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Contribution of Efflux to the Emergence of Isoniazid and Multidrug Resistance in Mycobacterium tuberculosis  [PDF]
Diana Machado, Isabel Couto, Jo?o Perdig?o, Liliana Rodrigues, Isabel Portugal, Pedro Baptista, Bruno Veigas, Leonard Amaral, Miguel Viveiros
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034538
Abstract: 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.
Clinical Characteristics and Treatment Outcomes of Patients with Low- and High-Concentration Isoniazid-Monoresistant Tuberculosis  [PDF]
Tsai-Yu Wang, Shu-Min Lin, Shian-Sen Shie, Pai-Chien Chou, Chien-Da Huang, Fu-Tsai Chung, Chih-His Kuo, Po-Jui Chang, Han-Pin Kuo
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086316
Abstract: Background Isoniazid (INH) resistance is now the most common type of tuberculosis (TB) infection resistance worldwide. The aim of this study was to evaluate the clinical characteristics and treatment outcomes of patients with low- and high-concentration INH-monoresistant TB. Methods One hundred and thirty-four patients with culture-confirmed INH-monoresistant TB during 2006 January to 2007 December were retrospectively enrolled. INH resistance was classified as either low-concentration or high-concentration resistance according to the critical concentrations of 0.2 μg/mL or 1 μg/mL of INH, respectively. The patients’ clinical outcomes, treatment regimens, and treatment duration were analyzed. Results The treatment success rates between low- and high-concentration INH-resistant TB were similar (81.8% vs. 86.7%). The treatment regimens and treatment duration were similar between both groups. Only a minor percentage of the patients in both groups received 6-month treatment regimens (low vs. high concentration resistance, 9.1% vs. 13.3%; respectively, p = 0.447) The most common reason for treatment duration longer than 6 months was pyrazinamide given for less than 6 months, followed by a delay in clinical response to treatment. Multivariable analysis showed that prior tuberculosis treatment (Odds ratio, 2.82, 95% C.I., 1.02–7.77, p = 0.045) was the only independent risk factor for unsuccessful treatment outcome. Conclusion Different levels of INH resistance did not affect the treatment outcomes of patients with INH-monoresistant tuberculosis. Prolonged Rifampin-containing regimens may achieve those good outcomes in patients with low- and high-concentration INH-monoresistant TB.
Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia
Belay Tessema, Joerg Beer, Frank Emmrich, Ulrich Sack, Arne C Rodloff
BMC Infectious Diseases , 2012, DOI: 10.1186/1471-2334-12-37
Abstract: Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany.Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType? MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType? MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance.The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType? MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH, RMP and MDR. However, the GenoType? MTBDRsl assay shows limitations in detecting resistance to EMB.According to the World Health Organization (WHO) report, the proportion of multidrug resistant tuberculosis (MDR-TB), resistant to at least isoniazid and rifampicin among new and previously treated TB cases globally ranges from 0% to 28.3% and from 0% to 61.6%, respectively [1]. In Ethiopia, the countrywide anti-TB drug resistance survey conducted in 2005 showed that the prevalence of MDR-TB
Molecular detection of rifampin and isoniazid resistance to guide chronic TB patient management in Burkina Faso
Paolo Miotto, Nuccia Saleri, Mathurin Dembelé, Martial Ouedraogo, Gisèle Badoum, Gabriele Pinsi, Giovanni B Migliori, Alberto Matteelli, Daniela M Cirillo
BMC Infectious Diseases , 2009, DOI: 10.1186/1471-2334-9-142
Abstract: Samples were collected in Burkina Faso where culture and DST are not currently available, and where chronic cases are therefore classified and treated based on clinical evaluation and sputum-smear microscopy results. One hundred and eight chronic TB patients (sputum smear-positive, after completing a re-treatment regimen for pulmonary TB under directly observed therapy) were enrolled in the study from December 2006 to October 2008. Two early morning sputum samples were collected from each patient, immediately frozen, and shipped to Italy in dry ice. Samples were decontaminated, processed for smear microscopy and DNA extraction. Culture was attempted on MGIT960 (Becton Dickinson, Cockeysville, USA) and decontaminated specimens were analyzed for the presence of mutations conferring resistance to rifampin and isoniazid by the molecular assay GenoType? MTBDRplus.We obtained a valid molecular test result in 60/61 smear-positive and 47/47 smear-negative patients.Among 108 chronic TB cases we identified patients who (i) harboured rifampin- and isoniazid-susceptible strains (n 24), (ii) were negative for MTB complex DNA (n 24), and (iii) had non-tuberculous mycobacteria infections (n 13). The most represented mutation conferring rifampin-resistance was the D516V substitution in the hotspot region of the rpoB gene (43.8% of cases). Other mutations recognized were the H526D (15.6%), the H526Y (15.6%), and the S531L (9.4%).All isoniazid-resistant cases (n 36) identified by the molecular assay were carrying a S315T substitution in the katG gene. In 41.7% of cases, a mutation affecting the promoter region of the inhA gene was also detected.The GenoType? MTBDRplus assay performed directly on sputum specimens improves the management of chronic TB cases allowing more appropriate anti-TB regimens.Emergence of drug-resistant Mycobacterium tuberculosis (MTB) strains is considered a real threat to achieving tuberculosis (TB) control [1-3]. Furthermore, multidrug-resistant TB (MDR-TB) a
Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR  [cached]
Hristea A,Otelea D,Paraschiv S,Macri A
Indian Journal of Medical Microbiology , 2010,
Abstract: Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR)-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible) using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100%) and 95% (95% CI: 89% to 100%), respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100%) and 74% (95% CI: 61% to 87%), respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.
Drug resistance-conferring mutations in Mycobacterium tuberculosis from Madang, Papua New Guinea
Ballif Marie,Harino Paul,Ley Serej,Coscolla Mireia
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-191
Abstract: Background Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. Results Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; Isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; Streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. Conclusions We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.
Multiple- mutations in the katG gene of Mycobacterium tuberculosis isolates correlate with high- level of resistance to isoniazid in patients with active pulmonary tuberculosis from Belarus
Saeed Zakerbostanabad
Iranian Journal of Microbiology , 2009,
Abstract: Background and Objectives: The aim of this study was to investigate the significance of multiple-mutations in the katG gene, predominant nucleotide changes and its correlation with high level of resistance to isoniazid in Mycobacterium tuberculosis isolates that were randomly collected from sputa of 42 patients with primary and secondary active pulmonary tuberculosis from different geographic regions of Belarus."nMaterials and Methods: Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA extraction, katG amplification, and DNA sequencing analysis were performed."nResults: Thirty four (80%) isolates were found to have multiple-mutations (composed of 2-5 mutations) in the katG. Increased number of predominant mutations and nucleotide changes were demonstrated in codons 315 (AGC→ACC) , 316 (GGC→AGC) , 309 (GGT→GTT) with a higher frequency among patients bearing secondary tuberculosis infection with elevated levels of resistance to isoniazid (MIC μg/ml ≥ 5-10) . Furthermore it was demonstrated that the combination of mutations with their predominant nucleotide changes were also observed in codons 315, 316, and 309 indicating higher frequencies of mutations among patients with secondary infection respectively."nConclusion: In this study 62% (n=21) of multi-mutated isolates found to have combination of mutations with predominant nucleotide changes in codons 315 (AGC→ACC) , 316 (GGC→GTT) , 309 (GGT→GGT) , and also demonstrated to be more frequent in isolates of patients with secondary infections, bearing higher level of resistance to isoniazid (≥ 5 -10μg/ml).
Screening for Streptomycin Resistance-Conferring Mutations in Mycobacterium tuberculosis Clinical Isolates from Poland  [PDF]
Tomasz Jagielski, Helena Ignatowska, Zofia Baku?a, ?ukasz Dziewit, Agnieszka Napiórkowska, Ewa Augustynowicz-Kope?, Zofia Zwolska, Jacek Bielecki
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0100078
Abstract: Currently, mutations in three genes, namely rrs, rpsL, and gidB, encoding 16S rRNA, ribosomal protein S12, and 16S rRNA-specific methyltransferase, respectively, are considered to be involved in conferring resistance to streptomycin (STR) in Mycobacterium tuberculosis. The aim of this study was to investigate the spectrum and frequency of these mutations in M. tuberculosis clinical isolates, both resistant and susceptible to STR. Sixty-four M. tuberculosis isolates recovered from as many TB patients from Poland in 2004 were included in the study. Within the sample were 50 multidrug-resistant (32 STR-resistant and 18 STR-susceptible) and 14 pan-susceptible isolates. Preliminary testing for STR resistance was performed with the 1% proportion method. The MICs of STR were determined by the Etest method. Mutation profiling was carried out by amplifying and sequencing the entire rrs, rpsL, and gidB genes. Non-synonymous mutations in either rrs or rpsL gene were detected in 23 (71.9%) of the STR-resistant and none of the STR-susceptible isolates. Mutations in the gidB gene were distributed among 12 (37.5%) STR-resistant and 13 (40.6%) STR-susceptible isolates. Four (12.5%) STR-resistant isolates were wild-type at all three loci examined. None of the rrs, rpsL or gidB mutations could be linked to low, intermediate or high level of STR resistance. In accordance with previous findings, the gidB 47T→G (L16R) mutation was associated with the Latin American-Mediterranean genotype family, whereas 276A→C (E92D) and 615A→G (A205A) mutations of the gidB gene were associated with the Beijing lineage. The study underlines the usefulness of rrs and rpsL mutations as molecular markers for STR resistance yet not indicative of its level. The gidB polymorphisms can serve as phylogenetic markers.
Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase
Vivian N Hawkins, Alyson Auliff, Surendra Prajapati, Kanchana Rungsihirunrat, Hapuarachchige C Hapuarachchi, Amanda Maestre, Michael T O'Neil, Qin Cheng, Hema Joshi, Kesara Na-Bangchang, Carol Sibley
Malaria Journal , 2008, DOI: 10.1186/1475-2875-7-72
Abstract: The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network.Synonymous and non-synonymous single nucleotide polymorphisms (SNPs) within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel). SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N) dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T) and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T) mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu.It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts sharply with the selective sweep of rare antifolate resistant alleles observed in the P. falciparum populations in Asia and Africa. The finding of multiple origins of resistance-conferring mutations has important implications for drug policy.In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and subsequently spread in natural populations. This is a major issue in Plasmodium falciparum, where resistance to two safe, inexpensive drugs, chloroquine and sulphadoxine-pyrimethamine, has spread widely in en
Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance
Ming Shi, Andrée Lougarre, Carole Alies, Isabelle Frémaux, Zhen Tang, Jure Stojan, Didier Fournier
BMC Evolutionary Biology , 2004, DOI: 10.1186/1471-2148-4-5
Abstract: To obtain information on the origin of the fitness of resistant alleles, we studied Drosophila melanogaster acetylcholinesterase, the target of organophosphate and carbamate insecticides. We produced in vitro 15 possible proteins resulting from the combination of the four most frequent mutations and we tested their catalytic activity and enzymatic stability. Mutations affected deacetylation of the enzyme, decreasing or increasing its catalytic efficiency and all mutations diminished the stability of the enzyme. Combination of mutations result to an additive alteration.Our findings suggest that the alteration of activity and stability of acetylcholinesterase are at the origin of the fitness cost associated with mutations providing resistance. Magnitude of the alterations was related to the allelic frequency in Drosophila populations suggesting that the fitness cost is the main driving force for the maintenance of resistant alleles in insecticide free conditions.Since the 1940's, the entire planet has been spread with insecticides and a lot of insects have developed resistance. As treatment is not continuous, insects have to adapt to be competitive in alternating periods with and without treatments. Thus, insecticide resistance offers the opportunity to study the adaptation of eukaryotes to variable environments. Resistance can be defined as the adaptation of a population from an environment free of insecticide to a new environment contaminated with new toxic molecules. Three main mechanisms of resistance to insecticides occur: reduction of insecticide penetration, increased degradation and modification of the insecticide target. One target is well documented: acetylcholinesterase (AChE, EC 3. 1. 1. 7). It is a key enzyme in the cholinergic synapses where it rapidly terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Organophosphates are substrates of AChE and their hydrolysis results in the phosphorylation of the active serin
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