经小鼠尾静脉注射诱导肺炎克雷伯菌血流感染模型的建立及评价

Establishment and evaluation of bloodstream infection model induced by Klebsiella pneumoniae via tail vein injection in mice

目的建立可靠的肺炎克雷伯菌血流感染模型,为细菌性血流感染早期诊断相关研究提供可靠的依据。方法选择肺炎克雷伯菌标准株ATCC 700603为实验菌株,将不同浓度的细菌悬液通过尾静脉注入小鼠体内,按Karber法计算LD50,以1/2LD50浓度注射小鼠,通过其体征变化、体重变化、血培养、分子生物学试验、H·E染色等指标综合评价模型,验证造模效果。结果小鼠LD50为1.11×109 CFU/ml,菌液感染小鼠1h后开始出现立毛,活动减少;感染前体重为(33.60±1.21)g,注射菌液后1天内体重下降至(28.86±1.42)g,跟对照组比较有明显差别(P<0.05),7天后恢复正常水平(33.17±1.72)g;正常小鼠白细胞为(1.42±0.66)×109/L,注射菌液3h后升至(2.52±1.01)×109/L,6h升至(3.08±0.85)×109/L,跟对照组比较有明显差别(P<0.05),2天后白细胞进一步升至(6.88±3.11)×109/L,7天后仍无明显下降;小鼠全血培养结果表明前2天任意时间血培养结果为阳性;毒力因子引物扩增疑似菌DNA,结果表明小鼠血流感染病原菌均为肺炎克雷伯菌;注射菌液1天后小鼠肺泡明显渗出、水肿、白细胞浸润;2天后肺结构逐渐恢复,肝恢复速度较肺慢。结论建立了小鼠肺炎克雷伯菌血流感染模型,能够为血流感染的早期诊断和不同病原菌感染的鉴别诊断等研究提供了可靠的动物模型。

OBJECTIVE To establish a reliable model of Klebsiellapneumoniaebloodstream infection,so as to provide a reliable experimental model for the study of early diagnosis of bacterial bloodstream infection.METHODS K.pneumoniae ATCC700603 was selected as experimental strain.Different concentrations of bacterial suspension were injected into mice via tail vein,and the LD50 was calculated by Karber method and 1/2LD50 was used as the injection concentration.The general condition,body weight changes,blood culture,molecular biological test and HE staining were observed to comprehensively evaluate the model,and verify the modeling effect.RESULTS The LD50 was 1.11×10^9 CFU/ml,when the bacterial fluid was infected after 1hour,and the mice＇s activity was decreased.The weight before infection was（33.60±1.21）g,1day after the injection,the weight decreased to（28.86±1.42）g,and there were significant differences compared with that of control group（P〈0.05）,and returned to normal level after 7days,which was（33.17±1.72）g.White blood cells in normal mice was（1.42±0.66）×10^9/L,after 3hours of injection of the bacteria,it increased to（2.52±1.01）×10^9/L,and increased to（3.08±0.85）×10^9/L after 6hours,and there were significant differences compared with control group（P〈0.05）.White blood cells increased to（6.88±3.11）×10^9/L after 2days,and there had no significant decline 7days later.The results of whole blood culture showed that the blood culture results were positive in the first 2days.The virulence factor PCR results showed that the pathogenic bacteria of the bloodstream in mice were K.pneumoniae.After 1daysof injection,the mice were infiltrated with obvious exudation,edema and leukocyte infiltration.The lung structure was gradual recovery after 2days,and liver recovery rate was slower than the lung.CONCLUSION A bloodstream infection model is successfully established,which provides a reliable animal model for the early diagnosis of bloodstream infection and the differential diagnosis of different pathogenic bacteria infection.