scholarly journals Fluoroquinolone Action against Clinical Isolates ofMycobacterium tuberculosis: Effects of a C-8 Methoxyl Group on Survival in Liquid Media and in Human Macrophages

1999 ◽  
Vol 43 (3) ◽  
pp. 661-666 ◽  
Author(s):  
Ben Yang Zhao ◽  
Richard Pine ◽  
John Domagala ◽  
Karl Drlica
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Lethal Dose ◽  
Clinical Isolates ◽  
Methoxyl Group ◽  
Human Macrophage ◽  
Wild Type ◽  
Liquid Media ◽  
Human Macrophages ◽  
Resistant Strains

ABSTRACT When the lethal action of a C-8 methoxyl fluoroquinolone against clinical isolates of Mycobacterium tuberculosis in liquid medium was measured, the compound was found to be three to four times more effective (as determined by measuring the 90% lethal dose) than a C-8-H control fluoroquinolone or ciprofloxacin against cells having a wild-type gyrA (gyrase) gene. Against ciprofloxacin-resistant strains, the C-8 methoxyl group enhanced lethality when alanine was replaced by valine at position 90 of the GyrA protein or when aspartic acid 94 was replaced by glycine, histidine, or tyrosine. During infection of a human macrophage model by wild-type Mycobacterium bovis BCG, the C-8 methoxyl group lowered survival 20- to 100-fold compared with the same concentration of a C-8-H fluoroquinolone. The C-8 methoxyl fluoroquinolone was also more effective than ciprofloxacin against a gyrA Asn94 mutant of M. bovis BCG. In an M. tuberculosis-macrophage system the C-8 methoxyl group improved fluoroquinolone action against both quinolone-susceptible and quinolone-resistant clinical isolates. Thus, a C-8 methoxyl group enhances the bactericidal activity of quinolones with N1-cyclopropyl substitutions; these data encourage further refinement of fluoroquinolones as antituberculosis agents.

scholarly journals Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

2011 ◽  
Vol 56 (3) ◽  
pp. 1253-1257 ◽  
Author(s):  
J. Werngren ◽  
E. Sturegård ◽  
P. Juréen ◽  
K. Ängeby ◽  
S. Hoffner ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Clinical Isolates ◽  
Wild Type ◽  
First Line ◽  
Content Type ◽  
Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

scholarly journals Relationship between Pyrazinamide Resistance, Loss of Pyrazinamidase Activity, and Mutations in the pncA Locus in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis

1999 ◽  
Vol 43 (9) ◽  
pp. 2317-2319 ◽  
Author(s):  
M. Mestdagh ◽  
P. A. Fonteyne ◽  
L. Realini ◽  
R. Rossau ◽  
G. Jannes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Radiometric Method ◽  
Clinical Isolates ◽  
Wild Type ◽  
Resistant Strains ◽  
Resistance Loss

ABSTRACT Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing ofpncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-typepncA might possess alternative resistance mechanisms.

scholarly journals Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruiqi Wang ◽  
Kun Li ◽  
Jifang Yu ◽  
Jiaoyu Deng ◽  
Yaokai Chen
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Clinical Isolates ◽  
Aminobenzoic Acid ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Expression Levels ◽  
Encoding Gene ◽  
Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

scholarly journals Insights into the Mechanism of Ethionamide Resistance in Mycobacterium tuberculosis through an in silico Structural Evaluation of EthA and Mutants Identified in Clinical Isolates

2020 ◽  
Author(s):  
Vinicius Carius de Souza ◽  
Deborah Antunes ◽  
Luciana H.S. Santos ◽  
Priscila Vanessa Zabala Capriles Goliatt ◽  
Ernesto Raul Caffarena ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Comparative Modeling ◽  
Clinical Isolates ◽  
Free Energies ◽  
Wild Type ◽  
Structural Evaluation ◽  
Type Complex ◽  
Computational Calculations ◽  
First Results ◽  
Delta G

Mutation in the ethionamide (ETH) activating enzyme, EthA, is the main factor determining resistance to this drug, used to treat TB patients infected with MDR and XDR Mycobacterium tuberculosis isolates. Many mutations in EthA of ETH resistant (ETH-R) isolates have been described but their roles in resistance remain uncharacterized, partly because structural studies on the enzyme are lacking. Thus, we took a two-tier approach to evaluate two mutations (Y50C and T453I) found in ETH-R clinical isolates. First, we used a combination of comparative modeling, molecular docking, and molecular dynamics to build an EthA model in complex with ETH that has hallmark features of structurally characterized homologs. Second, we used free energy computational calculations for the reliable prediction of relative free energies between the wild type and mutant enzymes. The ΔΔG values for Y50C and T453I mutant enzymes in complex with FADH2-NADP-ETH were 3.34 (+/−0.55) and 8.11 (+/−0.51) kcal/mol, respectively, compared to the wild type complex. The positive ΔΔG values indicate that the wild type complex is more stable than the mutants, with the T453I complex being the least stable. These are the first results shedding light on the molecular basis of ETH resistance, namely reduced complex stability of mutant EthA.

scholarly journals Insights into the Mechanism of Ethionamide Resistance in Mycobacterium tuberculosis through an in silico Structural Evaluation of EthA and Mutants Identified in Clinical Isolates

Catalysts ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 543 ◽  
Author(s):  
Vinicius Carius de Souza ◽  
Deborah Antunes ◽  
Lucianna H.S. Santos ◽  
Priscila Vanessa Zabala Capriles Goliatt ◽  
Ernesto Raul Caffarena ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Comparative Modeling ◽  
Clinical Isolates ◽  
Free Energies ◽  
Wild Type ◽  
Structural Evaluation ◽  
Type Complex ◽  
Computational Calculations ◽  
First Results ◽  
Main Factor

Mutation in the ethionamide (ETH) activating enzyme, EthA, is the main factor determining resistance to this drug, used to treat TB patients infected with MDR and XDR Mycobacterium tuberculosis isolates. Many mutations in EthA of ETH resistant (ETH-R) isolates have been described but their roles in resistance remain uncharacterized, partly because structural studies on the enzyme are lacking. Thus, we took a two-tier approach to evaluate two mutations (Y50C and T453I) found in ETH-R clinical isolates. First, we used a combination of comparative modeling, molecular docking, and molecular dynamics to build an EthA model in complex with ETH that has hallmark features of structurally characterized homologs. Second, we used free energy computational calculations for the reliable prediction of relative free energies between the wild type and mutant enzymes. The ΔΔG values for Y50C and T453I mutant enzymes in complex with FADH2-NADP-ETH were 3.34 (+/−0.55) and 8.11 (+/−0.51) kcal/mol, respectively, compared to the wild type complex. The positive ΔΔG values indicate that the wild type complex is more stable than the mutants, with the T453I complex being the least stable. These are the first results shedding light on the molecular basis of ETH resistance, namely reduced complex stability of mutant EthA.

scholarly journals Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages

2019 ◽  
Vol 221 (6) ◽  
pp. 989-999
Author(s):  
Jason D Simmons ◽  
Glenna J Peterson ◽  
Monica Campo ◽  
Jenny Lohmiller ◽  
Shawn J Skerrett ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nicotinic Acid ◽  
Mycobacterium Bovis ◽  
Related Compound ◽  
Antimicrobial Properties ◽  
Broth Culture ◽  
Wild Type ◽  
Bacille Calmette Guerin ◽  
Macrophage Function ◽  
Modest Activity

Abstract Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

scholarly journals A Novel Cecropin D-Derived Short Cationic Antimicrobial Peptide Exhibits Antibacterial Activity Against Wild-Type and Multidrug-Resistant Strains of Klebsiella pneumoniae and Pseudomonas aeruginosa

2020 ◽  
Vol 16 ◽  
pp. 117693432093626
Author(s):  
Iván Darío Ocampo-Ibáñez ◽  
Yamil Liscano ◽  
Sandra Patricia Rivera-Sánchez ◽  
José Oñate-Garzón ◽  
Ashley Dayan Lugo-Guevara ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Activity ◽  
Klebsiella Pneumoniae ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Health Concern ◽  
Wild Type ◽  
Cationic Antimicrobial Peptide ◽  
Promising Alternative ◽  
Resistant Strains

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa and Klebsiella pneumoniae are a serious worldwide public health concern due to the ineffectiveness of empirical antibiotic therapy. Therefore, research and the development of new antibiotic alternatives are urgently needed to control these bacteria. The use of cationic antimicrobial peptides (CAMPs) is a promising candidate alternative therapeutic strategy to antibiotics because they exhibit antibacterial activity against both antibiotic susceptible and MDR strains. In this study, we aimed to investigate the in vitro antibacterial effect of a short synthetic CAMP derived from the ΔM2 analog of Cec D-like (CAMP-CecD) against clinical isolates of K pneumoniae (n = 30) and P aeruginosa (n = 30), as well as its hemolytic activity. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of CAMP-CecD against wild-type and MDR strains were determined by the broth microdilution test. In addition, an in silico molecular dynamic simulation was performed to predict the interaction between CAMP-CecD and membrane models of K pneumoniae and P aeruginosa. The results revealed a bactericidal effect of CAMP-CecD against both wild-type and resistant strains, but MDR P aeruginosa showed higher susceptibility to this peptide with MIC values between 32 and >256 μg/mL. CAMP-CecD showed higher stability in the P aeruginosa membrane model compared with the K pneumoniae model due to the greater number of noncovalent interactions with phospholipid 1-Palmitoyl-2-oleyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). This may be related to the boosted effectiveness of the peptide against P aeruginosa clinical isolates. Given the antibacterial activity of CAMP-CecD against wild-type and MDR clinical isolates of P aeruginosa and K pneumoniae and its nonhemolytic effects on human erythrocytes, CAMP-CecD may be a promising alternative to conventional antibiotics.

scholarly journals β-Glucans inhibit intracellular growth of Mycobacterium bovis BCG but not virulent Mycobacterium tuberculosis in human macrophages

2011 ◽  
Vol 51 (4) ◽  
pp. 233-242 ◽  
Author(s):  
Bret E. Betz ◽  
Abul K. Azad ◽  
Jessica D. Morris ◽  
Murugesan V.S. Rajaram ◽  
Larry S. Schlesinger
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Intracellular Growth ◽  
Human Macrophages ◽  
Mycobacterium Bovis Bcg

scholarly journals The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis

2005 ◽  
Vol 202 (7) ◽  
pp. 987-999 ◽  
Author(s):  
Peter B. Kang ◽  
Abul K. Azad ◽  
Jordi B. Torrelles ◽  
Thomas M. Kaufman ◽  
Alison Beharka ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mannose Receptor ◽  
Human Macrophage ◽  
Phagosome Maturation ◽  
Human Macrophages ◽  
Phagocytic Process ◽  
Fusion Inhibition ◽  
Macrophage Mannose Receptor ◽  
Intracellular Adhesion Molecule ◽  
Intact Molecule

Mycobacterium tuberculosis (M.tb) survives in macrophages in part by limiting phagosome–lysosome (P-L) fusion. M.tb mannose-capped lipoarabinomannan (ManLAM) blocks phagosome maturation. The pattern recognition mannose receptor (MR) binds to the ManLAM mannose caps and mediates phagocytosis of bacilli by human macrophages. Using quantitative electron and confocal microscopy, we report that engagement of the MR by ManLAM during the phagocytic process is a key step in limiting P-L fusion. P-L fusion of ManLAM microspheres was significantly reduced in human macrophages and an MR-expressing cell line but not in monocytes that lack the receptor. Moreover, reversal of P-L fusion inhibition occurred with MR blockade. Inhibition of P-L fusion did not occur with entry via Fcγ receptors or dendritic cell–specific intracellular adhesion molecule 3 grabbing nonintegrin, or with phosphatidylinositol-capped lipoarabinomannan. The ManLAM mannose cap structures were necessary in limiting P-L fusion, and the intact molecule was required to maintain this phenotype. Finally, MR blockade during phagocytosis of virulent M.tb led to a reversal of P-L fusion inhibition in human macrophages (84.0 ± 5.1% vs. 38.6 ± 0.6%). Thus, engagement of the MR by ManLAM during the phagocytic process directs M.tb to its initial phagosomal niche, thereby enhancing survival in human macrophages.

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