生物标志物联合大蜡螟感染模型在鉴别临床高毒力肺炎克雷伯菌中的应用  

Application of biomarkers combined with Galleria mellonella infection model in the identification of clinical hypervirulent Klebsiella pneumoniae

作　　者：宋爽 赵树龙 孙静芳 徐银海 马萍 康海全 SONG Shuang;ZHAO Shulong;SUN Jingfang;XU Yinhai;MA Ping;KANG Haiquan(Department of Laboratory Medicine,Affiliated Hospital of Xuzhou Medical University,Xuzhou,Jiangsu 221002,China;College of Medical Technology,Xuzhou Medical University,Xuzhou,Jiangsu 221004,China)

机构地区：[1]徐州医科大学附属医院检验科,江苏徐州221002 [2]徐州医科大学医学技术学院,江苏徐州221004

出　　处：《检验医学与临床》2024年第20期3053-3058,共6页Laboratory Medicine and Clinic

基　　金：江苏省卫生健康委员会科研项目(Z2021009、Ym2023110);江苏省徐州市科技计划项目(KC23269);徐州医科大学附属医院院级科研项目(2023ZY07)。

摘　　要：目的探讨生物标志物检测联合大蜡螟感染模型准确鉴别临床高毒力肺炎克雷伯菌(hvKP)的应用价值。方法非重复收集2020年1月至2021年12月徐州医科大学附属医院分离自重症监护室患者的肺炎克雷伯菌,经拉丝实验和毒力基因检测鉴定出生物标志物阳性的肺炎克雷伯菌,并进行荚膜血清分型和多位点序列分型。使用标准菌株ATCC700603、ATCC43816建立大蜡螟感染模型,将临床分离的肺炎克雷伯菌分别制作4个不同浓度的菌液进行大蜡螟感染试验,每12小时记录死亡情况,绘制生存曲线并计算72 h 50%致死量(LD_(50))。结果共收集15株hvKP,检出3种血清型,分别为K1 ST23型(13.3%,2/15)、K64 ST11型(60.0%,9/15)、K112 ST15型(26.7%,4/15)。K1和K64型的菌株均含有rmpA、rmpA2、iucA和iroN 4个毒力基因,而K112型的4株均只含有rmpA2和iucA 2个毒力基因。K64 ST11型和K112 ST15型菌株均对碳青霉烯类抗菌药物耐药。大蜡螟能够区分出高毒力和低毒力肺炎克雷伯菌,当菌液浓度分别为1×10^(5) CFU/mL、1×10^(6) CFU/mL、1×10^(7) CFU/mL、1×10^(8) CFU/mL时,ATCC700603和ATCC43816对应浓度比较,差异有统计学意义(P=0.0001、0.0023、0.0245、0.0428)。未做任何处理的空白对照和注射了磷酸盐缓冲液的大蜡螟均存活,随着菌液浓度的升高,大蜡螟的病死率也随之升高。与高毒力标准菌株ATCC43816相同,K1、K64、K112型临床菌株均表现出高致死率,当菌液浓度在1×10^(7)~1×10^(8) CFU/mL时,所有大蜡螟在48 h内死亡。1×10^(6) CFU/mL的菌液可区分出hvKP和cKP,差异均有统计学意义(P<0.05)。ATCC700603、ATCC43816、K1型、K64型、K112型和cKP在72 h的LD_(50)分别为5.2×10^(6) CFU/mL、6.8×10^(4) CFU/mL、1.0×10^(4) CFU/mL、1.0×10^(4) CFU/mL、3.1×10^(4) CFU/mL、1.6×10^(6) CFU/mL,对应的Log10LD_(50)分别为6.718、4.834、3.987、3.987、4.504、6.212,临床分离的K1型、K64型、K112型肺炎克雷伯菌均可被定为高毒力菌株Objective To explore the application value of biomarker detection combined with Galleria mellonella infect ion model to accurately identify clinical hypervirulent Klebsiella pneumoniae(hvKP).Methods Kl ebsiella pneumoniae isolated from patients in the intensive care unit of the Affiliated Hospital of Xuzhou Medical University from January 2020 to December 2021 were collected in a non-repeated way.The biomarkers of Klebsiella pneumoniae were identified by string test and virulence gene detection,and capsule serotyping and multilocus sequence typing were performed.Standard strains ATCC700603 and ATCC43816 were used to establish Galleria mellonella infection models,and clinical isolates of Klebsiella pneumoniae were made into four different concentrations for Galleria mellonella infection test.The death rate was recorded every 12 hours,and the survival curve was drawn and the 72-hour 50%lethal dose(LD_(50))was calculated.Results A total of 15 hvKP strains were collected,and 3 serotypes were detected,including K1 ST23(13.3%,2/15),K64 ST11(60.0%,9/15),and K112 ST15(26.7%,4/15).The K1 and K64 strains all contained four virulence genes,rmpA,rmpA2,iucA and iroN,while the K112 strains all contained only two virulence genes.Both K64 ST11 and K112 ST15 strains were resistant to carbapenems.Galleria mellonella can distinguish high and low virulence Klebsiella pneumoniae.When the concentration of bacteria solution was 1×10^(5) CFU/mL,1×10^(6) CFU/mL,1×10^(7) CFU/mL and 1×10^(8) CFU/mL,the corresponding concentration of ATCC700603 and ATCC43816 was compared,and the differences were all statistically significant(P=0.0001,0.0023,0.0245,0.0428).The blank control group without any treatment and the phosphate buffer saline injected group survived.With the increase of bacterial concentration,the mortality of the galleria mellonella also increased.Similar to the highly virulent standard strain ATCC43816,the clinical strains of K1,K64 and K112 showed high lethality,and all strains died within 48 hours when the bacterial concentrat

关 键 词：大蜡螟感染模型 毒力基因检测 肺炎克雷伯菌 LD 50 高毒力 

分 类 号：R446.5[医药卫生—诊断学] R372[医药卫生—临床医学]