Abstract Persistent candidemia refers to the continued isolation of the same Candida species in the blood of a candidemic patient despite appropriate therapy. Despite the clinical importance of persistent candidemia, studies have superficially addressed the biological conditions behind this phenomenon. The aim of this study was to evaluate the correlation between the biofilm-forming ability by Candida bloodstream isolates and the persistence of infection. A total of 55 isolates of Candida were tested and characterized in two groups: (i) group I, which included seven patients with persistent candidemia, and (ii) group II, which included 18 patients with nonpersistent candidemia. Microorganisms were identified at the species level by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Biofilm quantification was evaluated by the crystal violet staining method and confocal scanning laser microscopy (CSLM). Molecular tests confirmed the identification of Candida albicans (92% group I and 94% group II) and Candida dubliniensis isolates (8% group I and 6% group II). All 55 isolates were able to form biofilms, but a higher biofilm mass was produced by C. albicans/C. dubliniensis strains cultured from the persistent group (P < .05). Our data suggest that Candida sp. biofilm production should be considered a relevant biologic variable in explaining patients who fail to clear a bloodstream infection despite adequate antifungal treatment with triazoles. Candida albicans, persistent candidemia, biofilm formation, virulence Introduction Candida bloodstream infections (BSIs) have become a major public health problem in tertiary hospitals all over the world. A recent study conducted in 183 US medical centers showed that Candida ranked as the most common cause of bloodstream infections.1 Despite all the advances in medical practices and diagnostic methods, hematogenous candidiasis has a crude mortality rate of approximately 50%.2–4 As observed in different clinical trials, a substantial number of patients develop persistent candidemia due to strains that show in vitro susceptibility despite more than 3 days of adequate antifungal therapy.5–7 The factors related to persistent candidemia are not completely understood, but this entity certainly is associated with a combination of conditions, including prematurity, impaired host immune response, the presence of infected implanted medical devices, pharmacodynamics aspects of the antifungal regimen, drug interactions, continuous exposure to broad-spectrum antibiotics, and endovascular infections.5,8 The presence of a central venous catheter (CVC) or other implanted medical device has been described as one of the most important risk factors for persistent candidemia due to the possibility of biofilm formation on the surface.5,9,10 The biofilm cells are significantly less susceptible to antimicrobial agents and may be able to evade the host immune system, possibly acting as a reservoir for reinfections.10–14 Candida biofilm production has been associated with increased virulence and mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.14–17 However, few studies have examined the role of the formation of biofilms by Candida isolates and the development of persistent candidemia. In the present study, we evaluated biofilm formation by Candida spp. bloodstream isolates recovered from patients who developed single or persistent episodes of candidemia despite appropriate antifungal therapy. Methods Definitions Persistent candidemia was defined as the persistence of sequential positive blood cultures after at least 3 days of antifungal treatment with in vitro activity against the pathogen. In counterpart, patients who were able to clear the bloodstream infections before 3 days of antifungal therapy were considered as having single episodes of candidemia.5,18 Microorganisms We selected two cohorts of patients with candidemia who were included in a multicenter study conducted to evaluate the epidemiology of candidemia in nine medical centers in Brazil:19 (i) group I, which included seven adult patients with persistent candidemia (37 isolates) despite the absence of neutropenia and endovascular infection, CVC removal within 72 h after diagnosis, and exposure to antifungal therapy with in vitro activity against the pathogen; and (ii) group II, which included 18 patients (18 isolates) exhibiting single episodes of candidemia. Identification of Candida spp. by sequencing of the ITS region of rDNA All isolates were identified at the species level by ITS sequencing, as previously described by our group.20 Candida spp. in vitro antifungal susceptibility testing Antifungal susceptibility testing was performed using the Clinical and Laboratory Standards Institute (CLSI) microdilution assay.21 The following antifungal drugs were tested: fluconazole, voriconazole, anidulafungin (Pfizer Incorporated, New York, NY, USA) and amphotericin B (Sigma Chemical Corporation, St Louis, MO, USA). The CLSI-M27-S4 document was used as an interpretative guideline for classifying the isolates as susceptible, susceptible dose dependent or resistant to the antifungal.22 Biofilm production assays Growth conditions and biofilm formation Initially, biofilm formation was studied in all isolates (groups I and II) according to the protocol previously described by Melo et al. (2011).12 Briefly, Candida isolates were cultured into RPMI 1640 medium and grown for 24 h with shaking at 200 rpm at 37°C. The cell cultures were harvested, washed with phosphate-buffered saline (PBS), and the inoculum was adjusted to 0.4 O.D. (optical density) at 530 nm (A530), that corresponds a concentration of 107 cells/ml in RPMI 1640 medium. One hundred microliters of the cell suspension were transferred to each well of 96-well polystyrene flat-bottom plates (Corning Inc. Costar). Biofilms were grown with shaking at 75 rpm for 72 h at 37°C. Each experiment was performed in triplicate, and biofilm quantifications are expressed as the means of 15 readings (wells) per isolate ± standard deviation. For easy handling of selected Candida samples for confocal scanning laser microscopy (CSLM), the biofilms were developed on Thermanox coverslips (Thermo Scientific, Waltham, Massachusetts, USA) in 12-well microtiter plates. The general procedure for biofilm development was similar that for 96-well plates, with the exception the volume of medium containing cells (2 ml) used for the adhesion step.23 For CSLM, we tested all initial isolates recovered from patients with persistent candidemia and four isolates representative of the control group (group II). Biofilm quantification with crystal violet (CV) staining The culture medium was aspirated from mature biofilms, and they were washed twice with sterile PBS to remove non–biofilm-forming cells. For quantification using CV staining, plates were dried for 45 min at 35°C. Biofilms were stained with 110 μl of 0.4% aqueous CV solution. After 45 min, the CV solution was removed, and the wells were washed with sterile distilled water. To unstain the biofilms, 200 μl of 95% ethanol was added, and the plate was incubated for 45 min. One hundred microliters of the solution were transferred to another microplate, and the absorbance was read using a Microplate Reader 680 (BIO RAD, Hercules, USA) at 570 nm. Confocal scanning laser microscopy (CSLM) Biofilms grown in 12-well plates for 48 h were stained with Live/Dead yeast viability kit (Thermo Scientific, Waltham, Massachusetts, USA), which is composed by two fluorescent probes, FUN® 1 and Calcofluor® White M2R. FUN® 1 is a fluorescent dye taken up by fungal cells; in the presence of metabolic viability, it is converted from a diffuse yellow cytoplasmic stain to red. Calcofluor® White binds to cellulose and chitin in cell walls and gives blue fluorescence. The biofilms were stained with the fluorescent probes for 45 min, in the dark, at 37°C. Coverslips were mounted on glass microscope slides using a mounting medium (Fluoromount-G) and images captured with a confocal laser scanning microscope (Leica TCS SP8) equipped with a Plan-Apochromat 63 × objective (numerical aperture 1.4) under oil immersion. The images were generated using Image J software and assembled using Photoshop CS6 software. The images are represented in maximum intensity projections corresponding to the z-series of confocal stacks.24 Statistical analysis We compared the biofilm formation (A570 values) exhibited by the Candida spp. strains from the two groups (persistent versus single episodes of candidemia) by using Student's t test with GraphPad Prism version 5.0 for Windows (GraphPad Software, CA, USA). Differences between groups with p values < 0.05 were considered significant. Nucleotide sequence accession numbers The sequences of Candida spp. generated in this study were deposited in the GenBank (NCBI) database under the following accession numbers: KC905052-KC905081, KF241844-KF241849 and KY996536-KY996553. Results Clinical characteristics of the patients enrolled We selected isolates from two different groups of patients: group I, which included seven patients exhibiting episodes of persistent candidemia, and group II, which included 18 patients exhibiting single episodes of fungemia. At least 50% of the patients in both groups were over 60 years old (57% group I; 50% group II), and a large number of patients had received corticosteroid therapy (29% group I; 44% group II), surgery (71% group I; 61% group II) or mechanical ventilation (29% group I; 44% group II). None of the patients belonging to either group had a previous history of AIDS, cancer, neutropenia (500 cells/mm3), cirrhosis, or organ transplantation. Regarding the antifungal treatment adopted, fluconazole was the initial treatment for 100% of patients in the persistent group and 83% of control patients. Of note, all patients from group I had the central venous catheter removed within 72 h after the diagnosis of candidemia, and all patients with diagnosis of endocarditis were excluded by clinical and laboratorial evaluation. Yeast identification After ITS region sequencing, 34 (92%) out of 37 isolates from group I were identified as C. albicans, and three (8%) were identified as C. dubliniensis. Among the 18 Candida spp. isolates from group II, 17 (94%) isolates were identified as C. albicans, and one (6%) isolate was identified as C. dubliniensis. Antifungal susceptibility All isolates were considered susceptible to all antifungal agents tested. The minimum inhibitory concentration (MIC) ranges for fluconazole, voriconazole, anidulafungin and amphotericin B were 0.125–1, 0.03–0.125, 0.03–0.25, and 0.125–1 mg/l, respectively. Biofilm production Quantification of biofilm formation showed absorbance values at 570 nm (A570) ranging from 0.292 to 1.535 for group I and from 0.155 to 1.073 for group II. As shown in Figure 1, Candida isolates from group I showed higher biofilm formation capability than those from group II. Indeed, the isolates from group I exhibited an A570 value of 0.873 ± 0.36 (mean ± standard deviation [SD]), and the group II isolates had an A570 value of 0.489 ± 0.271 (P = .0002). Figure 1. View largeDownload slide Biofilm production by 55 Candida spp. bloodstream isolates. Isolates recovered from patients with persistent candidemia (group I) presented a greater ability to form biofilms than isolates from group II (***P = .0002). Each symbol depicts the result for one Candida isolate; the black lines show the mean values for the group. Figure 1. View largeDownload slide Biofilm production by 55 Candida spp. bloodstream isolates. Isolates recovered from patients with persistent candidemia (group I) presented a greater ability to form biofilms than isolates from group II (***P = .0002). Each symbol depicts the result for one Candida isolate; the black lines show the mean values for the group. To better illustrate the differences in biofilm formation capability among the Candida spp. isolates from groups I and II, we arbitrarily established three categories of biofilm production based on the quartile values of the full range of Candida spp. tested. Isolates were categorized as low biofilm formers (LBFs) or high biofilm formers (HBFs) if their A570 was <0.432 (first quartile) or >1.07 (third quartile), respectively. Isolates with absorbance values between 0.432 and 1.07 were classed as intermediate biofilm formers (IBFs). In this scenario, 12 out of 37 (32.4%) isolates from group I were found to be HBFs, while in group II only one of the 18 (5.5%) isolates was considered an HBF. To confirm the results generated by crystal violet biofilm assay, we selected 11 isolates to be tested by CSLM, including: all initial Candida isolates recovered from each patient with persistent candidemia (group I) and four isolates representative of the control group (group II). As illustrated by Figure 2, an increase of the area occupied by metabolically active fungal biomass was clearly observed with Candida isolates cultured from patients with persistent candidemia (group I, Fig. 2B, 2C), when contrasted to images provided by the control group (group II, Fig. 2A, 2B). Figure 2. View largeDownload slide Confocal microscopy images of Candida albicans and Candida dubliniensis. After 48 h of growth, biofilms were stained by FUN® 1 (red) and Calcofluor® White M2R (blue). Isolates from grupo II (control group) produced a limited amount of fungal biofilm represented mostly by yeast cell elements (A, B), while isolates from grupo I (persistent candidemia) produced abundant mass of metabolic active filamentous and yeast forms (C, D). This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Confocal microscopy images of Candida albicans and Candida dubliniensis. After 48 h of growth, biofilms were stained by FUN® 1 (red) and Calcofluor® White M2R (blue). Isolates from grupo II (control group) produced a limited amount of fungal biofilm represented mostly by yeast cell elements (A, B), while isolates from grupo I (persistent candidemia) produced abundant mass of metabolic active filamentous and yeast forms (C, D). This Figure is reproduced in color in the online version of Medical Mycology. Discussion In the present study we clearly demonstrated, by using two different assays (crystal violet and confocal microscopy), that C. albicans/ C. dubliniensis isolates obtained from patients with persistent candidemia produced substantially more biofilm mass when compared to a control group of isolates obtained from patients with single episodes of fungemia. Biofilm formation has been considered one of the major virulence attributes of Candida spp., including C. albicans. Using different in vitro and in vivo model systems to study Candida spp. biofilm formation, several authors have demonstrated that sessile cells are much more resistant to antimicrobial agents and host immune factors than planktonic cells.10,13,14 From a clinical perspective, Candida biofilms are associated with negative effects, as they provide a safe environment for cells, acting as reservoirs for persistent sources of infections and potentially adversely affecting the function of implanted devices.10,13,25 In addition, the correlation between biofilm formation and mortality has been suggested by some authors. Tumbarello et al. (2007) reported that the frequency of deaths of patients with infection due to biofilm-forming isolates was greater than that for patients with candidemia caused by non-biofilm-forming isolates.15 Recently, Rajendram et al. (2016) demonstrated that biofilm-forming ability was significantly associated with C. albicans mortality.17 In contrast, Guembe et al. (2014) were not able to correlate biofilm production and crude mortality by candidemia.26 The discrepancies in results related to the clinical impact of biofilm in the mortality rates of candidemia may be secondary to the fact that mortality in these patients is related not only to fungal sepsis but also to the multiple risk factors and severe underlying diseases documented in this population.2,27 Indeed, it seems almost impossible to match all factors than can potentially have an influence on mortality. In our study, instead of using mortality as a clinical outcome to be correlated with biofilm production, we evaluated whether this virulence factor could be associated with persistent fungemia in patients who received adequate antifungal therapy and source control. In addition, we also removed from our cohort all patients with clinical conditions that could impair their phagocyte function, such as cancer, neutropenia, and AIDS, as well as patients with diagnosis of endocarditis. In this cohort, our findings clearly suggest that patients who developed persistent candidemia were infected by Candida strains with strong biofilm-forming ability. The fact that all episodes of persistent candidemia were initially treated with fluconazole, a drug with no antifungal activity against Candida biofilms, confirms our hypothesis that biofilm production should be considered a relevant biologic variable in explaining patients who fail to clear their infection despite adequate antifungal treatment with triazoles. Acknowledgments This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), [grant 2012/04767-1]. A.C.R.S. received a post-doc scholarship from Comissão de Aperfeiçoamento de Pessoal do Nível Superior (CAPES) in the Programa Nacional de Pós-Doutorado (PNPD Program), Brazil. A.L.C. received grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil [CNPq, grant307510/2015-8]. We thank Dr. Fernando Cesar Bizerra for helpful suggestions on experimental design. Declaration of interest A.L.C. has received educational grants from Astellas, Gilead, Pfizer, and United Medical, and research grants from Astellas and Pfizer. All other authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Magill SS , Edwards JR , Bamberg W et al. Multistate point-prevalence survey of health care-associated infections . N Engl J Med . 2014 ; 370 : 1198 – 1208 . Google Scholar CrossRef Search ADS PubMed 2. Colombo AL , Guimaraes T , Sukienik T et al. Prognostic factors and historical trends in the epidemiology of candidemia in critically ill patients: an analysis of five multicenter studies sequentially conducted over a 9-year period . Intensive Care Med . 2014 ; 40 : 1489—1498 . 3. Lortholary O , Renaudat C , Sitbon K et al. 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Medical Mycology – Oxford University Press
Published: Dec 8, 2017
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