FUNCTION OF INTERCELLULAR ADHESION A, FIBRINOGEN BINDING PROTEIN, AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGXiaoyan ^1,2 , CHENYing ¹ ,  HUANGYunchao ¹ , ZHOUYouquan ³ , ZHAOGuangqiang ¹ , YELianhua ¹ , LEIYujie ¹ , TANGQi ⁴

1. Department of Cardiovascular Thoracic Surgery, the Third Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650118, P. R. China;
2. Department of Cardiovascular Surgery, the Affiliated Yan'an Hospital of Kunming Medical University;
3. Department of Clinical Laboratory, the Third Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650118, P. R. China;
4. Department of Brest Surgery, the Third Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650118, P. R. China;

Corresponding author：

HUANGYunchao, Email: huangych2001@aliyun.com

Keywords：

Staphylococcus epidermidis; Candida albicans; Mixed species biofilm; Intercellular adhesion A gene; Fibrinogen binding protein gene; Accumulation-associated protein gene

DOI：

10.7507/1002-1892.20150015

Video：

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Objective To explore the function of intercellular adhesion A (icaA), fibrinogen binding protein (fbe), and accumulation-associated protein (aap) genes in formation of Staphylococcus epidermidis-Candida albicans mixed species biofilms. Methods The experiment was divided into 3 groups:single culture of Staphylococcus epidermidis ATCC35984 (S. epidermidis group) or Candida albicans ATCC10231 (C. albicans group), and co-culture of two strains (mixed group) to build in vitro biofilm model. Biofilm mass was detected by crystal violet semi-quantitative adherence assay at 2, 4, 6, 8, 12, 24, 48, and 72 hours after incubation. XTT assay was performed to determine the growth kinetics in the same time. Scanning electron microscopy (SEM) was used to observe the ultrastructure of the biofilms after 24 and 72 hours of incubation. The expressions of icaA, fbe, and aap genes were analyzed by real-time fluorescent quantitative PCR. Results Crystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 hours in the S. epidermidis and mixed groups; after co-cultured for 72 hours the thickness of biofilm in mixed group was more than that in the S. epidermidis group, and there was significant difference between 2 groups at the other time (P<0.05) except at 72 hours (P>0.05). In C. albicans group, the biofilm started to grow at 12 hours of cultivation, but the thickness of the biofilm was significantly lower than that in the mixed group in all the time points (P<0.05). XTT assay showed that the overall growth speed in the mixed group was greater than that in the C. albicans group, and it was greater than that in the S. epidermidis group at 48 hours; there was no significant difference in the growth speed between the mixed groups and the S. epidermidis group in the other time points (P>0.05) except at 12 hours (P<0.05). The absorbance (A) value in the mixed group was lower than that in the S. epidermidis group at 2 and 4 hours, but no significant difference was shown (P>0.05); the A value of mixed group was significantly higher than that of the C. albicans group after 6 hours (P<0.05). SEM observation showed that mature biofilms with complex structure formed in all groups. The real-time fluorescent quantitative PCR showed the expressions of fbe, icaA, and aap genes in mixed group increased 1.93, 1.52, and 1.46 times respectively at 72 hours compared with the S. epidermidis group (P<0.05). Conclusion Mixed species biofilms have more complex structure and are thicker than single species biofilms of Staphylococcus epidermidis or Candida albicans, which is related to increased expressions of the icaA, fbe, and aap genes of Staphylococcus epidermidis.

Citation： WANGXiaoyan, CHENYing, HUANGYunchao, ZHOUYouquan, ZHAOGuangqiang, YELianhua, LEIYujie, TANGQi. FUNCTION OF INTERCELLULAR ADHESION A, FIBRINOGEN BINDING PROTEIN, AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(1): 63-68. doi: 10.7507/1002-1892.20150015 Copy

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2.	Ziebuhr W, Hennig S, Eckart M, et al. Nosocomial infections by Staphylococcus epidermidis:how a commensal bacterium turns into a pathogen. Int J Antimicrob Agents, 2006, 28 Suppl 1:S14-S20.
3.	França A, Melo LD, Cerca N. Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis. BMC Res Notes, 2011, (4):572.
4.	Chen L, Wen YM. The role of bacterial biofilm in persistent infection and control strategies. Int J Oral Sci, 2011, 3(2):66-73.
5.	Sutter D, Stagliano D, Braun L, et al. Polymicrobial bloodstream infection in pediatric patients:risk factors, microbiology, and antimicrobial management. Pediatr Infect Dis J, 2008, 27(5):400-405.
6.	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.
7.	McKenzie FE. Case mortality in polymicrobial bloodstream infections. J Clin Epidemiol, 2006, 59(7):760-761.
8.	Park SJ, Han KH, Park JY, et al. Influence of bacterial presence on biofilm formation of Candida albicans. Yonsei Med J, 2014, 55(2):449-458.
9.	Cerca N, Gomes F, Pereira S, et al. Confocal laser scanning microscopy analysis of S.epidermidis biofilms exposed to farnesol, vancomycin and rifampicin. BMC Res Notes, 2012, 5:244-250.
10.	刘卫娟, 左泽兰, 马荣华. 机械通气患儿气管导管表面细菌生物形成及病原分析. 中国微生态学杂志, 2009, 21(9):788-791.
11.	王醒, 江筱莉, 周昱薇, 等. 纳米银/聚氨酯覆膜新型抗菌气管导管在家兔体内的抑菌效果研究. 中国医疗设备, 2013, 28(8):20-22.
12.	龚凤云, 刘丽娜, 邢铭友, 等. 表皮葡萄球菌耐药与生物膜的相关研究. 中华医院感染学杂志, 2011, 21(1):20-23.
13.	郜向娜, 余加林, 芦起, 等. 四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型. 中国微生态学杂志, 2010, 22(12):1081-1088.
14.	França A, Freitas AI, Henriques AF, et al. Optimizing a qPCR gene expression quantification assay for S.epidermidis biofilms:A comparison between commercial kits and a customized protocol, 2012, 7(5):e37480.
15.	李燕, 李冬冬, 陶传敏, 等. 表皮葡萄球菌生物膜形成及相关基因的检测与评价. 中华医院感染学杂志, 2010, 20(4):473-476.
16.	Agarwal A, Jain A. Glucose & chloride induce biofilm production & ica operon in clinic isolates of Staphylococci. Indian J Med Res, 2013,138:262-266.
17.	叶联华, 黄云超, 许赓, 等. 医源性表皮葡萄球菌胞间黏附素基因操纵子在聚氯乙烯材料表面细菌生物膜形成中的作用研究. 中国修复重建外科杂志, 2011, 25(4):466-471.
18.	Rohde H, Buran dt EC, Siems sen N, et al. Polysaccharide intercellular adhesion or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infection. Biomaterials, 2007, 28(9):1711-1720.
19.	汤琦, 袁兵, 黄云超,等. 乳腺外科表皮葡萄球菌icaA、icaD及聚集相关蛋白对细菌生物膜形成的影响. 中国修复重建外科杂志, 2014, 28(2):244-249.
20.	宋超, 余加林, 艾青, 等. 机械通气新生儿气管导管表面生物膜细菌多样性研究. 中华儿科杂志, 2013, 51(8):602-606.
21.	Adam B, Baillie GS, Douglas LJ. Mixed species biofilms of Candida albicans and Staphylococcus epidermidis. J Med Microbiol, 2002, 51(4):344-349.
22.	丁进亚, 黄前川, 徐娟, 等. 置入性导管相关感染的病原菌与生物被膜形成. 医学综述, 2012, 18(6):933-935.

1. Otto M. Staphylococcus epidermidis-the 'accidental' pathogen. Nat Rev Microbiol, 2009, 7(8):555-567.
2. Ziebuhr W, Hennig S, Eckart M, et al. Nosocomial infections by Staphylococcus epidermidis:how a commensal bacterium turns into a pathogen. Int J Antimicrob Agents, 2006, 28 Suppl 1:S14-S20.
3. França A, Melo LD, Cerca N. Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis. BMC Res Notes, 2011, (4):572.
4. Chen L, Wen YM. The role of bacterial biofilm in persistent infection and control strategies. Int J Oral Sci, 2011, 3(2):66-73.
5. Sutter D, Stagliano D, Braun L, et al. Polymicrobial bloodstream infection in pediatric patients:risk factors, microbiology, and antimicrobial management. Pediatr Infect Dis J, 2008, 27(5):400-405.
6. 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.
7. McKenzie FE. Case mortality in polymicrobial bloodstream infections. J Clin Epidemiol, 2006, 59(7):760-761.
8. Park SJ, Han KH, Park JY, et al. Influence of bacterial presence on biofilm formation of Candida albicans. Yonsei Med J, 2014, 55(2):449-458.
9. Cerca N, Gomes F, Pereira S, et al. Confocal laser scanning microscopy analysis of S.epidermidis biofilms exposed to farnesol, vancomycin and rifampicin. BMC Res Notes, 2012, 5:244-250.
10. 刘卫娟, 左泽兰, 马荣华. 机械通气患儿气管导管表面细菌生物形成及病原分析. 中国微生态学杂志, 2009, 21(9):788-791.
11. 王醒, 江筱莉, 周昱薇, 等. 纳米银/聚氨酯覆膜新型抗菌气管导管在家兔体内的抑菌效果研究. 中国医疗设备, 2013, 28(8):20-22.
12. 龚凤云, 刘丽娜, 邢铭友, 等. 表皮葡萄球菌耐药与生物膜的相关研究. 中华医院感染学杂志, 2011, 21(1):20-23.
13. 郜向娜, 余加林, 芦起, 等. 四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型. 中国微生态学杂志, 2010, 22(12):1081-1088.
14. França A, Freitas AI, Henriques AF, et al. Optimizing a qPCR gene expression quantification assay for S.epidermidis biofilms:A comparison between commercial kits and a customized protocol, 2012, 7(5):e37480.
15. 李燕, 李冬冬, 陶传敏, 等. 表皮葡萄球菌生物膜形成及相关基因的检测与评价. 中华医院感染学杂志, 2010, 20(4):473-476.
16. Agarwal A, Jain A. Glucose & chloride induce biofilm production & ica operon in clinic isolates of Staphylococci. Indian J Med Res, 2013,138:262-266.
17. 叶联华, 黄云超, 许赓, 等. 医源性表皮葡萄球菌胞间黏附素基因操纵子在聚氯乙烯材料表面细菌生物膜形成中的作用研究. 中国修复重建外科杂志, 2011, 25(4):466-471.
18. Rohde H, Buran dt EC, Siems sen N, et al. Polysaccharide intercellular adhesion or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infection. Biomaterials, 2007, 28(9):1711-1720.
19. 汤琦, 袁兵, 黄云超,等. 乳腺外科表皮葡萄球菌icaA、icaD及聚集相关蛋白对细菌生物膜形成的影响. 中国修复重建外科杂志, 2014, 28(2):244-249.
20. 宋超, 余加林, 艾青, 等. 机械通气新生儿气管导管表面生物膜细菌多样性研究. 中华儿科杂志, 2013, 51(8):602-606.
21. Adam B, Baillie GS, Douglas LJ. Mixed species biofilms of Candida albicans and Staphylococcus epidermidis. J Med Microbiol, 2002, 51(4):344-349.
22. 丁进亚, 黄前川, 徐娟, 等. 置入性导管相关感染的病原菌与生物被膜形成. 医学综述, 2012, 18(6):933-935.

Chinese Journal of Reparative and Reconstructive Surgery

FUNCTION OF INTERCELLULAR ADHESION A, FIBRINOGEN BINDING PROTEIN, AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS

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