scholarly journals Pharmacodynamics of Voriconazole against Wild-Type and Azole-Resistant Aspergillus flavus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Shivaprakash M. Rudramurthy ◽  
Seyedmojtaba Seyedmousavi ◽  
Manpreet Dhaliwal ◽  
Arunaloke Chakrabarti ◽  
Jacques F. Meis ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Murine Model ◽  
Drug Exposure ◽  
Resistant Isolate ◽  
Dependent Manner ◽  
Wild Type ◽  
Time Curve ◽  
Content Type ◽  
Significant Difference ◽  
Susceptible Isolate

ABSTRACT Invasive aspergillosis (IA) due to Aspergillus flavus is associated with high mortality. Although voriconazole (VRC) is widely recommended as the first-line treatment for IA, emergence of azole resistance in Aspergillus spp. is translating to treatment failure. We evaluated the efficacy of voriconazole in a nonneutropenic murine model of disseminated A. flavus infection using two voriconazole-resistant isolates (one harboring the Y319H substitution in the cyp51C gene) and two wild-type isolates without mutations. All isolates exhibited a dose-response relationship, and voriconazole treatment improved mouse survival in a dose-dependent manner. At 40 mg/kg of body weight, 100% efficacy was observed for 1 susceptible isolate and 1 resistant isolate (with mutation), whereas for another susceptible isolate and resistant isolate (without mutation), survival rates were 81% and 72%, respectively. The Hill equation with a variable slope fitted the relationship between the area under the concentration-time curve (AUC)/MIC ratio and 14-day survival well for each strain. An F test showed the 50% effective doses to be significantly different from each other (P = 0.0023). However, contrary to expectation, there was a significant difference in exposure-response relationships between strains, and it appeared that the susceptible strains required a relatively higher exposure than the resistant ones to result in the same treatment effect, the 50% effective pharmacokinetic/pharmacodynamic (PK/PD) index (EI50) required being negatively and log-linearly related to the MIC (P = 0.04). We conclude that the efficacy of voriconazole depended on drug exposure and the voriconazole MIC of the isolates, but lower exposures are required for strains with higher MICs. These findings may have profound significance in clinical practice with respect to dosing and drug choice.

scholarly journals The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

2012 ◽  
Vol 11 (10) ◽  
pp. 1226-1238 ◽  
Author(s):  
Lorna Gallagher ◽  
Rebecca A. Owens ◽  
Stephen K. Dolan ◽  
Grainne O'Keeffe ◽  
Markus Schrettl ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Aspergillus Fumigatus ◽  
High Performance ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Disulfide Linkages ◽  
Significant Difference ◽  
Culture Supernatants

ABSTRACTThe function of a number of genes in the gliotoxin biosynthetic cluster (gli) inAspergillus fumigatusremains unknown. Here, we demonstrate thatgliKdeletion from two strains ofA. fumigatuscompletely abolished gliotoxin biosynthesis. Furthermore, exogenous H2O2(1 mM), but not gliotoxin, significantly inducedA. fumigatus gliKexpression (P= 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P< 0.001) and H2O2(P< 0.01), unexpectedly, exogenous gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived fromA. fumigatusATCC 26933 significantly inhibited (P< 0.05) the growth of the ΔgliK26933deletion mutant. TheA. fumigatusΔgliK26933mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry withm/z394 to 396. These metabolites (m/z394 to 396) were present at significantly higher levels in the culture supernatants of theA. fumigatusΔgliK26933mutant than in those of the wild type (P= 0.0024 [fold difference, 24] andP= 0.0003 [fold difference, 9.6], respectively) and were absent fromA. fumigatusΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of theA. fumigatusΔgliK26933mutant compared to the wild type (P< 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P= 0.0045) between that ofA. fumigatusATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK46645mutant (31.4 pg/mg mycelium/min), strongly suggesting thatgliKabsence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role forgliKin gliotoxin biosynthesis and reveal new insights into gliotoxin functionality inA. fumigatus.

scholarly journals Pharmacodynamics of Anidulafungin against Clinical Aspergillus fumigatus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis

2012 ◽  
Vol 57 (1) ◽  
pp. 303-308 ◽  
Author(s):  
Seyedmojtaba Seyedmousavi ◽  
Roger J. M. Brüggemann ◽  
Willem J. G. Melchers ◽  
Paul E. Verweij ◽  
Johan W. Mouton
Keyword(s):  
Aspergillus Fumigatus ◽  
Murine Model ◽  
Resistance Mechanism ◽  
Maximal Response ◽  
Dependent Manner ◽  
Time Curve ◽  
Content Type ◽  
Disseminated Aspergillosis ◽  
The Hill

ABSTRACTAzole resistance is an emerging increasing problem inAspergillus fumigatusthat results in treatment failure. Alternative treatments may improve the therapeutic outcome in patients with azole-resistant invasive aspergillosis (IA). Little is known about thein vivoefficacy of the echinocandin anidulafungin (AFG) in IA. Thein vivoefficacy of 2.5, 5, 10, and 20 mg/kg of body weight AFG was assessed against two clinicalAspergillus fumigatusisolates with identical AFG minimum effective concentrations (MECs; 0.03 mg/liter) in a murine model of IA: a wild-type voriconazole (VCZ)-susceptible (VCZs)A. fumigatusisolate (AZN 8196) and a VCZ-resistant (VCZr)A. fumigatusisolate (V52-35) harboring the TR34/L98H resistance mechanism (substitution at codon L98 in combination with a 34-bp tandem repeat in the promoter region of theCYP51Agene). The pharmacokinetics of AFG were also assessed for each dose. Increasing doses increased survival for both isolates in a manner dependent on the AFG dose level (R2= 0.99 and 0.95, respectively) up to a maximum of 72.7% and 45.45% for the VCZsand VCZrisolates, respectively. The area under the concentration-time curve (AUC) correlated significantly with the dose in a linear fashion over the entire dosing range (R2= 0.86). The Hill equation with a variable slope fitted the relationship between the 24-h AUC/MEC ratio and 14-day survival well (R2= 0.87;P< 0.05). The 50% effective AUC/MEC for total AFG was 126.5 (95% confidence interval, 79.09 to 202.03). AFG treatment improved the survival of mice in a dose-dependent manner; however, a maximal response was not achieved with either isolate even in those treated with the highest AFG dose.

scholarly journals Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis

2011 ◽  
Vol 55 (9) ◽  
pp. 4326-4337 ◽  
Author(s):  
Anna-Barbara Hachmann ◽  
Elif Sevim ◽  
Ahmed Gaballa ◽  
David L. Popham ◽  
Haike Antelmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Stringent Response ◽  
Resistant Isolate ◽  
Cross Resistance ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Wild Type ◽  
Content Type

ABSTRACTDaptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2+-dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, DapR1, from the model bacteriumBacillus subtilis168. DapR1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. DapR1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of DapR1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator genemreB, the stringent response generelA, and the phosphatidylglycerol synthase genepgsA. Genetic reconstruction studies indicated that thepgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-typepgsArestored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca2+-DAP complex.

scholarly journals Fulvestrant-3-Boronic Acid (ZB716) Demonstrates Oral Bioavailability and Favorable Pharmacokinetic Profile in Preclinical ADME Studies

2021 ◽  
Vol 14 (8) ◽  
pp. 719
Author(s):  
Jiawang Liu ◽  
Nirmal Rajasekaran ◽  
Ahamed Hossain ◽  
Changde Zhang ◽  
Shanchun Guo ◽  
...  
Keyword(s):  
Boronic Acid ◽  
Oral Bioavailability ◽  
Drug Exposure ◽  
Liver Microsomes ◽  
Pharmacokinetic Profile ◽  
Pharmacokinetic Studies ◽  
Dependent Manner ◽  
Mucosal Permeability ◽  
Time Curve ◽  
Concentration Dependent Manner

Fulvestrant-3-boronic acid (ZB716), an oral selective estrogen receptor degrader (SERD) under clinical development, has been investigated in ADME studies to characterize its absorption, metabolism, and pharmacokinetics. ZB716 was found to have high plasma protein binding in human and animal plasma, and low intestinal mucosal permeability. ZB716 had high clearance in hepatocytes of all species tested. ZB716 was metabolized primarily by CYP2D6 and CYP3A. In human liver microsomes, ZB716 demonstrated relatively low inhibition of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (when testosterone was used as the substrate), and no inhibition of CYP2B6 and 3A4 (when midazolam was used as the substrate). In assays for enzyme activity, ZB716 induced CYP1A2, 2B6, and 3A4 in a concentration-dependent manner. Single-dose and repeated-dose pharmacokinetic studies in rats and dogs showed oral bioavailability, dose-proportional drug exposure, and drug accumulation as measured by maximum concentration and area under the concentration–time curve (AUC).

scholarly journals The Helicobacter pylori Autotransporter ImaA (HP0289) Modulates the Immune Response and Contributes to Host Colonization

2012 ◽  
Vol 80 (7) ◽  
pp. 2286-2296 ◽  
Author(s):  
William E. Sause ◽  
Andrea R. Castillo ◽  
Karen M. Ottemann
Keyword(s):  
Immune Response ◽  
Helicobacter Pylori ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
H Pylori ◽  
Murine Infections

ABSTRACTThe human pathogenHelicobacter pyloriemploys a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced geneHP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenicH. pylorimutant that lacksHP0289and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-typeH. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that theHP0289promoter is upregulated in the mouse stomach, and here we demonstrate thatHP0289expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that theHP0289mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-typeH. pylori. On the basis of this phenotype, we renamed HP0289 ImaA forimmunomodulatoryautotransporter protein. Our work has revealed that genes inducedin vivoplay an important role inH. pyloripathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allowH. pylorito fine tune the host immune response based on ImaA expression.

scholarly journals Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

2014 ◽  
Vol 58 (6) ◽  
pp. 2997-3007 ◽  
Author(s):  
Rati Tandon ◽  
Sharat Chandra ◽  
Rajendra Kumar Baharia ◽  
Sanchita Das ◽  
Pragya Misra ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Proliferating Cell Nuclear Antigen ◽  
Leishmania Donovani ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Content Type ◽  
Sensitive Isolate ◽  
Proliferating Cell

ABSTRACTPreviously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate ofLeishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance inL. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate.L. donovaniPCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate ofL. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessedin vitroagainst promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV(from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII(from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIIItreatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance inL. donovaniclinical isolates, which warrants detailed investigations regarding its mechanism.

scholarly journals Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Raees A. Paul ◽  
Shivaprakash M. Rudramurthy ◽  
Manpreet Dhaliwal ◽  
Pankaj Singh ◽  
Anup K. Ghosh ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Environmental Isolates ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

scholarly journals Comparison of the postantibiotic and postantibiotic sub-MIC effects of levofloxacin and ciprofloxacin on Staphylococcus aureus and Streptococcus pneumoniae.

1997 ◽  
Vol 41 (5) ◽  
pp. 950-955 ◽  
Author(s):  
L Licata ◽  
C E Smith ◽  
R M Goldschmidt ◽  
J F Barrett ◽  
M Frosco
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Resistant Strain ◽  
Drug Exposure ◽  
Inhibitory Concentration ◽  
Resistant Isolate ◽  
Postantibiotic Effect ◽  
Methicillin Resistant ◽  
Susceptible Isolate

The postantibiotic subminimum inhibitory concentration effect (PA SME) may simulate in vivo drug exposure more accurately than the postantibiotic effect (PAE) since subinhibitory concentrations of drug persist between antibiotic dosings. In this study, we compared the PAEs and PA SMEs of levofloxacin and ciprofloxacin for clinical isolates of fluoroquinolone-susceptible Staphylococcus aureus and Streptococcus pneumoniae. At two times the MIC, PAEs of levofloxacin were an average of 0.6 h longer than the PAEs obtained for ciprofloxacin for methicillin-susceptible and methicillin-resistant S. aureus strains. The PAEs of levofloxacin and ciprofloxacin ranged from 1.8 to 3.1 and 1.1 to 2.4 h, respectively. Continued exposure of the methicillin-resistant strain to 1/16, 1/8, and 1/4 the MIC resulted in PA SMEs of 6.5, 15.3, and >22.3 h, respectively, for levofloxacin and 3.8, 8.0, and 12.3 h, respectively, for ciprofloxacin. For isolates of S. pneumoniae, at two times the MIC of both fluoroquinolones, the average PAEs of levofloxacin and ciprofloxacin were equivalent: 1.3 h for the penicillin-susceptible isolate and 0.6 h for the penicillin-resistant isolate. Continued exposure of the penicillin-susceptible S. pneumoniae strain to 1/16, 1/8, and 1/4 the MIC resulted in average PA SMEs of 1.0, 1.4, and 2.8 h, respectively, for levofloxacin and 1.8, 2.0, and 2.5 h, respectively, for ciprofloxacin. Continued exposure of penicillin-resistant S. pneumoniae to 1/16, 1/8, and 1/4 the MIC of the same fluoroquinolones resulted in average PA SMEs of 0.6, 1.1, and 2.9 h, respectively, for levofloxacin and 0.6, 1.1, and 1.5 h, respectively, for ciprofloxacin. The PA SMEs observed demonstrate the superior activity of levofloxacin against methicillin-susceptible or methicillin-resistant S. aureus. Although PAEs were similar for the penicillin-susceptible and penicillin-resistant S. pneumoniae strains, the PA SME of levofloxacin at one-fourth the MIC was longer for penicillin-resistant S. pneumoniae.

scholarly journals Implications of the EUCAST Trailing Phenomenon inCandida tropicalisfor theIn VivoSusceptibility in Invertebrate and Murine Models

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
K. M. T. Astvad ◽  
D. Sanglard ◽  
E. Delarze ◽  
R. K. Hare ◽  
M. C. Arendrup
Keyword(s):  
Susceptibility Testing ◽  
Galleria Mellonella ◽  
Growth Control ◽  
Resistant Isolate ◽  
Murine Models ◽  
Wild Type ◽  
Content Type ◽  
Gradual Loss

ABSTRACTCandida tropicalisisolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigatedin vitroandin vivo(in aGalleria mellonellasurvival model and two nonlethal murine models).CDR1expression levels andERG11sequences were characterized. The survival in fluconazole-treatedG. mellonellawas inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment.CDR1expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibitedERG11wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in allin vivomodels, indicating an impact on fluconazole efficacy.CDR1upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.

scholarly journals A Novel Y319H Substitution in CYP51C Associated with Azole Resistance in Aspergillus flavus

2015 ◽  
Vol 59 (10) ◽  
pp. 6615-6619 ◽  
Author(s):  
R. A. Paul ◽  
S. M. Rudramurthy ◽  
J. F. Meis ◽  
J. W. Mouton ◽  
A. Chakrabarti
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Aspergillus Flavus ◽  
Amino Acid Substitution ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Nucleotide Change ◽  
Content Type ◽  
Susceptibility Status

ABSTRACTThis study aimed to explore any mutation in theCYP51gene conferring azole resistance inAspergillus flavus. Two voriconazole-resistant and 45 voriconazole-susceptible isolates were included in the study. Sequence analysis demonstrated a T1025C nucleotide change inCYP51C, resulting in the Y319H amino acid substitution in one resistant isolate. However, the earlier described T788G mutation inCYP51Cconferring voriconazole resistance inA. flavusisolates was present in all isolates, irrespective of their susceptibility status.

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