乳腺外科表皮葡萄球菌icaA、icaD及聚集相关蛋白基因对细菌生物膜形成的影响

BIOFILM AND ROLE OF icaA, icaD, AND ACCUMULATION-ASSOCIATED PROTEIN IN STAPHYLOCOCCUS EPIDERMIDIS ISOLATED IN BREAST SURGERY

目的探讨乳腺外科表皮葡萄球菌icaA、icaD和聚集相关蛋白（accumulation．associatedprotein，aap）基因对细菌生物膜形成的影响。方法于2011年12月-2013年1月乳腺外科收治的无感染症状女性患者临床标本中分离获得44株表皮葡萄球菌。PCR检测菌株icaA、icaD、aap基因，并根据基因表达结果将其分为icaA- icaD＋／aap＋组（A组）、icaA＋icaD＋／aap-组（B组）、icaAicaD-／aap＋组（C组）及icaA-icaDTaap组（D组）。取4组菌株制备硅胶材料表面细菌生物膜体外模型，于孵育8、12、24、30、36h采用结晶紫染色法半定量检测细菌黏附能力，孵育12、24h激光共聚焦显微镜观测形成的生物膜厚度，孵育24h扫描电镜观察细菌生物膜超微结构。结果PCR检测结果示，44株表皮葡萄球菌中，13株为icaA＋ icaD＋／aap＋（A组），12株为icaAqcaD＋／aap（B组），16株为icaAicaD-／aap＋（C组），3株为icaAicaD-／aap-（D组）。共29株（65．9％）形成细菌生物膜，其中A组13株，B组7株，C组9株，D组0株。半定量黏附实验示：孵育8h，4组细菌黏附能力比较差异无统计学意义（P〉0．05）；12～36h时，A、B、C组黏附能力均显著高于D组，A组显著高于B、C组（P〈0．05），B、C组间比较差异均无统计学意义（P〉0．05）。激光共聚焦显微镜观察示，12、24h时A、B、C组细菌生物膜厚度均大于D组，A组明显大于B、C组（P〈0．05）；B、C组间比较差异无统计学意义（P〉0．05）。扫描电镜观察示，孵育24h时A、B、C组硅胶片表面均可见成熟的细菌生物膜结构，其中A组细菌生物膜分布最广泛、结构最致密、层次最丰富，B、C组细菌生物膜结构相似；D组无明显细菌生物膜形成。结论icaA、icaD基因及aap基因对表皮葡萄球菌在硅胶表面的细菌生物膜形成均起重要作用，其中aap基因与icaA、icaD基因同时表达的菌株具有最强的细菌生物膜形成能力。

Objective To investigate biofilm formation on the surface of silica gel by breast surgery clinical specimens of Staphylococcus epidermidis and to analyze the relationship between biofilm formation and icaA, icaD, and accumulation- associated protein （aap） gene. Methods Between December 2011 and January 2013, 44 strains of Staphylococcus epidermidis were isolated from the clinical specimens of the female patients who had no symptom of infection. The icaA, icaD, and aap genes were detected by PCR and 4 genotypic groups were divided： icaA＋icaD＋/aap＋ group （group A）, icaA＋icaD＋/aap group （group B）, icaA-icaD/aap＋ group （group C）, and icaA icaD/aap- group （group D）. Biofilms mass was semi-quantified by semi-quantitative adherence assay after 8, 12, 24, 30, and 36 hours of incubation. The thickness of biofilms was measured by confocal laser scanning microscope （CLSM） at 12 and 24 hours after incubation. The ultrastructure of biofilms was observed by scanning electron microscope （SEM） at 24 hours after incubation. Results PCR test showed that 13 strains were icaA＋icaD＋/ aap＋（group A）, 12 strains were icaA＋icaD＋/aap-（group B）, 16 strains were icaA icaD/aap＋（group C）, and 3 strains were icaA icaD/aap-（group D）. In 29 strains which had bacterial biofilm formation （65.9%）, there were 13 strains in group A, 7 strains in group B, 9 strains in group C, and 0 in group D. The result of semi-quantitative adherence assay showed no significant difference in the absorbance （A） values among 4 groups at 8 hours （P 〉 0.05）. The A values of groups A, B, and C were significantly higher than that of group D at 12-36 hours, and group A was significantly higher than groups B and C （P 〈 0.05）, but there was nosignificant difference between groups B and C （P 〉 0.05）. The results of CLSM showed that the thickness ofbiofilm in groups A, B, and C was significantly larger than that in group D at 12 and 24 hours after incubation （P 〈 0.05）, and the thickness of biofilm in group A was significantly larger than that in groups B and C （P 〈 0.05）, but there was no significant difference between groups B and C （P 〉 0.05）. The result of SEM showed that the mature biofilm could be observed on the surface of silica gel in groups A, B, and C, and the ultrastructure of biofdms in group A were the most abundant and extensive among 3 groups. The ultrastructure of biofilm in group B was similar to that in group C. No obvious biofilms formed in group D. Conclusion icaA, icaD, and aap genes all play key roles in the process for biofilm formation ofStaphflococcus epidermidis. Futhermore, aap gene enhance the ability of biofilm-forming when aap and ica genes coexist, so the biofilm-forming ability of icaA＋icaD＋/aap＋ is strongest.