一株食源性蜡样芽孢杆菌的分离及其对小鼠肠黏膜免疫相关因子和肠道菌群的影响

Isolation of a foodborne Bacillus cereus strain and its effect on intestinal mucosal immunity-associated factors and gut microbial community in mice

摘要:

蜡样芽孢杆菌(Bacillus cereus)是一种常见的食源性致病菌，误食被其污染的食品会引起呕吐或者腹泻，严重者还会死亡。本研究利用划线培养法从腐败米饭中分离培养出一株蜡样芽孢杆菌。通过药敏试验分析及毒力相关基因PCR扩增对其致病性及耐药性进行分析，并将细菌培养物经过腹腔注射进行小鼠感染试验，检测其对小鼠肠黏膜免疫相关因子和肠道菌群的影响，从而分析该病原菌的致病途径及防治手段。结果显示，本研究所分离的食源性蜡样芽孢杆菌，对诺氟沙星、呋喃妥因、四环素、米诺环素、环丙沙星、大观霉素、克林霉素、红霉素、克拉霉素、氯霉素、左氟沙星和万古霉素敏感，对复方新诺明、苯唑西林和青霉素G具有耐受性。该菌株携带hblA、hblC、hblD、nheA、nheB、nheC和entFM 7种毒力相关基因，可产生致腹泻型毒素。小鼠感染后，出现腹泻，肠黏膜免疫球蛋白和炎性因子表达水平显著上调；肠道菌群检测结果表明，经该蜡样芽孢杆菌感染后，小鼠肠道菌群组成发生变化，标志着机体健康的拟杆菌门(Bacteroidetes)中的uncultured_bacterium_f_Muribaculaceae丰度显著下降，而标志着菌群失调的变形菌门(Proteobacteria)条件致病菌uncultured_bacterium_f_Enterobacteriaceae丰度显著升高，且与IgM和IgG呈显著正相关(P<0.05)。这些研究结果表明，携带致腹泻型毒力相关基因的致病性蜡样芽孢杆菌感染机体后，可以通过影响肠道菌群激活免疫系统。

关键词:蜡样芽孢杆菌;耐药性;毒力相关基因;肠黏膜免疫;肠道菌群

Abstract:

Bacillus cereus is a common foodborne pathogen. Accidently eating food contaminated by B. cereus will cause vomiting or diarrhea, and even death in severe cases. In the present study, a B. cereus strain was isolated from spoiled rice by streak culture. The pathogenicity and drug resistance of the isolated strain were analyzed by drug sensitivity test and PCR amplification of virulence-associated gene respectively. Cultures of the purified strain were injected intraperitoneally into mice to examine their effects on intestinal immunity-associated factors and gut microbial communities, to provide references for the pathogenic mechanism and medication guidance of these spoilage microorganisms. The results showed that the isolated B. cereus strain was sensitive to norfloxacin, nitrofurantoin, tetracycline, minocycline, ciprofloxacin, spectinomycin, clindamycin, erythrocin, clarithromycin, chloramphenicol, levofloxacin, and vancomycin, but resistant to bactrim, oxacillin and penicillin G. The strain carries seven virulence-associated genes including hblA, hblC, hblD, nheA, nheB, nheC and entFM, which are involved in diarrhea-causing toxins production. After infecting mice, the isolated B. cereus strain was found to cause diarrhea in mice, and the expression levels of immunoglobulins and inflammatory factors in the intestinal mucosae of the challenged mice were significantly up-regulated. Gut microbiome analysis showed that the composition of gut microbial community in mice changed after infection with B. cereus. The abundance of the uncultured_bacterium_f_Muribaculaceae in Bacteroidetes, which is a marker of body health, was significantly decreased. On the other hand, the abundance of uncultured_bacterium_f_Enterobacteriaceae, which is an opportunistic pathogen in Proteobacteria and a marker of dysbacteriosis, was significantly increased and was significantly positively correlated with the concentrations of IgM and IgG. These results showed that the pathogenic B. cereus carrying diarrhea type virulence-associated gene can activate the immune system by altering the composition of gut microbiota upon infection.

Key words:Bacillus cereus;drug resistance;virulence-associated genes;intestinal mucosal immunity;gut microbiota