基于高通量测序的6类中药饮片污染微生物群落特征分析

目的：研究6类中药饮片中污染微生物的群落特征，为中药饮片微生物限度标准制定提供依据。方法：参照《中华人民共和国药典》2015年版，对6类中药饮片共60批样品进行耐胆盐革兰阴性菌和沙门菌检查，采用VITEK生化鉴定系统对选择性分离平板上的微生物菌落进行鉴定，并基于16S rRNA高通量测序方法研究中药饮片中污染微生物的群落特征。结果：共有28批样品检出耐胆盐革兰阴性菌，不同类别饮片中耐胆盐革兰阴性菌的检出率差异较大，而沙门菌均未检出；菌株生化鉴定结果表明，6类中药饮片中污染的微生物均属于变形菌门，分布于14个属，其中包含阪崎克罗诺杆菌、肺炎克雷伯菌、铜绿假单胞菌、奇异变形杆菌、鲍氏不动杆菌等致病菌或条件致病菌；16S rRNA高通量测序微生物多样性分析结果表明，中药饮片污染微生物主要分布于6个门、27个属，不同类别中药饮片中的优势菌属具有明显差异。结论：16S rRNA高通量测序方法比传统培养法能够获得更加全面的中药饮片中污染微生物群落信息，不同类别中药饮片中污染微生物群落的组成具有一定差异，多种致病菌的检出提示中药饮片污染微生物具有一定的致病风险。

Objective: To study the microbial community composition of six Chinese herbal pieces, and provide support for the establishment of microbial limit standards. Methods: According to the Pharmacopoeia of the People's Republic of China 2015 edition, a total of 60 batches Chinese herbal pieces were tested for bile-tolerant Gram-negative bacteria and Salmonella, the microbial colonies on selective isolation medium were identified by VITEK system, and the microbial community composition of six Chinese herbal pieces were further studied based on 16S rRNA high-throughput sequencing method. Results: A total of 28 batches were positive for bile-tolerant Gramnegative bacteria, the detection rates of bile-tolerant Gram-negative bacteria in different types of Chinese herbal pieces showed significant difference, but Salmonella was not detected in any samples. The results of biochemical identification of strains on selective isolation medium demonstrated that the microbes all belong to the Proteobacteria, distributed in 14 genera, including Enterobacter sakazakii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa;16S rRNA microbial diversity analysis showed that the contaminated microorganisms of six Chinese herbal pieces were mainly distributed in 6 phylum and 27 genera, and the dominant group in different types had significant differences. Conclusion: This study revealed that 16S rRNA high-throughput sequencing method can provide more comprehensive information on the microbial community composition of Chinese herbal pieces than the traditional culture method. The contaminated microbial communities in different types of Chinese herbal pieces show significant differences, and various pathogenic bacteria are detected in Chinese herbal pieces, implying the potential risk of contaminated microorganisms in Chinese herbal pieces.

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