Whole Genome Sequencing Reveals Complex Evolution Patterns of Multidrug-Resistant Mycobacterium tuberculosis Beijing Strains in Patients

Abstract

Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.

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“…In contrast, rapid whole-genome sequencing (WGS) by next-generation sequencing (NGS) approaches can be used to detect known molecular markers associated with resistance to all drugs simultaneously directly from a primary culture (9). Moreover, WGS provides the ultimate molecular resolution to guide contact tracing (10,11). In light of the ongoing improvements in ease of use, turnaround time, and cost, WGS is therefore destined to become routine in well-resourced countries for every case of culture-positive TB (12,13).…”

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“…The intra‐host evolution of bacterial resistance patterns is one of the key aspects of drug resistance emergence and spread (Eldholm et al., 2014; Meacci et al., 2005; Merker et al., 2013). The existence of genetically variable drug‐resistant bacterial populations within a single patient is now acknowledged and could affect drug resistance evolution (Black et al., 2015; Eldholm et al., 2014; Müller, Borrell, et al., 2013; Shamputa et al., 2004).…”

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“…However, TB infections are notorious for their heterogeneity within the same host as the lengthy infection period allows for microevolution of the bacilli within the duration of one infection cycle. 17 On the other hand, the pathogen has a slow-replication rate and a proclivity toward low genetic diversity. Thus the bacilli may either show no genetic diversity or the presence of multiple SNPs between contacts making it extremely difficult to trace transmission using their gene sequence alone.…”