Mycopathologia (2014) 178:207–215 DOI 10.1007/s11046-014-9802-0
Sub-inhibitory Concentrations of Antifungals Suppress Hemolysin Activity of Oral Candida albicans and Candida tropicalis Isolates from HIV-Infected Individuals Sukumaran Anil • Mohamed Hashem • Sajith Vellappally • Shankargouda Patil • H. M. H. N. Bandara • L. P. Samaranayake
Received: 3 April 2014 / Accepted: 9 August 2014 / Published online: 21 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Secretion of hydrolytic enzymes such as hemolysin is considered an important virulence attribute of the opportunistic pathogenic fungus Candida. It is known that Candida spp. isolated from HIV-infected patients produce copious hemolysins. As common antifungal agents may perturb the production of extracellular enzymes, we evaluated the effect of three antifungals nystatin, amphotericin B and fluconazole on the hemolytic activity of Candida albicans and Candida tropicalis isolates from HIV-infected individuals. The impact of antimycotics on hemolytic activity was assessed by a previously described in vitro plate assay, after exposing ten isolates each of C.
albicans and C. tropicalis recovered from HIVinfected individuals to sub-minimum inhibitory concentrations (sub-MIC) of nystatin, amphotericin B and fluconazole. All Candida isolates showed a significant reduction in hemolytic activity. The reduction was highest for amphotericin B-exposed C. albicans and C. tropicalis followed by nystatin and fluconazole. The effect of antimycotics was more pronounced on the hemolytic activity of C. tropicalis compared to that of C. albicans. Commonly used antifungal agents significantly suppress hemolysin activity of Candida species. This implies that the antifungals, in addition to their lethality, may modulate key virulence attributes
S. Anil Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia e-mail: [email protected]
H. M. H. N. Bandara College of Pharmacy, The University of Texas at Austin, Austin, TX, USA e-mail: [email protected]
M. Hashem S. Vellappally Dental Health Department, Dental Biomaterials Research Chair, College of Applied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia e-mail: [email protected]
L. P. Samaranayake (&) School of Dentistry, University of Queensland, Brisbane, Australia e-mail: [email protected]
S. Vellappally e-mail: [email protected] S. Patil Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, M. S. Ramaiah University of Applied Sciences, Bangalore, Karnataka, India e-mail: [email protected]
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of the yeast. The clinical relevance of this phenomenon in HIV disease and other similar pathologies remains to be determined. Keywords Candida Antifungal agents Virulence attributes Hemolysin HIV infection Nystatin
Introduction Candida albicans is the commonest dimorphic fungus isolated from the human oral cavity, both in health and disease [1]. However, the prevalence and incidence of disease due to other less common species of Candida including C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. guillermondii, C. lusitaniae, C. kefyr and C. dubliniensis are on the rise [2]. When the host status is compromised, these opportunistic fungi can cause mild to severe infections, in susceptible hosts. Individuals who are, on immunosuppressives, cytotoxics and/or broad spectrum antibiotic therapy, those with HIV disease and diabetes or indwelling medical devices are all susceptible to candidal infections [3, 4]. Interestingly, some studies have shown that despite acute or chronic candidal infection, approximately 30–50 % of individuals do not experience any symptoms of candidiasis [5, 6]. HIV-infected individuals are particularly at risk of candidiasis. It is estimated that approximately 90 % of those with HIV disease and AIDS suffer from oral mucosal candidiasis at least once during the course of the HIV infection [6–8]. Oropharyngeal candidiasis, which is the most frequent fungal infection in HIV patients, is also considered as a marker/predictor for HIV progression [9–16]. As mentioned above, C. albicans is the commonest causative agent; however, other non-albicans species such as C. glabrata, C. tropicalis and C. krusei frequently cause oropharyngeal candidiasis in the latter cohorts [17, 18]. The conventional view that the severity and chronicity of oral candidiasis in HIV infection is mainly due to the virus-induced immune deficiency has been recently challenged as more information on the role of the causative organism and its invasive arsenal, such as hydrolytic enzymes, has come to light. Indeed, it has been suggested that Candida strains with altered virulent determinants that prefer
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an immunocompromised niche is likely to be selected in HIV-infected patients [16, 19–22]. Thus, the virulence determinants of the organism itself appear to play a significant role in the etiopathology of oropharyngeal candidiasis in HIV disease [23]. The principle virulent determinants of Candida include tissue-degrading hydrolytic exoenzymes such as phospholipases and proteinases, morphologic dimorphism (blastospore–mycelial transformation) and phenotypic switching. These are known to help the fungus adhere to host epithelia, to resist both the antifungals as well as the fungicidal activity of neutrophils [24–29]. The exoenzymes secreted by Candida such as proteinases, phospholipases, esterases, phosphatases and hemolysins are known to mediate host invasion, particularly by facilitating hyphal invasion of the epithelium [30–33]. Hemolysin production and hemolytic activity has been shown to be significantly higher in C. albicans than in non-albicans species such as C. tropicalis, C. glabrata, C. krusei, C. parapsilosis and C. orthopsilosis [34, 35]. Some workers have also noted that the expression of hemolysins and hemolytic activity in Candida isolates from HIV-infected patients is greater compared with isolates from healthy individuals [23, 36]. In another study, Samaranayake et al. [37] reported a positive correlation between germ tube formation and hemolysin production in Candida isolated from HIV-infected individuals. Apart from the foregoing reports, hemolysin activity of Candida from HIV-infected individuals has received scant attention. Rossoni et al. [38] found that most nonalbicans Candida species had a similar ability to produce hemolysins when compared to C. albicans isolates from HIV-infected individuals. However, Ramesh et al. [39] observed a higher amount of hemolysin production by C. albicans compared to C. glabrata. Despite the extensive use of antifungal agents, their effect on candidal virulence determinants has been little studied. A number of workers have investigated the phenotypic switching of candida in response to antimycotics [40–42]. In fact, as mentioned above, HIV-infected individuals carry different phenotypes of Candida and exhibit greater hemolytic activity. Yet, the effect of antimycotics on hemolysin activity of Candida isolates from HIV-infected patients has not been investigated. Thus, the aim of this study was to investigate the
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effect of transient exposure of C. albicans and C. tropicalis isolates from HIV-infected individuals to sub-therapeutic concentrations of commonly used antifungals belonging to the polyene class (nystatin, amphotericin B) and an azole, fluconazole.
Materials and Methods Antifungal Agents Reagent grade nystatin and amphotericin B were purchased from Sigma (Sigma Chemicals Co. St.Louis, USA) and dissolved in dimethylsulfoxide (DMSO) and absolute ethanol (3:2 ratio), respectively. Fluconazole (Pfizer Inc. New York, USA) was dissolved in absolute methanol. All 3 antifungals were prepared initially as 10 mg/ml stock solutions and stored at -20 °C until used. Sample Collection A total of 10 oral isolate each of C. albicans and C. tropicalis from HIV-infected subjects attending the Queen Elizabeth Hospital, Hong Kong, were obtained by the oral rinse technique [43]. All subjects manifested oral candidiasis when the samples were collected. Briefly, the patients were instructed to rinse the mouth for 60 s with 10 ml of sterile phosphatebuffered saline (0.01 M, pH 7.2). Oral rinses were collected in sterile containers and transported to the laboratory. Identification of Candida and Storage Fifty microliters of oral rinse was inoculated on Sabouraud dextrose agar (SDA) using a spiral plater and incubated for 48 h at 37 °C. At the end of incubation, isolated colonies were subcultured for further purification and identity was confirmed using the germ tube test and the commercially available API-20C AUX (bioMe´rieux, Basingstoke, UK) [44]. All isolates were stored in multiple aliquots at -20 °C, after confirming their purity. One strain each of Streptococcus pyogenes (Lancefield group A) and Streptococcus sanguis that induces beta and alpha hemolysins, respectively, were used as positive controls.
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Candidal Inocula Prior to each experiment, Candida species were subcultured on SDA, for 18 h at 37 °C. A loopful of this overnight growth was inoculated into Yeast nitrogen base (Difco Laboratories, Detroit, Michigan) medium containing 2 % Dextrose and incubated for 18 h in an orbital shaker (75 r.p.m.) at 37 °C. The resultant growth was harvested, washed twice in PBS and resuspended in PBS. Concentrations of Candida species were adjusted to desired values by spectrophotometry and were confirmed by hemocytometric counting. Minimum Inhibitory Concentration (MIC) Determination Broth Micro Dilution Method Antifungal susceptibility to nystatin (MIC) was determined by a broth microdilution assay in accordance with the CLSI guidelines [45]. Briefly, fungal cell suspensions (1 9 103 Cells/ml) were prepared as mentioned above in RPMI 1640 supplemented with 0.165M 3-(Nmorpholino) propanesulfonic acid (MOPS). Candida were treated with nystatin in a concentration gradient and incubated in a 96-well microtiter plate for 24 h at 35 °C. At the end of the incubation, the optical density of the fungal growth was measured by a spectrophotometer at 595 nm. The lowest concentration of the antifungal at which the Candida demonstrates 80 % of visible growth inhibition compared to the solvent control was considered as the MIC80 of the antifungal against Candida. The assay was performed quadruplicates on three separate occasions. E-Test The MIC determinations of amphotericin B and fluconazole were performed using the E-test. The E-test (AB BIODISK, Solna, Sweden) is a patented commercial method for the quantitative determination of MICs of antimicrobial drugs. Comparisons of the E-test method with the CLSI broth dilution method have demonstrated high levels of agreement [45]. The inocula of Candida species were prepared as mentioned above. Candida cell concentrations corresponding to McFarland 0.5 at 520 nm were prepared in sterile distilled water. Candida suspensions were
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inoculated on to RPMI-1640 (American Biorganics, Buffalo, NY) supplemented with 1.5 % agar and 2 % glucose and buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma, St. Louis, Mo.) using a sterile swab by streaking it across the entire surface of the agar in three directions. The plates were dried at room temperature for 15 min and the E-test strips were applied. The agar plates were incubated for 24 h at 35 °C. The drug concentration at which the border of the elliptical inhibition zone intersected the scale on the antifungal test strip was considered as the MIC of the respective antifungals. Exposure to Antifungal Agents Yeast cells, maintained on Sabouraud’s dextrose agar (SDA), were inoculated onto fresh plates and incubated overnight for 24 h prior to use. The organisms were harvested and a cell suspension prepared in sterile phosphate-buffered saline (PBS) of pH 7.4, at 520 nm corresponding to an optical density 0.5 McFarland standard. From this cell suspension, 1 ml was added to tubes containing 4 ml of RPMI broth (control) and 4 ml of RPMI/drug solution (test) in which the drug concentrations were twice the MIC. This gave a cell suspension of 106–107 cells/ml in each assay tube. The tubes were then incubated at 37 °C for a period of 1 h in a rotary incubator. Following this limited exposure, the drugs were removed by two cycles of dilution with sterile PBS and centrifugation for 10 min at 3,0009g. Afterward the supernatant was completely decanted and the pellets were resuspended in 5 ml of sterile PBS. This washing procedure was then repeated and the pellets resuspended in 2.5 ml of sterile PBS. It has been found by previous investigators that removal of 90 % of the supernatant with two washings reduces antimicrobial concentration 100-fold, while complete decanting of the supernatant with two washings (as carried out in the current study) reduces the concentration 10,000-fold [46, 47]. Hence, this method virtually eliminates any ‘‘carryover effect’’ of the drug following its removal. Viable counts of the control and test were done by spiral plating after drug removal and control suspensions were reconstituted as needed to obtain a cell concentration comparable to the test. CFU Assay A portion of the resultant suspension was gently vortexed for 1 min to disrupt the
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aggregates, serially diluted and inoculated using a spiral plater onto SDA. The resultant colony-forming units/ml of Candida were quantified after 48 h incubation at 37 °C. Each assay was carried out quadruplicate on two different occasions. Remaining candidal suspensions were reconstituted as needed to obtain a standard cell concentration (1 9 108 cells/ ml). Determination of Hemolysin Activity Hemolysin production was evaluated using a modification of the plate assay described by Luo et al. [35]. Briefly, 10 ll of aforementioned antifungal treated and control suspensions were spot-inoculated on to blood agar supplemented with 3 % glucose (w/v, pH 5.6 ± 0.2) so as to yield a circular inoculation site of about 5 mm in diameter. The plates were incubated for 48 h (5 % CO2, 37 °C). The presence of a distinct translucent halo around the inoculum site, viewed with transmitted light, indicated a positive hemolytic activity. The diameters of the zone of lysis and the colony were measured using a computerized image analysis system (Quantimet 500 Qwin, Leica, Cambridge, UK) [48]. The hemolytic index was determined using the ratio between the diameters of translucent halo and the colony to represent the hemolysin production intensity by different Candida species. The assay was conducted in quadruplicate on two different occasions for each Candida isolate. Statistical Analysis The intra-and interspecies differences of the hemolytic activity were analyzed by ANOVA. Multiple comparisons between isolates exposed to the three drugs and control were analyzed using the Dunnet’s multiple comparison test (InStat, GraphPad Software Inc., San Diego, California, USA).
Results Effect of Antifungal Solvents on C. albicans and C. tropicalis Since the antifungal agents were dissolved in absolute methanol and DMSO/absolute ethanol, equivalent
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amounts of the latter chemicals were tested to ascertain their effect on the isolates tested. These chemicals had no significant effect on the survival/ growth of Candida when compared with the controls, evaluated at identical concentrations. Minimum Inhibitory Concentrations (MIC) The MICs of nystatin, amphotericin B and fluconazole values for the ten isolates of C. albicans were 0.78–1.56, 0.19–0.38 and 0.12–0.38 lg/ml, respectively, whereas for the ten isolates of C. tropicalis, it was 0.78, 0.25–0.38 and 0.25–0.50 lg/ml, respectively. Impact of Hemolytic Activity of Candida Isolates Exposed to Antimycotics
Fig. 1 The hemolysin activity of control and drug exposed isolates C. albicans (C control, N nystatin, A amphotericin B, F fluconazole)
Two different types of haemolysis could be observed circumscribing the yeast ‘‘colony’’ when viewed with transmitted light after 48 h of incubation. The first was a totally translucent ring identical to b haemolysis produced by the control strain of b hemolytic Streptococcus, whereas the second was a greenish-black halo similar to a-haemolysis observed with the control strain of Staphylococcus. Hence, the terms ‘‘a haemolysis’’ and ‘‘b haemolysis’’ were used as descriptive terms to indicate incomplete and complete haemolysis, respectively, associated with the Candida strains tested [35]. In order to obtain quantitative data, we attempted to measure the diameter of hemolytic zones relative to the inoculum size using an image analysis system. However, clear-cut zones of haemolysis were perceptible only in relation to b hemolytic activity. The margins of a haemolysis were rather diffuse. Hence, the diameter of the latter zones was not accurately quantifiable either by naked eye estimation or the
image analysis system. The mean hemolytic activity of the control C. albicans isolates was 1.49 ± 0.07. Brief exposure to nystatin, amphotericin B and fluconazole significantly reduced the hemolytic activity with a mean value of 1.34 ± 0.06 (P \ 0.001), 1.33 ± 0.06 (P \ 0.001) and 1.41 ± 0.06, respectively (P \ 0.001) (Table 1; Fig. 1). The mean hemolysin activity of the control C. tropicalis isolates was 1.45 ± 0.01. Similar to C. albicans, brief exposure to nystatin, amphotericin B and fluconazole significantly reduced the hemolytic activity of C. tropicalis with mean values of 1.31 ± 0.06 (P \ 0.001), 1.30 ± 0.06 (P \ 0.001) and 1.39 ± 0.06 (P \ 0.001), respectively, compared to the respective controls (Table 1; Fig. 2). Candida albicans isolates appeared to produce more hemolysin than C. tropicalis; however, these findings were not significant (Table 1; Fig. 3).
Table 1 Intra- and interspecies variation in hemolysin activity of C. albicans and C. tropicalis exposed to nystatin (NYS), amphotericin B (AMB) and fluconazole (FLU) Isolates
No
Control
NYS
AMB
FLU
C. albicans
10
1.49 ± 0.07
1.34 ± 0.06*
1.33 ± 0.06*
1.41 ± 0.06*
C. tropicalis
10
1.45 ± 0.01
1.31 ± 0.06*
1.30 ± 0.06*
1.39 ± 0.06*
P-level
–
NS
NS
NS
NS
Mean of two experiments, each conducted in quadruplicate * P \ 0.001
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Fig. 2 The hemolysin activity of control and drug exposed isolates C. tropicalis (C control, N nystatin, A amphotericin B, F fluconazole)
Fig. 3 The hemolysin activity of ten isolates of C. albicans and C. tropicalis exposed to half MIC of nystatin (NYS), amphotericin B (AMB) and fluconazole (FLU)
Discussion The ability of pathogenic organisms to acquire iron in the mammalian host has been shown to be of critical importance in establishing an infection [49, 50]. In humans, most of the iron is located intracellularly as ferritin or as heme-containing compounds. The small amount of extracellular iron is attached to iron-binding and transport proteins, transferrin and lactoferrin. Since there is essentially no free iron in the human
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body, pathogens must acquire it from one or more iron-containing compounds [50]. The ability to utilize hemoglobin as an iron source is described as a strategy of C. albicans to exploit elemental iron from the host. For instance, C. albicans hyphae are able to rosette erythrocytes via complement receptor like molecules [51] and releases a cell surface bound mannoprotein hemolytic factor, hemolysin to acquire iron from erythrocytes [52, 53]. Clearly, therefore, the colonization and proliferation of Candida are possible only if sufficient iron is accessible to the fungus [54]. Thus the aim of this study was to investigate the effect of limited exposure of sub-MIC concentrations of three commonly used antifungals, nystatin, amphotericin B and fluconazole in the synthesis/activity of Candida virulent factor, hemolysins. As mentioned above, there are no data on the extent to which antifungals modify the hemolytic properties of Candida spp. particularly those from HIV-infected individuals. In a previous investigation, Mane et al. [23] studied the expression of virulent attributes of C. albicans isolated from HIV-infected individuals including, proteinase, phospholipase and hemolytic activities, as well as their ability to adhere. They noted a significant enhancement of the foregoing virulent attributes in HIV-related isolates compared with C. albicans isolates from healthy individuals [23]. Our results concur with the latter findings as all of our C. albicans and C. tropicalis strains from HIV-infected patients demonstrated considerable hemolytic activity. In the current study, the hemolytic activity of ten C. albicans and ten C. tropicalis isolates from HIVinfected individuals was assessed. Although there was no significant inter-species differentiation in the hemolytic activity between the two Candida species, all isolates from both species demonstrated considerable hemolytic activity. This observation is in agreement with the other studies done on Candida isolates from HIV-infected patients [38, 39]. Similar findings were noted by Luo et al. [35] who compared 15 isolates of C. albicans and five isolates of C. tropicalis. Furthermore, after evaluation of seven different species they observed that the virulence of the Candida species is directly associated with the quantity of hemolysin produced; i.e., More virulent strains produced significantly higher amounts of hemolysin compared to less virulent counterparts. For instance
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in relative terms, the hemolytic activities of C. albicans, C. dubliniensis, C. kefyr and C. tropicalis were significantly higher than those of C. glabrata, C. krusei, and C. lusitaniae [35]. We report here, for the first time, that there was a significant reduction of the hemolysin activity of Candida species isolated from HIV-infected individuals upon transient exposure to sub-MIC concentrations of nystatin, amphotericin B and fluconazole. The greatest suppression of hemolysin activity was noted in amphotericin B-exposed yeasts followed by nystatin and fluconazole. In a recent study, using various clinical Candida isolates from blood, oral, vagina and the urinary tract, Negril et al. [55] also demonstrated that exposure to amphotericin B and fluconazole led to reduced hemolytic activity in C. albicans (oral isolate), C. glabrata (oral and vaginal isolates) C. parapsilosis (urine isolate) and C. tropicalis (all except urinary isolate) while they noted increased activity of hemolysins in C. glabrata (from urine and vaginal tract), and C. tropicalis (from urine) as a response to both antifungals. Thus, they concluded that the hemolysin activities were strain and species dependent without any correlation between activity profile and the site of isolation. Similarly, hemolysin activity of some of the strains used in this study was affected by fluconazole and amphotericin B [55]. The clinical relevance of the suppression of Candida hemolysin activity in the presence of polyenes and azoles is difficult to fathom. However, as Candida blood stream infections are the fourth most common cause of bloodstream infections in US hospitals, and the third commonest cause of bloodstream infections in the intensive care unit [56–58], it is likely that the low concentration of systemic antifungals in septicemia patients with Candida infections may impact on the disease progression due to reduction of hemolysis in such situations, apart from the lethality of the antifungal itself on blood stream Candida. Although hemolysins are well-known virulence factor that contribute in Candida pathogenesis, its genetic regulation is poorly understood. We have, however, previously shown that a gene coding for hemolysin-like protein (HLP) is associated with the hemolytic activity of C. glabrata [59]. Nevertheless, the mechanism by which the hemolysin activity is hampered by antifungal exposure is yet to be determined. Taken together, the data from our studies imply that, in clinical terms, the reduction of hemolysin
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activity of Candida species due to antifungal exposure is likely to affect Candida infections in HIV patients. It is tempting to speculate, therefore, that the latter phenomenon may reduce the erythrocyte lysis of anemic HIV patients preventing the possible worsening of their anemia. Hence, commonly prescribed polyenes and azole antifungal agents may have a dual beneficial effect in HIV disease by reducing the virulence of the organism in addition to killing this opportunistic invader. Further investigations are warranted to explore the molecular mechanisms underlying antifungal interference of hemolysin synthesis/ function in Candida species, their utility in diagnostic terms and, last but not the least, their putative effects in the human host. Acknowledgments The authors would also like to extend their appreciation to the Research Centre, College of Applied Medical Sciences and Deanship of Scientific Research at King Saud University for funding this research.
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215 51. Almeida RS, Wilson D, Hube B. Candida albicans iron acquisition within the host. FEMS Yeast Res. 2009;9(7): 1000–12. 52. Linares CE, de Loreto ES, Silveira CP, Pozzatti P, Scheid LA, Santurio JM, et al. Enzymatic and hemolytic activities of Candida dubliniensis strains. Rev Inst Med Trop Sao Paulo. 2007;49(4):203–6. 53. Watanabe T, Takano M, Murakami M, Tanaka H, Matsuhisa A, Nakao N, et al. Characterization of a haemolytic factor from Candida albicans. Microbiology. 1999;145(Pt 3):689–94. 54. Sutak R, Lesuisse E, Tachezy J, Richardson DR. Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence. Trends Microbiol. 2008;16(6): 261–8. 55. Negri M, Lourenc¸o T, Silva SC, Henriques M, Azeredo J, Oliveira R. Effect of antifungal agents on non-Candida albicans Candida species enzymes secretion International Conference on Antimicrobial Research (ICAR2010); 3–5 November Valladolid, Spain; 2010. p. 392. 56. Hajjeh RA, Sofair AN, Harrison LH, Lyon GM, ArthingtonSkaggs BA, Mirza SA, et al. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a populationbased active surveillance program. J Clin Microbiol. 2004;42(4):1519–27. 57. Pfaller MA, Jones RN, Doern GV, Sader HS, Hollis RJ, Messer SA. International surveillance of bloodstream infections due to Candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the United States, Canada, and South America for the SENTRY program. The SENTRY Participant Group. J Clin Microbiol. 1998;36(7):1886–9. 58. Lewis RE. Overview of the changing epidemiology of candidemia. Curr Med Res Opin. 2009;25(7):1732–40. 59. Luo G, Samaranayake LP, Cheung BP, Tang G. Reverse transcriptase polymerase chain reaction (RT-PCR) detection of HLP gene expression in Candida glabrata and its possible role in in vitro haemolysin production. APMIS Acta Pathol Microbiol Immunol Scand. 2004;112(4–5): 283–90.
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Sub-inhibitory Concentrations of Antifungals Suppress Hemolysin Activity of Oral Candida albicans and Candida tropicalis Isolates from HIV-Infected Individuals Sukumaran Anil • Mohamed Hashem • Sajith Vellappally • Shankargouda Patil • H. M. H. N. Bandara • L. P. Samaranayake
Received: 3 April 2014 / Accepted: 9 August 2014 / Published online: 21 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Secretion of hydrolytic enzymes such as hemolysin is considered an important virulence attribute of the opportunistic pathogenic fungus Candida. It is known that Candida spp. isolated from HIV-infected patients produce copious hemolysins. As common antifungal agents may perturb the production of extracellular enzymes, we evaluated the effect of three antifungals nystatin, amphotericin B and fluconazole on the hemolytic activity of Candida albicans and Candida tropicalis isolates from HIV-infected individuals. The impact of antimycotics on hemolytic activity was assessed by a previously described in vitro plate assay, after exposing ten isolates each of C.
albicans and C. tropicalis recovered from HIVinfected individuals to sub-minimum inhibitory concentrations (sub-MIC) of nystatin, amphotericin B and fluconazole. All Candida isolates showed a significant reduction in hemolytic activity. The reduction was highest for amphotericin B-exposed C. albicans and C. tropicalis followed by nystatin and fluconazole. The effect of antimycotics was more pronounced on the hemolytic activity of C. tropicalis compared to that of C. albicans. Commonly used antifungal agents significantly suppress hemolysin activity of Candida species. This implies that the antifungals, in addition to their lethality, may modulate key virulence attributes
S. Anil Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia e-mail: [email protected]
H. M. H. N. Bandara College of Pharmacy, The University of Texas at Austin, Austin, TX, USA e-mail: [email protected]
M. Hashem S. Vellappally Dental Health Department, Dental Biomaterials Research Chair, College of Applied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia e-mail: [email protected]
L. P. Samaranayake (&) School of Dentistry, University of Queensland, Brisbane, Australia e-mail: [email protected]
S. Vellappally e-mail: [email protected] S. Patil Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, M. S. Ramaiah University of Applied Sciences, Bangalore, Karnataka, India e-mail: [email protected]
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of the yeast. The clinical relevance of this phenomenon in HIV disease and other similar pathologies remains to be determined. Keywords Candida Antifungal agents Virulence attributes Hemolysin HIV infection Nystatin
Introduction Candida albicans is the commonest dimorphic fungus isolated from the human oral cavity, both in health and disease [1]. However, the prevalence and incidence of disease due to other less common species of Candida including C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. guillermondii, C. lusitaniae, C. kefyr and C. dubliniensis are on the rise [2]. When the host status is compromised, these opportunistic fungi can cause mild to severe infections, in susceptible hosts. Individuals who are, on immunosuppressives, cytotoxics and/or broad spectrum antibiotic therapy, those with HIV disease and diabetes or indwelling medical devices are all susceptible to candidal infections [3, 4]. Interestingly, some studies have shown that despite acute or chronic candidal infection, approximately 30–50 % of individuals do not experience any symptoms of candidiasis [5, 6]. HIV-infected individuals are particularly at risk of candidiasis. It is estimated that approximately 90 % of those with HIV disease and AIDS suffer from oral mucosal candidiasis at least once during the course of the HIV infection [6–8]. Oropharyngeal candidiasis, which is the most frequent fungal infection in HIV patients, is also considered as a marker/predictor for HIV progression [9–16]. As mentioned above, C. albicans is the commonest causative agent; however, other non-albicans species such as C. glabrata, C. tropicalis and C. krusei frequently cause oropharyngeal candidiasis in the latter cohorts [17, 18]. The conventional view that the severity and chronicity of oral candidiasis in HIV infection is mainly due to the virus-induced immune deficiency has been recently challenged as more information on the role of the causative organism and its invasive arsenal, such as hydrolytic enzymes, has come to light. Indeed, it has been suggested that Candida strains with altered virulent determinants that prefer
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an immunocompromised niche is likely to be selected in HIV-infected patients [16, 19–22]. Thus, the virulence determinants of the organism itself appear to play a significant role in the etiopathology of oropharyngeal candidiasis in HIV disease [23]. The principle virulent determinants of Candida include tissue-degrading hydrolytic exoenzymes such as phospholipases and proteinases, morphologic dimorphism (blastospore–mycelial transformation) and phenotypic switching. These are known to help the fungus adhere to host epithelia, to resist both the antifungals as well as the fungicidal activity of neutrophils [24–29]. The exoenzymes secreted by Candida such as proteinases, phospholipases, esterases, phosphatases and hemolysins are known to mediate host invasion, particularly by facilitating hyphal invasion of the epithelium [30–33]. Hemolysin production and hemolytic activity has been shown to be significantly higher in C. albicans than in non-albicans species such as C. tropicalis, C. glabrata, C. krusei, C. parapsilosis and C. orthopsilosis [34, 35]. Some workers have also noted that the expression of hemolysins and hemolytic activity in Candida isolates from HIV-infected patients is greater compared with isolates from healthy individuals [23, 36]. In another study, Samaranayake et al. [37] reported a positive correlation between germ tube formation and hemolysin production in Candida isolated from HIV-infected individuals. Apart from the foregoing reports, hemolysin activity of Candida from HIV-infected individuals has received scant attention. Rossoni et al. [38] found that most nonalbicans Candida species had a similar ability to produce hemolysins when compared to C. albicans isolates from HIV-infected individuals. However, Ramesh et al. [39] observed a higher amount of hemolysin production by C. albicans compared to C. glabrata. Despite the extensive use of antifungal agents, their effect on candidal virulence determinants has been little studied. A number of workers have investigated the phenotypic switching of candida in response to antimycotics [40–42]. In fact, as mentioned above, HIV-infected individuals carry different phenotypes of Candida and exhibit greater hemolytic activity. Yet, the effect of antimycotics on hemolysin activity of Candida isolates from HIV-infected patients has not been investigated. Thus, the aim of this study was to investigate the
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effect of transient exposure of C. albicans and C. tropicalis isolates from HIV-infected individuals to sub-therapeutic concentrations of commonly used antifungals belonging to the polyene class (nystatin, amphotericin B) and an azole, fluconazole.
Materials and Methods Antifungal Agents Reagent grade nystatin and amphotericin B were purchased from Sigma (Sigma Chemicals Co. St.Louis, USA) and dissolved in dimethylsulfoxide (DMSO) and absolute ethanol (3:2 ratio), respectively. Fluconazole (Pfizer Inc. New York, USA) was dissolved in absolute methanol. All 3 antifungals were prepared initially as 10 mg/ml stock solutions and stored at -20 °C until used. Sample Collection A total of 10 oral isolate each of C. albicans and C. tropicalis from HIV-infected subjects attending the Queen Elizabeth Hospital, Hong Kong, were obtained by the oral rinse technique [43]. All subjects manifested oral candidiasis when the samples were collected. Briefly, the patients were instructed to rinse the mouth for 60 s with 10 ml of sterile phosphatebuffered saline (0.01 M, pH 7.2). Oral rinses were collected in sterile containers and transported to the laboratory. Identification of Candida and Storage Fifty microliters of oral rinse was inoculated on Sabouraud dextrose agar (SDA) using a spiral plater and incubated for 48 h at 37 °C. At the end of incubation, isolated colonies were subcultured for further purification and identity was confirmed using the germ tube test and the commercially available API-20C AUX (bioMe´rieux, Basingstoke, UK) [44]. All isolates were stored in multiple aliquots at -20 °C, after confirming their purity. One strain each of Streptococcus pyogenes (Lancefield group A) and Streptococcus sanguis that induces beta and alpha hemolysins, respectively, were used as positive controls.
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Candidal Inocula Prior to each experiment, Candida species were subcultured on SDA, for 18 h at 37 °C. A loopful of this overnight growth was inoculated into Yeast nitrogen base (Difco Laboratories, Detroit, Michigan) medium containing 2 % Dextrose and incubated for 18 h in an orbital shaker (75 r.p.m.) at 37 °C. The resultant growth was harvested, washed twice in PBS and resuspended in PBS. Concentrations of Candida species were adjusted to desired values by spectrophotometry and were confirmed by hemocytometric counting. Minimum Inhibitory Concentration (MIC) Determination Broth Micro Dilution Method Antifungal susceptibility to nystatin (MIC) was determined by a broth microdilution assay in accordance with the CLSI guidelines [45]. Briefly, fungal cell suspensions (1 9 103 Cells/ml) were prepared as mentioned above in RPMI 1640 supplemented with 0.165M 3-(Nmorpholino) propanesulfonic acid (MOPS). Candida were treated with nystatin in a concentration gradient and incubated in a 96-well microtiter plate for 24 h at 35 °C. At the end of the incubation, the optical density of the fungal growth was measured by a spectrophotometer at 595 nm. The lowest concentration of the antifungal at which the Candida demonstrates 80 % of visible growth inhibition compared to the solvent control was considered as the MIC80 of the antifungal against Candida. The assay was performed quadruplicates on three separate occasions. E-Test The MIC determinations of amphotericin B and fluconazole were performed using the E-test. The E-test (AB BIODISK, Solna, Sweden) is a patented commercial method for the quantitative determination of MICs of antimicrobial drugs. Comparisons of the E-test method with the CLSI broth dilution method have demonstrated high levels of agreement [45]. The inocula of Candida species were prepared as mentioned above. Candida cell concentrations corresponding to McFarland 0.5 at 520 nm were prepared in sterile distilled water. Candida suspensions were
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inoculated on to RPMI-1640 (American Biorganics, Buffalo, NY) supplemented with 1.5 % agar and 2 % glucose and buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma, St. Louis, Mo.) using a sterile swab by streaking it across the entire surface of the agar in three directions. The plates were dried at room temperature for 15 min and the E-test strips were applied. The agar plates were incubated for 24 h at 35 °C. The drug concentration at which the border of the elliptical inhibition zone intersected the scale on the antifungal test strip was considered as the MIC of the respective antifungals. Exposure to Antifungal Agents Yeast cells, maintained on Sabouraud’s dextrose agar (SDA), were inoculated onto fresh plates and incubated overnight for 24 h prior to use. The organisms were harvested and a cell suspension prepared in sterile phosphate-buffered saline (PBS) of pH 7.4, at 520 nm corresponding to an optical density 0.5 McFarland standard. From this cell suspension, 1 ml was added to tubes containing 4 ml of RPMI broth (control) and 4 ml of RPMI/drug solution (test) in which the drug concentrations were twice the MIC. This gave a cell suspension of 106–107 cells/ml in each assay tube. The tubes were then incubated at 37 °C for a period of 1 h in a rotary incubator. Following this limited exposure, the drugs were removed by two cycles of dilution with sterile PBS and centrifugation for 10 min at 3,0009g. Afterward the supernatant was completely decanted and the pellets were resuspended in 5 ml of sterile PBS. This washing procedure was then repeated and the pellets resuspended in 2.5 ml of sterile PBS. It has been found by previous investigators that removal of 90 % of the supernatant with two washings reduces antimicrobial concentration 100-fold, while complete decanting of the supernatant with two washings (as carried out in the current study) reduces the concentration 10,000-fold [46, 47]. Hence, this method virtually eliminates any ‘‘carryover effect’’ of the drug following its removal. Viable counts of the control and test were done by spiral plating after drug removal and control suspensions were reconstituted as needed to obtain a cell concentration comparable to the test. CFU Assay A portion of the resultant suspension was gently vortexed for 1 min to disrupt the
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aggregates, serially diluted and inoculated using a spiral plater onto SDA. The resultant colony-forming units/ml of Candida were quantified after 48 h incubation at 37 °C. Each assay was carried out quadruplicate on two different occasions. Remaining candidal suspensions were reconstituted as needed to obtain a standard cell concentration (1 9 108 cells/ ml). Determination of Hemolysin Activity Hemolysin production was evaluated using a modification of the plate assay described by Luo et al. [35]. Briefly, 10 ll of aforementioned antifungal treated and control suspensions were spot-inoculated on to blood agar supplemented with 3 % glucose (w/v, pH 5.6 ± 0.2) so as to yield a circular inoculation site of about 5 mm in diameter. The plates were incubated for 48 h (5 % CO2, 37 °C). The presence of a distinct translucent halo around the inoculum site, viewed with transmitted light, indicated a positive hemolytic activity. The diameters of the zone of lysis and the colony were measured using a computerized image analysis system (Quantimet 500 Qwin, Leica, Cambridge, UK) [48]. The hemolytic index was determined using the ratio between the diameters of translucent halo and the colony to represent the hemolysin production intensity by different Candida species. The assay was conducted in quadruplicate on two different occasions for each Candida isolate. Statistical Analysis The intra-and interspecies differences of the hemolytic activity were analyzed by ANOVA. Multiple comparisons between isolates exposed to the three drugs and control were analyzed using the Dunnet’s multiple comparison test (InStat, GraphPad Software Inc., San Diego, California, USA).
Results Effect of Antifungal Solvents on C. albicans and C. tropicalis Since the antifungal agents were dissolved in absolute methanol and DMSO/absolute ethanol, equivalent
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amounts of the latter chemicals were tested to ascertain their effect on the isolates tested. These chemicals had no significant effect on the survival/ growth of Candida when compared with the controls, evaluated at identical concentrations. Minimum Inhibitory Concentrations (MIC) The MICs of nystatin, amphotericin B and fluconazole values for the ten isolates of C. albicans were 0.78–1.56, 0.19–0.38 and 0.12–0.38 lg/ml, respectively, whereas for the ten isolates of C. tropicalis, it was 0.78, 0.25–0.38 and 0.25–0.50 lg/ml, respectively. Impact of Hemolytic Activity of Candida Isolates Exposed to Antimycotics
Fig. 1 The hemolysin activity of control and drug exposed isolates C. albicans (C control, N nystatin, A amphotericin B, F fluconazole)
Two different types of haemolysis could be observed circumscribing the yeast ‘‘colony’’ when viewed with transmitted light after 48 h of incubation. The first was a totally translucent ring identical to b haemolysis produced by the control strain of b hemolytic Streptococcus, whereas the second was a greenish-black halo similar to a-haemolysis observed with the control strain of Staphylococcus. Hence, the terms ‘‘a haemolysis’’ and ‘‘b haemolysis’’ were used as descriptive terms to indicate incomplete and complete haemolysis, respectively, associated with the Candida strains tested [35]. In order to obtain quantitative data, we attempted to measure the diameter of hemolytic zones relative to the inoculum size using an image analysis system. However, clear-cut zones of haemolysis were perceptible only in relation to b hemolytic activity. The margins of a haemolysis were rather diffuse. Hence, the diameter of the latter zones was not accurately quantifiable either by naked eye estimation or the
image analysis system. The mean hemolytic activity of the control C. albicans isolates was 1.49 ± 0.07. Brief exposure to nystatin, amphotericin B and fluconazole significantly reduced the hemolytic activity with a mean value of 1.34 ± 0.06 (P \ 0.001), 1.33 ± 0.06 (P \ 0.001) and 1.41 ± 0.06, respectively (P \ 0.001) (Table 1; Fig. 1). The mean hemolysin activity of the control C. tropicalis isolates was 1.45 ± 0.01. Similar to C. albicans, brief exposure to nystatin, amphotericin B and fluconazole significantly reduced the hemolytic activity of C. tropicalis with mean values of 1.31 ± 0.06 (P \ 0.001), 1.30 ± 0.06 (P \ 0.001) and 1.39 ± 0.06 (P \ 0.001), respectively, compared to the respective controls (Table 1; Fig. 2). Candida albicans isolates appeared to produce more hemolysin than C. tropicalis; however, these findings were not significant (Table 1; Fig. 3).
Table 1 Intra- and interspecies variation in hemolysin activity of C. albicans and C. tropicalis exposed to nystatin (NYS), amphotericin B (AMB) and fluconazole (FLU) Isolates
No
Control
NYS
AMB
FLU
C. albicans
10
1.49 ± 0.07
1.34 ± 0.06*
1.33 ± 0.06*
1.41 ± 0.06*
C. tropicalis
10
1.45 ± 0.01
1.31 ± 0.06*
1.30 ± 0.06*
1.39 ± 0.06*
P-level
–
NS
NS
NS
NS
Mean of two experiments, each conducted in quadruplicate * P \ 0.001
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Fig. 2 The hemolysin activity of control and drug exposed isolates C. tropicalis (C control, N nystatin, A amphotericin B, F fluconazole)
Fig. 3 The hemolysin activity of ten isolates of C. albicans and C. tropicalis exposed to half MIC of nystatin (NYS), amphotericin B (AMB) and fluconazole (FLU)
Discussion The ability of pathogenic organisms to acquire iron in the mammalian host has been shown to be of critical importance in establishing an infection [49, 50]. In humans, most of the iron is located intracellularly as ferritin or as heme-containing compounds. The small amount of extracellular iron is attached to iron-binding and transport proteins, transferrin and lactoferrin. Since there is essentially no free iron in the human
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body, pathogens must acquire it from one or more iron-containing compounds [50]. The ability to utilize hemoglobin as an iron source is described as a strategy of C. albicans to exploit elemental iron from the host. For instance, C. albicans hyphae are able to rosette erythrocytes via complement receptor like molecules [51] and releases a cell surface bound mannoprotein hemolytic factor, hemolysin to acquire iron from erythrocytes [52, 53]. Clearly, therefore, the colonization and proliferation of Candida are possible only if sufficient iron is accessible to the fungus [54]. Thus the aim of this study was to investigate the effect of limited exposure of sub-MIC concentrations of three commonly used antifungals, nystatin, amphotericin B and fluconazole in the synthesis/activity of Candida virulent factor, hemolysins. As mentioned above, there are no data on the extent to which antifungals modify the hemolytic properties of Candida spp. particularly those from HIV-infected individuals. In a previous investigation, Mane et al. [23] studied the expression of virulent attributes of C. albicans isolated from HIV-infected individuals including, proteinase, phospholipase and hemolytic activities, as well as their ability to adhere. They noted a significant enhancement of the foregoing virulent attributes in HIV-related isolates compared with C. albicans isolates from healthy individuals [23]. Our results concur with the latter findings as all of our C. albicans and C. tropicalis strains from HIV-infected patients demonstrated considerable hemolytic activity. In the current study, the hemolytic activity of ten C. albicans and ten C. tropicalis isolates from HIVinfected individuals was assessed. Although there was no significant inter-species differentiation in the hemolytic activity between the two Candida species, all isolates from both species demonstrated considerable hemolytic activity. This observation is in agreement with the other studies done on Candida isolates from HIV-infected patients [38, 39]. Similar findings were noted by Luo et al. [35] who compared 15 isolates of C. albicans and five isolates of C. tropicalis. Furthermore, after evaluation of seven different species they observed that the virulence of the Candida species is directly associated with the quantity of hemolysin produced; i.e., More virulent strains produced significantly higher amounts of hemolysin compared to less virulent counterparts. For instance
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in relative terms, the hemolytic activities of C. albicans, C. dubliniensis, C. kefyr and C. tropicalis were significantly higher than those of C. glabrata, C. krusei, and C. lusitaniae [35]. We report here, for the first time, that there was a significant reduction of the hemolysin activity of Candida species isolated from HIV-infected individuals upon transient exposure to sub-MIC concentrations of nystatin, amphotericin B and fluconazole. The greatest suppression of hemolysin activity was noted in amphotericin B-exposed yeasts followed by nystatin and fluconazole. In a recent study, using various clinical Candida isolates from blood, oral, vagina and the urinary tract, Negril et al. [55] also demonstrated that exposure to amphotericin B and fluconazole led to reduced hemolytic activity in C. albicans (oral isolate), C. glabrata (oral and vaginal isolates) C. parapsilosis (urine isolate) and C. tropicalis (all except urinary isolate) while they noted increased activity of hemolysins in C. glabrata (from urine and vaginal tract), and C. tropicalis (from urine) as a response to both antifungals. Thus, they concluded that the hemolysin activities were strain and species dependent without any correlation between activity profile and the site of isolation. Similarly, hemolysin activity of some of the strains used in this study was affected by fluconazole and amphotericin B [55]. The clinical relevance of the suppression of Candida hemolysin activity in the presence of polyenes and azoles is difficult to fathom. However, as Candida blood stream infections are the fourth most common cause of bloodstream infections in US hospitals, and the third commonest cause of bloodstream infections in the intensive care unit [56–58], it is likely that the low concentration of systemic antifungals in septicemia patients with Candida infections may impact on the disease progression due to reduction of hemolysis in such situations, apart from the lethality of the antifungal itself on blood stream Candida. Although hemolysins are well-known virulence factor that contribute in Candida pathogenesis, its genetic regulation is poorly understood. We have, however, previously shown that a gene coding for hemolysin-like protein (HLP) is associated with the hemolytic activity of C. glabrata [59]. Nevertheless, the mechanism by which the hemolysin activity is hampered by antifungal exposure is yet to be determined. Taken together, the data from our studies imply that, in clinical terms, the reduction of hemolysin
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activity of Candida species due to antifungal exposure is likely to affect Candida infections in HIV patients. It is tempting to speculate, therefore, that the latter phenomenon may reduce the erythrocyte lysis of anemic HIV patients preventing the possible worsening of their anemia. Hence, commonly prescribed polyenes and azole antifungal agents may have a dual beneficial effect in HIV disease by reducing the virulence of the organism in addition to killing this opportunistic invader. Further investigations are warranted to explore the molecular mechanisms underlying antifungal interference of hemolysin synthesis/ function in Candida species, their utility in diagnostic terms and, last but not the least, their putative effects in the human host. Acknowledgments The authors would also like to extend their appreciation to the Research Centre, College of Applied Medical Sciences and Deanship of Scientific Research at King Saud University for funding this research.
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