血液来源携带psm-mec基因人葡萄球菌SCCmec型别分析与生物被膜能力的研究

Study on the Biofilm Formation and SCCmec typing of psm-mec Positive Staphylococcus Hominis from Blood Culture

目的研究血液来源携带psm-mec基因人葡萄球菌SCCmec型别和psm-mec基因与生物被膜的相关性,为其临床感染的防治提供依据。方法收集临床血液来源经全自动微生物鉴定仪确认的人葡萄球菌。通过PCR扩增mecA基因区分耐甲氧西林人葡萄球菌(methicillin-resistant Staphylococcus hominis,MRSHo)和甲氧西林敏感人葡萄球菌(methicillin-susceptive Staphylococcus hominis,MSSHo)。体外粘附实验(Microtite Plate Assay,TCP)检测生物被膜。PCR扩增psm-mec基因并测序,分析其与生物被膜的相关性。PCR扩增psm-mec与mecR1及xylR基因的间隔序列和对psm-mec基因阳性菌株做SCCmec、mec和ccr型别检测,探究其在SCCmec上的定位与分布特征。结果共收集菌株55株,其中46株检出mecA基因为MRSHo,18株携带psm-mec基因,35株产生物被膜。携带与未携带psm-mec基因的菌株形成生物被膜的比率分别为83.33%和54.1%,二者差异显著(P=0.03)。DNA测序显示,3株psm-mec阳性生物被膜阴性的菌株中,2株于psm-mec编码区上游-12处发现G>A的点突变。所有psm-mec与mecR1及xylR的基因间隔序列阳性。携带psm-mec基因菌株分为,2株SCCmecⅢ型,7株Ⅲ型的变异型,9株SCCmec新型别;所有菌株均为Class A类mec;6株为ccr type 1,1株为ccr type 1+2,2株为ccr type 1+3,2株为ccr type 3,7株未扩增出ccr。结论在血液来源人葡萄球菌中,psm-mec基因与生物被膜存在相关性,其定位于mecR1与xylR两个基因之间,主要分布于SCCmecⅢ型、Ⅲ型变异型和新型别中,ccr基因存在高度变异,是造成SCCmec多态性的原因。

Objective The aim of this study was to analyze the SCCmee typing of psm-mec positive Staphylococcus homi- nis from blood culture and the relationship between psmmec with biofilm formation, to provide the basis for prevention and control of nosoeomial infection caused by Staphylococcus hominis. Methods Staphylococcus hominis were isolated from positive blood culture and identified by full automation nficrobiologieal identification system. The mecA gene was detected by PCR to distinguish MR- SHo from MSSHo. Mierotite Plate Assay（TCP） was used to evaluate boifilm formation capacity of Staphylococcus honfinis, psm-mee was amplified by PCR, and DNA sequencing was made to measure the nmtant of psm-mec. The correlation of between psm-mee with biofilm were analyzed. For analyzing the genetic location characteristic of psm-mec in SCCmee, two pairs special PCR primers were used to measure meeR1/psm-mee and psm-mee/xylR respectively. SCCmee types of psm-mee positive stains were determined by the results of a special multiplex PCR assay directly and by the results of mee and eer types indirectly. Results 55 strains of Staphylo- coccus hominis were isolated from positive blood culture. Among those isolates ,46 isolates were tested positive for the meeA gene to be MRSHo,18 isolates were psm-mee positive, 35 isolates were biofilm producers. 83.33% psm-mee positive strains compared with 54. 1% psm-mee negative strains had the ability of forming biofilm, and psm-mee had a correlation with biofilm formation（ P 〈 0. 05 ）. The results of psm-mee DNA sequencing were completely matched with psm-mee ORF by Blast. 2 out of 3 psm-mee positive non-biofilm strains had a mutant（ G 〉 A）locating at-12 upstream of psm-mee ORF. All of the 18 psm-mee positive strains were meeR1/ psm-mee and psm-mec/xylR positive. The results of the multiplex PCR showed,among psm-mee positive strains,2 strains belonged to SCCmee Ⅲ ,7 strains belonged to SCCmee Ⅲ-like,and 9 strains belonged to new SCCmee types. Among psm-mee positive isolates, all isolates harbored class A mee complex,7 isolates were not positive for any- eer complex,6 isolates contained ccr type 1,1 isolate con- tained eer typel ＋2,2 isolates contained eer type 1 ＋ 3,and 2 isolates contained eer type 3. Conclusion In MRSHo isolated from positive blood culture, psm-mee gene has a correlation with biofilm, psm-mee gene locates between meeR1 and xylR gene,and distrib- utes mainly in SCCmee Ⅲ, SCCmee Ⅲ-like and new SCCmee types, and the genetic diversity- of cer complex leads the SCCmee types variable.