scholarly journals Characterization of Spontaneous, In Vitro-Selected, Rifampin-Resistant Mutants of Mycobacterium tuberculosisStrain H37Rv

2000 ◽  
Vol 44 (12) ◽  
pp. 3298-3301 ◽  
Author(s):  
Glenn P. Morlock ◽  
Bonnie B. Plikaytis ◽  
Jack T. Crawford
Keyword(s):  
Point Mutations ◽  
Laboratory Strain ◽  
Resistant Mutant ◽  
Small Region ◽  
Clinical Isolates ◽  
Gene Coding ◽  
Resistant Mutants ◽  
Strain H37rv

ABSTRACT Resistance to rifampin in Mycobacterium tuberculosisresults from mutations in the gene coding for the beta subunit of RNA polymerase (rpoB). At least 95% of rifampin-resistant isolates have mutations in rpoB, and the mutations are clustered in a small region. About 40 distinct point mutations and in-frame insertions and deletions in rpoB have been identified, but point mutations in two codons, those coding for Ser531 and His526, are seen in about 70% of rifampin-resistant clinical isolates, with Ser531-to-Leu (TCG-to-TGG) mutations being by far the most common. To explore this phenomenon, we isolated independent, spontaneous, rifampin-resistant mutant versions of well-characterized M. tuberculosislaboratory strain H37Rv by plating 100 separate cultures, derived from a single low-density inoculum, onto rifampin-containing medium. Rifampin-resistant mutants were obtained from 64 of these cultures. Although we anticipated that the various point mutations would occur with approximately equal frequencies, sequencing the rpoBgene from one colony per plate revealed that 39 (60.9%) were Ser531 to Leu. We conclude that, for unknown reasons, the associated rpoB mutation occurs at a substantially higher rate than other rpoB mutations. This higher mutation rate may contribute to the high percentage of this mutation seen in clinical isolates.

scholarly journals Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

2016 ◽  
Vol 113 (13) ◽  
pp. 3669-3674 ◽  
Author(s):  
Min-Suk Song ◽  
Gyanendra Kumar ◽  
William R. Shadrick ◽  
Wei Zhou ◽  
Trushar Jeevan ◽  
...  
Keyword(s):  
Point Mutations ◽  
Random Mutagenesis ◽  
Resistant Mutant ◽  
Inhibitor Binding ◽  
New Class ◽  
Identification And Characterization ◽  
Modest Reduction

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

scholarly journals Ceftazidime and Aztreonam Resistance inProvidencia stuartii: Characterization of a Natural TEM-Derived ExtendedSpectrum β-Lactamase, TEM-60

1998 ◽  
Vol 42 (6) ◽  
pp. 1459-1462 ◽  
Author(s):  
Nicola Franceschini ◽  
Mariagrazia Perilli ◽  
Bernardetta Segatore ◽  
Domenico Setacci ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Point Mutations ◽  
Clinical Strain ◽  
In Vitro Susceptibility ◽  
Extended Spectrum ◽  
Gene Coding ◽  
Providencia Stuartii

ABSTRACT A plasmid-encoded β-lactamase produced from a clinical strain ofProvidencia stuartii has been purified and characterized. The gene coding for the β-lactamase was cloned and sequenced. It appears to be a new natural TEM-derived enzyme, named TEM-60. Point mutations (Q39K, L51P, E104K, and R164S) are present with respect to the TEM-1 enzyme; the mutation L51P has never been previously reported, with the exception of the chromosomally encoded extended-spectrum β-lactamase PER-1. Kinetic parameters relative to penicillins, cephalosporins, and monobactams other than mechanism-based inactivators were related to the in vitro susceptibility phenotype.

Characterization of sparfloxacin-resistant mutants of Staphylococcus aureus obtained in vitro

2001 ◽  
Vol 18 (2) ◽  
pp. 107-112 ◽  
Author(s):  
Joaquim Ruiz ◽  
Josep M. Sierra ◽  
M.Teresa Jiménez De Anta ◽  
Jordi Vila
Keyword(s):  
Staphylococcus Aureus ◽  
Resistant Mutants

scholarly journals Effect of Fluorescent Dyes onIn Vitro-Differentiated, Late-Stage Plasmodium falciparum Gametocytes

2014 ◽  
Vol 58 (12) ◽  
pp. 7398-7404 ◽  
Author(s):  
Tamirat Gebru ◽  
Benjamin Mordmüller ◽  
Jana Held
Keyword(s):  
Plasmodium Falciparum ◽  
Late Stage ◽  
Fluorescent Dyes ◽  
Clinical Symptoms ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Synthetic Dyes ◽  
Content Type ◽  
Highly Active

ABSTRACTPlasmodium falciparumgametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates ofP. falciparumwith a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC50) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC50s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC50s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly highin vitroactivity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds.

scholarly journals Characterization of the pathogenesis and immune response to Listeria monocytogenes strains isolated from a sustained national outbreak

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Pallab Ghosh ◽  
Yan Zhou ◽  
Quentin Richardson ◽  
Darren E. Higgins
Keyword(s):  
Listeria Monocytogenes ◽  
Laboratory Strain ◽  
The United States ◽  
Clinical Isolates ◽  
T Cell Antigens ◽  
Life Threatening ◽  
Cellular Immune ◽  
The Brain

AbstractListeria monocytogenes is an intracellular pathogen responsible for listeriosis, a foodborne disease that can lead to life-threatening meningitis. The 2011 L. monocytogenes cantaloupe outbreak was among the deadliest foodborne outbreaks in the United States. We conducted in vitro and in vivo infection analyses to determine whether strains LS741 and LS743, two clinical isolates from the cantaloupe outbreak, differ significantly from the common laboratory strain 10403S. We showed that LS741 and LS743 exhibited increased virulence, characterized by higher colonization of the brain and other organs in mice. Assessment of cellular immune responses to known CD8+ T cell antigens was comparable between all strains. However, pre-existing immunity to 10403S did not confer protection in the brain against challenge with LS741. These studies provide insights into the pathogenesis of clinical isolates linked to the 2011 cantaloupe outbreak and also indicate that currently utilized laboratory strains are imperfect models for studying L. monocytogenes pathogenesis.

scholarly journals Use of Bacteriophage Ba1 To Identify Properties Associated with Bordetella avium Virulence

2002 ◽  
Vol 70 (3) ◽  
pp. 1219-1224 ◽  
Author(s):  
Celia B. Shelton ◽  
Louise M. Temple ◽  
Paul E. Orndorff
Keyword(s):  
Lipid A ◽  
Laboratory Strain ◽  
Tracheal Ring ◽  
Phage Resistance ◽  
Dependent Manner ◽  
O Antigen ◽  
Bordetella Avium ◽  
Resistant Mutants

ABSTRACT Bordetella avium causes bordetellosis, an upper respiratory disease of birds. Commercially raised turkeys are particularly susceptible. We report here on the use of a recently described B. avium bacteriophage, Ba1, as a tool for investigating the effects of lysogeny and phage resistance on virulence. We found that lysogeny had no effect on any of the in vivo or in vitro measurements of virulence we employed. However, two-thirds (six of nine) spontaneous phage-resistant mutants of our virulent laboratory strain, 197N, were attenuated. Phage resistance was associated, in all cases, with an inability of the mutants to bind phage. Further tests of the mutants revealed that all had increased sensitivities to surfactants, and increased amounts of incomplete (O-antigen-deficient) lipopolysaccharide (LPS) compared to 197N. Hot phenol-water-extracted 197N LPS inactivated phage in a specific and dose-dependent manner. Acid hydrolysis and removal of lipid A had little effect upon the ability of isolated LPS to inactivate Ba1, suggesting that the core region and possibly the O antigen were required for phage binding. All of the mutants, with one exception, were significantly more sensitive to naive turkey serum and, without exception, significantly less able to bind to tracheal rings in vitro than 197N. Interestingly, the three phage-resistant mutants that remained virulent appeared to be O antigen deficient and were among the mutants that were the most serum sensitive and least able to bind turkey tracheal rings in vitro. This observation allowed us to conclude that even severe defects in tracheal ring binding and serum resistance manifested in vitro were not necessarily indicative of attenuation and that complete LPS may not be required for virulence.

scholarly journals Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shayla Hesse ◽  
Manoj Rajaure ◽  
Erin Wall ◽  
Joy Johnson ◽  
Valery Bliskovsky ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Cell Surface ◽  
Phage Therapy ◽  
Surface Structures ◽  
Phage Resistance ◽  
Cell Surface Structures ◽  
Phage Cocktail ◽  
Resistant Mutants

ABSTRACT The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro. Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design. IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

scholarly journals Diverse Cytomegalovirus UL27 Mutations Adapt to Loss of Viral UL97 Kinase Activity under Maribavir

2008 ◽  
Vol 53 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Sunwen Chou
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Genetic Defect ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Drug Selection ◽  
Normal Expression ◽  
High Level ◽  
Level Resistance

ABSTRACT In vitro resistance to maribavir (MBV), a cytomegalovirus UL97 kinase inhibitor currently in clinical trials, is known to result from viral UL97 mutations that confer moderate to high-level resistance and UL27 mutations that confer low-level resistance. To add to the four reported UL27 mutations, cytomegalovirus isolates or strains were propagated under MBV. Four clinical isolates evolved UL27 mutations, which were first detected after 8 to 30 passages under drug selection. In three separate experiments, laboratory strain T2294, which contained an exonuclease mutation, developed UL27 mutations at 10 to 12 passages under MBV. Most of these isolates and strains also developed a UL97 mutation, commonly T409M, before or after the appearance of the UL27 mutation. The passage of two laboratory strains genetically defective in UL97, in the absence of MBV, likewise resulted in UL27 mutations. The nine UL27 mutations observed included multiple instances of point, stop, and frameshift mutations, which were individually transferred to a reference CMV strain and which were shown to confer two- to threefold increases in MBV inhibitory concentrations. In contrast, seven common UL27 amino acid changes found in baseline clinical isolates conferred no MBV resistance. The mutants with UL27 mutations had slightly attenuated growth. The frequent mutation of UL27 suggests that its normal expression is mildly disadvantageous to the virus in the absence of UL97 kinase activity, whether the latter results from MBV inhibition or a genetic defect. Although the function of UL27 is unknown, it does not appear to be a direct antiviral target for MBV.

scholarly journals Molecular Basis for Different Levels oftet(M) Expression in Streptococcus pneumoniae Clinical Isolates

2012 ◽  
Vol 56 (10) ◽  
pp. 5040-5045 ◽  
Author(s):  
Patrick Grohs ◽  
Patrick Trieu-Cuot ◽  
Isabelle Podglajen ◽  
Sophie Grondin ◽  
Arnaud Firon ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Molecular Basis ◽  
Clinical Isolates ◽  
M Gene ◽  
Content Type ◽  
Frameshift Mutations ◽  
Resistant Mutants ◽  
Promoter Mutations ◽  
Different Levels

ABSTRACTSeventy-four unrelated clinical isolates ofStreptococcus pneumoniaeharboring thetet(M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 μg/ml) were found to harbor truncatedtet(M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in thetet(M) promoter region, among which four displayed increased Tc MICs (16 to 64 μg/ml). The same promoter mutations were detected in Tc-resistant mutants selectedin vitrofrom various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream oftet(M). Expression inEnterococcus faecalisof atet(M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parentalS. pneumoniaestrains. These results show that different levels of Tc susceptibility found in clinical isolates ofS. pneumoniaecan be explained by frameshift mutations withintet(M) and by alterations of the upstream transcriptional attenuator.

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