Antifungal susceptibilities, biofilms, phospholipase and
proteinase activities in the Candida rugosa complex and Candida pararugosa isolated from tertiary teaching hospitals
T. Peremalo1, P. Madhavan2,*, S. Hamzah1, L. Than3, E. H. Wong2, M. D. Mohd Nasir4, P. P. Chong5 and K. P. Ng6
DOI 10.1099/jmm.0.000940
Received 09 August 2018; Accepted 21 January 2019; Published 06 February 2019
Author affiliations: 1School of Pharmacy, Taylor’s University Lakeside Campus, Subang Jaya, Selangor Darul Ehsan, Malaysia; 2School of Medicine, Taylor’s University Lakeside Campus, Subang Jaya, Selangor Darul Ehsan, Malaysia; 3Department of Medical Microbiology and Parasitology, University Putra Malaysia, Selangor Darul Ehsan, Malaysia; 4Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra Malaysia, Selangor Darul Ehsan, Malaysia; 5School of Biosciences, Faculty of Health and Medical Sciences, Taylor’s University Lakeside Campus, Subang Jaya, Selangor Darul Ehsan, Malaysia; 6Department of Biomedical Imaging, Faculty of Medicine, University Malaya Medical Centre, Kuala Lumpur, Malaysia.
*Correspondence: P. Madhavan, Priya. Madhavan@ taylors. edu. my; mpriya0905@ gmail. com Keywords: C. rugosa complex; C. pararugosa; biofilm; enzymes; antifungal drug resistance.
Abbreviations: AMB, amphotericin B; ANOVA, analysis of variance; ATCC, American Type Culture Collection; BSA, bovine serum albumin; CAS, Caspofungin; CO2, carbon dioxide; CV, crystal violet; DNA, deoxyribonucleic acid; FL, Fluconazole; HIV, human immunodeficiency virus; ITS, internal transcribed spacer; MIC, minimum inhibitory concentrations; na, not available; NCBI, National Center for Biotechnology Information; OD, optical density; PBS, phosphate buffered saline; RPMI, Roswell Park Memorial Institute Medium; SDA, sabouraud dextrose agar; SEM, standard error of mean; SPSS, statistical package for the social sciences; USA, United States of America; VO, Voriconazole; XTT, 2,3-Bis-(2-Methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt; YDP, yeast extract peptone dextrose.
InTRoduCTIon
Candidiasis is a fungal infection caused by Candida species.
Many species of Candida occupy diverse sites on the human body including skin, oral cavity, vagina, digestive tract and nails as normal flora but are able to switch to pathogenic strains under certain circumstances [1]. Immunocompro- mised conditions associated with HIV patients, diabetes mellitus, chemotherapy treatment, prolonged hospitaliza- tion, organ transplantation, extensive surgical procedure,
long-term antibiotic use are some of the most favourable risk factors for various types of candidiasis [2].
Although C. albicans is the predominant cause of candidiasis, the high morbidity and mortality attributed to non-albicans Candida were found to be increasing over the years [3]. In recent years, there has been a significant epidemiological shift from C. albicans towards non-albicans Candida species with greater antifungal resistance capabilities [4–7]. Thus, this has emerged as a major concern in the control and management Abstract
Purpose. Non-albicans Candida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species.
Methodology. Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.
Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C.
tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C.
pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activ- ity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers.
None of the isolates exhibited phospholipase activity.
Conclusion. Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs.
Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.
of fungal infections. Amran et al. [8] reported that 82% of Candida species isolates that were isolated from in-patients with invasive candidiasis at Kuala Lumpur General Hospital were non-albicans Candida species. A total of 40.6% non- albicans Candida species were isolated from patients with candiduria in another tertiary care centre in Malaysia [9].
In Asia, C. tropicalis has been reported as the most common species in patients with candidemia [4]. A comprehensive review of global incidence rates showed that C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei were the five most common species associated with candidemia;
whereas C. dubliniensis, C. famata, C. guilliermondii, C. lusi- taniae, C. pelliculosa and C. rugosa were categorized as newly emerging fungal pathogens [10]. After several typing studies, it was found that C. rugosa has been re-classified into four different species that include C. rugosa, C. pseudorugosa, C.
neorugosa and C. mesorugosa, which are also known collec- tively as the C. rugosa complex [11–13]. Candida rugosa is now addressed as a species complex and reassigned recently to a new genus known as Diutina [14]. While not technically part of the C. rugosa complex, C. pararugosa is closely related and often misidentified as C. rugosa [13].
C. rugosa is considered as an emerging fungal pathogen [15], which is able to trigger invasive candidiasis in immu- nocompromised patients from various geographical regions including Brazil and India [16]. It is able to form biofilms with increased resistance to amphotericin B and fluconazole [17]. A total of 78 C. rugosa isolates were isolated from clinical specimens collected over a 14-year laboratory-based surveil- lance study conducted in a leading tertiary care hospital in Malaysia between 2000 to 2013 [18]. C. pararugosa was also described as an uncommon bloodstream [19] and oral pathogen [20].
In Malaysia, C. rugosa and C. pararugosa are known to be occasional species related to candidiasis and are isolated in a very small percentage. Although both species are uncommon, the enhanced pathogenic nature of C. rugosa and C. para- rugosa attracts the interest of scientists and the medical communities to conduct epidemiological studies on infec- tions in humans and other mammals towards the goal of more efficient antifungal treatment. Moreover, there are a number of extracellular enzymes produced by various Candida species including phospholipase, proteinase, esterase and hemolysin, which play important roles in their virulence capability [21, 22]. These enzymes are able to help Candida species in nutrient acquisition, adhesion and destruction of host cell membrane in order to promote tissue invasion [23, 24].
A better understanding of the characteristics of C. rugosa and C. pararugosa regarding antifungal resistance and their virulence factors are essential for effective treatment and management of patients with candidiasis. Therefore, the aims of this study were to identify the antifungal susceptibility patterns, biofilm characteristics, as well as the phospholipase, proteinase, esterase and haemolysin activities in the C. rugosa complex and C. pararugosa isolates from tertiary teaching hospitals in Malaysia.
METHodS
Clinical isolates
Nine isolates were obtained from two tertiary referral hospital laboratories in Malaysia. All the isolates were isolated from 2007 to 2016 mainly from blood (n=7) and skin (n=2) specimens. All the isolates, designated as Cr2745, Cr2672, Cr2692, Cr3715, Cr3114, Cr2610, Cr25103, Cr2014 and Cr2354, were inoculated on Sabouraud Dextrose agar (SDA) (Oxoid, Hampshire, UK) before further strain confirmation.
As a reference strain, C. rugosa ATCC 10571 was used as a positive control in this investigation. All isolates and reference strains were maintained on SDA media plates at 4 °C and kept as glycerol stock cultures at −80 °C.
Culture identification CHRoMagar Candida
CHROMagar Candida was prepared based on the manufac- turer’s guidelines and incubated at 37 °C for 48 h.
Biochemical tests
Biochemical tests were performed using RapID Yeast Plus System (Remel, USA) by referring to the manufacturer’s guidelines.
Molecular identification
The DNA extraction of Candida isolates were performed according to the method described in the GeneAll DNA extraction kit manual (GeneAll Biotechnology, Korea). The primers used were ITS1 and ITS4, which amplify the internal transcribed spacer regions (ITS) of the ribosomal DNA [25].
The amplicons were sequenced in both directions using the primers ITS1 and ITS4. A consensus sequence was obtained for the forward and reverse sequences for each of the isolates, and then compared to the NCBI GenBank database using the blast tool.
Antifungal susceptibility test method
All the isolates were further characterized for their suscep- tibility patterns towards four different antifungal agents, which were amphotericin B (0.002–32 µg ml−1), caspofungin (0.002–32 µg ml−1), fluconazole (0.016–256 µg ml−1) and vori- conazole (0.002–32 µg ml−1), using the E-test method. These E-tests were incubated at 37 °C for 48 h on lawn cultures of Candida isolates. MICs were determined according to the MIC test strip yeast MTS24 technical sheet (Liofilchem, Italy).
The tests were performed in duplicates for each isolate and the mean was calculated and reported.
Biofilm activity
Biofilm activity was investigated to identify the ability of C. rugosa complex and C. pararugosa to form biofilms at different time points. This protocol was derived from a previ- ously described work by Pierce et al. [26, 27], with minor modifications.
Inoculum preparation
A loopful of colony was inoculated into a falcon tube containing 20 ml of yeast extract peptone dextrose broth (YDP) (Becton, Dickinson, France) liquid medium and incubated overnight in an orbital shaker (180 r.p.m.) at 37
°C. The overnight suspension cultures were centrifuged at 3000 r.p.m. for 5 min at 4 °C. The supernatant was removed and washed twice in 20 ml sterile ice-cold PBS buffer. The final cell pellets were re-suspended in approximately 20 ml of the RPMI-1640 that has been pre-warmed to 37 °C. Cell suspension containing 1×106 cells ml−1 were quantified using a spectrophotometer (Genesys, Thermo Scientific).
Biofilm formation
From the standardized inoculum, 100 µl suspension aliquots were pipetted into selected wells of 96-well plates. Each isolate was tested in triplicates with one set of broth controls for 1.5, 6, 18, 24, 48 and 72 h. Once all the selected wells were seeded, the 96-well plates were sealed with parafilm and incubated at 37 °C. The medium was aspirated without touching and disrupting the walls of the 96-well plates. The wells were washed three times with sterile PBS in order to eliminate planktonic or non-adherent cells from the wells. Plates were drained in an inverted position to remove the residual PBS.
Bioactivity assay
Then, 100 µl of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)−2H- tetrazolium-5-carboxanilide (XTT)/menadione solution was added to each well and covered with aluminium foil as XTT is a light sensitive solution. The plates were incubated for 3 h at 37 °C. After incubation, 80 µl of the solution was transferred into new 96-well plates and an OD reading was obtained using a microplate reader (Epoch 2, Bio Tek Instruments) at 490 nm.
Biomass assay
Initial steps were the same as for the biofilm formation described above. Once washed with PBS buffer, 125 µl of 0.1%
crystal violet solution was added to the wells and incubated at room temperature for 15 min. Plates were washed three times with PBS buffer, then left overnight to dry at room temperature. Subsequently, 125 µl of 30% acetic acid was added in order to solubilize the crystal violet attached to the wells. Once added, the plates were incubated for 15 min and then 125 µl of the solubilized crystal violet was transferred into new 96-well plates, followed by quantification using a microplate reader at 550 nm [28].
Assay for extracellular enzymatic activity
Extracellular phospholipase and proteinase activities were performed according to the techniques described by Kumar et al. [29], with minor modifications. Haemolysin and esterase activities were screened based on the method described by Fatahinia et al. [22] with minor modifications. C. albicans ATCC 10231 were used as a positive control in this part of the study. Standard Pz value was determined by dividing the diameter of the colony by the total diameter of the colony plus
the zone of enzymatic activity. All Pz values were measured in millimetre (mm).
Inoculum preparation
A single colony of the Candida species was inoculated in 10 ml of yeast, peptone, dextrose (YPD) broth and incubated at 37°C for 18 h. After the incubation period, cells were harvested by centrifuging at 3000 r.p.m. for 5 min. Then, the supernatant was discarded and the pellets were first washed with sterile distilled water and centrifuged at the same speed.
Later, the pellets were washed again with normal sterile saline and centrifuged to eliminate any remaining residual media.
Cell suspensions containing 1×106 cells were obtained, which is equivalent to a 1.25 OD reading at A550.
Phospholipase assay
Isolates were screened for phospholipase activity using egg yolk agar medium. Upon preparing the egg yolk agar medium, 2 µl of the Candida cells suspension was inoculated in triplicates at equidistant points onto the egg yolk medium surface and incubated at 37 °C for 4 days. The diameter of the colony plus the precipitation zone were measured.
Proteinase production
Isolates were screened for proteinase activity using bovine serum albumin (BSA) agar medium. Then, 2 µl of Candida cell suspension was inoculated in triplicates at equidistant points onto BSA medium surface and incubated at 37 °C for 4 days. The diameter of the colony plus the precipitation zone were measured.
Haemolysin assay
For this assay, SDA was prepared with supplementation of 3%
(w/w) glucose and 7% (v/w) sheep blood. Then, 2 µl of the Candida cell with suspension was inoculated in triplicates at equidistant points onto the prepared media and incubated at 37 °C in 5% CO2 for 48 h. The appearance of a greenish-black ring around each colony was considered as incomplete lysis (alpha, α) while a clear ring of lysis around the colony was considered as complete lysis (beta, β).
Esterase assay
Isolates were screened for esterase activity using tween 80 opacity agar medium. Then, 2 µl of the Candida cell suspen- sion was inoculated in triplicates at equidistant points onto tween 80 opacity test medium and incubated at 30°C for 10 days with daily recording of the colony diameter and zone of precipitation.
Statistical analysis and enzymatic score
Statistical analysis was completed using SPSS software version 16.0. One-way ANOVA and Tukey’s multiple comparison test were used to compare the bioactivity and biomass production among all Candida isolates. A P-value of <0.05 was considered statistically significant. As for the biofilm study, the isolates were grouped into three categories: isolates with biofilm capabilities below the reference strain, isolates with biofilm
capabilities similar to the reference strain and isolates with biofilm capabilities above the reference strain.
As described in previous research by Vinodhini et al. [30], enzymatic activities for phospholipase, proteinase and esterase activity were measured as Pz values (colony diameter/
diameter of the colony plus the precipitation zone). Low Pz value indicates high activity whereas high Pz value indicates low activity. The scores for phospholipase and proteinase were divided into four different ranges: Pz value of 1.0 refers to no activity (−); Pz value of between 0.999 to 0.700 refers to low activity (+); Pz value between 0.699 to 0.400 refers to moderate activity (++); and Pz value between 0.399 to 0.100 refers to high activity (+++). As for esterase activity, the enzymatic scores are divided into five different ranges: Pz value of 1.0 refers to no activity (−), Pz value between 0.90–0.99 refers to weak activity (+), Pz value between 0.80–0.89 refers to mild activity (++), Pz value between 0.70–0.79 refers to strong activity (+++) and Pz value <0.69 refers to very strong activity (++++).
RESuLTS
In this study, primary identification was performed using CHROMagar Candida medium, on which one isolate yielded purple-pinkish colonies, one isolate yielded pale violet colonies, and the remaining seven isolates including the C. rugosa reference strain yielded brilliant blue colo- nies. On this basis, about 77.7% of the isolates resembled C.
rugosa. C. rugosa appeared as small colonies of brilliant blue with pale border colonies (Fig. 1a, b) while C. pararugosa (Fig. 1c) appeared as pale violet colonies on CHROMagar Candida medium. Further species identification was performed using RapID Yeast Plus System for biochemical analysis in which six isolates were identified as C. rugosa, two could not be reliably identified and one isolate was identified as C. guilliermondii.
The ITS sequence blast search results using GenBank showed the presence of the C. rugosa complex, from which six isolates had the closest match and corresponded to the
sequence of C. mesorugosa. Based on the ITS region sequence analysis, isolates Cr2745, Cr2672, Cr2692, Cr3715, Cr3714 and Cr2610 were identified as C. mesorugosa with 100%
similarity (GenBank accession number HM641831); isolate Cr25103 was identified as C. rugosa with 100% similarity (GenBank accession number HM641832); Cr2354 was iden- tified as C. guilliermondii with 100% similarity (GenBank accession number MG367466); and Cr2014 was identified as C. pararugosa with 100% similarity (GenBank accession number JN675331). Six of these C. mesorugosa isolates, one C.
rugosa isolate and the C. pararugosa isolate were further char- acterized based on their antifungal susceptibilities, biofilm- producing abilities and extracellular enzymatic (proteinase, phospholipase, esterase and haemolysin) activities.
The MICs for the C. rugosa complex ranged from 1.8 to 2 µg ml−1 for amphotericin B, whereas the C. pararugosa isolate had an MIC of 0.75 µg ml−1. As for caspofungin, MICs for the C. rugosa complex ranged from 2 to 5 µg ml−1, whereas for C.
pararugosa it was at 0.5 µg ml−1. As for fluconazole, the MICs for the C. rugosa complex ranged from 7 to 48 µg ml−1 and C.
pararugosa was at 8 µg ml−1. As for voriconazole, the MICs of for the C. rugosa complex ranged from 0.125 to 0.5 µg ml−1 and C. pararugosa was at 0.19 µg ml−1. These results are presented as means of duplicates in Table 1. Currently, the antifungal susceptibility reference breakpoints for the C. rugosa complex and C. pararugosa have yet to be defined. E-test antifungal tests showed both these species have elevated MICs compared to C. albicans and C. tropicalis, but the clinical significance of the higher MICs are not known.
Investigation of virulence factors by screening for biofilm production for all the isolates using XTT reduction assay and crystal violet assay methods was performed. Biofilms were formed by growing the isolates on polystyrene 96-well microtitre plates using crystal violet (CV) and tetrazolium (XTT) reduction assay to quantify the formed biofilms. Time point reading was done on all isolates post-incubation at 1.5, 6, 18, 24, 48 and 72 h.
Based on the XTT assay analysis, the ATCC reference strain, C.
rugosa ATCC 10571 showed the highest bioactivity level at all
Fig. 1. (a) Growth of Cr2745 (C. mesorugosa), (b) growth of Cr25103 (C. rugosa) and (c) growth of Cr2014 (C. pararugosa) on CHROMagar Candida incubated at 37 °C for 48 h.
time points of incubation except at 6 h when the highest level of bioactivity was observed for C. pararugosa (Cr2014).
The optimum time point for Cr10571 was 24 h with an activity of 0.075, followed by C. pararugosa at 48 h with an activity of 0.048. Most of the isolates had optimum bioactivity at 48 h. The second highest bioactivity level was observed for
strain Cr25103 with an activity of 0.046 at 24 h. Overall, the isolates appear to have reduced biofilm-producing capabili- ties compared to the reference strain, C. rugosa ATCC 10571.
These results are displayed in Fig. 2.
Isolates Cr10571, Cr 2610, Cr3715, Cr3114, Cr2014 and Cr25103 showed optimum biomass production at 18 h (Fig.
Table 1. E-test results, proteinase, esterase and haemolysin activities for the C. rugosa complex and C. pararugosa isolates
Strain ID MIC (µg ml−1) Proteinase activity
Pz value (mean±sem) (activity ratings)
Esterase activity Pz value (mean±sem) (activity ratings)
Haemolysin activity (type of haemolytic
activity)
VO FL AMB CAS
C. parapsilosis ATCC
22019 0.048 2 0.50 2 na na na
C. rugosa ATCC 10571 0.064 3.5 0.75 0.75 0.24±0.01 (+++) 1.0 (-) Beta haemolytic
C. mesorugosa (Cr2745) 0.5 48 2 5 0.33±0.01(+++) 0.62±0.01 (++++) Alpha haemolytic
C. mesorugosa (Cr2672) 0.5 32 1.8 2.3 0.27±0.01(+++) 0.63±0.02 (++++) Alpha haemolytic
C. mesorugosa (Cr2692) 0.5 48 2 2 0.26±0.01(+++) 0.62±0.01 (++++) Alpha haemolytic
C. mesorugosa (Cr3715) 0.5 40 2 3 0.26±0.01(+++) 0.63±0.01 (++++) Alpha haemolytic
C. mesorugosa (Cr3114) 0.5 48 2 3.5 0.29±0.01(+++) 0.62±0.01 (++++) Alpha haemolytic
C. mesorugosa (Cr2610) 0.5 40 2 4 0.25±0.01(+++) 0.63±0.01 (++++) Alpha haemolytic
C. rugosa (Cr25103) 0.125 7 2 3.5 0.30±0.01(+++) 1.0 (−) Alpha haemolytic
C. pararugosa (Cr2014) 0.19 8 0.75 0.5 0.27±0.01(+++) 1.0 (−) Alpha haemolytic
The MIC shown is the mean of duplicates.
(−), no activity;(++++), very strong activity;(+++), high/strong activity;(++), moderate/mild activity;(+), low/weak activity;AMB, Amphotericin B;CAS, Caspofungin;FL, Fluconazole;VO, Voriconazole;na, Not available.
Fig. 2. Bioactivity of C. rugosa and C. pararugosa isolates derived from XTT assay. The higher the bioactivity, this indicates the higher the biofilm production. Measurements were taken at selected time points of 1.5, 6, 18, 25, 48 and 72 h. Error bars represent the sem. P<0.05 for comparison of values among the isolates and different superscript alphabets above the bar indicate significance at P<0.05.
3). C. rugosa (Cr25103) had the highest biomass production followed by C. pararugosa (Cr2014) with an activity of 0.206 at 18 h post-incubation. Fig. 3 shows biomass production of the C. rugosa and C. pararugosa isolates. Overall for biofilm activity measured based on biomass production of the isolates, our results showed that all the clinical isolates have biofilm-forming capabilities, which were higher than that of the reference strain at 24 h.
Virulence is also enhanced by production of extracellular enzymatic activity in Candida species. Therefore, all the clinical isolates were screened for four different extracellular enzymatic activity which are commonly present and play a significant role in the pathogenesis of other Candida species.
Secretion of the extracellular enzymes were grouped based on the enzymatic activity scored categories. All isolates including reference strain exhibited low Pz values between 0.399 and 0.100, and high proteinase activity. All C. rugosa isolates exhibited a considerable amount of proteinase activity with Pz +++ scores. None of the isolates in our study showed any phospholipase activity (Pz values=1.0). The detailed results are expressed in Table 1.
Out of eight isolates, esterase activity was determined only in six C. mesorugosa isolates, which showed 75% of all isolates exhibiting Pz values of <0.69. All the C. mesorugosa isolates showed esterase activity on the fourth day of the 10-day incu- bation period. No esterase activity was detected in C. rugosa (Cr25103) and C. pararugosa (Cr2014) isolates. However, none of these isolates showed phospholipase activity (Pz=1.0).
All isolates including reference strain, C. rugosa ATCC 10571 showed alpha haemolytic activity. Overall, the extracellular enzymatic activities in all the isolates showed that proteinase
and haemolysin enzymes were produced. All isolates exhib- ited haemolysin followed by esterase production in 67% of isolates used in this study. The results for esterase and haemo- lytic activities are expressed in Table 1.
dISCuSSIon
Characterization of Candida species is extremely important due to the rising number of non-albicans Candida species [31], which limits the therapeutic options [32, 33]. Candida is the most important genus of yeast species in causing life- threatening fungal infections, mainly in immunocompro- mised patients. In fact, C. rugosa have been associated with immunocompromised patients [29].
In Malaysia, C. rugosa is known to be an uncommon species related to candidiasis. Madhavan et al. reported six clinical isolates obtained from patients in local hospitals between 2004 and 2009 [34]. On the other hand, Tay and co-workers reported six clinical strains of the C. rugosa complex isolated from a local medical care centre between 2005 and 2007 [17]. Following that, Ng et al. also reported that there were 78 strains of the C. rugosa complex isolated from various body sites in the period spanning from 2000 to 2013 [18]. However, the source of C. rugosa infection still remains unknown.
In this study, six isolates of C. mesorugosa and one isolate of C.
rugosa were identified, mainly from blood. This indicates that C. rugosa complex species present in this study are most likely associated with candidemia infections. The C. rugosa complex had been previously reported to be associated with candi- daemia cases [17, 35]. To the best of our knowledge, the C.
rugosa complex, including C. mesorugosa and C. pararugosa
Fig. 3. Biomass production of C. rugosa and C. pararugosa isolates based on the incubation time (h). Error bars represent the sem. P<0.05 for comparison of values among the isolates and different superscript alphabets above the bar indicate significance at P<0.05.
associated with candidiasis has not been reported previously in Malaysia although this may be due to their misidentifica- tion as C. rugosa isolates. This is the first study reporting on the C. rugosa complex and C. pararugosa isolate along with characterization of their antifungal susceptibility patterns and virulence factors. C. pararugosa was previously reported to be isolated from dairy products, which could be the possible route of transmission in humans [20].
Moreover, C. pararugosa was not able to be identified using the biochemical test kit used (RapID Yeast Plus) because this particular species is not listed as a detectable entity by the manufacturer and it was reported that this species was previ- ously misidentified as C. rugosa using phenotypic methods [17]. Therefore, molecular identification using internal tran- scribed spacer (ITS) region sequence analysis was applied in this study for more reliable identification of the uncommon species that could not be correctly identified using phenotypic identification technique [36, 37]. Accurate identification of pathogenic Candida species will allow provision of efficient antifungal stewardship and treatment management.
Susceptibility of isolates of C. rugosa to voriconazole had been reported previously [4, 16, 38, 39]. Although fluconazole is widely used for treating candidiasis, decreased susceptibili- ties of C. rugosa isolates to fluconazole have been reported [4, 16, 17, 39, 40]. Amphotericin B is considered an active anti- fungal agent against non-albicans Candida species including C. glabrata, C. parapsilosis, C. tropicalis [8, 9], C. krusei [2, 41]
and C. rugosa [4, 42, 43]. However, the existence of C. rugosa isolates with decreased susceptibility to amphotericin B has been previously reported [17, 40]. Although caspofungin resistance is rare among Candida species, the existence of caspofungin resistance among non-albicans species including C. parapsilosis and C. tropicalis has been reported in Malaysia [8, 44]. In such cases, the selection of an antifungal drug for candidiasis treatment can be decided based on the accurate species identification, patients’ clinical background including site of infection and in vitro antifungal susceptibility patterns of the particular Candida species [13, 45].
Concerning virulence, we studied the production of proteinase, phospholipase, esterase and haemolysin enzymes in the C. rugosa complex and C. pararugosa as these enzymes are considered to be crucial in Candida’s pathogenesis.
Overall, in our study, most of the blood isolates are able to produce proteinase, esterase and haemolysin activity. In this study, isolates of the C. rugosa complex and C. pararugosa were capable of producing high levels of proteinase. Enzyme production level might be influenced by species, origin of isolates, host cell involvement, site of infection and type of infection [46].
In this study, most of the clinical isolates showed very high esterase activity and 100% of all the isolates were alpha- haemolytic producers. Haemolysin activity is a known viru- lence factor of Candida species, which enables the fungus to utilize iron derived from haemoglobin through the lysis of erythrocytes. C. rugosa has previously been shown to be capable of producing alpha-haemolytic activity [47]. This
study confirms that the C. rugosa complex has the ability to utilize haemolysin to gain access to haemoglobin as an iron source.
Phospholipase is an important virulence factor for C. albicans [48], which leads to the hydrolysis of phospholipids in human cell membranes. The absence of phospholipase in the C. rugosa complex suggests that this species may be less virulent than C.
albicans species, which possesses remarkable phospholipase activity. Information on extracellular enzyme activity of C.
albicans is widely available in previous reviews compared to non-albicans Candida species, especially uncommon species such as the C. rugosa complex, which is very limited. Data obtained from the current study will contribute to the body of information on the C. rugosa complex and C. pararugosa enzymatic activity virulence factors. Overall, the present study showed higher secretion of esterase and differential alpha- and beta-haemolysin among the clinically isolated C. rugosa complex and C. pararugosa in comparison to the ATCC strain. We postulate that these factors may contribute towards the ability of the C. rugosa complex and C. pararu- gosa strains to cause candidiasis in humans, albeit to a lesser extent than the other more predominant species including C.
albicans, C. parapsilosis and C. tropicalis.
We also investigated biofilm production as it is considered an important virulence capability of Candida species that enables the yeast to colonize host tissues as well as abiotic surfaces [49]. Biofilm formation could augment the resistance of the fungus to various antifungal agents used in candidiasis treat- ment [50, 51]. This is due to the complex biofilm structure and extracellular matrix components, which could prevent or reduce the penetration of the antifungal drugs. Previous studies on the C. rugosa complex and C. pararugosa biofilm formation are limited and results in difficulty to compare and evaluate this phenotype in both species. In this study, we found that the C. rugosa complex and C. pararugosa isolates showed high biomass production, which when coupled with the XTT assay results, suggest that they possess a reasonable amount of biofilm-forming capability.
Hydrolytic enzymes such as secretory aspartyl proteinases, phospholipases and adherence of Candida species to host tissues have been regarded as the major determinants of the pathogenicity of C. albicans. Investigation in a previous study showed that there was no obvious association of biofilm production with proteinase activity among the C.
rugosa isolates [17], although it was suggested that aspartyl proteinase production might be enhanced during the biofilm formation of C. albicans [52].
ConCLuSIon
In conclusion, both the C. rugosa complex and C. pararu- gosa were capable of producing virulence factors such as proteinase, esterase and haemolysin enzymes and biofilm.
Further investigations are required to elucidate the trend of antifungal susceptibility and virulence factors contributing to the pathogenecity in both the species. The use of appropriate
antifungal agents is critical for management of the infection especially among the immunocompromised individuals.
Thus, a routine antifungal susceptibility test should be carried out for all types of candidiasis in order to trace the antifungal resistance among isolates of Candida. Additionally, assessment on factors influencing the extracellular enzymes production and biofilm formation that lead to establishment of infections caused by the C. rugosa complex and C. para- rugosa is warranted for better understanding their virulence mechanisms.
Funding information
This work received no specific grant from any funding agency.
Acknowledgements
This study was supported by Ministry of Education Malaysia (FRGS/2/2014/SKK01/TAYLOR/02/1) and Taylor’s University (TU) for funding and technical support.
Conflict of interest
The authors declare that there are no conflicts of interest.
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