Genomic diversity among Beijing and non-Beijing Mycobacterium tuberculosis isolates from Myanmar

Abstract

Background: The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.

Methodology/principle findings: Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.

Conclusion: This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.

Figure 1. Genetic diversity among 22 Mycobacterium tuberculosis isolates tested by CGH.

The dendogram was produced by weighted pair group method (WPGMA) clustering using a Pearson correlation based distance measure defined on regional z-score values. The red color indicates deletions in the clinical isolates compared to the reference strain M. tuberculosis H37Rv, whereas the green color shows extra regions in the clinical isolates not present in the reference strain M. tuberculosis H37Rv.

Figure 2. Regions chosen for PCR verification based on the CGH array analysis.

The dendogram based on CGH array analysis of 22 M. tuberculosis isolates from Myanmar (11 Beijing, 4 ST42, 2 ST89 and 5 previously unreported [UR] shared-types [ST]) was produced by weighted pair group method (WPGMA) clustering using a Pearson correlation based distance measure defined on regional z-score values, corresponding to P-values between 1e-3 and 1e-16. The green color indicates extra regions that are present in the clinical isolates, but not in the reference strain M. tuberculosis H37Rv, whereas the red color depicts deletions in the clinical isolates compared to the reference strain M. tuberculosis H37Rv.

Figure 3. PCR results from the 22 regions chosen for verification in 60 clinical M. tuberculosis isolates.

The dendogram based on PCR results from 16 deletions and 6 extra regions chosen for verification on 60 clinical M. tuberculosis isolates (28 Beijing, 13 ST42 and 19 other non-Beijing) was produced by unweighted pair group method (UPGMA) clustering using a Pearson correlation based distance measure.

Figure 4. Gene graph of the M.tuberculosis H37Rv 1754c-1765c, Mb1785-1787, MT1808 and MT1812-1813 regions.

The graph is based on region z-scores, with each line representing one isolate analyzed by CGH. Region z-scores of more than ±4.42 (absolute value) occurring in one or more samples, corresponding to a standard normal P-value of 0.00001 (1e-5) indicate deletions (above the 0-line) or extra regions (below 0-line). Red lines represent isolates of the Beijing lineage, green lines; ST42 lineage and blue lines; non-Beijing isolates other than ST42.