Prevalence and Fluconazole Susceptibility Profile of Candida spp . Clinical Isolates in a Brazilian Tertiary Hospital in Minas Gerais , Brazil

Candidiasis has become an important concern for clinical practice, especially with the increasing incidence of immunocompromised patients. In this scenario, the development resistance to fluconazole presents a challenge for treating these opportunistic infections. The aim of this study was to evaluate some epidemiology features of Candida infections in a Brazilian University Hospital using data, previously unavailable. We observed that 44% of the 93 clinical isolates tested, belonged to Candida albicans species and 56% belonged to non-Candida albicans species (mainly Candida tropicalis and Candida glabrata). Most strains were isolated from urine samples where C. albicans was predominantly detected. 29 strains presented a fluconazole resistance phenotype and of these, 22 were chemosensitised by FK506, a classical inhibitor of ABC transporters related to azoles resistance. These data suggest the probable role of efflux pumps in this resistance phenotype. Our study highlights the need for developing effective control measures for fungal infections, rational use of antifungal drugs and development of new molecules able to abrogate the active transport of antifungals.


INTRODUCTION
Candida spp.infections are recognized as a major challenge in public health, commonly associated with high morbidity and mortality since its diagnosis and treatment present difficulties and high healthcare costs (Colombo et al. 2008, Gudlaugsson et al. 2003, Arnold et al. 2010).In Latin America, mortality rates are usually higher than those observed in the Northern Hemisphere; however, it is worth mentioning that epidemiology of candidemia is poorly studied in this region in comparison to the United States and Europe (Nucci et al. 2013, Colombo et al. 2008, Santolaya et al. 2014).
Fluconazole (FCZ) is the first option for prophylaxis and treatment, due to its good tolerance, few side effects and low costs (Li et al. 2014).However, the widespread and prolonged use of antifungal agents induces tolerance development as well as collateral resistance to other drugs (Prasad and Rawal 2014).Some species, such as Candida glabrata and Candida krusei, already present decreased susceptibility or resistance to FCZ (Wingard et al. 1991, Marr et al. 2000).Preventing the intracellular accumulation of a drug by rapid extrusion of the antifungal agent is a known mechanism of resistance (Prasad et al. 2002).The overexpression of efflux pumps, such as ATP-binding cassette (ABC) family or major facilitator superfamily (MFS), has been shown to be the cause of resistance in some fungi (Cannon et al. 2009).
The aim of this study is to evaluate the frequency of yeast species isolated from urine, faeces, catheter, blood and other secretions, analyze the susceptibility to FCZ as well as investigate the possible mechanism of resistance of clinical strains obtained from the University Hospital of the Federal University of Juiz de Fora, Minas Gerais, Brazil, over a period of two years (2012)(2013)(2014).

Yeast strains and Patients
Ninety-three Candida spp.isolates were obtained from patients at the University Hospital of Universidade Federal de Juiz de Fora, Minas Gerais, The yeast strains grown in YPD medium (2% glucose, 2% peptone, 1% yeast extract) at 37 °C under agitation, were harvested in the exponential phase of growth and stored at 4 °C.

strain identifiCation
Isolate identification was performed by MALDI Microflex LT (Bruker Daltonics, Bremen, Germany) measurement, according to manufacturer formic acid extraction procedure.Briefly, in this identification method, a single colony of each strain grown overnight on YPD agar was suspended in 300µL of de-ionized water and 900µL of absolute ethanol and centrifuged at 14,462 xg for 2 min.The supernatant was discarded and the pellet was airdried.1:1 v 70% formic acid and 100% acetonitrile were added to the pellet and vortexed.The samples were centrifuged at 14,462 xg for 2 min, and 1µLof the supernatant was spotted in duplicate onto a steel target and air-dried at room temperature.Before identification, each spot was overlaid with 1µL of HCCA (α-Cyano-4-hydroxycinnamic acid, Bruker) matrix solution saturated with organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid) and air dried completely.The spectra were externally calibrated using standard ATCC Escherichia coli 25922, before plate identification.Raw spectra were processed using MALDI BIOTYPER Realtime Classification software version 3.1 (Bruker Daltonik MALDI Biotyper).Strains with score values ≥ 2 were indicated as reliable species identification.

antifunGal susCePtibilitY tests
In vitro susceptibility tests were performed using the broth microdiluition assay according to the protocol of the Clinical and Laboratory Standards Institute (CLSI) M27-A2 protocol.According to CLSI, FCZ MIC 50 (Minimal Inhibitory Concentration) end points ≤ 8µg/mL were categorized as susceptible.MIC 50 end points between 16 and 32µg/mL were classified as susceptible dose-dependent (SDD), and resistant strains obtained MIC 50 end points ≥ 64µg/mL.Cell growth was analyzed in a microplate reader at 600 nm (Fluostar Optima, BMG Labtech, Offenburg, Germany).

evaluation of fluConazole resistanCe reversion bY fK506
Since FK506 is a classical ABC transporter inhibitor (Egner et al. 2000), it was used as an identifier for resistance mediated by these transporters.The "spot test" was used as a measure of growth as previously described by (Reis de Sa et al. 2014).

flow CYtometrY assaY
The experiment was performed using eight representative resistant Candida spp.isolates.The cells were incubated overnight in 20 ml of YPD medium 1 x 10³ cells/mL at 37 °C under agitation.After incubation, with a value of 1.0 to 3.0 OD, the cells were centrifuged (5000 x g / 5 min) and washed four times with deionized water.After washing, the strains were maintained on ice for 2h.A concentration of 6 x 10 5 cells/mL were incubated with Rhodamine 6G (R6G) (5µM), an ABC transporter fluorescent substrate (Maesaki et al. 1999), in the presence or absence of glucose (2%) for 1h, at 37 °C under agitation.The cells were centrifuged (9000 x g / 2 min) and washed with PBS buffer (3 X) before analysis in duplicate by BD Accuri™ C6 Flow Cytometer, in order to measure of R6G efflux by strains.

data analYses
Data analysis was performed using the Statistical Package for the Social Sciences (SPSS © ) software (version 20).
The majority of resistant strains belonged to the C. tropicalis species (n=13, 45%), while C. albicans   was the most prevalent susceptible (n=27, 46.6%) and SDD (n=3, 50%) isolate (Table II).The MIC 50 values of each resistant strain are summarized in Table III.Seven Candida isolates presented MIC 50 higher than the maximum tested concentration: three of these strains were C. tropicalis, two C. albicans and two C. glabrata.Three isolates were resistant to concentrations higher than 500µg/ml, five for 250µg/ml, two for 125µg/ml and eleven for 64µg/ml.

reversion of fluConazole resistanCe
After the screening of clinical strains resistant to FCZ, we performed a chemosensitization assay using FK506 in order to evaluate the possible resistance mechanism of these Candida spp.isolates.The resistance phenotype of 22 clinical strains reverted upon treatment with the ABC transporter inhibitor, while the other seven (1016, 250i, 14A, 163A, 1027, 124i, 1114) isolates remained resistant, even in the presence of FK506 (Fig. 3).Among the 22 resistant Candida strains chemosentitized by FK506, we observed that 11 were C. tropicalis, seven were C. albicans, 4 C. glabrata.
flow CYtometrY Flow cytometry assay was performed in order to check the capability to extrude Rhodamine 6G (R6G), a fluorescent substrate pumped by ABC transporters, by resistant strains.For this test, we used four representative strains reverted by FK506 (Fig. 4a) and four that remained resistant even in the presence of FK506 (Fig. 4b).The data revealed that strains reverted by FK506 were able to pump out R6G in the presence of glucose whereas strains that displayed high level of resistance to FCZ, even in the presence of FK506, were not able to efflux the fluorescent probe in the presence or absence of glucose.These results confirm those obtained by chemosensitization assay (Fig. 3) since reverted strains also presented an efflux of R6G.

DISCUSSION
Regional differences in the incidence pattern of antifungal resistance and patient suceptibility can be found when monitoring fungal infections.Knowledge of the epidemiological characteristics of a certain community has both local and global importance, considering the constant flow of individuals within a country and around the world (Nucci et al. 2010).Since Candida spp.are the most common fungal nosocomial pathogens, contributing significantly to morbidity and mortality (Wisplinghoff et al. 2014, Pfaller et al. 2014), we decided to evaluate the prevalence of Candida infections at a Brazilian University Hospital located in a Southeast region, where no epidemiologic data relating to candidiasis had been previously collected.We also determined the susceptibility profile of Candida spp.strains -isolated from clinical material -as well as the possible role of ABC transporters in the FCZ resistance.
Our results revealed that the Candida species most commonly isolated were C. albicans (44.4%), C. tropicalis (28%) and C. glabrata (14%) (Fig. 1).These data were consistant with other Brazilian and international studies concerning nosocomial fungal infections, which show C. albicans as the most prevalent species isolated (Wille et al. 2013, da Costa et al. 2014, Corzo-Leon et al. 2014).Furthermore, we observed a higher proportion of non-albicans species (56%) compared with Candida albicans (44%), a trend which has also been observed in other studies.Oliveira and colleagues evaluated cases of fungemia from 2007 to 2010 and observed a higher prevalence of candidemia caused by Candida species other than C. albicans during a period of four years (Oliveira et al. 2014).Moretti and colleagues observed an increasing incidence of candidemia caused by C. tropicalis and C. glabrata between 2006C. tropicalis and C. glabrata between and 2010C. tropicalis and C. glabrata between (Moretti et al. 2013)) Urinary tract infections caused by Candida spp.are commonly observed in hospitalized patients, especially those in intensive care units (Kauffman 2014).The tendency of hospitalized patients to be predisposed to urinary tract infections caused by Candida spp is explained by factors such as instrumentation of the urinary tract, prolonged hospitalization, use of broad spectrum antibiotics and indwelling urinary tract devices (Sobel et al. 2011).The evaluation of bacteriuria and candiduria in an intensive care unit (ICU), conducted by Aubron and colleagues over a period of six years (2006)(2007)(2008)(2009)(2010)(2011), demonstrated that Candida spp.represented 55% of pathogens isolated from positive urine cultures (Aubron et al. 2015).We also observed this high Candida spp.prevalence in urinary tract infections, with 71 of the 93 isolates tested, coming from urine samples (Table I).Of these, the three most prevalent species were C. albicans (34 / 71 -48%), C. tropicalis (19 / 71 -27%) and C. glabrata (12 / 71 -17%) (Fig. 2).These data are confirmed by Sobel and colleagues who identify these three species as the main cause of candiduria (Sobel et al. 2011).
Azoles, specially FCZ, are by far the most commonly used antifungals in clinical practice and the emergence of the resistance to this chemotherapeutic is a concern in clinical practice (Vandeputte et al. 2012).When assessing the susceptibility profile to FCZ, we observed the resistance phenotype in 32.1% (29/93) of studied Candida strains (Table II).Our results differ from those obtained by many other studies, which detected a lower incidence of FCZ resistance (Wille et al. 2013, Tortorano et al. 2012, Lockhart et al. 2011, Pfaller et al. 2011, da Matta et al. 2007).These studies evaluated a higher number of samples compared to our study, which focused on a more specific population.This could explain the differences concerning the prevalence of resistance to FCZ.However, our results are consistent with other studies (Puig-Asensio et al. 2014 The active transport of azoles out of the cell, mediated by membrane ABC transporters is an important mechanism of resistance in Candida species (Sanglard et al. 2009).Therefore, we decided to evaluate the role of ABC transporters in the FCZ resistance expressed by the 29 Candida spp.strains.For this, we used the chemosinsitization experiment with FK506 (tacrolimus), a classic inhibitor of Candida ABC transporters (Cannon et al. 2009).We observed that in 75.8% (22/29) (Fig. 3) of these strains, FK506 could reverse the FCZ resistance, revealing a probable contribution of ABC transporters to this phenotype of resistance.Among the strains chemosensitized by FK506, we identified 11 C.tropicalis, seven C. albicans and four C. glabrata (Table III).Other studies also observed the participation of efflux pumps in FCZ resistance in Candida species.White et al. (2002) assessed the mechanism of resistance in C. albicans strains isolated from clinical material and showed that out of 13 strains with a FCZ MIC 50 for > 64µg/mL, six presented overexpression of ABC transporter genes related to resistance to azoles and CaCdr1/CaCdr2 (White et al. 2002).Evaluating the FCZ resistance mechanism of 20 strains of C. glabrata, Sanguinetti et al. (2005) noted that all FCZ resistant strains showed overexpression of at least one ABC transporter related to antifungal efflux (CgCdr1, CgCdr2 and CgSNQ), and that FK506 could chemosensitize all strains (Sanguinetti et al. 2005).Barchiesi et al. (2000) demonstrated that prolonged exposure of C. tropicalis to FCZ induced the expression of an ABC transporter that promotes the active efflux of this antifungal (Barchiesi et al. 2000).It is worth mentioning that seven of the clinical isolates did not present reversion of resistance phenotype using FK506.This data suggest that probably these strains possess other mechanism of resistance.Besides ABC transporters, Candida species can present an overexpression of major facilitator superfamily (MFS) proteins, another efflux pump that actively extrudes substrates using energy from proton gradient across membrane (Cannon et al. 2009).Additionally, resistance can be developed through different molecular mechanisms concerning: overexpression and/or mutation of ERG11 as well as alterations in ergosterol biosynthetic pathway (Lamping et al. 2010).In order to check and reinforce the possible involvement of ABC transporters on resistance phenotype, we also performed an efflux assay using R6G.Clinical isolates reverted by FK506 also extruded part of R6G, while the opposite was observed on strains that maintained resistance even in the presence of the inhibitor.These data corroborate the possible involvement of ABC transporters on resistant profile of those Candida spp.isolates reverted by FK506.
The data obtained so far, reinforce the emergence of FCZ resistant strains of Candida spp. in the hospital environment, as well as the involvement of active drug transporters in resistance phenotype.This phenomenon highlights the need for developing more effective control measures of fungal infections, rational use of antifungal drugs and development of new molecules able to abrogate the active transport of antifungals.

ACKNOWLEDGMENTS
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Instituto de Microbiologia Paulo de Góes (IMPG) and Universidade Federal do Rio de Janeiro (UFRJ).The authors thank their lab assistant, Mrs. Geralda Rodrigues Almeida for her great support, Dr. Ana Claudia Tessis (IFRJ -RJ/Brazil) for her help with flow citometry experiments and Dr. Louise Kemp for her critical reading of this manuscript.

Figure 2 -
Figure 2 -Number of species Candida isolated from each clinical source.The majority of clinical isolates were C. albicans in urine samples.

Figure 4 -
Figure 4 -Citometry efflux assay of Candida spp.strains using R6G as fluorescent substrate in the presence and absence of glucose.(a) Flow citometry conducted with clinical isolates that were chemosensitized by FK506.(b) Flow citometry assay of clinical isolates that retained resistance phenotype using FK506.Black bar = no glucose; Gray bar = with glucose.This experiment represents average of triplicate.

Figure 3 -
Figure 3 -Evaluation of FCZ reversion by FK506 on 29 resistant Candida spp.clinical strains.CTRL: yeast cells growth on Sabouraud solid medium in absence of FCZ for 48h.FCZ (+): yeast cell growth on Sabouraud solid medium containing 64µg/mL of FCZ.FCZ(+) and FK506: yeast growth in same conditions as CTRL plus FCZ (+) presenting FK506 10μM.Most clinical strains presented resistance reversion using FK506, a classic ABC transporter inhibitor.Only seven strains were still resistant to FCZ even in the presence of the inhibitor.

TABLE III Minimal inhibitory concentration (MIC 50 ) of resistant to FCZ Candida spp. isolates according to CLSI M27-A2 protocol.
. Pfaller et al. 2014 also pointed to the emergence of invasive fungal infections caused by non-albicans species of Candida in a study comprising 23 medical centers in the United States and two in Canada (Pfaller et al. 2014).