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Journal of Clinical Microbiology, April 2002, p. 1244-1248, Vol. 40, No. 4
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.4.1244-1248.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Departments of Clinical Pathology,1 Internal Medicine, Chonnam National University Medical School ,3 Department of Clinical Pathology, Seonam University, College of Medicine, Gwangju, Korea2
Received 8 August 2001/ Returned for modification 22 November 2001/ Accepted 22 January 2002
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ABSTRACT |
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INTRODUCTION |
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Factors that predispose patients to disseminated candidiasis include increased colonization of the gastrointestinal tract by Candida species resulting from prolonged use of broad-spectrum antibacterial agents, disruption of the gastrointestinal mucosal surfaces by cytotoxic agents or hypotension, and neutropenia (6). Central venous catheters (CVCs), however, appear to be the most common risk factor for the development of candidemia in patients without neutropenia or major immunodeficiencies (20; J. H. Rex, Editorial Response, Clin. Infect. Dis. 22:467-470, 1996). In addition, biofilm formation has been implicated as a potential virulence factor for at least one Candida species: C. parapsilosis. C. parapsilosis can proliferate in high concentrations of glucose and form biofilms on prosthetic materials. Biofilm formation has been associated with the enhanced capacity of C. parapsilosis to colonize indwelling CVCs (thus providing a reservoir from which the organism may enter the bloodstream) in individuals receiving intravenous hyperalimentation (3). Pfaller et al. (19) studied biofilm production by clinical isolates of C. parapsilosis grown in glucose-containing media. However, biofilm production by bloodstream isolates of Candida species other than C. parapsilosis and its potential relationship to CVC-related candidemia, as well as to other clinical characteristics, have rarely been assessed.
Therefore, we studied clinical bloodstream isolates of C. albicans and non-C. albicans Candida species recovered from nonneutropenic patients at Chonnam National University Hospital and compared Candida species to each other and to isolates from other anatomical sites for the capacity to produce biofilms. We also assessed the association between biofilm production and the clinical characteristics of candidemia, including the number of positive blood cultures, the presence of a CVC-related candidemia, the use of total parenteral nutrition (TPN), the clinical significance of candidemia, and the outcome of candidemia. The results of these investigations are presented here.
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MATERIALS AND METHODS |
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Determination of biofilm production. Biofilm production was assessed by using a modification of the method established by others (3, 10, 19). Briefly, biofilm formation was determined by both visual and spectrophotometric methods. Sabouraud dextrose broth (SDB) was prepared from powdered Sabouraud broth-modified antibiotic medium 13 (BBL, Cockeysville, Md.) according to the manufacturer's instructions, except for supplementation with 60 g of glucose per liter (final glucose concentration, 80 g/liter or 8%). Organisms were grown for 24 h at 35°C on Sabouraud dextrose agar plates (BBL), and saline-washed suspensions of each strain of Candida species were prepared. We used two strains (C. albicans ATCC 90028 and C. parapsilosis ATCC 96142) as controls in each experiment. The turbidity of each suspension was adjusted to the equivalent of 3 x 107 CFU/ml with SDB as determined by comparative plate counts and spectrophotometric readings. Next, 1 ml of suspension was inoculated into a polystyrene tube (Falcon #2095 17-by-120-mm conical tube with a screw cap [Becton Dickinson]) containing 9 ml of SDB. In addition, each well of microtitration plates (Nunclon; Nalge Nunc International, Roskilde, Denmark) was inoculated with aliquots of 20 µl of yeast cell suspension and 180 µl of SDB. Tubes and plates were then incubated at 35°C for 24 h without agitation. We observed that all Candida isolates tested grew well in this medium. In the microtiter plates, Candida isolates grown in this medium had A405 values of >1.0 at 24 h for all species with an initial inoculum of 106 CFU/ml.
After 24 h of incubation, the culture broth in the tube was aspirated gently, and tubes were washed once with distilled water. The walls of the tubes were stained with safranin after media and yeast cells were discarded. The adherent biofilm layer was scored as either negative or weakly (1+), moderately (2+ or 3+), or strongly (4+) positive as described by Pfaller et al. (19). Each isolate was tested at least three times, and two observers scored each tube independently in a blinded fashion. The intra- and interobserver reproducibility for deciding the presence or absence of biofilm by this method was 100% for all Candida species isolates. Since there was some disagreement (mean, 10%) regarding the intensity of positive results among samples, all positive results, including weak, moderate, or strong, were regarded as positive in this study.
After 24 h of incubation, the microtiter plate was also washed once with distilled water by using a microplate washer (ETL Testing Laboratories, Cortland, N.Y.). Spectrophotometric readings were performed at 405 nm with a microtiter plate reader (VERSAmax Tunable Microplate Reader; Molecular Devices Corp., Sunnyvale, Calif.) after 200 µl of distilled water was added to each well. As the percent transmittance (%T) was being measured, a wavelength of 405 nm was selected for reading the plates to minimize absorbance (7). The %T value for each test sample was subtracted from the %T value for the reagent blank to obtain a measure of the amount of light blocked when passing through the wells (%Tbloc). Biofilm production by each isolate was scored as either negative (%Tbloc, < 5), 1+ (%Tbloc, 5 to 20), 2+ (%Tbloc, 20 to 35), 3+ (%Tbloc, 35 to 50), or 4+ (%Tbloc, 
50). Each isolate was tested at least twice. No major discrepancies occurred between results obtained by the visual reading method and those obtained by the spectrophotometric method.
Clinical correlation studies. The hospital records for the 101 nonneutropenic patients from whom the Candida species bloodstream isolates were recovered were reviewed. Biofilm positivity of the isolates was compared with the clinical characteristics of the candidemic episode, including the number of positive blood cultures, the presence of CVC-related candidemia, the use of TPN, the presence of clinically significant infection, and the outcome of the fungemia (cleared or uncleared). Candidemia was defined as CVC-related if no other source of infection was found and if the semiquantitative catheter tip culture yielded more than 15 colonies of the same Candida species (9). A Candida species' isolate was regarded as clinically insignificant and not associated with disease (i) if the patient had only one positive blood culture; (ii) if the candidemia cleared without any specific interventions, such as catheter removal or antifungal therapy; and (iii) the patients did not show any definitive signs and symptoms associated with candidemia. Candidemia was judged as having a "cleared outcome" if blood cultures became negative at any time during a 3-month follow-up period and if signs and symptoms of bloodstream infection (fever, hypotension, or presence of a Candida infection) were resolved (20).
A Candida species isolate obtained from clinical specimens other than blood was defined as clinically significant if it was (i) isolated from closed, normally sterile sites without evidence of contamination; (ii) if multiple specimens of the same patient were positive for the same Candida species; or (iii) if Candida colonies were grown only from confirmed infection sites.
Statistical analysis. Chi-square analysis was used to compare biofilm positivity between different Candida species or between isolates recovered from blood cultures and those recovered from all other sites. Fisher's exact test or the chi-square test was used to compare the biofilm positivity of Candida species isolates with clinical characteristics and outcome. Differences between groups were considered to be significant when P was < 0.05.
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RESULTS |
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Comparison of biofilm production by bloodstream isolates and clinical characteristics of candidemia. Table 2 depicts the relationship between biofilm positivity of Candida species isolates and the presence or absence of clinical characteristics of candidemia. Of the Candida species recovered from the blood of nonneutropenic patients, C. parapsilosis was most frequently isolated (35%, 35 of 101), followed by C. albicans (30%, 30 of 101), C. tropicalis (18%, 18 of 101), C. glabrata (8%, 8 of 101), C. guilliermondii (5%, 5 of 101), C. pelliculosa (3%, 3 of 101), and C. lipolytica (2%, 2 of 101). Of 101 patients with candidemia, 59 (58%) were diagnosed as having CVC-related candidemia. The underlying risk factors for candidemia for 59 patients with CVC-related fungemia were recent operation (n = 30), neurological disease (n = 10), gastrointestinal disease (n = 5), dialysis treatment for chronic renal failure (n = 4), cardiovascular disease (n = 4), prematurity (n = 2), pulmonary disease (n = 2), and acute drug intoxication (n = 2). Of these 59 patients, 46 (78%) were receiving TPN via CVC. When individual Candida species were compared with all other Candida species within a given category, C. parapsilosis was the only species associated with a statistically significant cleared outcome (91% [32 of 35]; P = 0.0061).
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Of 35 bloodstream isolates of C. parapsilosis, 22 were isolated from patients with multiple positive blood cultures, and all 22 of these isolates (100%) were biofilm positive. In contrast, only 8 (62%) of 13 C. parapsilosis isolates that were recovered from blood only once were biofilm-positive (P = 0.0017). The average number of positive blood cultures per patient was 3.0 for C. parapsilosis, 2.7 for C. tropicalis, 2.6 for C. glabrata, 1.7 for C. albicans, and 1.6 for other Candida species. All 23 isolates (100%) of C. parapsilosis recovered from CVC-related candidemia were biofilm positive, but only 7 (53%) of 12 isolates recovered from non-CVC-related candidemia were biofilm positive (P = 0.0008). All 19 isolates (100%) of C. parapsilosis recovered from patients receiving TPN were biofilm positive, but only 11 (69%) of 16 isolates recovered from patients without use of TPN were biofilm positive (P < 0.01). All 27 C. parapsilosis isolates associated with clinically significant disease were biofilm positive, whereas only 3 (38%) of 8 isolates without clinical significance were biofilm positive (P < 0.0001). The biofilm positivity of non-C. albicans Candida species, excluding C. parapsilosis, associated with different clinical characteristics of candidemia was also analyzed. Biofilm positivity was higher for non-C. albicans Candida isolates, other than C. parapsilosis, from CVC-related candidemia (89%, 17 of 19) than from non-CVC-related candidemia (53% [9 of 17]; P < 0.05). In addition, biofilm positivity was higher for non-C. albicans Candida isolates, other than C. parapsilosis, from patients receiving TPN (88%; 15 of 17), than from patients not receiving TPN (58% [11 of 19]; P < 0.05). However, there was no significant association between biofilm positivity and the other three clinical characteristics of candidemia: number of positive blood cultures, clinical significance, and outcome (Table 2).
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DISCUSSION |
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In this study, we used SDB medium that contained high glucose (8%) and protein (1%), which has been used to induce biofilm formation by C. parapsilosis isolates in several studies (3, 10, 19). Although SDB is not a defined medium, it is more similar to the milieu found in vivo (especially within the CVC lumen) of patients receiving TPN via CVC. TPN solutions usually contain high glucose (10 to 70%) and amino acid (up to 50%) concentrations, as well as other nutrients (16). These components result in an acidic pH which varies according to the content and concentration of the amino acids present. In glucose-containing SDB medium, biofilm production by C. albicans isolates was significantly less frequent than that by other Candida isolates, regardless of whether the isolates were derived from blood or other sites. It is not known why C. albicans isolates recovered from blood, even in cases of CVC-related candidemia, demonstrated a lower percentage of biofilm positivity than other Candida species isolates in this study. However, our study suggests that high protein and glucose conditions, mimicking those found in TPN solutions, do not promote biofilm formation by C. albicans isolates. These results suggested that C. albicans isolates possess mechanisms other than biofilm production to establish bloodstream infections. C. albicans is a highly pathogenic Candida species and adhesion may be facilitated by a number of protein receptors on epithelial, endothelial, and foreign body surfaces, including fibronectin (14), fibrinogen (5), and vitronectin (13). Perhaps other virulence factors are more important for the pathogenicity of C. albicans.
We found that considerable differences in biofilm production existed among Candida species grown in high-glucose medium. Biofilm positivity occurred most frequently in isolates of C. tropicalis, followed by C. parapsilosis, C. glabrata, and C. albicans. In contrast, Hawser and Douglas (11) reported that isolates of C. parapsilosis and C. glabrata were significantly less likely to produce biofilms than the more pathogenic C. albicans. However, these studies examined only a few selected strains of different Candida species and used yeast nitrogen base medium containing little glucose (50 nM). Therefore, this medium would not mimic the high glucose content of TPN and may explain the difference in the results obtained by these authors compared to our work.
Our data provide evidence that the majority of non-C. albicans Candida species recovered from the blood of nonneutropenic patients have the capacity to produce significant amounts of biofilm when grown in high-glucose medium. The proportion of biofilm producers was much higher among isolates of non-C. albicans Candida species recovered from blood than it was among isolates recovered from other sites. Of non-C. albicans Candida species, blood isolates of C. parapsilosis were significantly more likely to be biofilm positive than isolates from other sites. Pfaller et al. (19), also by using SDB with 8% glucose, reported that 83% of C. parapsilosis isolates recovered from blood or cultured from catheters were biofilm positive versus 53% of isolates from all other sites, a result similar to our findings. The combined biofilm positivity of bloodstream isolates of all other non-C. albicans Candida species, excluding C. parapsilosis, was also significantly higher than for isolates from other sites. Therefore, the enhanced capacity of isolates of non-C. albicans Candida species from the blood to produce biofilms relative to isolates from other sites suggests that the ability to produce a biofilm may be important in allowing non-C. albicans Candida species to cause candidemia in patients receiving TPN.
We did not find any significant association between biofilm production and the clinical characteristics of candidemia due to C. albicans, since only 2 of 30 blood isolates of C. albicans were biofilm positive. Studies by Hawser and Douglas (11) also failed to reveal any correlation between biofilm formation by different isolates of C. albicans and pathogenicity, even though all of the C. albicans isolates in their study were biofilm producers.
In contrast to C. albicans candidemia, there was a significant association between biofilm positivity of non-C. albicans Candida species and clinical characteristics of candidemia, including the number of positive blood cultures, the presence of a CVC-related candidemia, the use of TPN, and clinical significance of candidemia. The associations between biofilm positivity with two clinical features (i.e., the presence of clinically significant disease and multiple positive blood cultures) appear to be derived from the high proportion of C. parapsilosis isolates in our data set since there were no significant associations between these two clinical features and other non-C. albicans Candida species if the C. parapsilosis isolates were removed from the analysis. The lack of significant association with clinically significant disease and multiple positive blood cultures for individual non-C. albicans Candida species, except C. parapsilosis, may be a reflection of the smaller numbers of bloodstream isolates obtained for each of these Candida species. It may be that significant relationships between clinical disease and biofilm production by non-C. albicans, non-C. parapsilosis Candida species may become apparent in a larger clinical study of isolates. However, despite the small numbers of non-C. albicans, non-C. parapsilosis Candida species isolates present, there was nonetheless a significant association between biofilm production and CVC or TPN use for candidemias caused by non-C. albicans Candida species other than C. parapsilosis, thus underscoring the relationship between biofilm production and CVC or TPN use.
The lack of a correlation between biofilm formation and clinical outcome of fungemia by C. albicans or non-C. albicans Candida species observed in our study suggests that factors other than biofilm formation are involved in the clearance of Candida species from the bloodstream. One factor affecting the outcome of candidemia is catheter removal, which has a powerful effect on the outcome of candidemia (Rex, Editorial Response). Since 1997, our hospital guidelines recommended removing a CVC at the first occurrence of candidemia. Of the 14 patients who died with catheters in place, 11 were admitted between 1994 and 1996. No deaths occurred among patients with CVC-related fungemia whose CVCs were removed except for three patients who died within 48 h of CVC removal (unpublished data). This suggests that early catheter removal has a powerful effect on the outcome of candidemia. Second, most cases of candidemia (80%) in our study had a favorable outcome, which was probably due to the high frequency of CVC-related candidemia in our patients (4; Rex, Editorial Response) or the high incidence of C. parapsilosis candidemia, which clears more easily than bloodstream infections by other Candida species (8, 12, 15, 21).
The spectrum of Candida species causing candidemia may vary by region and hospital (1, 10, 15, 17, 18). At Chonnam National University Hospital (an 850-bed tertiary care hospital), non-C. albicans Candida infections have increased during the past 5 years and account for approximately 70% of all Candida bloodstream infections. The Candida species recovered most frequently from blood was C. parapsilosis. The reason for the rising incidence of candidemia due to non-C. albicans Candida species is not completely understood. However, careful epidemiological studies of candidemia conducted at Chonnam National University Hospital have identified antibiotic exposure (100%), placement of CVCs (72%), and use of TPN (61%) as significant risk factors for the development of candidemia. Almost all (101 of 120) of the patients with candidemia were nonneutropenic (unpublished data). These results suggest that the increased incidence of candidemia due to non-C. albicans Candida species, including C. parapsilosis, in our hospital was mainly from CVC-related candidemia occurring in nonneutropenic patients receiving TPN. In addition, since no antifungal drug exposure occurred during patient hospitalization prior to the collection of bloodstream isolates, selective pressure favoring the growth of Candida species, such as C. glabrata or C. krusei, that are less susceptible to azole was not present.
Of the 59 patients with CVC-related fungemia described in this study, 46 (78%) were receiving TPN via CVC. The non-C. albicans Candida species recovered most frequently from blood at our hospital was C. parapsilosis, which also has a high frequency of biofilm production. Our study demonstrated that CVC-related candidemia due to non-C. albicans Candida species in nonneutropenic patients is much more likely to be associated with biofilm-producing strains than with biofilm-negative organisms. Furthermore, this study suggests that non-C. albicans Candida species, especially C. parapsilosis, have selective advantages for growth and biofilm formation if hyperalimentation fluid is present. These selective advantages appear to be independent of antifungal drug use. Future efforts directed at developing catheter materials that can resist biofilm formation may help to reduce the recent increase in candidemia caused by non-C. albicans Candida species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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