Rapid Identification of Clinically Relevant Nocardia Species to Genus Level by 16S rRNA Gene PCR

Two regions of the gene coding for 16S rRNA in Nocardia species were selected as genus-specific primer sequences for a PCR assay. The PCR protocol was tested with 60 strains of clinically relevant Nocardia isolates and type strains. It gave positive results for all strains tested. Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including Dietzia, Gordona, Mycobacterium, Rhodococcus, Streptomyces, and Tsukamurella. Besides, unlike the latter group of isolates, all Nocardia strains exhibited one MlnI recognition site but no SacI restriction site. This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of Nocardia spp. isolated from pathogenic samples.     

Application of SmartGene IDNS software to partial 16S rRNA gene sequences for a diverse group of bacteria in a clinical laboratory

Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bacterial clinical isolates was compared with gene sequences interrogated by the SmartGene and MicroSeq databases. Of 300 isolates, SmartGene identified 295 (98%) to the genus level and 262 (87%) to the species level, with 5 (2%) being inconclusive. MicroSeq identified 271 (90%) to the genus level and 223 (74%) to the species level, with 29 (10%) being inconclusive. SmartGene and MicroSeq agreed on the genus for 233 (78%) isolates and the species for 212 (71%) isolates. Conventional methods identified 291 (97%) isolates to the genus level and 208 (69%) to the species level, with 9 (3%) being inconclusive. SmartGene, MicroSeq, and conventional identifications agreed for 193 (64%) of the results. Twenty-seven microorganisms were not represented in MicroSeq, compared to only 2 not represented in SmartGene. Overall, SmartGene IDNS provides comprehensive and accurate identification of a diverse group of bacteria and has the added benefit of being a user-friendly program that can be modified to meet the unique needs of clinical laboratories.     

Determination of 16S rRNA Sequences of Enterococci and Application to Species Identification of Nonmotile Enterococcus gallinarum Isolates

The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.     

Identification of Fusarium Species Involved in Human Infections by 28S rRNA Gene Sequencing

Fusarium spp. have emerged as major opportunistic fungal agents. Since new antifungal agents exhibit variable activity against Fusarium isolates depending on the species, rapid identification at the species level is required. Conventional culture methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33 Fusarium isolates belonging to six Fusarium species associated with human infections. The data were then analyzed by the neighbor-joining method. By using distance matrix analysis and constructing the phylogram, we could easily distinguish the different species for all but one isolate. The method also allowed differentiation between the closely related genera Acremonium and Cylindrocarpon. In contrast to the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of Fusarium species isolated from patients.     

Description of an Unusual Neisseria meningitidis Isolate Containing and Expressing Neisseria gonorrhoeae-Specific 16S rRNA Gene Sequences

An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.     

secA1 Gene Sequence Polymorphisms for Species Identification of Nocardia Species and Recognition of Intraspecies Genetic Diversity

Sequence analysis of the Nocardia essential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5′-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica), Nocardia elegans (Nocardia africana), and Nocardia transvalensis (Nocardia blacklockiae); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana (n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.Nocardia spp. are Gram-positive saprophytic bacteria capable of causing suppurative infections, including pulmonary, cutaneous, central nervous system, and disseminated diseases. To date, approximately 90 species have been described (NCBI taxonomy for Nocardia [http://www.ncbi.nlm.nih.gov/Taxonomy/]; http://www.bacterio.cict.fr/n/nocardia.html), at least 33 of which have been implicated in human disease (2). Identification of clinical isolates to the species level is important to characterize associated disease manifestations and to predict antimicrobial susceptibility and for epidemiological and ecological purposes (2, 17).Because of the difficulty of identifying Nocardia isolates by standard phenotypically based methods and the inability of such methods to identify novel species (2, 17), various nucleic acid amplification methods targeting conserved Nocardia gene regions have been proposed to provide accurate species determination. Of these, sequence analysis of the 16S rRNA gene has become the gold standard for definitive species identification (2, 5, 6, 8, 19). Certain closely related species, however, may not be distinguished by this method due to insufficient interspecies polymorphisms within the 16S rRNA gene sequences (2, 5, 14). Other practical limitations include potential misidentifications as a result of multiple but different copies of the 16S rRNA gene in species such as Nocardia nova (7, 9) and/or the presence of intraspecies 16S rRNA gene sequence polymorphisms (or “sequence types” [STs]) in N. nova, Nocardia cyriacigeorgica, and other species (14, 21).As such, the continuing evaluation of alternate gene targets to facilitate species identification is important. Sequence polymorphisms within the Nocardia 65-kDa heat shock protein (hsp65), essential secretory protein SecA1 (secA1), gyrase B (gyrB), and 16S-23S rRNA intergenic spacer (ITS) region genes have been reported to enable species level identification (10, 18, 22-24). In particular, sequence variability within a portion (470 bp) of the secA1 gene locus (in conjunction with analysis of deduced amino acid sequences of the SecA1 protein) has shown promise in recognizing and discriminating between the major Nocardia spp. (10). However, data on the application of secA1 gene sequencing in the clinical microbiology laboratory for the identification of Nocardia isolates are few. In one report, reference (n = 30 species), and clinical Nocardia isolates were correctly identified by secA1 gene sequencing (10); in the only other published study, this approach assisted with identification of a novel Nocardia species from soil (16). Evaluation of larger numbers of clinical isolates is essential for establishing a robust repository of secA1 gene sequences.Our laboratory, which provides regional microbiology services to a large number of health care institutions, has undertaken routine species identification by partial (5′-end 606-bp) 16S rRNA gene sequencing of Nocardia isolates since 2005. In the course of evaluating this approach to providing species identification, we identified significant intraspecies sequence heterogeneity within certain species, such as N. nova and Nocardia brasiliensis (14), highlighting the need to recognize species-specific sequence-based genetic types, or sequence types. Here, to explore the potential of sequence analysis of the secA1 gene as an adjunct to, or a possible substitute for, 16S rRNA gene sequencing, we performed species identification of 120 Nocardia reference and clinical isolates representing the 16 most clinically relevant species by secA1 gene sequence analysis and compared the results with 5′-end 606-bp 16S rRNA gene sequencing. We also report on the genetic diversity of the Nocardia secA1 gene.     

Differentiation of Burkholderia Species by PCR-Restriction Fragment Length Polymorphism Analysis of the 16S rRNA Gene and Application to Cystic Fibrosis Isolates

Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (B. multivorans), III, and IV and B. vietnamiensis. B. gladioli is also involved, though rarely, in CF. Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiate Burkholderia species. This method was applied to 16 type and reference strains of the genus Burkholderia and to 51 presumed B. cepacia clinical isolates, each representative of one clone previously determined by PCR ribotyping. The 12 Burkholderia type strains tested were differentiated, including B. cepacia, B. multivorans, B. vietnamiensis, and B. gladioli, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and B. pyrrociniaT were distinguishable. CF clinical isolates were mainly distributed in RFLP group 2 (which includes B. multivoransT) and RFLP group 1 (which includes B. cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1. The remaining isolates either clustered with other Burkholderia species (B. cepacia genomovar IV or B. pyrrocinia, B. vietnamiensis, and B. gladioli) or harbored unique combinations of patterns. Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each Burkholderia species or genomovar in patients with CF.     

Comparison of MALDI-TOF MS,Housekeeping Gene Sequencing,and 16S rRNA Gene Sequencing for Identification of Aeromonas Clinical Isolates

PurposeThe genus Aeromonas is a pathogen that is well known to cause severe clinical illnesses, ranging from gastroenteritis to sepsis. Accurate identification of A. hydrophila, A. caviae, and A. veronii is important for the care of patients. However, species identification remains difficult using conventional methods. The aim of this study was to compare the accuracy of different methods of identifying Aeromonas at the species level: a biochemical method, matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS), 16S rRNA sequencing, and housekeeping gene sequencing (gyrB, rpoB).ResultsThe conventional biochemical method and 16S rRNA sequencing identified Aeromonas at the genus level very accurately, although species level identification was unsatisfactory. MALDI-TOF MS system correctly identified 60 (92.3%) isolates at the species level and an additional four (6.2%) at the genus level. Overall, housekeeping gene sequencing with phylogenetic analysis was found to be the most accurate in identifying Aeromonas at the species level.ConclusionThe most accurate method of identification of Aeromonas to species level is by housekeeping gene sequencing, although high cost and technical difficulty hinder its usage in clinical settings. An easy-to-use identification method is needed for clinical laboratories, for which MALDI-TOF MS could be a strong candidate.     

Multiple copies of the 16S rRNA gene in Nocardia nova isolates and implications for sequence-based identification procedures

Molecular investigation of two Nocardia patient isolates showed unusual restriction fragment length polymorphism patterns with restriction endonuclease assays (REA) using an amplified portion of the 16S rRNA gene. Patterns typical of Nocardia nova were obtained with REA of an amplified portion of the 65-kDa heat shock protein gene. Subsequent sequence analysis of the 16S rRNA gene regions of these isolates showed the presence of ambiguous bases within an expected restriction endonuclease recognition site which were not able to be resolved on repeat testing. Cloning of amplified regions of the 16S rRNA genes and subsequent sequencing of the resulting clones from the two patient isolates showed three different 16S rRNA gene sequences which corresponded to sequences found in N. nova, a molecular variant of N. nova, and a previously undescribed sequence. Hybridization studies using a DNA probe corresponding to an 89-bp conserved region of the 16S rRNA gene confirmed the presence of at least two copies of the 16S rRNA gene in the N. nova type strain, in a patient isolate identical to the molecular variant of N. nova, and in the two other patient isolates. All isolates were found to belong to the species N. nova as determined by DNA-DNA hybridization. Because minimal variation has been found in the 16S rRNA gene sequences of different species of Nocardia, those laboratories employing molecular methods for identification of these species must be aware of the potential identification complications that may be caused by the presence of differing 16S rRNA genes in the same isolate.     

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.     

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nocardia Species

The identification of Nocardia species, usually based on biochemical tests together with phenotypic in vitro susceptibility and resistance patterns, is a difficult and lengthy process owing to the slow growth and limited reactivity of these bacteria. In this study, a panel of 153 clinical and reference strains of Nocardia spp., altogether representing 19 different species, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). As reference methods for species identification, full-length 16S rRNA gene sequencing and phenotypical biochemical and enzymatic tests were used. In a first step, a complementary homemade reference database was established by the analysis of 110 Nocardia isolates (pretreated with 30 min of boiling and extraction) in the MALDI BioTyper software according to the manufacturer''s recommendations for microflex measurement (Bruker Daltonik GmbH, Leipzig, Germany), generating a dendrogram with species-specific cluster patterns. In a second step, the MALDI BioTyper database and the generated database were challenged with 43 blind-coded clinical isolates of Nocardia spp. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable identification to the species level for five species of which more than a single isolate was analyzed. Correct identification was achieved for 38 of the 43 isolates (88%), including 34 strains identified to the species level and 4 strains identified to the genus level according to the manufacturer''s log score specifications. These data suggest that MALDI-TOF MS has potential for use as a rapid (<1 h) and reliable method for the identification of Nocardia species without any substantial costs for consumables.Nocardia spp. are ubiquitous bacteria dispersed in vegetation, dust, soil, freshwater, and salt water that are isolated with increasing frequency from clinical specimens, especially those from immunocompromised patients (1).The taxonomy of the genus Nocardia has undergone major changes during the last decades, and currently more than 50 species have been characterized by phenotypic and molecular methods, besides a number of unnamed genomospecies (2). Not all of them have been found in humans, and Nocardia asteroides, previously considered the species most frequently isolated from clinical specimens, has been shown to be heterogeneous and has been divided into several species (2). Moreover, several additional species of human origin have been recently described and reported (5, 6).The routine identification of Nocardia strains to the species level by conventional phenotypical methods is a fastidious and time-consuming process owing to the limited biochemical reactivity of these organisms, often requiring 1 or more days to complete identification. Moreover, the available tests may be difficult to interpret and inconclusive and require dedicated trained staff. In order to overcome these drawbacks, molecular methods such as 16S rRNA gene sequencing and PCR-restriction fragment length polymorphism analysis of both the 65-kDa heat shock protein-encoding gene (hsp65) and the 16S rRNA gene have been recently advocated for Nocardia species identification (3). However, these methods remain accessible to reference laboratories only and are difficult to implement for routine bacterial identification in a clinical laboratory.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can analyze the protein composition of a bacterial cell and has emerged as a new technology for species identification. By measuring the exact sizes of peptides and small proteins, which are assumed to be characteristic for each bacterial species, it allows determination to the species level within a few minutes when the analysis is performed on whole cells, cell lysates, or crude bacterial extracts (8). Through the improvement of the technique, MALDI-TOF MS has proved over the recent years to be a rapid, accurate, easy-to-use, and inexpensive method for the universal identification of microorganisms (11). Until now, MALDI-TOF MS has been challenged for the identification of various groups of microorganisms, including Gram-positive bacteria, Enterobacteriaceae, Gram-negative nonfermenters, mycobacteria, anaerobes, and yeasts (8, 9, 11-13). In this respect, the use of MALDI-TOF MS as a tool for the identification of fastidious, slow-growing organisms such as Nocardia species, which are notoriously difficult to identify by conventional tests, in the routine laboratory appeared to us of major interest. One factor limiting the use of MALDI-TOF MS remains the limited availability of reference data sets for microorganisms that are infrequently isolated from clinical specimens, and it has been shown previously that the absence or the availability of only a small number of isolates of a given species in the reference database may account for most of the cases in which no identification can be obtained by the MALDI-TOF MS method (11).In this study, we therefore aimed to establish a large reference database for the MALDI-TOF MS-based identification of Nocardia species isolates. In a first step, we developed a simple modified extraction procedure based on boiling for 30 min, followed by ethanol-formic acid extraction, and we generated our own spectrum database issued from a large collection of clinical and reference Nocardia sp. isolates. Following the establishment of our reference database, we subsequently evaluated the methodology against 43 blind-coded clinical isolates of Nocardia species that were analyzed by phenotypical, biochemical, and enzymatic tests and by full-length 16S rRNA gene sequencing, which was used as the reference identification method.     

Phylogeny and Identification of Nocardia Species on the Basis of Multilocus Sequence Analysis

Nocardia species identification is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA and cellular fatty acid analysis to discriminate many species, and the unreliability of biochemical testing. Here, Nocardia species identification was achieved through multilocus sequence analysis (MLSA) of gyrase B of the β subunit of DNA topoisomerase (gyrB), 16S rRNA (16S), subunit A of SecA preprotein translocase (secA1), the 65-kDa heat shock protein (hsp65), and RNA polymerase (rpoB) applied to 190 clinical, 36 type, and 11 reference strains. Phylogenetic analysis resolved 30 sequence clusters with high (>85%) bootstrap support. Since most clusters contained a single type strain and the analysis corroborated current knowledge of Nocardia taxonomy, the sequence clusters were equated with species clusters and MLSA was deemed appropriate for species identification. By comparison, single-locus analysis was inadequate because it failed to resolve species clusters, partly due to the presence of foreign alleles in 22.1% of isolates. While MLSA identified the species of the majority (71.3%) of strains, it also identified clusters that may correspond to new species. The correlation of the identities by MLSA with those determined on the basis of microscopic examination, biochemical testing, and fatty acid analysis was 95%; however, MLSA was more discriminatory. Nocardia cyriacigeorgica (21.58%) and N. farcinica (14.74%) were the most frequently encountered species among clinical isolates. In summary, five-locus MLSA is a reliable method of elucidating taxonomic data to inform Nocardia species identification; however, three-locus (gyrB-16S-secA1) or four-locus (gyrB-16S-secA1-hsp65) MLSA was nearly as reliable, correctly identifying 98.5% and 99.5% of isolates, respectively, and would be more feasible for routine use in a clinical reference microbiology laboratory.As part of the aerobic actinomycetes, Nocardia is a group of filamentous branching bacilli that are characteristically Gram positive and modified acid fast. Although Nocardia species normally exist as soil saprophytes, they have increasingly been isolated as infectious agents in immunosuppressed patients and, in some cases, even healthy individuals. Infections range from pulmonary nocardiosis, characterized by necrotizing pneumonia, to cutaneous nocardiosis and even brain abscess (25).For nearly a century, since its inception in 1888 by Edmund Nocard, the genus Nocardia comprised only about a dozen species (26), largely because the somewhat biochemically inert nature of the group inhibited characterization (6). However, in 1988, Wallace et al. (38) uncovered latent diversity when they described six antimicrobial susceptibility pattern types among clinical isolates. DNA (e.g., 16S rRNA [16S] gene) sequencing confirmed and further expanded knowledge of the genetic diversity within the genus (6, 22). To date, the National Center for Biotechnology Information (NCBI) lists 86 recognized species (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi). However, the species differ in their abilities to cause human infection and their responses to antimicrobials (2, 6, 21, 25, 27, 33, 38). For this reason, species identification of Nocardia isolates from clinical specimens is relevant to patient treatment and provides important epidemiological information.Beyond Gram and modified-acid-fast staining, species identification of Nocardia relies heavily on biochemical tests and cellular fatty acid analysis, which are cumbersome, time-consuming, and not definitive. Various molecular identification schemes investigated to date represent promising alternatives (7, 10, 29, 32, 36). However, 16S rRNA gene sequencing, considered to be the “gold standard” for bacterial identification, fails to discriminate many species (7), and the reliability of identification methods on the basis of the DNA sequence of a single housekeeping gene suffers from stochastic genetic variation and horizontal gene transfer and recombination (12).Recently, multilocus sequence analysis (MLSA) has been suggested as a method to examine prokaryotic taxonomy. From phylogenetic analysis of a concatenated sequence typically consisting of 5 to 7 housekeeping genes, MLSA assigns a species designation on the basis of the assumption that sequence clusters represent species clusters (12). MLSA has been employed to identify the species of a number of genera with very promising results (1, 4, 5, 11, 14, 15, 16, 18, 20, 24, 28, 40). Furthermore, because of its ease of use, accuracy, and discriminatory power, MLSA may soon surpass DNA-DNA hybridization (DDH) as the gold standard for the investigation of prokaryotic taxonomy, species identification, and determination of genetic diversity (34).The purpose of this study was to develop an MLSA scheme for the species identification of Nocardia clinical isolates. Through phylogenetic analysis of concatenated sequences consisting of partial fragments of gyrase B, the β subunit of a type II DNA topoisomerase (gyrB), 16S, subunit A of the SecA preprotein translocase (secA1), the 65-kDa heat shock protein (hsp65), and RNA polymerase (rpoB) genes, we delineated 30 species clusters. Since most clusters contained a single type strain, the results correlated well with existing knowledge of Nocardia taxonomy and provided a means of species assignment for the clinical isolates on the basis of strain placement within the phylogenetic analysis. Furthermore, the MLSA identifications were consistent with, although more discriminatory than, species assignments based on traditional microscopic evaluation, biochemical testing, and cellular fatty acid analysis. We present MLSA as a practical tool for routine Nocardia species identification in a clinical reference microbiology laboratory.     

Analysis of the 16S rRNA Gene Sequence of Anaplasma centrale and Its Phylogenetic Relatedness to Other Ehrlichiae

The nucleotide sequence of the Anaplasma centrale 16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence of A. centrale was closely related to Anaplasma marginale by both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detect A. centrale DNA extracted from 10 infected bovine red blood cells in a reaction mixture. A. centrale DNA was amplified in the reaction, but not other related ehrlichial species.     

Detection of Kanamycin-Resistant Mycobacterium tuberculosis by Identifying Mutations in the 16S rRNA Gene

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, 200 μg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.     

Evaluation of 16S rRNA gene-based PCR assays for genus-level identification of Helicobacter species

The inclusivity, exclusivity, and detection limit of six 16S rRNA gene-based Helicobacter genus-specific PCR assays were examined. Five out of six assays were 100% inclusive, but the tests varied considerably in their exclusivity (9.1 to 95.5%). The clinical detection limit varied between 10(3) and 1 viable bacterial cell per reaction mixture.     

Identification by 16S rRNA Gene Sequencing of an Actinomyces hongkongensis Isolate Recovered from a Patient with Pelvic Actinomycosis

A case of Actinomyces hongkongensis pelvic actinomycosis in an adult woman is described. Conventional phenotypic tests failed to identify the Gram-positive bacillus isolated from a fluid aspirate of a pelvic abscess. The bacterium was identified by 16S rRNA gene sequencing and analysis using the SmartGene Integrated Database Network System software.     

Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene.

Chlamydia pneumoniae is an important cause of respiratory disease in humans, but diagnosis of C. pneumoniae is hindered by difficulties in the in vitro growth of the organism. In order to improve detection and identification, we recently developed a polymerase chain reaction (PCR) assay which uses oligonucleotide primers specific for C. pneumoniae. The nucleic acid sequence was determined for the 16S rRNA of C. pneumoniae, and regions in which C. pneumoniae differed from both Chlamydia psittaci and Chlamydia trachomatis were identified. Oligonucleotide primers corresponding to these unique regions were then synthesized and used in a PCR for the detection of C. pneumoniae. The C. pneumoniae-specific primers permitted the identification of six isolates of C. pneumoniae, but no reaction was observed with the 15 serovars of C. trachomatis or two strains of C. psittaci. PCR should prove to be valuable in confirming the identification of C. pneumoniae and in the diagnosis of C. pneumoniae infections.