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

Abstract

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

CLC Number:

S852.612

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.

Figures/Tables 11

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Table 2

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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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Isolation and Identification of Klebsiella pneumoniae of Sheep Origin and Establishment of a Method for the Extraction of Its Outer Membrane Vesicles

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