Echinocandin-Induced Microevolution of Candida parapsilosis Influences Virulence and Abiotic Stress Tolerance

ABSTRACT Candida species are a major cause of life-threatening bloodstream infections worldwide. Although Candida albicans is responsible for the vast majority of infections, the clinical relevance of other Candida species has also emerged over the last twenty years. This shift might be due in part to changes in clinical guidelines, as echinocandins became the first line of therapeutics for the treatment. Candida parapsilosis is an emerging non-albicans Candida species that exhibits lower susceptibility levels to these drugs. Candida species frequently display resistance to echinocandins, and the mechanism for this is well-known in C. albicans and Candida glabrata, where it is mediated by amino acid substitutions at defined locations of the β-1,3-glucan synthase, Fks1p. In C. parapsilosis isolates, Fks1p harbors an intrinsic amino acid change at position 660 of the hot spot 1 (HS1) region, which is thought to be responsible for the high MIC values. Less is known about acquired substitutions in this species. In this study, we used directed evolution experiments to generate C. parapsilosis strains with acquired resistance to caspofungin, anidulafungin, and micafungin. We showed that cross-resistance was dependent on the type of echinocandin used to generate the evolved strains. During their characterization, all mutant strains showed attenuated virulence in vivo and also displayed alterations in the exposure of inner cell wall components. The evolved strains harbored 251 amino acid changes, including three in the HS1, HS2, and HS3 regions of Fks1p. Altogether, our results demonstrate a direct connection between acquired antifungal resistance and virulence of C. parapsilosis. IMPORTANCE Candida parapsilosis is an opportunistic fungal pathogen with the ability to cause infections in immunocompromised patients. Echinocandins are the currently recommended first line of treatment for all Candida species. Resistance of Candida albicans to this drug type is well characterized. C. parapsilosis strains have the lowest in vitro susceptibility to echinocandins; however, patients with such infections typically respond well to echinocandin therapy. There is little knowledge of acquired resistance in C. parapsilosis and its consequences on other characteristics such as virulence properties. In this study, we aimed to dissect how acquired echinocandin resistance influences the pathogenicity of C. parapsilosis and to develop explanations for why echinocandins are clinically effective in the setting of acquired resistance.

Download Full-text

Genomic Analysis of Reduced Susceptibility to Tigecycline in Enterococcus faecium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04174-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 239-244 ◽

Cited By ~ 34

Author(s):

Vincent Cattoir ◽

Christophe Isnard ◽

Thibaud Cosquer ◽

Arlène Odhiambo ◽

Fiona Bucquet ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Enterococcus Faecium ◽

Efflux Pump ◽

Acquired Resistance ◽

Ribosomal Subunit ◽

Genomic Analysis ◽

Lactate Oxidase ◽

Content Type

ABSTRACTTigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported inEnterococcus faecium. Using the serial passage method,in vitromutant AusTig andin vitromutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 μg/ml) were obtained from strainsE. faeciumAus0004 and HM1070 (MICs, 0.03 μg/ml), respectively. In addition, two vancomycin-resistantE. faeciumclinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 μg/ml, respectively) were studied. Compared to the wild-type strains, thein vitromutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected therpsJgene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit),efau004_01228(encoding a cation transporter),efau004_01636(coding for a hypothetical protein), andefau004_02455(encoding thel-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci.

Download Full-text

Candida albicans Induces Arginine Biosynthetic Genes in Response to Host-Derived Reactive Oxygen Species

Eukaryotic Cell ◽

10.1128/ec.00290-12 ◽

2012 ◽

Vol 12 (1) ◽

pp. 91-100 ◽

Cited By ~ 40

Author(s):

Claudia Jiménez-López ◽

John R. Collette ◽

Kimberly M. Brothers ◽

Kelly M. Shepardson ◽

Robert A. Cramer ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Candida Albicans ◽

Amino Acid ◽

Reactive Oxygen ◽

Single Amino Acid ◽

Dependent Manner ◽

Oxygen Species ◽

Biosynthetic Genes ◽

Content Type

ABSTRACTThe interaction ofCandida albicanswith phagocytes of the host's innate immune system is highly dynamic, and its outcome directly impacts the progression of infection. While the switch to hyphal growth within the macrophage is the most obvious physiological response, much of the genetic response reflects nutrient starvation: translational repression and induction of alternative carbon metabolism. Changes in amino acid metabolism are not seen, with the striking exception of arginine biosynthesis, which is upregulated in its entirety during coculture with macrophages. Using single-cell reporters, we showed here that arginine biosynthetic genes are induced specifically in phagocytosed cells. This induction is lower in magnitude than during arginine starvationin vitroand is driven not by an arginine deficiency within the phagocyte but instead by exposure to reactive oxygen species (ROS). Curiously, these genes are induced in a narrow window of sublethal ROS concentrations.C. albicanscells phagocytosed by primary macrophages deficient in thegp91phoxsubunit of the phagocyte oxidase do not express theARGpathway, indicating that the induction is dependent on the phagocyte oxidative burst.C. albicans argpathway mutants are retarded in germ tube and hypha formation within macrophages but are not notably more sensitive to ROS. We also find that theARGpathway is regulated not by the general amino acid control response but by transcriptional regulators similar to theSaccharomyces cerevisiaeArgR complex. In summary, phagocytosis induces this single amino acid biosynthetic pathway in an ROS-dependent manner.

Download Full-text

Anidulafungin versus Caspofungin in a Mouse Model of Candidiasis Caused by Anidulafungin-Susceptible Candida parapsilosis Isolates with Different Degrees of Caspofungin Susceptibility

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01025-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 229-236 ◽

Cited By ~ 7

Author(s):

Dimitra Dimopoulou ◽

Georgios Hamilos ◽

Maria Tzardi ◽

Russell E. Lewis ◽

George Samonis ◽

...

Keyword(s):

Mouse Model ◽

Low Dose ◽

Candida Parapsilosis ◽

Cross Resistance ◽

High Dose ◽

Content Type ◽

Fungal Burden ◽

Altered Cell

ABSTRACTCandida parapsilosisisolates occasionally display resistancein vitroto echinocandins and cause breakthrough infections to echinocandins. The degree of thein vivocross-resistance among echinocandins and the fitness loss associated with caspofungin (CAS) resistance ofC. parapsilosisare not well studied. We compared the activities of CAS and anidulafungin (ANF), each given at 2 dosing schedules (high dose or low dose) in a nonneutropenic mouse model of invasive candidiasis (IC) caused by ANF-susceptible isolates ofC. parapsilosiswith different degrees of susceptibility to CAS (CAS resistant [CAS-R], MIC, >16 mg/liter; CAS intermediate [CAS-I], MIC, 4 mg/liter; and CAS susceptible [CAS-S], MIC, 2 mg/liter). We analyzed tissue fungal burden, histopathology, and weight loss patterns. Increasing CAS resistance was associated with reduced virulence ofC. parapsilosisisolates (mortality rates for CAS-S versus CAS-I versus CAS-R, 100% versus 11.1% versus 0%, respectively;P< 0.001). High doses of either echinocandin were active against infection with the CAS-I isolate when assessed by fungal burden reduction and weight gain. In contrast to CAS-S and CAS-I isolates, there was no reduction in fungal burden in mice infected with the CAS-R isolate following treatment with either echinocandin, each given at a high or low dose. Nevertheless, mice infected with the CAS-R isolate had reduced disease severity following echinocandin treatment, suggesting that echinocandins have activityin vivo, even against echinocandin-resistant strains. A complex interplay of residual echinocandin activity, decreased virulence, and/or fitness of isolates with altered cell wall and possible immunomodulatory effects can be encounteredin vivoduring infection with CAS-resistantC. parapsilosisisolates.

Download Full-text

Antifungal Susceptibility Profiles and Drug Resistance Mechanisms of Clinical Lomentospora prolificans Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00318-20 ◽

2020 ◽

Vol 64 (11) ◽

Cited By ~ 1

Author(s):

Yongqin Wu ◽

Nina Grossman ◽

Marissa Totten ◽

Warda Memon ◽

Anna Fitzgerald ◽

...

Keyword(s):

Amino Acid ◽

Tandem Repeats ◽

Hot Spot ◽

Antifungal Susceptibility ◽

Antifungal Drugs ◽

Upstream Region ◽

Amino Acid Residues ◽

Content Type ◽

Echinocandin Resistance

ABSTRACT Lomentospora prolificans is an opportunistic fungal pathogen with low susceptibility to current antifungal drugs. Here, we tested the in vitro susceptibility of 8 drugs against 42 clinical L. prolificans isolates. All isolates showed high MICs to voriconazole (MIC90>16 μg/ml), itraconazole (MIC90>16 μg/ml), posaconazole (MIC90>16 μg/ml), isavuconazole (MIC90>16 μg/ml), amphotericin B (MIC90>16 μg/ml), and terbinafine (MIC90>64 μg/ml) and high minimum effective concentrations (MECs) to micafungin (MEC90>8 μg/ml), with the exception of miltefosine showing an MIC90 value of 4 μg/ml. We examined six different in vitro drug combinations and found that the combination of voriconazole and terbinafine achieved the most synergistic effort against L. prolificans. We then annotated the L. prolificans whole genome and located its Cyp51 and Fks1 genes. We completely sequenced the two genes to determine if any mutation would be related to azole and echinocandin resistance in L. prolificans. We found no amino acid changes in Cyp51 protein and no tandem repeats in the 5′ upstream region of the Cyp51 gene. However, we identified three intrinsic amino acid residues (G138S, M220I, and T289A) in the Cyp51 protein that were linked to azole resistance. Likewise, two intrinsic amino acid residues (F639Y, W695F) that have reported to confer echinocandin resistance were found in Fks1 hot spot regions. In addition, three new amino acid alterations (D440A, S634R, and H1245R) were found outside Fks1 hot spot regions, and their contributions to echinocandin resistance need future investigation. Overall, our findings support the notion that L. prolificans is intrinsically resistant to azoles and echinocandins.

Download Full-text

Triazole Evolution of Candida parapsilosis Results in Cross-Resistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal Drug-Dependent Manner

mSphere ◽

10.1128/msphere.00821-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Csaba Papp ◽

Flóra Bohner ◽

Katica Kocsis ◽

Mónika Varga ◽

András Szekeres ◽

...

Keyword(s):

Amino Acid ◽

Candida Parapsilosis ◽

Treatment Strategies ◽

Antifungal Drugs ◽

Antifungal Treatment ◽

Cross Resistance ◽

Azole Resistance ◽

Resistance Development ◽

Content Type ◽

Invasive Infections

ABSTRACT The number of invasive infections caused by Candida species is increasing worldwide. The incidence of candidiasis cases caused by non-albicans Candida species, such as Candida parapsilosis, is also increasing, and non-albicans Candida species are currently responsible for more invasive infections than C. albicans. Additionally, while the development of azole resistance during invasive disease with C. albicans remains uncommon, azole-resistant C. parapsilosis strains are frequently isolated in the hospital setting. In this study, we applied direct selection to generate azole-adapted and azole-evolved C. parapsilosis strains in order to examine the effect of azole resistance development on fungal viability and pathogenesis progression. Depending on the drug applied, the different evolved strains developed distinct cross-resistance patterns: the fluconazole-evolved (FLUEVO) and voriconazole-evolved (VOREVO) strains gained resistance to fluconazole and voriconazole only, while posaconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POSEVO) strains showed higher echinocandin MIC values than the FLUEVO and VOREVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLUEVO and VOREVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POSEVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLUEVO and the VOREVO strains, along with the altered sterol composition of the POSEVO strain, we now highlight the impact of the above-mentioned amino acid changes in C. parapsilosis azole resistance development. We further revealed that the virulence of this species was only slightly or partially affected by fluconazole and voriconazole adaptation, while it significantly decreased after posaconazole adaptation. Our results suggest that triazole adaptation can result in azole cross-resistance and that this process may also result in virulence alterations in C. parapsilosis, depending on the applied drug. IMPORTANCE Candida parapsilosis causes life-threatening fungal infections. In the last 2 decades, the increasing number of azole-resistant C. parapsilosis clinical isolates has been attributable to the overuse and misuse of fluconazole, the first-line antifungal agent most commonly used in several countries. To date, the range of applicable antifungal drugs is limited. As a consequence, it is essential to understand the possible mechanisms of antifungal resistance development and their effect on virulence in order to optimize antifungal treatment strategies in the clinical setting. Our results revealed that the prolonged exposure to azoles resulted not only in azole resistance but also in cross-resistance development. Our data further indicate that resistance development may occur through different mechanisms that can also alter the virulence of C. parapsilosis. These results highlight the consequences of prolonged drug usage and suggest the need for developing alternative antifungal treatment strategies in clinical practice.

Download Full-text

Phagosomal Neutralization by the Fungal Pathogen Candida albicans Induces Macrophage Pyroptosis

Infection and Immunity ◽

10.1128/iai.00832-16 ◽

2016 ◽

Vol 85 (2) ◽

Cited By ~ 36

Author(s):

Slavena Vylkova ◽

Michael C. Lorenz

Keyword(s):

Amino Acids ◽

Transcription Factor ◽

Candida Albicans ◽

Amino Acid ◽

Content Type ◽

Caspase 1 ◽

Ammonia Release ◽

Hyphal Morphogenesis

ABSTRACT The interaction of Candida albicans with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that C. albicans co-opts amino acid catabolism to generate and excrete ammonia, which raises the extracellular pH, both in vitro and in vivo and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking STP2, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungus-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking AHR1 were defective in growth, alkalinization, and ammonia release on amino acid-rich media, similar to stp2Δ and ahr1Δ stp2Δ cells, and occupied more acidic phagosomes. Notably, ahr1Δ and stp2Δ strains did not induce pyroptosis, as measured by caspase-1-dependent interleukin-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that C. albicans-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of caspase-1-mediated macrophage lysis.

Download Full-text

In Vitro Activity of PBTZ169 against Multiple Mycobacterium Species

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01314-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 5

Author(s):

Jin Shi ◽

Jie Lu ◽

Shu'an Wen ◽

Zhaojing Zong ◽

Fengmin Huo ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Acquired Resistance ◽

Surrogate Marker ◽

Mycobacterium Fortuitum ◽

In Vitro Activity ◽

Mycobacterium Species ◽

Amino Acid Polymorphism ◽

Content Type

ABSTRACT In this study, we demonstrate that PBTZ169 exhibits significant differences in in vitro activity against multiple Mycobacterium species. The amino acid polymorphism at codon 387 of decaprenylphosphoryl-beta-d-ribose oxidase (DprE1) can be used as a surrogate marker for in vitro susceptibility to PBTZ169 in mycobacteria. In addition, the amino acid substitution at codon 154 in DprE1 may be associated with acquired resistance to PBTZ169 in the Mycobacterium fortuitum mutants.

Download Full-text

Detection of Cross-Resistance Between Methotrexate and Azoles in Candida albicans and Meyerozyma guilliermondii: An In Vitro Study

Acta Mycologica ◽

10.5586/am.566 ◽

2021 ◽

Vol 56 ◽

Author(s):

Filip Franciszek Karuga ◽

Katarzyna Góralska ◽

Ewa Brzeziańska-Lasota

Keyword(s):

Candida Albicans ◽

Acquired Resistance ◽

In Vitro Study ◽

Candida Guilliermondii ◽

Cross Resistance ◽

Meyerozyma Guilliermondii ◽

Before And After ◽

Significant Change ◽

Risk Of Treatment

Abstract In recent years, there has been a rapid increase in the incidence of Candida infections. The different species of the genus Candida vary in their virulence abilities and susceptibility to antifungal agents, depending on several external factors. The result of such modifications may be cross-resistance, which is understood as an acquired resistance to a certain antimicrobial agent after exposure to another drug. The aim of this study was to determine the possibility of cross-resistance between fluconazole, voriconazole, itraconazole, and methotrexate in Candida albicans and Meyerozyma guilliermondii (syn. Candida guilliermondii ). Fifteen strains of M. guilliermondii and eight strains of C. albicans , including the standard strains, were tested. For all strains, the minimum inhibitory concentrations (MICs) for fluconazole, voriconazole, and itraconazole were determined before and after stimulation with methotrexate. The median MICs in M. guilliermondii before and after stimulation were 9.333 and 64 mg/L ( p = 0.005) for fluconazole; 0.917 and 1.667 mg/L ( p = 0.001) for itraconazole, respectively. No significant change in MIC was observed for voriconazole. For C. albicans strains, the median MICs before and after stimulation were 0.917 and 64 mg/L ( p = 0.012) for fluconazole; 0.344 and 1.135 mg/L ( p = 0.018) for voriconazole, respectively. There was no significant change in MIC values for itraconazole. Thus, this study demonstrates the presence of cross-resistance between voriconazole, itraconazole, fluconazole, and methotrexate for the selected strains. Methotrexate exposure induces different responses when certain drugs are used for various species. Therefore, if a patient was previously exposed to methotrexate, there may be a higher risk of treatment failure with fluconazole than with other azoles such as voriconazole for fungemia caused by M. guilliermondii or itraconazole for C. albicans infection.

Download Full-text

Signaling Pathways Governing the Caspofungin Paradoxical Effect in Aspergillus fumigatus

mBio ◽

10.1128/mbio.01816-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Nicolas Papon ◽

Florent Morio ◽

Dominique Sanglard

Keyword(s):

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Acquired Resistance ◽

Paradoxical Effect ◽

Regulatory Pathway ◽

First Line ◽

Content Type ◽

Wide Range ◽

First Line Treatment

ABSTRACT Aspergillus fumigatus is responsible for a wide range of diseases affecting several million people worldwide. Currently, a few families of antifungals are available to fight aspergillosis, and we are facing a worrisome increase in resistance to azoles, the drugs used for both first-line treatment and prophylaxis of invasive aspergillosis. In this context, some of the latest antifungals, i.e., echinocandins, have gained attention. Even though acquired resistance to echinocandins is yet uncommon in A. fumigatus clinical isolates, some strains exhibit another characteristic that relies on their capacity to grow at suprainhibitory echinocandin concentrations in vitro. This intriguing phenomenon, especially observed with caspofungin and now referred to as the caspofungin paradoxical effect (CPE), relies on molecular mechanisms that were hitherto little understood. Here, we discuss the recent key findings of Valero and colleagues published in mBio (C. Valero, A. C. Colabardini, J. Chiaratto, L. Pardeshi, et al., mBio 11:e00816-20, 2020, https://doi.org/10.1128/mBio.00816-20) that will allow a better understanding of the complex regulatory pathway involved in governing the response of A. fumigatus to caspofungin.

Download Full-text

EUCAST Susceptibility Testing of Isavuconazole: MIC Data for Contemporary Clinical Mold and Yeast Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00073-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 10

Author(s):

Karin Meinike Jørgensen ◽

Karen Marie Thyssen Astvad ◽

Rasmus Krøger Hare ◽

Maiken Cavling Arendrup

Keyword(s):

Susceptibility Testing ◽

Pearson Correlation ◽

Acquired Resistance ◽

Cross Resistance ◽

Wild Type ◽

Content Type ◽

Wild Type Phenotype ◽

Mold And Yeast ◽

Gene Alterations

ABSTRACTIsavuconazole is the newest medical azole. We investigated EUCAST MICs for isavuconazole and seven comparators against 1,498 contemporary isolates (2016 to 2017). EUCAST susceptibility testing was performed. Isavuconazole MICs >2 dilution steps above the modal MIC were regarded as non-wild type for species without EUCAST epidemiological cutoff values (ECOFFs).CYP51Asequencing was performed when relevant. Pearson correlation analysis was adopted for comparing activity.Aspergillusaccounted for 90% of mold andCandidaaccounted for 97% of yeast isolates. Thirty (9.3%)Aspergillusfumigatusisolates were classified as resistant, and 10 (3.1%) were classified as non-wild type. Thirteen (4%) were cross-resistant to other mold-active azoles. Target gene alterations were found in 10 (76.9%) isolates, including 4 (30.8%) of environmental origin (TR34/L98H [n = 3] and Trip343/L98H [n = 1]). SixAspergillusterreusisolates were resistant, including two (17%) with MICs of >2 mg/liter and M217I alterations. Modal MICs/MIC50s (milligrams per liter) againstCandidaspp. were ≤0.004/≤0.004 forC. albicansandC. dubliniensis, 0.008/0.008 forC. tropicalis, 0.016/0.016 forC. parapsilosis, 0.06/0.06 forC. glabrata, and 0.125/0.125 forC. krusei. A non-wild-type phenotype was observed for 6.6% of isolates (C. glabrata[11.8%] andC. tropicalis[12.3%], specifically). All of these isolates were nonsusceptible/non-wild type to fluconazole (96.1%) or voriconazole (86.2%). Low MICs were found for several other species, exceptScedosporium apiospermumandFusarium. The best correlation was found between isavuconazole and voriconazole overall but forA. terreusand Mucorales to itraconazole and posaconazole, respectively. Isavuconazole displayed broadin vitroactivity. Acquired resistance was infrequent except inA. terreus,C. glabrata, andC. tropicalisand, when present, was associated with cross-resistance to other azoles. Revising the EUCAST breakpoints forA. fumigatus(defining an MIC of 2 mg/liter as intermediate [“I”]) would minimize major errors.

Download Full-text