大熊猫源肺炎克雷伯菌生物学特性

Isolation, identification, and biological characterization of Klebsiella pneumoniae from Ailuropoda melanoleuca

摘要:

【背景】肺炎克雷伯菌是仅次于大肠杆菌的常见条件致病菌之一，严重时可导致大熊猫发生出血性肠炎、全身性败血症等。【目的】明确大熊猫源肺炎克雷伯菌的生物学特性，对防控该病作出科学指导。【方法】分别采用结晶紫染色法、拉丝实验、K-B纸片法和PCR技术对46株大熊猫源肺炎克雷伯菌的生物被膜形成能力、高黏性表型、耐药表型和15种常见毒力基因等生物学特性进行研究，并根据以上生物学特性选择一株可能具有致病性的分离菌pneumoniae-X-5，研究其对小鼠的致病性。【结果】46株肺炎克雷伯菌均可形成荚膜；12株为高黏性表型肺炎克雷伯菌；能形成生物被膜的菌株占比为65%(30/46)；分离出的46株菌中多重耐药菌株占58%(27/46)，对氨苄西林、苯唑西林、青霉素、万古霉素呈100%耐药；毒力基因检出率最高的为ureA(91.30%，42/46)。pneumoniae-X-5菌株对小鼠的LD₅₀为8.9×10⁴CFU/mL；该菌株攻毒小鼠肺泡间隔增厚，炎性细胞浸润，肝细胞变性坏死，脾充血，十二指肠黏膜上皮和固有层分离，固有层部分细胞坏死。死亡小鼠脾脏含细菌量最多，其次为肝脏。【结论】本试验阐明了部分大熊猫源肺炎克雷伯菌的多重耐药性、能形成生物被膜、具有高黏表型等病原生物学特性，为大熊猫肺炎克雷伯杆菌病的防控及临床治疗提供了科学依据。

关键词:大熊猫;肺炎克雷伯菌;高黏性表型;毒力基因;耐药表型;致病性;病理变化

Abstract:

[Background] Klebsiella pneumoniae, one of the common opportunistic pathogens after Escherichia coli, can lead to hemorrhagic enteritis and systemic sepsis in giant pandas (Ailuropoda melanoleuca). [Objective] To identify the biological characteristics of K.pneumoniae from giant pandas and provide scientific guidance for the prevention and control of the disease. [Methods] The biofilm formation, high viscosity, drug resistance, and 15 common virulence genes of 46 strains of K.pneumoniae from A.melanoleuca were studied via crystal violet staining, string test, K-B disk diffusion method, and PCR, respectively. Further, we selected an isolate (pneumoniae-X-5) that may be pathogenic according to the above biological characteristics to study its pathogenicity in mice. [Results] All the 46 strains of K.pneumoniae could form capsule, among which 12 strains showed the phenotype of high viscosity and 65% (30/46) of the strains could form biofilm. Among the strains, 58% (27/46) were multi-drug resistant and 100% were resistant to ampicillin, oxacillin, penicillin, and vancomycin. Of the virulence genes, ureA had the highest detection rate of 91.30% (42/46). The LD₅₀ of pneumoniae-X-5 in mice was 8.9×10⁴ CFU/mL. The mice challenged with this strain had thickened alveolar septa, inflammatory cell infiltration, hepatocyte degeneration and necrosis, splenic congestion, separation of duodenal mucosal epithelium and lamina propria, and partial cell necrosis in the lamina propria. The spleen contained the highest amount of bacteria in the dead mice, followed by the liver. [Conclusion] This study elucidates the multi-drug resistance, biofilm formation, high viscosity and other pathogenic characteristics of K.pneumoniae from A.melanoleuca, providing a scientific basis for the prevention, control, and clinical treatment of K.pneumoniae disease in A.melanoleuca.

Key words:Ailuropoda melanoleuca;Klebsiella pneumoniae;high viscosity;virulence gene;drug resistance;pathogenicity;pathological change