Molecular and genetic characterization of rifampicin- and/or izoniazid-resistent strains of M. tuberculosis, isolated in Novosibirsk and Tomsk regions

The purpose of the work was to study rifampicin- and izoniazid-resistent strains of M. tuberculosis, circulating in Western Siberia, by VNTR and IS6110 typing. The authors also studied genetic causes of resistance to these antibiotics and undertook a search of new VNTR loci, displaying polymorphism in genomes of closely related clonally-disseminated variants of Mycobacterium tuberculosis in W-Beijing family model analysis.     

Fitness cost of drug resistance in Mycobacterium tuberculosis

Multidrug-resistant (MDR) - and extensively drug-resistant (XDR) - forms of tuberculosis are growing public health problems. Mathematical models predict that the future of the MDR and XDR tuberculosis epidemics depends in part on the competitive fitness of drug-resistant strains. Here, recent experimental and molecular epidemiological data that illustrate how heterogeneity among drug-resistant strains of Mycobacterium tuberculosis can influence the relative fitness and transmission of this pathogen are reviewed.     

Drug-resistant tuberculosis: resistance mechanisms and drug susceptibility of Mycobacterium tuberculosis

Mycobacterium (M.) tuberculosis is the most common individual causative agent of infectious disease in the world. It is responsible for 26% of preventable deaths in adulthood. Because the number of new cases grows at an annual rate of 2%, in 1993 WHO proclaimed tuberculosis a global health problem. The immediate cause for this was coinfection with causative agents of tuberculosis and human immunodeficiency virus, and spread of resistant and multiresistant strains of M. tuberculosis (MDR TB). It is estimated that 50 million people are infected with resistant strains of M. tuberculosis. Tuberculosis has emerged as a major public health problem for its high mortality (50%-80%) in the first 4-16 weeks of the disease and 100 times more expensive therapy for drug-resistant than for drug-susceptible tuberculosis. Mycobacteriologic laboratories play a fundamental role in the detection, combat and control of tuberculosis, especially in preventing the spread of drug-resistant tuberculosis. In this connection, there is an increased need of a rapid and reliable determination of the susceptibility of isolated strains of M. tuberculosis to the first- and second-line antituberculotic drugs.     

分析5种结核杆菌耐药基因突变与耐药水平的关系

目的：分析5种结核杆菌（M.tb)耐药基因突变的情况，了解基因突变和耐药水平的关系。方法：134例临床分离株均做传统梯度药敏试验和聚合酶链反应－单链构象多态性I（PCR－SSCP）试验。结果：耐PZA(pncA),SM(rpsL),REP(rpoB),INH(katG),EMB(embB)基因突变率分别为42．7％、72％、78％、69％和43．9％，其中，上述高耐株基因突变率分别为70％、87．2％、93．4％、80％、43．9％。低耐株分别为12．5％、28．5％、45．4％、18．7％，EMB在低耐区无基因突变，结论：M.tb耐药基因变与耐药水平联系密切，多数M.tb耐药基因突变易发生在高耐药区，也有少数菌基因突变易发生在低耐药区。     

Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance

The genome sequence of Mycobacterium tuberculosis revealed the presence of 12 membrane proteins proposed to have a function in the transport of lipids. Insertional inactivation of 11 of these has revealed that only 1 (MmpL3) is apparently essential for viability. Five of these proteins are conserved within the genome of Mycobacterium leprae. The drug susceptibilities of these 11 mutants to a broad spectrum of agents are unaltered, suggesting that unlike their function in other organisms, these proteins do not play a significant role in intrinsic drug resistance. Each of these mutants was assessed for growth kinetics and lethality in a murine low-dose aerosol model of tuberculosis, and four were found to be impaired in one or both measures of virulence. Two of these, with mutations of MmpL4 and the previously characterized MmpL7, which transports phthiocerol dimycocerosate, were found to have both impaired growth kinetics and impaired lethality. Mutants with inactivation of MmpL8, which transports a precursor of the sulfatides, or MmpL11, which transports an unknown substrate, were found to establish infection normally but to be significantly attenuated for lethality in time-to-death studies. These studies support the concept that MmpL-mediated lipid secretion both contributes to the innate ability of the pathogen to survive intracellularly and also contributes directly to the host-pathogen dialogue that determines the ultimate outcome of infection.     

Evaluation of fluoromycobacteriophages for detecting drug resistance in Mycobacterium tuberculosis

We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ～2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved.     

Mutations in the rpoB and katG genes leading to drug resistance in Mycobacterium tuberculosis in Latvia

To characterize the genetic basis of drug resistance in Mycobacterium tuberculosis in Latvia, mutations involved in rifampin (rpoB gene) and isoniazid (katG gene) resistance in DNA from 19 drug-susceptible and 51 multidrug-resistant M. tuberculosis complex isolates were analyzed. The most frequent rpoB gene mutations found by the Line Probe assay were the S531L (14 of 34 isolates), D516V (7 of 34), H526D (4 of 34), and D516Y plus P535S (4 of 34) mutations. Direct sequencing of seven isolates with unclear results from Line Probe assay showed the presence of the L533P mutation and the Q510H plus H526Y (1 of 34) and D516V plus P535S (4 of 34) double mutations, neither of which has been described previously. Single-strand conformation polymorphism analysis showed strand mobility differences between the rifampin-susceptible and -resistant samples for the D516V, H526D, and D516Y plus P535S mutations but not for the S531L mutation. Nucleotide substitution at codon 315 (AGC-->ACC) of the katG gene was found in 48 of 51 multidrug-resistant samples by sequencing. Furthermore, katG gene restriction fragment length polymorphism analysis with endonuclease AciI confirmed the nucleotide change in codon 315.     

Strain diversity,epistasis and the evolution of drug resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis harbours little DNA sequence diversity compared with other bacteria. However, there is mounting evidence that strain-to-strain variation in this organism has been underestimated. We review our current understanding of the genetic diversity among M. tuberculosis clinical strains and discuss the relevance of this diversity for the ongoing global epidemics of drug-resistant tuberculosis. Based on findings in other bacteria, we propose that epistatic interactions between pre-existing differences in strain genetic background, acquired drug-resistance-conferring mutations and compensatory changes could play a role in the emergence and spread of drug-resistant M. tuberculosis.     

Genetic polymorphism of clinical Mycobacterium tuberculosis strains circulating in the territory of Novosibirsk Region

Mycobacterium tuberculosis strains isolated from patients treated at TB dispensary branches in different districts of Novosibirsk were studied by genetic analysis. The below molecular methods were used: 1. PCR with random primers; 2. A method based on variable number of tandem repeats in loci; 3. IS6110 inverse PCR. Thirty-five samples of genome DNA of M. tuberculosis isolated were analyzed. Each of the 3 methods detected the main group of isolates, which comprised 61.8% of closely related strains revealed by method 1, 75.8%--by method 2, and 74.3%--by method 3. The remaining clusters were represented by 1 to 4 strains. The data obtained denote a relative homogeneity of M. tuberculosis strains circulating in Novosibirsk Region. No interplay was detected between the clustering of isolates and the presence or absence of mutation in genes conditioning the resistance to antibiotics.     

Molecular characterization and drug resistance testing of Mycobacterium tuberculosis isolates from Chad

The molecular characterizations of the first 40 Mycobacterium tuberculosis isolates from Chad revealed a high proportion of isolates of the Cameroon family (33%), of which one isolate showed a monodrug resistance. In total, 9/33 (27%) isolates were resistant to isoniazid. The implications of these findings are discussed.     

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide

The microscopic observation drug susceptibility assay (MODS) was evaluated to determine susceptibility to pyrazinamide in Mycobacterium tuberculosis, and compared with the broth microdilution method (BMM), absolute concentration method (ACM), and pyrazinamidase (PZase) determination. We tested 34 M. tuberculosis clinical isolates (24 sensitive and eight resistant to pyrazinamide) and the control strains M. tuberculosis H37Rv (ATCC 27294) and Mycobacterium bovis AN5. The MODS, BMM, ACM and PZase determination provided results in average times of 6, 18, 28 and 7 days, respectively. All methods showed excellent sensitivity and specificity (p <0.05). Of the methods studied, the MODS proved to be faster, efficient, inexpensive, and easy to perform. However, additional studies evaluating the MODS in differentiating pyrazinamide-resistant and pyrazinamide-susceptible M. tuberculosis must be conducted with a larger number of clinical isolates.     

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.     

Rapid drug resistance detection in Mycobacterium tuberculosis: a review of colourimetric methods

Several new methods to detect drug resistance in Mycobacterium tuberculosis have been proposed in recent years. Colourimetric methods that use redox indicators or the nitrate reduction assay have received increasing attention because of their simplicity and the absence of any requirement for sophisticated equipment or highly trained personnel. Several studies have evaluated their accuracy and performance in comparison with reference standard methods, particularly for the detection of resistance to rifampicin and isoniazid, which are the two most important drugs used for the treatment of tuberculosis. This review describes the development, evaluation and implementation of these methods as rapid alternative tests for the detection of multidrug resistance in M. tuberculosis. Based on published evidence and the high accuracy of colourimetric methods for detecting drug resistance in M. tuberculosis, these methods seem to be appropriate for implementation in high-burden low-resource countries.     

Mycobacterium tuberculosis mixed infections and drug resistance in sub-Saharan Africa: a systematic review

BackgroundSub-Saharan Africa, is a region that records high rates of TB infection. Mycobacterium tuberculosis mixed strain infection, especially when the strains involved are of different susceptibilities, is an area of great interest because it is linked with an increased risk of treatment failure and transmission of resistant strains within the population. This paper reviewed original studies that reported MTB mixed infection and heteroresistance in the region between 2010 and 2020 to understand the extent of mixed strain infection and heteroresistance in the region. This information is very critical in the control of TB and ending the TB epidemic by 2035 as per the World Health Organization''s vision.Methodspubmed, Scopus, JSTOR, AJOL, and Google Scholar databases were searched through both key terms and subject headings. The literature was screened, assessed for the quality and evidence synthesized.ResultsEighteen original articles were included in this review after having met the inclusion criteria. The frequency of mixed strain infection reported in these studies varied between 2.8% and 21.1% while drug resistance range between 0.06% to 19% depending on the study design and the drug susceptibility screening technique utilized. The majority of the studies (50%) utilized Spoligotyping in conjunction with MIRU-VNTR typing in the detection of mixed infections.ConclusionDespite the scarcity of data on mixed infections and heteroresistance in sub-Saharan Africa, various studies have revealed that these conditions are frequent in the region than previously thought. Given the evidence of the effect of mixed infections on drug resistance and treatment outcome, we conclude that mixed infection is an unavoidable topic for future studies.     

Multidrug resistance to Mycobacterium tuberculosis in a tertiary hospital

OBJECTIVE: The magnitude of drug-resistant Mycobacterium tuberculosis infection (MDR-TB) in Nigeria, the most populous country in sub-Saharan Africa, is largely unknown. This information would assist policymakers to develop intervention strategies against tuberculosis (TB) in the country. MATERIALS AND METHODS: This is a one-year laboratory-based study. Specimens from suspected new TB patients sent to the TB laboratory of the Department of Medical Microbiology, University College Hospital Ibadan, Nigeria from May 1, 2005 to April 27, 2006 were processed and analyzed. The specimens were stained with Ziehl-Neelsen (Z-N) reagents and cultured on Lowenstein-Jensen medium, incubated at 37 degrees C for 6-8 weeks. Isolates were confirmed as MDR-TB by Z-N reactions and biochemical methods. Drug susceptibility to streptomycin, ethambutol, rifampicin and isoniazid was done using Bactec 460 TB radiometric method. RESULTS: Of the 1,120 specimens processed, 80 (7.1%) were smear positive, while 56 (5.0%) were culture positive, even though the association was not statistically significant (p > 0.05). Culture contamination rate was 8.8%. Thirty (53.6%) of the culture positive isolates were resistant to both isoniazid and rifampicin, while 26 (46.4%) were susceptible. About half--53.3%--of the resistant isolates were from the antiretroviral clinic, while 10 (33.4%) were from peripheral centers. CONCLUSION: This study shows that MDR-TB is emerging in Nigeria. Further studies on MDR-TB are urgently needed in the country to ascertain the magnitude of the problem and to proffer solutions to it.     

Trained innate immunity and resistance to Mycobacterium tuberculosis infection

BackgroundSome individuals, even when heavily exposed to an infectious tuberculosis patient, develop neither active nor latent tuberculosis infection (LTBI). This ‘early clearance’ of Mycobacterium tuberculosis is associated with a history of bacillus Calmette–Guérin (BCG) vaccination. As BCG vaccination can boost innate immune responses through a process termed ‘trained immunity’, we hypothesize that BCG-induced trained innate immunity contributes to early clearance of M. tuberculosis.ObjectivesWe describe the epidemiological evidence and biological concepts of early clearance and trained immunity, and the possible relation between these two processes through BCG vaccination.SourcesRelevant data from published reports up to November 2018 were examined in the conduct of this review.ContentSeveral observational studies and one recent randomized trial support the concept that boosting innate immunity contributes to protection against M. tuberculosis infection, with BCG vaccination providing approximately 50% protection. The molecular mechanisms mediating early clearance remain largely unknown, but we propose that trained immunity, characterized by epigenetic and metabolic reprogramming of innate immune cells such as monocytes or macrophages, is at least partially responsible for eliminating the mycobacteria and inducing early clearance.ImplicationsFuture studies should examine if BCG revaccination increases early clearance of M. tuberculosis through induction of trained immunity. Epigenetic or metabolic modulation may further boost BCG-induced trained innate immunity to promote tuberculosis prevention. New tuberculosis vaccine candidates should also be examined for their capacity to improve protection against M. tuberculosis infection and induce trained immunity.     

Mixed infection with Beijing and non-Beijing strains and drug resistance pattern of Mycobacterium tuberculosis

Mixed infection with Beijing and non-Beijing strains of Mycobacterium tuberculosis has been reported and has been suggested to mediate elevation of the reinfection rate in regions with a high incidence of tuberculosis (TB). To evaluate the prevalence of infection with both Beijing and non-Beijing strains of M. tuberculosis in eastern Taiwan, the region with the highest TB incidence in Taiwan, 185 active pulmonary TB patients were enrolled at Tzu Chi General Hospital from October 2007 to September 2008. A modified multiplex PCR method was developed to distinguish Beijing and non-Beijing strains directly using the sputum of patients. Of the 185 patients, 46.5% were infected with a Beijing strain, 42.2% were infected with a non-Beijing strain, and 11.3% were infected with both strain types. Notably, mixed infection with both strain types was not associated with TB treatment history or the high-incidence race group, aborigines. In addition, the incidence rate of mixed infection before treatment with anti-TB medication was as high as that in patients with a history of anti-TB treatment. Further analysis of antibiotic susceptibility revealed that Beijing strains alone had the highest multidrug resistance rate (17.5%), mixed infection had the highest rate of resistance to at least one drug (23.8%), and non-Beijing strains had the highest rate of sensitivity to all drugs (79.5%), implying that Beijing strains are predominant in the development of drug resistance in tuberculosis.     

Detection of multidrug resistance in Mycobacterium tuberculosis

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.