Intragenic Distribution of IS 6110 in Clinical Mycobacterium tuberculosis Strains: Bioinformatic Evidence for Gene Disruption Leading to Underdiagnosed Antibiotic Resistance

Abstract

Antibiotic resistance is a global challenge for tuberculosis control, and accelerating its diagnosis is critical for therapy decisions and controlling transmission. Genotype-based molecular diagnostics now play an increasing role in accelerating the detection of such antibiotic resistance, but their accuracy depends on the instructed detection of genetic variations. Genetic mobile elements such as IS6110 are established sources of genetic variation in Mycobacterium tuberculosis, but their implication in clinical antibiotic resistance has thus far been unclear. Here, we describe the discovery of an intragenic IS6110 insertion into Rv0678 that caused antibiotic resistance in an in vitro-selected M. tuberculosis isolate. The subsequent development of bioinformatics scripts allowed genome-wide analysis of intragenic IS6110 insertions causing gene disruptions in 6,426 clinical M. tuberculosis strains. This analysis identified 10,070 intragenic IS6110 insertions distributed among 333 different genes. Focusing on genes whose disruption leads to antibiotic resistance, 12 clinical isolates were identified with high confidence to be resistant to bedaquiline, clofazimine, pyrazinamide, ethionamide, and para-aminosalicylic acid because of an IS6110-mediated gene disruption event. A number of these IS6110-mediated resistant strains had identical genomic distributions of IS6110 elements and likely represent transmission events of a single resistant isolate. These data provide strong evidence that IS6110-mediated gene disruption is a clinically relevant mechanism of antibiotic resistance in M. tuberculosis that should be considered for molecular diagnostics. Concomitantly, this analysis provides a list of 333 IS6110-disrupted genes in clinical tuberculosis isolates that can be deemed nonessential for human infection. IMPORTANCE To help control the spread of drug-resistant tuberculosis and to guide treatment choices, it is important that rapid and accurate molecular diagnostic tools are used. Current molecular diagnostic tools detect the most common antibiotic-resistance-conferring mutations in the form of single nucleotide changes, small deletions, or insertions. Mobile genetic elements, named IS6110, are also known to move within the M. tuberculosis genome and cause significant genetic variations, although the role of this variation in clinical drug resistance remains unclear. In this work, we show that both in vitro and in data analyzed from 6,426 clinical M. tuberculosis strains, IS6110 elements are found that disrupt specific genes essential for the function of a number of pivotal antituberculosis drugs. By providing ample evidence of clinically relevant IS6110-mediated drug resistance, we believe that this shows that this form of genetic variation must not be overlooked in molecular diagnostics of drug resistance.

Keywords: IS6110; Mycobacterium tuberculosis; antibiotic resistance; diagnostics; genetic polymorphisms.

FIG 1

(A and B) Representation of the sequencing reads mapped onto the H37Rv reference genome for parental H37Rv (A) and OH190-resistant clone 10.5 (B). Green and red sequencing reads represent mapping in the forward and reverse directions, respectively. While reads from the parental strain map fully to the reference genome, for the resistant strain, there is a 5-bp duplication of mapped sequencing reads (highlighted in the inset of panel B), a hallmark of a transposon insertion. In addition, sequencing reads of the OH190-resistant clone only partially map to this genomic region (soft clipping), with the nonaligned end of the reads (lighter color) aligning with IS6110. (C) Sequencing reads of the OH190-resistant clone mapped onto a “corrected” genome containing the intragenic IS6110 element in Rv0678 showing the now full alignment of the sequencing reads across the border of the IS6110 insertion sites.

FIG 2

Genome-wide analysis of intragenic IS6110 insertions found in 6,426 clinical M. tuberculosis isolates using a contig-based bioinformatics approach. (A) Representation of the genomic distribution of identified intragenic IS6110 insertions (outer circle), with the relative frequency for each intragenic insertion shown in the bar chart within the circle (linear scale). The top 10 most frequent genes found to be disrupted by IS6110 insertion are marked (1, mmpS1; 2, Rv3113; 3, Rv3128c; 4, idsB; 5, Rv1371; 6, Rv963c; 7, Rv1754c; 8, Rv2016; 9, ctpD; 10, mmpL12). The image was generated using Circos (39). (B) Bar chart showing the frequency of clinical isolates with different numbers of identified intragenic IS6110 insertions. The inset is a zoomed-in section of the main bar chart. Note that while all the isolates come from different patients, some may be classified as the same strain.

FIG 3

The IS6110 “barcode” of four identified clinical strains carrying an IS6110 insertion at base 71 of Rv0678 (marked with asterisks). This barcode was generated from the sequencing-read-based analysis and shows the distribution of IS6110 insertions along the H37Rv reference genome (x axis) against the coverage of each insertion event (y axis). The data clearly indicate that TB_RSA126, TB_RSA64, and TB_RSA63 have identical IS6110 barcodes, likely suggesting high similarity of the isolates and the potential transmission of this clinical strain, while the KT_0084 IS6110 barcode is different and likely not related.