Antimicrobial Drug Resistant Features of Mycobacterium Tuberculosis Associated With Treatment Failure

Abstract

Tuberculosis stands as a prominent cause of mortality in developing countries. The treatment of tuberculosis involves a complex procedure requiring the administration of a panel of at least four antimicrobial drugs for the duration of six months. The occurrence of treatment failure after the completion of a standard treatment course presents a serious medical problem. The purpose of this study was to evaluate antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure. Additionally, it aimed to evaluate the effectiveness of second line drugs such as amikacin, linezolid, moxifloxacin, and the efflux pump inhibitor verapamil against M. tuberculosis isolates associated with treatment failure. We monitored 1200 tuberculosis patients who visited TB centres in Lahore and found that 64 of them were not cured after six months of treatment. Among the M. tuberculosis isolates recovered from the sputum of these 64 patients, 46 (71.9%) isolates were simultaneously resistant to rifampicin and isoniazid (MDR), and 30 (46.9%) isolates were resistant to pyrazinamide, Resistance to amikacin was detected in 17 (26,5%) isolates whereas resistance to moxifloxacin and linezolid was detected in 1 (1.5%) and 2 (3.1%) isolates respectively. Among MDR isolates, the additional resistance to pyrazinamide, amikacin, and linezolid was detected in 15(23.4%), 4(2.6%) and 1(1.56%) isolates respectively. 

Introduction

Tuberculosis is one of the most challenging bacterial infections to treat. It is caused by a group of closely related organisms collectively referred to as the Mycobacterium tuberculosis complex (MTC). The MTC includes M. tuberculosis, M. africanum, M. orygis, M. bovis and the Bacillus Calmette–Guérin strain, M. microti, M. canetti, M. caprae, M. pinnipedii, M. suricattae and M. mungi [1]. More than 7 million people are infected with M. tuberculosis annually. Tuberculosis was the leading cause of mortality due to an infectious agent before the emergence of Covid-19 pandemic claiming the lives of 1.4 million people in 2019 (Global tuberculosis report, 2020). Pakistan ranks fifth among 22 high burden countries and fourth with respect to multi drug resistant cases of M. tuberculosis. Approximately 420,000 cases occur each year in Pakistan and out of these 9000 cases are of drug-resistant TB [2, 3]. Treating M. tuberculosis infection has always been very complicated. There is no single drug recommended for TB treatment. Instead, a combination of several drugs must be administrated simultaneously. The panel of 1st line drugs includes pyrazinamide (PZA), ethambutol, rifampicin (RIF) and isoniazid (INH), used in combination therapy for treating non MDR TB patients [4].

Materials and methods

Bacterial strains and growth conditions

The study includes a total of 1200 pulmonary tuberculosis patients undergoing treatment with first-line drugs including rifampicin, isoniazid, pyrazinamide, and ethambutol for six months in TB centres located at Mayo Hospital Lahore, and Gulab Devi Hospital Lahore during 2016–2017. A verbal informed consent was obtained from the participants following the guidelines of the institutional ethical review committee at University of Health Sciences, Lahore under letters No; UHS/Education/126-16/2611 and UHS/REG-18/ERC4176. The documentation of patient’s information was waved out by the ethical committee due to non-traceable identification of isolates and associated genotypic and phenotypic features with respect to identity of patients. The treatment of patients with first line drugs was based on identification of non-MDR tuberculosis using GeneXpert MTB/RIF analysis of sputum as a part of routine diagnostic protocol. The effectiveness of treatment in these patients was assessed after 6 months through ZN staining and GeneXpert MTB/RIF analysis of their sputum samples. 

Results

Hetero resistance to amikacin and pyrazinamide displayed by MDR M. tuberculosis cured from treatment failure patients as revealed from a comparative retro perspective cohort study
In our investigation of the treatment response in 1200 MTB patients, sixty-four patients remained positive for M. tuberculosis after six months of treatment with first line drugs as detected through ZN staining of sputum. Among these sixty-four isolates, 46 (71.9%) were resistant to rifampicin and isoniazid. Importantly, all rifampicin resistant isolates were also found to be resistant to isoniazid (Fig 1A, Table 1, S3 and S4 Tables in S1 File).

Discussion

Here, we present a detailed investigation of drug resistant features of M. tuberculosis associated with treatment failure. The incidence of treatment failure was observed in 5.33% (64 out of 1200) patients initially infected with non-MDR M. tuberculosis strains (Fig 1, Table 1). The course of treatment induced multi drug resistance in 81.5% (52/64) patients whereas the drug resistance was not the main cause of treatment failure in 12/64 patients. Interestingly, resistance to both rifampicin and isoniazid was found to coexist in MDR isolates (Fig 1), despite the different mechanisms of action of these two drugs. However, mutations associated with isoniazid resistance were relatively conserved and mostly located within the codon 315 of katG gene, while there was substantial variability in the mutation sites leading to rifampicin and pyrazinamide resistance (Table 2). Furthermore, our study identified several novel mutations in the pncA gene associated with pyrazinamide resistance (Table 2). Notably, three of these mutations caused frameshifts in the open reading frames, resulting in modified pncA gene products (Fig 2).

Acknowledgments

We are grateful to the TB centres at Mayo Hospital Lahore and Gulab Devi hospital Lahore and Unique Lahore Laboratory for giving access to clinical isolates.

Citation: Mushtaq F, Raza SM, Ahmad A, Aslam H, Adeel A, Saleem S, et al. (2023) Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure. PLoS ONE 18(10): e0293194. https://doi.org/10.1371/journal.pone.0293194

Editor: Atul Vashist, Bennett University, INDIA

Received: October 9, 2022; Accepted: October 8, 2023; Published: October 26, 2023

Copyright: © 2023 Mushtaq et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0293194#abstract0