Mechanism of azole resistance in Candida vulturna, an emerging multidrug resistant pathogen related with Candida haeumulonii and Candida auris.

BACKGROUND: Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. OBJECTIVES: This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. METHODS: We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. RESULTS: Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. CONCLUSIONS: This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.

Antifungal resistance: why are we losing this battle?

The emergence of fungal pathogens and changes in the epidemiological landscape are prevalent issues in clinical mycology. Reports of resistance to antifungals have been reported. This review aims to evaluate molecular and nonmolecular mechanisms related to antifungal resistance. Mutations in the ERG genes and overexpression of the efflux pump (MDR1, CDR1 and CDR2 genes) were the most reported molecular mechanisms of resistance in clinical isolates, mainly related to Azoles. For echinocandins, a molecular mechanism described was mutation in the FSK genes. Furthermore, nonmolecular virulence factors contributed to therapeutic failure, such as biofilm formation and selective pressure due to previous exposure to antifungals. Thus, there are many public health challenges in treating fungal infections.

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Comparative Genomics of the First Resistant Candida auris Strain Isolated in Mexico: Phylogenomic and Pan-Genomic Analysis and Mutations Associated with Antifungal Resistance.

Candida auris is an emerging multidrug-resistant and opportunistic pathogenic yeast. Whole-genome sequencing analysis has defined five major clades, each from a distinct geographic region. The current study aimed to examine the genome of the C. auris 20-1498 strain, which is the first isolate of this fungus identified in Mexico. Based on whole-genome sequencing, the draft genome was found to contain 70 contigs. It had a total genome size of 12.86 Mbp, an N50 value of 1.6 Mbp, and an average guanine-cytosine (GC) content of 45.5%. Genome annotation revealed a total of 5432 genes encoding 5515 proteins. According to the genomic analysis, the C. auris 20-1498 strain belongs to clade IV (containing strains endemic to South America). Of the two genes (ERG11 and FKS1) associated with drug resistance in C. auris, a mutation was detected in K143R, a gene located in a mutation hotspot of ERG11 (lanosterol 14-α-demethylase), an antifungal drug target. The focus on whole-genome sequencing and the identification of mutations linked to the drug resistance of fungi could lead to the discovery of new therapeutic targets and new antifungal compounds.

Challenging fungal infections in cystic fibrosis: a case of mixed Aspergillus species infection and antifungal combination testing.

Aspergillus stands as the predominant fungal genus in the airways of cystic fibrosis (CF) patients, significantly contributing to their morbidity and mortality. Aspergillus fumigatus represents the primary causative species for infections, though the emergence of rare species within the Aspergillus section Fumigati has become noteworthy. Among these, Aspergillus lentulus is particularly significant due to its frequent misidentification and intrinsic resistance to azole antifungal agents. In the management of invasive aspergillosis and resistant infections, combination antifungal therapy has proven to be an effective approach. This report documents a case involving the death of a CF patient due to a pulmonary exacerbation linked to the colonization of multiple Aspergillus species, including A. lentulus, A. fumigatus, and A. terreus, and treated with Itraconazole (ITC) monotherapy. We delineated the procedures used to characterize the Aspergillus isolates in clinical settings and simulated in vitro the impact of the combination antifungal therapy on the isolates obtained from the patient. We evaluated three different combinations: Amphotericin B (AMB)+Voriconazole (VRC), AMB+Anidulafungin (AND), and VRC+AND. Notably, all strains isolated from the patient exhibited a significant decrease in their minimum inhibitory concentration (MIC) or minimum effective concentration (MEC) values when treated with all antifungal combinations. The VRC+AMB combination demonstrated the most synergistic effects. This case report emphasizes the critical importance of susceptibility testing and precise identification of Aspergillus species to enhance patient prognosis. It also underscores the potential benefits of combined antifungal treatment, which, in this case, could have led to a more favourable patient outcome.

Uncovering a Novel cyp51A Mutation and Antifungal Resistance in Aspergillus fumigatus through Culture Collection Screening.

BACKGROUND: Aspergillus fumigatus is an important concern for immunocompromised individuals, often resulting in severe infections. With the emergence of resistance to azoles, which has been the therapeutic choice for Aspergillus infections, monitoring the resistance of these microorganisms becomes important, including the search for mutations in the cyp51A gene, which is the gene responsible for the mechanism of action of azoles. We conducted a retrospective analysis covering 478 A. fumigatus isolates. METHODS: This comprehensive dataset comprised 415 clinical isolates and 63 isolates from hospital environmental sources. For clinical isolates, they were evaluated in two different periods, from 1998 to 2004 and 2014 to 2021; for environmental strains, one strain was isolated in 1998, and 62 isolates were evaluated in 2015. Our primary objectives were to assess the epidemiological antifungal susceptibility profile; trace the evolution of resistance to azoles, Amphotericin B (AMB), and echinocandins; and monitor cyp51A mutations in resistant strains. We utilized the broth microdilution assay for susceptibility testing, coupled with cyp51A gene sequencing and microsatellite genotyping to evaluate genetic variability among resistant strains. RESULTS: Our findings reveal a progressive increase in Minimum Inhibitory Concentrations (MICs) for azoles and AMB over time. Notably, a discernible trend in cyp51A gene mutations emerged in clinical isolates starting in 2014. Moreover, our study marks a significant discovery as we detected, for the first time, an A. fumigatus isolate carrying the recently identified TR46/F495I mutation within a sample obtained from a hospital environment. The observed cyp51A mutations underscore the ongoing necessity for surveillance, particularly as MICs for various antifungal classes continue to rise. CONCLUSIONS: By conducting resistance surveillance within our institution's culture collection, we successfully identified a novel TR46/F495I mutation in an isolate retrieved from the hospital environment which had been preserved since 1998. Moreover, clinical isolates were found to exhibit TR34/L98H/S297T/F495I mutations. In addition, we observed an increase in MIC patterns for Amphotericin B and azoles, signaling a change in the resistance pattern, emphasizing the urgent need for the development of new antifungal drugs. Our study highlights the importance of continued monitoring and research in understanding the evolving challenges in managing A. fumigatus infections.

Species distribution and antifungal susceptibility profiles of clinical and environmental Fusarium isolates from Mexico: A multicenter study.

Fusarium spp. has emerged as an opportunistic etiological agent with clinical manifestations varying from localized infections to deep-seated systemic disease. It is also a phytopathogen of economic impact. There are few reports on the species diversity of this genus, and no comprehensive studies on the epidemiology nor the antifungal susceptibility of Fusarium in Mexico. The present multicentric study aims to shed light on the species distribution and antifungal susceptibility patterns of 116 strains of Fusarium isolated from clinical and environmental samples. Isolates were identified by standard phenotypic characteristics and by sequencing of the ITS (internal transcribed spacer), TEF1 (translation elongation factor 1-α), RPB2 (RNA polymerase II core subunit), and/or CAM1 (calmodulin) regions. Susceptibility tests were carried out against 15 antifungals of clinical and agricultural use. Regarding Fusarium distribution, we identified 27 species belonging to eight different species complexes. The most frequently isolated species for both clinical and environmental samples were F. falciforme (34%), F. oxysporum sensu stricto (12%), F. keratoplasticum (8%), and F. solani sensu stricto (8%). All Fusarium isolates showed minimum inhibitory concentrations (MICs) equal to or above the maximum concentration evaluated for fluconazole, 5-fluocytosine, caspofungin, micafungin, and anidulafungin. All isolates had a MIC of ≤16 µg/mL for voriconazole, with a mode of 4 µg/mL. F. verticillioides appeared to be the most susceptible to all antifungals tested.

Anti-Trypanosoma cruzi Activity, Mutagenicity, Hepatocytotoxicity and Nitroreductase Enzyme Evaluation of 3-Nitrotriazole, 2-Nitroimidazole and Triazole Derivatives.

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.

Analysis of CDR1 and MDR1 Gene Expression and ERG11 Substitutions in Clinical Candida tropicalis Isolates from Alexandria, Egypt.

INTRODUCTION: Candida tropicalis is a common non-albicans Candida (NAC) species that causes numerous fungal infections. Increasing antifungal resistance to azoles in NAC is becoming a major health problem worldwide; however, in Egypt, almost no data is available regarding fluconazole resistance mechanisms in C. tropicalis. The current study aims to investigate two possible important molecular mechanisms involved in fluconazole resistance in C. tropicalis isolates. MATERIALS: Fifty-four clinical C. tropicalis isolates were included. Identification and antifungal susceptibility profiles of the isolates were carried out using the VITEK 2 compact system. The molecular investigation of fluconazole resistance included the expression of the CDR1 and MDR1 genes by quantitative real-time RT-PCR as well as the sequence analysis of the ERG11 gene. RESULTS: Antifungal susceptibility testing identified 30 fluconazole-non-susceptible isolates. Statistically, CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher than that in fluconazole-susceptible isolates, with MDR1 gene expression levels that were similar in both non-susceptible and susceptible isolates. Sequence analysis of the ERG11 gene of 26 fluconazole-resistant isolates identified two missense mutations: A395T (Y132F) and G1390A (G464S). CONCLUSIONS: This study has highlighted the role of overexpression of the CDR1 gene and ERG11 gene mutations in fluconazole non-susceptibility. Further studies in Egypt are required to investigate other possible molecular mechanisms involved in azole resistance.

Reduced Susceptibility to Azoles in Cryptococcus gattii Correlates with the Substitution R258L in a Substrate Recognition Site of the Lanosterol 14-α-Demethylase.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a life-threatening fungal infection affecting mostly immunocompromised patients. In fact, cryptococcal meningitis accounts for about 19% of AIDS-related deaths in the world. Because of long-term azole therapies to treat this mycosis, resistance to fluconazole leading to treatment failure and poor prognosis has long been reported for both fungal species. Among the mechanisms implicated in resistance to azoles, mutations in the ERG11 gene, encoding the azole target enzyme lanosterol 14-α-demethylase, have been described. This study aimed to establish the amino acid composition of ERG11 of Colombian clinical isolates of C. neoformans and C. gattii and to correlate any possible substitution with the in vitro susceptibility profile of the isolates to fluconazole, voriconazole, and itraconazole. Antifungal susceptibility testing results showed that C. gattii isolates are less susceptible to azoles than C. neoformans isolates, which could correlate with differences in the amino acid composition and structure of ERG11 of each species. In addition, in a C. gattii isolate with high MICs for fluconazole (64 µg/mL) and voriconazole (1 µg/mL), a G973T mutation resulting in the substitution R258L, located in substrate recognition site 3 of ERG11, was identified. This finding suggests the association of the newly reported substitution with the azole resistance phenotype in C. gattii. Further investigations are needed to determine the exact role that R258L plays in the decreased susceptibility to fluconazole and voriconazole, as well as to determine the participation of additional mechanisms of resistance to azole drugs. IMPORTANCE The fungal species Cryptococcus neoformans and C. gattii are human pathogens for which drug resistance or other treatment and management challenges exist. Here, we report differential susceptibility to azoles among both species, with some isolates displaying resistant phenotypes. Azoles are among the most commonly used drugs to treat cryptococcal infections. Our findings underscore the necessity of testing antifungal susceptibility in the clinical setting in order to assist patient management and beneficial outcomes. In addition, we report an amino acid change in the sequence of the target protein of azoles, which suggests that this change might be implicated in resistance to these drugs. Identifying and understanding possible mechanisms that affect drug affinity will eventually aid the design of new drugs that overcome the global growing concern of antifungal resistance.

Giving a Hand: Synthetic Peptides Boost the Antifungal Activity of Itraconazole against Cryptococcus neoformans.

Cryptococcus neoformans is a multidrug-resistant pathogen responsible for infections in immunocompromised patients. Here, itraconazole (ITR), a commercial antifungal drug with low effectiveness against C. neoformans, was combined with different synthetic antimicrobial peptides (SAMPs), Mo-CBP3-PepII, RcAlb-PepII, RcAlb-PepIII, PepGAT, and PepKAA. The Mo-CBP3-PepII was designed based on the sequence of MoCBP3, purified from Moringa oleifera seeds. RcAlb-PepII and RcAlb-PepIII were designed using Rc-2S-Alb, purified from Ricinus communis seed cakes. The putative sequence of a chitinase from Arabidopsis thaliana was used to design PepGAT and PepKAA. All SAMPs have a positive liquid charge and a hydrophobic potential ranging from 41-65%. The mechanisms of action responsible for the combined effect were evaluated for the best combinations using fluorescence microscopy (FM). The synthetic peptides enhanced the activity of ITR by 10-fold against C. neoformans. Our results demonstrated that the combinations could induce pore formation in the membrane and the overaccumulation of ROS on C. neoformans cells. Our findings indicate that our peptides successfully potentialize the activity of ITR against C. neoformans. Therefore, synthetic peptides are potential molecules to assist antifungal agents in treating Cryptococcal infections.

Evaluation of the Sensititre YeastOne and Etest in Comparison with CLSI M38-A2 for Antifungal Susceptibility Testing of Three Azoles, Amphotericin B, Caspofungin, and Anidulafungin, against Aspergillusfumigatus and Other Species, Using New Clinical Breakpoints and Epidemiological Cutoff Values.

Aspergillosis is an invasive fungal disease associated with high mortality. Antifungal susceptibility testing (AFST) is receiving increasing consideration for managing patients, as well as for surveilling emerging drug resistance, despite having time-consuming and technically complex reference methodologies. The Sensititre YeastOne (SYO) and Etest methods are widely utilized for yeasts but have not been extensively evaluated for Aspergillus isolates. We obtained Posaconazole (POS), Voriconazole (VCZ), Itraconazole (ITC), Amphotericin B (AMB), Caspofungin (CAS), and Anidulafungin (AND) minimum inhibitory concentrations (MICs) for both the Etest (n = 330) and SYO (n = 339) methods for 106 sequenced clinical strains. For 84 A. fumigatus, we analyzed the performance of both commercial methods in comparison with the CLSI-AFST, using available cutoff values. An excellent correlation could be demonstrated for Etest-AMB and Etest-VCZ (p < 0.01). SYO-MICs of AMB, VCZ, and POS resulted in excellent essential agreement (>93%), and >80% for AMB, VCZ, and ITC Etest-MICs. High categoric agreement was found for AMB, ITC, and CAS Etest-MICs (>85%) and AMB SYO-MICs (>90%). The considerable number of major/very major errors found using Etest and SYO, possibly related to the proposed cutoffs and associated with the less time-consuming processes, support the need for the improvement of commercial methods for Aspergillus strains.

Decoding the antifungal resistance mechanisms in biofilms of emerging, ubiquitous and multidrug-resistant species belonging to the Scedosporium/Lomentospora genera.

The opportunistic filamentous fungi belonging to the Scedosporium and Lomentospora genera are highly tolerant to all classes of available antifungal drugs. Moreover, the mature biofilm formed by these fungi presents higher antifungal resistance when compared to planktonic cells. Nevertheless, the resistance mechanisms developed by the biofilm lifestyle are not completely elucidated. In the current study, we have investigated the mainly known resistance mechanisms to azoles (voriconazole and fluconazole) and polyenes (amphotericin B [AMB]) in S. apiospermum, S. minutisporum, S. aurantiacum, and L. prolificans (formerly S. prolificans) biofilms. Both classes of antifungals can physically bind to the extracellular matrix of mature biofilms, preventing the drugs from reaching their targets on biofilm-forming cells, which precludes their activity and toxicity. In addition, the activity of efflux pumps, measured by Rhodamine 6 G, was increased along with the maturation of the biofilm. The efflux pump's inhibition by L-Phe-L-Arg-ß-naphthylamide culminated in a 2- to 16-fold increase in azole susceptibility in conidial cells, but not in mature biofilms. Finally, we demonstrated by using specific inhibitors that in conidia, but not in biofilms, AMB induced the production of reactive oxygen species through the activity of the oxidative phosphorylation system (complex I-IV and alternative oxidases). However, the cellular redox imbalance caused by AMB was well-coped with the high activity of antioxidative enzymes, such as superoxide dismutase and catalase. Altogether, our results revealed that Scedosporium/Lomentospora biofilm resistance occurs through various mechanisms that operate concomitantly, which could explain the huge challenge in the clinical treatment of scedosporiosis/lomentosporiosis. LAY SUMMARY: Scedosporium/Lomentospora spp. are multidrug-resistant pathogens able to cause diverse types of infections with typical biofilm characteristics, which makes the treatment a hard issue. We deciphered the resistance mechanisms to classical antifungals developed in the biofilm formed by these fungi.

Selection of Aspergillus fumigatus isolates carrying the G448S substitution in CYP51A gene after long-term treatment with voriconazole in an immunocompromised patient.

We present a case of a 55-year-old man with a heart transplant who acquired Invasive Aspergillosis by Aspergillus fumigatus with the focus in the kidney. During about two years of antifungal treatment, most of the time with voriconazole, it was possible to obtain nine isolates of A. fumigatus, with the same genotypic characteristics, but with an increase in MIC for several azoles. The two last isolates presented high MICs for Voriconazole (>8 µg/mL>). Sequencing of the CYP51A gene showed G448S amino acid substitution in the same two isolates. In long-term treatments with antifungals, it would be important to regularly evaluate the susceptibility of isolated strains, as resistance to azoles has been increasingly described around the world.

A Brazilian Inter-Hospital Candidemia Outbreak Caused by Fluconazole-Resistant Candida parapsilosis in the COVID-19 Era.

Horizontal transmission of fluconazole-resistant Candida parapsilosis (FRCP) through healthcare workers' hands has contributed to the occurrence of candidemia outbreaks worldwide. Since the first COVID-19 case in Brazil was detected in early 2020, hospitals have reinforced hand hygiene and disinfection practices to minimize SARS-CoV-2 contamination. However, a Brazilian cardiology center, which shares ICU patients with a cancer center under a FRCP outbreak since 2019, reported an increased FRCP candidemia incidence in May 2020. Therefore, the purpose of this study was to investigate an inter-hospital candidemia outbreak caused by FRCP isolates during the first year of the COVID-19 pandemic in Brazil. C. parapsilosis bloodstream isolates obtained from the cancer (n = 35) and cardiology (n = 30) centers in 2020 were submitted to microsatellite genotyping and fluconazole susceptibility testing. The ERG11 gene of all isolates from the cardiology center was sequenced and compared to the corresponding sequences of the FRCP genotype responsible for the cancer center outbreak in 2019. Unprecedentedly, most of the FRCP isolates from the cardiology center presented the same genetic profile and Erg11-Y132F mutation detected in the strain that has been causing the persistent outbreak in the cancer center, highlighting the uninterrupted horizontal transmission of clonal isolates in our hospitals during the COVID-19 pandemic.

Fluconazol intra-estromal - eficácia na cirurgia de abscesso estromal em equino / Intrastromal fluconazole - effectiveness in the surgery of stromal abscess in a horse

Background: Due to its ocular microflora, the equine species is predisposed to develop mycotic ulcers which, when notproperly treated, can lead to the formation of a stromal abscess. A stromal abscess occurs through the introduction ofmicroorganisms into the corneal stroma. During re-epithelialization, the foreign body is encapsulated, thus creating abarrier that protects bacteria or fungi from treatment with antimicrobial medication. This framework can end up resultingin blindness due to chronic iridocyclitis, putting the animal’s vision at risk. The current work aims to report a case of corrective surgery for stromal abscess in a mare with the administration of intraoperative intrastromal fluconazole, in orderto corroborate the effectiveness of the technique.Case: A 9-year-old mare was evaluated, with the complaint that her right eye was closed and “yellowish” and that she hadalready been treated with intramuscular injectable anti-inflammatory drugs based on flunexin meglumine (Banamine® -50 mg) for 15 days, referring to a possible ulcer in the right eye. Ophthalmic screening resulted in a negative direct reflexand no threat response in the right eye. Examination of the conjunctiva showed congestion and chemosis. Examination ofthe cornea of the right eye was negative for Fluorescein and Green Lissamine tests, and opacity and corneal neovascularization were noted. The final diagnosis was a corneal abscess of probable fungal origin secondary to a keratomycosis. Afterthe consultation, complementary blood and biochemical tests were performed, which showed normal results for the speciesin question, and treatment was started with eye drops based on atropine 1% (Fagra® - 20 mL), ciprofloxacin antimicrobialeye drops (Ciprovet Colirio® - 5 mL), and antifungal eye drops based on ketoconazole...

The Medicinal Chemistry of 3-nitro-1,2,4-triazoles: Focus on Infectious Diseases.

Infectious diseases are among the leading causes of death worldwide, especially in developing countries. The historical lack of interest of the pharmaceutical industry in developing new drugs against many of these diseases, such as tuberculosis, leishmaniasis, Chagas disease, sleeping sickness, and fungal infections, has left millions of individuals dependent on old treatments that are often ineffective and present different adverse effects. In this sense, new substances against these diseases must be identified. A class of substances that has stood out in the search for new drugs against these diseases is azole derivatives. Within this class, the 3-nitro-1,2,4-triazole nucleus has attracted increasing interest due to its potential, specifically when compared to the 1,2,4-triazole nucleus without the presence of the nitro group, and also in relation to the 2-nitroimidazole nucleus, showing greater potency and selectivity against different etiological agents. This is even more relevant considering that 3-nitro-1,2,4-triazolic substances can promote their activity through different mechanisms of action, such as the inhibition of ergosterol biosynthesis and also via activation by the nitroreductase enzyme, which can avoid the development of cross-resistance. Therefore, in this review, the medicinal chemistry of nitrotriazoles is discussed through the analysis of their potential in terms of biological activity against the etiological agents of several diseases, such as Chagas disease, sleeping sickness and leishmaniasis, caused by kinetoplastid parasites, tuberculosis, caused by the mycobacteria Mycobacterium tuberculosis, and against different species of pathogenic fungi. In addition, aspects related to enzymatic activities, molecular modeling and organic synthesis of these substances are also addressed.

Species-Specific Differences in C-5 Sterol Desaturase Function Influence the Outcome of Azole Antifungal Exposure.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), leading to depletion of cellular ergosterol and the synthesis of an aberrant sterol diol that disrupts membrane function. In Candida albicans, sterol diol production is catalyzed by the C-5 sterol desaturase enzyme encoded by ERG3. Accordingly, mutations that inactivate ERG3 enable the fungus to grow in the presence of the azoles. The purpose of this study was to compare the propensities of C-5 sterol desaturases from different fungal pathogens to produce the toxic diol upon S14DM inhibition and thus contribute to antifungal efficacy. The coding sequences of ERG3 homologs from C. albicans (CaERG3), Candida glabrata (CgERG3), Candida auris (CaurERG3), Cryptococcus neoformans (CnERG3), Aspergillus fumigatus (AfERG3A-C) and Rhizopus delemar (RdERG3A/B) were expressed in a C. albicans erg3Δ/Δ mutant to facilitate comparative analysis. All but one of the Erg3p-like proteins (AfErg3C) at least partially restored C-5 sterol desaturase activity and to corresponding degrees rescued the stress and hyphal growth defects of the C. albicans erg3Δ/Δ mutant, confirming functional equivalence. Each C-5 desaturase enzyme conferred markedly different responses to fluconazole exposure in terms of the MIC and residual growth observed at supra-MICs. Upon fluconazole-mediated inhibition of S14DM, the strains expressing each homolog also produced various levels of 14α-methylergosta-8,24(28)-dien-3ß,6α-diol. The RdErg3A and AfErg3A proteins are notable for low levels of sterol diol production and failing to confer appreciable azole sensitivity upon the C. albicans erg3Δ/Δ mutant. These findings suggest that species-specific properties of C-5 sterol desaturase may be an important determinant of intrinsic azole sensitivity.

Colombian Essential Oil of Ruta graveolens against Nosocomial Antifungal Resistant Candida Strains.

Drug resistance in antifungal therapy, a problem unknown until a few years ago, is increasingly assuming importance especially in immunosuppressed patients and patients receiving chemotherapy and radiotherapy. In the past years, the use of essential oils as an approach to improve the effectiveness of antifungal agents and to reduce antifungal resistance levels has been proposed. Our research aimed to evaluate the antifungal activity of Colombian rue, Ruta graveolens, essential oil (REO) against clinical strains of Candida albicans, Candida parapsilopsis, Candida glabrata, and Candida tropicalis. Data obtained showed that C. tropicalis and C. albicans were the most sensitive strains showing minimum inhibitory concentrations (MIC) of 4.1 and 8.2 µg/mL of REO. Time-kill kinetics assay demonstrated that REO showed a fungicidal effect against C. tropicalis and a fungistatic effect against C. albicans. In addition, an amount of 40% of the biofilm formed by C. albicans was eradicated using 8.2 µg/mL of REO after 1 h of exposure. The synergistic effect of REO together with some antifungal compounds was also investigated. Fractional inhibitory concentration index (FICI) showed synergic effects of REO combined with amphotericin B. REO Lead a disruption in the cellular membrane integrity, consequently resulting in increased intracellular leakage of the macromolecules, thus confirming that the plasma membrane is a target of the mode of action of REO against C. albicans and C. tropicalis.

Azole Resistance in Clinical and Environmental Aspergillus Isolates from the French West Indies (Martinique).

The emergence of azole resistant Aspergillus spp., especially Aspergillus fumigatus, has been described in several countries around the world with varying prevalence depending on the country. To our knowledge, azole resistance in Aspergillus spp. has not been reported in the West Indies yet. In this study, we investigated the antifungal susceptibility of clinical and environmental isolates of Aspergillus spp. from Martinique, and the potential resistance mechanisms associated with mutations in cyp51A gene. Overall, 208 Aspergillus isolates were recovered from clinical samples (n = 45) and environmental soil samples (n = 163). They were screened for resistance to azole drugs using selective culture media. The Minimum Inhibitory Concentrations (MIC) towards voriconazole, itraconazole, posaconazole and isavuconazole, as shown by the resistant isolates, were determined using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) microdilution broth method. Eight isolates (A. fumigatus, n = 6 and A. terreus, n = 2) had high MIC for at least one azole drug. The sequencing of cyp51A gene revealed the mutations G54R and TR34/L98H in two A. fumigatus clinical isolates. Our study showed for the first time the presence of azole resistance in A. fumigatus and A. terreus isolates in the French West Indies.

Environmental Clonal Spread of Azole-Resistant Candida parapsilosis with Erg11-Y132F Mutation Causing a Large Candidemia Outbreak in a Brazilian Cancer Referral Center.

Clonal outbreaks due to azole-resistant Candida parapsilosis (ARCP) isolates have been reported in numerous studies, but the environmental niche of such isolates has yet to be defined. Herein, we aimed to identify the environmental niche of ARCP isolates causing unremitting clonal outbreaks in an adult ICU from a Brazilian cancer referral center. C. parapsilosis sensu stricto isolates recovered from blood cultures, pericatheter skins, healthcare workers (HCW), and nosocomial surfaces were genotyped by multilocus microsatellite typing (MLMT). Antifungal susceptibility testing was performed by the EUCAST (European Committee for Antimicrobial Susceptibility Testing) broth microdilution reference method and ERG11 was sequenced to determine the azole resistance mechanism. Approximately 68% of isolates were fluconazole-resistant (76/112), including pericatheter skins (3/3, 100%), blood cultures (63/70, 90%), nosocomial surfaces (6/11, 54.5%), and HCW's hands (4/28, 14.2%). MLMT revealed five clusters: the major cluster contained 88.2% of ARCP isolates (67/76) collected from blood (57/70), bed (2/2), pericatheter skin (2/3), from carts (3/7), and HCW's hands (3/27). ARCP isolates were associated with a higher 30 day crude mortality rate (63.8%) than non-ARCP ones (20%, p = 0.008), and resisted two environmental decontamination attempts using quaternary ammonium. This study for the first time identified ARCP isolates harboring the Erg11-Y132F mutation from nosocomial surfaces and HCW's hands, which were genetically identical to ARCP blood isolates. Therefore, it is likely that persisting clonal outbreak due to ARCP isolates was fueled by environmental sources. The resistance of Y132F ARCP isolates to disinfectants, and their potential association with a high mortality rate, warrant vigilant source control using effective environmental decontamination.