scholarly journals Contributions ofyap1Mutation and SubsequentatrFUpregulation to Voriconazole Resistance inAspergillus flavus

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Yuuta Ukai ◽  
Miho Kuroiwa ◽  
Naoko Kurihara ◽  
Hiroki Naruse ◽  
Tomoyuki Homma ◽  
...  
Keyword(s):  
Point Mutation ◽  
Nuclear Export ◽  
Resistance Mechanism ◽  
Resistant Mutant ◽  
Gene Replacement ◽  
Bzip Transcription Factor ◽  
Homologous Gene ◽  
Sequencing Analysis ◽  
Content Type

ABSTRACTAspergillus flavusis the second most significant pathogenic cause of invasive aspergillosis; however, its emergence risks and mechanisms of voriconazole (VRC) resistance have not yet been elucidated in detail. Here, we demonstrate that repeated exposure ofA. flavusto subinhibitory concentrations of VRCin vitrocauses the emergence of a VRC-resistant mutant with a novel resistance mechanism. The VRC-resistant mutant shows a MIC of 16 μg/ml for VRC and of 0.5 μg/ml for itraconazole (ITC). Whole-genome sequencing analysis showed that the mutant possesses a point mutation inyap1, which encodes a bZIP transcription factor working as the master regulator of the oxidative stress response, but no mutations in thecyp51genes. This point mutation inyap1caused alteration of Leu558 to Trp (Yap1Leu558Trp) in the putative nuclear export sequence in the carboxy-terminal cysteine-rich domain of Yap1. This Yap1Leu558Trpsubstitution was confirmed as being responsible for the VRC-resistant phenotype, but not for that of ITC, by the revertant to Yap1wild typewith homologous gene replacement. Furthermore, Yap1Leu558Trpcaused marked upregulation of theatrFATP-binding cassette transporter, and the deletion ofatrFrestored susceptibility to VRC inA. flavus. These findings provide new insights into VRC resistance mechanisms via a transcriptional factor mutation that is independent of thecyp51gene mutation inA. flavus.

scholarly journals In VitroAntibacterial Activity of NB-003 against Propionibacterium acnes

2011 ◽  
Vol 55 (9) ◽  
pp. 4211-4217 ◽  
Author(s):  
J. Pannu ◽  
A. McCarthy ◽  
A. Martin ◽  
T. Hamouda ◽  
S. Ciotti ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Propionibacterium Acnes ◽  
Resistant Mutant ◽  
Microtiter Plate ◽  
Content Type ◽  
Oil In Water ◽  
Complete Disruption ◽  
Kinetics Of ◽  
Gel Formulations

ABSTRACTNB-003 and NB-003 gel formulations are oil-in-water nanoemulsions designed for use in bacterial infections.In vitrosusceptibility ofPropionibacterium acnesto NB-003 formulations and comparator drugs was evaluated. Both NB-003 formulations were bactericidal against allP. acnesisolates, including those that were erythromycin, clindamycin, and/or tetracycline resistant. In the absence of sebum, the MIC90s/minimum bactericidal concentrations (MBC90s) for NB-003, NB-003 gel, salicylic acid (SA), and benzoyl peroxide (BPO) were 0.5/2.0, 1.0/2.0, 1,000/2,000, and 50/200 μg/ml, respectively. In the presence of 50% sebum, the MIC90s/MBC90s of NB003 and BPOs increased to 128/1,024 and 400/1,600 μg/ml, respectively. The MIC90s/MBC90s of SA were not significantly impacted by the presence of sebum. A reduction in the MBC90s for NB-003 and BPO was observed when 2% SA or 0.5% BPO was integrated into the formulation, resulting in MIC90s/MBC90s of 128/256 μg/ml for NB003 and 214/428 μg/ml for BPO. The addition of EDTA enhanced thein vitroefficacy of 0.5% NB-003 in the presence or absence of 25% sebum. The addition of 5 mM EDTA to each well of the microtiter plate resulted in a >16- and >256-fold decrease in MIC90and MBC90, yielding a more potent MIC90/MBC90of ≤1/<1 μg/ml. The kinetics of bactericidal activity of NB-003 againstP. acneswere compared to those of a commercially available product of BPO. Electron micrographs ofP. acnestreated with NB-003 showed complete disruption of bacteria. Assessment of spontaneous resistance ofP. acnesrevealed no stably resistant mutant strains.

scholarly journals Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

2015 ◽  
Vol 59 (6) ◽  
pp. 3059-3065 ◽  
Author(s):  
C. Pitart ◽  
F. Marco ◽  
T. A. Keating ◽  
W. W. Nichols ◽  
J. Vila
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistant Mutant ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Content Type ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Impact

ABSTRACTCeftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200Enterobacteriaceaeand 25Pseudomonas aeruginosastrains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistantEnterobacteriaceaestrains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBLEscherichia coli(MIC90of 0.25 mg/liter), ESBLKlebsiella pneumoniae(MIC90of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90of 1 mg/liter), non-ESBLE. coli(MIC90of ≤0.125 mg/liter), non-ESBLK. pneumoniae(MIC90of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistantP. aeruginosastrains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtainedin vitrofrom two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains ofEnterobacteriaceaeandP. aeruginosawere ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affectEnterobacteriaceaeandP. aeruginosasusceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.

scholarly journals Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

2014 ◽  
Vol 58 (9) ◽  
pp. 5191-5201 ◽  
Author(s):  
Giorgia Letizia Marcone ◽  
Elisa Binda ◽  
Lucia Carrano ◽  
Mervyn Bibb ◽  
Flavia Marinelli
Keyword(s):  
Antibiotic Resistance ◽  
Gene Dosage ◽  
Antibiotic Production ◽  
Resistance Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Penicillin G ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
High Level

ABSTRACTGlycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens.Nonomuraeasp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonicalvanHAXgenes. Consequently, we investigated the role of the β-lactam-sensitived,d-peptidase/d,d-carboxypeptidase encoded byvanYn, the onlyvan-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic inNonomuraeasp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we variedvanYngene dosage and expressedvanHatAatXatfrom the teicoplanin producerActinoplanes teichomyceticusinNonomuraeasp. ATCC 39727. Knocking outvanYn, complementing avanYnmutant, or duplicatingvanYnhad no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production.Nonomuraeasp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYnactivity. The heterologous expression ofvanHatAatXatincreased A40926 resistance inNonomuraeasp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. ThevanYn-based self-resistance inNonomuraeasp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants ofEnterococcus faeciumselectedin vitrofor high-level resistance to glycopeptides and penicillins.

scholarly journals Transcription Factor ADS-4 Regulates Adaptive Responses and Resistance to Antifungal Azole Stress

2015 ◽  
Vol 59 (9) ◽  
pp. 5396-5404 ◽  
Author(s):  
Kangji Wang ◽  
Zhenying Zhang ◽  
Xi Chen ◽  
Xianyun Sun ◽  
Cheng Jin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Efflux Pump ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Bzip Transcription Factor ◽  
Azole Resistance ◽  
Homologous Gene ◽  
Adaptive Responses ◽  
Transcriptional Responses ◽  
Content Type

ABSTRACTAzoles are commonly used as antifungal drugs or pesticides to control fungal infections in medicine and agriculture. Fungi adapt to azole stress by rapidly activating the transcription of a number of genes, and transcriptional increases in some azole-responsive genes can elevate azole resistance. The regulatory mechanisms that control transcriptional responses to azole stress in filamentous fungi are not well understood. This study identified a bZIP transcription factor, ADS-4 (antifungaldrugsensitive-4), as a new regulator of adaptive responses and resistance to antifungal azoles. Transcription ofads-4inNeurospora crassacells increased when they were subjected to ketoconazole treatment, whereas the deletion ofads-4resulted in hypersensitivity to ketoconazole and fluconazole. In contrast, the overexpression ofads-4increased resistance to fluconazole and ketoconazole inN. crassa. Transcriptome sequencing (RNA-seq) analysis, followed by quantitative reverse transcription (qRT)-PCR confirmation, showed that ADS-4 positively regulated the transcriptional responses of at least six genes to ketoconazole stress inN. crassa. The gene products of four ADS-4-regulated genes are known contributors to azole resistance, including the major efflux pump CDR4 (Pdr5p ortholog), an ABC multidrug transporter (NcAbcB), sterol C-22 desaturase (ERG5), and a lipid transporter (NcRTA2) that is involved in calcineurin-mediated azole resistance. Deletion of theads-4-homologous gene Afads-4inAspergillus fumigatuscaused hypersensitivity to itraconazole and ketoconazole, which suggested that ADS-4 is a functionally conserved regulator of adaptive responses to azoles. This study provides important information on a new azole resistance factor that could be targeted by a new range of antifungal pesticides and drugs.

scholarly journals TCP1γ Subunit Is Indispensable for Growth and Infectivity of Leishmania donovani

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Shailendra Yadav ◽  
Jitendra Kuldeep ◽  
Mohammad I. Siddiqi ◽  
Neena Goyal
Keyword(s):  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Gene Replacement ◽  
Titration Calorimetry ◽  
Content Type ◽  
T Complex ◽  
Complex Protein ◽  
Oligomeric Complex

ABSTRACT T-complex protein-1 (TCP1) is a ubiquitous group II chaperonin and is known to fold various proteins, such as actin and tubulin. In Leishmania donovani, the γ subunit of TCP1 (LdTCP1γ) has been cloned and characterized. It forms a high-molecular-weight homo-oligomeric complex that performs ATP-dependent protein folding. In the present study, we evaluated the essentiality of the LdTCP1γ gene. Gene replacement studies indicated that LdTCP1γ is essential for parasite survival. The LdTCP1γ single-allele-replacement mutants exhibited slowed growth and decreased infectivity in mouse macrophages compared to the growth and infectivity of the wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, an antitrypanosomal drug, not only inhibited the luciferase refolding activity of the recombinant LdTCP1γ (rLdTCP1γ) homo-oligomeric complex but also exhibited potential antileishmanial efficacy both in vitro and in vivo. The interaction of suramin and LdTCP1γ was further validated by isothermal titration calorimetry. The study suggests LdTCP1γ as a potential drug target and also provides a framework for the development of a new class of drugs.

scholarly journals Role of the GapA and CrmA Cytadhesins of Mycoplasma gallisepticum in Promoting Virulence and Host Colonization

2013 ◽  
Vol 81 (5) ◽  
pp. 1618-1624 ◽  
Author(s):  
Ivana Indiková ◽  
Peter Much ◽  
László Stipkovits ◽  
Karin Siebert-Gulle ◽  
Michael P. Szostak ◽  
...  
Keyword(s):  
Point Mutation ◽  
Mycoplasma Gallisepticum ◽  
Chronic Respiratory Disease ◽  
Host Colonization ◽  
Content Type ◽  
Inner Organs ◽  
Avian Pathogen ◽  
Low Passage

ABSTRACTMycoplasma gallisepticumis an important avian pathogen that commonly induces chronic respiratory disease in chicken. To better understand the mycoplasma factors involved in host colonization, chickens were infected via aerosol with two hemadsorption-negative (HA−) mutants, mHAD3 and RCL2, that were derived from a low passage of the pathogenic strain R (Rlow) and are both deficient in the two major cytadhesins GapA and CrmA. After 9 days of infection, chickens were monitored for air sac lesions and for the presence of mycoplasmas in various organs. The data showed that mHAD3, in which thecrmAgene has been disrupted, did not promote efficient colonization or significant air sac lesions. In contrast, the spontaneous HA−RCL2 mutant, which contains a point mutation in thegapAstructural gene, successfully colonized the respiratory tract and displayed an attenuated virulence compared to that of Rlow. It has previously been shownin vitrothat the point mutation of RCL2 spontaneously reverts with a high frequency, resulting in on-and-off switching of the HA phenotype. Detailed analyses further revealed that such an event is not responsible for the observedin vivooutcome, since 98.4% of the mycoplasma populations recovered from RCL2-infected chickens still display the mutation and the associated phenotype. Unlike Rlow, however, RCL2 was unable to colonize inner organs. These findings demonstrate the major role played by the GapA and CrmA proteins inM. gallisepticumhost colonization and virulence.

scholarly journals A Novel Point Mutation Promotes Growth Phase-Dependent Daptomycin Tolerance in Staphylococcus aureus

2015 ◽  
Vol 59 (9) ◽  
pp. 5366-5376 ◽  
Author(s):  
Lukas Mechler ◽  
Alexander Herbig ◽  
Kerstin Paprotka ◽  
Martin Fraunholz ◽  
Kay Nieselt ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Point Mutation ◽  
Growth Phase ◽  
Drug Tolerance ◽  
Stationary Growth Phase ◽  
Cellular Functions ◽  
Upstream Gene ◽  
Content Type ◽  
High Degree

ABSTRACTRecalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters, and also planktonic cells and is associated with chronic or relapsing infections with pathogens such asStaphylococcus aureus. Here we report thein vitroevolution of anS. aureusstrain that exhibits a high degree of nonsusceptibility to daptomycin as a result of cyclic challenges with bactericidal concentrations of the drug. This phenotype was attributed to stationary growth phase-dependent drug tolerance and was clearly distinguished from resistance. The underlying genetic basis was revealed to be an adaptive point mutation in the putative inorganic phosphate (Pi) transporter genepitA. Drug tolerance caused by this allele, termedpitA6, was abrogated when the upstream genepitRwas inactivated. Enhanced tolerance toward daptomycin, as well as the acyldepsipeptide antibiotic ADEP4 and various combinations of other drugs, was accompanied by elevated intracellular concentrations of Piand polyphosphate, which may reversibly interfere with critical cellular functions. The evolved strain displayed increased rates of survival within human endothelial cells, demonstrating the correlation of intracellular persistence and drug tolerance. These findings will be useful for further investigations ofS. aureusdrug tolerance, toward the development of additional antipersister compounds and strategies.

scholarly journals Contribution of Resistance-Nodulation-Division Efflux Pump OperonsmeU1-V-W-U2-Xto Multidrug Resistance of Stenotrophomonas maltophilia

2011 ◽  
Vol 55 (12) ◽  
pp. 5826-5833 ◽  
Author(s):  
Chao-Hsien Chen ◽  
Chiang-Ching Huang ◽  
Tsao-Chuen Chung ◽  
Rouh-Mei Hu ◽  
Yi-Wei Huang ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Acquired Resistance ◽  
Type Systems ◽  
Resistant Mutant ◽  
Multidrug Resistant ◽  
Content Type ◽  
Membrane Fusion Protein ◽  
Resistance Nodulation Division

ABSTRACTKJ09C, a multidrug-resistant mutant ofStenotrophomonas maltophiliaKJ, was generated byin vitroselection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes:smeU1,smeV,smeW,smeU2, andsmeX. Proteins encoded bysmeV,smeW, andsmeXwere similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded bysmeU1andsmeU2were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to β-lactams and erythromycin was not affected. The expression of thesmeU1-V-W-U2-Xoperon was regulated by the divergently transcribed LysR-type regulator genesmeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation ofsmeVandsmeWcompletely abolished the activity of the SmeVWX pump, whereas inactivation ofsmeXalone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression.

scholarly journals Influence of High Mutation Rates on the Mechanisms and Dynamics of In Vitro and In Vivo Resistance Development to Single or Combined Antipseudomonal Agents

2007 ◽  
Vol 51 (7) ◽  
pp. 2574-2581 ◽  
Author(s):  
V. Plasencia ◽  
N. Borrell ◽  
M. D. Maciá ◽  
B. Moya ◽  
J. L. Pérez ◽  
...  
Keyword(s):  
Combined Therapy ◽  
Bacterial Load ◽  
Resistance Mechanism ◽  
Resistant Mutant ◽  
Mutation Rates ◽  
Aminoglycoside Resistance ◽  
Resistance Development ◽  
Mutant Cells

ABSTRACT We studied the mechanisms and dynamics of the development of resistance to ceftazidime (CAZ) alone or combined with tobramycin (TOB) or ciprofloxacin (CIP) in vitro and in vivo (using a mouse model of lung infection with human antibiotic regimens). Pseudomonas aeruginosa strain PAO1 and its hypermutable derivative PAOΔmutS were used, and the results were compared with those previously obtained with CIP, TOB, and CIP plus TOB (CIP-TOB) under the same conditions. An important (200-fold) amplification of the number of resistant mutant cells was documented for PAOΔmutS-infected mice that were under CAZ treatment compared to the number for mice that received placebo, whereas the median number of resistant mutant cells was below the detection limits for mice infected by PAO1. These results were intermediate between the high amplification with CIP (50,000-fold) and the low amplification with TOB (10-fold). All CAZ-resistant single mutant cells selected in vitro or in vivo hyperproduced AmpC. On the other hand, the three combinations studied were found to be highly effective in the prevention of in vivo resistance development in mice infected with PAOΔmutS, although the highest therapeutic efficacy (in terms of mortality and total bacterial load reduction) compared to those of the individual regimens was obtained with CIP-TOB and the lowest was with CAZ-CIP. Nevertheless, mutant cells that were resistant to the three combinations tested were readily selected in vitro for PAOΔmutS (mutation rates from 1.2 × 10−9 to 5.8 × 10−11) but not for PAO1, highlighting the potential risk for antimicrobial resistance development associated with the presence of hypermutable strains, even when combined therapy was used. All five independent CAZ-TOB-resistant PAOΔmutS double mutants studied presented the same resistance mechanism (AmpC hyperproduction plus an aminoglycoside resistance mechanism not related to MexXY), whereas four different combinations of resistance mechanisms were documented for the five CAZ-CIP-resistant double mutants.

scholarly journals OmpK26, a Novel Porin Associated with Carbapenem Resistance in Klebsiella pneumoniae

2011 ◽  
Vol 55 (10) ◽  
pp. 4742-4747 ◽  
Author(s):  
Laura García-Sureda ◽  
Antonio Doménech-Sánchez ◽  
Mariette Barbier ◽  
Carlos Juan ◽  
Joan Gascó ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane Proteins ◽  
Systemic Infection ◽  
Carbapenem Resistance ◽  
Resistant Isolate ◽  
Identical Result ◽  
Sequencing Analysis ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACTClinical isolates ofKlebsiella pneumoniaeresistant to carbapenems are being isolated with increasing frequency. Loss of the expression of the major nonspecific porins OmpK35/36 is a frequent feature in these isolates. In this study, we looked for porins that could compensate for the loss of the major porins in carbapenem-resistant organisms. Comparison of the outer membrane proteins from twoK. pneumoniaeclinical isogenic isolates that are susceptible (KpCS-1) and resistant (KpCR-1) to carbapenems revealed the absence of OmpK35/36 and the presence of a new 26-kDa protein in the resistant isolate. An identical result was obtained when another pair of isogenic isolates that are homoresistant (Kpn-3) and heteroresistant (Kpn-17) to carbapenems were compared. Mass spectrometry and DNA sequencing analysis demonstrated that this new protein, designated OmpK26, is a small monomeric oligogalacturonate-specific porin that belongs to the KdgM family of porins. Insertion-duplication mutagenesis of the OmpK26 coding gene,yjhA, in the carbapenem-resistant, porin-deficient isolate KpCR-1 caused the expression of OmpK36 and the reversion to the carbapenem-susceptible phenotype, suggesting that OmpK26 is indispensable for KpCR-1 to lose OmpK36 and become resistant to these antibiotics. Moreover, loss of the major porin and expression of OmpK26 reducedin vitrofitness and attenuated virulence in a murine model of acute systemic infection. Altogether, these results indicate that expression of the oligogalacturonate-specific porin OmpK26 compensates for the absence of OmpK35/36 and allows carbapenem resistance inK. pneumoniaebut cannot restore the fitness of the microorganism.

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