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Evaluation of culture conditions for mixed biofilm formation with clinically isolated non-albicans Candida species and Staphylococcus epidermidis on silicone
Microbial Pathogenesis 112 (2017) 215–220
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Evaluation of culture conditions for mixed biofilm formation with clinically isolated non-albicans Candida species and Staphylococcus epidermidis on silicone
MARK
Yulong Tan∗,1, Matthias Leonhard1, Berit Schneider-Stickler Department of Otorhinolaryngology and Head and Neck Surgery, Medical University of Vienna, Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Keywords: Mixed biofilm Non-albicans Candida species Clinical isolates
Silicone is frequently used in clinical and medical fields for medical devices. Mixed biofilms composed of Candida and bacterial species causes frequently failure of medical silicone devices, In this in vitro study, we analyzed mixed biofilm formation of clinically isolated non-albicans Candida species and Staphylococcus epidermidis, including Candida tropicalis, Candida krusei and Candida parapsilosis under the influence of different growth media (RPMI 1640, BHI and TSB) and several culture variables (incubation period, feeding conditions and FBS). Our results showed that culture conditions strongly influence mixed biofilm formation. TSB and BHI resulted in larger amount of biofilm formations with stronger metabolic activity of biofilms. Growth conditions may also influence the biofilm formation, which was enhanced by longer incubation period, using a fed-batch system and FBS. Therefore, the potential influences of external environmental factors are very important for mixed biofilm formation with clinically isolated non-albicans Candida species and S. epidermidis, which should be considered when designing or studying the mixed biofilm under in vitro conditions.
1. Introduction A biofilm is described as a well-structured population of microbial cells that are enclosed in a self-produced extracellular polymer matrix and adherent to a surface [1]. These structured communities on medical devices significantly increase the risk of infection [2,3]. Although many implant-associated infections are caused by a single pathogenic microorganism, which have been extensively studied in the past, it has become clear that polymicrobial biofilms are the dominant form in nature. Mixed bacterial-fungal biofilms infections involving medical devices have been attracting more attention [4,5]. Most cases of candidiasis have been attributed to Candida albicans, an opportunistic resident species in the oral cavity, but also to nonalbicans Candida species. Furthermore, the non-albicans Candida species are adept in forming biofilms of medical devices in clinical practice [4,6,7]. Staphylococci, especially the species of Staphylococcus epidermidis, are known as a most prevalent opportunistic pathogen, which cause the majority of implant-associated infections [8,9]. Notably, approximately 20–40% of cases of candidaemia were accompanied by bacteraemia, with Staphylococcus species being the main pathogens [10,11].
∗
1
Silicone is widely used in clinical and medical fields due to its biocompatibility and mechanical and moulding properties [12,13]. However, silicone becomes rapidly colonized by microorganisms that form a biofilm, which limits the devices life time and increases the risk of infection [5,14]. Although biofilm-associated implant infections involving Candida or bacteria are common, Candida/bacteria mixed biofilms have still been studied little, especially non-albicans Candida biofilms. There have been several studies reporting an in vitro effect of growth media, substrates and techniques on biofilms [15–17]. In our previous work [18], we found that different growth media and several culture variables (inoculum concentration, incubation period and feeding conditions) can strongly influence non-albicans Candida species biofilm formation. However, up to now, only few in vitro studies have investigated the mixed species biofilm formation with non-albicans Candida species and Staphylococcus. Therefore, it is important to know the conditions under which mixed biofilms of non-albicans Candida species and Staphylococcus are able to adhere to surfaces of silicone and form biofilms. In this study, we assess the impact of the culture condition factors on mixed species biofilm with non-albicans Candida species and Staphylococcus on silicone in vitro.
Corresponding author. E-mail address: [email protected] (Y. Tan). Both authors contributed equally to this manuscript.
http://dx.doi.org/10.1016/j.micpath.2017.10.002 Received 8 September 2017; Received in revised form 3 October 2017; Accepted 4 October 2017 Available online 05 October 2017 0882-4010/ © 2017 Published by Elsevier Ltd.
Microbial Pathogenesis 112 (2017) 215–220
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2. Material and methods 2.1. Strains and growth media The strains used in this study were Candida tropicalis, Candida krusei, Candida parapsilosis and S. epidermidis, isolated from voice prostheses of laryngectomized patients in routine follow up examinations. The strains were stored in −80 °C and thawed before use. Tryptic Soy Broth (TSB) (Sigma-Aldrich, Austria) was utilized for bacteria while yeast peptone dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose [Sigma-Aldrich, Austria]) was used for Candida species. Dilutions of TSB, RPMI-1640 (Life Technologies, America) and Brain Heart Infusion Broth (BHI, Sigma-Aldrich, Austria) were used for culturing the mixed biofilms. 2.2. Biofilm formation on medical grade silicone Biofilms were formed on silicone as described previously [19]. In brief, medical grade silicone plates (diameter: 8 mm, thickness: 3 mm, Websinger, Austria) were steam sterilized at 121 °C (20 min) and placed into wells of 96-well microplate. Overnight bacterial and fungal cultures were diluted to a cell density of 1 × 106 cfu/ml in RPMI-1640, TSB or BHI. Equal volumes of bacterial and each of fungal suspensions were mixed. 100 μl diluted mixed microbial suspensions were pipetted into wells of a 96-well microplate. After an adhesion period (90 min) at 37 °C, non-attached cells were removed and fresh media was added. The plates were incubated at 37 °C for 24 h or 48 h without shaking. In order to evaluate the effect of fed-batch growth on 48 h biofilms, the culture medium was replaced by a fresh medium after 24 h of growth. The culture medium with or without 10% fetal bovine serum (FBS) was used to evaluate the serum affection on biofilm formation. 2.3. Crystal violet assay Biofilms were grown and biofilm biomass was evaluated with crystal violet (CV) staining. The plates were washed and stained with 150 μl of 0.1% (w/v) crystal violet for 30 min. The amount of biofilm biomass was determined by measuring its OD590. 2.4. XTT assay The metabolic activity of biofilms was calculated using a 2, 3-bis(2methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5 carboxanilide (XTT) reduction assay [20]. Silicone were washed and incubated for 2 h with 150 μl XTT working reagent (XTT 180 mg/l; AppliChem, Darmstadt, Germany) at 37 °C. The resulting absorbance was test with reading at OD490. 2.5. Statistical analysis All the experiments were done in triplicate. Means ± standard deviations (SD) had been calculated for each experiment. Statistical analysis was performed by analysis of variance (ANOVA) with Tukey post hoc test. Statistical significance was accepted at p < 0.05. 3. Results 3.1. Mixed biofilm formation under different growth media
Fig. 1. Biofilm formation by clinically isolated non-albicans Candida species and S. epidermidis in different media. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation in different media are marked with * (p < 0.05).
Mixed biofilms out of three Candida species, C. tropicalis, C. krusei and C. parapsilosis and S. epidermidis were grown in three standard growth media, namely RPMI 1640, BHI and TSB, to evaluate their ability to form biofilms. Results obtained after 24 h (Fig. 1) revealed that mixed biofilm with C. tropicalis and S. epidermidis exhibited significantly more biofilm formation when grown in BHI medium than the 216
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Fig. 2. The metabolic activity of biofilms by clinically isolated non-albicans Candida species and S. epidermidis in different media. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the metabolic activity of biofilms in different media are marked with * (p < 0.05).
other two media. However, both mixed biofilms with C. krusei or C. parapsilosis manifested significantly enhanced biofilm formations in BHI and TSB media without significant difference (p > 0.05). 3.2. The metabolic activity of biofilms in different growth media The metabolic activities of three species of Candida and S. epidermidis mixed biofilm in the different growth media were tested with XTT reduction method (Fig. 2). Similar to CV assay, the metabolic activity of all the biofilms were significantly reduced in RPMI. However, there are significant differences of the metabolic activity when using BHI medium or TSB medium. 3.3. Influence of incubation time on biofilm formation To determine incubation time influence on the biofilm accumulation, mixed suspension was incubated for 24 h and 48 h. As can be seen in Fig. 3, all the tested strains increased biofilm biomass from 24 h to 48 h. 3.4. Influence of feeding conditions on biofilm formation Next, we tested whether changing the culture media, after 24 h of growth, would affect the biomass at 48 h biofilms. As shown in Fig. 4, the biofilm biomass increased with changing the culture media after 24 h of growth. 3.5. Influence of FBS on biofilm formation Since treatment of different material surfaces with serum is better for cell attachment and biofilm production [21,22], we examined the effect of 10% FBS in the growth medium on the formation of mixed biofilm. The results showed that mixed biofilms displayed significantly higher biofilm biomass on FBS-treated surfaces compared with the untreated surfaces (Fig. 5). 4. Discussion Mixed biofilms of Candida and bacteria are the most common mode of growth in nature [23–25]. The purpose of this study was to evaluate the effect of growth media and culture conditions on mixed biofilm production by clinical isolates of Candida species and S. epidermidis. RPMI 1640, BHI and TSB media are commonly used for growing mixed biofilms with Candida species and bacteria [26–29]. However, no studies focused strictly on the testing of these media on the Candida/ bacteria mixed biofilm development. Here, we compared the effect of these three growth media on biofilm formation with clinical isolates of Candida species and S. epidermidis. BHI medium is one of the nutrientrich media that well supports biofilm formation. TSB medium is lessrich (compared to BHI) and also frequently used in biofilm investigation [30–32]. RPMI 1640 medium is chemically defined, iron-limited media [26,33]. Our results showed that development of mixed biofilms consistently yielded the lowest biofilm formation when grown in RPMI 1640 medium, which suggests that composition of the media affected the biofilm formation and poorer medium resulted in the less amount of biofilm formation. Moreover, the results show a correlation between biofilm formation and the metabolic activity of biofilms, which suggests that the growth media tested were not only able to influence the mass of biofilm, but also to impact the metabolic activity of cells in maturing 217
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Fig. 4. Influence of feeding conditions on biofilm formation. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation with different feeding conditions are marked with * (p < 0.05).
Fig. 3. Influence of incubation time on biofilm formation. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation with different inoculum time are marked with * (p < 0.05).
time until mature biofilms developed and the cells have been released to the surrounding environment from the mature biofilms [35,36]. As expected, longer inoculum time resulted in more biofilm formation in this study. This observation could be explained by the higher induction of extracellular materials. With incubation time, extracellular
biofilms. A general overview of the data revealed that incubation time plays a crucial role in biofilm development [34,35]. Former studies revealed that the amount of biofilm normally increases with longer incubation 218
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are based on a batch culture mode. However, biofilms develop in nature are not similar to batch systems. In contrast, the condition is more like fed-batch mode. Our results showed that fed-batch conditions increased biofilm formation. In this mode, the substrate and nutrients can be added continuously, which leads to biofilms metabolically more active [17,37,38]. These results demonstrated that the fed-batch growth was a favorable culture condition for the mixed biofilm formation of Candida species and S. epidermidis. Although the effect of serum on biofilm formation is poorly understood, both Candida and Staphylococcus have receptors to various proteins in serum [39]. Previous reports have demonstrated that serum components can increase Candida biofilm [40,41]. Serum may stimulate Candida biofilm growth through multiple mechanisms such as adhesion, aggregation, germ tube or hyphal emergence [42,43]. Harriott and Noverr [31] showed S. aureus forms poor monoculture biofilms in serum, but it forms a substantial polymicrobial biofilm in the presence of C. albicans in serum, although there is not an exact mechanism to explain. Our results also confirmed that mixed biofilm formation can be enhanced by serum conditioning. 5. Conclusion From this study, we conclude that medium composition, incubation period, or feeding conditions affected mixed biofilms. Although results in this study show optimized culture conditions for mixed biofilms of non-albicans Candida species and S. epidermidis, it may be necessary to evaluate more factors to understand the exact mechanism. Overall, establishment of in vitro conditions to allow optimal biofilm growth may lead to more robust model systems to test the efficacy of novel antimicrobial and antibiofilm agents. Conflict of interest The authors declare that they have no conflict of interest. References [1] X. Wang, X. Yao, Z. Zhu, T. Tang, K. Dai, I. Sadovskaya, S. Flahaut, S. Jabbouri, Effect of berberine on Staphylococcus epidermidis biofilm formation, Int. J. Antimicrob. Agents 34 (2009) 60–66. [2] H. Hanna, P. Bahna, R. Reitzel, T. Dvorak, G. Chaiban, R. Hachem, I. Raad, Comparative in vitro efficacies and antimicrobial durabilities of novel antimicrobial central venous catheters, Antimicrob. Agents Chemother. 50 (2006) 3283–3288. [3] I. Veiga-Malta, Preventing healthcare-associated infections by monitoring the cleanliness of medical devices and other critical points in a sterilization service, Biomed. Instrum. Technol. 50 (Suppl 3) (2016) 45–52. [4] M. Leonhard, B. Schneider-Stickler, Voice prostheses, microbial colonization and biofilm formation, Adv. Exp. Med. Biol. 830 (2015) 123–136. [5] J.J. Oosterhof, K.J. Buijssen, H.J. Busscher, B.F. van der Laan, H.C. van der Mei, Effects of quaternary ammonium silane coatings on mixed fungal and bacterial biofilms on tracheoesophageal shunt prostheses, Appl. Environ. Microbiol. 72 (2006) 3673–3677. [6] R.M. Donlan, J.W. Costerton, Biofilms: survival mechanisms of clinically relevant microorganisms, Clin. Microbiol. Rev. 15 (2002) 167-+. [7] L.J. Douglas, Candida biofilms and their role in infection, Trends Microbiol. 11 (2003) 30–36. [8] V. Vadyvaloo, M. Otto, Molecular genetics of Staphylococcus epidermidis biofilms on indwelling medical devices, Int. J. Artif. Organs 28 (2005) 1069–1078. [9] C. von Eiff, B. Jansen, W. Kohnen, K. Becker, Infections associated with medical devices: pathogenesis, management and prophylaxis, Drugs 65 (2005) 179–214. [10] E. Bouza, A. Burillo, P. Munoz, J. Guinea, M. Marin, M. Rodriguez-Creixems, Mixed bloodstream infections involving bacteria and Candida spp, J. Antimicrob. Chemother. 68 (2013) 1881–1888. [11] S.H. Kim, Y.K. Yoon, M.J. Kim, J.W. Sohn, Risk factors for and clinical implications of mixed Candida/bacterial bloodstream infections, Clin. Microbiol. Infect. 19 (2013) 62–68. [12] L. Rodrigues, I.M. Banat, J. Teixeira, R. Oliveira, Strategies for the prevention of microbial biofilm formation on silicone rubber voice prostheses, J. Biomed. Mater. Res. B Appl. Biomater. 81 (2007) 358–370. [13] Y. Tan, M. Leonhard, D. Moser, S. Ma, B. Schneider-Stickler, Inhibition of mixed fungal and bacterial biofilms on silicone by carboxymethyl chitosan, Colloids. Surf. B Biointerfaces 148 (2016) 193–199. [14] F.J. Hilgers, A.J. Balm, Long-term results of vocal rehabilitation after total laryngectomy with the low-resistance, indwelling ProvoxTM Voice prosthesis system,
Fig. 5. Influence of FBS on biofilm formation. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation with different feeding conditions are marked with * (p < 0.05).
polymeric substances increases, so that more biofilm biomass formed at 48 h. Moreover, different growth media also impact on biofilm development even though the incubation times are different. Most of the methods that have been developed for forming biofilm
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[29] F.L. Brighenti, A.C. Medeiros, B.M. Matos, Z.E. Ribeiro, C.Y. Koga-Ito, Evaluation of caries-associated virulence of biofilms from Candida albicans isolated from saliva of pediatric patients with sickle-cell anemia, J. Appl. Oral. Sci. 22 (2014) 484–489. [30] M.E. Nyenje, E. Green, R.N. Ndip, Evaluation of the effect of different growth media and temperature on the suitability of biofilm formation by Enterobacter cloacae strains isolated from food samples in South Africa, Molecules 18 (2013) 9582–9593. [31] M.M. Harriott, M.C. Noverr, Candida albicans and Staphylococcus aureus form polymicrobial biofilms: effects on antimicrobial resistance, Antimicrob. Agents Chemother. 53 (2009) 3914–3922. [32] C.A. Kennedy, J.P. O'Gara, Contribution of culture media and chemical properties of polystyrene tissue culture plates to biofilm development by Staphylococcus aureus, J. Med. Microbiol. 53 (2004) 1171–1173. [33] M. Zapotoczna, H. McCarthy, J.K. Rudkin, J.P. O'Gara, E. O'Neill, An essential role for coagulase in Staphylococcus aureus biofilm development reveals new therapeutic possibilities for device-related infections, J. Infect. Dis. 212 (2015) 1883–1893. [34] J.J. Cotter, J.P. O'Gara, E. Casey, Rapid depletion of dissolved oxygen in 96-well microtiter plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration, Biotechnol. Bioeng. 103 (2009) 1042–1047. [35] T. Mathur, S. Singhal, S. Khan, D. Upadhyay, T. Fatma, A. Rattan, Detection of biofilm formation among the clinical isolates of staphylococci: an evaluation of three different screening methods, Indian J. Med. Microbi. 24 (2006) 25. [36] B.R. Boles, M. Thoendel, P.K. Singh, Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms, Mol. Microbiol. 57 (2005) 1210–1223. [37] T. Pongtharangkul, A. Demirci, Effects of fed-batch fermentation and pH profiles on nisin production in suspended-cell and biofilm reactors, Appl. Microbiol. Biot. 73 (2006) 73–79. [38] D.A. Rodrigues, M.A. Almeida, P.A. Teixeira, R.T. Oliveira, J.C. Azeredo, Effect of batch and fed-batch growth modes on biofilm formation by Listeria monocytogenes at different temperatures, Curr. Microbiol. 59 (2009) 457–462. [39] P. Moreillon, J.M. Entenza, P. Francioli, D. McDevitt, T.J. Foster, P. Francois, P. Vaudaux, Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis, Infect. Immun. 63 (1995) 4738–4743. [40] L.P. Samaranayake, J. McCourtie, T.W. MacFarlane, Factors affecting the in-vitro adherence of Candida albicans to acrylic surfaces, Arch. Oral. Biol. 25 (1980) 611–615. [41] H. Nikawa, H. Nishimura, S. Makihira, T. Hamada, S. Sadamori, L.P. Samaranayake, Effect of serum concentration on Candida biofilm formation on acrylic surfaces, Mycoses 43 (2000) 139–143. [42] H. Nikawa, H. Nishimura, T. Yamamoto, T. Hamada, L. Samaranayake, The role of saliva and serum in Candida albicans biofilm formation on denture acrylic surfaces, Microb. Ecol. Health. Dis. 9 (1996) 35–48. [43] Y.H. Samaranayake, B.P. Cheung, J.Y. Yau, S.K. Yeung, L.P. Samaranayake, Human serum promotes Candida albicans biofilm growth and virulence gene expression on silicone biomaterial, PLoS One 8 (2013) e62902.
Clin. Otolaryngol. Allied Sci. 18 (1993) 517–523. [15] S. Lee, K.H. Choi, Y. Yoon, Effect of NaCl on biofilm formation of the isolate from Staphylococcus aureus outbreak linked to ham, Korean J. Food Sci. Anim. Resour. 34 (2014) 257–261. [16] M. Abdallah, G. Chataigne, P. Ferreira-Theret, C. Benoliel, D. Drider, P. Dhulster, N.E. Chihib, Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants, Appl. Microbiol. Biotechnol. 98 (2014) 2597–2607. [17] N. Cerca, G.B. Pier, M. Vilanova, R. Oliveira, J. Azeredo, Influence of batch or fedbatch growth on Staphylococcus epidermidis biofilm formation, Lett. Appl. Microbiol. 39 (2004) 420–424. [18] Y. Tan, M. Leonhard, S. Ma, B. Schneider-Stickler, Influence of culture conditions for clinically isolated non-albicans Candida biofilm formation, J. Microbiol. Methods 130 (2016) 123–128. [19] Y. Tan, M. Leonhard, D. Moser, S. Ma, B. Schneider-Stickler, Long-term antibiofilm activity of carboxymethyl chitosan on mixed biofilm on silicone, Laryngoscope 126 (2016) E404–E408. [20] J.E. Nett, M.T. Cain, K. Crawford, D.R. Andes, Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay, J. Clin. Microbiol. 49 (2011) 1426–1433. [21] D.M. Kuhn, J. Chandra, P.K. Mukherjee, M.A. Ghannoum, Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces, Infect. Immun. 70 (2002) 878–888. [22] J.P. Frade, B.A. Arthington-Skaggs, Effect of serum and surface characteristics on Candida albicans biofilm formation, Mycoses 54 (2011) e154–162. [23] P. Schmidt, J. Walker, L. Selway, D. Stead, Z. Yin, B. Enjalbert, M. Weig, A.J.P. Brown, Proteomic analysis of the pH response in the fungal pathogen Candida glabrata, Proteomics 8 (2008) 534–544. [24] T. Pereira-Cenci, D.M. Deng, E.A. Kraneveld, E.M. Manders, A.A. Del Bel Cury, J.M. Ten Cate, W. Crielaard, The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces, Arch. Oral. Biol. 53 (2008) 755–764. [25] H.M. Bandara, J.Y. Yau, R.M. Watt, L.J. Jin, L.P. Samaranayake, Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation, J. Med. Microbiol. 58 (2009) 1623–1631. [26] S. Kucharikova, H. Tournu, K. Lagrou, P. Van Dijck, H. Bujdakova, Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin, J. Med. Microbiol. 60 (2011) 1261–1269. [27] I. Serrano-Fujarte, E. Lopez-Romero, G.E. Reyna-Lopez, M.A. Martinez-Gamez, A. Vega-Gonzalez, M. Cuellar-Cruz, Influence of culture media on biofilm formation by Candida species and response of sessile cells to antifungals and oxidative stress, Biomed. Res. Int. 2015 (2015) 783639. [28] E.P. Fox, E.S. Cowley, C.J. Nobile, N. Hartooni, D.K. Newman, A.D. Johnson, Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures, Curr. Biol. 24 (2014) 2411–2416.
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Evaluation of culture conditions for mixed biofilm formation with clinically isolated non-albicans Candida species and Staphylococcus epidermidis on silicone
MARK
Yulong Tan∗,1, Matthias Leonhard1, Berit Schneider-Stickler Department of Otorhinolaryngology and Head and Neck Surgery, Medical University of Vienna, Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Keywords: Mixed biofilm Non-albicans Candida species Clinical isolates
Silicone is frequently used in clinical and medical fields for medical devices. Mixed biofilms composed of Candida and bacterial species causes frequently failure of medical silicone devices, In this in vitro study, we analyzed mixed biofilm formation of clinically isolated non-albicans Candida species and Staphylococcus epidermidis, including Candida tropicalis, Candida krusei and Candida parapsilosis under the influence of different growth media (RPMI 1640, BHI and TSB) and several culture variables (incubation period, feeding conditions and FBS). Our results showed that culture conditions strongly influence mixed biofilm formation. TSB and BHI resulted in larger amount of biofilm formations with stronger metabolic activity of biofilms. Growth conditions may also influence the biofilm formation, which was enhanced by longer incubation period, using a fed-batch system and FBS. Therefore, the potential influences of external environmental factors are very important for mixed biofilm formation with clinically isolated non-albicans Candida species and S. epidermidis, which should be considered when designing or studying the mixed biofilm under in vitro conditions.
1. Introduction A biofilm is described as a well-structured population of microbial cells that are enclosed in a self-produced extracellular polymer matrix and adherent to a surface [1]. These structured communities on medical devices significantly increase the risk of infection [2,3]. Although many implant-associated infections are caused by a single pathogenic microorganism, which have been extensively studied in the past, it has become clear that polymicrobial biofilms are the dominant form in nature. Mixed bacterial-fungal biofilms infections involving medical devices have been attracting more attention [4,5]. Most cases of candidiasis have been attributed to Candida albicans, an opportunistic resident species in the oral cavity, but also to nonalbicans Candida species. Furthermore, the non-albicans Candida species are adept in forming biofilms of medical devices in clinical practice [4,6,7]. Staphylococci, especially the species of Staphylococcus epidermidis, are known as a most prevalent opportunistic pathogen, which cause the majority of implant-associated infections [8,9]. Notably, approximately 20–40% of cases of candidaemia were accompanied by bacteraemia, with Staphylococcus species being the main pathogens [10,11].
∗
1
Silicone is widely used in clinical and medical fields due to its biocompatibility and mechanical and moulding properties [12,13]. However, silicone becomes rapidly colonized by microorganisms that form a biofilm, which limits the devices life time and increases the risk of infection [5,14]. Although biofilm-associated implant infections involving Candida or bacteria are common, Candida/bacteria mixed biofilms have still been studied little, especially non-albicans Candida biofilms. There have been several studies reporting an in vitro effect of growth media, substrates and techniques on biofilms [15–17]. In our previous work [18], we found that different growth media and several culture variables (inoculum concentration, incubation period and feeding conditions) can strongly influence non-albicans Candida species biofilm formation. However, up to now, only few in vitro studies have investigated the mixed species biofilm formation with non-albicans Candida species and Staphylococcus. Therefore, it is important to know the conditions under which mixed biofilms of non-albicans Candida species and Staphylococcus are able to adhere to surfaces of silicone and form biofilms. In this study, we assess the impact of the culture condition factors on mixed species biofilm with non-albicans Candida species and Staphylococcus on silicone in vitro.
Corresponding author. E-mail address: [email protected] (Y. Tan). Both authors contributed equally to this manuscript.
http://dx.doi.org/10.1016/j.micpath.2017.10.002 Received 8 September 2017; Received in revised form 3 October 2017; Accepted 4 October 2017 Available online 05 October 2017 0882-4010/ © 2017 Published by Elsevier Ltd.
Microbial Pathogenesis 112 (2017) 215–220
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2. Material and methods 2.1. Strains and growth media The strains used in this study were Candida tropicalis, Candida krusei, Candida parapsilosis and S. epidermidis, isolated from voice prostheses of laryngectomized patients in routine follow up examinations. The strains were stored in −80 °C and thawed before use. Tryptic Soy Broth (TSB) (Sigma-Aldrich, Austria) was utilized for bacteria while yeast peptone dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose [Sigma-Aldrich, Austria]) was used for Candida species. Dilutions of TSB, RPMI-1640 (Life Technologies, America) and Brain Heart Infusion Broth (BHI, Sigma-Aldrich, Austria) were used for culturing the mixed biofilms. 2.2. Biofilm formation on medical grade silicone Biofilms were formed on silicone as described previously [19]. In brief, medical grade silicone plates (diameter: 8 mm, thickness: 3 mm, Websinger, Austria) were steam sterilized at 121 °C (20 min) and placed into wells of 96-well microplate. Overnight bacterial and fungal cultures were diluted to a cell density of 1 × 106 cfu/ml in RPMI-1640, TSB or BHI. Equal volumes of bacterial and each of fungal suspensions were mixed. 100 μl diluted mixed microbial suspensions were pipetted into wells of a 96-well microplate. After an adhesion period (90 min) at 37 °C, non-attached cells were removed and fresh media was added. The plates were incubated at 37 °C for 24 h or 48 h without shaking. In order to evaluate the effect of fed-batch growth on 48 h biofilms, the culture medium was replaced by a fresh medium after 24 h of growth. The culture medium with or without 10% fetal bovine serum (FBS) was used to evaluate the serum affection on biofilm formation. 2.3. Crystal violet assay Biofilms were grown and biofilm biomass was evaluated with crystal violet (CV) staining. The plates were washed and stained with 150 μl of 0.1% (w/v) crystal violet for 30 min. The amount of biofilm biomass was determined by measuring its OD590. 2.4. XTT assay The metabolic activity of biofilms was calculated using a 2, 3-bis(2methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5 carboxanilide (XTT) reduction assay [20]. Silicone were washed and incubated for 2 h with 150 μl XTT working reagent (XTT 180 mg/l; AppliChem, Darmstadt, Germany) at 37 °C. The resulting absorbance was test with reading at OD490. 2.5. Statistical analysis All the experiments were done in triplicate. Means ± standard deviations (SD) had been calculated for each experiment. Statistical analysis was performed by analysis of variance (ANOVA) with Tukey post hoc test. Statistical significance was accepted at p < 0.05. 3. Results 3.1. Mixed biofilm formation under different growth media
Fig. 1. Biofilm formation by clinically isolated non-albicans Candida species and S. epidermidis in different media. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation in different media are marked with * (p < 0.05).
Mixed biofilms out of three Candida species, C. tropicalis, C. krusei and C. parapsilosis and S. epidermidis were grown in three standard growth media, namely RPMI 1640, BHI and TSB, to evaluate their ability to form biofilms. Results obtained after 24 h (Fig. 1) revealed that mixed biofilm with C. tropicalis and S. epidermidis exhibited significantly more biofilm formation when grown in BHI medium than the 216
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Fig. 2. The metabolic activity of biofilms by clinically isolated non-albicans Candida species and S. epidermidis in different media. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the metabolic activity of biofilms in different media are marked with * (p < 0.05).
other two media. However, both mixed biofilms with C. krusei or C. parapsilosis manifested significantly enhanced biofilm formations in BHI and TSB media without significant difference (p > 0.05). 3.2. The metabolic activity of biofilms in different growth media The metabolic activities of three species of Candida and S. epidermidis mixed biofilm in the different growth media were tested with XTT reduction method (Fig. 2). Similar to CV assay, the metabolic activity of all the biofilms were significantly reduced in RPMI. However, there are significant differences of the metabolic activity when using BHI medium or TSB medium. 3.3. Influence of incubation time on biofilm formation To determine incubation time influence on the biofilm accumulation, mixed suspension was incubated for 24 h and 48 h. As can be seen in Fig. 3, all the tested strains increased biofilm biomass from 24 h to 48 h. 3.4. Influence of feeding conditions on biofilm formation Next, we tested whether changing the culture media, after 24 h of growth, would affect the biomass at 48 h biofilms. As shown in Fig. 4, the biofilm biomass increased with changing the culture media after 24 h of growth. 3.5. Influence of FBS on biofilm formation Since treatment of different material surfaces with serum is better for cell attachment and biofilm production [21,22], we examined the effect of 10% FBS in the growth medium on the formation of mixed biofilm. The results showed that mixed biofilms displayed significantly higher biofilm biomass on FBS-treated surfaces compared with the untreated surfaces (Fig. 5). 4. Discussion Mixed biofilms of Candida and bacteria are the most common mode of growth in nature [23–25]. The purpose of this study was to evaluate the effect of growth media and culture conditions on mixed biofilm production by clinical isolates of Candida species and S. epidermidis. RPMI 1640, BHI and TSB media are commonly used for growing mixed biofilms with Candida species and bacteria [26–29]. However, no studies focused strictly on the testing of these media on the Candida/ bacteria mixed biofilm development. Here, we compared the effect of these three growth media on biofilm formation with clinical isolates of Candida species and S. epidermidis. BHI medium is one of the nutrientrich media that well supports biofilm formation. TSB medium is lessrich (compared to BHI) and also frequently used in biofilm investigation [30–32]. RPMI 1640 medium is chemically defined, iron-limited media [26,33]. Our results showed that development of mixed biofilms consistently yielded the lowest biofilm formation when grown in RPMI 1640 medium, which suggests that composition of the media affected the biofilm formation and poorer medium resulted in the less amount of biofilm formation. Moreover, the results show a correlation between biofilm formation and the metabolic activity of biofilms, which suggests that the growth media tested were not only able to influence the mass of biofilm, but also to impact the metabolic activity of cells in maturing 217
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Fig. 4. Influence of feeding conditions on biofilm formation. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation with different feeding conditions are marked with * (p < 0.05).
Fig. 3. Influence of incubation time on biofilm formation. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation with different inoculum time are marked with * (p < 0.05).
time until mature biofilms developed and the cells have been released to the surrounding environment from the mature biofilms [35,36]. As expected, longer inoculum time resulted in more biofilm formation in this study. This observation could be explained by the higher induction of extracellular materials. With incubation time, extracellular
biofilms. A general overview of the data revealed that incubation time plays a crucial role in biofilm development [34,35]. Former studies revealed that the amount of biofilm normally increases with longer incubation 218
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are based on a batch culture mode. However, biofilms develop in nature are not similar to batch systems. In contrast, the condition is more like fed-batch mode. Our results showed that fed-batch conditions increased biofilm formation. In this mode, the substrate and nutrients can be added continuously, which leads to biofilms metabolically more active [17,37,38]. These results demonstrated that the fed-batch growth was a favorable culture condition for the mixed biofilm formation of Candida species and S. epidermidis. Although the effect of serum on biofilm formation is poorly understood, both Candida and Staphylococcus have receptors to various proteins in serum [39]. Previous reports have demonstrated that serum components can increase Candida biofilm [40,41]. Serum may stimulate Candida biofilm growth through multiple mechanisms such as adhesion, aggregation, germ tube or hyphal emergence [42,43]. Harriott and Noverr [31] showed S. aureus forms poor monoculture biofilms in serum, but it forms a substantial polymicrobial biofilm in the presence of C. albicans in serum, although there is not an exact mechanism to explain. Our results also confirmed that mixed biofilm formation can be enhanced by serum conditioning. 5. Conclusion From this study, we conclude that medium composition, incubation period, or feeding conditions affected mixed biofilms. Although results in this study show optimized culture conditions for mixed biofilms of non-albicans Candida species and S. epidermidis, it may be necessary to evaluate more factors to understand the exact mechanism. Overall, establishment of in vitro conditions to allow optimal biofilm growth may lead to more robust model systems to test the efficacy of novel antimicrobial and antibiofilm agents. Conflict of interest The authors declare that they have no conflict of interest. References [1] X. Wang, X. Yao, Z. Zhu, T. Tang, K. Dai, I. Sadovskaya, S. Flahaut, S. Jabbouri, Effect of berberine on Staphylococcus epidermidis biofilm formation, Int. J. Antimicrob. Agents 34 (2009) 60–66. [2] H. Hanna, P. Bahna, R. Reitzel, T. Dvorak, G. Chaiban, R. Hachem, I. Raad, Comparative in vitro efficacies and antimicrobial durabilities of novel antimicrobial central venous catheters, Antimicrob. Agents Chemother. 50 (2006) 3283–3288. [3] I. Veiga-Malta, Preventing healthcare-associated infections by monitoring the cleanliness of medical devices and other critical points in a sterilization service, Biomed. Instrum. Technol. 50 (Suppl 3) (2016) 45–52. [4] M. Leonhard, B. Schneider-Stickler, Voice prostheses, microbial colonization and biofilm formation, Adv. Exp. Med. Biol. 830 (2015) 123–136. [5] J.J. Oosterhof, K.J. Buijssen, H.J. Busscher, B.F. van der Laan, H.C. van der Mei, Effects of quaternary ammonium silane coatings on mixed fungal and bacterial biofilms on tracheoesophageal shunt prostheses, Appl. Environ. Microbiol. 72 (2006) 3673–3677. [6] R.M. Donlan, J.W. Costerton, Biofilms: survival mechanisms of clinically relevant microorganisms, Clin. Microbiol. Rev. 15 (2002) 167-+. [7] L.J. Douglas, Candida biofilms and their role in infection, Trends Microbiol. 11 (2003) 30–36. [8] V. Vadyvaloo, M. Otto, Molecular genetics of Staphylococcus epidermidis biofilms on indwelling medical devices, Int. J. Artif. Organs 28 (2005) 1069–1078. [9] C. von Eiff, B. Jansen, W. Kohnen, K. Becker, Infections associated with medical devices: pathogenesis, management and prophylaxis, Drugs 65 (2005) 179–214. [10] E. Bouza, A. Burillo, P. Munoz, J. Guinea, M. Marin, M. Rodriguez-Creixems, Mixed bloodstream infections involving bacteria and Candida spp, J. Antimicrob. Chemother. 68 (2013) 1881–1888. [11] S.H. Kim, Y.K. Yoon, M.J. Kim, J.W. Sohn, Risk factors for and clinical implications of mixed Candida/bacterial bloodstream infections, Clin. Microbiol. Infect. 19 (2013) 62–68. [12] L. Rodrigues, I.M. Banat, J. Teixeira, R. Oliveira, Strategies for the prevention of microbial biofilm formation on silicone rubber voice prostheses, J. Biomed. Mater. Res. B Appl. Biomater. 81 (2007) 358–370. [13] Y. Tan, M. Leonhard, D. Moser, S. Ma, B. Schneider-Stickler, Inhibition of mixed fungal and bacterial biofilms on silicone by carboxymethyl chitosan, Colloids. Surf. B Biointerfaces 148 (2016) 193–199. [14] F.J. Hilgers, A.J. Balm, Long-term results of vocal rehabilitation after total laryngectomy with the low-resistance, indwelling ProvoxTM Voice prosthesis system,
Fig. 5. Influence of FBS on biofilm formation. (A) C. parapsilosis and S. epidermidis; (B) C. krusei and S. epidermidis; (C) C. tropicalis and S. epidermidis. The results shown represent the means and standard deviations (error bars) of three independent experiments. Statistical differences in the biofilm formation with different feeding conditions are marked with * (p < 0.05).
polymeric substances increases, so that more biofilm biomass formed at 48 h. Moreover, different growth media also impact on biofilm development even though the incubation times are different. Most of the methods that have been developed for forming biofilm
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