Multiple drug resistance and conservative amplification of the H region in Leishmania major

Amplification of the H region has been previously observed in methotrexate (MTX)-resistant strains of Leishmania major and in unselected laboratory stocks of L. tarentolae. We now show that selection of L. major with the structurally unrelated drugs primaquine or terbinafine generated resistant lines exhibiting H region amplification and 23- and 12-fold cross-resistance to MTX, respectively. These and other drug-resistant lines bearing H region amplification also exhibited weak cross-resistance to primaquine and terbinafine, associating the amplified H region with pleiotropic resistance to MTX and other drugs. In contrast, lines selected for chloroquine or pentamidine resistance did not show H region amplification or this pattern of drug cross-resistance. The primaquine- and terbinafine-selected lines exhibited wild-type levels of dihydrofolate reductase-thymidylate synthase and normal uptake and accumulation of MTX, and the MTX resistance of these lines was not reversed by verapamil. These data suggest that the mechanism of MTX cross-resistance associated with H region amplification is novel and distinct from that mediated by overexpression of MDR genes in multidrug-resistant mammalian cells. Structural studies indicated that the amplified H region DNA in these L. major lines was largely (possibly exclusively) extra-chromosomal and consisted of circular inverted repeats joined at two DNA rearrangement junctions. Southern blot analyses showed that these rearrangement junctions were identical in four independent cell lines, suggesting that these sites are "hotspots" for DNA rearrangement. H region amplification in all of these lines was conservative, defined as retention of the chromosomal H region locus without structural alteration or reduction in copy number. This finding is consistent with an over-replication/recombination model for amplification of the H region.     

Modeling the emergence of the 'hot zones': tuberculosis and the amplification dynamics of drug resistance

'Hot zones' are areas that have >5% prevalence (or incidence) of multidrug-resistant tuberculosis (MDRTB). We present a new mathematical model (the amplifier model) that tracks the emergence and evolution of multiple (pre-MDR, MDR and post-MDR) strains of drug-resistant Mycobacterium tuberculosis. We reconstruct possible evolutionary trajectories that generated hot zones over the past three decades, and identify the key causal factors. Results are consistent with recently reported World Health Organization (WHO) data. Our analyses yield three important insights. First, paradoxically we found that areas with programs that successfully reduced wild-type pansensitive strains often evolved into hot zones. Second, some hot zones emerged even when MDR strains were substantially less fit (and thus less transmissible) than wild-type pansensitive strains. Third, levels of MDR are driven by case-finding rates, cure rates and amplification probabilities. To effectively control MDRTB in the hot zones, it is essential that the WHO specify a goal for minimizing the amplification probability.     

AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis

The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.     

Molecular characterization of Mycobacterium tuberculosis isolates in a region of Brazil with a high incidence of tuberculosis

One hundred and seventy Mycobacterium tuberculosis clinical isolates were characterized by spoligotyping to evaluate the biodiversity of tubercle bacilli in a region of Brazil with a high incidence of tuberculosis (Pelotas and Rio Grande cities - Rio Grande do Sul State). The spoligotyping results were compared to the World Spoligotyping Database (Institut Pasteur de Guadeloupe), which contains data from >14,000 worldwide isolates of M. tuberculosis. The isolates clustered by spoligotyping were further characterized by IS6110-RFLP to confirm the clonal relationship. Sixty-six different spoligotypes were identified, grouping 125 of the isolates (74%). Approximately half of the isolates belonged to seven of the most frequently occurring spoligotypes in the database. Three shared types (with two or more isolates) not previously identified were given the type numbers 826, 827 and 863. An additional 45 spoligotypes were identified that did not match any existing database pattern. RFLP characterization reduced the number of isolates in most of the clusters, thereby showing a higher differentiation capacity than spoligotyping. These results highlight the importance of molecular epidemiology studies of tuberculosis in insufficiently studied regions with a high TB burden, in order to uncover the true extent of genetic diversity of the pathogen.     

Treating tuberculosis with high doses of anti-TB drugs: mechanisms and outcomes

Tuberculosis (TB) is considered as one of the most serious threats to public health in many parts of the world. The threat is even more severe in the developing countries where there is a lack of advanced medical amenities and contemporary anti-TB drugs. In such situations, dosage optimization of existing medication regimens seems to be the only viable option. Therapeutic drug monitoring study results suggest that high-dose treatment regimens can compensate the low serum concentration of anti-TB drugs and shorten the therapy duration. The article presents a critical review on the possible changes that occur in the host and the pathogen upon the administration of standard and high-dose regimens. Some of the most common factors that are responsible for low anti-TB drug concentrations in the serum are differences in hosts’ body weight, metabolic processing of the drug, malabsorption and/or drug–drug interaction. Furthermore, failure to reach the cavitary pulmonary and extrapulmonary tissues also contributes to the therapeutic inefficiency of the drugs. In such conditions, administration of higher doses can help in compensating the pathogenic outcomes of enhancement of the pathogen’s physical barriers, efflux pumps and genetic mutations. The present article also presents a summary of the recorded treatment outcomes of clinical trials that were conducted to test the efficacy of administration of high dose of anti-tuberculosis drugs. This review will help physicians across the globe to understand the underlying pathophysiological changes (including side effects) that dictate the clinical outcomes in patients administered with standard and/or high dose anti-TB drugs.     

Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis

This minireview presents recent developments in molecular methods for the diagnosis of tuberculosis, including detection, identification and determination of drug resistance of Mycobacterium tuberculosis . Tuberculosis remains one of the major causes of global death from a single infectious agent. This situation is worsened by the HIV/AIDS pandemic because one-third of HIV/AIDS patients are coinfected with M. tuberculosis . Also of great concern is the emergence of drug-resistant tuberculosis because there are almost no treatment options available for patients affected by highly resistant strains of M. tuberculosis . Advances in molecular biology techniques and a better knowledge of the molecular mechanisms of drug resistance have provided new tools for the rapid diagnosis of tuberculosis. Several nucleic acid amplification technologies have been developed and evaluated. New molecular approaches are being introduced continuously. This minireview will also comment on the future perspectives for the molecular diagnosis of tuberculosis and the feasibility for the implementation of these newer techniques in the clinical diagnostic laboratory.     

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.     

Efflux as a mechanism for drug resistance in Mycobacterium tuberculosis

Tuberculosis remains an important global public health problem, with an estimated prevalence of 14 million individuals with tuberculosis worldwide in 2007. Because antibiotic treatment is one of the main tools for tuberculosis control, knowledge of Mycobacterium tuberculosis drug resistance is an important component for the disease control strategy. Although several gene mutations in specific loci of the M. tuberculosis genome have been reported as the basis for drug resistance, additional resistance mechanisms are now believed to exist. Efflux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic and eukaryotic cells. Mycobacterium tuberculosis presents one of the largest numbers of putative drug efflux pumps compared with its genome size. Bioinformatics as well as direct and indirect evidence have established relationships among drug efflux with intrinsic or acquired resistance in M. tuberculosis. This minireview describes the current knowledge on drug efflux in M. tuberculosis.     

Recombination sites in plasmid drug resistance gene amplification.

The resistance plasmid NR1 derivative pRR330 consists of a neomycin-kanamycin resistance gene (neo-kan) flanked by directly repeated sequences of both insertion element IS1 DNA (768 base pairs) and 840 base pairs of DNA which are a part of the chloramphenicol acetyltransferase (cam) gene. Most Escherichia coli cell populations that were cultured in high neomycin concentrations carried plasmids whose neo-kan gene amplification was mediated either by IS1 DNA or by cam DNA as homologous recombination sites. This suggests that the final amplified cell populations were the descendants of a single cell.     

Gene amplification in murine leukemia cells with multiple drug resistance acquired in vivo

The P388rm and P388rx cell lines resistant to antracycline antibiotics were obtained as a result of chemotherapy of mice bearing P388 leukemia, by means of increasing dosages of rubomycin and ruboxyl, respectively. These cell lines possessed cross-resistance to vinblastine, vincristine, colchicine, actinomycin D and some other drugs. Multidrug resistance (MDR) of P388rm and P388rx is due to decreased uptake of different cytotoxic compounds by the cells. Development of resistance to rubomycin and ruboxyl was accompanied by the appearance of additional chromosomal structures--long homogeneously staining regions (HSRs), double minute chromosomes and others usually containing amplified DNA sequences. Southern blot-hybridization with cloned DNA fragments amplified in Djungarian and Chinese hamster cell lines having MDR has revealed in P388rm and P388rx cells approximately 50-fold amplification of mdr and pC52 genes. Thus, in mouse leukemia cells which acquired MDR in vivo, as a result of chemotherapy, amplification is observed of the same genes that undergo amplification during selection of cell cultures for MDR in vitro.     

The road to drug resistance in Mycobacterium tuberculosis

Sequencing of serial isolates of extensively drug-resistant tuberculosis highlights how drug resistance develops within a single patient and reveals unexpected levels of pathogen diversity.Tuberculosis (TB) remains a crucial public health problem, with increasing drug resistance posing a challenge to current control efforts. Treatment regimens for drug-susceptible TB are onerous, requiring a minimum of six months of treatment with four antitubercular drugs. There are patients who develop multi-drug-resistant (MDR), extensively drug-resistant (XDR) and totally drug-resistant (TDR) forms, which are successively more difficult to treat. In these circumstances, treatment regimens involve the use of a larger number of less-effective drugs, which have a narrower therapeutic margin.In many bacteria, drug-resistance determinants are carried on mobile genetic elements. However, in Mycobacterium tuberculosis (Mtb), drug resistance is exclusively associated with point mutations and chromosomal rearrangements. Poor or intermittent therapy has long been thought to be the major explanation for drug resistance, and it is believed that drug-resistant strains develop through the sequential fixation of a small set of mutations, such that the pathogen samples only a small proportion of possible evolutionary paths [1].The application of whole-genome sequencing (WGS) has revealed previously underappreciated levels of genetic diversity within circulating Mtb populations, and the implications of this diversity for transmission and disease outcomes are increasingly being acknowledged. By contrast, mycobacterial heterogeneity within a single host, and any concomitant biological or clinical significance, has been explored but seldom documented.In a study published in this issue of Genome Biology, Eldholm and colleagues apply WGS to investigate the evolution from drug-sensitive to XDR-TB within a single patient [2]. This adds to an emerging body of evidence that suggests that intra-host microbial diversity is substantial and might have significant consequences when inferring transmission. There are few instances, if any, in the literature where this has been investigated in such detail.     

Recent updates on drug resistance in Mycobacterium tuberculosis

Tuberculosis (TB) along with acquired immune deficiency syndrome and malaria rank among the top three fatal infectious diseases which pose threat to global public health, especially in middle and low income countries. TB caused by Mycobacterium tuberculosis (Mtb) is an airborne infectious disease and one-third of the world's population gets infected with TB leading to nearly 1·6 million deaths annually. TB drugs are administered in different combinations of four first-line drugs (rifampicin, isoniazid, pyrazinamide and ethambutol) which form the core of treatment regimens in the initial treatment phase of 6–9 months. Several reasons account for the failure of TB therapy such as (i) late diagnosis, (ii) lack of timely and proper administration of effective drugs, (iii) lower availability of less toxic, inexpensive and effective drugs, (iv) long treatment duration, (v) nonadherence to drug regimen and (vi) evolution of drug-resistant TB strains. Drug-resistant TB poses a significant challenge to TB therapy and control programs. In the background of worldwide emergence of 558 000 new TB cases with resistance to rifampicin in the year 2017 and of them, 82% becoming multidrug-resistant TB (MDR-TB), it is essential to continuously update the knowledge on the mechanisms and molecular basis for evolution of Mtb drug resistance. This narrative and traditional review summarizes the progress on the anti-tubercular agents, their mode of action and drug resistance mechanisms in Mtb. The aim of this review is to provide recent updates on drug resistance mechanisms, newly developed/repurposed anti-TB agents in pipeline and international recommendations to manage MDR-TB. It is based on recent literature and WHO guidelines and aims to facilitate better understanding of drug resistance for effective TB therapy and clinical management.     

Mutations linked with antimicrobial resistance in Mycobacterium tuberculosis isolated from tuberculosis patients in the Samara region

A total of 234 M. tuberculosis isolates were used to demonstrate the leading role of mutations in, respectively, codon 531 of gene rpoB (90.0%) and codon 315 of gene katG (92.9%), in the development of resistance to rifampicin and isoniazid by the methods of reverse hybridization with oligonucleotide probes and the sequencing of gene stretches. The levels of primary resistance of M. tuberculosis to rifampicin, isoniazid and multiresistance, according to the molecular-genetic analysis, were 41.0%, 57.7% and 37.2% respectively. The coincidence of the results of the bacteriological and molecular-genetic analyses of the antimicrobial resistance of the isolates was 90.4% and 95.3% for isoniazid and rifampicin respectively. The prevalence of individual types of mutations, linked with antimicrobial resistance, in the presence of a considerable spread of strains of the family Beijing in the region may be indicative of the limited number of M. tuberculosis clones circulating in the region.     

Pleiotropic drug resistance and gene amplification in a SEWA mouse tumor cell line : Complex relations revealed by drug uptake data, and lipid and protein analysis

SEWA mouse lines resistant to actinomycin D (AMD) or vincristine (VCR) exhibit the pleiotropic drug resistance (PDR) phenotype, and express a low-MW protein (p21) and numerous double minutes (DM). In drug uptake studies these lines were compared with the non-resistant parental line and with a methotrexate (MTX)-resistant line, not exhibiting PDR. On treatment with labelled AMD or VCR the two PDR lines displayed a highly reduced intracellular content of drug, whereas uptake of MTX was unchanged. Uptake of AMD was shown to be temperature-dependent. The MTX-resistant line did not exhibit any significant change in AMD or VCR uptake. Other workers have emphasized the role of a high-MW glycoprotein in the development of PDR. A search for a similar glycoprotein in our cells was unsuccessful. Since all indications point to membrane factors being important in the development of PDR, the lines were also subjected to lipid analysis. Compared with control cells distinct differences were detected in the lipid composition of all resistant lines (including the MTX-resistant line). In the course of our experiments, the DM in our most AMD-resistant line were replaced by two homogeneously staining regions (HSR). Simultaneously, the overproduction of p21 ceased, but the PDR phenotype persisted. This event tends to implicate a minor role for the p21 protein in PDR, but similar transitions from DM to HSR in other AMD-resistant SEWA lines were not accompanied by a decrease in p21 over-production. Our data point to a complex genetic control of multi-drug resistance.     

结核分枝杆菌的耐药基因及其相关分子机制

结核分枝杆菌原发性和继发性耐药是当前控制和治疗结核病面临的重要问题,随着分子遗传学的发展,已经阐明了结核分枝杆菌耐药的分子基础是染色体的突变,影响了药靶本身或激活了药物前体的细菌酶,造成MTB的耐药。本文主要就MTB对其常用药物的耐药机制展开讨论,以便正确认识MTB对不同药物的耐药机制,建立快速检测耐药结核分枝杆菌基因型的分子生物学方法。     

结核分枝杆菌的耐药基因及其相关分子机制

结核分枝杆菌原发性和继发性耐药是当前控帝】和治疗结核病面临的重要问题,随着分子遗传学的发展,已经阐明了结核分枝杆菌耐药的分子基础是染色体的突变,影响了药靶本身或激活了药物前体的细菌酶,造成MTB的耐药。本文主要就MTB对其常用药物的耐药机制展开讨论,以便正确认识MTB对不同药物的耐药机制,建立快速检测耐药结核分枝杆菌基因型的分子生物学方法。