2株鹌鹑源肺炎克雷伯菌的分离鉴定及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2025.05.039

摘要/Abstract

摘要： 【目的】从腹泻鹌鹑粪便样本中分离并鉴定肺炎克雷伯菌，以期为鹌鹑肺炎克雷伯菌疾病防控提供基础。【方法】从8份腹泻鹌鹑粪便样本中筛选纯化菌落，通过平板划线法对细菌进行分离纯化培养，利用PCR扩增16S rDNA序列并测序，使用Mega 11.0软件构建系统进化树。采用K-B法检测菌株对常见抗菌药物的敏感性，并检测菌株中耐药基因和毒力基因分布情况，探究其生物学特性，通过动物试验评估菌株的致病性。【结果】本研究从腹泻鹌鹑粪便样本中分离并鉴定出2株肺炎克雷伯菌，分别命名为Ksp-A1和Ksp-A2，均属于K57血清型。系统进化树结果显示，2株分离株与其他已知肺炎克雷伯菌株相似性较高，且与肺炎克雷伯菌DB-4和WGT-31株属于同一进化分支。药敏试验结果显示，分离株对氨苄西林、苯唑西林和红霉素等表现耐药。分离株Ksp-A1和Ksp-A2均携带多种耐药基因，包括bla_SHV、aacC2、oqxAB、tetA、tetM和cat1基因。2株分离株均携带毒力基因K88、hlyE、iucD、irp2、ompA和ompC。生物学特性分析显示，在pH 7.0的LB肉汤培养基中，分离株Ksp-A1和Ksp-A2均在2 h内进入对数生长期，并分别在12和9 h达到峰值。小鼠致病性试验结果显示，注射菌液后，小鼠表现被毛粗乱、精神沉郁、呼吸急促及腹泻等感染症状，且随着菌液浓度增加，小鼠死亡率逐渐上升，菌液浓度为1×10⁹ CFU/mL时，小鼠死亡率均达100%，Ksp-A1和Ksp-A2对小鼠的半数致死剂量(LD₅₀)分别为3.3×10⁷和2.6×10⁶ CFU。【结论】试验成功分离获得2株鹌鹑源K57型肺炎克雷伯菌，其对β-内酰胺类药物表现耐药，生长速度较快，对酸碱耐受能力强。研究结果为鹌鹑源肺炎克雷伯菌疾病的防治提供了基础数据。

关键词: 鹌鹑; 肺炎克雷伯菌; 分离鉴定; 耐药性; 毒力基因

Abstract: 【Objective】 The study was conducted to isolate and identify Klebsiella pneumoniae from fecal samples of quail with diarrhea,in order to provide a basis for the prevention and control of Klebsiella pneumoniae disease in quail.【Method】 The purified colonies were screened from 8 fecal samples of quail with diarrhea,and the bacteria were isolated and purified by plate scribing method. 16S rDNA sequence was amplified by PCR and sequenced,and the phylogenetic tree was constructed using Mega 11.0 software.K-B method was used to detect the sensitivity of strains to common antibiotics, the distribution of drug resistance genes and virulence genes in strains was detected,and the biological characteristics was explore.The pathogenicity of the strains was evaluated by animal tests.【Result】 In this study,two strains of Klebsiella pneumoniae were isolated and identified from fecal samples of diarrhea quail,named Ksp-A1 and Ksp-A2,respectively,both belonging to K57 serotype.Phylogenetic tree showed that the two isolates were similar to other known Klebsiella pneumoniae strains,and belonged to the same evolutionary clade as Klebsiella pneumoniae strains DB-4 and WGT-31.The results of drug sensitivity test showed that the isolates were resistant to ampicillin,oxacillin,erythromycin,and so on.Isolates Ksp-A1 and Ksp-A2 carried multiple resistance genes,including bla_SHV,aacC2,oqxAB,tetA,tetM and cat1 genes.The two isolates carried virulence genes such as K88,hlyE, iucD,irp2,ompA and ompC.Biological characteristics analysis showed that in LB broth medium with pH 7.0,isolates Ksp-A1 and Ksp-A2 entered the logarithmic growth phase within 2 h,and reached the peak at 12 and 9 h,respectively.The results of pathogenicity test in mice showed that after the injection of the bacterial solution,the mice showed infection symptoms such as coarse hair,depression,shortness of breath and diarrhea,and the mortality rate of the mice gradually increased with the increase of the bacterial solution concentration,and the mortality rate of the mice reached 100% when the bacterial solution concentration was 1×10⁹ CFU/mL. The median lethal dose (LD₅₀) of isolates Ksp-A1 and Ksp-A2 to mice were 3.3×10⁷ and 2.6×10⁶ CFU,respectively. 【Conclusion】 Two strains of Klebsiella pneumoniae K57 from quail were successfully isolated in the experiment，which showed resistance to β-lactam drugs,had rapid growth rates,and showed high tolerance to acidic and alkaline conditions.The results provided basic data for the prevention and treatment of Klebsiella pneumoniae disease in quail.

Key words: quails; Klebsiella pneumoniae; isolation and identification; drug resistance; virulence genes

中图分类号:

S852.61

龙宝琴, 王辉翔, 余林金, 哈尔勒哈·阿曼太, 陈浩然, 许梦娇, 史陇兴, 李有文. 2株鹌鹑源肺炎克雷伯菌的分离鉴定及生物学特性分析[J]. 中国畜牧兽医, 2025, 52(5): 2364-2378.

LONG Baoqin, WANG Huixiang, YU Linjin, HAERLEHA·Amantai, CHEN Haoran, XU Mengjiao, SHI Longxing, LI Youwen. Isolation,Identification and Biological Characteristics Analysis of Two Strains of Klebsiella pneumoniae from Quail[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2364-2378.

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