Resistance and virulence genes characteristic of a South Asia Clade (I) Candida auris strain isolated from blood in Beijing.

INTRODUCTION: Candida auris is a globally disseminated invasive ascomycetous yeast, that imposes a substantial burden on healthcare systems. It has been documented to have spread to over 40 countries across six continents, necessitating in-depth comprehension through advanced techniques like Whole-Genome Sequencing. METHOD: This study entailed the isolation and Whole-Genome Sequencing of a fluconazole-resistant C. auris strain (CA01) obtained from a patient's blood in Beijing. Genome analysis was conducted to classify the strain, and molecular docking was performed to understand the impact of mutations on drug resistance. RESULTS: Genome analysis revealed that CA01 belongs to the South Asia Clade (I) and shares the closest genetic relationship with previously reported strains BJCA001 and BJCA002. Notably, unlike BJCA001, CA01 exhibits significant resistance to fluconazole primarily due to the A395T mutation in the ERG11 gene. Molecular docking studies demonstrated that this mutation leads to geometric changes in the active site where fluconazole binds, resulting in decreased binding affinity. Additionally, the present findings have identified several core virulence genes in C. auris, such as RBF1. DISCUSSION: The findings from this study expand the understanding of the genetic diversity and adaptive mechanisms of C. auris within the South Asia Clade (I). The observed fluconazole resistance driven by the ERG11 mutation A395T highlights the need for heightened awareness and adaptation in clinical treatment strategies in China. This study provides critical insights into drug resistance and virulence profiles at a genetic level, which could guide future therapeutic and management strategies for C. auris infections.

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.

Emergence and circulation of azole-resistant C. albicans, C. auris and C. parapsilosis bloodstream isolates carrying Y132F, K143R or T220L Erg11p substitutions in Colombia.

Methods: Over a four-year period, 123 Candida bloodstream isolates were collected at a quaternary care hospital. The isolates were identified by MALDI-TOF MS and their fluconazole (FLC) susceptibility patterns were assessed according to CLSI guidelines. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pump activity were performed for resistant isolates. Results: Out of 123 clinical strains,C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%; in addition, a high proportion of isolates were cross-resistant to voriconazole. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R, or T220L) were found in 11/19 (58%) of FLCresistant isolates. Furthermore, novel mutations were found in all genes evaluated. Regarding efflux pumps, 8/19 (42%) of FLC-resistant Candida spp strains showed significant efflux activity. Finally, 6/19 (31%) of FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Among FLC-resistant species, C. auris 7/10 (70%) and C. parapsilosis 6/24 (25%) displayed the highest percentages of resistance (C. albicans 6/46, 13%). Discussion: Overall, 68% of FLC-resistant isolates exhibited a mechanism that could explain their phenotype (e.g. mutations, efflux pump activity, or both). We provide evidence that isolates from patients admitted to a Colombian hospital harbor amino acid substitutions related to resistance to one of the most commonly used molecules in the hospital setting, with Y132F being the most frequently detected.

Determinants of fluconazole resistance and echinocandin tolerance in C. parapsilosis isolates causing a large clonal candidemia outbreak among COVID-19 patients in a Brazilian ICU.

Patients presenting with severe COVID-19 are predisposed to acquire secondary fungal infections such as COVID-19-associated candidemia (CAC), which are associated with poor clinical outcomes despite antifungal treatment. The extreme burden imposed on clinical facilities during the COVID-19 pandemic has provided a permissive environment for the emergence of clonal outbreaks of multiple Candida species, including C. auris and C. parapsilosis. Here we report the largest clonal CAC outbreak to date caused by fluconazole resistant (FLZR) and echinocandin tolerant (ECT) C. parapsilosis. Sixty C. parapsilosis strains were obtained from 57 patients at a tertiary care hospital in Brazil, 90% of them were FLZR and ECT. Although only 35.8% of FLZR isolates contained an ERG11 mutation, all of them contained the TAC1L518F mutation and significantly overexpressed CDR1. Introduction of TAC1L518F into a susceptible background increased the MIC of fluconazole and voriconazole 8-fold and resulted in significant basal overexpression of CDR1. Additionally, FLZR isolates exclusively harboured E1939G outside of Fks1 hotspot-2, which did not confer echinocandin resistance, but significantly increased ECT. Multilocus microsatellite typing showed that 51/60 (85%) of the FLZR isolates belonged to the same cluster, while the susceptible isolates each represented a distinct lineage. Finally, biofilm production in FLZR isolates was significantly lower than in susceptible counterparts Suggesting that it may not be an outbreak determinant. In summary, we show that TAC1L518F and FKS1E1393G confer FLZR and ECT, respectively, in CAC-associated C. parapsilosis. Our study underscores the importance of antifungal stewardship and effective infection control strategies to mitigate clonal C. parapsilosis outbreaks.

Effects of ß-lapachone and ß-nor-lapachone on multidrug efflux transporters and biofilms of Candida glabrata.

Infections caused particularly by Candida glabrata are hard to treat due to the development of antifungal resistance that occurs mainly through the production of efflux pumps and biofilm. Thus, a promising strategy to overcome infections caused by C. glabrata could be to use a substance able to inhibit efflux pumps and eradicate biofilms. Lapachones are natural naphthoquinones that possess a variety of pharmacological properties. Previous studies show that these substances inhibit the growth, virulence factors and efflux pumps of C. albicans. The aim of the present study was to evaluate whether lapachones are able to inhibit efflux pumps related to antifungal resistance in C. glabrata and either prevent biofilm formation or affect mature biofilms. Assays were performed with Saccharomyces cerevisiae strains that overexpress C. glabrata transporters (CgCdr1p and CgCdr2p). One C. glabrata clinical isolate that overexpresses CgCdr1p was also used. Both ß-lapachone and ß-nor-lapachone affected the growth of S. cerevisiae and C. glabrata when combined to fluconazole, and this action was inhibited by ascorbic acid. Both lapachones stimulated ROS production, inhibited efflux activity, adhesion, biofilm formation and the metabolism of mature biofilms of C. glabrata. Data obtained on the present study point to the potential use of ß-lapachone and ß-nor-lapachone as antibiofilm agents and adjuvants on the antifungal therapy related to resistant infections caused by C. glabrata.

Determinants of fluconazole resistance and the efficacy of fluconazole and milbemycin oxim combination against Candida parapsilosis clinical isolates from Brazil and Turkey.

Fluconazole-resistant Candida parapsilosis (FLZR-CP) outbreaks are a growing public health concern and have been reported in numerous countries. Patients infected with FLZR-CP isolates show fluconazole therapeutic failure and have a significantly increased mortality rate. Because fluconazole is the most widely used antifungal agent in most regions with outbreaks, it is paramount to restore its antifungal activity. Milbemycin oxim (MOX), a well-known canine endectocide, is a potent efflux pump inhibitor that significantly potentiates the activity of fluconazole against FLZR C. glabrata and C. albicans. However, the FLZ-MOX combination has not been tested against FLZR-CP isolates, nor is it known whether MOX may also potentiate the activity of echinocandins, a different class of antifungal drugs. Furthermore, the extent of involvement of efflux pumps CDR1 and MDR1 and ergosterol biosynthesis enzyme ERG11 and their link with gain-of-function (GOF) mutations in their transcription regulators (TAC1, MRR1, and UPC2) are poorly characterized among FLZR-CP isolates. We analyzed 25 C. parapsilosis isolates collected from outbreaks in Turkey and Brazil by determining the expression levels of CDR1, MDR1, and ERG11, examining the presence of potential GOF mutations in their transcriptional regulators, and assessing the antifungal activity of FLZ-MOX and micafungin-MOX against FLZR and multidrug-resistant (MDR) C. parapsilosis isolates. ERG11 was found to be universally induced by fluconazole in all isolates, while expression of MDR1 was unchanged. Whereas mutations in MRR1 and UPC2 were not detected, CDR1 was overexpressed in three Brazilian FLZR-CP isolates, which also carried a novel TAC1L518F mutation. Of these three isolates, one showed increased basal expression of CDR1, while the other two overexpressed CDR1 only in the presence of fluconazole. Interestingly, MOX showed promising antifungal activity against FLZR isolates, reducing the FLZ MIC 8- to 32-fold. However, the MOX and micafungin combination did not exert activity against an MDR C. parapsilosis isolate. Collectively, our study documents that the mechanisms underpinning FLZR are region specific, where ERG11 mutations were the sole mechanism of FLZR in Turkish FLZR-CP isolates, while simultaneous overexpression of CDR1 was observed in some Brazilian counterparts. Moreover, MOX and fluconazole showed potent synergistic activity, while the MOX-micafungin combination showed no synergy.

Transcriptome Analysis Unveils Gln3 Role in Amino Acids Assimilation and Fluconazole Resistance in Candida glabrata.

After Candida albicans, Candida glabrata is one of the most common fungal species associated with candidemia in nosocomial infections. Rapid acquisition of nutrients from the host is important for the survival of pathogens which possess the metabolic flexibility to assimilate different carbon and nitrogen compounds. In Saccharomyces cerevisiae, nitrogen assimilation is controlled through a mechanism known as Nitrogen Catabolite Repression (NCR). NCR is coordinated by the action of four GATA factors; two positive regulators, Gat1 and Gln3, and two negative regulators, Gzf3 and Dal80. A mechanism in C. glabrata similar to NCR in S. cerevisiae has not been broadly studied. We previously showed that in C. glabrata, Gln3, and not Gat1, has a major role in nitrogen assimilation as opposed to what has been observed in S. cerevisiae in which both factors regulate NCR-sensitive genes. Here, we expand the knowledge about the role of Gln3 from C. glabrata through the transcriptional analysis of BG14 and gln3Δ strains. Approximately, 53.5% of the detected genes were differentially expressed (DEG). From these DEG, amino acid metabolism and ABC transporters were two of the most enriched KEGG categories in our analysis (Up-DEG and Down-DEG, respectively). Furthermore, a positive role of Gln3 in AAA assimilation was described, as was its role in the transcriptional regulation of ARO8. Finally, an unexpected negative role of Gln3 in the gene regulation of ABC transporters CDR1 and CDR2 and its associated transcriptional regulator PDR1 was found. This observation was confirmed by a decreased susceptibility of the gln3Δ strain to fluconazole.

Trends towards lower azole susceptibility among 200 Candida tropicalis bloodstream isolates from Brazilian medical centres.

OBJECTIVES: Candida tropicalis is one of the three most frequent species causing candidaemia in Latin America. Despite the high prevalence of C. tropicalis in candidaemia cases in Brazil, little is known about the trends in fluconazole susceptibility over time. The objective of this study was to evaluate temporal trends in azole resistance rates among C. tropicalis bloodstream isolates from patients treated in six Brazilian medical centres over a 12-year period. METHODS: We selected 200 C. tropicalis bloodstream isolates from six medical centres in Brazil collected between 2007 and 2018. Species identification was confirmed by MALDI-TOF/MS. Antifungal susceptibility testing for four antifungal agents was performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. RESULTS: Overall, rates of non-susceptibility were 4% and 3.5% to fluconazole and voriconazole, respectively. All isolates were susceptible to amphotericin B and only one isolate was resistant to echinocandins. CONCLUSION: Although we failed to demonstrate statistical differences in the rates of azole resistance documented during the period of analysis, trends towards lower susceptibility to fluconazole and voriconazole were shown.

In vitro evaluation of anti-fungal activity of tropicamide against strains of Candida spp. resistant to fluconazole in planktonic and biofilm form.

Candida spp. is considered to be the third or fourth most common cause of bloodstream infections associated with healthcare services in the world. Currently, several strains exhibit resistance to the traditional treatments, making the development of new therapeutic molecules necessary. Drug repositioning is an alternative that can be used to work around problems such as toxicity, cost and time in the development of new drugs. This study aims to evaluate the in vitro antifungal effect of tropicamide, molecule of anticholinergic action, against planktonic cells of Candida spp. and biofilm of C. albicans. Six strains of different Candida species were used to determine the minimum inhibitory concentration (MIC) of tropicamide and fluconazole according to CLSI document M27-A3 and one strain of C. albicans was used to evaluate the activity of tropicamide against biofilms. In concentrations of 64µg/mL, the tropicamide exhibited 50% of inhibitory activity in planktonic cell and in concentrations of 128µg/mL is able to inhibit the formation of C. albicans biofilm. Despite the inhibitory activity shown at the present study, the use of a larger number of strains, as well as in vivo cytotoxicity assays, is necessary to confirm the hypothesis that tropicamide can be used as an adjuvant agent in the treatment of infections by the Candida genus.

Batzelladine D and norbatzelladine L purified from marine sponge Monanchora arbuscula induce the reversal of fluconazole.

ATP-Binding Cassette (ABC) transporters are the main class of transmembrane transporters involved in pathogenic fungal resistance against chemotherapeutic agents. Herein we report results which show that batzelladine D (1) and norbatzelladine L (2) reverse the fluconazole resistance phenotype mediated by Pdr5p transporter on Saccharomyces cerevisiae. Both alkaloids were able to chemosensitize the Pdr5p-overexpressing strain by synergistic interaction with fluconazole. Both compounds also showed an inhibitory effect on the catalytic activity and on the intracellular accumulation of rhodamine 6G, and did not show significant in vitro mammalian cells toxicity.

Synergistic interactions between ß-lapachone and fluconazole in the inhibition of CaCdr2p and CaMdr1p in Candida albicans.

BACKGROUND: Mortality rate of invasive Candida infections is raising mainly amongst immunocompromised patients. These infections are hard-to-treat mainly due to the increasing incidence of resistance. The overexpression of ATP-binding cassette and major facilitator superfamily transporters is the main responsible for the failure of antifungal therapies. In a Saccharomyces cerevisiae model, ß-lapachone inhibited Pdr5p, a transporter homologous to those found in Candida albicans. AIMS: To determine whether ß-lapachone reverses the resistance phenotype mediated by efflux transporters in C. albicans clinical isolates. METHODS: The antifungal activity of ß-lapachone combined with fluconazole was measured by agarose chemosensitization and microdilution assays. CaCdr2p and CaMdr1p activities were evaluated through fluorescent dyes accumulation. ATPase activity was assessed using transporter-enriched plasma membranes. RESULTS: ß-lapachone reverted antifungal resistance of S. cerevisiae and C. albicans strains overexpressing CaCdr2p and CaMdr1p transporters by inhibiting these proteins activities. CaCdr2p ATPase activity was not impaired by the compound. CONCLUSIONS: ß-lapachone is a promising drug candidate to be used as an adjuvant in the treatment of candidiasis caused by fluconazole-resistant C. albicans strains.

Synergistic effects of ketamine and azole derivatives on Candida spp. resistance to fluconazole.

The emergence of Candida spp. with resistance to antifungal molecules, mainly the azole class, is an increasing complication in hospitals around the globe. Aim: In the present research, we evaluated the synergistic effects of ketamine with two azole derivatives, itraconazole and fluconazole, on strains of Candida spp. to fluconazole. Materials & methods: The drug synergy was evaluated by quantifying the fractional inhibitory concentration index and by fluorescence microscopy and flow cytometry techniques. Results: Our achievements showed a synergistic effect between ketamine in addition to the two antifungal agents (fluconazole and itraconazole) against planktonic cells and biofilms of Candida spp. Conclusion: This combination promoted alteration of membrane integrity, generation of reactive oxygen species, damage to and DNA and externalization of phosphatidylserine.

In vitro activity of diphenyl diselenide and ebselen alone and in combination with antifungal agents against Trichosporon asahii.

This study evaluated the in vitro susceptibility of Trichosporon asahii strains to diphenyl diselenide (DPDS) and ebselen (EBS) alone and in combination with amphotericin B (AMB), fluconazole (FCZ), itraconazole (ITZ) and caspofungin (CAS) using the microdilution method. EBS showed in vitro activity against T asahii strains with minimal inhibitory concentration (MIC) ranged from 0.25 to 8.0 µg/mL. For DPDS, the MIC ranged from 8.0 to 64 µg/mL. The combinations demonstrating the greatest synergism rate against fluconazole-resistant T asahii strains were the following: CAS + DPDS (96.67%), AMB + DPDS (93.33%), FCZ + DPDS (86.67%) and ITZ + DPDS (83.33%). The combinations AMB + DPDS and AMB + EBS exhibited the highest synergism rate against the fluconazole-susceptible (FS) T asahii strains (90%). Antagonism was observed in the following combinations: FCZ + EBS (80%) and FCZ + DPDS (13.33%) against the FS strains, and ITZ + EBS (20%) against the FR strains. Our findings suggest that the antimicrobial activity of DPDS and EBS against T. asahii and its use as an adjuvant therapy with antifungal agents warrant in vivo experimental investigation.

Expression of fluconazole resistance-associated genes in biofilm from 23 clinical isolates of Candida albicans.

This study aimed to establish the influence of biofilm from clinical isolates of Candida albicans on fluconazole resistance, focusing on efflux pumps and azole-targeted enzymes. Twenty-three C. albicans clinical isolates were collected from two hospitals in Shanghai, China. Antifungal susceptibility tests were performed on biofilm and planktonic cells. A crystal violet assay was used to monitor biofilm growth. Real-time RT-PCR was performed to quantify the expression of the transporter-related genes MDR1, CDR1, and CDR2 as well as ERG11, a gene encoding an enzyme targeted by antifungal drugs. Fluconazole resistance was shown to increase in biofilm in a time-dependent manner. No significant differences were observed between different strains of C. albicans. Genes encoding efflux pumps were overexpressed in early stages of biofilm formation and could also be induced by fluconazole. While ERG11 was not upregulated in biofilm, it was overexpressed upon the addition of fluconazole to biofilm and planktonic cells. Gene expression also appeared to be related to the original genotype of the strain. The upregulation of genes encoding efflux pumps demonstrates their role in the development of fluconazole resistance during the early stages of C. albicans biofilm formation.

Candida glabrata's Genome Plasticity Confers a Unique Pattern of Expressed Cell Wall Proteins.

Candida glabrata is the second most common cause of candidemia, and its ability to adhere to different host cell types, to microorganisms, and to medical devices are important virulence factors. Here, we consider three characteristics that confer extraordinary advantages to C. glabrata within the host. (1) C. glabrata has a large number of genes encoding for adhesins most of which are localized at subtelomeric regions. The number and sequence of these genes varies substantially depending on the strain, indicating that C. glabrata can tolerate high genomic plasticity; (2) The largest family of CWPs (cell wall proteins) is the EPA (epithelial adhesin) family of adhesins. Epa1 is the major adhesin and mediates adherence to epithelial, endothelial and immune cells. Several layers of regulation like subtelomeric silencing, cis-acting regulatory regions, activators, nutritional signaling, and stress conditions tightly regulate the expression of many adhesin-encoding genes in C. glabrata, while many others are not expressed. Importantly, there is a connection between acquired resistance to xenobiotics and increased adherence; (3) Other subfamilies of adhesins mediate adherence to Candida albicans, allowing C. glabrata to efficiently invade the oral epithelium and form robust biofilms. It is noteworthy that every C. glabrata strain analyzed presents a unique pattern of CWPs at the cell surface.

Impact of inappropriate antifungal therapy according to current susceptibility breakpoints on Candida bloodstream infection mortality, a retrospective analysis.

BACKGROUND: The mortality of Candida Bloodstream Infection (CBSI) remains high. Antifungal susceptibility breakpoints were recently updated for Candida species, the impact remains unknown. In this study we evaluated the impact of inappropriate antifungal treatment according to recent breakpoints on 30-day mortality of CBSI. METHODS: From June 2008 to July 2014, data on CBSI episodes from two tertiary-care centers, treated > 72 h were analyzed. Antifungal therapy and 30-day mortality were registered. Inappropriate antifungal treatment according to current Clinical & Laboratory Standards Institute (CLSI) breakpoints was adjusted with 30-day mortality-related co-variates. RESULTS: One hundred forty-nine episodes of CBSI were analyzed. The most frequent species were: C. albicans (40%), C. tropicalis (23%) and C. glabrata complex (20%). According to the 2012 CLSI, 10.7% received inappropriate treatment. The 30-day mortality was 38%; severe sepsis [Odds ratio (OR) 3.4; 95% CI 1.3-8.4], cirrhosis (OR 36; 95% CI 12.2-605), early central venous catheter removal (OR 0.23; 95% CI 0.08-0.66) and previous antifungal therapy (OR 0.15; 95%CI 0.03-0.62), were associated with 30-day mortality by multivariate analysis. Inappropriate antifungal treatment was not (OR 0.19; 95% CI 0.03-1.2). CONCLUSIONS: Appropriate antifungal therapy according to CLSI 2012 did not have an impact on mortality. Mortality of CBSI remains high due to disease severity and comorbidities; early antifungal therapy and catheter removal may reduce it.

Outbreak of candidemia caused by fluconazole resistant Candida parapsilosis strains in an intensive care unit.

BACKGROUND: Candidemia is an increasing problem in tertiary care hospitals worldwide. Here, we report the first outbreak of candidemia caused by fluconazole-resistant C. parapsilosis (FRCP) strains in Brazil. METHODS: This was a cross-sectional study of clinical and microbiological data of all candidemic episodes diagnosed from July 2011 to February 2012 in a 200-bed tertiary care hospital. Initial yeast identification and susceptibility testing were performed using the VITEK 2 - System. Isolates of Candida spp. resistant to fluconazole were sent to a reference laboratory (LEMI-UNIFESP) for further molecular identification and confirmation of resistance by CLSI microdilution test. A multivariate analysis was conducted to identify factors associated with FRCP infection. RESULTS: We identified a total of 40 critically ill patients with candidemia (15 women) with a median age of 70 years. The incidence of candidemia was 6 cases/1,000 patients admissions, including 28 cases (70 %) of infection with C. parapsilosis, 21 of which (75 %) were resistant to fluconazole. In only 19 % of FRCP candidemia cases had fluconazole been used previously. The results of our study indicated that diabetes is a risk factor for FRCP candidemia (p = 0.002). Overall, mortality from candidemia was 45 %, and mortality from episodes of FRCP infections was 42.9 %. CONCLUSIONS: The clustering of incident cases in the ICU and molecular typing of strains suggest horizontal transmission of FRCP. Accurate vigilant monitoring for new nosocomial strains of FRCP is required.

First report of Candida auris in America: Clinical and microbiological aspects of 18 episodes of candidemia.

OBJECTIVES: Characterization of a hospital outbreak of Candida auris candidemia that involved 18 critically ill patients in Venezuela. METHOD: Bloodstream isolates of C. auris obtained from 18 patients admitted at a medical center in Maracaibo, between March, 2012 and July, 2013 were included. Species identification was confirmed by ITS rDNA sequencing. Isolates were subsequently typed by amplified fragment length polymorphism fingerprinting (AFLP). Susceptibility testing was performed according to CLSI. Clinical data were collected from all cases by using a standard clinical form. RESULTS: A total of 13 critically ill pediatric and 5 adult patients, with a median age of 26 days, were included. All were previously exposed to antibiotics and multiple invasive medical procedures. Clinical management included prompt catheter removal and antifungal therapy. Thirteen patients (72%) survived up to 30 days after onset of candidemia. AFLP fingerprinting of all C. auris isolates suggested a clonal outbreak. The isolates were considered resistant to azoles, but susceptible to anidulafungin and 50% of isolates exhibited amphotericin B MIC values of >1 µg/ml. CONCLUSIONS: The study demonstrated that C. auris is a multiresistant yeast pathogen that can be a source of health-care associated infections in tertiary care hospitals with a high potential for nosocomial horizontal transmission.

Amino acid substitution in Cryptococcus neoformans lanosterol 14-α-demethylase involved in fluconazole resistance in clinical isolates.

The molecular basis of fluconazole resistance in Cryptococcus neoformans has been poorly studied. A common azole resistance mechanism in Candida species is the acquisition of point mutations in the ERG11 gene encoding the enzyme lanosterol 14-α-demethylase, target of the azole class of drugs. In C. neoformans only two mutations were described in this gene. In order to evaluate other mutations that could be implicated in fluconazole resistance in C. neoformans we studied the genomic sequence of the ERG11 gene in 11 clinical isolates with minimal inhibitory concentration (MIC) values to fluconazole of ≥16µg/ml. The sequencing revealed the G1855A mutation in 3 isolates, resulting in the enzyme amino acid substitution G484S. These strains were isolated from two fluconazole-treated patients. This mutation would not intervene in the susceptibility to itraconazole and voriconazole.

Combination of fluconazole with silver nanoparticles produced by Fusarium oxysporum improves antifungal effect against planktonic cells and biofilm of drug-resistant Candida albicans.

Silver nanoparticles (AgNPs) have been extensively studied because of their anti-microbial potential. Here, we evaluated the effect of biologically synthesized silver nanoparticles (AgNPbio) alone and in combination with fluconazole (FLC) against planktonic cells and biofilms of FLC-resistant Candida albicans AgNPbio exhibited a fungicidal effect, with a minimal inhibitory concentration (MIC) and fungicidal concentration ranging from 2.17 to 4.35 µg/ml. The combination of AgNPbio and FLC reduced the MIC of FLC around 16 to 64 times against planktonic cells of allC. albicans There was no significant inhibitory effect of AgNPbio on biofilm cells. However, FLC combined with AgNPbio caused a significant dose-dependent decrease in the viability of both initial and mature biofilm. All concentrations of AgNPbio, alone or in combination with FLC, were not cytotoxic to mammalian cells.The results highlight the effectiveness of the combination of AgNPbio with FLC against FLC-resistant C. albicans.