Mycopathologia (2013) 175:265–272 DOI 10.1007/s11046-013-9638-z
Candida albicans and Non-C. albicans Candida Species: Comparison of Biofilm Production and Metabolic Activity in Biofilms, and Putative Virulence Properties of Isolates from Hospital Environments and Infections A. V. Ferreira • C. G. Prado • R. R. Carvalho K. S. T. Dias • A. L. T. Dias
•
Received: 8 November 2012 / Accepted: 11 March 2013 / Published online: 27 March 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Candida albicans and, more recently, non-C. albicans Candida spp. are considered the most frequent fungi in hospitals. This study analyzed Candida spp. isolates and compared the frequency of different species, that is, C. albicans and nonC. albicans Candida spp., and the origins of isolates, that is, from hospital environments or infections. Yeast virulence factors were evaluated based on biofilm production and metabolic activity. Hemolysin production and the antifungal susceptibility profiles of isolates were also evaluated. Candida spp. were highly prevalent in samples collected from hospital environments, which may provide a reservoir for continuous infections with these yeasts. There were no differences in the biofilm productivity levels and metabolic activities of the environmental and clinical isolates, although the metabolic activities of non-C. albicans Candida spp. biofilms were greater than those of the C. albicans biofilms (p \ 0.05). Clinical samples had higher hemolysin production (p \ 0.05) and lower
A. V. Ferreira C. G. Prado R. R. Carvalho A. L. T. Dias (&) Microbiology and Immunology Department, Biomedical Sciences Institute, Federal University of Alfenas (UNIFAL-MG), Street Gabriel Monteiro da Silva, 700, Alfenas, MG 37130-000, Brazil e-mail:
[email protected];
[email protected] K. S. T. Dias Federal University of Alfenas, Alfenas, MG, Brazil
susceptibility to fluconazole (p \ 0.05). Non-C. albicans Candida spp. predominated in samples collected from hospital environments and infections (p \ 0.05). These species had a lower susceptibility to fluconazole and amphotericin B, and their biofilms had higher metabolic activities than those produced by C. albicans, which may explain the increased incidence of fungal infections with these yeasts during recent years. Keywords Candida spp. Hospital environments Biofilm Hemolysin Antifungals Virulence
Introduction The search for new extracorporeal reservoirs colonized by Candida spp. has increased greatly in recent years. Natural environments, inanimate surfaces, the hands of health professionals, and hospitals have been studied to clarify the epidemiological cycle of hospital infections caused by Candida spp. of exogenous origin [1]. Colonization of inanimate surfaces was reported for some Candida spp., which could survive for 3–14 days on dry surfaces [2]. The hands of health professionals may be colonized by yeasts, and they also serve as reservoirs for hospital-acquired infections (HIs) or nosocomial infections. The use of catheters and other medical devices is also related to the development of HIs with Candida spp. via exogenous routes [3]. Fungal HIs have emerged in recent years and are currently the subject of numerous studies. The rapid
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increase in the incidence of these infections has led to a high mortality rate, which has reached 60 % in some hospital infections [4]. There has been an increased incidence of HIs caused by Aspergillus spp., Zygomyces spp., Fusarium spp., Scedosporium spp., Cryptococcus spp., Trichosporon spp., Geotrichum spp., and Rhodotorula spp. [5], but the Candida spp. yeasts are still the most frequent, accounting for 80 % of isolates from fungal infections. Candida spp. are the fourth leading cause of hematogenous infections (candidemia) [6]. Candida spp. are not related exclusively to hospital candidemia, because hematogenous infections also occur in the community [7]. In candidemia, the balance between C. albicans and non-C. albicans Candida spp. may be related to fungal virulence [7–9]. This study aimed to assess the prevalence of C. albicans and non-C. albicans Candida spp. in HIs and hospital environments and to determine the profiles associated with virulence. Thus, the biofilm productivity, metabolic activity, hemolysin production, and the amphotericin B and fluconazole susceptibility profiles of isolates were also investigated.
Materials and Methods Samples Yeast samples were obtained from two different sources in a tertiary care hospital, and only those that contained yeasts were maintained. The samples were compiled once a week during a 1-year period from 2010 to 2011. (a)
Sources in hospital environments: a total of 103 samples were collected, that is, 42 from a neonatal intensive care center and 61 from a surgical center. (b) Hospital infection sources: a total of 677 samples were collected from hospitalized patients, that is, 49 from catheter tips and 628 from blood cultures that were negative for bacterial growth. Sample collection Samples were collected from hospital environments, that is, trays used for the preparation of drugs, switches, sheets, door knobs, and the right hands of health professionals, using sterile swabs soaked in physiological solution supplemented with 0.5 g/L
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chloramphenicol. Samples were collected from selected surface layer areas of 4 cm 9 4 cm. The swabs with samples were placed in brain–heart infusion (BHI) broth supplemented with 0.5 g/L chloramphenicol. Samples from the laboratory coats of health professionals were collected by pressing a petri dish, measuring 4 cm in diameter and containing BHI agar supplemented with 0.5 g/L chloramphenicol, onto the front of the laboratory coats, about 5 cm from the pockets. Environmental air samples were collected in 4-cm-diameter petri dishes containing Sabouraud agar, which were exposed to the environment for 25 min. Blood samples were previously inoculated in a blood culture broth at 35 °C for 5 days and after seeded directly into Sabouraud agar. The catheter tips were rolled back and forth on blood agar plates, and putative yeast colonies were plated onto Sabouraud agar. All plates were incubated at 35 °C for 48 h. Sample identification Analysis of growth in a chromogenic medium culture (CHROMagar CandidaÒ) facilitated the determination of colony purity and the identification of Candida spp. Macromorphological/micromorphological analyses and physiological tests were also performed, that is, zymograms and auxanograms, to confirm the chromogenic medium culture results [10]. Discrimination between presumptive C. albicans and C. dubliniensis isolates was based on the growth of C. albicans at 45 °C for 48 h [11]. All the strains were cryopreserved at -80 °C and added to the mycological collection of our research group NEMIC. Factors Associated with the Virulence of Candida spp. Biofilm Production Biofilm production was evaluated using the method of Shin et al. [12] with some modifications. Polystyrene microtiter plates were treated with 100 lL of human fetal serum in each well. After incubating for 24 h at 37 °C, the serum was removed carefully and 300 lL of cell suspension containing 3 9 107 cells/mL in yeast nitrogen base (YNB) broth supplemented with 100 mM glucose was added to the wells. The plates were incubated at 37 °C for 72 h at 75 rpm.
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The sample plates containing biofilms were washed twice with phosphate-buffered saline (PBS), and the presence or absence of biofilms was evaluated based on spectrophotometer readings at 405 nm. In the controls, a well of each microtiter plate was treated in the same way, but the Candida suspension was omitted. The non-biofilm producer strain C. lusitaniae NEMIC 219 and the biofilm producer strain C. lusitaniae NEMIC 1034 were used as negative and positive biofilm production controls, respectively. Metabolic Activity of Biofilms This analysis used a method to test the reduction of XTT (Sigma, USA) [13, 14] but with some modifications. Before each test, 1 mg/mL XTT solution (Sigma, St. Louis, MO) in PBS was thawed and mixed with 0.4 mM menadione at a ratio of 5:1 (XTT:menadione) to prepare the reagent solution. After spectrophotometric determination of biofilm formation, 200 lL of PBS and 12 lL of the reagent solution (XTT-menadione) were added to each well. After incubating for 2 h, the color change was measured spectrophotometrically at 490 nm (Zenith 200 rt; Anthos Labtec, Cambridge, UK). Activity of Hemolytic Enzymes For each sample, a 2.0-mL inoculum of sample, containing 1.5 9 107 cells/mL, was placed at a predetermined point on the surface of a blood agar plate, which was supplemented with 7 % sheep blood and 3 % glucose. The plates were incubated at 37 °C. At 48 h postinoculation, the samples were classified based on the hemolytic activity [15]. b-Hemolytic Staphylococcus aureus (ATCC 6538) was used as the positive control. Determination of the Pattern of Response to Antifungal Drugs Antifungal tests were conducted using strips with a continuous antifungal concentration gradient (EtestÒ) to determine the minimum inhibitory concentration (MIC) of planktonic cells (free cells), which were interpreted based on documents M44-A2 (CLSI) [16], M27-A3 (CLSI) [17], and the manufacturer’s criteria (AB Biodisk Solna, Sweden). Candida krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were used as
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the controls. Assessments of the response profiles with different antifungal agents were performed in triplicate and on different days. The results were expressed as the geometric mean values of MIC50 for fluconazole and MIC90 for amphotericin B (Table 4). Statistical Analysis The results were compared by analysis of variance (ANOVA). However, the hemolytic activity was compared using Fisher’s exact test. Differences between groups were considered to be significant at p \ 0.05.
Results Distribution of Candida spp. in Hospitals Among the 780 samples, the prevalence of Candida spp. in catheter tip samples was 28.6 %, and 1.4 % in blood cultures. It was found that 43.7 % of the samples from hospital environments were positive for Candida spp. Samples from environment and sources of hospital infection were collected at the same frequency, and Candida isolates accounted for approximately 8 % of isolates each week. Among the isolates from hospital environments, 33.4 % came from the laboratory coats of professionals, 24.5 % from door knobs, 20 % from switches, 11.1 % from the hands of professionals involved in Table 1 Distribution of Candida spp. isolates from hospital environments and infections Species
Hospital environment percentage (number of isolates)
Hospital infection percentage (number of isolates)
Candida albicans
11.1 (5)
30.5 (7)
Candida ciferri
8.9 (4)
–
Candida famata
4.4 (2)
–
Candida guilliermondii
2.2 (1)
4.3 (1)
Candida krusei
–
4.3 (1)
Candida lusitaniae
35.6 (16)
26.1 (6)
Candida parapsilosis
13.3 (6)
17.4 (4)
Candida tropicalis
24.5 (11)
17.4 (4)
100 (45)
100 (23)
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hospital environments, that is, 65.2 and 28.9 %, respectively (p \ 0.05). Table 2 shows a comparison of hemolysin production by C. albicans and non-C. albicans Candida spp. Biofilm Formation and Metabolic Activity
Fig. 1 Prevalence of C. albicans and non-C. albicans Candida spp. in isolates from hospital environments and infections. *Indicates significant differences between C. albicans and non-C. albicans Candida spp. from hospital environments (p \ 0.05). **Indicates significant differences between C. albicans and non-C. albicans Candida spp. from hospital infections (p \ 0.05)
patient care, 4.4 % from bed sheets, 4.4 % from the air, and 2.2 % from medicine trays. Table 1 shows the Candida spp. isolated from hospital environments and infections. The most common species were C. lusitaniae and C. tropicalis in hospital environments (35.6 and 24.5 % of species, respectively), whereas C. albicans was the most prevalent species in HIs (30.5 % of species). However, most of the isolates were non-C. albicans Candida spp. (Fig. 1). Hemolytic Activity Evaluation The production of hemolysin by Candida spp. from HIs was higher than that by samples isolated from Table 2 Comparison of hemolysin production by Candida spp. isolated from different sources
Candida spp.
Susceptibility Profiles Using the Antifungal Drugs Amphotericin B and Fluconazole All Candida spp. samples were susceptible to amphotericin B. Isolates from hospital environments were 100 % susceptible to fluconazole, whereas 100 % of C. albicans and 94.75 % of non-C. albicans Candida isolates from HIs were susceptible. The fluconazole MIC50 values were higher in non-C. albicans Candida species (Table 4), whereas C. krusei and C. lusitaniae isolates from infections had MICs that were equal to or lower than C. albicans isolates. A comparison based on the origins of samples showed that C. albicans and non-C. albicans Candida spp. isolated from HIs had higher fluconazole MICs. Percentage (no. hemolysin production/total no.) Hospital environment
Candida albicans
60 (3/5)
non-C. albicans Candida spp.
25 (10/40)
Candida ciferri Candida famata Candida guilliermondii Candida krusei Candida lusitaniae
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All of the isolates produced biofilms. There were no differences in the biofilm production and metabolic activity of isolates from hospital environments and hospitalized patients (Table 3). Although the likelihood of biofilm production was the same, the biofilms produced by non-C. albicans Candida spp. had higher absolute metabolic activities than C. albicans biofilms irrespective of the isolation source (Fig. 2), with the exception of C. tropicalis. Despite the higher absolute metabolic activity of C. krusei, it was not possible to reach any conclusions about this species as there was only one isolate.
25 (1/4) 100 (2/2) 0 (0/1) – 25 (4/16)
Hospital infection 100 (7/7) 50 (8/16) – – 0 (0/1) 100 (1/1) 20 (1/5)
Candida parapsilosis
16.7 (1/6)
60 (3/5)
Candida tropicalis
27.3 (3/11)
75 (3/4)
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Table 3 Comparison of biomass production and the metabolic activity in biofilms produced by C. albicans and non-C. albicans Candida spp. isolated from different sources (mean optical density ± SD) Biofilm production Hospital environment
Metabolic activity a
Hospital infection
p value (source)
Hospital environment
Hospital infection
p valuea (source)
All Candida species
0.137 ± 0.038
0.121 ± 0.049
0.1605
0.139 ± 0.078
0.127 ± 0.083
0.5800
Candida albicans
0.136 ± 0.014
0.109 ± 0.047
0.2332
0.063 ± 0.029
0.072 ± 0.039
0.6743
non-C. albicans Candida species
0.137 ± 0.040
0.127 ± 0.051
0.5472
0.149 ± 0.076
0.152 ± 0.086
0.9079
p value (species)
0.9697
0.5454
Candida ciferri
0.140 ± 0.017
–
0.184 ± 0.089
–
Candida famata Candida guilliermondii
0.133 ± 0.002 0.138
– 0.136
0.173 ± 0.106 0.205
– 0.166
Candida krusei
–
0.141
–
0.312
b
\0.05
\0.05
Candida lusitaniae
0.123 ± 0.054
0.091 ± 0.063
0.221 ± 0.182
0.172 ± 0.074
Candida parapsilosis
0.137 ± 0.035
0.128 ± 0.043
0.192 ± 0.090
0.171 ± 0.073
Candida tropicalis
0.155 ± 0.026
0.164 ± 0.036
0.093 ± 0.060
0.059 ± 0.036
a
Comparison among different sources
b
Comparison among Candida albicans and non-C. albicans Candida spp.
Discussion
Fig. 2 Comparison of the metabolic activity in biofilms produced by C. albicans and non-C. albicans Candida spp. isolates from hospital environments and infections. *Indicates significant differences between C. albicans and non-C. albicans Candida spp. from hospital environments (p \ 0.05). **Indicates significant differences between C. albicans and non-C. albicans Candida spp. from hospital infections (p \ 0.05)
The amphotericin B MIC90 tests detected no differences related to the origins of samples. However, the absolute MICs of non-C. albicans Candida spp. were equal to or higher than those of C. albicans (except environmental isolates of C. parapsilosis).
HIs related to vascular access are extremely worrying and challenging, mainly because of their high rates of mortality. The current study detected a high positive rate of Candida spp. isolates from catheter tips, which is an important entry point for systemic infections [18]. Catheter tip surfaces can also promote the formation of biofilms [19], which are considered one of the most important virulence factors associated with yeasts. A combination of an increasingly aging population, a high proportion of immunosuppressed patients, and the increasing number of medical devices is probably related to the increased frequency of infectious complications related to catheters [20]. The high frequencies of Candida spp. in samples collected from hospital environments, including laboratory coats, door handles, switches, and the hands of professionals, are a cause for concern because they can provide a reservoir for the colonization and infection of hospitalized patients, especially immunocompromised patients and those experiencing venous access. Thus, yeasts may be transmitted directly or indirectly via the hands of professionals, thereby maintaining a continuous supply of yeasts for infections. Thus, it is important to ensure that basic techniques for the prevention of HIs are maintained, because the hands of
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Table 4 Comparison of the susceptibility profiles of C. albicans and non-C. albicans Candida spp. isolated from hospital environments and infections Fluconazole MIC50 (lg/mL) Environment
Infection
Amphotericin B MIC90 (lg/mL) a
p value (source)
Environment
Infection
p valuea (source)
Candida albicans
0.094
0.500
\0.05
0.190
0.125
0.1180
non-C. albicans Candida species
0.750
1.000
\0.05
0.250
0.500
0.6116
p valueb (species)
\0.05
\0.05
0.6545
\0.05
Candida ciferri
0.250
–
0.750
–
Candida famata
0.500
–
0.190
–
Candida guilliermondii
1.500
0.750
0.380
0.190
Candida krusei
–
0.380
–
0.125
Candida lusitaniae
1.000
0.500
0.250
0.125
Candida parapsilosis
1.500
4.000
0.125
0.500
Candida tropicalis
0.500
2.000
0.190
0.500
a
Comparison of different sources (statistical analysis based on the means of the MIC50 or MIC90 values)
b
Comparison of C. albicans and non-C. albicans Candida spp. (statistical analysis based on the means of the MIC50 or MIC90 values)
health professionals become reservoirs for the infection of patients if they are colonized by yeasts. The predominance of non-C. albicans Candida spp. has been reported in several studies [5, 21], although the current study found a higher proportion among isolates from the environment (88.9 %) compared with samples from hospitalized patients (69.5 %). The prevalence of C. albicans infections in hospitals may be attributable to endogenous infections, although they may have exogenous origins because yeasts can be carried from several critical areas in hospitals. This was particularly important in the current study because there was a high rate of Candida spp. colonization in the environment and on the hands of professionals who provided patient care. Among the factors associated with the virulence of Candida spp. isolates, the presence of hemolytic exoenzymes in microorganisms is fundamentally important because it allows yeast to acquire iron ions from hemoglobin [22]. Studies have shown that this metal may be related to the establishment and dissemination of infections [23, 24]. In the current study, a comparison of hemolysin production by different species of Candida showed that C. albicans produced more hemolytic factors than non-C. albicans Candida spp., irrespective of their source, which agreed with a report by Luo et al. [15]. The production of hemolysin by isolates from HIs was high, but there was also high enzyme production by environmental
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isolates, which suggested that these environmental strains may have been the sources of infections. This also showed that the yeasts present in hospital environments had virulence attributes that facilitated the establishment of exogenous infections in patients, including the possibility of systemic infections. This also demonstrated the complexity of the processes involved in the epidemiological chain of colonization– infection processes. This study found that all Candida spp. isolates were capable of biofilm production. Indeed, most infections caused by Candida are associated with the formation of biofilms on surfaces rather than the presence of planktonic cells [25, 26]. Douglas [27] reported that the biofilm-forming capacity was directly related to microbial virulence because the cells in biofilms appeared to be more resistant to antifungal drugs than planktonic cells [25], including amphotericin B and fluconazole [28], which may contribute to the pathogenesis of superficial and systemic candidiasis [29]. This makes it difficult to remove and eliminate microorganisms from contaminated environmental sources which are associated with increased rates of morbidity and mortality during infections. Biofilms confer resistance to host defenses, as well as high-level resistance to antifungal therapy [30]. Implantable medical devices may facilitate microbial colonization and biofilm formation [31]. Thus, the high prevalence of Candida isolates in samples from catheter tips in the
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current study shows that catheters may have an important role in the spread, progression, and persistence of HIs. It is also noteworthy that systemic catheter-related infections caused by Candida spp. colonization are associated with longer-duration infections, high rates of treatment failure, and an increased risk of dissemination and death [19, 31]. Mohandas et al. [32] suggested that biofilm production is more important for non-C. albicans Candida spp.. The current study detected no difference in the biofilm productivity and metabolic activity of biofilms derived from environmental and clinical sources, although the biofilms formed by non-C. albicans Candida spp. had higher metabolic activities than C. albicans biofilms. The metabolic activity appears to be an important correlate of pathogenicity because the metabolic activity of biofilms may be related to the level of virulence factor expression and the level of resistance to antifungal agents [33]. The higher metabolic activity of non-C. albicans Candida spp. may be a cause of the increased frequency of fungal infections associated with these yeasts. The range of antifungal therapies available for treating HIs is still low compared with the diversity of antibacterial drugs. The antifungal drugs amphotericin B and fluconazole are traditional drugs used for treatment and prophylaxis in hospital settings. The accurate identification of yeasts and their virulenceassociated factors is essential for predicting the response to antifungal agents, and for detecting the emergence of yeasts with increased tolerance or antifungal resistance mechanisms. The current study found that non-C. albicans Candida spp. had higher fluconazole MICs. It was also shown that continuous exposure to low doses of antifungal agents and the capacity for high-intensity biofilm formation with high numbers of viable cells may explain this increased tolerance and the possible development of resistance mechanisms. Franc¸a et al. [34] reported 56 % mortality in cases of candidemia, although 68 % of these patients had received antifungal treatments and most strains had a favorable susceptibility profile. This suggested that other factors are involved in the patient’s prognosis [34], two of which may be the capacity for biofilm formation and the expression of a diversity of virulence-associated factors. Given the results of this study, it is remarkable that C. albicans remains the only widely studied Candida species, although this high clinical interest is because it is the
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main source of fungal infections in humans. However, infections caused by Candida non-C. albicans spp. have been increasing in several parts of the world, and the current data support this trend. The increased prevalence of these agents in infections may be associated with a higher frequency of virulenceassociated factors. There is also an emerging need for health surveillance programs to monitor possible changes in the distributions of yeast species and their susceptibility profiles and to establish the epidemiological chain during infection, which may facilitate successful interventions in the infection–environmental contamination cycle. Further studies should be conducted in hospitals, and investments must be made in training to prevent and control fungal contamination, colonization, and HIs with or without any apparent risk of morbidity and mortality. In conclusion, the results of the current study showed that the Candida genus was highly prevalent in samples collected from hospital environments. These contaminants may provide a continuous reservoir for infection with these yeasts. Candida non-C. albicans spp. predominated in samples collected from hospital environments and in infections. These species had higher fluconazole and amphotericin B MICs, and their biofilms had higher metabolic activities than biofilms produced by C. albicans, which may explain the higher frequency of fungal infections with these yeasts. Furthermore, Candida spp. were frequently isolated from catheter tips, which may contribute to the spread, progression, and persistence of HIs. Acknowledgments Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais-FAPEMIG (APQ-01684-08 e APQ00518-11). Conflict of interest All authors report no conflicts of interest relevant to this study.
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