Genetic factors associated with acquired phenotypic drug resistance and its compensatory evolution during tuberculosis treatment.

OBJECTIVES: We elucidated the factors, evolution, and compensation of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (MTB) isolates under dual pressure from the intra-host environment and anti-tuberculosis (anti-TB) drugs. METHODS: This retrospective case-control study included 337 patients with pulmonary tuberculosis from 15 clinics in Tianjin, China, with phenotypic drug susceptibility testing results available for at least two time points between January 1, 2009 and December 31, 2016. Patients in the case group exhibited acquired AMR to isoniazid (INH) or rifampicin (RIF), while those in the control group lacked acquired AMR. The whole-genome sequencing (WGS) was conducted on 149 serial longitudinal MTB isolates from 46 patients who acquired or reversed phenotypic INH/RIF-resistance during treatment. The genetic basis, associated factors, and intra-host evolution of acquired phenotypic INH/RIF-resistance were elucidated using a combined analysis. RESULTS: Anti-TB interruption duration of ≥30 days showed association with acquired phenotypic INH/RIF resistance (aOR = 2·2, 95% CI, 1·0-5·1) and new rpoB mutations (p = 0·024). The MTB evolution was 1·2 (95% CI, 1·02-1·38) single nucleotide polymorphisms per genome per year under dual pressure from the intra-host environment and anti-TB drugs. AMR-associated mutations occurred before phenotypic AMR appearance in cases with acquired phenotypic INH (10 of 16) and RIF (9 of 22) resistances. DISCUSSION: Compensatory evolution may promote the fixation of INH/RIF-resistance mutations and affect phenotypic AMR. The TB treatment should be adjusted based on gene sequencing results, especially in persistent culture positivity during treatment, which highlights the clinical importance of WGS in identifying reinfection and AMR acquisition before phenotypic drug susceptibility testing.

Characterization of Fluoroquinolone-Resistant and Multidrug-Resistant Mycobacterium tuberculosis Isolates Using Whole-Genome Sequencing in Tianjin, China.

Objective: Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a global concern. This study aimed to determine the molecular characteristics of fluoroquinolone-resistant and multidrug-resistant M. tuberculosis strains using whole-genome sequencing to predict drug resistance in M. tuberculosis in Tianjin, China, which has not been established previously. Methods: Twenty-one fluoroquinolone-resistant and multidrug-resistant M. tuberculosis strains were isolated from sputum samples. Phenotypic drug resistance against 12 anti-tuberculosis drugs was determined using drug susceptibility testing. Whole-genome sequencing was performed to predict drug resistance in M. tuberculosis based on genome regions associated with drug resistance. The sensitivity of whole-genome sequencing for predicting drug resistance was calculated based on phenotypic drug susceptibility testing information. Results: Among the 21 isolates, mutations in 15 genome regions associated with drug resistance, including rpoB for rifampicin; katG and inhA promoter for isoniazid; gyrA and gyrB for ofloxacin and moxifloxacin; rpsL for streptomycin; rrs for streptomycin, amikacin, kanamycin and capreomycin; pncA and panD for pyrazinamide; embB, embC-embA, aftA, and ubiA for ethambutol; ethA for protionamide; and folC for para-aminosalicylic acid, were detected. Compared with traditional drug susceptibility testing results, the sensitivities for whole-genome sequencing of rifampin, isoniazid, ofloxacin, moxifloxacin, streptomycin, ethambutol, pyrazinamide, kanamycin, and amikacin resistance were 100%, 90.48%, 95.24%, 92.86%, 95.27%, 85.71%, 66.67%, 50%, and 50%, respectively. The sensitivities for whole-genome sequencing of capreomycin, protionamide, and para-aminosalicylic acid were not calculated because only one isolate showed phenotypic drug resistance. Mutations determined in drug susceptibility-associated genes can explain phenotypic drug resistance in most isolates. Notably, these mutations were absent in certain drug-resistant isolates, indicating other drug resistance mechanisms. Conclusion: Whole-genome sequencing represents an effective diagnostic tool for fluoroquinolone-resistant and multidrug-resistant TB though it has some obstacles. Whole-genome sequencing should be used to predict drug resistance prior to performing traditional phenotypic drug susceptibility testing in Tianjin, China.

Performance of Sequencing in Predicting Ofloxacin Resistance in Mycobacterium tuberculosis from Positive Bactec MGIT 960 Cultures.

Ofloxacin (OFX) resistant Mycobacterium tuberculosis (MTB) isolates have been increasingly observed worldwide, complicating tuberculosis control. This study investigated the performance of sequencing which could predict OFX resistance in MTB isolates from positive Bactec MGIT 960 cultures. A total of 214 Bactec MGIT 960 positive cultures were identified by p-nitrobenzoic-acid and thiophene-2-carboxylic hydrazide. Drug susceptibility testing was performed using the proportion method and sequencing. The sequence results were compared with the results of the proportion method to analyze the consistency between the results. We identified a total of 204 MTB and 10 nontuberculous mycobacteria (NTM) isolates. Among the MTB isolates, 98% (200/204) carried gyrA and gyrB genes, whereas 20% (2/10) of the NTM isolates had gyrA and gyrB genes. Of the 202 MTB isolates, 7.4% (15/202) and 92.6% (187/202) were OFX-resistant and susceptible isolates, respectively. Among the OFX resistant isolates, 80% (12/15) had mutations in gyrA (n=9), gyrB (n=1), or both gyrA and gyrB (n=2). Among the OFX-susceptible isolates, 2.7% (5/187) had mutations in gyrA (n=2) or gyrB (n=3). Compared with the proportion method, the sensitivity and specificity of sequencing were 78.6% and 99.5%, respectively. Sequencing had good performance for detecting OFX resistance and could be used for predicting OFX susceptibility.

Molecular characteristics of ofloxacin mono-resistant Mycobacterium tuberculosis isolates from new and previously treated tuberculosis patients.

BACKGROUND: Ofloxacin (OFX) resistant Mycobacterium tuberculosis (MTB) isolates have been increasingly observed and are a major concern in recent years. This study investigated the genetic mutations associated with OFX resistance among clinical OFX mono-resistant MTB isolates from new and previously treated tuberculosis patients. METHODS: A total of 50 unrelated OFX mono-resistant MTB isolates were analyzed. For all isolates, the quinolone resistance determining regions of gyrA and gyrB were PCR amplified and sequenced. RESULTS: Single mutations in the quinolone resistance determining regions of gyrA (positions D94A, G, N, and Y; A90V; and S91P) and gyrB (positions T539A and E540D) were observed in 62% (31/50) and 4% (2/50) of all OFX mono-resistant isolates, respectively. No differences were detected between the proportions of isolates with mutations in gyrA/gyrB from new and previously treated tuberculosis patients (P=.820). CONCLUSIONS: Although mutations in gyrB were rare, they were as important as mutations in gyrA in predicting OFX resistance in MTB in Tianjin, China.

[Characteristics of Mycobacterium tuberculosis genotype and the relationship between Beijing genotype and drug-resistant phenotypes in Tianjin].

OBJECTIVE: To explore the distribution and characteristics on genotype of Mycobacterium tuberculosis and the relationship between Beijing genotype and drug-resistant phenotypes in Tianjin city. METHODS: 656 clinical strains were collected from Tianjin Center for Tuberculosis Control and ten other Tuberculosis Institute in Tianjin from January 2008 to June 2009. Information regarding administration, clinical as well as laboratory findings of patients were collected. Proportion method was adopted to detect the susceptibility on four anti-tuberculosis drugs, namely streptomycin (SM), isoniazid (INH), rifampicin (RFP) and ethambutol (EMB). Both Beijing and non-Beijing genotypes were differentiated by multiplex PCR. The relationship between Beijing genotype and drug-resistant phenotypes was analyzed. RESULTS: In this study, the overall resistance rate of MTB was 26.98%, with multidrug-resistant rate was 6.25%. Among 656 MTB strains, 600 isolates (91.46%) belonged to Beijing genotype. There was significant difference between Beijing and non-Beijing genotype (χ(2) = 4.26, P = 0.039) among the Tianjin household registered population. Concerning the drug resistance, there was no significant difference between the two groups. CONCLUSION: Beijing genotype strains were the predominant one in Tianjin. The proportion of people infected with the Beijing genotype strains in Tianjin household registration of patients was significantly higher than the proportion of patients in the floating population in the same region. Results from the statistical analysis did not reveal any statistically significant association between Beijing genotype and drug resistance.

Molecular characterization of drug-resistant Beijing family isolates of Mycobacterium tuberculosis from Tianjin, China.

OBJECTIVE: Tuberculosis remains a severe public health issue, and the Beijing family of mycobacterium tuberculosis (M. tuberculosis) is widespread in East Asia, especially in some areas in China, like Beijing and Tianjin. This study aimed at determining the mutation patterns of drug-resistant Beijing strains of M. tuberculosis isolated from Tianjin, China. METHODS: A total of 822 M. tuberculosis isolates were screened for drug resistance by an absolute concentration method and the genotype was identified by PCR. 169 drug-resistant isolates of the Beijing family were analyzed for the potential mutations in the rpoB, katG, inhA promoter region and in rpsL, rrs and embB genes, which are associated with resistance to rifampin (RFP), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) respectively by PCR and DNA sequencing. RESULTS: Fifty-eight out of 63 RFP-resistant isolates were found to carry the mutations within the 81-bp RFP resistance determining region (RRDR) of the rpoB gene and the most frequent mutations occurred at codon 531 (44.4%), 526 (28.6%), and 516 (7.9%) respectively. 16 mutation patterns affecting 12 different codons around the RRDR of rpoB were found. Of 116 INH-resistant isolates, 56 (48.3%) had the mutation of katG 315 (AGC-->ACC) (Ser-->Thr), 3 (2.6%) carried S315N (AGC-->AAC) and 27 (16.0%) had the mutation of inhA-15A-->T. 84 out of 122 SM-resistant isolates (68.9%) displayed mutations at the codons 43 or 88 with AAG-->AGG (Lys-->Arg) of the rpsL gene and 22 (18.0%) with the mutations at positions 513A-->C, 516C-->T or 905 A-->G in the rrs gene. Of 34 EMB-resistant isolates, 6 had mutation with M306V (ATG-->GTG), 3 with M306I (ATG-->ATT), 1 with M306I (ATG-->ATA), 1 with D328Y (GAT-->TAT), 1 with V348L (GTC-->CTC), and 1 with G406S (GGC-->AGC) in the embB gene. CONCLUSION: These novel findings extended our understanding of resistance-related mutations in the Beijing strains of M. tuberculosis and may provide a scientific basis for development of new strategies for diagnosis and control of tuberculosis in China and other countries where Beijing strains are prevalent.