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Journal of Clinical Microbiology, March 2006, p. 1081-1083, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.1081-1083.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Insertion Sequence IS900 Revisited

Makeda Semret, Christine Y. Turenne, and Marcel A. Behr*

McGill University Health Center, Montreal, Quebec, Canada

Received 22 November 2005/ Returned for modification 28 December 2005/ Accepted 1 January 2006


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ABSTRACT
 
Many studies investigating Mycobacterium avium subsp. paratuberculosis in Crohn's disease have used molecular detection of IS900 in clinical samples, but some have described polymorphisms in IS900 as variants of this organism. Analysis of 23 M. avium subsp. paratuberculosis isolates revealed that IS900 is highly conserved, with only two sequevars distinguishing sheep and cattle lineages. Amplification of IS900-like sequences is not sufficient as a proxy for M. avium subsp. paratuberculosis.


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TEXT
 
Mycobacterium avium subsp. paratuberculosis is a pathogen of many mammals, in which it causes a severe inflammatory bowel disease called Johne's disease (8). Similarities between this and Crohn's disease of humans have fueled investigations on the zoonotic potential of M. avium subsp. paratuberculosis. Because of the limitations of culture-based diagnostics, including slow growth and the presumed low abundance of the organism, PCR-based testing of a multicopy insertion element, IS900, has been used in a number of studies as the sole criterion for the detection of M. avium subsp. paratuberculosis in clinical and environmental samples. This approach raises two critical issues: first, false-positive results may be obtained if isolates other than M. avium subsp. paratuberculosis bear sequences with similarity to the IS900 element (5, 6), and second, there is the risk of laboratory cross-contamination, which is of special concern in PCR-based assays.

Sequencing of IS900 amplicons has been performed in different studies to address both of these concerns (1, 4, 11, 13, 14), but predictably, this has led to confusion. The determination that IS900 sequences from different samples are invariant and identical to a reference sequence would suggest the presence of M. avium subsp. paratuberculosis rather than another species but cannot address the issue of contamination. Conversely, the finding of polymorphisms within IS900 would suggest that cross-contamination did not occur (11); however, this raises the possibility that some amplicons may not represent M. avium subsp. paratuberculosis.

Sequence comparisons have further been confounded by the fact that several different IS900 sequences are deposited in GenBank, with no clear indication of which should be the referent sequence. The original IS900 sequence (GenBank accession no. X16293) is a 1,451-bp sequence (7) and differs by 4 single-nucleotide polymorphisms from the 15 IS900 elements of the sequenced strain, M. avium subsp. paratuberculosis strain K10 (GenBank accession no. NC002944) (10). Additionally, several groups have described unique variants of IS900 with sequences that differ from the original sequence and from the M. avium subsp. paratuberculosis strain K10 sequence by one or more single-nucleotide polymorphisms, with a total of eight such variants at the time of this writing (2, 12, 17).

Our goal was to determine the IS900 sequence conservation in a panel of isolates confirmed to be M. avium subsp. paratuberculosis by the use of IS900-independent markers. The use of strains classified as M. avium subsp. paratuberculosis by other markers permitted us to overcome the inherent tautology of IS900 and M. avium subsp. paratuberculosis (since the former has been used as a proxy for the latter) and formally study the IS900 sequences from clinical isolates. We selected 23 isolates which reflect a broad host range (including human isolates), broad geographic origins, different genomic clades (16), different hsp65 sequevars (18), and different molecular fingerprints. The isolates had been cultured and the DNA was extracted by standard methods (16, 19). The isolates were identified as M. avium subsp. paratuberculosis based on (i) PCR for large sequence polymorphism A8 (LSPA 8), a region that is specifically missing from M. avium subsp. paratuberculosis (15), and (ii) hsp65 sequencing (18). Furthermore, they were determined to be sheep (n = 10) or cattle (n = 13) types by using a three-primer PCR for LSPA 20 (which is missing from sheep strains) (16) and by determination of the hsp65 sequevars (code 5 for cattle, code 6 for sheep types) (18) (Table 1). Most of the isolates were also characterized by restriction fragment length polymorphism analysis with IS900 and IS1311 and were determined to bear nonidentical fingerprints.


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  		TABLE 1. M. avium paratuberculosis isolates studieda

The isolates thus assembled as well as two negative control isolates (M. avium subsp. avium strain 104 and a water isolate) were subjected to PCR amplification of the IS900 sequence. Three sets of primers were used to cover nearly the entire length of IS900: IS900L1 (5'-CCT TTC TTG AAG GGT GTT CG-3') and IS900R1 (3'-GTC TTT GGC GTC GGT C-5') to amplify the sequence from nucleotide positions 7 to 555, IS900L2 (5'-CAG CGG CTG CTT TAT ATT CC-3') and IS900R2 (3'-ATG CTG TGT TGG GCG TTA-5') to amplify the sequence from nucleotide positions 472 to 918, and IS900L3 (5'-CGC CTT CGA CTA CAA CAA GA-3') and IS900R3 (3'-GGG TGG TAG ACA GCG TGG T-5') to amplify the sequence from nucleotide positions 739 to 1413 of IS900.

Amplification reactions were performed as described previously (15). The PCR products were sequenced on a 3730XL DNA analyzer system in a core facility (McGill University and Génome Québec Innovation Centre), using the PCR primers for sequencing of forward and reverse fragments. Chromatograms were manually edited to ensure sequence accuracy and were added to the alignment component of MEGA 3 (9). Sequence comparisons of each IS900 fragment were performed by BLAST analysis against sequences in the NCBI database (http://www.ncbi.nlm.nih.gov/).

Sequences spanning nucleotide positions 26 to 1400 of IS900 were obtained for each isolate under study. All 13 isolates of the cattle type gave uniform sequences, with 100% identity to IS900 elements of the published genome sequence of M. avium subsp. paratuberculosis strain K10 (GenBank accession no. NC002944) (10), as well as to a sequence obtained by an independent group from a bovine field isolate of M. avium subsp. paratuberculosis from New Zealand (GenBank accession no. AF305073). Sequences obtained from sheep type isolates exhibited two ambiguities with respect to those of the cattle type, manifested by double peaks on the electropherograms at two separate loci. At position 216, all 10 sheep strains showed a predominant G peak and a smaller A peak, in contrast to cattle strains, for which only an A peak was noted. At position 169, a C was present for all cattle strains, whereas for 5 of the 10 sheep strains a predominant T peak with a smaller C peak was seen at the same locus (Fig. 1). No other ambiguities or differences were noted. This suggests that some copies of IS900 in sheep strains have undergone polymorphisms at these two loci.


Figure 1
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  		FIG. 1. Sequence electropherogram of two sections of the IS900 element from M. avium subsp. paratuberculosis of the sheep type showing sequence ambiguities (depicted by the codes R and Y) at base pair position 216 (A) and base pair position 169 (B).

Comparisons of other sequences deposited in NCBI, notably, the original IS900 sequence (GenBank accession no. X16293) and several others (3, 7), consistently revealed polymorphisms at four different loci in reference to the genome sequence of M. avium subsp. paratuberculosis strain K10: positions 36 and 37 (a 2-nucleotide gap, corresponding to GC in the sequences of M. avium subsp. paratuberculosis strain K10), positions 122 and 123 (CG changed to GC in the M. avium subsp. paratuberculosis K10 sequence), position 726 (a 1-nucleotide gap corresponding to a G in the sequence of M. avium subsp. paratuberculosis K10), and position 690 (insertion of a G in these sequences compared to the sequence of M. avium subsp. paratuberculosis K10). Our isolates did not manifest these or other polymorphisms described in unusual isolates from water buffaloes, goats, sheep, and cows (2, 17).

We therefore observed that a small but diverse panel of M. avium subsp. paratuberculosis isolates had highly uniform IS900 sequences, and the polymorphisms described in other IS900 sequences deposited in public databases were not found in our isolates. As many studies have relied solely on the PCR detection of IS900 to identify M. avium subsp. paratuberculosis, it is difficult to determine whether these polymorphisms reflect true variants, such as those that have been described in Mycobacterium isolates other than M. avium subsp. paratuberculosis (5, 6), or result instead from technical sequencing errors.

Based on this analysis we propose that future IS900 sequence comparisons be made in reference to the sequenced strain, M. avium subsp. paratuberculosis strain K10, shown here to be identical to a broad range of clinical isolates. Furthermore, we suggest that sequence discrepancies in comparison to this reference sequence should prompt further investigations and should be interpreted as suggestive of a Mycobacterium organism other than M. avium subsp. paratuberculosis until proven otherwise through conventional microbiologic or alternate genetic methods.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the Natural Science and Engineering Research Council (grant GEN2282399). M.S. is funded by the Fonds de la Recherche en Sante du Quebec (FRSQ), and M.A.B. is a New Investigator of the Canadian Institutes of Health Research.

None of the authors have a conflict of interest or any commercial association that may pose a conflict of interest.

We thank Desmond M. Collins for providing samples and Elizabeth Fidalgo for assistance with culturing of the isolates for this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases and Medical Microbiology, A5-156, Montreal General Hospital, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada. Phone: (514) 934-1934, ext. 42815. Fax: (514) 934-8423. E-mail: marcel.behr{at}mcgill.ca. Back


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Journal of Clinical Microbiology, March 2006, p. 1081-1083, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.1081-1083.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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