羊源肠球菌溶血性的检测

Detecting hematolysis of Enterococcus from sheep

【目的】了解羊源肠球菌溶血素的特性。【方法】以平板法、接触法、培养法、上清法及PCR法,对11株肠球菌临床分离株、30株健康羊分离株、肠球菌参考株和G群链球菌参考株进行了溶血性检测。【结果】接触法和上清法均不能检测到11株肠球菌临床分离株对兔血和羊血的溶血;平板法和培养法测得11株肠球菌临床分离株中,63.6%对兔血呈现β溶血,36.4%对羊血平板呈现α溶血;基于检测cylA基因的PCR法,63.6%溶兔血菌能扩增出特异性条带,扩增产物序列与GenBank(L37110)中肠球菌同源性达99.3%。平板法测定30株健康羊分离株,初次分离培养53.3%对兔血β溶血,53.3%对羊血α溶血,43.3%对羊血β溶血,但二次传代后只有6%对兔血仍有溶血能力,且30株均不能检测到cylA。标准肠球菌对羊血平板有α溶血,而对兔血没有溶血性。【结论】提示肠球菌溶血性具有一定的溶血谱,不同检测方法检测的溶血情况不同;并且肠球菌溶血素必须在红细胞诱导下,通过细菌的生长繁殖产生;溶血素表型和基因型的检测不完全一致,对二者同时检测能提高肠球菌溶血素检测的准确性。

[Objective] To characterize hematolysis of Enterococcus from sheep. [Methods] Using plate assay （PA）, contact hemolysis （CH）, supernatant assay （SA）, culture hemolysis （CLH） and PCR, we studied hemolysis characteristics of 11 Enterococcus clinical isolates, 30 isolates from healthy sheep, a standard G-Streptococcus and a standard Enterococcus [Results] Rabbit blood and sheep blood were not hemolysising in the 11 clinical isolates analyzed by SA and CH. Of the clinical isolates 63.6% had β-hemolysis with rabbit blood and 36.4% had α-hemolysis with sheep blood analyzed by PA and CLH assay. Of the cylA gene 63.6% was detected in clinical isolates, the sequence of cylA gene was 99.3% homologous with cylA of plasmid pADl（GenBank accession number： L37110）. β-hemolysis had 53.3% in rabbit blood, α-hemolysis and β-hemolysis in sheep blood had 53.3% and 43.3% respectively in 30 healthy sheep initial isolates with PA. Only 6% had hemolytic capacity in rabbit blood after second generations. The cylA gene was not detected in 30 healthy sheep isolates by PCR. Standard Enterococcus strain of ix-hemolysis of sheep blood had no hemolysis of rabbit blood. [Conclusion] The red blood cells could induce enterococci hemolysis secreting in the bacteria growth. The result was different with the Phenotype and the Genotype.