scholarly journals Multiple Resistance Mechanisms among Aspergillus fumigatus Mutants with High-Level Resistance to Itraconazole

2003 ◽  
Vol 47 (5) ◽  
pp. 1719-1726 ◽  
Author(s):  
Adriana M. Nascimento ◽  
Gustavo H. Goldman ◽  
Steven Park ◽  
Salvatore A. E. Marras ◽  
Guillaume Delmas ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Molecular Beacon ◽  
Efflux Pump ◽  
Pcr Amplification ◽  
Resistance Mechanisms ◽  
Major Facilitator Superfamily ◽  
Expression Levels ◽  
Clinical Resistance ◽  
High Level

ABSTRACT A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 μg ml−1 were selected in vitro (following UV irradiation as a preliminary step) to investigate mechanisms of drug resistance in this clinically important pathogen. Eight of the RIT mutants were found to have a mutation at Gly54 (G54E, -K, or -R) in the azole target gene CYP51A. Primers designed for highly conserved regions of multidrug resistance (MDR) pumps were used in reverse transcriptase PCR amplification reactions to identify novel genes encoding potential MDR efflux pumps in A. fumigatus. Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail. Analysis of the deduced amino acid sequence encoded by AfuMDR3 revealed high similarity to major facilitator superfamily transporters, while AfuMDR4 was a typical member of the ATP-binding cassette superfamily. Real-time quantitative PCR with molecular beacon probes was used to assess expression levels of AfuMDR3 and AfuMDR4. Most RIT mutants showed either constitutive high-level expression of both genes or induction of expression upon exposure to itraconazole. Our results suggest that overexpression of one or both of these newly identified drug efflux pump genes of A. fumigatus and/or selection of drug target site mutations are linked to high-level itraconazole resistance and are mechanistic considerations for the emergence of clinical resistance to itraconazole.

scholarly journals In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa

2020 ◽  
Vol 75 (9) ◽  
pp. 2508-2515 ◽  
Author(s):  
María A Gomis-Font ◽  
Gabriel Cabot ◽  
Irina Sánchez-Diener ◽  
Pablo A Fraile-Ribot ◽  
Carlos Juan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Infection Model ◽  
Mechanisms Of Resistance ◽  
Resistance Development ◽  
C Elegans ◽  
Imipenem Resistance ◽  
High Level

Abstract Objectives We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. Methods PAO1 or PAOMS strains were incubated for 24 h in Mueller–Hinton Broth with 0.125–64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. Results Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. Conclusions Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

scholarly journals In Vitro Evolution of Itraconazole Resistance in Aspergillus fumigatus Involves Multiple Mechanisms of Resistance

2004 ◽  
Vol 48 (11) ◽  
pp. 4405-4413 ◽  
Author(s):  
Márcia Eliana da Silva Ferreira ◽  
José Luiz Capellaro ◽  
Everaldo dos Reis Marques ◽  
Iran Malavazi ◽  
David Perlin ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistant Mutant ◽  
Marker Genes ◽  
Reverse Transcription Pcr ◽  
Selectable Marker Genes ◽  
Clinical Resistance ◽  
Mutant Strains ◽  
Drug Efflux Pumps ◽  
High Level

ABSTRACT We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.

scholarly journals High Levels of Intrinsic Tetracycline Resistance inMycobacterium abscessusAre Conferred by a Tetracycline-Modifying Monooxygenase

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Paulami Rudra ◽  
Kelley Hurst-Hess ◽  
Pascal Lappierre ◽  
Pallavi Ghosh
Keyword(s):  
Bacterial Infections ◽  
Efflux Pump ◽  
Tetracycline Resistance ◽  
Resistance Mechanisms ◽  
Mycobacterium Abscessus ◽  
Repeat Sequence ◽  
Content Type ◽  
High Level

ABSTRACTTetracyclines have been one of the most successful classes of antibiotics. However, its extensive use has led to the emergence of widespread drug resistance, resulting in discontinuation of use against several bacterial infections. Prominent resistance mechanisms include drug efflux and the use of ribosome protection proteins. Infrequently, tetracyclines can be inactivated by the TetX class of enzymes, also referred to as tetracycline destructases. Low levels of tolerance to tetracycline inMycobacterium smegmatisandMycobacterium tuberculosishave been previously attributed to the WhiB7-dependent TetV/Tap efflux pump. However,Mycobacterium abscessusis ∼500-fold more resistant to tetracycline thanM. smegmatisandM. tuberculosis. In this report, we show that this high level of resistance to tetracycline and doxycycline inM. abscessusis conferred by a WhiB7-independent tetracycline-inactivating monooxygenase, MabTetX (MAB_1496c). The presence of sublethal doses of tetracycline and doxycycline results in a >200-fold induction of MabTetX, and an isogenic deletion strain is highly sensitive to both antibiotics. Further, purified MabTetX can rapidly monooxygenate both antibiotics. We also demonstrate that expression of MabTetX is repressed by MabTetRx, by binding to an inverted repeat sequence upstream of MabTetRx; the presence of either antibiotic relieves this repression. Moreover, anhydrotetracycline (ATc) can effectively inhibit MabTetX activityin vitroand decreases the MICs of both tetracycline and doxycyclinein vivo. Finally, we show that tigecycline, a glycylcycline tetracycline, not only is a poor substrate of MabTetX but also is incapable of inducing the expression of MabTetX. This is therefore the first demonstration of a tetracycline-inactivating enzyme in mycobacteria. It (i) elucidates the mechanism of tetracycline resistance inM. abscessus, (ii) demonstrates the use of an inhibitor that can potentially reclaim the use of tetracycline and doxycycline, and (iii) identifies two sequential bottlenecks—MabTetX and MabTetRx—for acquiring resistance to tigecycline, thereby reiterating its use againstM. abscessus.

scholarly journals Identification of novel mutations contributing to azole tolerance of Aspergillus fumigatus through in vitro exposure to tebuconazole

2021 ◽  
Author(s):  
Takahito Toyotome ◽  
Kenji Onishi ◽  
Mio Sato ◽  
Yoko Kusuya ◽  
Daisuke Hagiwara ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Glutamate Transporter ◽  
Resistance Mechanisms ◽  
Major Facilitator Superfamily ◽  
Mutant Selection ◽  
Synonymous Mutations ◽  
Mitochondrial Inner Membrane ◽  
Azole Tolerance ◽  
Major Facilitator

Azole resistance of Aspergillus fumigatus is a global problem. The major resistant mechanism is a cytochrome P 450 14-α sterol demethylase Cyp51A alteration such as mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerances and resistant mechanisms such as hmg1 (a 3-hydroxy-3-methylglutaryl-coenzyme-A reductase gene) mutation are known, few reports have described studies elucidating non-Cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three-round selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the obtained isolates and the parental strain revealed a non-synonymous mutation in MfsD for a major facilitator superfamily protein (Afu1g11820, R337L mutation) in all isolates. Furthermore, non-synonymous mutations in AgcA for a mitochondrial inner membrane aspartate/glutamate transporter (Afu7g05220, E535Stop mutation), UbcD for a ubiquitin-conjugating enzyme E2 (Afu3g06030, T98K mutation), AbcJ for an ABC transporter (Afu3g12220, G297E mutation), and RttA for a putative protein r esponsible for t ebuconazole t olerance (Afu7g04740, A83T mutation), were found in at least one isolate. Disruption of the agcA gene led to decreased susceptibility to azoles. Reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. Reversion of T98K mutation to wild type in UbcD led to decreased susceptibility to azoles. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and agricultural azole fungicides.

scholarly journals Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

2001 ◽  
Vol 45 (12) ◽  
pp. 3422-3426 ◽  
Author(s):  
Siddhartha Roychoudhury ◽  
Tracy L. Twinem ◽  
Kelly M. Makin ◽  
Mark A. Nienaber ◽  
Chuiying Li ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Efflux Pump ◽  
Serial Passage ◽  
Efflux Pump Inhibitor ◽  
In Vitro Development ◽  
Development Of Resistance ◽  
High Level ◽  
Vitro Development

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru

2019 ◽  
Vol 58 (1) ◽  
pp. 54-60 ◽  
Author(s):  
Beatriz Bustamante ◽  
Luis Ricardo Illescas ◽  
Andrés Posadas ◽  
Pablo E Campos
Keyword(s):  
Aspergillus Fumigatus ◽  
Latin American ◽  
Pcr Amplification ◽  
Sensu Stricto ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Molecular Testing ◽  
Naive Patient ◽  
In Vitro Susceptibility

Abstract Azole resistance among Aspergillus fumigatus isolates, which is mainly related to mutations in the cyp51A gene, is a concern because it is rising, worldwide disseminated, and associated with treatment failure and death. Data on azole resistance of aspergillus from Latin American countries is very scarce and do not exist for Peru. Two hundred and seven Aspergillus clinical isolates collected prospectively underwent mycology and molecular testing for specie identification, and 143 isolates were confirmed as A. fumigatus sensu stricto (AFSS). All AFSS were tested for in vitro azole susceptibility, and resistant isolates underwent PCR amplification and sequencing of the whole cyp51A gene and its promoter. The in vitro susceptibility showed a minimal inhibitory concentration (MIC) range, MIC50 and MIC90 of 0.125 to &gt;16, 0.25, and 0.5 μg/ml for itraconazole; 0.25 to 2, 0.5, and 0.5 μg/ml for voriconazole; and 0.003 to 1, 0.06, and 0.125 μg/ml for posaconazole. Three isolates (2%) showed resistance to itraconazole and exhibited different mutations of the cyp51A gene. One isolate harbored the mutation M220K, while a second one exhibited the G54 mutation plus a modification in the cyp51A gene promoter. The third isolate, from an azole naive patient, presented an integration of a 34-bp tandem repeat (TR34) in the promoter region of the gene and a substitution of leucine 98 by histidine (L98H). The three source patients had a diagnosis or suspicion of chronic pulmonary aspergillosis.

scholarly journals Evaluation of amoxicillin-clavulanic acid resistance in the Bifidobacterium genus

2021 ◽  
Author(s):  
Leonardo Mancabelli ◽  
Walter Mancino ◽  
Gabriele Andrea Lugli ◽  
Chiara Argentini ◽  
Giulia Longhi ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Clavulanic Acid ◽  
Acid Resistance ◽  
Resistance Mechanisms ◽  
Western World ◽  
Beneficial Effects ◽  
Amoxicillin Clavulanic Acid ◽  
High Level ◽  
The Impact

Amoxicillin-Clavulanic acid (AMC) is one of the most frequently prescribed antibiotic formulations in the Western world. Extensive oral use of this antimicrobial combination influences the gut microbiota. One of the most abundant early colonizers of the human gut microbiota is represented by different taxa of the Bifidobacterium genus, which include many members that are considered to bestow beneficial effects upon their host. In the current study, we investigated the impact of AMC administration on the gut microbiota composition, comparing the gut microbiota of 23 children that had undergone AMC antibiotic therapy to that of 19 children that had not been treated with antibiotics during the preceding six months. Moreover, we evaluated AMC sensitivity by Minimal Inhibitory Concentration (MIC) test of 261 bifidobacterial strains, including reference strains for the currently recognized 64 bifidobacterial (sub)species, as well as 197 bifidobacterial isolates of human origin. These assessments allowed the identification of four bifidobacterial strains, which exhibit a high level of AMC insensitivity, and which were subjected to genomic and transcriptomic analyses to identify the putative genetic determinants responsible for this AMC insensitivity. Furthermore, we investigated the ecological role of AMC-resistant bifidobacterial strains by in vitro batch-cultures. Importance Based on our results, we observed a drastic reduction in gut microbiota diversity of children treated with antibiotics, also affecting the abundance of Bifidobacterium, a bacterial genus commonly found in the infant gut. MIC experiments revealed that more than 98% of bifidobacterial strains tested were shown to be inhibited by the AMC antibiotic. Isolation of four insensitive strains and sequencing of their genome revealed the identity of possible genes involved in AMC resistance mechanisms. Moreover, gut-simulating in-vitro experiments revealed that one strain, i.e. B. breve PRL2020, is able to persist in the presence of a complex microbiota combined with AMC antibiotic.

scholarly journals The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

2019 ◽  
Author(s):  
Seth A. Wander ◽  
Ofir Cohen ◽  
Xueqian Gong ◽  
Gabriela N. Johnson ◽  
Jorge Buendia-Buendia ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Acquired Resistance ◽  
Metastatic Breast ◽  
Resistance Mechanisms ◽  
Akt Activation ◽  
Hormone Receptor Positive ◽  
Ras Activation ◽  
Whole Exome ◽  
Clinical Resistance

AbstractClinical resistance mechanisms to CDK4/6 inhibitors in HR+ breast cancer have not been clearly defined. Whole exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including RB1 loss, activating alterations in AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of ER expression. In vitro experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired RB1, KRAS, AURKA, or CCNE2 alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Besides inactivation of RB1, which accounts for ∼5% of resistance, seven of these mechanisms have not been previously identified as clinical mediators of resistance to CDK4/6 inhibitors in patients. Three of these—RAS activation, AKT activation, and AURKA activation—have not to our knowledge been previously demonstrated preclinically. Together, these eight mechanisms were present in 80% of resistant tumors profiled and may define therapeutic opportunities in patients.SignificanceWe identified eight distinct mechanisms of resistance to CDK4/6 inhibitors present in 80% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR+ MBC.

scholarly journals Identification of novel mutations contributing to azole tolerance of Aspergillus fumigatus through in vitro exposure to tebuconazole

2020 ◽  
Author(s):  
Takahito Toyotome ◽  
Kenji Onishi ◽  
Mio Sato ◽  
Yoko Kusuya ◽  
Daisuke Hagiwara ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Parental Strain ◽  
Resistance Mechanisms ◽  
Wild Type ◽  
Genome Sequences ◽  
Novel Mutations ◽  
Mutant Selection ◽  
Synonymous Mutations ◽  
Azole Tolerance

AbstractAzole resistance of Aspergillus fumigatus is a global problem. The major resistant mechanism is a cyp51A alteration such as mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerances and resistant mechanisms such as hmg1 mutation are known, few reports describe studies elucidating non-cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three-round selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the obtained isolates and the parental strain revealed a non-synonymous mutation in MfsD (Afu1g11820, R337L mutation) in all isolates. Furthermore, non-synonymous mutations in AgcA (Afu7g05220, E535Stop mutation), UbcD (Afu3g06030, T98K mutation), AbcJ (Afu3g12220, G297E mutation), and RttA (Afu7g04740, A83T mutation), a protein responsible for tebuconazole tolerance, were found in at least one isolate. Clarification by constructing the MfsD R337L mutant suggests that the mutation contributes to azole tolerance. Disruption of the agcA gene and reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. The reversion of T98K mutation to wild type in UbcD led to the level of azole susceptibility comparable to the parental strain. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and to agricultural azole fungicides.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

2021 ◽  
Author(s):  
Marion Aruanno ◽  
Samantha Gozel ◽  
Isabelle Mouyna ◽  
Josie E Parker ◽  
Daniel Bachmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Drug Transporters ◽  
Major Facilitator Superfamily ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

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