Journal of Microbiological Methods 98 (2014) 59–63
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A simple and inexpensive device for biofilm analysis Hala Almshawit ⁎, Ian Macreadie, Danilla Grando School of Applied Sciences, RMIT University, Australia
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 24 December 2013 Accepted 24 December 2013 Available online 31 December 2013 Keywords: Biofilm Calgary Biofilm Device Candida glabrata
a b s t r a c t The Calgary Biofilm Device (CBD) has been described as a technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. In this study a simple and inexpensive alternative to the CBD was developed from polypropylene (PP) microcentrifuge tubes and pipette tip boxes. The utility of the device was demonstrated using Candida glabrata, a yeast that can develop antimicrobial-resistant biofilm communities. Biofilms of C. glabrata were formed on the outside surface of microcentrifuge tubes and examined by quantitative analysis and scanning electron microscopy. Growth of three C. glabrata strains, including a clinical isolate, demonstrated that biofilms could be formed on the microcentrifuge tubes. After 24 h incubation the three C. glabrata strains produced biofilms that were recovered into cell suspension and quantified. The method was found to produce uniform and reproducible results with no significant differences between biofilms formed on PP tubes incubated in various compartments of the device. In addition, the difference between maximum and minimum counts for each strain was comparable to those which have been reported for the CBD device. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Many microorganisms exist in different environments as biofilms. Biofilms are a community of microbial cells that are surface-attached and embedded in a self produced extracellular polymeric matrix (Flemming and Wingender, 2010). These communities can cause significant problems in both medical (e.g. persistent and recurrent infections, device-related infections) and industrial (e.g. biofouling and biocorrosion) settings. However, they can be beneficial as well (e.g. bioremediation) (Singh et al., 2006; Niveditha et al., 2012; Santopolo et al., 2012). In the medical field, adhesion and dispersion of cells from biofilms are an important part of the biofilm developmental cycle as it is associated with disseminated invasive diseases such as candidaemia (Uppuluri et al., 2010). Like many other microorganisms, Candida glabrata cells are able to adhere, surround themselves by an external matrix and establish biofilms on surfaces of medical devices such as central venous and urinary catheters (Nett et al., 2007; Mohammadi et al., 2012; Toulet et al., 2012). C. glabrata is the second most prevalent Candida species after C. albicans to cause candidiasis in immunocompromised patients (Li et al., 2007). The increase in C. glabrata infections has been sustained over recent decades (Fidel et al., 1999; Li et al., 2007; Liang and Zhou, 2007). In addition, the yeast's ability to rapidly develop resistance to fluconazole and develop or acquire high-level resistance to other azoles
Abbreviations: PP, Polypropylene; CBD, Calgary Biofilm Device; CFU, Colony Forming Units; SEM, scanning electron microscope. ⁎ Corresponding author. Tel.: +61 3 9925 7148; fax: +61 3 9925 7110. E-mail addresses: [email protected], [email protected] (H. Almshawit). 0167-7012/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.12.020
following exposure to fluconazole makes the treatment of this infection difficult. C. glabrata has been reported to develop resistance to echinocandins which are considered a useful antifungal agent due to their low toxicity (Posteraro et al., 2006; Pfaller et al., 2012; Alexander et al., 2013). Moreover, the ability of many clinical strains to produce biofilms protects fungal cells from antifungal drugs and thus leads to their persistence as important clinical pathogens (Domergue et al., 2005; Kaur et al., 2005; Calderone and Clancy, 2012). The composition and quantity of the biofilm matrix may vary at different sites of infection and may differ between species. Environmental conditions such as nutrient availability and mechanical stimuli also affect biofilm formation. For instance, it has been demonstrated that matrix synthesis by Candida biofilm cells is minimal in static conditions in comparison to dynamic environments (Al-Fattani and Douglas, 2006; Tournu and Van Dijck, 2012). Several in vitro models have been described that demonstrate the impact of different types of nutritional supplies and surface materials (in flow or static conditions) on adhesion and biofilm properties of several Candida species. These models include a 96-well polystyrene microtiter plate, as well as discs of catheters made from different substrates in 6- or 24-well plates and the Calgary Biofilm Device (CBD) (Ceri et al., 1999 and reviewed by Tournu and Van Dijck (2012)). These models show good reproducibility and some of them have been assembled from readily available laboratory materials (Uppuluri and Lopez-Ribot, 2010). The CBD is one of the most widely used methods for testing the susceptibility of microbial biofilm to antibiotics and toxic compounds (Coenye et al., 2011). It consists of two parts: a lid with 96 pegs, which sits in the wells of a standard 96-well microtiter plate (MBEC Assay System, Innovotech, Canada (Harrison et al., 2006)). The CBD facilitates the production of 96 biofilms grown on pegs. These biofilms
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are used in standard experiments for biofilm susceptibility and minimum biofilm eradication concentration (MBEC) assays after exposure to toxic chemicals (Ceri et al., 2001; ASTM, 2012). Polypropylene (PP) is a chemically inert polymer with highly versatile mechanical properties, which makes it useful in a wide range of medical devices (e.g. catheters, hernia meshes and sutures) (NavaOrtiz et al., 2010). Therefore, development of reproducible and cost effective in vitro PP models to study Candida biofilms is important. This study describes a simple polypropylene (PP) biofilm model that can be easily built from materials commonly available in most microbiological laboratories. This model mimics the CBD and is cheaper than a CBD. Our device uses up to 96 PP tubes in place of pegs used in the CBD. The tubes sit within a recycled pipette tip box instead of the microtiter plate used in the CBD. 2. Materials and methods 2.1. Organisms and media C. glabrata ATCC2001, ATCC90030 and C. glabrata clinical isolate (A2a2) (obtained from a woman infected with vaginal candidiasis (Watson et al., 2013) were used in this study. The strains were characterised using CHROMagar plates (Thermo Fisher, Adelaide, Australia) and their identity confirmed using API ID 32C™ (bioMerieux, Lyon, France). Isolates were retrieved from frozen glycerol stock cultures, streaked to YEPD agar (1% (w/v) yeast extract, 2% (w/v) bacteriological peptone, 2% (w/v) dextrose solidified with 1.5% agar (Cell Biosciences)). The same medium was used for determination of colony forming units (CFUs). YEPD liquid medium was used in the reaction vessel to initiate biofilm formation. 2.2. Biofilm reactor The reactor was assembled manually using commercially available laboratory materials. It consisted of a two-part reaction vessel (Fig. 1a) which was a recycled empty 200 μl pipette tip box (Barrier) with added capped microcentrifuge tubes (400 μl, 7 mm × 47 mm, Beckman). The device can accommodate 96 tubes (Fig. 1a). These parts are made from polypropylene (PP) and can be autoclaved. The box is divided internally into nine compartments (6 large compartments (105 cm3) and 3 small ones (23.8 cm3)), allowing for nine simultaneous separate treatments (Fig. 1b). The surface pipette tip locator plate was modified by enlarging the holes by drilling with an 8 mm drill bit. This enabled enough space around the added microcentrifuge tubes so that they could be inserted and removed without contacting the surface of the tubes. The tubes were stabilised in their positions by the addition of tape on the top 5 mm of the tube (Fig. 4). The box with added tubes was autoclaved before use. In addition, empty tip boxes were also autoclaved for use in second growth rounds (see 2.3). 2.3. Biofilm establishment An inoculum of approximately 107 CFU/ml (OD600 = 1) was prepared by obtaining a cell suspension from colonies formed after growth overnight on solidified YEPD media. The large compartments of the box were filled with 8 ml of the cell suspension by aseptically lifting the tip locator plate complete with tubes or by pipetting directly through an empty tip locator plate opening (the small compartments were not used in this step). The complete biofilm reactor with culture was then incubated at 35 °C with shaking at 220 rpm on a rotary shaker for 2 h (adhesion phase) and then left for ~ 30 min to sediment planktonic cells. The shaking during incubation caused fluid flow around the tubes, generating shear forces across all tubes and allowing cells to either stay in suspension or attach to tubes. Tubes were lifted from the reactor and the surface of the tube was observed microscopically using a Leica DM2500 microscope and photographed with a Leica DFC310 FX camera.
Fig. 1. Biofilm reactor mimicking CBD consists of a 200 μl pipette tips box and 400 μl microcentrifuge tubes. (a) The tip rack without tip locator showing the separate compartments, indicates large compartments (*) and small compartments (**). A & B indicate the compartments that were used for Fig. 3. (b) The complete reactor with PP tubes. A, B and numbers indicate the compartments and rows which were used for Fig. 3. Refer to Section 2.2 for additional description.
A second growth cycle was performed in fresh YEPD to mature biofilm formation. Briefly, the clean sterile compartments of the PP device were filled with YEPD liquid media, 12 to 15 ml for small compartments, 18 ml in the case of larger compartments. The volume of the media depends on the desired amount of biofilm that is wanted to be produced. The most important issue is the similarity in the height of the media in the compartments. Thus equal areas of the used PP tubes will be covered, leading to similar growth. Tubes with established biofilms (2 h) were transferred (by holding them by their caps using sterile forceps) to the new reactor with fresh YEPD. The box was covered by its lid and incubated at 35 °C for 24 h. All these steps were performed aseptically. 2.4. Biofilm enumeration Cells were removed from the surface of the tubes by a 30 s vortex in 3 ml sterile MilliQ water in a 15 ml centrifuge tube (Buck and Andrews, 1999). Viable cell numbers (CFUs) were determined by subculture after
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measuring turbidity of the resulting suspension using a spectrophotometer (Eppendorf). CFU/ml was calculated for the bulk solution which is the 3 ml of MilliQ water. 2.5. Environmental scanning electron microscopy (ESEM) The PP tubes incubated in the tip box (24 h) were washed in MilliQ water to remove media. Washing was performed by dipping the PP tube in and out of 8 ml of sterile MilliQ water in a 10 ml centrifuge tube. The tubes were then immersed in 4% formaldehyde and stored at 4 °C. For ESEM, tubes were dried at room temperature and then visualized using a ESEM (RMIT facility, FEI Quanta 200 ESEM (2002) in low vacuum mode at 10 kV). 2.6. Statistical analysis GraphPad Prism 6 was used to analyse the data statistically. A one way ANOVA test combined with Tukey's multiple comparisons test and e-test were used to analyse the data. 3. Results and discussion 3.1. Biofilm formation and reproducibility Adhesion of cells of C. glabrata clinical isolates on the surface of PP tubes after 2 h was observed by light microscopy (Fig. 2). The clinical isolate had more apparent adhesion than the two control strains after a 2 h incubation. Fig. 3 shows a comparison between cell numbers in biofilms that were formed after 24 h on different tubes in two different compartments, and between different rows of one compartment
Fig. 3. Comparisons between biofilm formation of A2a2 strain at different sites in the PP reactor (a) difference in cell number between two big compartments (b) comparison between cell numbers between different rows in each compartment. ns — no significant difference (P N 0.05), error bars represent the standard error of the mean (SEM).
Fig. 2. Establishment of biofilm by the isolates on the surface of PP microcentrifuge tubes after shaking for 2 h in media. (a) C. glabrata ATCC2001; (b) C. glabrata A2a2.
(Fig. 3). The results show reproducibility of cell counts in different compartments with means of 2.5 × 107 and 2.2 × 107 CFU/ml for compartments A and B respectively and for counts between rows of a single compartment (P N 0.05, Fig. 3A & B). Counting cells from biofilms by vortex disruption has been used in a previous study (Buck and Andrews, 1999). We checked that this process was successful by repeating a further wash and vortex and found that less than 0.1% of the original CFU/ml was obtained through this extra wash (data not shown). The experiment was repeated three times and there was no significant difference in the total number of cells based on location within the reactor. The ability of the clinical A2a2 strain to produce biofilm was compared with the control strains (ATCC2001 and ATCC90030) by counting colonies. Table 1 compares the mean, standard error of the mean, Pvalue, maximum and minimum counts found between biofilms of these strains in the same conditions. The cell concentrations after detachment from biofilms were 2.3 × 107, 5.8 × 105 and 1.1 × 105 CFU/ml for C. glabrata strains A2a2, ATCC2001 and ATCC90030, respectively. The variation between maximum and minimum counts for each strain is comparable to those which were achieved by CBD device (Ceri et al., 1999). This indicates that the reproducibility of this reactor is comparable with the CBD device. Fig. 4 demonstrates a visual difference in biofilm producing ability of the clinical and a control strain. A2a2 was able to build a thick white layer of biofilm whereas the control strain tubes looked clear. This demonstrates that the PP reactor shows a difference between strong biofilm producing strains and weak or non-biofilm forming strains. The experiment was repeated with RPMI 1640 medium
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Table 1 Statistical data on the colonization on PP tubes surface by different C. glabrata strains after 24 h. Means and standard deviations (SD) were calculated based on log10 of CFU/ml per tube. C. glabrata strain
Mean (log10 of CFU/ml per tube)
SD (log10 of CFU/ml per tube)
Maximum count (log10 of CFU/ml per tube)
Minimum count (log10 of CFU/ml per tube)
P value
A2a2 CBS 138 ATCC 90030
7.34 5.61 4.84
0.05 0.50 0.45
7.42 6.07 5.36
7.28 5.07 4.28
A2a2 vs CBS138 (P b 0.01) A2a2 vs ATCC 90030 (P b 0.0001) CBS 138 vs ATCC 90030 (P N 0.05)
supplemented with 2% dextrose (Kucharikova et al., 2011), and coating the tubes with lysine to increase the positive charge on the PP tubes surface (Harrison et al., 2006) or by using foetal calf serum. However, the attachment of C. glabrata ATCC2001 and 90030 could not be improved and the attachment of the clinical isolate was optimum when YEPD medium was used. Scanning electron microscopy of biofilms formed by C. glabrata A2a2 (Fig. 5) showed a discontinuous layer of large blastospores in clusters adhering to the PP tube surface and only small amounts of extracellular polymeric substances (EPS). This means that the biomass of C. glabrata A2a2 in the biofilm is due to the high density of cells rather than production of large amounts of the external matrix. The major advantages offered by the biofilm reactor developed for this study are its affordability and being a partly re-usable device, it leads to less environmental waste. While the CBD device costs 8.5–19 USD (Nunc-Immuno TSP; Innovatech), the reactor described here costs about 3 USD. PP tubes are available for 3 USD per 100 (California Pacific Labs) and PP tips boxes are a negligible cost since these have been recycled. Media is an additional cost for the reactors. The CBD uses 20 ml of media but the device described here needs 168 ml to be filled completely at a cost of 1.3 USD (if using YEPD medium). In addition, this reactor allows the handling of each biofilm separately without disruption to other biofilms and more important is the reproducibility of the quantitative counts of the biofilms produced in this reactor which make it a promising method for measuring the susceptibility of biofilms to antifungal or toxic materials. It is well recognized that the chemical nature of the implant material plays a critical role in initial Candida colonization and thus helps define
the overall characteristics of the resultant Candida biofilms (Tournu and Van Dijck, 2012). The adherence of different C. glabrata strains to polyvinyl chloride (PVC), Teflon, polyurethane, polystyrene, polymethylmetacrylate, hydroxyapatite, silicone elastomer and soft denture liner surfaces has been studied (Hawser and Douglas, 1994; Kuhn et al., 2002; Estivill et al., 2011). Since PP is used widely in medical devices (Nava-Ortiz et al., 2010), we examined the adhesion of C. glabrata cells to PP. The results demonstrate that C. glabrata strains vary in biofilm production on polypropylene under the same conditions of nutrient, temperature and flow. Biofilms that can be formed on discs that are made from urine catheters or other clinical implemented devices (Hawser and Douglas, 1994) can be easily disrupted because of the transfer process using forceps. The tubes used in this study have a top section separate from the biofilm forming site that can be used to handle the tubes without disturbing the biofilm. The washing after the formation of biofilm may not be necessary since unattached cells will be lost under the effect of gravity. However, the washing procedure in our PP reactor is very gentle compared with the washing procedure used in published methods using microtiter plates (Tournu and Van Dijck, 2012). Furthermore, the separate sections (compartments) in the box allow different treatments simultaneously for at least eight replicates of one treatment. We conclude that this reactor is a useful, simple, low cost miniature device for parallel study of Candida glabrata biofilms and might be useful for other microbial species which produce biofilms. Due to the reproducibility of formed biofilms on each tube in the reactor, we believe that this assay will enable rapid and reproductive screening of Candida biofilms for antifungal resistance testing under static or flow conditions.
Fig. 4. Difference between ability of C. glabrata strains to produce biofilms. PP microcentrifuge tubes after incubation for 24 h in the biofilm reactor. C. glabrata ATCC2001 on the left, the clinical isolate A2a2 on the right. The arrow indicates the tape which was used to stabilise the tube in the rack.
Fig. 5. Scanning electron micrograph of Candida glabrata clinical isolate (A2a2) biofilm formed on the biofilm reactor. PP microcentrifuge tubes were removed from the device washed and fixed as described in materials and methods. Note the discontinuous layer of large blastospores in clusters adheres to the PP surface. The inset at right corner on the top indicates the small amounts of extra polymeric substances (white arrows).
H. Almshawit et al. / Journal of Microbiological Methods 98 (2014) 59–63
Acknowledgment The authors acknowledge the facilities, the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the RMIT Microscopy & Microanalysis Facility, at RMIT University. References Alexander, B.D., Johnson, M.D., Pfeiffer, C.D., Jimenez-Ortigosa, C., Catania, J., Booker, R., Castanheira, M., Messer, S.A., Perlin, D.S., Pfaller, M.A., 2013. Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations. Clin. Infect. Dis. 56, 1724–1732. Al-Fattani, M.A., Douglas, L.J., 2006. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J. Med. Microbiol. 55, 999–1008. ASTM Standard E2799, 2012. Standard Test Method for Testing Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Using the MBEC Assay. ASTM International, West Conshohocken, PA. http://dx.doi.org/10.1520/E2799-12, www.astm.org. Buck, J.W., Andrews, J.H., 1999. Attachment of the yeast Rhodosporidium toruloides is mediated by adhesives localized at sites of bud cell development. Appl. Environ. Microbiol. 65, 465–471. Calderone, R.A., Clancy, C.J., 2012. Candida and Candidiasis, Second edition. John Wiley & Sons. Ceri, H., Olson, M.E., Stremick, C., Read, R.R., Morck, D., Buret, A., 1999. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J. Clin. Microbiol. 37, 1771–1776. Ceri, H., Olson, M., Morck, D., Storey, D., Read, R., Buret, A., Olson, B., 2001. The MBEC assay system: multiple equivalent biofilms for antibiotic and biocide susceptibility testing. Methods Enzymol. 337, 377–385. Coenye, T., De Prijck, K., Nailis, H., Nelis, H.J., 2011. Prevention of Candida albicans biofilm formation. Open Mycol. J. 5, 9–20. Domergue, R., Castaño, I., De Las, Peñas A., Zupancic, M., Lockatell, V., Hebel, J.R., Johnson, D., Cormack, B.P., 2005. Nicotinic acid limitation regulates silencing of Candida adhesins during UTI. Science 308, 866–870. Estivill, D., Arias, A., Torres-Lana, A., Carrillo-Muñoz, A.J., Arévalo, M.P., 2011. Biofilm formation by five species of Candida on three clinical materials. J. Microbiol. Meth. 86, 238–242. Fidel, P.L., Vazquez, J.A., Sobel, J.D., 1999. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin. Microbiol. Rev. 12, 80–96. Flemming, H.C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633. Harrison, J.J., Ceri, H., Yerly, J., Stremick, C.A., Hu, Y., Martinuzzi, R., Turner, R.J., 2006. The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device. Biol. Proced. Online 8, 194–215. Hawser, S.P., Douglas, L.J., 1994. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect. Immun. 62, 915–921. Kaur, R., Domergue, R., Zupancic, M.L., Cormack, B.P., 2005. A yeast by any other name: Candida glabrata and its interaction with the host. Curr. Opin. Microbiol. 8, 378–384.
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Kucharikova, S., Tournu, H., Lagrou, K., Van Dijck, P., Bujdakova, H., 2011. Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. J. Med. Microbiol. 60, 1261–1269. Kuhn, D.M., Chandra, J., Mukherjee, P.K., Ghannoum, M.A., 2002. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect. Immun. 70, 878–888. Li, L., Redding, S., Dongari-Bagtzoglou, A., 2007. Candida glabrata: an emerging oral opportunistic pathogen. J. Dent. Res. 86, 204–215. Liang, Q., Zhou, B., 2007. Copper and manganese induce yeast apoptosis via different pathways. Mol. Biol. Cell 18, 4741–4749. Mohammadi, P., Shoaie, N., Roudbar Mohammadi, S., 2012. Isolation and detection of yeast biofilms from urine catheters of infectious patients. Jundishapur J. Microbiol. 5, 533–536. Nava-Ortiz, C.A.B., Burillo, G., Concheiro, A., Bucio, E., Matthijs, N., Nelis, H., Coenyc, T., Alvarez-Lorenzo, C., 2010. Cyclodextrin-functionalized biomaterials loaded with miconazole prevent Candida albicans biofilm formation in vitro. Acta Biomater. 6, 1398–1404. Nett, J., Lincoln, L., Marchillo, K., Andes, D., 2007. β-1,3 glucan as a test for central venous catheter biofilm infection. J. Infect. Dis. 195, 1705–1712. Niveditha, S., Pramodhini, S., Umadevi, S., Kumar, S., Stephen, S., 2012. The isolation and the biofilm formation of uropathogens in the patients with catheter associated urinary tract infections (UTIs). J. Clin. Diagn. Res. 6, 1478–1482. Pfaller, M.A., Castanheira, M., Lockhart, S.R., Ahlquist, A.M., Messer, S.A., Jones, R.N., 2012. Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrata. J. Clin. Microbiol. 50, 1199–1203. Posteraro, B., Sanguinetti, M., Fiori, B., La Sorda, M., Spanu, T., Sanglard, D., Fadda, G., 2006. Caspofungin activity against clinical isolates of azole cross-resistant Candida glabrata overexpressing efflux pump genes. J. Antimicrob. Chemother. 58, 458–461. Santopolo, L., Marchi, E., Frediani, L., Decorosi, F., Viti, C., Giovannetti, L., 2012. A novel approach combining the Calgary Biofilm Device and Phenotype MicroArray for the characterization of the chemical sensitivity of bacterial biofilms. Biofouling 28, 1023–1032. Singh, R., Paul, D., Jain, R.K., 2006. Biofilms: implications in bioremediation. Trends Microbiol. 14, 389–397. Toulet, D., Debarre, C., Imbert, C., 2012. Could liposomal amphotericin B (L-AMB) lock solutions be useful to inhibit Candida spp. biofilms on silicone biomaterials? J. Antimicrob. Chemother. 67, 430–432. Tournu, H., Van Dijck, P., 2012. Candida biofilms and the host: models and new concepts for eradication. Int. J. Microbiol. 845352. Uppuluri, P., Lopez-Ribot, J.L., 2010. An easy and economical in vitro method for the formation of Candida albicans biofilms under continuous conditions of flow. Virulence. 1, 483–487. Uppuluri, P., Chaturvedi, A.K., Srinivasan, A., Banerjee, M., Ramaubramaniam, A.K., Koehler, J.R., Kadosh, D., Lopez-Ribot, J.L., 2010. Dispersion as an important step in the Candida albicans biofilm developmental cycle. PLoS Pathog. 6, 1000828. Watson, C.J., Grando, D., Fairley, C.K., Chondros, P., Garland, S.M., Myers, S., Pirotta, M.A., 2013. Randomized placebo controlled double blind trial of the effects of oral garlic supplements on vaginal Candida colony counts. Brit. J. Obstet. Gynaecol. http://dx.doi.org/ 10.1111/1471-0528.12518.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A simple and inexpensive device for biofilm analysis Hala Almshawit ⁎, Ian Macreadie, Danilla Grando School of Applied Sciences, RMIT University, Australia
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 24 December 2013 Accepted 24 December 2013 Available online 31 December 2013 Keywords: Biofilm Calgary Biofilm Device Candida glabrata
a b s t r a c t The Calgary Biofilm Device (CBD) has been described as a technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. In this study a simple and inexpensive alternative to the CBD was developed from polypropylene (PP) microcentrifuge tubes and pipette tip boxes. The utility of the device was demonstrated using Candida glabrata, a yeast that can develop antimicrobial-resistant biofilm communities. Biofilms of C. glabrata were formed on the outside surface of microcentrifuge tubes and examined by quantitative analysis and scanning electron microscopy. Growth of three C. glabrata strains, including a clinical isolate, demonstrated that biofilms could be formed on the microcentrifuge tubes. After 24 h incubation the three C. glabrata strains produced biofilms that were recovered into cell suspension and quantified. The method was found to produce uniform and reproducible results with no significant differences between biofilms formed on PP tubes incubated in various compartments of the device. In addition, the difference between maximum and minimum counts for each strain was comparable to those which have been reported for the CBD device. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Many microorganisms exist in different environments as biofilms. Biofilms are a community of microbial cells that are surface-attached and embedded in a self produced extracellular polymeric matrix (Flemming and Wingender, 2010). These communities can cause significant problems in both medical (e.g. persistent and recurrent infections, device-related infections) and industrial (e.g. biofouling and biocorrosion) settings. However, they can be beneficial as well (e.g. bioremediation) (Singh et al., 2006; Niveditha et al., 2012; Santopolo et al., 2012). In the medical field, adhesion and dispersion of cells from biofilms are an important part of the biofilm developmental cycle as it is associated with disseminated invasive diseases such as candidaemia (Uppuluri et al., 2010). Like many other microorganisms, Candida glabrata cells are able to adhere, surround themselves by an external matrix and establish biofilms on surfaces of medical devices such as central venous and urinary catheters (Nett et al., 2007; Mohammadi et al., 2012; Toulet et al., 2012). C. glabrata is the second most prevalent Candida species after C. albicans to cause candidiasis in immunocompromised patients (Li et al., 2007). The increase in C. glabrata infections has been sustained over recent decades (Fidel et al., 1999; Li et al., 2007; Liang and Zhou, 2007). In addition, the yeast's ability to rapidly develop resistance to fluconazole and develop or acquire high-level resistance to other azoles
Abbreviations: PP, Polypropylene; CBD, Calgary Biofilm Device; CFU, Colony Forming Units; SEM, scanning electron microscope. ⁎ Corresponding author. Tel.: +61 3 9925 7148; fax: +61 3 9925 7110. E-mail addresses: [email protected], [email protected] (H. Almshawit). 0167-7012/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.12.020
following exposure to fluconazole makes the treatment of this infection difficult. C. glabrata has been reported to develop resistance to echinocandins which are considered a useful antifungal agent due to their low toxicity (Posteraro et al., 2006; Pfaller et al., 2012; Alexander et al., 2013). Moreover, the ability of many clinical strains to produce biofilms protects fungal cells from antifungal drugs and thus leads to their persistence as important clinical pathogens (Domergue et al., 2005; Kaur et al., 2005; Calderone and Clancy, 2012). The composition and quantity of the biofilm matrix may vary at different sites of infection and may differ between species. Environmental conditions such as nutrient availability and mechanical stimuli also affect biofilm formation. For instance, it has been demonstrated that matrix synthesis by Candida biofilm cells is minimal in static conditions in comparison to dynamic environments (Al-Fattani and Douglas, 2006; Tournu and Van Dijck, 2012). Several in vitro models have been described that demonstrate the impact of different types of nutritional supplies and surface materials (in flow or static conditions) on adhesion and biofilm properties of several Candida species. These models include a 96-well polystyrene microtiter plate, as well as discs of catheters made from different substrates in 6- or 24-well plates and the Calgary Biofilm Device (CBD) (Ceri et al., 1999 and reviewed by Tournu and Van Dijck (2012)). These models show good reproducibility and some of them have been assembled from readily available laboratory materials (Uppuluri and Lopez-Ribot, 2010). The CBD is one of the most widely used methods for testing the susceptibility of microbial biofilm to antibiotics and toxic compounds (Coenye et al., 2011). It consists of two parts: a lid with 96 pegs, which sits in the wells of a standard 96-well microtiter plate (MBEC Assay System, Innovotech, Canada (Harrison et al., 2006)). The CBD facilitates the production of 96 biofilms grown on pegs. These biofilms
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are used in standard experiments for biofilm susceptibility and minimum biofilm eradication concentration (MBEC) assays after exposure to toxic chemicals (Ceri et al., 2001; ASTM, 2012). Polypropylene (PP) is a chemically inert polymer with highly versatile mechanical properties, which makes it useful in a wide range of medical devices (e.g. catheters, hernia meshes and sutures) (NavaOrtiz et al., 2010). Therefore, development of reproducible and cost effective in vitro PP models to study Candida biofilms is important. This study describes a simple polypropylene (PP) biofilm model that can be easily built from materials commonly available in most microbiological laboratories. This model mimics the CBD and is cheaper than a CBD. Our device uses up to 96 PP tubes in place of pegs used in the CBD. The tubes sit within a recycled pipette tip box instead of the microtiter plate used in the CBD. 2. Materials and methods 2.1. Organisms and media C. glabrata ATCC2001, ATCC90030 and C. glabrata clinical isolate (A2a2) (obtained from a woman infected with vaginal candidiasis (Watson et al., 2013) were used in this study. The strains were characterised using CHROMagar plates (Thermo Fisher, Adelaide, Australia) and their identity confirmed using API ID 32C™ (bioMerieux, Lyon, France). Isolates were retrieved from frozen glycerol stock cultures, streaked to YEPD agar (1% (w/v) yeast extract, 2% (w/v) bacteriological peptone, 2% (w/v) dextrose solidified with 1.5% agar (Cell Biosciences)). The same medium was used for determination of colony forming units (CFUs). YEPD liquid medium was used in the reaction vessel to initiate biofilm formation. 2.2. Biofilm reactor The reactor was assembled manually using commercially available laboratory materials. It consisted of a two-part reaction vessel (Fig. 1a) which was a recycled empty 200 μl pipette tip box (Barrier) with added capped microcentrifuge tubes (400 μl, 7 mm × 47 mm, Beckman). The device can accommodate 96 tubes (Fig. 1a). These parts are made from polypropylene (PP) and can be autoclaved. The box is divided internally into nine compartments (6 large compartments (105 cm3) and 3 small ones (23.8 cm3)), allowing for nine simultaneous separate treatments (Fig. 1b). The surface pipette tip locator plate was modified by enlarging the holes by drilling with an 8 mm drill bit. This enabled enough space around the added microcentrifuge tubes so that they could be inserted and removed without contacting the surface of the tubes. The tubes were stabilised in their positions by the addition of tape on the top 5 mm of the tube (Fig. 4). The box with added tubes was autoclaved before use. In addition, empty tip boxes were also autoclaved for use in second growth rounds (see 2.3). 2.3. Biofilm establishment An inoculum of approximately 107 CFU/ml (OD600 = 1) was prepared by obtaining a cell suspension from colonies formed after growth overnight on solidified YEPD media. The large compartments of the box were filled with 8 ml of the cell suspension by aseptically lifting the tip locator plate complete with tubes or by pipetting directly through an empty tip locator plate opening (the small compartments were not used in this step). The complete biofilm reactor with culture was then incubated at 35 °C with shaking at 220 rpm on a rotary shaker for 2 h (adhesion phase) and then left for ~ 30 min to sediment planktonic cells. The shaking during incubation caused fluid flow around the tubes, generating shear forces across all tubes and allowing cells to either stay in suspension or attach to tubes. Tubes were lifted from the reactor and the surface of the tube was observed microscopically using a Leica DM2500 microscope and photographed with a Leica DFC310 FX camera.
Fig. 1. Biofilm reactor mimicking CBD consists of a 200 μl pipette tips box and 400 μl microcentrifuge tubes. (a) The tip rack without tip locator showing the separate compartments, indicates large compartments (*) and small compartments (**). A & B indicate the compartments that were used for Fig. 3. (b) The complete reactor with PP tubes. A, B and numbers indicate the compartments and rows which were used for Fig. 3. Refer to Section 2.2 for additional description.
A second growth cycle was performed in fresh YEPD to mature biofilm formation. Briefly, the clean sterile compartments of the PP device were filled with YEPD liquid media, 12 to 15 ml for small compartments, 18 ml in the case of larger compartments. The volume of the media depends on the desired amount of biofilm that is wanted to be produced. The most important issue is the similarity in the height of the media in the compartments. Thus equal areas of the used PP tubes will be covered, leading to similar growth. Tubes with established biofilms (2 h) were transferred (by holding them by their caps using sterile forceps) to the new reactor with fresh YEPD. The box was covered by its lid and incubated at 35 °C for 24 h. All these steps were performed aseptically. 2.4. Biofilm enumeration Cells were removed from the surface of the tubes by a 30 s vortex in 3 ml sterile MilliQ water in a 15 ml centrifuge tube (Buck and Andrews, 1999). Viable cell numbers (CFUs) were determined by subculture after
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measuring turbidity of the resulting suspension using a spectrophotometer (Eppendorf). CFU/ml was calculated for the bulk solution which is the 3 ml of MilliQ water. 2.5. Environmental scanning electron microscopy (ESEM) The PP tubes incubated in the tip box (24 h) were washed in MilliQ water to remove media. Washing was performed by dipping the PP tube in and out of 8 ml of sterile MilliQ water in a 10 ml centrifuge tube. The tubes were then immersed in 4% formaldehyde and stored at 4 °C. For ESEM, tubes were dried at room temperature and then visualized using a ESEM (RMIT facility, FEI Quanta 200 ESEM (2002) in low vacuum mode at 10 kV). 2.6. Statistical analysis GraphPad Prism 6 was used to analyse the data statistically. A one way ANOVA test combined with Tukey's multiple comparisons test and e-test were used to analyse the data. 3. Results and discussion 3.1. Biofilm formation and reproducibility Adhesion of cells of C. glabrata clinical isolates on the surface of PP tubes after 2 h was observed by light microscopy (Fig. 2). The clinical isolate had more apparent adhesion than the two control strains after a 2 h incubation. Fig. 3 shows a comparison between cell numbers in biofilms that were formed after 24 h on different tubes in two different compartments, and between different rows of one compartment
Fig. 3. Comparisons between biofilm formation of A2a2 strain at different sites in the PP reactor (a) difference in cell number between two big compartments (b) comparison between cell numbers between different rows in each compartment. ns — no significant difference (P N 0.05), error bars represent the standard error of the mean (SEM).
Fig. 2. Establishment of biofilm by the isolates on the surface of PP microcentrifuge tubes after shaking for 2 h in media. (a) C. glabrata ATCC2001; (b) C. glabrata A2a2.
(Fig. 3). The results show reproducibility of cell counts in different compartments with means of 2.5 × 107 and 2.2 × 107 CFU/ml for compartments A and B respectively and for counts between rows of a single compartment (P N 0.05, Fig. 3A & B). Counting cells from biofilms by vortex disruption has been used in a previous study (Buck and Andrews, 1999). We checked that this process was successful by repeating a further wash and vortex and found that less than 0.1% of the original CFU/ml was obtained through this extra wash (data not shown). The experiment was repeated three times and there was no significant difference in the total number of cells based on location within the reactor. The ability of the clinical A2a2 strain to produce biofilm was compared with the control strains (ATCC2001 and ATCC90030) by counting colonies. Table 1 compares the mean, standard error of the mean, Pvalue, maximum and minimum counts found between biofilms of these strains in the same conditions. The cell concentrations after detachment from biofilms were 2.3 × 107, 5.8 × 105 and 1.1 × 105 CFU/ml for C. glabrata strains A2a2, ATCC2001 and ATCC90030, respectively. The variation between maximum and minimum counts for each strain is comparable to those which were achieved by CBD device (Ceri et al., 1999). This indicates that the reproducibility of this reactor is comparable with the CBD device. Fig. 4 demonstrates a visual difference in biofilm producing ability of the clinical and a control strain. A2a2 was able to build a thick white layer of biofilm whereas the control strain tubes looked clear. This demonstrates that the PP reactor shows a difference between strong biofilm producing strains and weak or non-biofilm forming strains. The experiment was repeated with RPMI 1640 medium
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Table 1 Statistical data on the colonization on PP tubes surface by different C. glabrata strains after 24 h. Means and standard deviations (SD) were calculated based on log10 of CFU/ml per tube. C. glabrata strain
Mean (log10 of CFU/ml per tube)
SD (log10 of CFU/ml per tube)
Maximum count (log10 of CFU/ml per tube)
Minimum count (log10 of CFU/ml per tube)
P value
A2a2 CBS 138 ATCC 90030
7.34 5.61 4.84
0.05 0.50 0.45
7.42 6.07 5.36
7.28 5.07 4.28
A2a2 vs CBS138 (P b 0.01) A2a2 vs ATCC 90030 (P b 0.0001) CBS 138 vs ATCC 90030 (P N 0.05)
supplemented with 2% dextrose (Kucharikova et al., 2011), and coating the tubes with lysine to increase the positive charge on the PP tubes surface (Harrison et al., 2006) or by using foetal calf serum. However, the attachment of C. glabrata ATCC2001 and 90030 could not be improved and the attachment of the clinical isolate was optimum when YEPD medium was used. Scanning electron microscopy of biofilms formed by C. glabrata A2a2 (Fig. 5) showed a discontinuous layer of large blastospores in clusters adhering to the PP tube surface and only small amounts of extracellular polymeric substances (EPS). This means that the biomass of C. glabrata A2a2 in the biofilm is due to the high density of cells rather than production of large amounts of the external matrix. The major advantages offered by the biofilm reactor developed for this study are its affordability and being a partly re-usable device, it leads to less environmental waste. While the CBD device costs 8.5–19 USD (Nunc-Immuno TSP; Innovatech), the reactor described here costs about 3 USD. PP tubes are available for 3 USD per 100 (California Pacific Labs) and PP tips boxes are a negligible cost since these have been recycled. Media is an additional cost for the reactors. The CBD uses 20 ml of media but the device described here needs 168 ml to be filled completely at a cost of 1.3 USD (if using YEPD medium). In addition, this reactor allows the handling of each biofilm separately without disruption to other biofilms and more important is the reproducibility of the quantitative counts of the biofilms produced in this reactor which make it a promising method for measuring the susceptibility of biofilms to antifungal or toxic materials. It is well recognized that the chemical nature of the implant material plays a critical role in initial Candida colonization and thus helps define
the overall characteristics of the resultant Candida biofilms (Tournu and Van Dijck, 2012). The adherence of different C. glabrata strains to polyvinyl chloride (PVC), Teflon, polyurethane, polystyrene, polymethylmetacrylate, hydroxyapatite, silicone elastomer and soft denture liner surfaces has been studied (Hawser and Douglas, 1994; Kuhn et al., 2002; Estivill et al., 2011). Since PP is used widely in medical devices (Nava-Ortiz et al., 2010), we examined the adhesion of C. glabrata cells to PP. The results demonstrate that C. glabrata strains vary in biofilm production on polypropylene under the same conditions of nutrient, temperature and flow. Biofilms that can be formed on discs that are made from urine catheters or other clinical implemented devices (Hawser and Douglas, 1994) can be easily disrupted because of the transfer process using forceps. The tubes used in this study have a top section separate from the biofilm forming site that can be used to handle the tubes without disturbing the biofilm. The washing after the formation of biofilm may not be necessary since unattached cells will be lost under the effect of gravity. However, the washing procedure in our PP reactor is very gentle compared with the washing procedure used in published methods using microtiter plates (Tournu and Van Dijck, 2012). Furthermore, the separate sections (compartments) in the box allow different treatments simultaneously for at least eight replicates of one treatment. We conclude that this reactor is a useful, simple, low cost miniature device for parallel study of Candida glabrata biofilms and might be useful for other microbial species which produce biofilms. Due to the reproducibility of formed biofilms on each tube in the reactor, we believe that this assay will enable rapid and reproductive screening of Candida biofilms for antifungal resistance testing under static or flow conditions.
Fig. 4. Difference between ability of C. glabrata strains to produce biofilms. PP microcentrifuge tubes after incubation for 24 h in the biofilm reactor. C. glabrata ATCC2001 on the left, the clinical isolate A2a2 on the right. The arrow indicates the tape which was used to stabilise the tube in the rack.
Fig. 5. Scanning electron micrograph of Candida glabrata clinical isolate (A2a2) biofilm formed on the biofilm reactor. PP microcentrifuge tubes were removed from the device washed and fixed as described in materials and methods. Note the discontinuous layer of large blastospores in clusters adheres to the PP surface. The inset at right corner on the top indicates the small amounts of extra polymeric substances (white arrows).
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Acknowledgment The authors acknowledge the facilities, the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the RMIT Microscopy & Microanalysis Facility, at RMIT University. References Alexander, B.D., Johnson, M.D., Pfeiffer, C.D., Jimenez-Ortigosa, C., Catania, J., Booker, R., Castanheira, M., Messer, S.A., Perlin, D.S., Pfaller, M.A., 2013. Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations. Clin. Infect. Dis. 56, 1724–1732. Al-Fattani, M.A., Douglas, L.J., 2006. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J. Med. Microbiol. 55, 999–1008. ASTM Standard E2799, 2012. Standard Test Method for Testing Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Using the MBEC Assay. ASTM International, West Conshohocken, PA. http://dx.doi.org/10.1520/E2799-12, www.astm.org. Buck, J.W., Andrews, J.H., 1999. Attachment of the yeast Rhodosporidium toruloides is mediated by adhesives localized at sites of bud cell development. Appl. Environ. Microbiol. 65, 465–471. Calderone, R.A., Clancy, C.J., 2012. Candida and Candidiasis, Second edition. John Wiley & Sons. Ceri, H., Olson, M.E., Stremick, C., Read, R.R., Morck, D., Buret, A., 1999. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J. Clin. Microbiol. 37, 1771–1776. Ceri, H., Olson, M., Morck, D., Storey, D., Read, R., Buret, A., Olson, B., 2001. The MBEC assay system: multiple equivalent biofilms for antibiotic and biocide susceptibility testing. Methods Enzymol. 337, 377–385. Coenye, T., De Prijck, K., Nailis, H., Nelis, H.J., 2011. Prevention of Candida albicans biofilm formation. Open Mycol. J. 5, 9–20. Domergue, R., Castaño, I., De Las, Peñas A., Zupancic, M., Lockatell, V., Hebel, J.R., Johnson, D., Cormack, B.P., 2005. Nicotinic acid limitation regulates silencing of Candida adhesins during UTI. Science 308, 866–870. Estivill, D., Arias, A., Torres-Lana, A., Carrillo-Muñoz, A.J., Arévalo, M.P., 2011. Biofilm formation by five species of Candida on three clinical materials. J. Microbiol. Meth. 86, 238–242. Fidel, P.L., Vazquez, J.A., Sobel, J.D., 1999. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin. Microbiol. Rev. 12, 80–96. Flemming, H.C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633. Harrison, J.J., Ceri, H., Yerly, J., Stremick, C.A., Hu, Y., Martinuzzi, R., Turner, R.J., 2006. The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device. Biol. Proced. Online 8, 194–215. Hawser, S.P., Douglas, L.J., 1994. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect. Immun. 62, 915–921. Kaur, R., Domergue, R., Zupancic, M.L., Cormack, B.P., 2005. A yeast by any other name: Candida glabrata and its interaction with the host. Curr. Opin. Microbiol. 8, 378–384.
63
Kucharikova, S., Tournu, H., Lagrou, K., Van Dijck, P., Bujdakova, H., 2011. Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. J. Med. Microbiol. 60, 1261–1269. Kuhn, D.M., Chandra, J., Mukherjee, P.K., Ghannoum, M.A., 2002. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect. Immun. 70, 878–888. Li, L., Redding, S., Dongari-Bagtzoglou, A., 2007. Candida glabrata: an emerging oral opportunistic pathogen. J. Dent. Res. 86, 204–215. Liang, Q., Zhou, B., 2007. Copper and manganese induce yeast apoptosis via different pathways. Mol. Biol. Cell 18, 4741–4749. Mohammadi, P., Shoaie, N., Roudbar Mohammadi, S., 2012. Isolation and detection of yeast biofilms from urine catheters of infectious patients. Jundishapur J. Microbiol. 5, 533–536. Nava-Ortiz, C.A.B., Burillo, G., Concheiro, A., Bucio, E., Matthijs, N., Nelis, H., Coenyc, T., Alvarez-Lorenzo, C., 2010. Cyclodextrin-functionalized biomaterials loaded with miconazole prevent Candida albicans biofilm formation in vitro. Acta Biomater. 6, 1398–1404. Nett, J., Lincoln, L., Marchillo, K., Andes, D., 2007. β-1,3 glucan as a test for central venous catheter biofilm infection. J. Infect. Dis. 195, 1705–1712. Niveditha, S., Pramodhini, S., Umadevi, S., Kumar, S., Stephen, S., 2012. The isolation and the biofilm formation of uropathogens in the patients with catheter associated urinary tract infections (UTIs). J. Clin. Diagn. Res. 6, 1478–1482. Pfaller, M.A., Castanheira, M., Lockhart, S.R., Ahlquist, A.M., Messer, S.A., Jones, R.N., 2012. Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrata. J. Clin. Microbiol. 50, 1199–1203. Posteraro, B., Sanguinetti, M., Fiori, B., La Sorda, M., Spanu, T., Sanglard, D., Fadda, G., 2006. Caspofungin activity against clinical isolates of azole cross-resistant Candida glabrata overexpressing efflux pump genes. J. Antimicrob. Chemother. 58, 458–461. Santopolo, L., Marchi, E., Frediani, L., Decorosi, F., Viti, C., Giovannetti, L., 2012. A novel approach combining the Calgary Biofilm Device and Phenotype MicroArray for the characterization of the chemical sensitivity of bacterial biofilms. Biofouling 28, 1023–1032. Singh, R., Paul, D., Jain, R.K., 2006. Biofilms: implications in bioremediation. Trends Microbiol. 14, 389–397. Toulet, D., Debarre, C., Imbert, C., 2012. Could liposomal amphotericin B (L-AMB) lock solutions be useful to inhibit Candida spp. biofilms on silicone biomaterials? J. Antimicrob. Chemother. 67, 430–432. Tournu, H., Van Dijck, P., 2012. Candida biofilms and the host: models and new concepts for eradication. Int. J. Microbiol. 845352. Uppuluri, P., Lopez-Ribot, J.L., 2010. An easy and economical in vitro method for the formation of Candida albicans biofilms under continuous conditions of flow. Virulence. 1, 483–487. Uppuluri, P., Chaturvedi, A.K., Srinivasan, A., Banerjee, M., Ramaubramaniam, A.K., Koehler, J.R., Kadosh, D., Lopez-Ribot, J.L., 2010. Dispersion as an important step in the Candida albicans biofilm developmental cycle. PLoS Pathog. 6, 1000828. Watson, C.J., Grando, D., Fairley, C.K., Chondros, P., Garland, S.M., Myers, S., Pirotta, M.A., 2013. Randomized placebo controlled double blind trial of the effects of oral garlic supplements on vaginal Candida colony counts. Brit. J. Obstet. Gynaecol. http://dx.doi.org/ 10.1111/1471-0528.12518.
