包装饮用水中检出铜绿假单胞菌的同源性分析

Homology Analysis of Pseudomonas Aeruginosa Detected in Packaged Drinking Water

【目的】为研究包装饮用水中检出铜绿假单胞菌种群特征,同时验证各方法在包装饮用水铜绿假单胞菌检验分析中的适用效果。【方法】采用飞行质谱(MALDI TOF MS)、16S rRNA基因序列测定、rpo B基因序列测定3种方法,对30株广东省东莞市包装饮用水食品安全监督抽检中检出的铜绿假单胞菌进行同源性分析。同时与国标GB 8538-2016中生化鉴定方法结果和菌株表型进行比对,确认常规检验结果。【结果】东莞地区包装饮用水中污染的铜绿在CN平板上菌落形态主要为浅绿色,其次为深绿色,极少数为黄色。飞行质谱法、16S rRNA法都能100%符合生化鉴定结果。飞行质谱结果与表型存在一定相关性,深绿色菌株基本集中在进化树的上方,且10株深绿色菌株中有8株集中在一个分支上。16S rRNA分型结果能识别到独特的黄色菌落菌株18A3074-1,其与其他菌株区别明显,处在进化树的最边缘位置。rpo B基因序列分型在菌株亲缘关系较近时具备一定的分辨率,来源于同一生产企业的4株菌株呈现聚集状态。【结论】质谱和16S rRNA同源分析结果都与菌株表型相关,初步积累了本地包装饮用水中的铜绿菌株库,为监管和污染溯源调查提供数据基础。3种方法各自具备独特优势:质谱法适用于日常检验鉴定,16S rRNA法适合疑难菌株鉴定,rpo B基因法可以用于近亲缘关系菌株的分型和溯源调查。

[Objective] In order to study the application value of various methods in the inspection of Pseudomonas aeruginosa in packaged drinking water and improve the efficiency of food safety supervision and inspection. [Methods] 30 strains of P.aeruginosa were detected in packaged drinking water drawn by food safety supervised sampling in Dongguan City, Guangdong Province. And the homology analysis was carried out by using three methods: mass spectrometry(MALDI TOF MS), 16S rRNA gene sequence determination, and rpo B gene sequence determination. These results are compared with the results of biochemical identification methods in the national standard GB 8538-2016 and strain phenotype. [Results] The results showed that the colony morphology on CN plates of P.aeruginosa in Dongguan packaged drinking water was mainly light green, followed by dark green, and very few were yellow. Both the MALDI TOF MS and 16S rRNA methods were 100% consistent with the biochemical identification results. There was a certain correlation between the MALDI TOF MS results and the phenotype. The dark green strains were basically concentrated at the top of the evolutionary tree, and 8 out of 10 dark green strains were concentrated on one branch. The 16S rRNA results could identify unique yellow colony strains 18A3074-1. However, the difference between dark and light color of colonies did not show strong correlation with 16S rRNA affinities. The rpo B gene sequence typing has a certain resolution when the strains are closely related, which is suitable as the basis for subspecies typing. [Conclusion] The results of mass spectrometry and 16S rRNA homology analysis were both related to the strains phenotype, and a library of P.aeruginosa in local packaged drinking water was preliminarily established, which provides a data basis for supervision and pollution traceability investigations. The three methods have their own unique advantages: Mass spectrometry is suitable for daily inspection and identification, 16S rRNA method is suitable for identification of confusing strains, and rpo B gene method can be used for typing and traceability investigation of closely related strains.