scholarly journals Azole Resistance of Aspergillus fumigatus Biofilms Is Partly Associated with Efflux Pump Activity

2011 ◽  
Vol 55 (5) ◽  
pp. 2092-2097 ◽  
Author(s):  
Ranjith Rajendran ◽  
Eilidh Mowat ◽  
Elaine McCulloch ◽  
David F. Lappin ◽  
Brian Jones ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Dependent Manner ◽  
Content Type ◽  
Quantitative Expression ◽  
Pump Activity ◽  
Competitive Substrate

ABSTRACTThis study investigated the phase-dependent expression and activity of efflux pumps inAspergillus fumigatustreated with voriconazole. Fourteen strains were shown to become increasingly resistant in the 12-h (16- to 128-fold) and 24-h (>512-fold) phases compared to 8-h germlings. An Ala-Nap uptake assay demonstrated a significant increase in efflux pump activity in the 12-h and 24-h phases (P< 0.0001). The efflux pump activity of the 8-h germling cells was also significantly induced by voriconazole (P< 0.001) after 24 h of treatment. Inhibition of efflux pump activity with the competitive substrate MC-207,110 reduced the voriconazole MIC values for theA. fumigatusgermling cells by 2- to 8-fold. Quantitative expression analysis ofAfuMDR4mRNA transcripts showed a phase-dependent increase as the mycelial complexity increased, which was coincidental with a strain-dependent increase in azole resistance. Voriconazole also significantly induced this in a time-dependent manner (P< 0.001). Finally, anin vivomouse biofilm model was used to evaluate efflux pump expression, and it was shown thatAfuMDR4was constitutively expressed and significantly induced by treatment with voriconazole after 24 h (P< 0.01). Our results demonstrate that efflux pumps are expressed in complexA. fumigatusbiofilm populations and that this contributes to azole resistance. Moreover, voriconazole treatment induces efflux pump expression. Collectively, these data may provide evidence for azole treatment failures in clinical cases of aspergillosis.

scholarly journals A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

2016 ◽  
Vol 60 (10) ◽  
pp. 5858-5866 ◽  
Author(s):  
Somanon Bhattacharya ◽  
Jack D. Sobel ◽  
Theodore C. White
Keyword(s):  
Candida Albicans ◽  
Efflux Pump ◽  
Pathogenic Fungus ◽  
Resistance Mechanism ◽  
Efflux Pumps ◽  
Resistant Isolate ◽  
Vaginal Infections ◽  
Content Type ◽  
Qrt Pcr ◽  
Pump Activity

ABSTRACTCandida albicansis a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginalC. albicansisolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginalC. albicansisolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginalC. albicansisolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginalC. albicansisolates.

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 Aspergillus fumigatusAfssn3-Afssn8Pair Reverse Regulates Azole Resistance by Conferring Extracellular Polysaccharide, Sphingolipid Pathway Intermediates, and Efflux Pumps to Biofilm

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Nanbiao Long ◽  
Liping Zeng ◽  
Shanlei Qiao ◽  
Lei Li ◽  
Guowei Zhong
Keyword(s):  
Amino Acids ◽  
Aspergillus Fumigatus ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Extracellular Polysaccharide ◽  
Azole Resistance ◽  
Drug Penetration ◽  
Reverse Genetic ◽  
Content Type ◽  
Barrier Layers

ABSTRACTAntifungal treatment is often ineffectual, partly because of biofilm formation. In this study, by using a combined forward and reverse genetic strategy, we identified that nucleus-localized AfSsn3 and its partner AfSsn8, which constitute a Cdk8-cyclin pair, are required for azole resistance inAspergillus fumigatus. Deletion ofAfssn3led to increased absorption and utilization of glucose and amino acids. Interestingly, absorption and utilization of glucose accelerated the extracellular polysaccharide formation, while utilization of the amino acids serine, threonine, and glycine increased sphingolipid pathway intermediate accumulation. In addition, the absence ofAfssn3induced the activity of the efflux pump proteins. These factors indicate the mature biofilm is responsible for the major mechanisms ofA. fumigatusresistance to azoles in the ΔAfssn3mutant. Collectively, the loss ofAfssn3led to two “barrier” layers between the intracellular and extracellular spaces, which consequently decreased drug penetration into the cell.

scholarly journals In Vivo Biomarker Analysis of the Effects of Intranasally Dosed PC945, a Novel Antifungal Triazole, on Aspergillus fumigatus Infection in Immunocompromised Mice

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Genki Kimura ◽  
Takahiro Nakaoki ◽  
Thomas Colley ◽  
Garth Rapeport ◽  
Pete Strong ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Interleukin 17 ◽  
Dependent Manner ◽  
Necrosis Factor Alpha ◽  
Once Daily ◽  
Content Type ◽  
Treatment Regimens ◽  
Biomarker Analysis ◽  
Immunocompromised Mice

ABSTRACT PC945 is a novel triazole optimized for lung delivery, and the objective of this study is to determine the effects of intranasally dosed PC945 on Aspergillus fumigatus infection and associated biomarkers in immunocompromised mice. PC945, posaconazole, or voriconazole was administered intranasally once daily on days 0 to 3 (early intervention) or days 1 to 3 (late intervention) postinfection in temporarily neutropenic A/J mice infected intranasally with A. fumigatus, and bronchoalveolar lavage fluid (BALF) and serum were collected on day 3. The effects of extended prophylaxis treatment (daily from days −7 to +3 or days −7 to 0) were also compared with those of the shorter treatment regimens (days −1 to +3 or days −1 and 0). Early and late interventions with PC945 (2.8 to 350 μg/mouse; approximately 0.11 to ∼14 mg/kg of body weight) were found to inhibit lung fungal loads and to decrease the concentrations of galactomannan (GM) in both BALF and serum as well as several biomarkers in BALF (interferon gamma [IFN-γ], interleukin-17 [IL-17], and malondialdehyde) and serum (tumor necrosis factor alpha [TNF-α] and IL-6) in a dose-dependent manner and were >3- and >47-fold more potent than intranasally dosed posaconazole and voriconazole, respectively. Furthermore, extended prophylaxis with low-dose PC945 (0.56 μg/mouse; 0.022 mg/kg) was found to inhibit fungal loads and to decrease the concentrations biomarkers more potently than did the shorter treatment regimens. Thus, PC945 dosed intranasally once daily showed potent antifungal effects, and the effects of PC945 accumulated upon repeat dosing and were persistent. Therefore, PC945 has the potential to be a novel inhaled therapy for the treatment of A. fumigatus infection in humans.

scholarly journals Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities

2012 ◽  
Vol 56 (9) ◽  
pp. 4771-4778 ◽  
Author(s):  
Bartolomé Moyá ◽  
Alejandro Beceiro ◽  
Gabriel Cabot ◽  
Carlos Juan ◽  
Laura Zamorano ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Binding Protein ◽  
Efflux Pumps ◽  
Binding Affinities ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Reverse Transcription Pcr

ABSTRACTWe investigated the mechanisms leading toPseudomonas aeruginosapan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence ofoprD,ampD, anddacBmutations was explored by PCR followed by sequencing and the expression ofampCand efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC50s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressedampCand one or several efflux pumps, particularlymexBand/ormexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n= 1) or diminished expression (n= 1) of PBP1a and a lack of PBP4 expression (n= 4), which correlated with AmpC overexpression driven bydacBmutation. Analysis of the essential PBP IC50s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC50s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in thein vivoemergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.

scholarly journals Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

2012 ◽  
Vol 56 (4) ◽  
pp. 2084-2090 ◽  
Author(s):  
Astrid Pérez ◽  
Margarita Poza ◽  
Ana Fernández ◽  
Maria del Carmen Fernández ◽  
Susana Mallo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type

ABSTRACTMultidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence inEnterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates ofE. cloacaewere used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). TheacrAandtolCgenes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrAand EcΔtolCand JcΔacrAand JcΔtolCknockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance ofE. cloacaeto several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that theacrAandtolCgenes both affect the fitness ofE. cloacae, as fitness was clearly reduced in theacrAandtolCKO strains. The median CI values obtainedin vitroandin vivowere, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in bothE. cloacaeclinical strains when either theacrAortolCgene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates ofE. cloacae.

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 Antileishmanial Aminopyrazoles: Studies into Mechanisms and Stability of Experimental Drug Resistance

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
M. Van den Kerkhof ◽  
D. Mabille ◽  
S. Hendrickx ◽  
P. Leprohon ◽  
C. E. Mowbray ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Copy Number ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Copy Number Variations ◽  
Content Type ◽  
Development Of Resistance ◽  
Efflux Pump Inhibitors

ABSTRACT Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases initiative (DNDi) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of Leishmania infantum promastigotes (in vitro) and intracellular amastigotes (both in vitro and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and Lutzomyia longipalpis sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either in vitro or in vivo. Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after in vivo passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.

scholarly journals MAPK1 of Leishmania donovani Modulates Antimony Susceptibility by Downregulating P-Glycoprotein Efflux Pumps

2015 ◽  
Vol 59 (7) ◽  
pp. 3853-3863 ◽  
Author(s):  
Mansi Garg ◽  
Neena Goyal
Keyword(s):  
Leishmania Donovani ◽  
Drug Sensitivity ◽  
Efflux Pump ◽  
Indian Subcontinent ◽  
Normal Growth ◽  
Efflux Pumps ◽  
Content Type ◽  
P Glycoprotein ◽  
Pump Activity ◽  
Antimony Resistance

ABSTRACTEmergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. InLeishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance inLeishmania donovani. TheL. donovaniMAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug.

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