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Year : 2015  |  Volume : 33  |  Issue : 5  |  Page : 97--101

Effects of fluconazole treatment of mice infected with fluconazole-susceptible and -resistant Candida tropicalis on fungal cell surface hydrophobicity, adhesion and biofilm formation

RL Kanoshiki1, SB de Paula1, JP Santos1, AT Morey1, NB Souza1, LM Yamauchi1, BP Dias Filho2, SF Yamada-Ogatta1,  
1 Department of Microbiology, Biological Sciences Center, State University of Londrina, Londrina, Brazil
2 Department of Basic Health Sciences, Health Sciences Center, University of Maringá, Maringá, Brazil

Correspondence Address:
S F Yamada-Ogatta
Department of Microbiology, Biological Sciences Center, State University of Londrina, Londrina
Brazil

Abstract

Background : The incidence of Candida tropicalis less susceptible to fluconazole (FLC) has been reported in many parts of the world. Objectives : The aim of this study was to examine the changes of putative virulence attributes of Candida tropicalis accompanying the development of resistance to FLC in vitro and in vivo. Materials and Methods : A FLC-resistant strain (FLC-R) was obtained after sequential exposure of a clinical isolate FLC-sensitive (FLC-S) to increasing concentrations of the antifungal. The course of infection by both strains was analyzed in BALB/c mice. Analyses of gene expression were performed by real-time polymerase chain reaction PCR. The cell surface hydrophobicity, adhesion and biofilm formation were also determined. Results : Development of resistance to FLC could be observed after 15 days of subculture in azole-containing medium. Overexpression of MDR1 and ERG11 genes were observed in FLC-R, and this strain exhibited enhanced virulence in mice, as assessed by the mortality rate. All mice challenged with the FLC-R died and FLC-treatment caused earlier death in mice infected with this strain. All animals challenged with FLC-S survived the experiment, regardless of FLC-treatment. Overall, FLC-R derivatives strains were significantly more hydrophobic than FLC-S strains and showed greater adherence and higher capacity to form biofilm on polystyrene surface. Conclusions : The expression of virulence factors was higher in FLC-R-C. tropicalis and it was enhanced after FLC-exposure. These data alert us to the importance of identifying microorganisms that show resistance to the antifungals to establish an appropriate management of candidiasis therapy.

How to cite this article:
Kanoshiki R L, de Paula S B, Santos J P, Morey A T, Souza N B, Yamauchi L M, Dias Filho B P, Yamada-Ogatta S F. Effects of fluconazole treatment of mice infected with fluconazole-susceptible and -resistant Candida tropicalis on fungal cell surface hydrophobicity, adhesion and biofilm formation .Indian J Med Microbiol 2015;33:97-101

How to cite this URL:
Kanoshiki R L, de Paula S B, Santos J P, Morey A T, Souza N B, Yamauchi L M, Dias Filho B P, Yamada-Ogatta S F. Effects of fluconazole treatment of mice infected with fluconazole-susceptible and -resistant Candida tropicalis on fungal cell surface hydrophobicity, adhesion and biofilm formation . Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 12 ];33:97-101
Available from: http://www.ijmm.org/text.asp?2015/33/5/97/148834

Full Text

 Introduction



Species of the genus Candida are among the most common opportunistic fungal pathogens. The incidence of bloodstream Candida spp. infections has increased dramatically over the years and C. albicans has been regarded as the major causative agent of candidemia. [1],[2],[3],[4] However, other Candida species have become a significant cause of such infections and Candida tropicalis is among the most common agents, mainly in Latin America and Asia-Pacific. [3],[4] The reasons for this epidemiological change are unclear, but the widespread use of azoles for the treatment of candidiasis might have contributed to this scenario. [1] Indeed, Candida species that are less susceptible to fluconazole (FLC), including C. tropicalis, have been reported elsewhere. [2],[5]

FLC is still currently used for the treatment of candidiasis. [1],[4] Besides the well-known antimicrobial effects of FLC, this antifungal can interfere with the expression of putative virulence factors of Candida spp. in vitro.[6],[7] Moreover, previous studies have shown that the development of FLC-resistance, due to recurrent exposure to the antifungal, may be associated with morphological and physiological changes in Candida spp. These changes can influence virulence in animal models of candidiasis. [8],[9],[10],[11]

In contrast to C. albicans, little is known about the influence of FLC on the expression of virulence factors in other Candida species, either in vitro or in vivo. In one study, it was shown that FLC induces an increase in Candida dubliniensis adherence to mammalian epithelial cells. [7] In view of this, we compared in this study the expression of virulence attributes in fluconazole-susceptible and in vitro induced fluconazole-resistant C. tropicalis in vitro and in an intravenous mouse infection model.

 Materials and Methods



Reagents

The reagents used in this study were obtained from the following companies. Himedia, Mumbai, India; Sabouraud dextrose (SD) broth and agar; Invitrogen, Auckland, New Zealand: Agarose, Platinum® SYBR® Green qPCR SuperMix-UDG, Trizol®, 1 kb plus DNA molecular weight; Sigma-Aldrich Chemical Co, São Paulo, Brazil: Fluconazole, menadione, RPMI 1640, xylene, 2,3-bis (2-methoxy-4-nitro -5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT); Integrated DNA Technologies, Iowa, USA: Oligonucleotide primers.

Microorganisms

C. tropicalis, originally recovered from the blood of a patient with candidemia, was used throughout this study. The species identification was carried out by standard mycological methods. The FLC susceptibility profile was determined by the broth microdilution method as described by the Clinical Laboratory Standards Institute. [12] Candida parapsilosis ATCC 22019 was used as the quality control organism in antifungal susceptibility assays.

Induction of fluconazole resistance

A single FLC-susceptible C. tropicalis strain (S) colony was inoculated into 5.0 ml of RPMI 1640, and the culture was incubated at 37°C for 18 h. An aliquot of this culture with 5 × 10 3 cells/ml was transferred to fresh medium supplemented with 4 μg/ml FLC, and the cells were incubated at 37°C for 48 h. An aliquot of this culture containing the same inoculum as described above was further subcultured twice in fresh medium containing 8, 16, 32 and 64 μg/ml FLC. Following each subculture, the tubes were incubated at 37°C for 48 h or until detectable yeast growth. At each passage, a 1.0 ml aliquot was used to assess the minimal inhibitory concentration (MIC) by the broth microdilution method. [12] The FLC-resistant strain (R) was serially cultured in SD broth without the antifungal to determine the stability of the resistance. The genetic relatedness of the R strain to S strain was determined by random amplified polymorphic DNA (RAPD) as described elsewhere. [13]

Animals and Candida tropicalis infection

BALB/c mice (male, eight- to 12-week-old) were maintained under standard conditions. Commercial rodent diet and sterilized water were available ad libitum. All procedures with the animals were in accordance with the guidelines of the Brazilian Code for the Use of Laboratory Animals (CEEA number 101/09). Groups of seven mice were used in all experiments. S and R were grown in SD broth medium at 37°C for 18 h, and 100 μl of 0.15 M phosphate-buffered saline pH 7.4 (PBS)-suspension of 1.0 × 10 7 yeasts were injected intravenously (i.v.) via the lateral tail vein. [14] The infected mice were divided into four experimental groups, where the first two groups of mice were infected with the S and R strains, respectively, and did not receive FLC therapy. For the third and fourth groups of mice infected with S and R, respectively, FLC at 5 mg/kg/day was administered by oral gavage once daily for five days beginning 2 h after Candida inoculation. Negative control animals were inoculated i.v. with 100 μl of PBS. The animals were observed daily and survival rates were determined up to 30 days post-infection. Kidneys, taken as representative organs [14] , were removed from two randomly chosen animals at 5 days post-infection. The organs were weighed and homogenized in sterile PBS. Aliquots of 100 μl from 10-fold serial dilutions of organ homogenates were inoculated onto SD agar, in duplicate. Plates were incubated at 37°C, and the colonies were counted after 24 h. Results were expressed as number of colony-forming units (CFU) per gram of tissue ± standard error. The C. tropicalis strains were named as follows: S1 and R1 for the S and R counterpart strains recovered from the animals without FLC treatment; S2 and R2 for the S and R counterpart strains recovered from the animals after FLC treatment.

Cell surface hydrophobicity (CSH) determination

The hydrophobicity of the yeast was determined by the biphasic hydrocarbon/aqueous method. [6] CSH was expressed as the percentage decrease in absorbance of the aqueous phase of the test as compared with the control, where the greater the change in absorbance of the aqueous phase, the more hydrophobic the yeast sample.

Adhesion and biofilm formation on polystyrene assays

C. tropicalis strains were grown in SD broth at 37°C for 18 h. The yeasts were harvested by centrifugation, and the cells were washed twice with sterile PBS and counted. A 20-μl SD broth suspension of 6 × 10 5 yeasts was placed in each well of flat-bottomed 96-well microtitre plates (Techno Plastic Products, Switzerland) containing 180 μl of SD broth. The plates were incubated at 37°C for 1 h and 24 h, for adhesion and biofilm formation assays, respectively. After the incubation time, the plates were washed once with sterile distilled water, and the metabolic activity of the cells was quantified using the XTT-reduction assay as described previously [15]

Analysis of ERG11 and MDR1 gene expression

Real-time PCR was performed to determine the relative mRNA levels of ERG11 (coding for lanosterol 14 a-demethylase) and MDR1 (coding for an efflux protein belonging to the major facilitator superfamily) of the C. tropicalis strains, with ACT1 (coding for actin) used as a reference housekeeping gene to normalize the data, as described previously. [15] Thermal dissociation confirmed that RT-PCR generated a single amplicon. The relative quantification of gene expression was performed by the ∆∆C T method and differences in expression are reported, using FLC-susceptible cells as the reference population.

Statistical analysis

The results were analyzed by Student's t-test using the software GRAPHPAD PRISM version 5.0 (GRAPHPAD Software, San Diego, CA). Comparative analysis of the samples was carried out using Tukey's test. P values less than 0.05 were considered significant.

 Results and Discussion



Development of fluconazole resistance

The frequent use of FLC for the treatment and prophylaxis of fungal diseases has contributed to the isolation of Candida spp. displaying reduced azole-susceptibility. [1] Moreover, the in vitro and in vivo induction of resistance to FLC for these microorganisms have been reported. .[8],[11],[16] To determine the effects of FLC on expression of virulence attributes of C. tropicalis, we first obtained a FLC-resistant derivative by exposing the yeast to increasing concentrations of the antifungal. The MIC of FLC for the S strain was 2.0 μg/ml. After 15 days exposure to the antifungal (64 μg/ml), high levels of FLC resistance (MIC > 128 μg/ml) were observed for C. tropicalis isolate. No change in FLC MIC was detected following 60 subcultures in antifungal-free medium. RAPD analysis confirmed the genetic relatedness between the S and R strains [[Figure 1]a].{Figure 1}

The in vitro induction of FLC resistance in Candida spp. has been previously reported. Barchiesi et al. [8] found that subculture in azole-containing broth medium (8.0, 32.0 and 128 μg/ml) induced a rapid development of C. tropicalis ATCC 750 resistant cells, and the MIC values of these cells varied depending on the FLC concentrations used in the induction medium. The FLC MIC values of the resistant cells obtained in the presence of 8.0 and 32.0 μg/ml reverted to the initial sensitive cells (1 μg/ml) after 12 and 11 successive subcultures in FLC-free medium, respectively. For the resistant cells obtained with 128 μg/ml FLC, MIC was significantly reduced (512 to 16 μg/ml) but remained stable over 60 subcultures in FLC-free medium. On the other hand, the in vitro-induced resistant C. dubliniensis clinical isolates (which FLC MIC ranged from 16 to 64 μg/ml) remained stable over 10 subcultures in FLC-free medium. [16] Overexpression of MDR1 and CDR1 [ATP-binding cassette pump [8] and ERG11[17] has been associated with fluconazole resistance in C. tropicalis. We observed an increased expression of 6.5- and 3.1-fold for ERG11 and MDR1 genes, respectively, in the R strain [[Figure 1]b].

FLC-resistant Candida tropicalis caused high mortality rate in intravenous mouse infection model

There was a significant difference in the survival rate between BALB/c mice infected with S and R strains. All mice infected with R died before 16 days post-infection. For the R1 group, mortality started on the 7 th day post-infection reaching 100% by the 15 th day. In the R2 group, no animals survived after the 7 th day post-infection. On the other hand, all mice infected with C. tropicalis S strain survived the infection, regardless of FLC treatment [[Figure 1]c].

The virulence of FLC-susceptible and FLC-resistant Candida, especially C. albicans strains, has been investigated in several studies using intravenous mouse infection models. Fekete-Forgαc et al. [9] observed that the in vitro induced FLC-resistance was related to an increase in virulence of C. albicans in pathogen-free NMRI mice. No mortality was observed in animals infected with FLC-susceptible strain at 12 days post-infection, whereas all animals infected with FLC-resistant strain died within the same period. Similarly, high mortality rate (70%) of BALB/c mice infected with in vitro induced FLC-resistant C. albicans compared to parental FLC-susceptible strain (10% mortality) was observed after 7 days of infection by Angiolella et al. [10] On the other hand, Schulz et al. [11] showed that the FLC-resistant C. albicans strain developed in vivo was not more virulent than the FLC-susceptible parental counterpart in a disseminated CFW-1 mouse model. Around 60% of the animals infected with the resistant strain died at 7 days post-infection, regardless of FLC treatment. The susceptible strain produced 100% mortality in infected animals after 3 days of infection. However, all animals survived the infection with the susceptible strain after oral FLC therapy.

In contrast to C. albicans, few studies have examined the correlation between FLC-resistance and virulence in C. tropicalis. It has been shown that the development of in vitro FLC-resistance was related to a loss of virulence, since mortality was not observed in female BALB/c mice infected with an in vitro induced FLC-resistant C. tropicalis strain. [8] Nevertheless, around 60% mortality was observed in animals infected with the parental susceptible strain, and it was associated with higher kidney fungal burden of the animals. These data differ from our results, which may be a reflection of the genetic background that influences the pathogenic mechanisms of C. tropicalis strains, as shown previously by others. [14],[18] In the present study, there was a significant difference (P < 0.05) in kidneys fungal load from infected and FLC-treated mice with the R strain (R2) on day five post-infection compared to the other groups [Table 1].{Table 1}

Higher virulence of FLC-resistant Candida tropicalis may be related to changes in cell surface hydrophobicity, adhesion and biofilm formation

The increase of virulence in FLC-resistant Candida strains, both in vitro and in vivo, has been associated with changes in its phenotypic features. [9],[10] A significant difference in CSH of C. tropicalis strains was observed using an aqueous/hydrocarbon method [Table 1]. The mean relative CSH of the S strain was 46.77 ± 2.41, while the corresponding value for the R strain was 70.54 ± 3.82. After mouse infection, the mean relative CSH was significantly lower in S2 compared to S1. In contrast, an increased CSH was observed in R2 compared to R1 (P < 0.05). The R1 and R2 strains were significantly more hydrophobic than the S1 and S2 strains counterparts. These results agree with an earlier observation that the hydrophobic C. albicans cells were more virulent in CD1 mice. [19] In fact, previous studies reported that CSH is correlated with increased virulence in C. albicans. The hydrophobic cells exhibited greater adherence to acrylic surfaces and host cells and decreased susceptibility to killing by polymorphonuclear neutrophils, which can contribute to the colonization and dissemination of the yeast. [19]

No significant correlation was found between CSH and adhesion to polystyrene surface in this study. The development of in vitro C. tropicalis FLC-resistance was accompanied by increased cell adhesion to polystyrene compared to the parental susceptible strain (S, 0.2133 ± 0.026 vs. R, 0.2908 ± 0.016, P < 0.05). There was a tendency towards increased cell adhesion when comparing the S1 and R1 strains (P > 0.05). Moreover, R2 showed greater adhesion to polystyrene compared to the S2 strain (P < 0.05), which is consistent with mortality rate of the animals. The association of FLC resistance, developed in vitro or in vivo conditions, with increased adherence of C. albicans to different surfaces was previously reported. [9],[10],[11] Except for Schulz et al. [11] , other authors have showed that increased adherence is associated with increased virulence, as judged by the mortality of the animals during experimental infection with C. albicans.

Biofilm formation is an important virulence trait of Candida spp. Sessile cells in this community display a phenotype that is markedly different from their free-floating (planktonic) counterparts. Candida spp. biofilms are notoriously resistant to a variety of antifungals [15] , which contributes to the persistence of the fungi in their environment. Biofilm formation can vary among Candida spp. and the degree of biofilm formation correlates with virulence in BALB/c disseminated infection. [5] In this study, a significant correlation (P < 0.05) was observed between CSH and biofilm formation in all C. tropicalis strains. Although all C. tropicalis strains were able to form biofilms on polystyrene surface, a higher metabolic activity was observed in biofilm from the R compared to the S derivative strains. R2 exhibited the greatest metabolic activity of biofilm, which is consistent with the high mortality rate in the shortest time of animals infected with this strain. Similarly, Angiolella et al. [10] observed that the virulence of the in vitro-induced resistant C. albicans strain in mice was associated with increased biofilm formation.

In conclusion, the results of this study showed that the in vitro-induced resistance to fluconazole was accompanied by an increased virulence of C. tropicalis. Accordingly, FLC-R was more hydrophobic and showed greater adherence and higher capability to form biofilm on polystyrene surface. These results corroborate the importance of monitoring the susceptibilities of C. tropicalis to FLC for appropriate management of infections caused by this yeast.

 Acknowledgements



We thank Dr. A. Leyva for English editing of the manuscript. This work was supported by grants from Pro-Reitoria de Pesquisa e Pós-Graduação (PROPPG) of Universidade Estadual de Londrina and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES.

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