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In vitro inhibitory activity of EDTA against planktonic and adherent cells ofCandida sp.

In vitro inhibitory activity of EDTA against planktonic and adherent cells ofCandida sp. Annals of Microbiology, 57 (1) 115-119 (2007) In vitro inhibitory activity of EDTA against planktonic and adherent cells of Candida sp. 1 1 2 2 Beata CHUDZIK *, Anna MALM , Barbara RAJTAR , Malgorzata POLZ-DACEWICZ 1 2 Department of Pharmaceutical Microbiology, Department of Virology, Medical University of Lublin, 1 Chodzki Street, 20-093 Lublin, Poland Received 13 September 2006 / Accepted 10 January 2007 Abstract - Candida sp. can cause infections of indwelling medical devices associated with biofilm formation, which are difficult to treat due to insensitivity of adherent microorganisms to host defence mechanisms and standard antimicrobial therapy. The aim of this paper was to determine the effect of EDTA (disodium salt) on the adhesion of Candida sp. to some catheters and also on biofilm formation by the yeasts and its eradication in relation to cytotoxicity of this chelating agent to the cell cultures. The adhesion process and biofilm for- mation, and also EDTA cytotoxicity to green monkey kidney (GMK) cell culture were determined using MTT tetrazolium salt [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] reduction assay. EDTA inhibited the growth of free-floating forms of Candida sp. strains with minimal inhibitory concentration (MIC) from 0.06 to 0.25 mM; the minimal fungicidal concentration (MFC) values ranged from 64 to 128 mM. The prevention of Candida sp. adhesion on the catheters used or eradication of the adherent cells was achieved at 0.5 to 4.0 mM EDTA. Also biofilm formation was prevented by 0.5 to 4.0 mM EDTA. Much higher concentration of EDTA (32 to 128 mM) was needed to eradicate the mature biofilm. EDTA at concentration up to 1 mM was not toxic for GMK cells. At higher concentration, toxicity of EDTA to GMK cells was correlated with the concentration of this agent and the time of exposure. Summing up, EDTA may be regarded as a useful agent rather in prophylaxis of candidal infections of medical devices. Key words: Candida sp., biofilm, EDTA, biomaterials. INTRODUCTION Several authors have studied the efficacy of EDTA (disodi- um or tetrasodium salt) alone or in combination with other The yeast-like fungi belonging to the genus of Candida can agents (e.g. minocycline) against clinically relevant microor- cause a variety of superficial and deep-seated mycoses. ganisms, most commonly isolated from hospital required Recently, these microorganisms have been involved in medical device-related infections, including Candida sp. nosocomial infections associated with indwelling medical (Kite et al., 2004; Percival et al., 2005; Raad et al., 1997, devices, complicated by catheter-related blood-stream 2003, 2006). The aim of this paper was to determine the infections (Raad et al., 2003; Ramage et al., 2005). The effect of disodium EDTA on adhesion of cells of various devices become colonised by the yeast-like fungi that form species of Candida sp. to surfaces of some catheters and a biofilm – an universal, complex, interdependent commu- also on biofilm formation by the yeasts and its eradication nity of surface-linked microbial cells embedded in the matrix in relation to cytotoxicity of this chelating agent to the cell of extracellular polymeric substances (Hawser and Douglas, line cultures. 1994; Jabra-Rizk et al., 2004). Biofilm-associated infections are difficult to treat due to the inherent resistance of adher- ent microorganisms both to host defence mechanisms and MATERIALS AND METHODS standard antimicrobial therapy (Baillie and Douglas, 1999). Although, Candida albicans is predominant etiologic agent of Microorganisms and culture conditions. A total of 7 candidiasis, other species that tend to be less susceptible to strains of Candida sp., possessing hydrophilic or hydropho- the commonly used antifungal drugs such as Candida kru- bic cell surface, were used in this study. The hydrophobici- sei, Candida glabrata or Candida famata have emerged as ty of cell surface was assessed using Salt Aggregation Test opportunistic pathogens (Chandra et al., 2001; Jabra-Rizk (SAT) according to Lindahl et al. (1981). The collection et al., 2004). included the following isolates: C. albicans (1 hydrophilic EDTA is a known metal chelator, showing in vitro and 2 hydrophobic isolates), C. famata (1 hydrophilic and 1 inhibitory activity against Candida sp. (Gil et al., 1994). hydrophobic isolates), C. glabrata (1 hydrophobic isolate), C. krusei (1 hydrophilic isolate). The isolates were obtained from nasopharynx of patients with lung cancer undergoing pulmonary resection, stored at –20 °C in 50% glycerol and cultured on Sabouraud dextrose agar at 30 ºC for 48 h; * * Corresponding author. E-mail: BeChudz@poczta.fm 116 B. Chudzik et al. before each experiment, the isolates were subcultured on fresh Sabouraud medium. Medium changing and catheters Sabouraud glucose broth (further called Sabouraud medi- washing procedures after overnight incubation at 35°C were um) at 30 ºC for 48 h. repeated three times (total incubation period lasted 72 h). (iv) In order to assay the effect of EDTA on biofilm erad- EDTA. Standard powder of EDTA (ethylenediaminete- ication, the mature 72 h-biofilms were incubated in the traacetic acid disodium salt dihydrate, approx. 99% titra- presence of EDTA for 24 h. In all assays a drop of 1% MTT tion) was examined (Sigma). solution was added to each dish. After incubation at 35 °C for 24 h, in the presence of Candida sp. viable cells, MTT Determination of minimal inhibitory concentration was reduced to the violet tetrazolium formazan product, (MIC) and minimal fungicidal concentration (MFC) for accompanied by violet colour of the medium. In each exper- EDTA. Determination of MIC for EDTA was performed by a iment, the control free-EDTA assays were carried out. All broth microdilution method in accordance with the guide- experiments were done in triplicates. The representative lines recommended by CLSI (Clinical and Laboratory data are presented. Standards Institute, 2002), using serial two-fold dilutions of EDTA in Sabouraud medium. Final concentrations of EDTA Cytotoxicity assay. EDTA cytotoxicity to green monkey ranged from 0.03 to 128 mM. Stock inoculum suspensions kidney (GMK) cell was determined by MTT toxicity assay of yeasts were prepared in Sabouraud medium and adjust- (Takenouchi and Munekata, 1998). Monolayers of GMK cells ed to optical density corresponding to 0.5 Mc Farland stan- were grown in Eagle’s Minimal Essential Medium (MEM) sup- dard, i.e. 1.5 x 10 CFU (Colony Forming Units)/ml. After plemented with 100 µg/ml of streptomycin and 100 U/ml of incubation at 35 °C for 48 h, the MICs were assessed visu- penicillin. After overnight incubation at 37 ºC, the medium ally as the lowest EDTA concentration showing complete above the cell culture was removed. To the 2 ml samples of growth inhibition. In order to determine the MFC for EDTA, MEM without calf serum EDTA was added to obtain final con- 10 µl from each tube that showed thorough growth inhibi- centrations ranging from 0.12 to 64 mM. Control assays tion, from the last positive one and from the growth control containing only cells of GMK in MEM were also carried out. was streaked onto Sabouraud dextrose agar plates. After Cell cultures were incubated at 37 ºC for 24 or 72 h with the incubation at 35 °C for 48 h, the MFCs were assessed changing medium every day. After incubation, medium visually as the lowest EDTA concentration from ≤ 1 colony above the cell culture was removed and 1 ml of MEM with- was visible on the agar plate (Pujol et al., 2000). All exper- out calf serum and 100 µl of 5 mg/ml MTT solution in PBS iments were done in triplicates. The representative data are were added to the samples and the incubation was contin- presented. ued for another 4 h at 37 ºC before addition of 1 ml of aque- ous solution containing 50% dimethylformamide and 20% Biomaterials. All assays were carried out on two types of solution of SDS to solubilise the insoluble formazan precipi- catheters that differed in unevenness of surface from each tates produced by MTT reduction. The absorbance of con- other, silicone elastomer-coated latex urinary Foley catheter verted dye was measured at two wavelengths: 540 nm and PCV Thorax catheter. (A ) and 620 nm (A ), using ELISA plate reader 540 620 (Organon Technika Microwell System Reader 530). All The effect of EDTA on adhesion of Candida sp. and experiments were done in triplicates. The mean values (± biofilm formation on the biomaterials. The adhesion SD) were presented. process and biofilm formation were determined by using MTT tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] reduction assay (Levitz et al., RESULTS 1985). The used catheters were cut aseptically into ca 0.5 cm fragments and placed into Petri dishes. The standard- As shown by scanning electron micrographs (data not pre- ised yeast suspensions (optical density of 0.5 Mc Farland sented), the structures of both catheters used in this study standard) were prepared. In each experiment, various con- were different in the degree of surface roughness. However, centrations of EDTA (0.03-128 mM) were used. we have found biofilm formation by all Candida sp. strains, (i) In order to assay the effect of EDTA on adhesion both hydrophilic and hydrophobic isolates, on two catheters. process, the yeast suspensions in sterile PBS (phosphate- This was monitored by formation of violet tetrazolium for- buffered saline) containing EDTA were incubated with bio- mazan product on inside and outside surfaces of catheters materials at 35 °C for 1 h. Nonadherent cells were removed and violet coloured medium after addition of MTT solution. by careful rinsing catheter discs with sterile phosphate- According to Table 1, EDTA inhibited growth of all buffered saline (PBS) and then resuspended in Sabouraud Candida sp. strains (free-floating forms) with MIC ranging medium, followed by overnight incubation at 35 °C. from 0.06 to 0.25 mM. The MFC values for EDTA were much (ii) In order to assay the effect of EDTA on eradication of higher, from 64 to 128 mM, resulting in the high MFC/MIC adherent cells, the yeast suspensions in sterile PBS were values. incubated with biomaterials at 35 °C for 1 h. Nonadherent As shown Table 2, adhesion process of Candida sp. on cells were removed by careful rinsing catheter discs with both catheters was prevented from 0.5 to 4 mM EDTA. The sterile PBS and then resuspended in Sabouraud medium adherent cells of Candida sp. were eradicated from both containing EDTA, followed by overnight incubation at 35 °C. catheters at similar EDTA concentrations applied for 24 h. (iii) In order to assay the effect of EDTA on biofilm for- Prevention of adhesion process and eradication of adherent mation, the yeast suspensions in Sabouraud medium con- cells were monitored by lack of formation of violet tetrazoli- taining EDTA were incubated with biomaterials at 35 °C for um formazan product after addition of MTT. 24 h. Nonadherent cells were removed by careful rinsing Data presented in Table 3 showed that biofilm formation catheter discs with sterile PBS and then resuspended in by Candida sp. on both catheters was prevented between Ann. Microbiol., 57 (1), 115-119 (2007) 117 TABLE 1 - In vitro activity of EDTA against planktonic cells of DISCUSSION Candida sp. Candida sp. infections can be regarded as an important Strain MIC (mM) MFC (mM) MFC/MIC medical problem, especially in the immunocompromised patients, e.g. in lung cancer patients (Chandra et al., 2001). C. albicans 0.062 64 1032 hydrophilic Besides, according to our unpublished data, Candida sp. iso- C. albicans 0.25 128 (70)* 512 (280) lates (e.g. C. krusei) have shown to possess a potential abil- hydrophobic ity to colonise pleural drains in patients undergoing pul- C. albicans 0.25 128 512 monary resection. Although fungal infections of medical hydrophobic devices are less common than bacterial infections, they are C. famata 0.125 128 1024 most difficult to treat (Chandra et al., 2001). hydrophobic Using MTT method, we have found out that all assayed C. famata 0.125 128 (85)* 1024 (680) strains of Candida sp. were able to form biofilm on both hydrophilic used catheters, irrespective of the kind of polymer (silicone, C.glabrata 0.125 64 512 PCV) and hydrophobic or hydrophilic cell surface of the hydrophobic yeasts. It is known (Strevett and Chen, 2003) that several C. krusei 0.25 64 256 non-specific or specific interactions between microorgan- hydrophilic isms and an array of surfaces, including biomaterials, are involved in microbial cell attachment and biofilm develop- * In parentheses detailed MFC values determined by titration at ment. One of the most important factors appears to be EDTA concentrations between 64 and 128 mM were presented. microbial cell hydrophobicity (Liu et al., 2004). However, our data suggest that there is no correlation between cell surface hydrophobicity of the assayed Candida sp. strains 0.5 and 4.0 mM of EDTA. In contrast, much higher concen- and their adherent properties to the used catheters. The trations of EDTA (32 to 128 mM) were needed to eradicate obtained data confirm that various species of Candida may the mature biofilm of Candida sp. during 24-h treatment. be regarded as potential etiologic agents in infections asso- Prevention of biofilm formation and eradication of the ciated with indwelling medical devices. mature biofilm was also monitored by the lack of formation EDTA is known to possess anticoagulant properties. of violet tetrazolium formazan product after addition of MTT. Besides, this chelating agent is recommended for some Table 4 shows data concerning EDTA cytotoxicity at con- therapeutic purposes, e.g. hypercalcaemia at lead poisoning centration affecting adhesion of Candida sp. or biofilm for- (Raad et al., 1997) or endodontic therapy in patients with mation and eradicating adherent cells or the mature biofilm. oral candidiasis (Sen et al., 2000). In addition, EDTA, pos- Using MTT cytotoxicity assay, it was found that EDTA at low sessing anticandidal activity in vitro (Gil et al., 1994), may concentration (up to 1 mM) was not toxic for GMK cells after be regarded as a potential agent in prophylaxis and treat- 24 or 72 h treatment, as evidenced by the absorbance of ment of infections of medical devices. the converted dye compared to that in control assay. Data presented in this paper indicate that growth of Microscopy did not reveal any changes in the monolayer. At planktonic (free-floating) Candida sp. was inhibited by EDTA higher EDTA concentration, the rate of toxic effect on GMK at concentration ranging from 0.06 to 0.25 mM. It should be cells monitored by decreased absorbance of the converted noted that MIC values for EDTA for the assayed Candida sp. dye, accompanied by the destroyment of the monolayer, clinical isolates were comparable to those described by was proportional with concentration of the chelating agent other authors for clinical isolates of the yeasts (Raad et al., and the time of cell treatment. 1997). In contrast, the obtained MFC values were much TABLE 2 - In vitro effect of EDTA on adhesion process of Candida sp. to biomaterials determined by MTT assay Strain Minimal concentration preventing Minimal concentration eradicating adhesion process (mM) adhesion process (mM) Thorax catheter Foley catheter Thorax catheter Foley catheter C. albicans 22 22 hydrophilic C. albicans 0.5 2 0.5 1 hydrophobic C. albicans 44 44 hydrophobic C. famata 24 24 hydrophobic C. famata 0.5 0.5 1 1 hydrophilic C.glabrata 0.5 2 2 2 hydrophobic C. krusei 12 12 hydrophilic 118 B. Chudzik et al. TABLE 3 - In vitro effect of EDTA on biofilm-embedded cells of Candida sp. determined by MTT assay Strain Minimal concentration preventing Minimal concentration eradicating biofilm formation (mM) biofilm (mM) Thorax catheter Foley catheter Thorax catheter Foley catheter C. albicans 2 2 64 64 hydrophilic C. albicans 0.5 0.5 64 64 hydrophobic C. albicans 2 4 128 128 hydrophobic C. famata 2 4 64 64 hydrophobic C. famata 2 2 128 (80)* 32 hydrophilic C.glabrata 2 2 32 32 hydrophobic C. krusei 2 2 64 64 hydrophilic * In parenthesis EDTA concentration determined by titration between 64 and 128 mM were pre- sented. TABLE 4 - Effect of EDTA on viability of GMK cells determined by MTT assay EDTA conc. Viability (nM) After 24 h After 72 h 540 nm 620 nm 540 nm 620 nm None *1.502 ± 0.067* 1.005 ± 0.045 1.593 ± 0.168 1.032 ± 0.095 0.25 1.581 ± 0.196 1.041 ± 0.124 1.614 ± 0.111 1.046 ± 0.067 0.5 1.421 ± 0.020 0.947 ± 0.015 1.481 ± 0.179 0.918 ± 0.089 1 1.458 ± 0.032 0.962 ± 0.006 1.530 ± 0.044 1.005 ± 0.029 2 0.924 ± 0.216 0.581 ± 0.129 0.649 ± 0.159 0.394 ± 0.096 4 0.496 ± 0.058 0.300 ± 0.042 0.242 ± 0.011 0.117 ± 0.005 32 0.395 ± 0.013 0.264 ± 0.004 0.103 ± 0.004 0.077 ± 0.022 64 0.259 ± 0.061 0.185 ± 0.041 0.049 ± 0.025 0.006 ± 0.002 128 0.231 ± 0.073 0.170 ± 0.037 0.045 ± 0.005 0.005 ± 0.002 * Values are mean ± SD. higher in comparison to those presented in the literature centration 30 mg/ml (about 81 mM) or 40 mg/ml (about (Raad et al., 1997). This suggests differential, strain- 108 mM). However, according to our data, the high con- dependent fungicidal effect of EDTA against Candida sp. centration of EDTA showed toxic effect on viability of cell The growth of the assayed Candida sp. in presence of cultures. On the other hand, in special clinical cases, e.g. the fragments of catheters was inhibited at concentration treatment of hypercalcaemia at lead poisoning EDTA was somewhat higher than MIC values, ranging from 0.5 to 4.0 used at much higher concentration – about 8.1 M (Raad et mM. This is in agreement with the general view that the al., 1997). It should be noted that eradicating EDTA effect adherent forms of microorganisms become resistant to against microorganisms (e.g. C. parapsilosis) embedded in agents that are active against the planktonic forms of the biofilm could be potentiated by combination of this agent same organism (Hawser, 1996; Baillie et al., 1999; Chandra with minocycline or with minocycline and 25% ethanol due et al., 2001; Raad et al., 2003; Jabra-Rizk et al., 2004). Our to their synergistic activity (Raad et al., 1997, 2006). data indicate that eradication of the preformed, mature It is possible that inhibitory activity of EDTA against biofilm of Candida sp. required high concentrations of EDTA adherent cells of Candida sp. is due to its inhibitory effect on (32 to 128 mM) comparable to those showing fungicidal the yeast-to-mycelium transition (Gil et al., 1994; Percival activity for planktonic cells of the yeasts. Also other authors et al., 2005). Moreover, the well-known metal-chelating (Percival et al., 2005; Raad et al., 1997, 2003) found that activity of EDTA may be primarily involved in inhibition of di- or tetra-sodium EDTA alone might be effective in eradi- Candida sp. attachment to biomaterials. Our data suggest cation of catheter-associated biofilms of C. albicans at con- that there is no correlation between cell surface hydropho- Ann. Microbiol., 57 (1), 115-119 (2007) 119 bicity or hydrophilicity of the assayed Candida sp. strains, Hawser S. (1996). Comparisons of the susceptibilities of plank- tonic and adherent Candida albicans to antifungal agents: a even among strains belonging to the same species, e.g. two modified XTT tetrazolium assay using synchronised C. albi- hydrophobic isolates of C. albicans. Also, the sensitivity to cans cells. J. Med. Vet. Mycol., 34: 149-152. EDTA of the yeasts in the form of planktonic, adherent or Jabra-Rizk M.A., Falkler W.A., Meiller T.F. (2004). Fungal biofilms embedded in biofilm cells is not correlated with cell surface and drug resistance. Emerg. Infect. Dis., 10: 14-19. properties. However, these problems require further stud- Kite P., Eastwood K., Sugden S., Percival S.L. (2004). Use of in ies. vivo-generated biofilms from hemodialysis catheters to test Several studies were undertaken to manage microbial the efficacy of a novel antimicrobial catheter lock for biofilm biofilm formation on biomaterials, including the incorpora- eradication in vitro. J. Clin. Microbiol., 42: 3073-3076. tion of antimicrobial agents into biomaterials, changing sur- Levitz S.M., Diamond R.D. (1985). A rapid colorimetric assay of face chemistry of biomaterial or lock treatment (Raad et al., fungal viability with the tetrazolium salt MTT. J. Infect. Dis., 1997, 2003, 2006; Percival et al., 2005). Our data and 152: 938-944. those from literature indicate that EDTA, at relatively low Lindahl M., Faris A., Wadstrom T., Hjerten S. (1981). A new test concentration, may be useful in preventing colonisation by based on ‘salting out’ to measure relative surface hydropho- both C. albicans and non-albicans Candida sp. of various bicity of bacterial cells. Biochim. Biophys. Acta, 677: 471-476. type catheters, and hence in prophylaxis of candidal infec- Liu Y., Yang S.F., Li Y., Xu H., Qin L., Tay J.H. (2004). The influ- tions associated with indwelling medical devices. Besides, ence of cell and substratum surface hydrophobicities on the MTT tetrazolium assay is a simple and useful method in microbial attachment. J. Biotechnol., 110: 251-256. the estimation of adhesion and biofilm formation by Candida Percival S.L., Kite P., Eastwood K., Murga R. (2005). Tetrasodium sp. and in screening the assessment of effects of various EDTA as a novel central venous catheter lock solution against agents on these processes. biofilm. Infect. Control Hosp. Epidemiol., 26: 515-519. Data presented in this paper indicate that EDTA may be Pujol I., Aguilar C., Fernándes-Ballart J., Guarro J. (2000). regarded as a useful agent rather in prophylaxis of candidal Comparison of the minimum fungicidal concentration of infections of medical devices but not in the eradication of amphotericin B determined in filamentous fungi by macrodi- the preformed Candida sp. biofilm due to its cytotoxicity to lution and microdilution methods. Med. Mycol., 38: 23-26. cell cultures at higher concentration. Raad I., Buzaid A., Rhyne J., Hachem R., Darouiche R., Safar H., Albitar M., Sherertz R.J. (1997). Minocycline and ethylenedi- Acknowlegments aminetetraacetate for the prevention of recurrent vascular catheter infections. Clin. Infect. Dis., 25: 149-151. This work was supported by a grant from European Social Found (Agreement No. Z/2.06/II/2.6/09/04/U/06/04). Raad I., Chatzinikolaou I., Chaiban G., Hanna H., Hachem R., Dvorak T., Cook G., Costerton W. (2003). In vitro and ex vivo activities of minocycline and EDTA against microorganisms embedded in biofilm on catheter surfaces. Antimicrob. Agents REFERENCES Chemother., 47: 3580-3585. Raad I., Hanna H., Dvorak T., Chaiban G., Hachem R. (2006). Baillie G.S., Douglas L.J. (1999). Candida biofilms and their sus- ceptibility to antifungal agents. Method Enzymol., 310: 644- Optimal antimicrobial catheter lock solution, using different 656. combination of miocycline, EDTA and 25% ethanol: rapid eradication of organisms embedded in biofilm. Antimicrob. Chandra J., Kuhn D.M., Mukherjee P.K., McCormick T., Agents Chemother. Published online ahead of print on 30 Ghannoum M.A. (2001). Biofilm formation by the fungal October 2006. pathogen Candida albicans: development, architecture, and drug resistance. J. Bacteriol., 183: 5385-5394. Ramage G., Saville S.P., Thomas D.P., López-Ribot J.L. (2005). Candida biofilms: an update. Eukaryot. Cell, 4: 633-638. CLSI - Clinical and Laboratory Standards Institute (2002). NCCLS document M27-A2. Reference method for broth dilution anti- Sen B.H., Akdeniz B.G., Denizici A.A. (2000). The effect of ethyl- fungal susceptibility testing of yeasts; Approved standard- enediamine-tetraacetic acid on Candida albicans. Oral Surg. Second Edition, Wayne, Pennsylvania, USA. Oral Med. Oral Pathol. Radiol. Endod., 90: 651-655. Gil M.L., Casanova M., Martinez J.P. (1994). Changes in the cell Strevett K.A., Chen G. (2003). Microbial surface termodynamics wall glycoprotein composition of Candida albicans associated and applications. Res. Microbiol., 154: 329-335. to the inhibition of germ tube formation by EDTA. Arch. Microbiol., 161: 489-494. Takenouchi T., Munekata E. (1998). Amyloid beta-peptide- induced inhibition of MTT reduction in PC12h and C1300 neu- Hawser S.P., Douglas L.J. (1994). Biofilm formation by Candida roblastoma cells: effect of nitroprusside. Peptides, 19: 365- species on the surface of catheter materials in vitro. Infect. Immun., 62: 915-921. 372. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

In vitro inhibitory activity of EDTA against planktonic and adherent cells ofCandida sp.

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Springer Journals
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Copyright © 2007 by University of Milan and Springer
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 57 (1) 115-119 (2007) In vitro inhibitory activity of EDTA against planktonic and adherent cells of Candida sp. 1 1 2 2 Beata CHUDZIK *, Anna MALM , Barbara RAJTAR , Malgorzata POLZ-DACEWICZ 1 2 Department of Pharmaceutical Microbiology, Department of Virology, Medical University of Lublin, 1 Chodzki Street, 20-093 Lublin, Poland Received 13 September 2006 / Accepted 10 January 2007 Abstract - Candida sp. can cause infections of indwelling medical devices associated with biofilm formation, which are difficult to treat due to insensitivity of adherent microorganisms to host defence mechanisms and standard antimicrobial therapy. The aim of this paper was to determine the effect of EDTA (disodium salt) on the adhesion of Candida sp. to some catheters and also on biofilm formation by the yeasts and its eradication in relation to cytotoxicity of this chelating agent to the cell cultures. The adhesion process and biofilm for- mation, and also EDTA cytotoxicity to green monkey kidney (GMK) cell culture were determined using MTT tetrazolium salt [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] reduction assay. EDTA inhibited the growth of free-floating forms of Candida sp. strains with minimal inhibitory concentration (MIC) from 0.06 to 0.25 mM; the minimal fungicidal concentration (MFC) values ranged from 64 to 128 mM. The prevention of Candida sp. adhesion on the catheters used or eradication of the adherent cells was achieved at 0.5 to 4.0 mM EDTA. Also biofilm formation was prevented by 0.5 to 4.0 mM EDTA. Much higher concentration of EDTA (32 to 128 mM) was needed to eradicate the mature biofilm. EDTA at concentration up to 1 mM was not toxic for GMK cells. At higher concentration, toxicity of EDTA to GMK cells was correlated with the concentration of this agent and the time of exposure. Summing up, EDTA may be regarded as a useful agent rather in prophylaxis of candidal infections of medical devices. Key words: Candida sp., biofilm, EDTA, biomaterials. INTRODUCTION Several authors have studied the efficacy of EDTA (disodi- um or tetrasodium salt) alone or in combination with other The yeast-like fungi belonging to the genus of Candida can agents (e.g. minocycline) against clinically relevant microor- cause a variety of superficial and deep-seated mycoses. ganisms, most commonly isolated from hospital required Recently, these microorganisms have been involved in medical device-related infections, including Candida sp. nosocomial infections associated with indwelling medical (Kite et al., 2004; Percival et al., 2005; Raad et al., 1997, devices, complicated by catheter-related blood-stream 2003, 2006). The aim of this paper was to determine the infections (Raad et al., 2003; Ramage et al., 2005). The effect of disodium EDTA on adhesion of cells of various devices become colonised by the yeast-like fungi that form species of Candida sp. to surfaces of some catheters and a biofilm – an universal, complex, interdependent commu- also on biofilm formation by the yeasts and its eradication nity of surface-linked microbial cells embedded in the matrix in relation to cytotoxicity of this chelating agent to the cell of extracellular polymeric substances (Hawser and Douglas, line cultures. 1994; Jabra-Rizk et al., 2004). Biofilm-associated infections are difficult to treat due to the inherent resistance of adher- ent microorganisms both to host defence mechanisms and MATERIALS AND METHODS standard antimicrobial therapy (Baillie and Douglas, 1999). Although, Candida albicans is predominant etiologic agent of Microorganisms and culture conditions. A total of 7 candidiasis, other species that tend to be less susceptible to strains of Candida sp., possessing hydrophilic or hydropho- the commonly used antifungal drugs such as Candida kru- bic cell surface, were used in this study. The hydrophobici- sei, Candida glabrata or Candida famata have emerged as ty of cell surface was assessed using Salt Aggregation Test opportunistic pathogens (Chandra et al., 2001; Jabra-Rizk (SAT) according to Lindahl et al. (1981). The collection et al., 2004). included the following isolates: C. albicans (1 hydrophilic EDTA is a known metal chelator, showing in vitro and 2 hydrophobic isolates), C. famata (1 hydrophilic and 1 inhibitory activity against Candida sp. (Gil et al., 1994). hydrophobic isolates), C. glabrata (1 hydrophobic isolate), C. krusei (1 hydrophilic isolate). The isolates were obtained from nasopharynx of patients with lung cancer undergoing pulmonary resection, stored at –20 °C in 50% glycerol and cultured on Sabouraud dextrose agar at 30 ºC for 48 h; * * Corresponding author. E-mail: BeChudz@poczta.fm 116 B. Chudzik et al. before each experiment, the isolates were subcultured on fresh Sabouraud medium. Medium changing and catheters Sabouraud glucose broth (further called Sabouraud medi- washing procedures after overnight incubation at 35°C were um) at 30 ºC for 48 h. repeated three times (total incubation period lasted 72 h). (iv) In order to assay the effect of EDTA on biofilm erad- EDTA. Standard powder of EDTA (ethylenediaminete- ication, the mature 72 h-biofilms were incubated in the traacetic acid disodium salt dihydrate, approx. 99% titra- presence of EDTA for 24 h. In all assays a drop of 1% MTT tion) was examined (Sigma). solution was added to each dish. After incubation at 35 °C for 24 h, in the presence of Candida sp. viable cells, MTT Determination of minimal inhibitory concentration was reduced to the violet tetrazolium formazan product, (MIC) and minimal fungicidal concentration (MFC) for accompanied by violet colour of the medium. In each exper- EDTA. Determination of MIC for EDTA was performed by a iment, the control free-EDTA assays were carried out. All broth microdilution method in accordance with the guide- experiments were done in triplicates. The representative lines recommended by CLSI (Clinical and Laboratory data are presented. Standards Institute, 2002), using serial two-fold dilutions of EDTA in Sabouraud medium. Final concentrations of EDTA Cytotoxicity assay. EDTA cytotoxicity to green monkey ranged from 0.03 to 128 mM. Stock inoculum suspensions kidney (GMK) cell was determined by MTT toxicity assay of yeasts were prepared in Sabouraud medium and adjust- (Takenouchi and Munekata, 1998). Monolayers of GMK cells ed to optical density corresponding to 0.5 Mc Farland stan- were grown in Eagle’s Minimal Essential Medium (MEM) sup- dard, i.e. 1.5 x 10 CFU (Colony Forming Units)/ml. After plemented with 100 µg/ml of streptomycin and 100 U/ml of incubation at 35 °C for 48 h, the MICs were assessed visu- penicillin. After overnight incubation at 37 ºC, the medium ally as the lowest EDTA concentration showing complete above the cell culture was removed. To the 2 ml samples of growth inhibition. In order to determine the MFC for EDTA, MEM without calf serum EDTA was added to obtain final con- 10 µl from each tube that showed thorough growth inhibi- centrations ranging from 0.12 to 64 mM. Control assays tion, from the last positive one and from the growth control containing only cells of GMK in MEM were also carried out. was streaked onto Sabouraud dextrose agar plates. After Cell cultures were incubated at 37 ºC for 24 or 72 h with the incubation at 35 °C for 48 h, the MFCs were assessed changing medium every day. After incubation, medium visually as the lowest EDTA concentration from ≤ 1 colony above the cell culture was removed and 1 ml of MEM with- was visible on the agar plate (Pujol et al., 2000). All exper- out calf serum and 100 µl of 5 mg/ml MTT solution in PBS iments were done in triplicates. The representative data are were added to the samples and the incubation was contin- presented. ued for another 4 h at 37 ºC before addition of 1 ml of aque- ous solution containing 50% dimethylformamide and 20% Biomaterials. All assays were carried out on two types of solution of SDS to solubilise the insoluble formazan precipi- catheters that differed in unevenness of surface from each tates produced by MTT reduction. The absorbance of con- other, silicone elastomer-coated latex urinary Foley catheter verted dye was measured at two wavelengths: 540 nm and PCV Thorax catheter. (A ) and 620 nm (A ), using ELISA plate reader 540 620 (Organon Technika Microwell System Reader 530). All The effect of EDTA on adhesion of Candida sp. and experiments were done in triplicates. The mean values (± biofilm formation on the biomaterials. The adhesion SD) were presented. process and biofilm formation were determined by using MTT tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] reduction assay (Levitz et al., RESULTS 1985). The used catheters were cut aseptically into ca 0.5 cm fragments and placed into Petri dishes. The standard- As shown by scanning electron micrographs (data not pre- ised yeast suspensions (optical density of 0.5 Mc Farland sented), the structures of both catheters used in this study standard) were prepared. In each experiment, various con- were different in the degree of surface roughness. However, centrations of EDTA (0.03-128 mM) were used. we have found biofilm formation by all Candida sp. strains, (i) In order to assay the effect of EDTA on adhesion both hydrophilic and hydrophobic isolates, on two catheters. process, the yeast suspensions in sterile PBS (phosphate- This was monitored by formation of violet tetrazolium for- buffered saline) containing EDTA were incubated with bio- mazan product on inside and outside surfaces of catheters materials at 35 °C for 1 h. Nonadherent cells were removed and violet coloured medium after addition of MTT solution. by careful rinsing catheter discs with sterile phosphate- According to Table 1, EDTA inhibited growth of all buffered saline (PBS) and then resuspended in Sabouraud Candida sp. strains (free-floating forms) with MIC ranging medium, followed by overnight incubation at 35 °C. from 0.06 to 0.25 mM. The MFC values for EDTA were much (ii) In order to assay the effect of EDTA on eradication of higher, from 64 to 128 mM, resulting in the high MFC/MIC adherent cells, the yeast suspensions in sterile PBS were values. incubated with biomaterials at 35 °C for 1 h. Nonadherent As shown Table 2, adhesion process of Candida sp. on cells were removed by careful rinsing catheter discs with both catheters was prevented from 0.5 to 4 mM EDTA. The sterile PBS and then resuspended in Sabouraud medium adherent cells of Candida sp. were eradicated from both containing EDTA, followed by overnight incubation at 35 °C. catheters at similar EDTA concentrations applied for 24 h. (iii) In order to assay the effect of EDTA on biofilm for- Prevention of adhesion process and eradication of adherent mation, the yeast suspensions in Sabouraud medium con- cells were monitored by lack of formation of violet tetrazoli- taining EDTA were incubated with biomaterials at 35 °C for um formazan product after addition of MTT. 24 h. Nonadherent cells were removed by careful rinsing Data presented in Table 3 showed that biofilm formation catheter discs with sterile PBS and then resuspended in by Candida sp. on both catheters was prevented between Ann. Microbiol., 57 (1), 115-119 (2007) 117 TABLE 1 - In vitro activity of EDTA against planktonic cells of DISCUSSION Candida sp. Candida sp. infections can be regarded as an important Strain MIC (mM) MFC (mM) MFC/MIC medical problem, especially in the immunocompromised patients, e.g. in lung cancer patients (Chandra et al., 2001). C. albicans 0.062 64 1032 hydrophilic Besides, according to our unpublished data, Candida sp. iso- C. albicans 0.25 128 (70)* 512 (280) lates (e.g. C. krusei) have shown to possess a potential abil- hydrophobic ity to colonise pleural drains in patients undergoing pul- C. albicans 0.25 128 512 monary resection. Although fungal infections of medical hydrophobic devices are less common than bacterial infections, they are C. famata 0.125 128 1024 most difficult to treat (Chandra et al., 2001). hydrophobic Using MTT method, we have found out that all assayed C. famata 0.125 128 (85)* 1024 (680) strains of Candida sp. were able to form biofilm on both hydrophilic used catheters, irrespective of the kind of polymer (silicone, C.glabrata 0.125 64 512 PCV) and hydrophobic or hydrophilic cell surface of the hydrophobic yeasts. It is known (Strevett and Chen, 2003) that several C. krusei 0.25 64 256 non-specific or specific interactions between microorgan- hydrophilic isms and an array of surfaces, including biomaterials, are involved in microbial cell attachment and biofilm develop- * In parentheses detailed MFC values determined by titration at ment. One of the most important factors appears to be EDTA concentrations between 64 and 128 mM were presented. microbial cell hydrophobicity (Liu et al., 2004). However, our data suggest that there is no correlation between cell surface hydrophobicity of the assayed Candida sp. strains 0.5 and 4.0 mM of EDTA. In contrast, much higher concen- and their adherent properties to the used catheters. The trations of EDTA (32 to 128 mM) were needed to eradicate obtained data confirm that various species of Candida may the mature biofilm of Candida sp. during 24-h treatment. be regarded as potential etiologic agents in infections asso- Prevention of biofilm formation and eradication of the ciated with indwelling medical devices. mature biofilm was also monitored by the lack of formation EDTA is known to possess anticoagulant properties. of violet tetrazolium formazan product after addition of MTT. Besides, this chelating agent is recommended for some Table 4 shows data concerning EDTA cytotoxicity at con- therapeutic purposes, e.g. hypercalcaemia at lead poisoning centration affecting adhesion of Candida sp. or biofilm for- (Raad et al., 1997) or endodontic therapy in patients with mation and eradicating adherent cells or the mature biofilm. oral candidiasis (Sen et al., 2000). In addition, EDTA, pos- Using MTT cytotoxicity assay, it was found that EDTA at low sessing anticandidal activity in vitro (Gil et al., 1994), may concentration (up to 1 mM) was not toxic for GMK cells after be regarded as a potential agent in prophylaxis and treat- 24 or 72 h treatment, as evidenced by the absorbance of ment of infections of medical devices. the converted dye compared to that in control assay. Data presented in this paper indicate that growth of Microscopy did not reveal any changes in the monolayer. At planktonic (free-floating) Candida sp. was inhibited by EDTA higher EDTA concentration, the rate of toxic effect on GMK at concentration ranging from 0.06 to 0.25 mM. It should be cells monitored by decreased absorbance of the converted noted that MIC values for EDTA for the assayed Candida sp. dye, accompanied by the destroyment of the monolayer, clinical isolates were comparable to those described by was proportional with concentration of the chelating agent other authors for clinical isolates of the yeasts (Raad et al., and the time of cell treatment. 1997). In contrast, the obtained MFC values were much TABLE 2 - In vitro effect of EDTA on adhesion process of Candida sp. to biomaterials determined by MTT assay Strain Minimal concentration preventing Minimal concentration eradicating adhesion process (mM) adhesion process (mM) Thorax catheter Foley catheter Thorax catheter Foley catheter C. albicans 22 22 hydrophilic C. albicans 0.5 2 0.5 1 hydrophobic C. albicans 44 44 hydrophobic C. famata 24 24 hydrophobic C. famata 0.5 0.5 1 1 hydrophilic C.glabrata 0.5 2 2 2 hydrophobic C. krusei 12 12 hydrophilic 118 B. Chudzik et al. TABLE 3 - In vitro effect of EDTA on biofilm-embedded cells of Candida sp. determined by MTT assay Strain Minimal concentration preventing Minimal concentration eradicating biofilm formation (mM) biofilm (mM) Thorax catheter Foley catheter Thorax catheter Foley catheter C. albicans 2 2 64 64 hydrophilic C. albicans 0.5 0.5 64 64 hydrophobic C. albicans 2 4 128 128 hydrophobic C. famata 2 4 64 64 hydrophobic C. famata 2 2 128 (80)* 32 hydrophilic C.glabrata 2 2 32 32 hydrophobic C. krusei 2 2 64 64 hydrophilic * In parenthesis EDTA concentration determined by titration between 64 and 128 mM were pre- sented. TABLE 4 - Effect of EDTA on viability of GMK cells determined by MTT assay EDTA conc. Viability (nM) After 24 h After 72 h 540 nm 620 nm 540 nm 620 nm None *1.502 ± 0.067* 1.005 ± 0.045 1.593 ± 0.168 1.032 ± 0.095 0.25 1.581 ± 0.196 1.041 ± 0.124 1.614 ± 0.111 1.046 ± 0.067 0.5 1.421 ± 0.020 0.947 ± 0.015 1.481 ± 0.179 0.918 ± 0.089 1 1.458 ± 0.032 0.962 ± 0.006 1.530 ± 0.044 1.005 ± 0.029 2 0.924 ± 0.216 0.581 ± 0.129 0.649 ± 0.159 0.394 ± 0.096 4 0.496 ± 0.058 0.300 ± 0.042 0.242 ± 0.011 0.117 ± 0.005 32 0.395 ± 0.013 0.264 ± 0.004 0.103 ± 0.004 0.077 ± 0.022 64 0.259 ± 0.061 0.185 ± 0.041 0.049 ± 0.025 0.006 ± 0.002 128 0.231 ± 0.073 0.170 ± 0.037 0.045 ± 0.005 0.005 ± 0.002 * Values are mean ± SD. higher in comparison to those presented in the literature centration 30 mg/ml (about 81 mM) or 40 mg/ml (about (Raad et al., 1997). This suggests differential, strain- 108 mM). However, according to our data, the high con- dependent fungicidal effect of EDTA against Candida sp. centration of EDTA showed toxic effect on viability of cell The growth of the assayed Candida sp. in presence of cultures. On the other hand, in special clinical cases, e.g. the fragments of catheters was inhibited at concentration treatment of hypercalcaemia at lead poisoning EDTA was somewhat higher than MIC values, ranging from 0.5 to 4.0 used at much higher concentration – about 8.1 M (Raad et mM. This is in agreement with the general view that the al., 1997). It should be noted that eradicating EDTA effect adherent forms of microorganisms become resistant to against microorganisms (e.g. C. parapsilosis) embedded in agents that are active against the planktonic forms of the biofilm could be potentiated by combination of this agent same organism (Hawser, 1996; Baillie et al., 1999; Chandra with minocycline or with minocycline and 25% ethanol due et al., 2001; Raad et al., 2003; Jabra-Rizk et al., 2004). Our to their synergistic activity (Raad et al., 1997, 2006). data indicate that eradication of the preformed, mature It is possible that inhibitory activity of EDTA against biofilm of Candida sp. required high concentrations of EDTA adherent cells of Candida sp. is due to its inhibitory effect on (32 to 128 mM) comparable to those showing fungicidal the yeast-to-mycelium transition (Gil et al., 1994; Percival activity for planktonic cells of the yeasts. Also other authors et al., 2005). Moreover, the well-known metal-chelating (Percival et al., 2005; Raad et al., 1997, 2003) found that activity of EDTA may be primarily involved in inhibition of di- or tetra-sodium EDTA alone might be effective in eradi- Candida sp. attachment to biomaterials. Our data suggest cation of catheter-associated biofilms of C. albicans at con- that there is no correlation between cell surface hydropho- Ann. Microbiol., 57 (1), 115-119 (2007) 119 bicity or hydrophilicity of the assayed Candida sp. strains, Hawser S. (1996). Comparisons of the susceptibilities of plank- tonic and adherent Candida albicans to antifungal agents: a even among strains belonging to the same species, e.g. two modified XTT tetrazolium assay using synchronised C. albi- hydrophobic isolates of C. albicans. Also, the sensitivity to cans cells. J. Med. Vet. Mycol., 34: 149-152. EDTA of the yeasts in the form of planktonic, adherent or Jabra-Rizk M.A., Falkler W.A., Meiller T.F. (2004). Fungal biofilms embedded in biofilm cells is not correlated with cell surface and drug resistance. Emerg. Infect. Dis., 10: 14-19. properties. However, these problems require further stud- Kite P., Eastwood K., Sugden S., Percival S.L. (2004). Use of in ies. vivo-generated biofilms from hemodialysis catheters to test Several studies were undertaken to manage microbial the efficacy of a novel antimicrobial catheter lock for biofilm biofilm formation on biomaterials, including the incorpora- eradication in vitro. J. Clin. Microbiol., 42: 3073-3076. tion of antimicrobial agents into biomaterials, changing sur- Levitz S.M., Diamond R.D. (1985). A rapid colorimetric assay of face chemistry of biomaterial or lock treatment (Raad et al., fungal viability with the tetrazolium salt MTT. J. Infect. Dis., 1997, 2003, 2006; Percival et al., 2005). Our data and 152: 938-944. those from literature indicate that EDTA, at relatively low Lindahl M., Faris A., Wadstrom T., Hjerten S. (1981). A new test concentration, may be useful in preventing colonisation by based on ‘salting out’ to measure relative surface hydropho- both C. albicans and non-albicans Candida sp. of various bicity of bacterial cells. Biochim. Biophys. Acta, 677: 471-476. type catheters, and hence in prophylaxis of candidal infec- Liu Y., Yang S.F., Li Y., Xu H., Qin L., Tay J.H. (2004). The influ- tions associated with indwelling medical devices. Besides, ence of cell and substratum surface hydrophobicities on the MTT tetrazolium assay is a simple and useful method in microbial attachment. J. Biotechnol., 110: 251-256. the estimation of adhesion and biofilm formation by Candida Percival S.L., Kite P., Eastwood K., Murga R. (2005). Tetrasodium sp. and in screening the assessment of effects of various EDTA as a novel central venous catheter lock solution against agents on these processes. biofilm. Infect. Control Hosp. Epidemiol., 26: 515-519. Data presented in this paper indicate that EDTA may be Pujol I., Aguilar C., Fernándes-Ballart J., Guarro J. (2000). regarded as a useful agent rather in prophylaxis of candidal Comparison of the minimum fungicidal concentration of infections of medical devices but not in the eradication of amphotericin B determined in filamentous fungi by macrodi- the preformed Candida sp. biofilm due to its cytotoxicity to lution and microdilution methods. Med. Mycol., 38: 23-26. cell cultures at higher concentration. Raad I., Buzaid A., Rhyne J., Hachem R., Darouiche R., Safar H., Albitar M., Sherertz R.J. (1997). Minocycline and ethylenedi- Acknowlegments aminetetraacetate for the prevention of recurrent vascular catheter infections. Clin. Infect. Dis., 25: 149-151. This work was supported by a grant from European Social Found (Agreement No. Z/2.06/II/2.6/09/04/U/06/04). Raad I., Chatzinikolaou I., Chaiban G., Hanna H., Hachem R., Dvorak T., Cook G., Costerton W. (2003). 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Published: Nov 20, 2009

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