scholarly journals Fluconazole Treatment Is Effective against a Candida albicans erg3/erg3 Mutant In Vivo Despite In Vitro Resistance

2006 ◽  
Vol 50 (2) ◽  
pp. 580-586 ◽  
Author(s):  
Taiga Miyazaki ◽  
Yoshitsugu Miyazaki ◽  
Koichi Izumikawa ◽  
Hiroshi Kakeya ◽  
Shunichi Miyakoshi ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
In Vivo Studies ◽  
Azole Resistance ◽  
In Vitro Testing ◽  
Fluconazole Resistance ◽  
Hyphal Formation

ABSTRACT Candida albicans ERG3 encodes a sterol C5,6-desaturase which is essential for synthesis of ergosterol. Defective sterol C5,6 desaturation has been considered to be one of the azole resistance mechanisms in this species. However, the clinical relevance of this resistance mechanism is still unclear. In this study, we created a C. albicans erg3/erg3 mutant by the “Ura-blaster” method and confirmed the expected azole resistance using standard in vitro testing and the presence of ergosta-7,22-dien-3β-ol instead of ergosterol. For in vivo studies, a wild-type URA3 was placed back into its native locus in the erg3 homozygote to avoid positional effects on URA3 expression. Defective hyphal formation of the erg3 homozygote was observed not only in vitro but in kidney tissues. A marked attenuation of virulence was shown by the longer survival and the lower kidney burdens of mice inoculated with the reconstituted Ura+ erg3 homozygote relative to the control. To assess fluconazole efficacy in a murine model of disseminated candidiasis, inoculum sizes of the control and the erg3 homozygote were chosen which provided a similar organ burden. Under these conditions, fluconazole was highly effective in reducing the organ burden in both groups. This study demonstrates that an ERG3 mutation causing inactivation of sterol C5,6-desaturase cannot confer fluconazole resistance in vivo by itself regardless of resistance measured by standard in vitro testing. The finding questions the clinical significance of this resistance mechanism.

scholarly journals Impact of the Order of Initiation of Fluconazole and Amphotericin B in Sequential or Combination Therapy on Killing of Candida albicans In Vitro and in a Rabbit Model of Endocarditis and Pyelonephritis

2001 ◽  
Vol 45 (2) ◽  
pp. 485-494 ◽  
Author(s):  
Arnold Louie ◽  
Pamela Kaw ◽  
Partha Banerjee ◽  
Weiguo Liu ◽  
George Chen ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amphotericin B ◽  
Induced Resistance ◽  
Rabbit Model ◽  
In Vivo Studies ◽  
Infection Model ◽  
Simultaneous Exposure ◽  
The Impact

ABSTRACT In vitro time-kill studies and a rabbit model of endocarditis and pyelonephritis were used to define the impact that the order of exposure of Candida albicans to fluconazole (FLC) and amphotericin B (AMB), as sequential and combination therapies, had on the susceptibility of C. albicans to AMB and on the outcome. The contribution of FLC-induced resistance to AMB for C. albicans also was assessed. In vitro, AMB monotherapy rapidly killed each of four C. albicans strains; FLC alone was fungistatic. Preincubation of these fungi with FLC for 18 h prior to exposure to AMB decreased their susceptibilities to AMB for 8 to >40 h. Induced resistance to AMB was transient, but the duration of resistance increased with the length of FLC preincubation. Yeast sequentially incubated with FLC followed by AMB plus FLC (FLC→AMB+FLC) showed fungistatic growth kinetics similar to that of fungi that were exposed to FLC alone. This antagonistic effect persisted for at least 24 h. Simultaneous exposure of C. albicans to AMB and FLC [AMB+FLC(simult)] demonstrated activity similar to that with AMB alone for AMB concentrations of ≥1 μg/ml; antagonism was seen using an AMB concentration of 0.5 μg/ml. The in vitro findings accurately predicted outcomes in our rabbit infection model. In vivo, AMB monotherapy and treatment with AMB for 24 h followed by AMB plus FLC (AMB→AMB+FLC) rapidly sterilized kidneys and cardiac vegetations. AMB+FLC(simult) and FLC→AMB treatments were slower in clearing fungi from infected tissues. FLC monotherapy and FLC→AMB+FLC were both fungistatic and were the least active regimens. No adverse interaction was observed between AMB and FLC for the AMB→FLC regimen. However, FLC→AMB treatment was slower than AMB alone in clearing fungi from tissues. Thus, our in vitro and in vivo studies both demonstrate that preexposure of C. albicans to FLC reduces fungal susceptibility to AMB. The length of FLC preexposure and whether AMB is subsequently used alone or in combination with FLC determine the duration of induced resistance to AMB.

scholarly journals Advanced Resistance Studies Identify Two Discrete Mechanisms in Staphylococcus aureus to Overcome Antibacterial Compounds that Target Biotin Protein Ligase

Antibiotics ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 165 ◽  
Author(s):  
Andrew J. Hayes ◽  
Jiulia Satiaputra ◽  
Louise M. Sternicki ◽  
Ashleigh S. Paparella ◽  
Zikai Feng ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Resistance Rate ◽  
Biotin Protein Ligase ◽  
Essential Enzyme

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

scholarly journals Azole Resistance by Loss of Function of the Sterol Δ5,6-Desaturase Gene (ERG3) in Candida albicans Does Not Necessarily Decrease Virulence

2012 ◽  
Vol 56 (4) ◽  
pp. 1960-1968 ◽  
Author(s):  
L. A. Vale-Silva ◽  
A. T. Coste ◽  
F. Ischer ◽  
J. E. Parker ◽  
S. L. Kelly ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Premature Stop Codon ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Wild Type ◽  
Disseminated Candidiasis ◽  
Double Base

ABSTRACTThe inactivation ofERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism ofin vitroresistance to azole antifungal drugs in the human pathogenCandida albicans. ERG3inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified aC. albicansclinical isolate (VSY2) with high-level resistance to azole drugsin vitroand an absence of ergosterol but normal filamentation. Sequencing ofERG3in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming thatERG3inactivation was the mechanism of azole resistance. Additionally, the replacement of bothERG3alleles byerg3-1in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinicalERG3mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole bothin vitroandin vivo, theERG3-derived mutant of SC5314 was resistant onlyin vitroand was less virulent than the wild type. This suggests that VSY2 compensated for thein vivofitness defect ofERG3inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation ofERG3does not necessarily affect filamentation and virulence.

Influence of Streptococcus mitis and Streptococcus sanguinis on virulence of Candida albicans: in vitro and in vivo studies

2018 ◽  
Vol 64 (2) ◽  
pp. 215-222 ◽  
Author(s):  
Ana Luiza do Rosário Palma ◽  
Nádia Domingues ◽  
Patrícia Pimentel de Barros ◽  
Graziella Nuernberg Back Brito ◽  
Antônio Olavo Cardoso Jorge
Keyword(s):  
Candida Albicans ◽  
In Vivo Studies ◽  
Streptococcus Mitis ◽  
Streptococcus Sanguinis

scholarly journals Cancer Stem Cells: Powerful Targets to Improve Current Anticancer Therapeutics

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Rayana L. Bighetti-Trevisan ◽  
Lucas O. Sousa ◽  
Rogerio M. Castilho ◽  
Luciana O. Almeida
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Cancer Cells ◽  
Resistance Mechanisms ◽  
In Vivo Studies ◽  
Tumor Resistance ◽  
Therapeutic Implications ◽  
Anticancer Therapeutics

A frequent observation in several malignancies is the development of resistance to therapy that results in frequent tumor relapse and metastasis. Much of the tumor resistance phenotype comes from its heterogeneity that halts the ability of therapeutic agents to eliminate all cancer cells effectively. Tumor heterogeneity is, in part, controlled by cancer stem cells (CSC). CSC may be considered the reservoir of cancer cells as they exhibit properties of self-renewal and plasticity and the capability of reestablishing a heterogeneous tumor cell population. The endowed resistance mechanisms of CSC are mainly attributed to several factors including cellular quiescence, accumulation of ABC transporters, disruption of apoptosis, epigenetic reprogramming, and metabolism. There is a current need to develop new therapeutic drugs capable of targeting CSC to overcome tumor resistance. Emerging in vitro and in vivo studies strongly support the potential benefits of combination therapies capable of targeting cancer stem cell-targeting agents. Clinical trials are still underway to address the pharmacokinetics, safety, and efficacy of combination treatment. This review will address the main characteristics, therapeutic implications, and perspectives of targeting CSC to improve current anticancer therapeutics.

Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis

2019 ◽  
Vol 74 (8) ◽  
pp. 2230-2238 ◽  
Author(s):  
Florent Morio ◽  
Lisa Lombardi ◽  
Ulrike Binder ◽  
Cédric Loge ◽  
Estelle Robert ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Clinical Isolate ◽  
Antifungal Susceptibility ◽  
Azole Resistance ◽  
Amino Acid Substitutions ◽  
Fluconazole Resistance ◽  
Candida Orthopsilosis

AbstractBackgroundAzoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis.ObjectivesWe investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis.MethodsAntifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate.ResultsCompared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole.ConclusionsOur data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.

scholarly journals KY-62, a Polyene Analog of Amphotericin B, for Treatment of Murine Candidiasis

1998 ◽  
Vol 42 (1) ◽  
pp. 147-150 ◽  
Author(s):  
John R. Graybill ◽  
Laura K. Najvar ◽  
Annette Fothergill ◽  
Thomas Hardin ◽  
Michael Rinaldi ◽  
...  
Keyword(s):  
Body Weight ◽  
Candida Albicans ◽  
Amphotericin B ◽  
Clinical Development ◽  
Water Soluble ◽  
Clinical Isolates ◽  
In Vitro Testing ◽  
Immunocompetent Mice

ABSTRACT KY-62 is a water-soluble analog of amphotericin B. In vitro testing of five clinical isolates of Candida albicans showed KY-62 to have potency similar to that of amphotericin B. KY-62 was administered to mice infected intravenously with C. albicans. In vivo, KY-62 was effective in immunocompetent mice, with potency similar to that of amphotericin B. KY-62 was well tolerated up to 30 mg/kg of body weight per dose, an amount that would be lethal with amphotericin B. KY-62 was less effective in mice rendered neutropenic with 5-fluorouracil. The addition of flucytosine had little effect. KY-62 may have potential for clinical development.

scholarly journals The Strength of Synergistic Interaction between Posaconazole and Caspofungin Depends on the Underlying Azole Resistance Mechanism of Aspergillus fumigatus

2015 ◽  
Vol 59 (3) ◽  
pp. 1738-1744 ◽  
Author(s):  
Eleftheria Mavridou ◽  
Joseph Meletiadis ◽  
Antony Rijs ◽  
Johan W. Mouton ◽  
Paul E. Verweij
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanism ◽  
Fungal Growth ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Synergistic Interactions ◽  
Drug Concentrations

ABSTRACTThe majority of azole resistance mechanisms inAspergillus fumigatuscorrespond to mutations in thecyp51Agene. As azoles are less effective against infections caused by multiply azole-resistantA. fumigatusisolates, new therapeutic options are warranted for treating these infections. We therefore investigated thein vitrocombination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistantA. fumigatusisolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly (ttest,P< 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates (P< 0.01). FIC-2 values were inversely correlated with POSA MICs (rs= −0.52,P= 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA (rs= 0.76,P< 0.0001) and CAS (rs= 0.52,P= 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) againstA. fumigatusisolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

scholarly journals An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Brian M. Peters ◽  
Arturo Luna-Tapia ◽  
Hélène Tournu ◽  
Jeffrey M. Rybak ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mouse Model ◽  
Vaginal Candidiasis ◽  
Azole Resistance ◽  
Endosomal Trafficking ◽  
Wild Type ◽  
Content Type ◽  
Azole Antifungals

ABSTRACT We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro, it does not seem to affect azole susceptibility in vivo.

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