Search

Candida auris is a growing concern due to its resistance to antifungal drugs, particularly amphotericin B (AMB), detected in 30 to 60% of clinical isolates. However, the mechanisms of AMB resistance remain poorly understood. Here we investigated 441 in vitro- and in vivo-evolved C. auris lineages from 4 AMB-susceptible clinical strains of different clades. Genetic and sterol analyses revealed four major types of sterol alterations as a result of clinically rare variations in sterol biosynthesis genes ERG6, NCP1, ERG11, ERG3, HMG1, ERG10 and ERG12. In addition, aneuploidies in chromosomes 4 and 6 emerged during resistance evolution. Fitness trade-off phenotyping and mathematical modelling identified diverse strain- and mechanism-dependent fitness trade-offs. Variation in CDC25 rescued fitness trade-offs, thereby increasing the infection capacity. This possibly contributed to therapy-induced acquired AMB resistance in the clinic. Our findings highlight sterol-modulating mechanisms and fitness trade-off compensation as risks for AMB treatment failure in clinical settings., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target., (© 2024. The Author(s).)

Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite-green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z ' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a K i of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound., Importance: Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a K i value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase., Competing Interests: The authors declare no conflict of interest.

We used whole-genome sequencing to analyze a collection of 35 fluconazole-resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two persistent clinical lineages were identified. We identified copy number variation (CNV) of two genes, ERG11 and CDR1B , in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show that the annotated telomeric gene CDR1B is actually an artifactual in silico fusion of two highly similar neighboring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased the expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance., (© 2024. The Author(s).)

Objectives: The aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates., Methods: Sequences of CpERG11 and CpTAC1 were determined for 35 resistant Candida parapsilosis clinical isolates. A plasmid-based CRISPR-Cas9 system was used to introduce mutations leading to amino acid substitution in CpTac1 and CpErg11. Triazole susceptibility was determined by broth microdilution and E-test. Differential expression of genes mediated by CpTAC1 mutation was determined by RNA sequencing, and relative expression of individual transporter genes was assessed with RT-qPCR., Results: Six isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11 Y132F substitution. When introduced into susceptible isolates, this CpERG11 mutation led to a 4- to 8-fold increase in fluconazole minimum inhibitory concentrations (MIC; 0.125 μg/mL vs. 0.5 μg/mL, 0.125 μg/mL vs. 0.5 μg/mL, and 0.5 μg/mL vs. 4 μg/mL). When introduced into a susceptible isolate, the CpTAC1 mutation leading to the G650E substitution resulted in an 8-fold increase in fluconazole MIC (0.25 μg/mL vs. 2 μg/mL), whereas correction of this mutation in resistant isolates led to a 16-fold reduction in MIC (32 μg/mL vs. 2 μg/mL). CpCDR1, CpCDR1B, and CpCDR1C were overexpressed in the presence CpTac1 G650E . Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 μg/mL vs. 8 μg/mL)., Discussion: These results define the specific contribution made by the Y132F substitution in CpERG11 and demonstrate a role for activating mutations in CpTAC1 in triazole resistance in C. parapsilosis., (Copyright © 2023. Published by Elsevier Ltd.)

Aspergillus fumigatus is a ubiquitous environmental mold that can cause severe disease in immunocompromised patients and chronic disease in individuals with underlying lung conditions. Triazoles are the most widely used class of antifungal drugs to treat A. fumigatus infections, but their use in the clinic is threatened by the emergence of triazole-resistant isolates worldwide, reinforcing the need for a better understanding of resistance mechanisms. The predominant mechanisms of A. fumigatus triazole resistance involve mutations affecting the promoter region or coding sequence of the target enzyme of the triazoles, Cyp51A. However, triazole-resistant isolates without cyp51A -associated mutations are frequently identified. In this study, we investigate a pan-triazole-resistant clinical isolate, DI15-105, that simultaneously carries the mutations hapE P88L and hmg1 F262del , with no mutations in cyp51A . Using a Cas9-mediated gene-editing system, hapE P88L and hmg1 F262del mutations were reverted in DI15-105. Here, we show that the combination of these mutations accounts for pan-triazole resistance in DI15-105. To our knowledge, DI15-105 is the first clinical isolate reported to simultaneously carry mutations in hapE and hmg1 and only the second with the hapE P88L mutation. IMPORTANCE Triazole resistance is an important cause of treatment failure and high mortality rates for A. fumigatus human infections. Although Cyp51A-associated mutations are frequently identified as the cause of A. fumigatus triazole resistance, they do not explain the resistance phenotypes for several isolates. In this study, we demonstrate that hapE and hmg1 mutations additively contribute to pan-triazole resistance in an A. fumigatus clinical isolate lacking cyp51 -associated mutations. Our results exemplify the importance of and the need for a better understanding of cyp51A -independent triazole resistance mechanisms., Competing Interests: The authors declare no conflict of interest.

Fungal infections are responsible for significant morbidity and mortality. Resistance to the limited number of agents in the antifungal armamentarium among pathogenic fungi represents a growing public health threat. Particularly concerning is the emerging fungal pathogen Candida auris that frequently exhibits resistance to the triazole class of antifungals and amphotericin B, and for which isolates resistant to all of the major antifungal classes have been reported. In this brief review, we provide an overview of what is currently known about the molecular and genetic basis for antifungal resistance in this fungal pathogen., Competing Interests: Conflict of interest statement The authors report no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Candida parapsilosis is a common cause of invasive candidiasis worldwide and is the most commonly is7olated Candida species among pediatric and neonatal populations. Previous work has demonstrated that nonsynonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility. However, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates are poorly understood. We identified 7 nonsynonymous CpMRR1 mutations in 12 isolates from within a collection of 35 fluconazole-resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1 , we identified two other genes, one encoding a major facilitator superfamily (MFS) transporter ( CpMDR1B , CPAR2_603010) and one encoding an ATP-binding cassette (ABC) transporter ( CpCDR1B , CPAR2_304370), as being upregulated in isolates carrying CpMRR1 -activating mutations. Overexpression of CpMDR1 in a susceptible strain and disruption in resistant clinical isolates that overexpress CpMDR1 had little to no effect on fluconazole susceptibility. Conversely, overexpression of either CpMDR1B or CpCDR1B increased resistance, and disruption in clinical isolates overexpressing these genes decreased fluconazole resistance. Our findings suggest that activating mutations in CpMRR1 represent important genetic determinants of fluconazole resistance in clinical isolates of C. parapsilosis, and unlike what is observed in Candida albicans, this is primarily driven by upregulation of both MFS (CpMdr1B) and ABC (CpCdr1B) transporters.

As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1 -flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene ( CaHygB ). Cassettes were PCR-amplified from both SAT1 -flipper and CaHygB -flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the ADE2 gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 10 4 homozygous deletion mutants per μg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 neutral locus. A variety of single or compound mutants (Δ/Δ als3 , Δ/Δ cph1 Δ/Δ efg1 , Δ/Δ ece1 ) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1β response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates. IMPORTANCE Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The SAT1 -flipper was reengineered to contain CaHygB encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with in vitro ribonucleoprotein assembly with commercial reagents, both SAT1 - and CaHygB -flipper cassettes were simultaneously integrated at loci with high efficiency (10 4 transformants per μg DNA) and upward of 99% homozygous gene targeting across a collection of diverse isolates of various anatomical origin. Revertant strains were constructed by overlap extension PCR with CRISPR-Cas9 targeted integration at the NEUT5 locus. Thus, this facile system will aid in unraveling the genetic factors contributing to the complexity of intraspecies diversity.

Objective: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance., Methods: Whole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling., Results: A novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation., Conclusions: Together these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11 WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11 K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11 . However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

The ubiquitous fungal pathogen Aspergillus fumigatus is the primary cause of opportunistic mould infections in humans. Aspergilli disseminate via asexual conidia passively travelling through air currents to germinate within a broad range of environs, wherever suitable nutrients are found. Though the average human inhales hundreds of conidia daily, A. fumigatus invasive infections primarily affect the immunocompromised. At-risk individuals can develop often fatal invasive disease for which therapeutic options are limited. Regrettably, the global insurgence of isolates resistant to the triazoles, the frontline antifungal class used in medicine and agriculture to control A. fumigatus, is complicating the treatment of patients. Triazole antifungal resistance in A. fumigatus has become recognized as a global, yet poorly comprehended, problem. Due to a multitude of factors, the magnitude of resistant infections and their contribution to treatment outcomes are likely underestimated. Current studies suggest that human drug-resistant infections can be either environmentally acquired or de novo host selected during patient therapy. While much concerning development of resistance is yet unknown, recent investigations have revealed assorted underlying mechanisms enabling triazole resistance within individual clinical and environmental isolates. This review will provide an overview of triazole resistance as it is currently understood, as well as highlight some of the prominent biological mechanisms associated with clinical and environmental resistance to triazoles in A. fumigatus., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris , fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro Comparative analyses of comprehensive sterol profiles, [ 3 H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida , and relative expression levels of C. auris ERG11 , CDR1 , and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance. IMPORTANCE Candida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris ., (Copyright © 2020 Rybak et al.)

Imipenem-cilastatin-relebactam (IMI-REL) is a novel β-lactam-β-lactamase inhibitor combination recently approved for the treatment of complicated urinary tract infections (cUTIs) and complicated intraabdominal infections (cIAIs). Relebactam is a β-lactamase inhibitor with the ability to inhibit a broad spectrum of β-lactamases such as class A and class C β-lactamases, including carbapenemases. The addition of relebactam to imipenem restores imipenem activity against several imipenem-resistant bacteria, including Enterobacteriaceae and Pseudomonas aeruginosa. Clinical data demonstrate that IMI-REL is well tolerated and effective in the treatment of cUTIs and cIAIs due to imipenem-resistant bacteria. In a phase III trial comparing IMI-REL with imipenem plus colistin, favorable clinical response was achieved in 71% and 70% of patients, respectively. Available clinical and pharmacokinetic data support the approved dosage of a 30-minute infusion of imipenem 500 mg-cilastatin 500 mg-relebactam 250 mg every 6 hours, along with dosage adjustments based on renal function. In this review, we describe the chemistry, mechanism of action, spectrum of activity, pharmacokinetics and pharmacodynamics, and clinical efficacy, and safety and tolerability of this new agent. The approval of IMI-REL represents another important step in the ongoing fight against multidrug-resistant gram-negative pathogens., (© 2020 Pharmacotherapy Publications, Inc.)

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. Th e A. fumigatus genes abcA, abcC, abcF, abcG, abcH , and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5 -deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5 -deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs., (Copyright © 2020 Esquivel et al.)

Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections each year. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis, both as frontline and as salvage therapy. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of triazole resistance, including mutations in the sterol-demethylase-encoding gene cyp51A , overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC , among a large collection of highly triazole-resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of triazole resistance exhibited by this collection, we subsequently describe the identification and characterization of a novel genetic determinant of triazole resistance. Mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-encoding gene, hmg1 , were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different hmg1 mutations, predicted to encode residue alterations in the conserved sterol sensing domain of Hmg1, resulted in significantly increased resistance to the triazole class of agents. Additionally, correction of a hmg1 mutation in a pan-triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all triazole agents. Mutations in hmg1 were also shown to lead to the accumulation of ergosterol precursors, such as eburicol, by sterol profiling, while not altering the expression of sterol-demethylase genes. IMPORTANCE Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections annually. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical triazole resistance in A. fumigatus , by first demonstrating that previously characterized mechanisms of resistance have nominal impact on triazole susceptibility and subsequently identifying a novel mechanism of resistance with a profound impact on clinical triazole susceptibility. We demonstrate that mutations in the HMG-CoA reductase gene, hmg1 , are common among resistant clinical isolates and that hmg1 mutations confer resistance to all clinically available triazole antifungals., (Copyright © 2019 Rybak et al.)

Invasive aspergillosis is a leading cause of morbidity and mortality among immunocompromised populations and is predicted to cause more than 200 000 life-threatening infections each year. Aspergillus fumigatus is the most prevalent pathogen isolated from patients with invasive aspergillosis, accounting for more than 60% of all cases. Currently, the only antifungal agents available with consistent activity against A. fumigatus are the mould-active triazoles and amphotericin B, of which the triazoles commonly represent both front-line and salvage therapeutic options. Unfortunately, the treatment of infections caused by A. fumigatus has recently been further complicated by the global emergence of triazole resistance among both clinical and environmental isolates. Mutations in the A. fumigatus sterol-demethylase gene cyp51A, overexpression of cyp51A and overexpression of efflux pump genes are all known to contribute to resistance, yet much of the triazole resistance among A. fumigatus still remains unexplained. Also lacking is clinical experience with therapeutic options for the treatment of triazole-resistant A. fumigatus infections and mortality associated with these infections remains unacceptably high. Thus, further research is greatly needed to both better understand the emerging threat of triazole-resistant A. fumigatus and to develop novel therapeutic strategies to combat these resistant infections., (© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Candida auris has rapidly emerged as a health care-associated and multidrug-resistant pathogen of global concern. In this work, we examined the relative expression of the four C. auris genes with the highest degree of homology to Candida albicans CDR1 and MDR1 among three triazole-resistant clinical isolates as compared to the triazole-susceptible genome reference clinical isolate. We subsequently utilized a novel Cas9-mediated system for genetic manipulations to delete C. auris CDR1 and MDR1 in both a triazole-resistant clinical isolate and a susceptible reference strain and observed that MICs for all clinically available triazoles decreased as much as 128-fold in the CDR1 deletion strains. The findings of this work reveal for the first time that C. auris CDR1 and MDR1 are more highly expressed among triazole-resistant clinical isolates of C. auris and that the overexpression of CDR1 is a significant contributor to clinical triazole resistance., (Copyright © 2019 American Society for Microbiology.)

Among emerging non- albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2 , ERG5 , ERG6 , ERG11 , ERG24 , ERG25 , and UPC2 Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins., (Copyright © 2017 American Society for Microbiology.)

We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21 Δ/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 ., (Copyright © 2017 American Society for Microbiology.)

Within the limited antifungal armamentarium, the azole antifungals are the most frequent class used to treat Candida infections. Azole antifungals such as fluconazole are often preferred treatment for many Candida infections as they are inexpensive, exhibit limited toxicity, and are available for oral administration. There is, however, extensive documentation of intrinsic and developed resistance to azole antifungals among several Candida species. As the frequency of azole resistant Candida isolates in the clinical setting increases, it is essential to elucidate the mechanisms of such resistance in order to both preserve and improve upon the azole class of antifungals for the treatment of Candida infections. This review examines azole resistance in infections caused by C. albicans as well as the emerging non- albicans Candida species C. parapsilosis, C. tropicalis, C. krusei , and C. glabrata and in particular, describes the current understanding of molecular basis of azole resistance in these fungal species.

Resistance among Gram-positive organisms has been steadily increasing over the last several years; however, the development of new antibiotics to treat infections caused from these organisms has fallen short of the emergent need. Specifically, resistance among Staphylococcus aureus and Enterococcus spp. to essential antibiotics is considered a major problem. Oritavancin is a semisynthetic lipoglycopeptide antibiotic that was recently approved for the treatment of acute bacterial skin and skin structure infections (ABSSSI). While structurally related to vancomycin, oritavancin also possesses unique mechanisms of action that greatly enhance its antimicrobial potency against multi-drug resistant pathogens including both VanA- and VanB-mediated vancomycin-resistant enterococci. Owing to the addition of the highly hydrophobic tail group, oritavancin possesses a prolonged half-life ranging from 200-300 h. Although oritavancin is only currently Food and Drug Administration approved for ABSSSI, this agent may eventually play a role in additional indications where new innovative therapy is needed including bacteremia and deep-seeded, Gram-positive infections such as infective endocarditis or osteomyelitis. This review will focus on oritavancin's spectrum of activity, mechanisms of action and resistance, pharmacokinetic and pharmacodynamic properties, and the completed and ongoing clinical studies evaluating its use.

Study Objective: To evaluate the pharmacokinetics and pharmacodynamics of ceftaroline in adults with cystic fibrosis (CF)., Design: Open-label, single-center, prospective study., Setting: University-affiliated teaching institution., Patients: Eight patients with a diagnosis of CF and a history of methicillin-resistant Staphylococcus aureus who were treated with ceftaroline between November 2013 and September 2014., Intervention: All patients received at least three doses of intravenous ceftaroline 600 mg every 12 hours, administered as a 60-minute infusion, to achieve steady-state concentrations before blood sample collection. After an interim analysis of the first four patients' pharmacokinetic data, the remaining four patients received a change in dosage of ceftaroline to 600 mg every 8 hours., Measurements and Main Results: Patients' blood samples were collected at two time points, 2 and 6 hours after infusion initiation, after administration of at least three doses of ceftaroline. Serum ceftaroline concentrations were determined by using a validated mass spectrometry, with a lower limit of detection of 20 ng/ml. These ceftaroline concentrations were used to estimate patient-specific pharmacokinetic parameters, and 10,000-patient Monte Carlo simulations were performed to determine the pharmacodynamic probability of target attainment (PTA) for ceftaroline in adults with CF. A PTA of 90% or higher for the desired pharmacodynamic target was considered adequate. The PTA for 60% or higher of the dosing interval during which free (unbound) drug concentrations exceed the minimum inhibitory concentration (%fT > MIC) was simulated for various MICs. Compared with values previously reported in other populations, the volume of distribution was increased in the study patients, and the estimated half-life was shorter. Monte Carlo simulations revealed that a dose of ceftaroline 600 mg every 8 hours, infused over 60 minutes, maintained a higher than 90% PTA for %fT > MIC of 60% or higher for an MIC at the susceptibility breakpoint of 1 mg/L., Conclusion: The pharmacokinetics of ceftaroline is altered in adults with CF, which suggests the need for modified dosing in this patient population to achieve adequate %fT > MIC. A dosage of intravenous ceftaroline 600 mg every 8 hours administered as a 60-minute infusion should be considered to achieve 60% fT > MIC., (© 2016 Pharmacotherapy Publications, Inc.)

Pseudomonas aeruginosa is a common pathogen implicated in nosocomial infections with increasing resistance to a limited arsenal of antibiotics. Monte Carlo simulation provides antimicrobial stewardship teams with an additional tool to guide empiric therapy. We modeled empiric therapies with antipseudomonal β-lactam antibiotic regimens to determine which were most likely to achieve probability of target attainment (PTA) of ≥90%. Microbiological data for P. aeruginosa was reviewed for 2012. Antibiotics modeled for intermittent and prolonged infusion were aztreonam, cefepime, meropenem, and piperacillin/tazobactam. Using minimum inhibitory concentrations (MICs) from institution-specific isolates, and pharmacokinetic and pharmacodynamic parameters from previously published studies, a 10,000-subject Monte Carlo simulation was performed for each regimen to determine PTA. MICs from 272 isolates were included in this analysis. No intermittent infusion regimens achieved PTA ≥90%. Prolonged infusions of cefepime 2000 mg Q8 h, meropenem 1000 mg Q8 h, and meropenem 2000 mg Q8 h demonstrated PTA of 93%, 92%, and 100%, respectively. Prolonged infusions of piperacillin/tazobactam 4.5 g Q6 h and aztreonam 2 g Q8 h failed to achieved PTA ≥90% but demonstrated PTA of 81% and 73%, respectively. Standard doses of β-lactam antibiotics as intermittent infusion did not achieve 90% PTA against P. aeruginosa isolated at our institution; however, some prolonged infusions were able to achieve these targets.

Coinciding with the continually increasing population of immunocompromised patients worldwide, the incidence of invasive fungal infections has grown over the past 4 decades. Unfortunately, infections caused by both yeasts such as Candida and molds such as Aspergillus or Mucorales remain associated with unacceptably high morbidity and mortality. In addition, the available antifungals with proven efficacy in the treatment of these infections remain severely limited. Although previously available second-generation triazole antifungals have significantly expanded the spectrum of the triazole antifungal class, these agents are laden with shortcomings in their safety profiles as well as formulation and pharmacokinetic challenges. Isavuconazole, administered as the prodrug isavuconazonium, is the latest second-generation triazole antifungal to receive U.S. Food and Drug Administration approval. Approved for the treatment of both invasive aspergillosis and invasive mucormycosis, and currently under investigation for the treatment of candidemia and invasive candidiasis, isavuconazole may have therapeutic advantages over its predecessors. With clinically relevant antifungal potency against a broad range of yeasts, dimorphic fungi, and molds, isavuconazole has a spectrum of activity reminiscent of the polyene amphotericin B. Moreover, clinical experience thus far has revealed isavuconazole to be associated with fewer toxicities than voriconazole, even when administered without therapeutic drug monitoring. These characteristics, in an agent available in both a highly bioavailable oral and a β-cyclodextrin-free intravenous formulation, will likely make isavuconazole a welcome addition to the triazole class of antifungals., (© 2015 Pharmacotherapy Publications, Inc.)

Gram-negative resistance has reached a crucial point, with emergence of pathogens resistant to most or all available antibiotics. Ceftazidime-avibactam is a newly approved agent combining ceftazidime and a novel β-lactamase inhibitor with activity against multidrug-resistant gram-negative bacteria. Avibactam has increased potency and expanded spectrum of inhibition of class A and C β-lactamases relative to available β-lactamase inhibitors, including extended-spectrum β-lactamases, AmpC, and Klebsiella pneumoniae carbapenemase (KPC) enzymes. Avibactam expands ceftazidime's spectrum of activity to include many ceftazidime- and carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa. Early clinical data indicate that ceftazidime-avibactam is effective and well tolerated in patients with complicated urinary tract infections (cUTIs) and complicated intraabdominal infections (cIAI). In a phase II trial of patients with cUTIs, ceftazidime-avibactam produced similar rates of clinical and microbiologic success compared with imipenem-cilastatin (70.5% and 71.4% microbiologic success rates, respectively). Likewise, patients receiving ceftazidime-avibactam plus metronidazole in a phase II study of patients with cIAI had similar response rates to those receiving meropenem (91.2% and 93.4% clinical success rates, respectively). Based on available in vitro, in vivo, and phase II trial data, as well as preliminary phase III trial results in ceftazidime-resistant, gram-negative cUTI and cIAI, ceftazidime-avibactam received U.S. Food and Drug Administration approval for treatment of cUTI, including pyelonephritis, and cIAI, in combination with metronidazole, in adult patients with limited or no alternative treatment options. The approved dosage, ceftazidime 2 g-avibactam 0.5 g administered as a 2-hour infusion every 8 hours, was selected based on pharmacodynamic analysis and available clinical data. This dosage is under further investigation in patients with cUTI, cIAI, and nosocomial or ventilator-associated pneumonia. The current body of evidence suggests that ceftazidime-avibactam is a promising addition to our therapeutic armamentarium with potential to answer an urgent unmet medical need. Further data in highly resistant gram-negative infections, particularly those caused by KPC-producing Enterobacteriaceae, are needed. As it is introduced into clinical use, careful stewardship and rational use are essential to preserve ceftazidime-avibactam's potential utility., (© 2015 Pharmacotherapy Publications, Inc.)

Treatment of multidrug-resistant Gram-positive infections continues to challenge clinicians as the emergence of new resistance mechanisms outpaces introduction of novel antimicrobial agents. Tedizolid phosphate is a next-generation oxazolidinone with activity against both methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. Tedizolid has consistently shown potency advantages over linezolid against Gram-positive microorganisms including those with reduced susceptibility to linezolid. Of particular significance, minimum inhibitory concentrations of tedizolid appear to be largely unaffected by the chloramphenicol-florfenicol resistance (cfr) gene, which has been implicated in a number of published linezolid-resistant organism outbreaks. Tedizolid phosphate also has been found to have a favorable pharmacokinetic profile allowing for once-daily dosing in both oral and intravenous forms. Potency and pharmacokinetic advantages have allowed for lower total daily doses of tedizolid, compared to linezolid, being needed for clinical efficacy in the treatment of acute bacterial skin and skin structure infections (ABSSSI). The decreased total drug exposure produced may in part be responsible for a decrease in the observed adverse effects including thrombocytopenia. Tedizolid phosphate is currently indicated for the treatment of ABSSSI and under investigation for the treatment of nosocomial pneumonia. Although much of the role of tedizolid remains to be defined by expanding clinical experience, tedizolid is likely a welcomed addition to the mere handful of agents available for the treatment of multidrug-resistant Gram-positive infections.

Antimicrobial resistance among gram-positive organisms such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) continues to limit therapeutic options. The oxazolidinones are a synthetic class of agents now commonly relied on for the treatment of serious MRSA and VRE infections. With increasing utilization of linezolid, resistant pathogens have once again begun to emerge. Tedizolid, a next-generation oxazolidinone, possesses a spectrum of activity including MRSA and VRE, with significantly enhanced potency also against linezolid-resistant strains. Preclinical and early clinical studies have reported positive results, demonstrating a favorable pharmacokinetic profile in combination with key potential safety advantages. In two phase III clinical trials, tedizolid was found noninferior to linezolid in the treatment of acute bacterial skin and skin structure infections. Investigations for treatment of ventilator-acquired and health care-associated pneumonia are currently underway. Tedizolid has been subjected to pharmacodynamics studies throughout its development that have highlighted properties unique to this agent. Considerable accumulations in epithelial lining fluid and antimicrobial activity greatly augmented by the presence of granulocytes suggest that slow but bactericidal activity may be possible in some clinical scenarios. Structural distinctions between tedizolid and linezolid suggest that tedizolid has decreased vulnerability to oxazolidinone resistance mechanisms. Tedizolid minimum inhibitory concentrations are essentially unchanged in organisms possessing the chloramphenicol-florfenicol resistance gene, a horizontally transferable linezolid resistance mechanism. Although the clinical experience with tedizolid remains limited, early data suggest a potential role in the treatment of serious infections due to multidrug-resistant gram-positive pathogens., (© 2014 Pharmacotherapy Publications, Inc.)

Introduction: Staphylococcus aureus and Enterococcus spp. are two of the most common organisms causing nosocomial infections today; and are consistently associated with high mortality rates (approximately 20 and 44%, respectively). Resistance among these pathogens to first line agents such as methicillin and vancomycin continues to rise while isolates with reduced susceptibility to newer agents including linezolid and daptomycin continue to emerge, representing a serious concern for clinicians., Areas Covered: Mechanisms of action and resistance as well as in vitro and clinical experience in the treatment of resistant staphylococci and enterococci with currently available agents are discussed. Additionally, novel combination regimens showing enhanced efficacy and available data pertaining to prospective therapies including solithromycin, tedizolid, dalbavancin and oritavancin will be covered., Expert Opinion: With an increase in organisms displaying reduced susceptibility to vancomycin and the associated treatment failures, the significance of alternative therapies such as daptomycin, linezolid, ceftaroline, and prospective anti-gram-positive agents is on the rise. As our understanding of antimicrobial pharmacokinetic-pharmacodynamics principles continues to evolve, the selection of highly effective agents and optimization of dosages may lead to improved patient outcomes and delay the development of resistance.