我国首例由丁酸梭菌引起婴儿E型肉毒中毒实验室诊断研究

Laboratory investigation of the first infant botulism case caused by type E botulinum neurotoxin producing Clostridium butyricum in China

目的对一起疑似婴儿肉毒中毒进行实验室诊断研究。方法对1例疑似婴儿肉毒中毒病例的粪便、食用过的食品和生活环境涂抹共计30份标本/样品进行梭菌分离鉴定及肉毒毒素检测,对分离菌株进行产毒试验。结果将患儿粪便标本培养物上清液腹腔注射小鼠后可致小鼠出现典型肉毒毒素中毒表现(竖毛、呼吸困难并出现典型的蜂腰、四肢麻痹),继而死亡,且培养物上清液经胰酶处理后毒性增强,表现为小鼠出现中毒及死亡时间较未处理组明显缩短。但培养物上清液经100℃加热处理后再次染毒动物,小鼠未出现中毒和死亡。混合型肉毒毒素诊断血清及单价抗E型肉毒毒素诊断血清可对小鼠起到保护作用。从患儿的粪便标本中分离到G^+芽胞杆菌,该菌在哥伦比亚血平板上呈不规则半透明扁平菌落,边缘根状生长,并携带E型肉毒毒素产毒基因,16S rRNA将其鉴定为丁酸梭菌,产毒试验结果显示该菌株可产E型肉毒毒素。结论该起婴儿中毒事件是由感染产E型肉毒毒素的丁酸梭菌引起。

Objective Laboratory investigation was performed on a suspected case of infant botulism.Methods Thirty samples of stool,left-over food and environment swabs related to the case were collected,Clostridium spp.isolation,identification and toxicity determination by mouse assay were carried out,and toxin production for isolate was conducted.Results Mice injected with the stool culture supernatant showed the typical signs of botulism including irritable,dyspnea,bellows breathing and quadriplegia followed by death.The toxicity of the stool culture supernatant was enhanced after the treatment by trypsinization but ceased after being heated at 100℃.The polyvalent antibody against botulinum neurotoxins(BoNTs)and the monovalent antibody against BoNT type E could protect mice from death.One gram-positive Clostridium isolate was cultured from infant stool sample.The morphology of the colony on the Columbia blood agar plate showed characteristics of irregular,translucent and flat with rootlike growth.It was positive for type E BoNT-encoding gene and identified as C.butyricum by 16 S rRNA sequencing.Toxin production test illustrated that the C.butyricum isolate could produce type E BoNT.Conclusion This was an infant botulism caused by type E BoNT-producing C.butyricum.