<Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> mutants with multidrug resistance: History of origin,genetic and molecular mechanisms of resistance,and emerging challenges

The review summarizes the data on the Mycobacterium tuberculosis mutations that lead to multidrug resistance (MDR) to various antibiotics. MDR strains arose over the past 30 years as a variety of antituberculosis drugs were introduced in medicine, and they largely discount the results of chemotherapy for tuberculosis. The most dangerous of them are strains with extensive drug resistance (XDR), which are resistant to four or five different drugs on average. The molecular mechanisms that make a strain resistant are considered. XDR and MDR strains result from successive and usually independent resistance mutations, which arise in various regions of the mycobacterial genome. In addition, the formation of resistant strains is affected by the phenomenon of tolerance and mycobacterial latency in infected tissues.     

Tellurite: history, oxidative stress, and molecular mechanisms of resistance

The perceived importance of tellurium (Te) in biological systems has lagged behind selenium (Se), its lighter sister in the Group 16 chalcogens, because of tellurium's lower crustal abundance, lower oxyanion solubility and biospheric mobility and the fact that, unlike Se, Te has yet to be found to be an essential trace element. Te applications in electronics, optics, batteries and mining industries have expanded during the last few years, leading to an increase in environmental Te contamination, thus renewing biological interest in Te toxicity. This chalcogen is rarely found in the nontoxic, elemental state (Te⁰), but its soluble oxyanions, tellurite (TeO₃²⁻) and tellurate (TeO₄²⁻), are toxic for most forms of life even at very low concentrations. Although a number of Te resistance determinants (Tel^R) have been identified in plasmids or in the bacterial chromosome of different species of bacteria, the genetic and/or biochemical basis underlying bacterial TeO₃²⁻ toxicity is still poorly understood. This review traces the history of Te in its biological interactions, its enigmatic toxicity, importance in cellular oxidative stress, and interaction in cysteine metabolism.     

Analysis of the genetic determinants of multidrug and extensive drug resistance in Mycobacterium tuberculosis with the use of an oligonucleotide microchip

Steadily growing resistance of the tuberculosis causative agent towards a broad spectrum of antituberculosis drugs calls for rapid and reliable methods for identifying the genetic determinants responsible for this resistance. In this study, we present a biochip-based method for simultaneous identification of mutations within rpoB gene associated with rifampin resistance, mutations in katG, inhA, ahpC genes responsible for isoniazid resistance, mutations within the regions of gyrA and gyrB genes leading to fluoroquinolones resistance, and mutations in the rrs gene and the eis promoter region associated with the resistance to kanamycin, capreomycin and amikacin. The oligonucleotide microchip, as the core element of this assay, provides simultaneous identification of 99 mutations in the format “one sample—one PCR—one microchip”, and it makes it possible to complete analysis of multidrug-resistant and extensively drug-resistant tuberculosis within a single day. The tests on 63 Mycobacterium tuberculosis clinical isolates with different resistance profiles using the developed approach allows us to reveal the spectrum of drug-resistance associated mutations, and to estimate the significance of the inclusion of extra genetic loci in the determination of M. tuberculosis drug resistance.     

结核分枝杆菌对异烟肼耐药的分子机制

抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述.     

Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

Prediction of drug resistance in M. tuberculosis: molecular mechanisms,tools, and applications

Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis. These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis.     

结核分枝杆菌耐药性的分子生物学研究进展

结核病一直是世界性问题,我国其发病情况尤为严重,是亚洲的第二大结核病发病国家。结核病治疗方面常使用抗生素作为首选药物,随着抗菌药的滥用,结核杆菌对多种抗菌药产生耐药性,结核病耐药患者增多,治疗难度增加。因此,结核杆菌耐药分子机制的研究更加重要,新型抗结核药物研制更加迫切。结核分枝杆菌的基因突变是引起耐药的主要分子学依据,因此基于结核分枝杆菌耐药性相关基因的深入探索,对于预防结核病的传播及治疗皆具有深远影响。本文从分子生物学角度分析了近年来结核分枝杆菌耐药性产生的原因及相关研究进展。     

The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis

Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have emerged worldwide. In many countries and regions, these resistant strains constitute a serious threat to the efficacy of tuberculosis control programs. An important element in gaining control of this epidemic is developing an understanding of the molecular basis of resistance to the most important antituberculosis drugs: isoniazid, rifampin, and pyrazinamide. On the basis of this information, more exacting laboratory testing, and ultimately more appropriate and timely treatment regimens, can be developed.     

Advances in the development of molecular genetic tools for Mycobacterium tuberculosis

Mycobacterium tuberculosis, a Gram-positive bacterium of great clinical relevance, is a lethal pathogen owing to its complex physiological characteristics and development of drug resistance. Several molecular genetic tools have been developed in the past few decades to study this microorganism. These tools have been instrumental in understanding how M. tuberculosis became a successful pathogen. Advanced molecular genetic tools have played a significant role in exploring the complex pathways involved in M. tuberculosis pathogenesis. Here, we review various molecular genetic tools used in the study of M. tuberculosis. Further, we discuss the applications of clustered regularly interspaced short palindromic repeat interference (CRISPRi), a novel technology recently applied in M. tuberculosis research to study target gene functions. Finally, prospective outcomes of the applications of molecular techniques in the field of M. tuberculosis genetic research are also discussed.     

Aphid resistance in Brassica crops: challenges, biotechnological progress and emerging possibilities

Aphids, (Hemiptera: Aphidoidea) a nefarious insect pest of Brassicaceae members including major vegetable and oilseed crops have coevolved with their host plant and emerged as most economically important insect pest of crop Brassicas. Their atypical feeding mechanism and unusual reproductive biology made them intractable to control below economic threshold level of damage to the crops. To a large extent aphid infestation is controlled by spraying agrochemicals of systemic mode of action and rarely by biological control. Use of systemic insecticides is highly cost intensive as well poses bigger threat of their incorporation in dietary chain. Breeding for genetic resistance against aphids has not been possible owing to the non-availability of resistance source within the crossable germplasms and lack of knowledge of the genetics of the trait. Genetic engineering with insect resistant transgenes seems to be the only potential avenue to address this difficult-to-accomplish breeding objective. Some success had been achieved in terms of developing aphid resistant cultivars through genetic engineering however, commercial utilization of such crops are still awaited. Thus protection of crops against aphids necessarily requires more research to identify either more effective insecticidal transgenes or biological phenomenon that can usher to new mechanism of resistance. The present review is an attempt to highlight the current status and possible avenues to develop aphid resistance in Brassicaceae crops.     

Molecular mechanisms of adefovir sensitivity and resistance in HBV polymerase mutants: a molecular dynamics study

Molecular modeling studies of adefovir diphosphate with the wild type and the mutant HBV polymerase-DNA complex demonstrated that the increase in adefovir sensitivity toward HBV polymerase mutants (rtL180M, rtM204V/I, rtL180M-M204V/I) is a result of increased van der Waals interaction and is supplemented by the decreased affinity of natural substrate toward the mutant HBV polymerase. In the case of rtN236T mutant, loss of two hydrogen bonds accompanied by significant decrease in electrostatic interactions is observed, which explains the observed decrease in drug sensitivity and binding affinity of adefovir diphosphate toward the rtN236T mutant HBV polymerase.     

Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics

Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major beta-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most beta-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based beta-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from beta-lactamase deletion mutants) that are hypersusceptible to beta-lactam antibiotics might reveal novel genes involved with other mechanisms of beta-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine beta-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode beta-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.     

New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.