中性粒细胞杀菌活性试验检测高毒力肺炎克雷伯菌毒力的研究

Neutrophil bactericidal activity assay for detecting the virulence of hypervirulent Klebsiella pneumoniae

目的 利用中性粒细胞杀菌活性试验,检测毒力基因rmpA、rmpA2阳性和拉丝试验阳性的高毒力肺炎克雷伯菌抗中性粒细胞杀菌能力,验证该试验鉴定高毒力肺炎克雷伯菌的可行性.方法 收集浙江省2016年1月至2017年7月期间临床血流感染分离的150株非重复肺炎克雷伯菌.采用PCR检测碳青霉烯类抗生素耐药基因（blaKPC,blaNDM,blaIMP-1,blaIMP-2）、荚膜血清型（K1,K2,K5,K20,K54,K57）和毒力基因（rmpA,rmpA2,iucA,iroN）.将拉丝试验阳性和毒力基因rmpA、rmpA2阳性的菌株定义为高毒力肺炎克雷伯菌（hvKP）,拉丝试验和毒力基因rmpA、rmpA2阴性的菌株定义为经典肺炎克雷伯菌（cKP）.采用中性粒细胞杀菌活性试验检测肺炎克雷伯菌的毒力,细菌的存活率计算方式为：加中性粒细胞共孵育的实验组菌落形成单位（CFU）/不加中性粒细胞共孵育的对照组CFU.结果 150株肺炎克雷伯菌中检测到65株携带rmpA2毒力基因,占43.3％.在rmpA2阳性菌株中,rmpA、iroN、blaKPC和K2的检出率分别为73.8％、80.0％、75.4％和40.0％,拉丝试验阳性率为36.9％;中性粒细胞杀菌试验中,hvKP组菌株和cKP组菌株的存活率分别为0.866±0.056和0.368±0.058,两组存活率差异有统计学意义（P〈0.001）.结论 分离自浙江省携带rmpA、rmpA2毒力基因和拉丝试验阳性的hvKP经中性粒细胞处理后仍有非常高的存活率,中性粒细胞杀菌活性试验阳性,该试验是一个鉴定肺炎克雷伯菌毒力简单有效的方法.

Objective To evaluate the feasibility of using neutrophil bactericidal activity assay for analyzing the anti-bactericidal ability of hypervirulent Klebsiella pneumoniae （ hvKP） strains that harbored the virulence genes of rmpA and rmpA2 and were positive for string test .Methods A total of 150 non-duplicate blood-borne Klebsiella pneumoniae strains were collected from Zhejiang Province from January 2016 to July 2017.PCR was performed to detect carbapenem resistance genes （blaKPC, blaNDM, blaIMP-1, blaIMP-2）, cap-sule genotypes （K1, K2, K5, K20, K54 and K57） and virulence genes （rmpA, rmpA2, iucA and iroN）. Klebsiella pneumoniae strains that were positive for string test and harbored rmpA and rmpA2 genes were iden-tified as hvKP strains, while classic Klebsiella pneumoniae （cKP） strains were negative for string test, rmpA or rmpA2 gene.Neutrophil bactericidal activity assay was performed to analyze the virulence of Klebsiella pneumoniae strains and the survival rate was determined by using the following equation： the number of colony-forming units （ CFUs） in experimental group divided by the number of CFUs in control group .Re-sults Of the 150 Klebsiella pneumoniae strains, 43.3% （65/150） harbored the rmpA2 gene and among them, strains positive for genes of rmpA, iroN and blaKPC and K2 respectively accounted for 73.8%, 80.0%, 75.4%and 40.0%.Twenty-four （36.9%） rmpA2 gene-positive strains showed positive result of string test.The survival rates of hvKP and cKP groups were respectively 0.866±0.056 and 0.368±0.058 and the difference between them was statistically significant （P〈0.001）.Conclusion Most of the hvKP strains that carry rmpA and rmpA2 genes and are positive for string test in Zhejiang Province survive the neu-trophil treatment , which indicates that the neutrophil bactericidal activity assay is an effective and simple method for identifying the virulence of Klebsiella pneumoniae.