动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌的分离及安全性

Toxicity and antimicrobial resistance of Bacillus cereus group from microecological preparations for animal use

摘要:

[背景] 芽孢杆菌是仅次于乳酸菌常用于微生态制剂中的菌种，然而部分芽孢杆菌微生态制剂规范不严，应用存在安全隐患。[目的] 调查我国在售动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌携带情况，揭示蜡样芽孢杆菌应用的潜在风险。[方法] 对微生态制剂预处理，选择性筛选分离蜡样芽孢杆菌，通过全基因组测序测绘细菌毒素基因谱与耐药基因谱，细胞计数试剂盒-8法测定菌株对细胞的毒性，利用微量肉汤稀释法确定菌株耐药值。[结果] 从50份微生态制剂产品中筛选分离得到23株蜡样芽孢杆菌群细菌，它们对氨苄西林、林可霉素和泰妙菌素3种抗生素均耐药，主要毒力基因nhe、hbl、cytK、ces的检出率分别为100%、30%、39%和4%，分离株均有溶血性且39%菌株产生热稳定毒素，不同菌株对非洲绿猴肾细胞呈现出不同程度的毒性。[结论] 微生态制剂来源的蜡样芽孢杆菌毒性与耐药性严重，携带毒素基因与耐药基因广泛，多株菌株呈高细胞毒性且产生热稳定毒素。芽孢杆菌微生态制剂存在安全性问题，应加强对蜡样芽孢杆菌的质量安全监管力度，规范微生态制剂的市场秩序，杜绝安全隐患。

Abstract:

[Background] Bacillus is commonly used in microecological preparations next to lactic acid bacteria. However, some Bacillus spp. are not strictly regulated and pose potential safety hazards in application. [Objective] To study the prevalence of Bacillus cereus in Bacillus microecological preparations on sale from all over the country, and reveal the potential risks of the application of B. cereus. [Methods] B. cereus group strains were isolated and identified from the pretreated microecological preparations. Toxicity and antimicrobial resistance of B. cereus group strains were analyzed using whole-genome sequence. The cytotoxicity was evaluated via Cell Counting Kit-8 assay. Antimicrobial susceptibility was determined by broth microdilution methods. [Results] Twenty-three B. cereus group strains were isolated from 50 microecological preparations, which are resistant to ampicillin, lincomycin and tiamulin, and the detection rates of major virulence genes nhe, hbl, cytK, ces were 100%, 30%, 39% and 4%, respectively. All isolates were hemolytic and showed varying degrees of toxicity to Vero cell, 9 of which produced heat-stable toxins. [Conclusion] The widespread of toxin and resistance genes, and apparent observations of hemolytic activity, cytotoxic, and heat-stable toxin production in B. cereus group isolates from Bacillus microecological preparations, pose great threat to food safety and human safety. The findings highlights need for enhanced efforts required in the surveillance of risk factors, virulence factors and antimicrobial resistance in B. cereus from microecological preparations.