Classification of Leptospira genomospecies 1, 3, 4 and 5 as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively.

The genus Leptospira currently comprises 16 named species. In addition, four unnamed hybridization groups were designated Leptospira genomospecies 1, 3, 4 and 5. These groups represent valid species-level taxa, but were not assigned names in the original description by Brenner et al. [Int J Syst Bacteriol 49, 839-858 (1999)]. To rectify this situation, it is proposed that Leptospira genomospecies 1, genomospecies 3, genomospecies 4 and genomospecies 5 should be classified as Leptospira alstonii sp. nov., Leptospira vanthielii sp. nov., Leptospira terpstrae sp. nov. and Leptospira yanagawae sp. nov., respectively, with strains L. alstonii 79601(T) ( = ATCC BAA-2439(T)), L. vanthielii WaZ Holland(T) ( = ATCC 700522(T)), L. terpstrae LT 11-33(T) ( = ATCC 700639(T)) and L. yanagawae Sao Paulo(T) ( = ATCC 700523(T)) as the type strains. The type strains are also available from the culture collections of the WHO Collaborating Centres in Amsterdam, The Netherlands, and Brisbane, Australia.

Mycobacterium parascrofulaceum sp. nov., novel slowly growing, scotochromogenic clinical isolates related to Mycobacterium simiae.

A group of pigmented, slowly growing mycobacteria identified by 16S rRNA gene sequencing as 'MCRO 33' (GenBank accession no. AF152559) have been isolated from several clinical specimens in various laboratories across Canada. Genotypically, the organism is most closely related to Mycobacterium simiae. However, it presents with a similar phenotypic profile to Mycobacterium scrofulaceum. Several reference strains obtained from ATCC and TMC culture collections, previously identified as M. scrofulaceum or M. simiae, have also been found to possess the MCRO 33 16S rRNA gene sequence. Biochemical testing, susceptibility testing, HPLC, hsp65 gene and 16S-23S spacer (ITS1) sequencing were performed on clinical and reference strains to characterize further this unique species. Of the clinical strains, one was isolated from a cervix biopsy whereas all other clinical isolates were obtained from respiratory samples. In one patient, symptoms, imaging and repeat clinical specimens positive on culture for this organism were suggestive of active clinical disease. The description of this species, for which the name Mycobacterium parascrofulaceum sp. nov. is proposed, follows the present trend of a large number of novel Mycobacterium species identified due in great part to sequence-based methods. The type strain is HSC68T (= ATCC BAA-614T = DSM 44648T).

Mycobacterium saskatchewanense sp. nov., a novel slowly growing scotochromogenic species from human clinical isolates related to Mycobacterium interjectum and Accuprobe-positive for Mycobacterium avium complex.

A pigmented, slowly growing Mycobacterium avium complex AccuProbe-positive organism was isolated from the sputum and pleural fluid of a 72-year-old female with bronchiectasis. The unusual morphology of the organism prompted further identification by 16S rRNA gene sequencing, revealing a perfect identity with previously uncharacterized strain Mycobacterium sp. MCRO 8 (GenBank accession no. X93034), with the closest established species by 16S rDNA analysis being Mycobacterium interjectum. HPLC of the organism corresponded to previously obtained patterns identified as M. interjectum-like and, upon sequence evaluation of a selection of strains with a similar profile, more were subsequently identified as MCRO 8. A total of 16 strains isolated from human respiratory samples were evaluated in the characterization of this novel species, for which the name Mycobacterium saskatchewanense sp. nov. is proposed. The type strain is strain 00-250(T) (=ATCC BAA-544(T)=DSM 44616(T)=CIP 108114(T)).

Reassessment of sequence-based targets for identification of bacillus species.

The Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevant B. cereus and B. anthracis as well as nonpathogenic B. psychrotolerans and B. psychrodurans. Consequently, 27 type and clinical strains from the B. cereus group were used to test alternate targets (rpoB, vrrA, and the 16S-23S spacer region) for identification. The rpoB gene proved the best alternate target, with a conserved 4-nucleotide difference between B. cereus and B. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database at http://www.ridom_rdna.de/.

Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries.

Current methods for identification of Mycobacterium spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify Mycobacterium isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of Mycobacterium spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.

Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species.

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing approximately 440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.

Identification of Mycobacterium species by multiple-fluorescence PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapid and sensitive techniques that can accurately identify all mycobacterial species. Multiple-fluorescence-based PCR and subsequent single-strand conformation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolates. The species-specific SSCP patterns generated were then entered into a database by using BioNumerics, version 1.5, software with a pattern-recognition capability, among its multiple uses. Patient specimens previously identified by 16S rRNA gene sequencing were subsequently tested by this method and were identified by comparing their patterns with those in the reference database. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identified as unique species or were identified by their closest relative, as they were not in the database. We propose that MF-PCR-SSCP offers a rapid, specific, and relatively inexpensive identification tool for the differentiation of mycobacterial species.

Evaluation of recA sequences for identification of Mycobacterium species.

16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genus Mycobacterium. Previous studies have shown that Mycobacterium species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e., M. gastri and M. kansasii. In the present study, we have used the recA gene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The recA genes of 30 Mycobacterium species were amplified by PCR, sequenced, and compared with the published recA sequences of M. tuberculosis, M. smegmatis, and M. leprae available from GenBank. By recA sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% between M. gastri and M. kansasii to 75.7% between M. aurum and M. leprae. Exceptions to this were members of the M. tuberculosis complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new Mycobacterium species that contain a protein intron in their recA genes, similar to M. tuberculosis and M. leprae. We propose that recA gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of the Mycobacterium species.

Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR-single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections.

Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system.

Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.

Screening of stool samples for identification of vancomycin-resistant Enterococcus isolates should include the methyl-alpha-D-glucopyranoside test to differentiate nonmotile Enterococcus gallinarum from E. faecium.

The methyl-alpha-D-glucopyranoside (MDG) test has been shown to be superior to motility testing in differentiating Enterococcus faecium from E. gallinarum. In the present study, 33 vancomycin-resistant enterococcus (VRE) isolates collected as part of a stool surveillance study were compared by using motility and MDG. Motility testing identified all 33 isolates as E. faecium, whereas MDG identified 11 of the 33 isolates as nonmotile E. gallinarum. The MDG results were confirmed by sequencing the 16S rDNA V6-to-V8 region. We conclude that the MDG test is a necessary component of routine VRE screening.