Molecular Identification of Rare Clinical Mycobacteria by Application of 16S-23S Spacer Region Sequencing.

Objective(s) In addition to several molecular methods and in particular 16S rDNA analysis, the application of a more discriminatory genetic marker, i.e., 16S-23S internal transcribed spacer gene sequence has had a great impact on identification and classification of mycobacteria. In the current study we aimed to apply this sequencing power to conclusive identification of some Iranian clinical strains of mycobacteria. Materials and Methods The test strains consisted of nineteen mycobacterial isolates which were initially identified by the use of conventional phenotypic techniques and molecular methods and subjected to further definitive identification using the 16S-23S internal transcribed spacer gene sequencing. Results Out of 19 studied strains, 7 isolates were found to be rapidly growing and 12 isolates as slowly growing mycobacteria. With the exception of one isolate, i.e., the isolate HNTM87, which yielded a distinct ITS sequence incomparable with all previously identified mycobacteria, the remaining isolates produced the sequences similar to the established mycobacteria and were clearly identified and differentiated from closely related taxa. A phylogenetic tree based on maximum parsimony analysis of 16S-23S internal transcribed spacer gene sequences constructed showing the relatedness of Iranian clinical isolates with the closely related type species of mycobacteria. Conclusion This study showed that the 16S-23S internal transcribed spacer gene of the genus Mycobacterium exhibits a high variation which is of value for discriminating closely related taxa and could be used independently or in combination with 16S rDNA sequencing to delineate the true identity of rare mycobacterial species.

In the last decade, the rapid development of molecular techniques has led to a great increase in our knowledge of mycobacterial identification and taxonomy (2). Methods for the detection and identification of mycobacteria include nucleic acid probes (3), PCR hybridization with species-specific probes (4,5), PCR RFLP analysis (6,7) and nucleic acid sequencing (8).
In recent years a well-established sequencing analysis using 16S rRNA gene (rDNA) has greatly contributed to an accurate identification and classification of mycobacteria (8). However, some members of clinically significant mycobacterial complex groups e.g., M. terrae complex (M terrae, M nonchromogenicum, and M triviale) are difficult to be differentiated from each other due to a very high degree of 16S rDNA sequence similarity (9,10).
Several studies have shown that sequencing of 16S-23S internal transcribed spacer (ITS) gene could help to differentiate and identify the closely related mycobacterial species (11)(12)(13). In the current study we aimed to apply this sequencing power to the precise diagnosis of clinical strains of mycobacterial species which were difficult to correctly delineate their identity by conventional microbiological testing or even 16S rDNA sequencing.

Initial identification and classification
The isolates were initially screened by the main conventional phenotypic tests including standard morphological and biochemical assays previously established for identification of mycobacteria (14). The preliminary identification was confirmed by the key molecular markers including a genus specific segment of 65-kDa heat shock protein gene (hsp) and 16S rDNA sequencing following standard procedures (15,16).

Molecular identification by ITS gene sequencing
All isolates which were preliminarily identified and classified into the previously known mycobacterial clusters according to phenotypic and molecular features (Table 1) were subjected to more definitive identification by ITS gene sequencing following the procedures described herein.

DNA extraction and purification
Chromosomal DNA was extracted using a method of Pitcher et al (17) with a slight modification to facilitate the susceptibility of cells to the standard digestion (16). In brief, after thermal inactivation, a pretreatment of biomass with lipase (Type VII; final concentration, 2 mg/ml [Sigma]) and a further treatment with proteinase K (100 pg/ml) and sodium dodecyl sulfate (final concentration, 0.5% [wt/vol]) were applied. The DNA was purified by phenol chloroform-isoamyl alcohol and precipitated with isopropanol. The precipitate was washed in 70% ethanol, dehydrated and dissolved in 100 µl of Milli-Q water and stored in 20 °C freezer until use.

Nucleotide sequencing
The amplified ITS gene generated by PCR reaction was purified using a QIA quick PCR purification kit (Qiagen, Chatsworth, Calif) as described in the instructions of the manufacturer. The purified PCR products were directly sequenced with the forward 16S-1511f and reverse 23S-23r primers using an ABI 3100 genetic analyzer and a Big Dye Terminator cycle sequencing kit by SEQLAB Company (Germany).

Data analysis of ITS gene sequences
The obtained sequences were aligned with the published ITS gene sequences of the type strains of mycobacteria (retrieved from GenBank TM database) using the jPhydit software package according to primarystructure (18). Comparative analyses of ITS gene sequences were performed with distance matrix, maximum-parsimony, and maximumlikelihood methods (19) as implemented in the Mega4 program (20). Tree topologies were tested by bootstrap analysis on 1000 resampling (21).

Nucleotide sequence accession numbers
The GenBank accession numbers of the ITS gene for mycobacterial isolates determined in this work are: HQ406781-98 and HM536981.

Results
On the basis of growth characteristics (Table 1), 7 isolates were rapidly growing mycobacteria (RGM) and the remaining 12 clinical isolates were slowly growing mycobacteria (SGM). The isolates were initially classified into four Runyon's groups (22) and further speciated by 16S rDNA sequencing (Table 1).
PCR amplification of the ITS region with the primers 16S-1511f and 23S-23r resulted in detection of a single band of approximately 550 bp and 400 bp for RGM and SGM respectively. The variation in product length was considerable between slow and rapid growing mycobacteria showing that the ITS gene of the SGM are shorter than that of RGM. This is due to the features that clearly differentiate SGM from RGM on the basis of PCR product of ITS gene.
With the exception of HNTM87 which yielded a distinct ITS gene sequence which initially failed to produce any significant matches to sequences within genus Mycobacterium, the remaining isolates produced sequences that were clearly identified and differentiated from closely related taxa (Table 1, Figure 1). These are the isolates HNTM19 and HNTM20 as M. avium and M. interacellurae, the isolate HNTM2 as M. Kansasii, the isolate HNTM95 as M. fortuitum, the isolate HNTM71 as M. porcinum and the isolates HNTM70, HNTM80 and M137 as M. conceptionense ( Table 1). The ITS gene sequence analysis reconfirmed the identity of the remaining isolates i.e., HNTM11, HNTM64, HNTM65, HNTM75, HNTM79, HNTM83, HNTM92, HNTM104 and HNTM105 which were initially well identified by 16S rDNA sequencing ( Table 1).
The isolate HNTM72 which was placed within NTM due to a negative niacin test found to be a member of M. tuberculosis complex (MTBC) by 16S rDNA sequencing. This was reconfirmed by ITS gene sequencing. A Phylogenetic tree based on maximum parsimony analysis of the ITS gene sequences was constructed to find the relatedness of Iranian clinical isolates with the closely related type species of mycobacteria ( Figure 2). The resulting dendrogram showed that the slowly and rapidly growing isolates were clearly grouped into two separate clusters.

Discussion
Identification of mycobacterial species by conventional methods including growth characteristics and biochemical tests are timeconsuming and often not unequivocal in their interpretation (2,8). Currently, analysis of the gene encoding 16S rDNA is widely accepted for identification of mycobacterial isolates to the species level; however this technique is unable to differentiate members of several closely related species called complex groups (5,7,(9)(10)(11).
In the current study we intended to use a more discriminatory DNA sequencing technology, i.e., the ITS gene sequence analysis, to specify the exact identity of some Iranian clinical isolates. These isolates were formerly classified as a member of mycobacterial complex groups or their identity was yet to be resolved more definitely or reconfirmed due to ambiguity in a few phenotypic and biochemical features. By using ITS sequencing, these strains showed a significant nucleotide differences with the closely related taxa. Furthermore, they formed a distinct node in phylogenetic tree representing a previously defined species. With 16S rDNA analysis these isolates had shown a high nucleotide similarity with the closely related mycobacteria which in turn resulted in the formation of a joint line of descent in phylogenetic analysis.  Taxon  1  47  50  51  53  54  56  58  68  70  71  72  208  209  210  211  227  230  239  240  241  249  250  251  252  253  254  255  256  257  258  259  260  261 M. avium (ATCC 35766 T ) .

Conclusion
Our study revealed that the ITS gene of the genus Mycobacterium exhibits a high variation which is of value for discriminating closely related taxa. However, in terms of MTBC, this is not the case since the technique lacks a sufficient power to differentiate and separate members of the group from each other.