基于16S rRNA序列分析的动物饮片典型菌污染特征研究  

Characterization of typical bacteria isolation from animal decoction spieces based on 16S rRNA sequence analysis

作　　者：于志君[1] 李腊梅[2] 刘婧[1,3] 江珍玉 邓海英 YU Zhi-jun;LI La-mei;LIU Jing;JIANG Zhen-yu;DENG Hai-ying(Hubei Province Key Laboratory of Occupational Hazard Identification and Control,Wuhan University of Science and Technology,Wuhan 430065,China;Jingmen Center for Disease Control and Prevention,Jingmen 448000,China;Yichang Humanwell Pharmaceutical CO.,LTD.,Yichang 443005,China)

机构地区：[1]武汉科技大学职业危害识别与控制湖北省重点实验室,武汉430065 [2]荆门市疾病预防控制中心,荆门448000 [3]宜昌人福药业有限责任公司,宜昌443005

出　　处：《药物分析杂志》2023年第10期1737-1750,共14页Chinese Journal of Pharmaceutical Analysis

基　　金：武汉科技大学大学生创新基金项目(20ZA064);职业危害识别与控制湖北省重点实验室开放基金(OHIC2022G07)。

摘　　要：目的:探讨以动物组织为药用部位的饮片微生物污染模式及典型菌污染特点。方法:收集16味46批常见动物类饮片,依据2020年版《中华人民共和国药典》规定方法,对样品进行需氧菌总数(TAMC)、酵母霉菌总数(TYMC)、耐热菌及规定控制菌检验;采用多因素线性回归分析方法探究与样品微生物污染载量相关的因素;运用16S rRNA序列分析方法鉴定饮片污染典型菌。结果:37.0%的样品TAMC超过10^(4)CFU·g^(-1),32.6%的样品TYMC超过10^(3)CFU·g^(-1),60.9%的样品可检出耐热菌;不含内脏组织动物饮片lg TYMC均值高于含内脏组织饮片(P=0.01),其TYMC检出不合格率高于含内脏组织组(χ^(2)=7.59,P=0.00);采收加工炮制过程中未经热处理样品lg TYMC均值高于热处理组(P=0.02),TYMC检出不合格率高于热处理组(χ^(2)=3.99,P=0.04)。13.0%(6/46)的样品检出耐胆盐革兰阴性菌,8.7%(4/46)的样品耐胆盐革兰阴性菌载量超过10^(4)MPN·g^(-1),8.7%(4/46)样品中检出大肠埃希菌,1批蝉蜕样品中检测到铜绿假单胞菌。样品TAMC载量与耐热菌载量呈相关性(P=0.00)。样品中共分离到55株典型菌,芽胞杆菌属细菌占47.3%,肠杆菌属细菌占23.6%,肠球菌占16.4%,样品中检出蜡样芽胞杆菌、阴沟肠杆菌复合体成员等条件致病菌。结论:含动物组织类饮片微生物负载水平高,耐热菌污染严重,耐胆盐革兰阴性菌污染率低,需氧菌负载与耐热菌负载水平具有相关性,饮片典型菌以芽胞杆菌属细菌污染最常见,但污染频率高的条件致病菌在动物饮片微生物控制中应引起足够重视。Objective:To investigate the characterization of the microbe contamination of common animal decoction pieces and typical bacteria isolated from animal decoction pieces.Methods:16 kinds including 46 batches of animal decoction pieces were collected.In light of the methods described as Chinese Pharmacopoeia(2020),the total aerobic microbial count(TAMC),total yeast count(TYMC),and thermophilic bacteria of samples were detected,the control bacteria were isolated from the samples.Multiple linear regression analysis was used to explore the factors related to microbe contamination load.Typical bacteria in the samples on the selective isolation mediums were isolated,and identified by the 16S rRNA amplicon sequencing analysis.Results:The TAMC of 37.0%samples exceeded 10^(4)CFU·g^(-1),the TYMC of 32.6% samples were higher than 10^(3)CFU·g^(-1),and the thermophilic bacteria were detected in 60.9% of the samples.The mean of lg TYMC of samples without guts was higher than those containing guts(P=0.01),the unqualified rate of TYMC of the samples without guts was higher than those containing guts(χ^(2)=7.59,P=0.00).The mean value of lg TYMC of unheated samples was higher than those heated(P=0.02),the unqualified rate of TYMC of unheated samples was higher than heated samples(χ^(2)=3.99,P=0.04).Bile-resistant gram-negative bacteria were detected in 13.0%(6/46)of the samples,the bile-resistant gram-negative bacteria load of 8.7%(4/46)samples exceeded 10^(4)MPN·g^(-1).Escherichia coli was detected in 8.7%(4/46)of the samples,and Pseudomonas aeruginosa was detected in one batch of Cicadae Periostracum.The results of multivariate linear regression analysis indicated that the TAMC load was correlated with the load of thermophilic bacteria(P=0.00).The morphology and Gram's staining properties of typical bacteria were observed under a microscope,the results of 16S rRNA sequence analysis indicated that a total of 55 typical bacteria strains were obtained including 47.3% of Bacillus,23.6% of Enterobacter,and 16.4% of Enterococcus fr

关 键 词：动物饮片 微生物污染 典型菌 16S rRNA序列分析 细菌鉴定 聚类分析 

分 类 号：R917[医药卫生—药物分析学]