Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units

Abstract

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.

FIG. 1.

IS6110 RFLP patterns of isolates in this study. The genetic similarity of the isolates depicted in the dendrogram is based on IS6110 RFLP analysis as determined by the BioImage Whole Band Analyzer. RFLP patterns were compared using the Jaccard matching method with an allowed band size deviation of 1%, and the dendrogram showing the relationships between patterns was generated by the unweighted pair group method using arithmetic averages. The IS6110 RFLP patterns were numbered in the order they appear in the dendrogram, with the NTGSN pattern number in parentheses. The numbers 1, 2, and 3 indicate that an isolate with this IS6110 RFLP pattern was PCR positive for the corresponding IS6110 insertion site in strain CDC1551 (INS 1, INS 3, or INS 4); 3? and 4? indicate that the isolate was PCR positive but the predicted RFLP fragment was not apparent in the pattern.

FIG.2.

MIRU-VNTR patterns of isolates in this study. The genetic distance of the isolates depicted in the dendrogram is based on MIRU-VNTR analysis as determined by the SAS/GRAPH module (ET100.exe and cluster.SAS) by the unweighted pair group method using arithmetic averages (7). The patterns were compared by using unweighted loci and treating the allele numbers as nominal values. The MIRU-VNTR patterns were numbered in the order they appear in the dendrogram for cross-reference with Table 2. The MIRU-VNTR patterns of the group 1, Beijing, and M. bovis isolates are labeled, and the patterns of all isolates with a copy of IS6110 at INS 1, INS 3, and INS 4 are indicated with asterisks. The dash in pattern 45 represents the missing MIRU locus 27, which was not amplified from one isolate; the asterisk in pattern 13 indicates the missing 3′ 53-bp unit in MIRU locus 4 in one of our isolates; and the zero in pattern 4 indicates that the isolate is characterized as having zero units at MIRU locus 24 (see the text).