Evidence of the Presence of IS 1245 and IS 1311 or Closely Related Insertion Elements in Nontuberculous Mycobacteria outside of the Mycobacterium avium Complex

Abstract

A PCR assay based on the simultaneous detection of IS1245 and IS1311 was developed and used to determine the host range of these insertion elements. Specific PCR products were observed in Mycobacterium malmoense, Mycobacterium scrofulaceum, and Mycobacterium nonchromogenicum, indicating that IS1245 and IS1311 are not limited to the Mycobacterium avium complex.The 1,414-bp insertion element IS1245 belongs to the Staphylococcus aureus IS256 family of insertion sequences. It is present in up to 27 copies in Mycobacterium avium (8) and was found to be stable during in vivo and in vitro passage (1, 14), making it a popular target for restriction fragment length polymorphism strain typing in recent years (5,6,12,19). The host range was originally demonstrated to be limited to the M. avium group (M. avium and subspecies paratuberculosis and silvaticum) by PCR amplification of a 427-bp target sequence within IS1245, leading to its use as a species-specific target for diagnostic detection (11).Recently, Beggs et al. found IS1245 in strains of Mycobacterium intracellulare, demonstrating that the element is present in further species of the M. avium complex (MAC) (2).A closely related insertion element, IS1311 (85% sequence identity to IS1245 at the DNA level), has likewise been used as a target for restriction fragment length polymorphism strain typing of M. avium (5,15,16).In our laboratory, a PCR assay based on the simultaneous detection of a highly homologous 130-bp portion of IS1245 and IS1311 (91% sequence identity) was developed as a diagnostic tool for detection of MAC in clinical specimens and used to determine the host range of these elements.Mycobacterial strains were received from five collaborating mycobacterial laboratories. Species of nontuberculous mycobacteria (NTM) were identified by 16S ribosomal DNA (rDNA) sequencing (10) and/or 23S rDNA probes (GenoType; Hain Diagnostika, Nehren, Germany). Twelve NTM strains (1 Mycobacterium shimoidei strain, 1 Mycobacterium scrofulaceum strain, 6 M. intracellulare strains, 2 Mycobacterium malmoense strains, 1 Mycobacterium chelonae strain, and 1 Mycobacterium szulgai strain) were identified by a combination of standard biochemical methods (9) and high-performance liquid chromatography (4).Cells were lysed by incubation at 100°C for 5 min in the presence of 2 M NaOH and 4% Triton. Crude lysates or DNA purified using QIAamp spin columns (Qiagen, Hilden, Germany) were used as PCR templates.PCR analysis of NTM strains was performed by two independent laboratories (Lab 1 and Lab 2). Amplification was performed on GeneAmp PCR systems 9600 and 2400 (PerkinElmer, Weiterstadt, Germany). A 100-l reaction mixture contained 1 mM MgCl 2, 320 mol of deoxynucleoside triphosphates, 5 U of AmpliTaq Gold (GeneAmp; Perkin-Elmer), 100 pmol of each primer, and 5 to 10 l of lysate with 0.1 to 1 ng of template DNA in 1ϫ PCR Buffer II (Perkin-Elmer). The sense primer N3 (5Ј ACTTCCTGCGCAACGTGCT 3Ј) recognized positions 885 to 903 of IS1245 (GenBank accession no. L33879) and positions 823 to 8...

Mycobacterium avium in the Postgenomic Era

“…As for any widely tested insertion element, the "presence" of the IS1245 element in species outside its typical host has been documented, although this was not confirmed by sequencing and may have been related elements, such as IS1311 (12,134). Some M. avium isolates have been documented as being IS1245 negative, but only a few such reports have presented further documentation of strain identity by a sequence-based method (12).…”

Distribution of IS 1311 and IS 1245 in Mycobacterium avium Subspecies Revisited

“…avium and M. avium subsp. paratuberculosis (21) and has been detected in strains of M. intracellulare, Mycobacterium malmoense, and Mycobacterium scrofulaceum (9). There is, however, considerably discrepancy in the literature about the presence and the copy number of IS1245 and IS1311 in the M. avium subspecies (4,7,16).…”

“…silvaticum by PCR amplification of a 427-bp target sequence (7) and has also been found in some other mycobacteria (9). A closely related IS element is IS1311, which shows 85% sequence identity with IS1245 at the DNA level (9,16,17). The element is present in M. avium subsp.…”

Mycobacterial Typing Systems: The Good, the Bad and the Unattainable

“…However, the use of VNTR for typing M. avium described shows potential as a discriminatory typing tool. The use of IS units for a myriad of NTM isolates has also been described [19][20][21]; however, it appears that none of the traditional typing systems are robust enough to facilitate typing of both MTC and NTM isolates.…”