Cryptic Microheteroresistance Explains Mycobacterium tuberculosis Phenotypic Resistance

Area of Science:

• Microbiology
• Genomics
• Infectious Diseases

Background:

• Minority drug-resistant Mycobacterium tuberculosis subpopulations are difficult to detect with current methods.
• Phenotypic resistance in tuberculosis can be linked to these minor variants, leading to diagnostic challenges.

Purpose of the Study:

• To evaluate the effectiveness of targeted next-generation sequencing (NGS) in identifying minor drug-resistant Mycobacterium tuberculosis subpopulations.
• To assess the role of single molecule overlapping reads (SMOR) in resolving these variants.

Main Methods:

• Analyzed primary cultured Mycobacterium tuberculosis isolates using SMOR, a targeted NGS approach designed to minimize sequencing errors.
• Compared SMOR results with Sanger sequencing and single-colony selection on antibiotic-containing media.
• Included isolates phenotypically resistant but lacking resistance-associated variants (RAVs) by Sanger sequencing.

Main Results:

• SMOR detected numerous heteroresistant RAVs at low frequencies (<5% and <1%) missed by Sanger sequencing.
• In isolates with no Sanger-identified RAVs, SMOR detected minor resistant subpopulations in 80% of cases.
• Single-colony selection corroborated SMOR findings for 90% of RAV-containing specimens.

Conclusions:

• Undetected minor variant Mycobacterium tuberculosis subpopulations contribute to false-negative drug resistance determinations.
• Targeted NGS methods like SMOR are crucial for resolving cryptic resistant subpopulations below current detection limits.