method of differentiation. Consequently, it may be a useful tool for epidemiologic studies of tuberculosis.     

16S～23S rDNA间隔区序列PCR和RFLP分析对分枝杆菌复合菌群鉴定的研究

目的　对结核分枝杆菌复合菌群、鸟 胞内分枝杆菌复合菌群以及龟分枝杆菌龟亚种及脓肿亚种等几种快速生长分枝杆菌进行鉴定。方法　应用通用引物a及结核分枝杆菌特异的引物b ,对受试菌种的 16S～ 2 3SrDNA间隔区序列进行PCR扩增 ,并对引物a扩增产物进行限制性内切酶HaeⅢ、MSPⅠ消化反应。结果　应用引物aPCR扩增及RFLP分析能将除结核分枝杆菌复合菌群外的受试的其它菌群中的各菌种区别开 ;而引物b对受试各菌进行扩增 ,只有结核分枝杆菌有扩增条带出现 ,可将结核分枝杆菌与结核分枝杆菌复合菌群的其它菌区别开 ,达到菌种鉴定目的。结论　应用16S～ 2 3SrDNA间隔区序列PCR和RFLP分析能将结核分枝杆菌复合菌群等几组常规方法难鉴定的菌群加以区别。     

Identification of streptococcus mitis group species by RFLP of the PCR-amplified 16S-23S rDNA intergenic spacer

Mitis group streptococci are pioneer colonizers of tooth surfaces and are implicated in various pathologies. Thus, accurate identification of oral mitis group strains would be valuable for studies of plaque ecology and dental caries and for diagnostic use in endocarditis or sepsis patients. The aim of this study was to evaluate the usefulness of PCR-RFLP analysis of the 16S-23S intergenic spacer for differentiating and identifying streptococcus mitis group species. The 16S-23S rDNA spacer regions of 27 type and reference Streptococcus strains, representing 8 species, were studied by PCR-mediated amplification by using oligonucleotide primers FGPS 1490-72 and FGPL 132'-38. PCR products were digested, independently, with 14 restriction enzymes. Only AluI, MboI, CfoI, HinfI and MaeII distinguished some species, particularly AluI and CfoI, but not all the species. Eight clusters were clearly generated, corresponding to currently recognized species, but only with the addition of five ITS restriction patterns, generated by AluI + MboI + CfoI + HinfI + MaeII, then clustered by UPGMA, on a distance consensus matrix. The combination of these five ITS RFLP tests allowed a relatively conclusive genomic group differentiation of mitis group species. Despite this observation, more strains of each species will need to be analyzed, particularly clinical isolates, before arriving at general conclusions about the utility of ITS restrictions for identification of strains at the species level. An ITS PCR-RFLP-based identifying method for streptococcus mitis group species would provide significant advantages over other molecular taxonomic methods which require DNA extraction and DNA-DNA hybridization.     

rrn operons in Haemophilus parainfluenzae and mosaicism of conserved and species-specific sequences in the 16S-23S rDNA long spacer.

The mosaic organisation of short-sequence boxes was analysed in the cloned and sequenced long ribosomal spacer (547 bp) of Haemophilus parainfluenzae GR. Comparison and alignment of both the long and the short spacer were performed in H. parainfluenzae and H. influenzae Rd. The long spacer contained two tRNA genes (tRNA(Ala) and tRNA(Ile)) which are highly homologous to the corresponding genes found in the spacers of other species, such as Haemophilus spp., Actinobacillus spp., and Plesiomonas shigelloides. At the 3' end of tRNA(Ala) a putative ribosomal spacer loop was found, showing a strong secondary structure. Pulsed field gel electrophoresis (PFGE) analysis after restriction of the genome of H. parainfluenzae GR with I-Ceu I and subsequent polymerase chain reaction (PCR) analysis of PFGE-separated DNA fragments demonstrated that the H. parainfluenzae genome contained six operons and that the long spacer was present in three copies of them. Two short DNA segments were identified as being species-specific, allowing us to design PCR primers which were useful in the molecular identification of H. parainfluenzae isolates.     

Molecular typing of papillomatous digital dermatitis-associated Treponema isolates based on analysis of 16S-23S ribosomal DNA intergenic spacer regions

Papillomatous digital dermatitis (PDD), an emerging infectious disease of cattle, is characterized by painful, ulcerative foot lesions. The detection of high numbers of invasive spirochetes in PDD lesions suggests an important role for these organisms in the pathogenesis of PDD. PDD-associated spirochetes have phenotypic characteristics consistent with members of the genus TREPONEMA: Partial 16S ribosomal DNA (rDNA) sequence analysis of clonal isolates from California cattle showed that they comprise three phylotypes which cluster closely with human-associated Treponema spp. of the oral cavity (T. denticola and T. medium/T. vincentii) or genital area (T. phagedenis). The goal of our study was to apply 16S-23S rDNA intergenic spacer region (ISR) sequence analysis to the molecular typing of U.S. PDD-associated Treponema isolates. This methodology has potentially greater discriminatory power for differentiation of closely related bacteria than 16S rDNA analysis. We PCR amplified, cloned, and sequenced the ISRs from six California PDD-associated Treponema isolates and, for comparative purposes, one strain each of T. denticola, T. medium, T. vincentii, and T. phagedenis. Two ISRs that varied in length and composition were present in all the PDD-associated Treponema isolates and in T. denticola, T. medium, and T. phagedenis. ISR1 contained a tRNA(Ala) gene, while ISR2 contained a tRNA(Ile) gene. Only a single ISR (ISR1) was identified in T. vincentii. Comparative analyses of the ISR1 and ISR2 sequences indicated that the California PDD-associated Treponema isolates comprised three phylotypes, in agreement with the results of 16S rDNA analysis. PCR amplification of the 16S-tRNA(Ile) region of ISR2 permitted rapid phylotyping of California and Iowa PDD-associated Treponema isolates based on product length polymorphisms.     

Population diversity of Staphylococcus intermedius isolates from various host species: typing by 16S-23S intergenic ribosomal DNA spacer polymorphism analysis

Twelve 16S-23S ribosomal DNA intergenic spacer (ITS-PCR) types were identified among 57 Staphylococcus intermedius isolates from humans and other animals. Six ITS-PCR types were host specific, and most human and canine strains belonged to the same types (A and J). Pigeon, horse, and mink strains appeared more heterogeneous.     

Identification of three clinically relevant Borrelia burgdorferi sensu lato genospecies by PCR-restriction fragment length polymorphism analysis of 16S-23S ribosomal DNA spacer amplicons

We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.     

Molecular typing of Mycobacterium avium isolates by sequencing of the 16S-23S rDNA internal transcribed spacer and comparison with IS1245-based fingerprinting

The nucleotide sequence of the variable 16S-23S rDNA internal transcribed spacer (ITS) was determined in 32 strains of Mycobacterium avium, including 29 clinical isolates, two environmental isolates and the reference strain M. avium ATCC 35712. The results were compared with those obtained by the IS1245-based restriction fragment length polymorphism (RFLP) assay. The strains belonged to three distinct ITS sequevars, Mav-A, Mav-B and Mav-C. Sixteen of 17 isolates of the Mav-B sequevar were from HIV-positive patients; the Mav-A sequevar included six and five isolates from HIV-positive and HIV-negative individuals, respectively, as well as the two environmental isolates and the M. avium reference strain ATCC 35712; only one isolate, from a HIV-infected patient, belonged to the Mav-C sequevar. IS1245-RFLP analysis of M. avium isolates of sequevars Mav-A and Mav-B showed that isolates occurring in clusters of identical or highly related RFLP patterns generally belong to the same sequevar, and that M. avium strains belonging to the same sequevar may present distinct and unrelated IS1245-RFLP patterns. The question of the molecular markers specific for M. avium clones pathogenic for man is discussed.     

Analysis of the 16S-23S rRNA gene internal transcribed spacer region in Klebsiella species

The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types—ITS^none (without tRNA genes), ITS^glu [with a tRNA^{Glu (UUC)} gene], and ITS^ile+ala [with tRNA^{Ile (GAU)} and tRNA^{Ala (UGC)} genes]—were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITS^glu and ITS^ile+ala. The presence of ITS^none in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITS^none, from 0.967 to 1.000 for ITS^glu, and from 0.968 to 1.000 for ITS^ile+ala. Interspecies sequence identities range from 0.775 to 0.989 for ITS^none, from 0.798 to 0.997 for ITS^glu, and from 0.712 to 0.985 for ITS^ile+ala. Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.     

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.     

Analysis of the 16S to 23S rRNA intergenic spacer region of Mycoplasma synoviae field strains

Mycoplasma synoviae has been associated with economic loss in the chicken and turkey industries. The molecular characterization of M. synoviae at strain level allows the analysis of relationships between strains that may be valuable in epidemiological investigations. In the present study, the intergenic spacer region (ISR) between the 16S and 23S rRNA genes was examined to see whether useful information about strains could be derived. M. synoviae has two copies of this region, which may not be exactly the same (intercistronic heterogeneity). Sequencing of the ISRs of 21 M. synoviae isolates and the type strain revealed that 19 of them had such heterogeneity so DNA cloning was performed where necessary. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and a dendrogram was constructed. The length of the ISRs varied between 305 and 309 base pairs. Apart from having extra A/Ts in poly-A or poly-T regions and the presence of a few polymorphisms, the sequences of the M. synoviae strains were similar. Based on phylogenetic analysis, the strains were assigned to 10 groups—taking into account that within each group the DNA similarity was 100%, while the lowest similarity between groups was 95.8%. The results were compared with those obtained with the vlhA gene, resulting in very similar M. synoviae groups. Although the ISR could be a good target for strain typing, as has been shown by others for Mycoplasma gallisepticum, the method may be too cumbersome for routine use with M. synoviae because of complications with intercistronic heterogeneity. However, if the ISR sequence information was to be combined with other mutation detection techniques it could increase the discriminatory power.     

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.     

Association between 16S-23S internal transcribed spacer sequence groups of Mycobacterium avium complex and pulmonary disease

Organisms within the Mycobacterium avium complex (MAC) may have differential virulence. We compared 33 subjects with MAC pulmonary disease to 75 subjects with a single positive culture without disease. M. avium isolates were significantly more likely to be associated with MAC pulmonary disease (odds ratio = 5.14, 95% confidence interval = 1.25 to 22.73) than M. intracellulare.     

Identification of Acinetobacter clinical isolates by polymerase chain reaction analysis of 16S-23S ribosomal ribonucleic acid internal transcribed spacer

Background: The genus Acinetobacter is a diverse group of Gram-negative bacteria involve at least 33 species using the molecular methods. Although the genus Acinetobacter comprises a number of definite bacterial species, some of these species are of clinical importance. Therefore, it is of vital importance to use a method which is able to reliably and efficiently differentiate the numerous Acinetobacter species. Objectives: This study aims to identify Acinetobacter of clinical isolates from Assir region to the species level by 16S-23S intergenic spacers internal transcribed spacer (ITS) of ribosomal ribonucleic acid (rRNA). Materials and Methods: Deoxyribonucleic acid extraction, polymerase chain reaction amplification of 16S-23S intergenic spacer sequences (ITS) was performed using the bacterium-specific universal primers. Results: Based on the 16S-23S intergenic spacers (ITS) of rRNA sequences, all isolates tested were identified as Acinetobacter baumannii. The isolates shared a common ancestral lineage with the prototypes A. baumannii U60279 and U60280 with 99% sequence similarities. Conclusion: These findings confirmed 16S-23S rRNA ITS for the identification of A. baumannii of different genotypes among patients.     

PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level

The genus Acinetobacter is phenotypically rather homogeneous, but genotypicaliy heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplrfied large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypicaliy related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.