羊源肺炎克雷伯菌分离鉴定及其外膜囊泡提取方法的建立

doi:10.11843/j.issn.0366-6964.2025.01.033

摘要/Abstract

摘要：

旨在探究羊源肺炎克雷伯菌的致病性及其外膜囊泡的提取方法。本研究对伴有咳嗽、腹泻症状死亡的绵羊剖检并采集肺，肝，空肠等病变器官，采用形态观察、生化特性鉴定，分子生物学鉴定及测序方法对病原菌进行分离鉴定; 通过药敏试验、拉丝试验、毒力基因检测、致病性试验及病理组织学观察分析其致病性和耐药性; 使用改良沉淀法提取其外膜囊泡，通过透射电镜，纳米粒径测定及SDS-PAGE进行鉴定。结果显示病原菌经分离纯化后镜下呈现卵圆形革兰阴性杆菌，结合生化试验及16S rRNA鉴定结果表明该病原菌为肺炎克雷伯菌；药敏试验结果显示病原菌对阿米卡星，头孢他啶，亚胺培南，哌拉西林四种药物敏感；拉丝试验结果符合阳性特征及毒力基因扩增显示荚膜多糖基因wzy-K1及代谢基因peg-344为阳性，确定该病原菌为高毒力肺炎克雷伯菌；小鼠致病性试验结果表明病原菌对小鼠半数致死量(LD₅₀)浓度为1.8×10⁵ CFU，经病理组织学观察肝脏、脾脏淤血且有大量炎性细胞浸润；透射电镜，纳米粒径测定显示沉淀物形态结构、粒径大小均符合细菌外膜囊泡特征，SDS-PAGE显示存在特征性条带。本试验成功从病死绵羊体内分离出高毒力肺炎克雷伯菌并通过沉淀法提取其外膜囊泡，为预防和治疗羊源肺炎克雷伯菌病提供参考及肺炎克雷伯菌外膜囊泡的基础研究提供帮助。

关键词: 绵羊, 肺炎克雷伯菌, 致病性, 外膜囊泡

Abstract:

This experiment was conducted to investigate the pathogenicity of highly virulent Klebsiella pneumoniae of sheep origin and the extraction of its outer membrane vesicles. In this study, sheep that died with cough and diarrhea symptoms were dissected and the diseased organs such as lungs, livers, and jejunum were collected. Morphological observation, biochemical characterization, molecular biological identification, and sequencing methods were used to isolate and identify the pathogenic bacteria; the pathogenic bacteria were identified by drug sensitivity test, pulling test, virulence gene test, pathogenicity test, and pathohistological observation, and analyzed for pathogenicity and resistance; and the modified precipitation method was used to Its outer membrane vesicles were extracted and identified by transmission electron microscopy, nanoparticle size determination and SDS-PAGE. The results showed that the pathogen was isolated and purified, and the microscope showed oval-shaped gram-negative bacteria, and the biochemical test and 16S rRNA identification results indicated that the pathogen was Klebsiella pneumoniae; the results of drug sensitivity test showed that the pathogen was sensitive to four drugs, namely, amikacin, ceftazidime, imipenem, and piperacillin; the results of the pulling test showed the positive characteristics, and the virulence gene amplification showed that the pod polysaccharide gene wzy-K1 and the metabolism gene peg-344 were positive; the results of mouse pathogenicity test showed that the pathogen was positive to the LD₅₀ of mice. metabolic gene peg-344 was positive, the pathogen was identified as highly virulent Klebsiella pneumoniae; the results of the mouse pathogenicity test showed that the pathogen had a concentration of 1.8×10⁵ CFU in the mouse LD_50, and the liver and spleen were bruised with a large number of inflammatory cell infiltration; transmission electron microscopy and nano-particle sizing showed that the morphologic structure of the sediment and the particle size were consistent with the characteristics of the outer membrane vesicles of the bacteria. Transmission electron microscopy, nanoparticle size determination showed that the morphology and structure of the precipitate and the size of the particles were consistent with the characteristics of bacterial outer membrane vesicles, and SDS-PAGE showed the presence of characteristic bands. In this experiment, highly pathogenic Klebsiella pneumoniae was successfully isolated from sick and dead sheep and an improved method of extracting its outer membrane vesicles was established to provide reference for the prevention and treatment of Klebsiella pneumoniae disease of sheep origin and help for basic research on the outer membrane vesicles of Klebsiella pneumoniae.

Key words: goats, Klebsiella pneumoniae, pathogenicity, outer membrane vesicles

中图分类号:

S852.612

范维, 刘昕昕, 翟艺禄, 张新玉, 王唯, 付佳棋, 孙福亮. 羊源肺炎克雷伯菌分离鉴定及其外膜囊泡提取方法的建立[J]. 畜牧兽医学报, 2025, 56(1): 353-364.

FAN Wei, LIU Xinxin, ZHAI Yilu, ZHANG Xinyu, WANG Wei, FU Jiaqi, SUN Fuliang. Isolation and Identification of Klebsiella pneumoniae of Sheep Origin and Establishment of a Method for the Extraction of Its Outer Membrane Vesicles[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 353-364.

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基因 Genes	引物序列(5′→3′) Primer sequence(5′→3′)	片段大小/bp Product size	退火温度/℃ Annealing temperature
WABG	F：ACCATCGGCCATTTGATAGA R：CGGACTGGCAGATCCATATC	683	55
Uge	F: TCTTCACGCCTTCCTTCACT R: GATCATCCGGTCTCCCTGTA	534	57
Wyz-K1	F: GGTGCTCTTTACATCATTGC R: GCAATGGCCATTTGCGTTAG	1 200	57
Urea	F: GCTGACTTAAGAGAACGTTATG R: GATCATGGCGCTACCTCA	337	56
Alls	F: CCGAAACATTACGCACCTTT R: ATCACGAAGAGCCAGGTCAC	508	57
Peg-344-1	F: CTTGAAACTATCCCTCCAGTC R: CCAGCGAAAGAATAACCCC	508	58
ENTB	F: GTCAACTGGGCCTTTGAGCCGTC R: TATGGGCGTAAACGCCGGTGAT	400	59
Fimh	F: TGCTGCTGGGCTGGTCGATG R: GGGAGGGTGACGGTGACATC	550	55
Mrkd	F: AAGCTATCGCTGTACTTCCGGCA R: GGCGTTGGCGCTCAGATAGG	340	62
Irp2	F: GCTACAATGGGACAGCAACGAC R: GCAGAGCGATACGGAAAATGC	230	57
p-rmpA	F: GAGTAGTTAATAAATCAATAGCAAT R: CAGTAGGCATTGCAGCA	325	55
p-rmpA2	F: GTGCAATAAGGATGTTACATT R: GGATGCCCTCCTCCTG	430	59
iroN	F: GTCCGGCGGTAACTTCAGCC R: TCAGAATGAAACTACCGCCC	829	58
iucA-1	F: AATCAATGGCTATTCCCGCTG R: CGCTTCACTTCTTTCACTGACAGG	239	55

生化试验 Biochemical test	结果 Results	生化试验 Biochemical test	结果 Results
硫化氢 Hydrogen Sulfide	-	尿素 Carbamide urea ureophil	+
苯丙氨酸 Phenylalanine	-	半固体 Semi-solid	-
葡萄糖酸盐 Gluconate	+	葡萄糖产气 Glucose gas production	+
蛋白胨水 peptone water	-	赖氨酸 Lysine	+
葡磷胨水 Dextran Peptone Water	-	鸟氨酸 Ornithine	-
枸橼酸盐 Citrate	+	棉子糖 Marshmallow	+
木糖 Xylose	+	山梨醇 Sorbitol C₆H₁₄O₆	-
侧金盏花醇 lateral marigold alcohol	+

抗菌药物 Antibacterial drugs	抑菌圈直径/mm Inhibitory zone Diameter	抑菌圈判定标准/mm			敏感性 Sensitivity
抗菌药物 Antibacterial drugs	抑菌圈直径/mm Inhibitory zone Diameter	耐药 Resistent	中介 Intermediate	敏感 Sensitive	敏感性 Sensitivity
阿米卡星 Amikacin	20	≤14	15~18	≥19	S
头孢他啶 Ceftazidime	19	≤14	14~18	≥18	S
诺氟沙星 Norfloxacin	14	≤12	13~17	≥18	I
氟苯尼考 Florfenicol	7	≤12	13~17	≥18	R
红霉素 Eythromycin	11	≤15	16~20	≥21	R
四环素 Tetracycline	16	≤14	15~18	≥19	I
环丙沙星 Ciprofloxacin	12	≤15	16~20	≥21	R
米诺环素 Minocycline	9	≤14	15~18	≥19	R
亚胺培南 Imipenem	18	≤10	11~15	≥16	S
头孢哌酮 Cefoperazone	17	≤15	16~20	≥21	I
庆大霉素 Gentamycin	3	≤6	7~9	≥10	R
哌拉西林 Piperacillin	23	≤17	18~20	≥21	S
头孢曲松 Ceftriaxone	20	≤14	15~22	≥23	I
青霉素 Penicillin	11	≤13	13~17	≥17	R

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