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.     

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.     

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.     

Use of PCR and reverse line blot hybridization macroarray based on 16S-23S rRNA gene internal transcribed spacer sequences for rapid identification of 34 mycobacterium species

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.     

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.     

A new PCR primer for the identification of Paracoccidioides brasiliensis based on rRNA sequences coding the internal transcribed spacers (ITS) and 5 x 8S regions.

Internal transcribed spacer (ITS) genes including the 5.8S ribosomal (r)RNA of Paracoccidioides brasiliensis were amplified and the DNA sequences were determined. Based on a comparison of the sequence information, a new polymerase chain reaction (PCR) primer pair was designed for specific amplification of DNA for P. brasiliensis. This primer pair amplified a 418-bp DNA sequence and was 100% successful in identifying 29 strains of P. brasiliensis (including the reference strains) isolated from the regions of Brazil, Costa Rica, Japan, Argentina or from different sources. The results of specificity tests of these primers to compare the fungus with those of Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei are also reported.     

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.     

Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes

A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.     

Genetic diversity of the internal transcribed spacers (ITS) and 5.8S rRNA genes among the clinical isolates of Candida parapsilosis in Brazil and Japan.

The internal transcribed spacer (ITS) region including 5.8S rDNA sequences of 58 isolates of Candida parapsilosis in Brazil and Japan was analyzed. Although most of the C. parapsilosis strains tested were confirmed to belong to three already reported genetically distinct groups (I, II and III) based on their ITS region sequences, 5 strains of the Brazilian isolates showed different sequences from those heretofore reported and suggested a presence of new genotype. For these strains of C. parapsilosis, we proposed a new genetic group (IV). The sequence similarities of this new group of IV to I, II and III were 87.4%, 94.7% and 87.3% in the ITS1 region, respectively. Genetic diversity in ITS regions of the remaining C. parapsilosis strains in Brazil and Japan was also discussed.     

Typing of Pneumocystis carinii strains with type-specific oligonucleotide probes derived from nucleotide sequences of internal transcribed spacers of rRNA genes.

We have recently developed a method for typing Pneumocystis carinii strains that infect humans. The method takes advantage of nucleotide sequence variations in internal transcribed spacers (ITSs) of the rRNA genes of P. carinii. To date, two types of nucleotide sequences (designated types A and B) have been found in the ITS1 region, and three types of nucleotide sequences (designated types a, b and c) have been found in the ITS2 region. Of the six potential combination types, we have detected four, designated types Ac, Bb, Ba, and Bc. To simplify typing, we have designed five oligonucleotide probes, probes 1-A, 1-B, 2-a, 2-b, and 2-c, which are specific to ITS1 type A and type B and ITS2 type a, type b, and type c, respectively, of P. carinii strains that infect humans. We also have designed an oligonucleotide which reacts specifically with P. carinii strains that infect rats. The ITS region were amplified by PCR, and the PCR products were then probed with these type-specific oligonucleotide probes. Typing with the type-specific oligonucleotide probes was found to be effective with specimens containing only one type of P. carinii. These methods are rapid and simple to perform and will be useful for studying the epidemiology of P. carinii infections.     

Potential limitations of the 16S-23S rRNA intergenic region for molecular detection of Bartonella species

PCR targeting the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region has been proposed as a rapid and reliable method for the detection of Bartonella species DNA in clinical samples. Because of variation in ITS sequences among Bartonella species, a single PCR amplification can be used to detect different species within this genus. Therefore, by targeting the ITS region, multiple PCRs or additional sample-processing steps beyond the primary amplification can be avoided when attempting to achieve molecular diagnostic detection of Bartonella species. Although PCR amplification targeting this region is considered highly sensitive, amplification specificity obviously depends on primer design. We report evidence of nonspecific PCR amplification of Mesorhizobium species with previously published primers that were designed to amplify the Bartonella consensus ITS region. Use of these or other, less species-specific, primers could lead to a false-positive diagnostic test result when evaluating clinical samples. We also report the presence of Mesorhizobium species DNA as a contaminant in molecular-grade water, a series of homologous sequences in the ITS region that are common to Bartonella and Mesorhizobium species, the amplification of Mesorhizobium DNA with unpublished primers designed in our laboratory targeting the ITS region, and the subsequent design of unambiguous ITS primers that avoid nonspecific amplification of Mesorhizobium species. Our results define some potential limitations associated with the molecular detection of Bartonella species in patient samples and indicate that primer specificity is of critical importance if the ITS region is used as a diagnostic target for detection of Bartonella species.     

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.     

Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA.

Previous genetic analysis of Haemophilus influenzae strains isolated from genital and neonatal infections identified a group of biotype IV that constitutes a cryptic genospecies only distantly related to H. influenzae and H. Haemolyticus. Small-subunit rRNA genes of two representative strains of this genital Haemophilus genospecies (strains 16N and 2406) were sequenced. The analysis indicated that these strains form a monophyletic unit with H. haemolyticus and H. influenzae biogroups Influenzae and Aegyptius and are more closely related to H. haemolyticus than to H. influenzae biogroups Influenzae and Aegyptius. 16S rRNA gene sequences were used to formulate primers for PCR-based identification of cryptic genital Haemophilus organisms. A 242-bp fragment was amplified from strains belonging to the genital Haemophilus genospecies but not from strains of 12 other Haemophilus species, including strains of H. influenzae biotype IV sensu stricto.     

Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer rRNA genes

Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (kappa, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples.     

Development of Specific Nested Oligonucleotide PCR Primers for the Streptococcus iniae 16S-23S Ribosomal DNA Intergenic Spacer

Streptococcus iniae is a cause of septicemia, meningoencephalitis, and death in farmed fish and of cellulitis in human beings. A set of nested oligonucleotide PCR primers that specifically amplified a 373-bp subunit from a variety of clinical isolates from farmed fish and human patients were constructed from a 524-bp consensus sequence of the S. iniae 16S-23S ribosomal DNA intergenic spacer.     

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.     

PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.

A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.