Genetic heterogeneity in Mycobacterium tuberculosis isolates reflected in IS6110 restriction fragment length polymorphism patterns as low-intensity bands

Abstract

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS6110 RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple M. tuberculosis strains. We did not find any multiple LIBs, suggesting that multiple infections are rare in the Netherlands. However, we did observe a few LIBs in 94 patterns (7.4%) and examined the nature of this phenomenon. With single-colony cultures it was found that LIBs mostly represent mixed bacterial populations with slightly different RFLP patterns. Mixtures were expressed in RFLP patterns as LIBs when 10 to 30% of the DNA analyzed originated from a bacterial population with another RFLP pattern. Presumably, a part of the LIBs did not represent mixed bacterial populations, as in some clusters all strains exhibited LIBs in their RFLP patterns. The occurrence of LIBs was associated with increased age in patients. This may reflect either a gradual change of the bacterial population in the human body over time or IS6110-mediated genetic adaptation of M. tuberculosis to changes in the environmental conditions during the dormant state or reactivation thereafter.

FIG. 1

IS6110 RFLP patterns of isolates, showing LIBs, and SCCs, from three different patients (A to C). Lanes 1 show the banding patterns of the isolates, with LIBs indicated by arrows. Lanes 2 to 5 show the banding patterns of SCCs of these isolates. The numbers on the right indicate the sizes of standard DNA fragments in kilobase pairs.

FIG. 2

IS6110 RFLP patterns of different mixtures of the DNAs of two M. tuberculosis strains. Lanes 1 and 21 show the RFLP patterns of the pure DNAs of the two strains. Lanes 2 to 20 show the patterns of mixtures of the DNAs of these strains. The numbers in the second horizontal row indicate the ratios of the DNA mixtures. The numbers on the left indicate the sizes of standard DNA fragments in kilobase pairs.

FIG. 3

IS6110 RFLP patterns of different mixtures of the DNAs of two SCCs of an M. tuberculosis strain differing in a single IS6110 element. Lane 1 shows the pattern of pure DNA of one SCC. Lanes 2 to 21 depict patterns of mixtures of this DNA with an increasing amount of DNA of another SCC containing an additional IS6110 copy at a PvuII restriction fragment of approximately 3.5 kb. The numbers in the second horizontal row indicate the ratios of the DNA mixtures. The numbers on the left indicate the sizes of standard DNA fragments in kilobase pairs.

FIG. 4

IS6110 RFLP patterns of three patient isolates of a cluster showing an LIB (indicated by the arrow) at the same PvuII restriction fragment. The numbers on the right indicate the sizes of standard DNA fragments in kilobase pairs.

FIG. 5

Frequency distribution of 6,189 normal-intensity bands (NIB) and 69 LIBs per band position category (GelCompar band position and sizes of standard DNA fragments in kilobase pairs).