Biofilm formation and analysis of associated genes involved in staphylococcus isolates from bovine mastitis | 奶牛乳腺炎葡萄球菌生物被膜形成及相关基因分析

Chinese. 【目的】研究临床分离的奶牛乳腺炎葡萄球菌生物被膜形成能力,相关基因的分布及二者之间的关系;【方法】利用通用硅胶片法培养葡萄球菌生物被膜,经充分洗涤后对生物被膜进行银染定性判断菌株形成生物被膜的能力,通过生物被膜菌落结晶紫染色法定量检测菌株生物被膜的形成能力,并用扫描电镜观察被膜结构,对葡萄球菌bap、icaAD、icaBC、sar、agr、sigB、clfaA、clfaB、fnbpA和fnbpB进行PCR扩增检测。【结果】 银染法定性结果显示137株葡萄球菌中有120株形成肉眼可见的生物被膜,生物被膜形成率为87.6%;结晶紫染色定量检测与硅胶粘附的有132株,不粘附的有5株。试验所用的137株葡萄球菌中,57株能扩增出bap基因;有43株和54株分别能扩增出icaAD和icaBC;有73株、49株和38株分别扩增出sigB、sar和agr;分别有76株和50株染色体中存在clfaA和clfaB;fnbpA和fnbpB分别在52株和26株菌株中扩增出。【结论】推测bap、sigB、sar、icaAD和icaBC基因是生物被膜形成的重要相关基因;agr及粘附素基因clfaA、clfaB、fnbpA和fnbpB对生物被膜形成的作用不明显。

English. 【Objective】The objective of the study was to investigate biofilm forming ability, distribution of biofilm-associated genes of the clinically isolated bovine mastitis Staphylococcus and the correlation of them. 【Method】Staphylococcus biofilm formation was conducted using a silicone elastomer slices plate assay in 24-well plates, after rinsing planktonic bacteria away, the biofilm forming abilities of them were determined by silver staining qualitatively and by crystal violet staining quantitatively, and the structure of biofilm was observed by using scanning electron microscopy. bap, icaAD, icaBC, sar, agr, sigB, clfaA, clfaB, fnbpA and fnbpB were amplified by PCR.【Result】 Formation of biofilm could be found macroscopically in 120 out of 137 strains by silver staining, and the biofilm formation rate was 87.6%. It showed that 5 strains didn’t adhere to the surface of silica gel by crystal violet staining, while the rest 132 isolates did. bap was amplified in 57 isolates and icaAD and icaBC in 43 and 54 strains, respectively. sigB, sar and agr were amplified in 73, 49 and 38 isolates, and clfaA and clfaB in 76 and 50 strains, respectively. fnbpA was present in 52 strains and fnbpB in 26 isolates. 【Conclusion】 It reveals that bap, sigB, sar, icaAD and icaBC may be significant biofilm-associated genes, for these genes are present more in biofilm-positive strains than in biofilm-negative strains. The roles of agr, clfaA, clfaB, fnbpA, and fnbpB genes in biofilm development are unassured.