Evolutionary study and characterization of multi-drug resistant mycobacterium tuberculosis under antibiotic stress

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has been a public health concern for centuries, causing nearly 2 million deaths annually worldwide. The situation was worsened with multidrug-resistant MTB (MDR-TB) emergence, especially in developing countries with high TB burdens. New cost-effective molecular assays for rapid detection and identification of TB from direct specimens are therefore in urgent need. In addition, understanding the molecular basis of drug resistance and the transmission of MDR-TB is critical for developing new therapeutic and preventive strategies against the disease. This study can be divided into three sections. In the first section we introduced a new in-house developed IS6110 real-time PCR (IS6110-qPCR) assay for TB diagnosis. Optimal cut-off Cycle-threshold (Ct) value was determined by Receiver Operating Characteristic (ROC) curve, which was set at Ct=24.140. Prospective analysis of IS6110 qPCR assay among 503 respiratory specimens showed a high overall sensitivity and specificity at 97.2% and 99.7%, respectively, with no cross reactivity observed among non-tuberculous mycobacteria specimens. Limit-of-detection analysis on MTB-spiked sputum also demonstrated an average detection limit of 5.0CFU/mL at Ct=23.18 (±SD, 0.57). With an average cost less than one-fourth of commercial TB diagnostic platform, IS6110-qPCR was suggested to be an accurate and cost-effective assay for suspected TB cases screening, especially for regions with limited resources settings. In the second part of the study, we focused on a pair of consecutive MDR-TB isolated from the same patient before (pre-treatment strain) and 4-months after treatment initiation (post-treatment strain). The post-treatment strain demonstrated reduced phenotypic susceptibility towards isoniazid, ethambutol and rifabutin while carrying identical missense mutations at rpoB S531L, inhA C-15T and embB M306V when compared to pre-treatment strain. Furthermore, post-treatment strain demonstrated 2-fold elevation in in vitro bacterial growth rate when compared to pre-treatment strain. To better understand the genomic characteristics in MDR-TB strains, single molecule real-time sequencing was performed to identify mutations that contribute to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Using the whole genome sequencing data, comparative genomic analysis identified three polymorphisms between the clinical isolates. These included a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, together with a missense mutation at lpdA V44I and a 6bp in-frame deletion at cobM codon 67-68 in post-treatment strains. Using structural-based and sequence-based algorithms, we further predicted that the mutations could be deleterious on protein functions. In the last part of the study, we characterized the phenotypic effects conferred by the polymorphisms between the two MDR-TB clinical isolates. Cloning, transformation and over-expression of both wild-type and mutated lpdA reduced the phenotypic susceptibility of post-treatment strains towards isoniazid and ethambutol. Combined with in-silico X-ray crystallography result, lpdA mutation could be a lost-in-function that partially impaired the activity of the protein, which disturbed NAD/NADH+ equilibrium and resulted in the alteration of EMB and INH phenotypic susceptibility. Meanwhile, over-expression of wild-type rv0888 in pre-treatment strain was able to restore the in vitro growth rate of the pre-treatment strain, suggesting the mutation was involved in the regulation of MTB growth fitness.