Isolation,Identification and Biological Characteristics of Carbapenem-resistant Pseudomonas aeruginosa from Tupaia belangeri
[Objective]This experiment was aimed to determine the cause of death of Tupaia belangeri during the breeding process,analyze the biological characteristics,pathogenicity and drug resistance of the pathogen,and develop prevention and control methods.[Method]Pathological dissection was performed on the dead Tupaia belangeri and the pathological changes of each major organ were recorded,including lung,liver,spleen and intestine.Single colonies were isolated from the main diseased organs using the streak plate method,and the morphology of the colonies was observed.At the same time,Gram staining was used to observe under the microscope,followed by biochemical and drug resistance tests,and pathogenicity test.Further analysis was conducted on the isolated strains using 16S rRNA gene sequencing and bacteria genome sequencing.[Result]A carbapenem-resistant Pseudomonas aeruginosa strain was isolated from the spleen and intestine of the deceased Tupaia belangeri chinensis.The isolate presented a gray-white colony producing green water-soluble pigments in LB agar medium,and was Gram-negative under microscope.Biochemical tests further confirmed that it was a non-fermenting bacterium.Sequence evolution analysis showed that it had a high similarity of 99％with the soil source Pseudomonas aeruginosa F4 in GenBank.The drug sensitivity test results showed that the isolated strain had certain resistance to carbapenem antibiotics such as imipenem and meropenem,as well as quinolone antibiotics such as levofloxacin.The pathogenicity test result showed that the median lethal dose(LD50)of the isolated strain to adult Kunming mice was 3.382 ×108 CFU.The organs and tissues of dead mice were isolated for pathological observation,and inflammatory damage and necrosis of the lungs,liver,spleen,intestine,etc.were observed.Bacterial isolation was conducted again on various organs,and based on 16S rRNA sequence analysis,Pseudomonas aeruginosa could be found in the spleen,intestine and blood of dead mice,with high pathogenicity.The whole genome sequencing results confirmed that the isolate strain had genes related to carbapenem resistance and shared similar virulence factor genes with the highly pathogenic reference strain of Pseudomonas aeruginosa.[Conclusion]This study isolated one carbapenem-resistant Pseudomonas aeruginosa from the deceased Tupaia belangeri,providing a new theoretical basis for the study of microbial pathogenicity and domestication of Tupaia belangeri.
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维普
