scholarly journals In Vitro Study of Stepwise Acquisition of rv0678 and atpE Mutations Conferring Bedaquiline Resistance

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Nabila Ismail ◽  
Nazir A. Ismail ◽  
Shaheed V. Omar ◽  
Remco P. H. Peters
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Solid Medium ◽  
In Vitro Study ◽  
Whole Genome ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Atcc Strain ◽  
High Level ◽  
Level Resistance

ABSTRACT Bedaquiline resistance within Mycobacterium tuberculosis may arise through efflux-based (rv0678) or target-based (atpE) pathway mutations. M. tuberculosis mutant populations from each of five sequential steps in a passaging approach, using a pyrazinamide-resistant ATCC strain, were subjected to MIC determinations and whole-genome sequencing. Exposure to increasing bedaquiline concentrations resulted in increasing phenotypic resistance (up to >2 μg/ml) through MIC determination on solid medium (Middlebrook 7H10). rv0678 mutations were dynamic, while atpE mutations were fixed, once occurring. We present the following hypothesis for in vitro emergence of bedaquiline resistance: rv0678 mutations may be the first transient step in low-level resistance acquisition, followed by high-level resistance due to fixed atpE mutations.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Isoniazid Resistance in Mycobacterium tuberculosis Is a Heterogeneous Phenotype Composed of Overlapping MIC Distributions with Different Underlying Resistance Mechanisms

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Arash Ghodousi ◽  
Elisa Tagliani ◽  
Eranga Karunaratne ◽  
Stefan Niemann ◽  
Jennifer Perera ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanisms ◽  
Whole Genome ◽  
Tube System ◽  
Isoniazid Resistance ◽  
Content Type ◽  
Indicator Tube ◽  
Promoter Mutations ◽  
Level Resistance ◽  
Growth Indicator

ABSTRACT MIC testing using the Bactec mycobacteria growth indicator tube system 960 of 70 phylogenetically diverse, isoniazid-resistant clinical strains of Mycobacterium tuberculosis revealed a complex pattern of overlapping MIC distributions. Whole-genome sequencing explained most of the levels of resistance observed. The MIC distribution of strains with only inhA promoter mutations was split by the current concentration endorsed by the Clinical and Laboratory Standards Institute to detect low-level resistance to isoniazid and is, consequently, likely not optimally set.

scholarly journals Contribution of rpoB Mutations to Development of Rifamycin Cross-Resistance in Mycobacterium tuberculosis

1998 ◽  
Vol 42 (7) ◽  
pp. 1853-1857 ◽  
Author(s):  
D. L. Williams ◽  
L. Spring ◽  
L. Collins ◽  
L. P. Miller ◽  
L. B. Heifets ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cross Resistance ◽  
In Vitro Mutagenesis ◽  
Missense Mutations ◽  
Resistance Mutations ◽  
Development Of Resistance ◽  
High Level ◽  
The Impact ◽  
Level Resistance

ABSTRACT The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the β-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutantrpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoBmutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specificrpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

scholarly journals Plasmodium falciparum Resistance to a Lead Benzoxaborole Due to Blocked Compound Activation and Altered Ubiquitination or Sumoylation

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kirthana M. V. Sindhe ◽  
Wesley Wu ◽  
Jenny Legac ◽  
Yong-Kang Zhang ◽  
Eric E. Easom ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Stress Responses ◽  
Mechanisms Of Action ◽  
Antimalarial Drugs ◽  
New Drugs ◽  
Loss Of Function ◽  
Content Type ◽  
High Level ◽  
Level Resistance

ABSTRACT New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured Plasmodium falciparum, against fresh Ugandan P. falciparum isolates, and in murine malaria models. To gain mechanistic insights, we selected in vitro for P. falciparum isolates resistant to AN13762. In all of 11 independent selections with 100 to 200 nM AN13762, the 50% inhibitory concentration (IC50) increased from 18–118 nM to 180–890 nM, and whole-genome sequencing of resistant parasites demonstrated mutations in prodrug activation and resistance esterase (PfPARE). The introduction of PfPARE mutations led to a similar level of resistance, and recombinant PfPARE hydrolyzed AN13762 to the benzoxaborole AN10248, which has activity similar to that of AN13762 but for which selection of resistance was not readily achieved. Parasites further selected with micromolar concentrations of AN13762 developed higher-level resistance (IC50, 1.9 to 5.0 μM), and sequencing revealed additional mutations in any of 5 genes, 4 of which were associated with ubiquitination/sumoylation enzyme cascades; the introduction of one of these mutations, in SUMO-activating enzyme subunit 2, led to a similar level of resistance. The other gene mutated in highly resistant parasites encodes the P. falciparum cleavage and specificity factor homolog PfCPSF3, previously identified as the antimalarial target of another benzoxaborole. Parasites selected for resistance to AN13762 were cross-resistant with a close analog, AN13956, but not with standard antimalarials, AN10248, or other benzoxaboroles known to have different P. falciparum targets. Thus, AN13762 appears to have a novel mechanism of antimalarial action and multiple mechanisms of resistance, including loss of function of PfPARE preventing activation to AN10248, followed by alterations in ubiquitination/sumoylation pathways or PfCPSF3. IMPORTANCE Benzoxaboroles are under study as potential new drugs to treat malaria. One benzoxaborole, AN13762, has potent activity and promising features, but its mechanisms of action and resistance are unknown. To gain insights into these mechanisms, we cultured malaria parasites with nonlethal concentrations of AN13762 and generated parasites with varied levels of resistance. Parasites with low-level resistance had mutations in PfPARE, which processes AN13762 into an active metabolite; PfPARE mutations prevented this processing. Parasites with high-level resistance had mutations in any of a number of enzymes, mostly those involved in stress responses. Parasites selected for AN13762 resistance were not resistant to other antimalarials, suggesting novel mechanisms of action and resistance for AN13762, a valuable feature for a new class of antimalarial drugs.

scholarly journals Point Mutations within the Fatty Acid Synthase Type II Dehydratase Components HadA or HadC Contribute to Isoxyl Resistance in Mycobacterium tuberculosis

2012 ◽  
Vol 57 (1) ◽  
pp. 629-632 ◽  
Author(s):  
Laila Gannoun-Zaki ◽  
Laeticia Alibaud ◽  
Laurent Kremer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Point Mutations ◽  
Mycolic Acid ◽  
Type Ii ◽  
Content Type ◽  
High Level ◽  
Fas Ii ◽  
Level Resistance

ABSTRACTThe mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO inMycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

scholarly journals Identification of FosA8, a Plasmid-Encoded Fosfomycin Resistance Determinant from Escherichia coli, and Its Origin in Leclercia adecarboxylata

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Laurent Poirel ◽  
Xavier Vuillemin ◽  
Nicolas Kieffer ◽  
Linda Mueller ◽  
Marie-Christine Descombes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Natural Resistance ◽  
Whole Genome ◽  
Content Type ◽  
Acid Identity ◽  
E Coli ◽  
Fosfomycin Resistance ◽  
High Level ◽  
Level Resistance

ABSTRACT A plasmid-located fosfomycin resistance gene, fosA8, was identified from a CTX-M-15-producing Escherichia coli isolate recovered from urine. Identification of this gene was obtained by whole-genome sequencing. It encoded FosA8, which shares 79% and 78% amino acid identity with the most closely related FosA2 and FosA1 enzymes, respectively. The fosA8 gene was located on a transferable 50-kb plasmid of IncN type encoding high-level resistance to fosfomycin. In silico analysis and cloning experiments identified fosA8 analogues (99% identity) in the genome of Leclercia decarboxylata, which is an enterobacterial species with natural resistance to fosfomycin. This finding adds L. decarboxylata to the list of enterobacterial species that are a reservoir of fosA-like genes which have been captured from the chromosome of a progenitor and are then acquired by E. coli.

scholarly journals Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

2006 ◽  
Vol 50 (8) ◽  
pp. 2860-2862 ◽  
Author(s):  
Emma Huitric ◽  
Jim Werngren ◽  
Pontus Juréen ◽  
Sven Hoffner
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Point Mutations ◽  
Gene Mutations ◽  
Rpob Gene ◽  
Clinical Isolates ◽  
Large Deletions ◽  
High Level ◽  
Level Resistance

ABSTRACT The distribution and resistance levels of 189 in vitro-selected rifampin-resistant Mycobacterium tuberculosis mutants of Beijing and other genotypes were determined. Apart from a higher amount of codon 522 point mutations and large deletions, a spread of mutations similar to that reported for clinical isolates was seen. Most mutations were correlated with high-level resistance; a lower level, or a MIC of <16 mg/liter, was associated with codon 522 mutations. Multiple mutations were detected in two Beijing mutants.

scholarly journals Origins of a 350-Kilobase Genomic Duplication in Mycobacterium tuberculosis and Its Impact on Virulence

2014 ◽  
Vol 82 (7) ◽  
pp. 2902-2912 ◽  
Author(s):  
Pilar Domenech ◽  
Anya Rog ◽  
Jalal-ud-din Moolji ◽  
Nicolas Radomski ◽  
Ashley Fallow ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Selective Pressure ◽  
East Asian ◽  
Whole Genome ◽  
Content Type ◽  
Asian Lineage ◽  
Mouse Model Of Infection ◽  
Genomic Duplication

ABSTRACTIn the present study, we have investigated the evolution and impact on virulence of a 350-kb genomic duplication present in the most recently evolved members of theMycobacterium tuberculosisEast Asian lineage. In a mouse model of infection, comparing HN878 subclones HN878-27 (no duplication) and HN878-45 (with the 350-kb duplication) revealed that the latter is impaired forin vivogrowth during the initial 3 weeks of infection. Furthermore, the median survival time of mice infected with isolate HN878-45 is significantly longer (77 days) than that of mice infected with HN878-27. Whole-genome sequencing of both isolates failed to reveal any mutational events other than the duplication that could account for such a substantial difference in virulence. Although we and others had previously speculated that the 350-kb duplication arose in response to some form of host-applied selective pressure (P. Domenech, G. S. Kolly, L. Leon-Solis, A. Fallow, M. B. Reed, J. Bacteriol.192:4562–4570, 2010, and B. Weiner, J. Gomez, T. C. Victor, R. M. Warren, A. Sloutsky, B. B. Plikaytis, J. E. Posey, P. D. van Helden, N. C. Gey van Pittius, M. Koehrsen, P. Sisk, C. Stolte, J. White, S. Gagneux, B. Birren, D. Hung, M. Murray, J. Galagan, PLoS One7:e26038, 2012), here we show that this large chromosomal amplification event is very rapidly selected within standardin vitrobroth cultures in a range of isolates. Indeed, subclones harboring the duplication were detectable after just five rounds ofin vitropassage. In contrast, the duplication appears to be highly unstablein vivoand is negatively selected during the later stages of infection in mice. We believe that the rapidin vitroevolution ofM. tuberculosisis an underappreciated aspect of its biology that is often ignored, despite the fact that it has the potential to confound the data and conclusions arising from comparative studies of isolates at both the genotypic and phenotypic levels.

scholarly journals Correlation of molecular resistance mechanisms and phenotypic resistance levels in streptomycin-resistant Mycobacterium tuberculosis.

1996 ◽  
Vol 40 (11) ◽  
pp. 2452-2454 ◽  
Author(s):  
A Meier ◽  
P Sander ◽  
K J Schaper ◽  
M Scholz ◽  
E C Böttger
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanism ◽  
Active Agent ◽  
Cross Resistance ◽  
Permeability Barrier ◽  
Wild Type ◽  
Resistance Mutations ◽  
Phenotypic Resistance ◽  
Level Resistance

Quantitative susceptibility testing of clinical isolates of streptomycin-resistant Mycobacterium tuberculosis demonstrated that there is a close correlation between the molecular resistance mechanism and the in vitro activity of streptomycin: mutations in rpsL were mainly associated with high-level resistance, mutations in rrs were associated with an intermediate level of resistance, and streptomycin-resistant isolates with wild-type rpsL and rrs exhibited a low-level resistance phenotype. Investigations of streptomycin-resistant isolates with wild-type rpsL and rrs revealed that (i) there is no cross-resistance to other drugs and (ii) a permeability barrier may contribute to resistance, because resistance was significantly lowered in the presence of a membrane-active agent.

scholarly journals Screening the Pathogen Box for Identification of Candida albicans Biofilm Inhibitors

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Taissa Vila ◽  
Jose L. Lopez-Ribot
Keyword(s):  
Candida Albicans ◽  
Biofilm Formation ◽  
Antibiofilm Activity ◽  
Potential Candidate ◽  
Predominant Role ◽  
Chemical Library ◽  
Content Type ◽  
High Level ◽  
Level Resistance

ABSTRACT Candida albicans remains the main causative agent of candidiasis, one of the most frequent nosocomial infections, with unacceptably high mortality rates. Biofilm formation is a major risk factor for invasive candidiasis, as Candida biofilms display high-level resistance to most antifungal agents. In this work we have screened the Pathogen Box chemical library (Medicines for Malaria Venture [MMV], Switzerland) in search for inhibitors of C. albicans biofilm formation. Our initial screen identified seven hits, and additional dose-response assays confirmed the biofilm-inhibitory activity of six of these small molecules. Three compounds, MMV688768, MMV687273, and MMV687807, were also able to reduce the metabolic activity of cells within preformed biofilms. Interestingly, the most potent of these, compound MMV688768, displayed increased antibiofilm activity compared to its activity against planktonic cultures, indicating that it may affect processes with a predominant role during the biofilm mode of growth. This compound demonstrated a high selectivity index when its antibiofilm activity was compared with its toxicity in liver hepatocellular cells. In vitro combination assays showed a synergistic interaction between compound MMV688768 and fluconazole against preformed biofilms. Overall, our results indicate that this compound may constitute a potential candidate for further clinical development.

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