STRUCTURE OF CANDIDA ALBICANS-STAPHYLOCOCCUS EPIDERMIDIS MIXED SPECIES BIOFILM ON POLYVINYL CHLORIDE BIOMATERIAL_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHENYing , WANGXiaoyan , ZHAOGuangqiang , LEIYujie , YELianhua , HUANGQiubo , DUANWanshi ,  HUANGYunchao

Department of Thoracic Surgery, the Third Affiliated Hospital of Kunming Medical University, Tumor Hospital of Yunnan Province, Kunming Yunnan, 650118, P. R. China;

Corresponding author：

HUANGYunchao, Email: huangych2001@aliyun.com

Keywords：

Polyvinyl chlorid; Candida albicans; Staphylococcus epidermidis; Mixed species biofilm

DOI：

10.7507/1002-1892.20150131

Video：

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Objective To establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm on polyvinyl chloride (PVC) material, and to observe mixed species biofilm formation and its microstructure. Methods Staphylococcus epidermidis bacteria (ATCC35984) and Candida albicans fungal (ATCC10231)were co-incubated with 0.5 cm diameter PVC pieces in tryptic soy broth (TSB) to form mixed specie biofilms (experimental group). At 2, 6, 12, 24, 48, and 72 hours, the thicknesses of the biofilms, the number of bacteria per sight, and the percentage of viable cells in biofilms were measured, and three-dimensional images of biofilms were obtained using confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) at 48 hours. PVC material cultured in the TSB medium served as control group. Results In control group, there was no pathogenic bacteria adhesion on the PVC material surface. In experimental group, CLSM showed that colonies and biofilm formation were found at 6 hours after co-culture, and gradually increased with time. The pathogenic bacteria colonies reached the peak at 24 hours, and biofilm thickness attained peak value at 48 hours. In experimental group, the number of colony was significantly different among 2, 6, and 24 hours, and between 2, 6 hours and 48, 72 hours (P<0.05), but no significant difference was found among 24, 48, and 72 hours (P>0.05). The biofilm thickness showed significant difference between the other time points (P<0.05) except between 48 and 72 hours (P>0.05). The percentage of viable cells in the outer layers of the biofilm was significantly higher than that in inner and middle layers at 48 hours (P<0.05). Three-dimensional reconstruction displayed that the surface of mixd species was uneven; living bacterium mainly located at the protuberance, and dead bacteria mainly located at the concaves. SEM image showed that Staphylococcus epidermidis attached to various forms of Candida albicans (spores, pseudohyphae, hyphae) gradually, and formed multilayer reticulate sophisticated structure on the surface of PVC with time. Conclusion Candida albicans-Staphylococcus epidermidis mixed species biofilm is sophisticated in structure. The combination of CLSM, SEM, and three-dimensional image reconstruction technology is ideal for investigation of mixed species biofilm on PVC material.

Citation： CHENYing, WANGXiaoyan, ZHAOGuangqiang, LEIYujie, YELianhua, HUANGQiubo, DUANWanshi, HUANGYunchao. STRUCTURE OF CANDIDA ALBICANS-STAPHYLOCOCCUS EPIDERMIDIS MIXED SPECIES BIOFILM ON POLYVINYL CHLORIDE BIOMATERIAL. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(5): 609-614. doi: 10.7507/1002-1892.20150131 Copy

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2.	Maldonado NA, Cano LE, Bedout C, et al. Association of clinical and demographic factors in invasive candidiasis caused by fluconazole-resistant Candida species:a study in 15 hospitals, Medellín, Colombia 2010-2011. Diagn Microbiol Infect Dis, 2014, 79(2):280-286.
3.	Li D, Zhang W, Zheng S, et al. Surveillance study of candidemia in cancer patients in North China. Med Mycol, 2013, 51(4):378-384.
4.	Grim SA, Berger K, Teng C, et al. Timing of susceptibility-based antifungal drug administration in patients with Candida bloodstream infection:correlation with outcomes. J Antimicrob Chemother, 2012, 67(3):707-714.
5.	Klotz SA, Chasin BS, Powell B, et al. Polymicrobial bloodstream infections involving Candida species:analysis of patients and review of the literature. Diagn Microbiol Infect Dis, 2007, 59(4):401-406.
6.	Lin YJ, Alsad L, Vogel F, et al. Interactions between Candida albicans and Staphylococcus aureus within mixed species biofilms. Bios, 2013, 84(1):30-39.
7.	Harriott MM, Noverr MC. Importance of Candida-bacterial polymicrobial biofilms in disease. Trends Microbiol, 2011, 19(11):557-563.
8.	Valle J, Solano C, García B, et al. Biofilm switch and immune response determinants at early stages of infection. Trends Microbiol, 2013, 21(8):364-371.
9.	Yujie L, Geng X, Huang YC, et al. The effect of brominated furanones on the formation of Staphylococcus aureus biofilm on PVC. Cell Biochem Biophys, 2013, 67(3):1501-1505.
10.	Zhao GQ, Ye LH, Huang YC, et al. In vitro model of bacterial biofilm formation on polyvinyl chloridebiomaterial. Cell Biochem Biophys, 2011, 61(2):371-376.
11.	Pammi M, Liang R, Hicks J, et al. Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans. BMC Microbiol, 2013, 13:257.
12.	Dominiak DM, Nielsen JL, Nielsen PH. Extracellular DNA is abundant and important for microcolony strength in mixed microbial biofilms. Environ Microbiol, 2011, 13(3):710-721.
13.	Zhang S, Zhang T, Yan M, et al. Crystal structure of the WOPR-DNA complex and implications for Wor1 function in white-opaque switching of Candida albicans. Cell Res, 2014, 24(9):1108-1120.
14.	Chandra J, McCormick TS, Imamura Y, et al. Interaction of Candida albicans with adherent human peripheral blood mononuclear cells increases C. albicans biofilm formation and results in differential expression of pro-and anti-inflammatory cytokines. Infect Immun, 2007, 75(5):2612-2620.
15.	Samaranayake YH, Cheung BP, Yau JY, et al. Human serum promotes Candida albicans biofilm growth and virulence gene expression on silicone biomaterial. PLoS One, 2013, 8(5):e62902.
16.	陈曦, 张洪勋. 微生物膜研究方法和潜在的控制技术. 科技导报, 2011, 29(4):74-79.
17.	Almeida C, Azevedo NF, Santos S, et al. Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). PLoS One, 2011, 6(3):e14786.
18.	Karcz J, Bernas T, Nowak A, et al. Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy. Scanning, 2012, 34(1):26-36.
19.	Alhede M, Qvortrup K, Liebrechts R, et al. Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition. FEMS Immunol Med Microbiol, 2012, 65(2):335-342.
20.	Xiao J, Klein MI, Falsetta ML, et al. The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm. PLoS Pathog, 2012, 8(4):e1002623.

1. Stoodley P, Sidhu S, Nistico L, et al. Kinetics and morphology of polymicrobial biofilm formation on polypropylene mesh. FEMS Immunol Med Microbiol, 2012, 65(2):283-290.
2. Maldonado NA, Cano LE, Bedout C, et al. Association of clinical and demographic factors in invasive candidiasis caused by fluconazole-resistant Candida species:a study in 15 hospitals, Medellín, Colombia 2010-2011. Diagn Microbiol Infect Dis, 2014, 79(2):280-286.
3. Li D, Zhang W, Zheng S, et al. Surveillance study of candidemia in cancer patients in North China. Med Mycol, 2013, 51(4):378-384.
4. Grim SA, Berger K, Teng C, et al. Timing of susceptibility-based antifungal drug administration in patients with Candida bloodstream infection:correlation with outcomes. J Antimicrob Chemother, 2012, 67(3):707-714.
5. Klotz SA, Chasin BS, Powell B, et al. Polymicrobial bloodstream infections involving Candida species:analysis of patients and review of the literature. Diagn Microbiol Infect Dis, 2007, 59(4):401-406.
6. Lin YJ, Alsad L, Vogel F, et al. Interactions between Candida albicans and Staphylococcus aureus within mixed species biofilms. Bios, 2013, 84(1):30-39.
7. Harriott MM, Noverr MC. Importance of Candida-bacterial polymicrobial biofilms in disease. Trends Microbiol, 2011, 19(11):557-563.
8. Valle J, Solano C, García B, et al. Biofilm switch and immune response determinants at early stages of infection. Trends Microbiol, 2013, 21(8):364-371.
9. Yujie L, Geng X, Huang YC, et al. The effect of brominated furanones on the formation of Staphylococcus aureus biofilm on PVC. Cell Biochem Biophys, 2013, 67(3):1501-1505.
10. Zhao GQ, Ye LH, Huang YC, et al. In vitro model of bacterial biofilm formation on polyvinyl chloridebiomaterial. Cell Biochem Biophys, 2011, 61(2):371-376.
11. Pammi M, Liang R, Hicks J, et al. Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans. BMC Microbiol, 2013, 13:257.
12. Dominiak DM, Nielsen JL, Nielsen PH. Extracellular DNA is abundant and important for microcolony strength in mixed microbial biofilms. Environ Microbiol, 2011, 13(3):710-721.
13. Zhang S, Zhang T, Yan M, et al. Crystal structure of the WOPR-DNA complex and implications for Wor1 function in white-opaque switching of Candida albicans. Cell Res, 2014, 24(9):1108-1120.
14. Chandra J, McCormick TS, Imamura Y, et al. Interaction of Candida albicans with adherent human peripheral blood mononuclear cells increases C. albicans biofilm formation and results in differential expression of pro-and anti-inflammatory cytokines. Infect Immun, 2007, 75(5):2612-2620.
15. Samaranayake YH, Cheung BP, Yau JY, et al. Human serum promotes Candida albicans biofilm growth and virulence gene expression on silicone biomaterial. PLoS One, 2013, 8(5):e62902.
16. 陈曦, 张洪勋. 微生物膜研究方法和潜在的控制技术. 科技导报, 2011, 29(4):74-79.
17. Almeida C, Azevedo NF, Santos S, et al. Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). PLoS One, 2011, 6(3):e14786.
18. Karcz J, Bernas T, Nowak A, et al. Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy. Scanning, 2012, 34(1):26-36.
19. Alhede M, Qvortrup K, Liebrechts R, et al. Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition. FEMS Immunol Med Microbiol, 2012, 65(2):335-342.
20. Xiao J, Klein MI, Falsetta ML, et al. The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm. PLoS Pathog, 2012, 8(4):e1002623.

Chinese Journal of Reparative and Reconstructive Surgery

STRUCTURE OF CANDIDA ALBICANS-STAPHYLOCOCCUS EPIDERMIDIS MIXED SPECIES BIOFILM ON POLYVINYL CHLORIDE BIOMATERIAL

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