scholarly journals In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Matthew B. McNeil ◽  
Devon D. Dennison ◽  
Catherine D. Shelton ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
23S Rrna ◽  
Clinical Settings ◽  
Content Type ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Ribosomal Protein L3 ◽  
Resistant Strains

ABSTRACT Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA (rrl). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings.

scholarly journals Characterization of Mutations Conferring Resistance to Rifampin inMycobacterium tuberculosisClinical Strains

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Tomasz Jagielski ◽  
Zofia Bakuła ◽  
Anna Brzostek ◽  
Alina Minias ◽  
Radosław Stachowiak ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Content Type ◽  
Control Programs ◽  
Gene Coding ◽  
H37rv Strain ◽  
Different Types ◽  
Resistant Strains

ABSTRACTResistance ofMycobacterium tuberculosisto rifampin (RMP), mediated by mutations in therpoBgene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individualrpoBmutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout therpoBgene among 115Mycobacterium tuberculosisclinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discoveredrpoBmutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results fromin vitromutagenesis experiments were combined with the assessment of the prevalence ofrpoBmutations, and their reciprocal co-occurrences, across globalM. tuberculosispopulations. Twenty-two different types of mutations in therpoBgene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50and MIC90values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenicM. tuberculosisH37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

scholarly journals Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Zhaojing Zong ◽  
Wei Jing ◽  
Jin Shi ◽  
Shu'an Wen ◽  
Tingting Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Novel Mutation ◽  
Multidrug Resistant ◽  
23S Rrna ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Significant Difference ◽  
Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

scholarly journals Isolation and characterization of arsenic resistant soil bacteria and their effects on germination of rice under arsenic contamination

2015 ◽  
Vol 2 (2) ◽  
pp. 229-237
Author(s):  
Istiaq Ahmed ◽  
Md Tofazzal Islam ◽  
Md Akhter Hossain Chowdhury ◽  
Md Kamruzzaman
Keyword(s):  
Contaminated Soils ◽  
Sodium Arsenite ◽  
Agar Plate ◽  
Rice Seedling ◽  
Resistant Bacteria ◽  
Gram Staining ◽  
Isolation And Characterization ◽  
Resistant Strains

This study was carried out to isolate, screen and characterize arsenic (As) resistant bacteria from As contaminated soils of Dumrakandi and Matlab under Faridpur and Chandpur districts and to evaluate their efficiency in reducing As toxicity against rice seedlings during germination. Thirteen strains were isolated from the soils which showed resistance to different levels of sodium arsenite (viz. 5, 10, 20 and 40 mM) in both agar plate and broth assay using BSMY I media. Among the isolates, BTL0011, BTL0012, BTL0015 and BTL0022 showed highest resistance to 40 mM sodium arsenite. Gram staining and KOH solubility test revealed that five strains were gram positive and rest eight was gram negative. They grew well in the liquid media at pH 5.5 to 8.5. In-vitro rice seedling bioassay with two superior isolates (BTL0011 and BTL0022) revealed that As resistant strains significantly enhanced seed germination of BRRI dhan29 and BRRI dhan47 at 60 ppm As. This study was laid out in CRD with three replications. The performance of BTL 0022 was superior to BTL0011. The overall results suggest that BTL0011 and BTL0022 can be used for bioremediation of As contaminated soils and to increase the germination and seedling growth of rice in As contaminated soils.Res. Agric., Livest. Fish.2(2): 229-237, August 2015

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

2015 ◽  
Vol 59 (8) ◽  
pp. 4446-4452 ◽  
Author(s):  
Vadim Makarov ◽  
João Neres ◽  
Ruben C. Hartkoorn ◽  
Olga B. Ryabova ◽  
Elena Kazakova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitro Group ◽  
Covalent Bond ◽  
Antimycobacterial Activity ◽  
Content Type ◽  
Sar Studies ◽  
Covalent Bond Formation

ABSTRACT8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity againstMycobacterium tuberculosisin vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml againstM. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorablein vitroabsorption-distribution-metabolism-excretion/toxicity (ADME/T) andin vivopharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

scholarly journals In Vitro Activity and MIC of Sitafloxacin against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis Isolated in Thailand

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Manoon Leechawengwongs ◽  
Therdsak Prammananan ◽  
Sarinya Jaitrong ◽  
Pamaree Billamas ◽  
Nampueng Makhao ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Resistant Strains ◽  
Drug Resistant Strains

ABSTRACT New fluoroquinolones (FQs) have been shown to be more active against drug-resistant Mycobacterium tuberculosis strains than early FQs, such as ofloxacin. Sitafloxacin (STFX) is a new fluoroquinolone with in vitro activity against a broad range of bacteria, including M. tuberculosis. This study aimed to determine the in vitro activity of STFX against all groups of drug-resistant strains, including multidrug-resistant M. tuberculosis (MDR M. tuberculosis), MDR M. tuberculosis with quinolone resistance (pre-XDR), and extensively drug-resistant (XDR) strains. A total of 374 drug-resistant M. tuberculosis strains were tested for drug susceptibility by the conventional proportion method, and 95 strains were randomly submitted for MIC determination using the microplate alamarBlue assay (MABA). The results revealed that all the drug-resistant strains were susceptible to STFX at a critical concentration of 2 μg/ml. Determination of the MIC90s of the strains showed different MIC levels; MDR M. tuberculosis strains had a MIC90 of 0.0625 μg/ml, whereas pre-XDR and XDR M. tuberculosis strains had identical MIC90s of 0.5 μg/ml. Common mutations within the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB did not confer resistance to STFX, except that double mutations of GyrA at Ala90Val and Asp94Ala were found in strains with a MIC of 1.0 μg/ml. The results indicated that STFX had potent in vitro activity against all the groups of drug-resistant M. tuberculosis strains and should be considered a new repurposed drug for treatment of multidrug-resistant and extensively drug-resistant TB.

scholarly journals Isolation and characterization of fluconazole- and amphotericin B-resistant Candida albicans from blood of two patients with leukemia.

1997 ◽  
Vol 41 (1) ◽  
pp. 196-199 ◽  
Author(s):  
F S Nolte ◽  
T Parkinson ◽  
D J Falconer ◽  
S Dix ◽  
J Williams ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amphotericin B ◽  
High Dose ◽  
Clinical Settings ◽  
Desaturase Gene ◽  
Isolation And Characterization ◽  
Fluconazole Resistant ◽  
Resistant Strains ◽  
Fluconazole Prophylaxis

Infections with fluconazole-resistant Candida albicans isolate have rarely been described in clinical settings other than oropharyngeal candidiasis in patients with late-stage AIDS. We report on two patients with leukemia who developed fungemia caused by fluconazole-resistant C. albicans after receiving fluconazole prophylaxis (400 mg/day) and empiric amphotericin B therapy (0.5 mg/kg of body weight per day). The fluconazole MICs for the isolates were > or = 64 micrograms/ml, and the isolates were resistant to other azoles and had membrane sterol changes consistent with a mutation in the delta 5,6-sterol desaturase gene. The lack of ergosterol in the cytoplasmic membrane of the fluconazole-resistant strains also imparted resistance to amphotericin B. Both patients were successfully treated with high-dose amphotericin B (1 to 1.25 mg/kg/day) and flucytosine (150 mg/kg/day).

Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae

1988 ◽  
Vol 8 (10) ◽  
pp. 3997-4008
Author(s):  
M Wittekind ◽  
J Dodd ◽  
L Vu ◽  
J M Kolb ◽  
J M Buhler ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Polymerase I

The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon.

scholarly journals Molecular Characterization of Fluoroquinolone-Resistant Moraxella catarrhalis Variants Generated In Vitro by Stepwise Selection

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Kageto Yamada ◽  
Ryoichi Saito ◽  
Saori Muto ◽  
Machiko Kashiwa ◽  
Yoshiko Tamamori ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Moraxella Catarrhalis ◽  
Respiratory Infections ◽  
Fluoroquinolone Resistance ◽  
Novel Mutations ◽  
Content Type ◽  
Stepwise Selection ◽  
Resistant Strains

ABSTRACT Moraxella catarrhalis causes respiratory infections. In this study, fluoroquinolone-resistant strains were selected in vitro to evaluate the mechanism of fluoroquinolone resistance. Strains with reduced fluoroquinolone susceptibility were obtained by stepwise selection in levofloxacin, and fluoroquinolone targets gyr and par were sequenced. Six novel mutations in GyrA (D84Y, T594dup, and A722dup), GyrB (E479K and D439N), and ParE (Q395R) involved in M. catarrhalis resistance to fluoroquinolones were revealed.

scholarly journals In VitroActivity of Solithromycin against Erythromycin-Resistant Streptococcus agalactiae

2013 ◽  
Vol 58 (3) ◽  
pp. 1693-1698 ◽  
Author(s):  
Giorgio Piccinelli ◽  
Prabhavathi Fernandes ◽  
Carlo Bonfanti ◽  
Francesca Caccuri ◽  
Arnaldo Caruso ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Group B Streptococcus ◽  
Phenotypic Characterization ◽  
Content Type ◽  
Resistant Strains ◽  
Group B ◽  
Microdilution Broth

ABSTRACTThein vitroantibacterial activity of solithromycin (CEM-101) against macrolide-resistant isolates (n= 62) ofStreptococcus agalactiae(group B streptococcus [GBS]) was determined. Phenotypic characterization of macrolide-resistant strains was performed by double-disc diffusion testing. A multiplex PCR was used to identify theerm(B),erm(TR), andmef(A/E) genes, capsular genotypes, and alpha-like (Alp) protein genes from the GBS strains. Determination of MIC was carried out using the microdilution broth method. The Etest method was used for penicillin, azithromycin, clarithromycin, and erythromycin. Solithromycin had a MIC50of ≤0.008 μg/ml and a MIC90of 0.015 μg/ml against macrolide-susceptibleS. agalactiae. These MICs were lower than those displayed by penicillin (MIC50of 0.032 μg/ml and MIC90of 0.047 μg/ml), the antibiotic agent of choice for prophylaxis and treatment of GBS infections. Against macrolide-resistantS. agalactiae, solithromycin had a MIC50of 0.03 μg/ml and a MIC90of 0.125 μg/ml. Againsterm(B) strains, solithromycin had a MIC50of 0.03 μg/ml and a MIC90of 0.06 μg/ml, while againstmef(A) strains, it had a MIC50of 0.03 μg/ml and a MIC90of 0.125 μg/ml. Most erythromycin-resistant GBS strains were of serotype V (64.5%) and associated significantly withalp2-3. Moreover, a statistically significant association was observed between the constitutive macrolide-lincosamide-streptogramin B resistance (cMLSB) phenotype and theerm(B) gene-carrying strains, thealp2-3gene and the M phenotype, and themef(A/E) gene andepsilon. Overall, our results show that solithromycin had lower or similar MICs than penicillin and potent activity against macrolide-resistant strains independent of their genotype or phenotype, representing a valid therapeutic alternative where β-lactams cannot be used.

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