Resistant Clinical Isolates Of Mycobacterium Tuberculosis Research Articles

Investigation of bedaquiline resistance and genetic mutations in multi-drug resistant Mycobacterium tuberculosis clinical isolates in Chongqing, China

BackgroundTo investigate the prevalence and molecular characterization of bedaquiline resistance among MDR-TB isolates collected from Chongqing, China.MethodsA total of 205 MDR-TB isolates were collected from Chongqing Tuberculosis Control Institute between March 2019 and June 2020. The MICs of BDQ were determined by microplate alamarblue assay. All strains were genotyped by melting curve spoligotyping, and were subjected to WGS.ResultsAmong the 205 MDR isolates, the resistance rate of BDQ was 4.4% (9/205). The 55 (26.8%) were from male patients and 50 (24.4%) were new cases. Furthermore, 81 (39.5%) of these patients exhibited lung cavitation, 13 (6.3%) patients afflicted with diabetes mellitus, and 170 (82.9%) isolates belonged to Beijing family. However, the distribution of BDQ resistant isolates showed no significant difference among these characteristics. Of the 86 OFX resistant isolates, 8 isolates were XDR (9.3%, 8/86). Six BDQ resistant isolates (66.7%, 6/9) and two BDQ susceptible isolates (1.0%, 2/196) carried mutations in Rv0678. A total of 4 mutations types were identified in BDQ resistant isolates, including mutation in A152G (50%, 3/6), T56C (16.7%, 1/6), GA492 insertion (16.7%, 1/6), and A274 insertion (16.7%, 1/6). BDQ showed excellent activity against MDR-TB in Chongqing.ConclusionsBDQ showed excellent activity against MDR-TB in Chongqing. The resistance rate of BDQ was not related to demographic and clinical characteristics. Mutations in Rv0678 gene were the major mechanism to BDQ resistance, with A152G as the most common mutation type. WGS has a good popularize value and application prospect in the rapid detection of BDQ resistance.

Bedaquiline resistant Mycobacterium tuberculosis clinical isolates with and without rv0678 mutations have similar growth patterns under varying BDQ drug pressure

Resistance associated mutations have been reported to alter the growth of Mycobacterium tuberculosis (MTB) isolates under drug pressure. However, there is little information on the growth characteristics of bedaquiline (BDQ) resistant isolates in the presence of BDQ. To further understand the role of rv0678, we aimed to study whether the presence of rv0678 variants in BDQ resistant isolates alters the killing effect of BDQ. We, therefore, selected BDQ resistant clinical MTB isolates with (n = 6) and without (n = 3) variants in rv0678 gene. Using time kill assays, growth inhibition; taken as the relative change in log average colony forming unit (CFU)/ml at selected time points (24–96 h), was studied at Minimum Inhibitory Concentrations (MICs): 0x, 1x, 2.5x, 5x, 7.5x, 10x for these isolates. Growth inhibition was then analyzed using Kruskal Wallis and Kolmogorov Smirnov tests in PRISM vr.9. During the 24–96 h lag phase isolates with and without variants in rv0678 showed a similar growth inhibition pattern. No difference was noted in growth inhibition between BDQ resistant isolates and H37Rv. These findings suggest that role of alternate mechanisms in contributing to BDQ tolerance needs to be explored.

Pyrazinamide resistance and pncA mutations in drug resistant Mycobacterium tuberculosis clinical isolates from Myanmar

Pyrazinamide (PZA) is an important anti-tuberculosis drug, which is active against semi-dormant bacilli and used as a component of first-line drugs and drug-resistant tuberculosis regimens. Mutations in pncA and its promoter region are main cause of PZA resistance. There are limited PZA susceptibility data as there is no routine drug susceptibility testing (DST) for PZA. This study was aimed to determine the proportion of PZA resistance among rifampicin-resistant tuberculosis patients and to identify mutations which are responsible for PZA resistance in pncA and its promoter region. Liquid-based DST was performed to detect PZA susceptibility on 192 culture positive rifampicin-resistant isolates collected from National Tuberculosis Reference Laboratory. Sequencing on pncA including its promoter region was performed and analysis was done on 157 isolates. Phenotypic PZA resistance was detected in 58.9% of isolates. Sixty-five different mutations were distributed in pncA or promoter region of 82 isolates. Sensitivity and specificity of pncA sequencing in detection of PZA resistance showed 89.8% and 95.6% respectively. High proportion of PZA resistance among rifampicin-resistant cases highlighted the need for effective treatment regimen development for PZA-resistant MDR-TB. It is also suggested that routine PZA susceptibility test should be incorporated to treatment monitoring regimen and National Drug Resistance surveys.

Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil.

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide.OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution.METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB).FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression.MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDRplus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil.

BACKGROUNDThe accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB.METHODWe studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible.FINDINGSAmong the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively.MAIN CONCLUSIONSBoth methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.

Mutations in catalase-peroxidase KatG from isoniazid resistant Mycobacterium tuberculosis clinical isolates: insights from molecular dynamics simulations.

The current multidrug therapy for tuberculosis (TB) is based on the use of isoniazid (INH) in combination with other antibiotics such as rifampin, ethambutol and pyrazinamide. Literature reports have shown that Mycobacterium tuberculosis, the causative agent of TB, has become resistant to this treatment by means of point mutations in the target enzymes of these drugs, such as catalase-peroxidase (KatG). By means of equilibrium molecular dynamics in the presence of the ligand, this work evaluated ten point mutations described in the enzyme KatG that are related to resistance to INH . The results showed that the resistance mechanism is related to stereochemical modifications at the N-terminal domain of the protein, which restrict INH access to its catalytic site, not involving mechanisms of electrostatic nature. These results show insights that can be useful for the identification of new anti-TB drugs which may be able to circumvent this mechanism of resistance.

Molecular characterization of Isoniazid and Rifampicin target genes in multi-drug resistant Mycobacterium tuberculosis isolates from southwest of Iran

Mycobacterium tuberculosis is a slow growing bacterium and the main reason of tuberculosis in the world. Despite numerous health policy on TB this disease remains a public health problem according to the vast mechanisms of resistant specially mutations in the related genes of this bacterium. In the present study other than allele-specific mutation detection occurrence of mutations were either evaluated in the katG regulatory and katG, inhA and rpoB structural regions in Isoniazid and Rifampicin resistant Mycobacterium tuberculosis clinical isolates from TB suspected individuals referred to the southwest TB center of Iran. The results of the study showed that about 65% of Isoniazid resistant isolates have mutation in katG or inhA related genes. Either about 60% of Rifampicin resistant isolates have mutation at least in one of rpoB516,526,531 codons. Mutation analysis in Isoniazid resistant isolates which not detected in previous step showed that seven of thirteen (53.84%) isolates have mutation in regulatory and structural inhA gene. Either mutation analysis in katG structural gene showed a point mutation in three of thirteen isolates (23.08%). In rpoB structural evaluation it has been deduced that seven of twelve (53.84%) of isolates have a different mutation algorithm in compare with sensitive isolates. In the study we have found that some isolates of our region don't follow the routine mutation algorithm like other studies. This may be related to different geographical field of study and immigration from different parts of the world to Iran. Based on these results we highly recommended in the situations where isolates detected sensitive according to the genetic results proportional anti-biogram methods must be implemented.

Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil.

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

Proteomics of multidrug resistant Mycobacterium tuberculosis clinical isolates: a peep show on mechanism of drug resistance & perhaps more.

Proteomics of multidrug resistant Mycobacterium tuberculosis clinical isolates: a peep show on mechanism of drug resistance & perhaps more.

Characterization of the genetic diversity of extensively-drug resistant Mycobacterium tuberculosis clinical isolates from pulmonary tuberculosis patients in Peru.

BackgroundPeru holds the fourth highest burden of tuberculosis in the Americas. Despite an apparently well-functioning DOTS control program, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase. To worsen this situation, cases of extensively drug resistance tuberculosis (XDR-TB) have been detected. Little information exists about the genetic diversity of drug-susceptible vs. MDR-TB and XDR-TB.MethodsCryopreserved samples of XDR strains from 2007 to 2009 (second semester), were identified and collected. Starting from 227 frozen samples, a total of 142 XDR-TB strains of Mycobacterium tuberculosis complex (MTBC; 1 isolate per patient) were retained for this study. Each strain DNA was analyzed by spoligotyping and the 15-loci Mycobacterial Interspersed Repetitive Unit (MIRU-15).ResultsAmong the 142 isolates analyzed, only 2 samples (1.41%) could not be matched to any lineage. The most prevalent sublineage was Haarlem (43.66%), followed by T (27.46%), LAM (16.2%), Beijing (9.15%), and X clade (1.41%). Spoligotype analysis identified clustering for 128/142 (90.1%) isolates vs. 49/142 (34.5%) with MIRUs. Of the samples, 90.85% belonged to retreated patients. The drug resistant profile demonstrated that 62.67% showed resistance to injectable drugs capreomycin (CAP) and kanamycin (KAN) vs. 15.5% to CAP alone and 21.8% to KAN alone. The SIT219/T1 and SIT50/H3 were the most prevalent patterns in our study. The spoligoforest analysis showed that SIT53/T1 was at the origin of many of the T lineage strains as well as a big proportion of Haarlem lineage strains (SIT50/H3, followed by SIT47/H1, SIT49/H3, and SIT2375/H1), as opposed to the SIT1/Beijing strains that did not appear to evolve into minor Beijing sublineages among the XDR-TB strains.ConclusionIn contrast with other Latin-American countries where LAM sublineage is the most predominant, we found the Haarlem to be the most common followed by T sublineage among the XDR-TB strains.

Publisher's Expression of Concern: Geographical Differences Associated with SNPs in Nine Gene Targets among Resistant Clinical Isolates of Mycobacterium tuberculosis

The American Society for Microbiology (ASM) and the Journal of Clinical Microbiology (JCM) are issuing this Expression of Concern to alert readers to questions about the ownership of the data in the following publication: M. Hoshide, L. Qian, C. Rodrigues, R. Warren, T. Victor, H. B. Evasco II, T. Tupasi, V. Crudu, and J. T. Douglas. Geographic Differences Associated with SNPs in Nine Gene Targets among Resistant Clinical Isolates of Mycobacterium tuberculosis. J. Clin. Microbiol. Published ahead of print 19 June 2013. doi:10.1128/JCM.00857-13. JCM has been notified that substantial portions of the data presented in the manuscript were derived from results of a multicenter study centered at the University of California in San Diego (UCSD) and funded by the National Institutes of Health, and that the manuscript by Hoshide et al. was submitted for publication without prior approval of the UCSD study center. This matter is now under discussion by the University of Hawaii, Dr. Douglas's institution, and UCSD. Pending resolution of the questions raised regarding the right of the authors to publish these data, JCM has placed publication of the final version of the manuscript on hold. It should be noted that, according to ASM policies prohibiting duplicate publication, while the Hoshide et al. manuscript remains published, JCM cannot consider papers from other participants in the multicenter study if they include essentially the same data.

A primary investigation on the isoniazid-induced alterations in efflux gene expression among the isoniazid resistant Mycobacterium tuberculosis clinical isolates

To detect the changes on the expression of putative drug efflux genes caused by isoniazid-inducement in single resistance to the isoniazid Mycobacterium tuberculosis (M. tuberculosis) clinical isolates, for exploring the putative efflux genes which causing M. tuberculosis isoniazid resistance as well as the mechanism related to high expression of the putative efflux genes. We selected 35 M. tuberculosis clinical isolates which were only resistant to isoniazid as well as 10 sensitive M. tuberculosis clinical isolates and using H37Rv as control. Each strain was cultured in 7H9 liquid medium without isoniazid and with subinhibitory isoniazid concentration (1/4 MIC) induction. After RNA extraction and reverse transcription, real-time PCR was conducted to assess the expression changes of 27 putative drug efflux pump genes with formula 2(-ΔΔCT) to calculate the expression of each putative drug efflux pump genes. Of the 27 putative genes, 13 of them were expressed at high level. High expression of Rv1258c gene had the maximum number of 6 strains, followed by high expression of Rv0849 and Rv2265 which both had 5 strains. Fourteen strains (40.00%) out of the 35 strains had high expression pump genes. Six strains (17.14%) had only one highly expressed putative efflux pump gene. Eight strains (22.86%) had two or more highly expressed putative efflux pump genes, including two, four, five, seven genes that highly expressed in 4, 2, 1, 1 strains respectively. For the 27 putative genes, ten sensitive strains and H37Rv did not show highly expressed genes. Rv1258c, Rv2265, Rv0849, etc. genes might be the putative efflux pumps genes of M. tuberculosis resistant to isoniazid. Isoniazid might serve as the inducer of M. tuberculosis part putative efflux pump genes, inducing activation and causing high expression of these putative efflux pump genes.

Geographical Profile ofrpoBGene Mutations in Rifampicin ResistantMycobacterium tuberculosisIsolates in Sri Lanka

The nature and frequency of mutations in the rpoB gene of rifampicin (RIF) resistant Mycobacterium tuberculosis clinical isolates varies considerably between different geographical regions. The objective of the present study was the identification of rpoB gene mutations responsible for RIF resistance in M. tuberculosis isolates in Sri Lanka. Three regions of the rpoB gene of M. tuberculosis, one corresponding to a 437-bp region, including the rifampicin resistance-determining region (RRDR) and two other regions (1395 bp and 872 bp) spanning the RRDR, were polymerase chain reaction amplified, and were subjected to DNA sequencing. The two mutations found within the RRDR in the 31 RIF resistant strains isolated in this study were at codon 526 (n=15, 48.4%) CAC (His)→TAC (Tyr) and codon 531 (n=3, 9.7%) TCG (Ser)→TTG (Leu). A significant proportion (n=15, 48.3%) showed mutations spanning the RRDR, including two novel mutations at codon 626 (n=13, 41.9%) GAC (Asp)→GAG (Glu) and 184 (n=2, 6.4%) GAC (Asp)→GAT (Asp), a silent mutation. Two isolates revealed double mutations (codons 626+526 and 626+184). The presence of a high frequency of new mutations, and the different frequencies of the universally prevailing mutations, as reported here, emphasizes the need for expanding the geographical database of mutations for effective application of an rpoB-based diagnosis of multidrug resistant tuberculosis.

Genotypic detection of mutations in rifampicin resistant clinical isolates ofMycobacterium tuberculosis.

The resistance of majority ofM. tuberculosis strains to rifampicin a key drug in the tuberculosis therapy, has been cause of major alarm world-wide. Such an association of strain with the drug takes a long time i.e. 2-3 months. We have modified a method for rapid detection of rifampicin resistant strains by screening them through PCR-SSCP.We have identified mutations in rpo, β region of the gene encoding, β sub-unit of RNA polymerase in rifampicin resistant 67 clinical isolates ofM. tuberculosis. Mutations were screened in these isolates by single strand confirmation polymorphism (SSCP). When DNA sequencing data was compared with the result of SSCP analysis, direct PCR sequencing results were more easily interpreted and contatined more sequence dependent information. These findings provide the basis for rapid detection of rifampicin esistance, a marker of multidrug resistance.

Trifluoperazine and CEF-allicin from garlic (Allium sativum) as potential new antitubercular drugs active against drug resistantMycobacterium tuberculosis

Trifluoperazine (TFP) and a compound called CEF-allicin purified from garlic (Allium sativum) possess antitubercular activity against both drug susceptible and resistant clinical isolates ofMycobacterium tuberculosis. They are bactericidal in nature with multiple sites of primary action. This new use for known drug TFP was based on our observation that mycobacteria have calmodulin like protein which regulates their metabolism and a calmodulin antagonist has antitubercular activity. The minimum inhibitory concentration (MIC) of TFP againstM. tuberculosis was 4-5 μg/ml. It inhibited considerably by 6hrs, the synthesis of total lipids from(14)C-acetate and proteins and DNA as judged by the uptake of(14)C-glycine and(3)H-thymidine respectively by the bacilli. With 50 clinical isolates from our hospital at Delhi, the MIC was 4μg/ml, for 40% and 8μg/ml, for 50% of the isolates susceptible as well as resistant to one or more of the five drugs isoniazid, rifampicin, streptomycin, ethambutol and pyrazinamide. The MIC of CEF-allicin was 25μg/ml, for bothMycobacterium tuberculosis and isoniazid resistant clinical isolate TRC-C 1193. It inhibited in 6hrs or less the synthesis of total lipids completely and proteins and DNA ofM. tuberculosis from its labeled precursors almost completely.