Characterisation of an ABC transporter of a resistant Candida glabrata clinical isolate

BACKGROUND Candida glabrata ranks second in epidemiological surveillance studies, and is considered one of the main human yeast pathogens. Treatment of Candida infections represents a contemporary public health problem due to the limited availability of an antifungal arsenal, toxicity effects and increasing cases of resistance. C. glabrata presents intrinsic fluconazole resistance and is a significant concern in clinical practice and in hospital environments. OBJECTIVE The aim of this study was to characterise the azole resistance mechanism presented by a C. glabrata clinical isolate from a Brazilian university hospital. METHODS Azole susceptibility assays, chemosensitisation, flow cytometry and mass spectrometry were performed. FINDINGS Our study demonstrated extremely high resistance to all azoles tested: fluconazole, voriconazole, posaconazole and itraconazole. This isolate was chemosensitised by FK506, a classical inhibitor of ABC transporters related to azole resistance, and Rhodamine 6G extrusion was observed. A mass spectrometry assay confirmed the ABC protein identification suggesting the probable role of efflux pumps in this resistance phenotype. MAIN CONCLUSIONS This study emphasizes the importance of ABC proteins and their relation to the resistance mechanism in hospital environments and they may be an important target for the development of compounds able to unsettle drug extrusion.

Candida glabrata among Candida spp. from environmental health practitioners of a Brazilian Hospital

Abstract The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided.

Especies de levaduras aisladas en muestras de orina en un hospital regional de España / Isolated yeast species in urine samples in a Spanish regional hospital

La detección de candiduria en pacientes hospitalizados o inmunodeprimidos tiene una gran relevancia clínica. El objetivo de nuestro trabajo fue describir la frecuencia de aislamiento de diferentes especies significativas de levaduras en las muestras de orina procesadas en nuestro hospital en el periodo 2010-2013 y analizar su sensibilidad a los antifúngicos de uso habitual. La identificación de especies se realizó por siembra en un medio cromogénico, prueba de filamentación y sistemas automatizados (ASM Vitek y MALDI Biotyper), mientras que la sensibilidad a los antifúngicos se determinó con el sistema ASM Vitek. De los 632 aislamientos de levaduras obtenidos, 371 fueron Candida albicans y 261 especies de Candida no C. albicans. Las especies con mayor número de aislados resistentes fueron Candida glabrata y Candida krusei. Basados en nuestros resultados, consideramos que la identificación de la especie y el estudio de la sensibilidad a los antifúngicos deberían ser prácticas habituales por parte de los laboratorios cuando se aíslan especies diferentes a C. albicans

Candiduria detection in hospitalized or immunocompromised patients is of great clinical significance. The aim of our study was to describe the isolation frequency of significant species of yeasts in urine samples processed in our hospital during the period 2010- 2013, and to analyze their susceptibility to commonly used antifungal agents. Species identification was performed by seeding on a chromogenic medium, the filamentation test and automated systems (ASM Vitek and MALDI Biotyper), while susceptibility was determined using the ASM Vitek system. Of the 632 yeast isolates in urine, 371 were Candida albicans species and 261 non-C. albicans Candida spp. The species with the highest number of resistant isolates were Candida glabrata and Candida krusei. Based on the results obtained, we believe that species identification and the susceptibility study should be current practice in the laboratories when species other than C. albicans are isolated

Synergistic effects of tacrolimus and azole antifungal compounds in fluconazole-susceptible and fluconazole-resistant Candida glabrata isolates

In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata, a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro, notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.

Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland

Over the last decades, Candida spp have been responsible for an increasing number of infections, especially in patients requiring intensive care. Knowledge of local epidemiology and analysis of the spread of these pathogens is important in understanding and controlling their transmission. The aim of this study was to evaluate the genetic diversity of 31 Candida albicans and 17 Candida glabrata isolates recovered from intensive care unit patients from the tertiary hospital in Krakow between 2011-2012. The strains were typed by random amplified polymorphic DNA (RAPD) polymerase chain reaction using five primers (CD16AS, HP1247, ERIC-2, OPE-3 and OPE-18). The results of the present investigation revealed a high degree of genetic diversity among the isolates. No clonal relationship was found among the C. albicans strains, whereas two C. glabrata isolates were identical. The source of Candida infection appeared to be mostly endogenous; however, the presence of two clonal C. glabrata strains suggested the possibility of cross-transmission of these pathogens. Our study confirmed the high discriminatory power of the RAPD technique in the molecular typing of Candida clinical isolates. This method may be applied to the evaluation of transmission routes of pathogenic fungi on a local level.

The activity of echinocandins, amphotericin B and voriconazole against fluconazole-susceptible and fluconazole-resistant Brazilian Candida glabrata isolates

The extensive use of azole antifungal agents has promoted the resistance of Candida spp to these drugs. Candida glabrata is a problematic yeast because it presents a high degree of primary or secondary resistance to fluconazole. In Brazil, C. glabrata has been less studied than other species. In this paper, we compared the activity of three major classes of antifungal agents (azoles, echinocandins and polyenes) against fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains. Cross-resistance between fluconazole and voriconazole was remarkable. Among the antifungal agents, the echinocandins were the most effective against FS and FR C. glabrata and micafungin showed the lowest minimal inhibitory concentrations.

Susceptibility of clinical Candida species isolates to antifungal agents by E-test, Southern Iran: a five year study

The incidence of fungal infections in immunocompromised patients, especially by Candida species, has increased in recent years. This study was designed to identify Candida species and determine antifungal susceptibility patterns of 595 yeast strains isolated from various clinical specimens. Identification of the isolates were determined by the API 20 C AUX kit and antifungal susceptibilities of the species to fluconazole, amphotericin B, ketoconazole, itraconazole, voriconazole, and caspofungin were determined by the agar-based E-test method. Candida albicans [48%] was the most frequently isolated species, followed by Candida kruzei [16.1%], Candida glabrata [13.5%], Candida kefyr [7.4%], Candida parapsilosis [4.8%], Candida tropicalis [1.7%] and other species [8.5%]. Resistance varies depending on the species and the respective antifungal agents. Comparing the MIC90 for all the strains, the lower MIC90 was observed for caspofungin [0.5 microg/ml]. The MIC90 for all Candida species were 64 microg/ml for fluconazole, 0.75 microg/ml for amphotericin B, 4 microg/ml for ketoconazole, 4 microg/ml for itraconazole, and 2 microg/ml for voriconazole. Species definition and determination of antifungal susceptibility patterns are advised for the proper management and treatment of patients at risk for systemic candidiasis. Resistance to antifungal agents is an alarming sign for the emerging common nosocomial fungal infections

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

In vitro antifungal sensitivity of fluconazole, clotrimazole and nystatin against vaginal candidiasis in females of childbearing age

Vaginal candidiasis is the most common infection of females. A large variety of antifungal drugs are used for treatment. The objective of this study was isolation and identification of Candida from high vaginal swabs and in vitro antifungal activity of Clotrimazole, Fluconazole and Nystatin against Candida. Two hundred and fifty high vaginal swabs were collected from females reporting at different hospitals of Karachi. Wet mount was performed to observe the budding cells of Candida. Vaginal swabs were cultured on Sabouraud's dextrose agar with added antibiotics. Plates were incubated at room temperature for seven days. Chlamydospores of Candida albicans were identified on corn meal agar. Species of Candida were identified on Biggy agar. In vitro antifungal activity of Clotrimazole, Fluconazole and Nystatin was performed by MIC [Minimum inhibitory concentration], well diffusion method and disc diffusion method. Out of 250 high vaginal swabs, Candida species were isolated in 100 [40%] of cases. Out of 100, C. albican 30 [30%], C. tropicalis 21 [21%], C. parapsillosis 10 [10%], C. parakrusi 8 [8%], C.glabrata 8 [8%], C. krusei 3 [3%] were isolated. In vitro antifungal activity indicated Clotrimazole [MIC 16 and 8 microg/ml] effective against 68 [70%] of Candida SPP, Fluconazole [MIC 64 and 32 microg/ml] effective against 29 [36.2%] and Nystatin disc [100 units] was 51 [63.5%] effective. C. albicans was mainly isolated. Clotrimazole was more effective as compared to Fluconazole and Nystatin. Antifungal susceptibility testing should be determined before therapy to avoid treatment failures

In vitro Evaluation of Antibiotic Lock Technique for the Treatment of Candida albicans, C. glabrata, and C. tropicalis Biofilms

Candidaemia associated with intravascular catheter-associated infections is of great concern due to the resulting high morbidity and mortality. The antibiotic lock technique (ALT) was previously introduced to treat catheter-associated bacterial infections without removal of catheter. So far, the efficacy of ALT against Candida infections has not been rigorously evaluated. We investigated in vitro activity of ALT against Candida biofilms formed by C. albicans, C. glabrata, and C. tropicalis using five antifungal agents (caspofungin, amphotericin B, itraconazole, fluconazole, and voriconazole). The effectiveness of antifungal treatment was assayed by monitoring viable cell counts after exposure to 1 mg/mL solutions of each antibiotic. Fluconazole, itraconazole, and voriconazole eliminated detectable viability in the biofilms of all Candida species within 7, 10, and 14 days, respectively, while caspofungin and amphotericin B did not completely kill fungi in C. albicans and C. glabrata biofilms within 14 days. For C. tropicalis biofilm, caspofungin lock achieved eradication more rapidly than amphotericin B and three azoles. Our study suggests that azoles may be useful ALT agents in the treatment of catheter-related candidemia.

Genotyping of the MTL loci and susceptibility to two antifungal agents of Candida glabrata clinical isolates

The opportunistic fungal pathogen Candida glabrata is the second most common isolate from bloodstream infections worldwide and is naturally less susceptible to the antifungal drug fluconazole than other Candida species. C. glabrata is a haploid yeast that contains three mating-type like loci (MTL), although no sexual cycle has been described. Strains containing both types of mating information at the MTL1 locus are found in clinical isolates, but it is thought that strains containing type a information are more common. Here we investigated if a particular combination of mating type information at each MTLlocus is more prevalent in clinical isolates from hospitalized patients in Mexico and if there is a correlation between mating information and resistance to fluconazole and 5-fluorocytosine. We found that while both types of information at MTL1 are equally represented in a collection of 64 clinical isolates, the vast majority of isolates contain a-type information at MTL2 and α-type at MTL3. We also found no correlation of the particular combination of mating type information at the three MTL loci and resistance to fluconazole.

Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata

Candida glabrata is an opportunistic fungal pathogen that can cause severe invasive infections and can evade phagocytic cell clearance. We are interested in understanding the virulence of this fungal pathogen, in particular its oxidative stress response. Here we investigated C. glabrata, Saccharomyces cerevisiae and Candida albicans responses to two different oxidants: menadione and cumene hydroperoxide (CHP). In log-phase, in the presence of menadione, C. glabrata requires Cta1p (catalase), while in a stationary phase (SP), Cta1p is dispensable. In addition, C. glabrata is less resistant to menadione than C. albicans in SP. The S. cerevisiae laboratory reference strain is less resistant to menadione than C. glabrata and C. albicans; however S. cerevisiaeclinical isolates (CIs) are more resistant than the lab reference strain. Furthermore, S. cerevisiae CIs showed an increased catalase activity. Interestingly, in SP C. glabrata and S. cerevisiae are more resistant to CHP than C. albicans and Cta1p plays no apparent role in detoxifying this oxidant.

Vinegar as an antimicrobialagent for control of Candida spp. in complete denture wearers

The use of denture is known to increase the carriage of Candida in healthy patients, and the proliferation of Candida albicans strains can be associated with denture-induced stomatitis. The aim of this study was to evaluate the use of vinegar as an antimicrobial agent for control of Candida spp. in complete upper denture wearers. Fifty-five patients were submitted to a detailed clinical interview and oral clinical examination, and were instructed to keep their dentures immersed in a 10%vinegar solution (pH less than 3) overnight for 45 days. Before and after the experimental period, saliva samples were collected for detection of Candida, counting of cfu/mL and identification of species by phenotypical tests (germ tube formation, chlamidoconidia production, and carbohydrate fermentation and assimilation). The results were analyzed using Spearman's correlation and Student's t-test (p£0.05). Candida yeasts were present in 87.3% of saliva samples before the treatment. A significant reduction was verified in CFU/mL counts of Candida after treatment. A positive correlation between Candida and denture stomatitis was verified, since the decrease of cfu/mL counts was correlated with a reduction in cases of denture stomatitis. Although it was not able to eliminate C. albicans, the immersion of the complete denture in 10% vinegar solution, during the night, reduced the amounts (cfu/mL) of Candida spp. in the saliva and the presence of denture stomatitis in the studied patients.

Prevalencia de Candida albicans y Candida no albicans en diferentes muestras clínicas: Período 1999-2001 / Prevalence of Candida albicansand Candida no albicans inclinical samples during 1999-2001

Las levaduras implicadas en procesos patológicos son de indiscutible importancia debido al incremento experimentado por estas infecciones en las últimas décadas, a los cambios observados en las especies causales y al uso empírico de antifúngicos. En el Centro de Micología se estudiaron 1006 aislamientos provenientes de una amplia gama de muestras clínicas durante el periodo 1999-2001. Candida albicans con 40,3% resultó la especie de mayor frecuencia de aislamiento, pero las especies de Candida no albicans con 54,9% resultaron de mayor prevalencia y el 4,8% fueron otras levaduras. En los hemocultivos Candida parapsilosis con 34,9%, C. albicans con 30,2% y C. tropicalis con 25,6% resultaron las más recuperadas, mientras que C. glabrata se presentó con un 2,3%. En las secreciones mucosas C.albicans con 60%-80% fue la especie preponderante. Hemos detectado especies de Candida causantes de mediastinitis, lo que nos alerta sobre su importancia en estos procesos. Las infecciones del tracto urinario por levaduras se detectaron en mayor frecuencia en individuos hospitalizados, resultando C. albicans con 47,7% la especie más aislada, y dentro de Candida no albicans, C. glabrata con 24,8% y C. tropicalis con 20,0%. En las onixis candidiásicas C. parapsilosis con 37,7% desplazó a C.albicans con 22,0% de este lugar anatómico. Los estudios de sensiblidad al fluconazol de las especies de Candida nos permiten concluir que C.albicans es una especie sensible y que los mayores porcentajes de resistencia se observaron en C. glabrata (21,41%) y and C. krusei (69,23%).

The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3%) was the most isolated species, although, the Candida no albicans species with 54.9% showed the major prevalence. In blood cultures Candida parapsilosis (34.9%), C. albicans (30.2%) and C. tropicalis (25.6%) were recovered most frequently while C. glabrata represented only 2.3%. C. albicans with 60%-80% was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7%), the most commonly isolated, followed by C. glabrata (24.8%) and C. tropicalis (20.0%). In the candidal onychomycoses, C. parapsilosis (37.7%) outplaced C. albicans (22.0%). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41%) and C. krusei (69.23%).