半褶织纹螺(Nassarius semiplicatus)及其生活环境中分离培养的细菌毒性测试与种类分析

Toxicity screening and identification of bacteria isolated from snails Nassarius semiplicatus and their habitat

在中国沿海地区,因食用织纹螺导致的中毒事件时有发生。最近的研究表明,河豚毒素及其衍生物是织纹螺中主要的致毒成分。但是,对于织纹螺中河豚毒素的来源还不清楚。【目的】本研究尝试分离、培养和鉴定织纹螺及其生活环境中的细菌,并对其毒性进行分析,为探明织纹螺中河豚毒素的可能来源提供科学依据。【方法】先后于2006年6月13日和19日在江苏省盐城采集织纹螺样品,应用小鼠生物法对织纹螺样品的毒性进行了测试;从织纹螺体内及其生活环境中分离细菌,并选择部分菌株进行了室内培养;以直接竞争酶联免疫分析方法(ELISA)对培养菌株中的河豚毒素进行了检测;通过对细菌16S核糖体DNA(rDNA)部分序列的测定,对有毒菌株进行了初步的种类分析。【结果】实验结果表明,采集的织纹螺为半褶织纹螺,两次采集样品的毒性分别为247MU(mouseunit,小鼠单位)和270MU/100g组织(湿重)。对14个菌株进行了毒性检测,其中有毒细菌9株。产毒菌株的毒性普遍较低,毒性范围为15～96ng/g。有毒菌株核糖体序列与弧菌(Vibrio)、希瓦氏菌(Shewanella)、动性球菌(Planococcus)、海单胞菌(Marinomonas)、发光杆菌(Photobacterium)等菌属有较高的相似性,可能具有较近的亲缘关系。【结论】研究发现半褶织纹螺体内及其生活环境中存在能够产生河豚毒素的细菌,说明织纹螺中的河豚毒素可能与其体内及其生活环境中的细菌有关,有必要进行深入研究。

[Objective] Tetrodotoxin and its analogues （TTXs） were responsible for the poisoning incidents associated with snail Nassarius spp. We studied bacteria isolated from toxic snails as well as their habitat to probe into the relationship between bacteria and toxicity of nassariid gastropod. [Methods] Two snail samples were collected from Sheyang, Jiangsu Province on June 13 and 19, 2006, and the toxicity of the snail samples was tested with mouse bioassay method. Bacteria were isolated from the snail samples and from their habitat. A part of isolated bacteria were then cultured in the lab, and TTX in bacteria were screened with an ELISA method. [Results] The snails collected were identified as Nassarius semiplicatus, and the toxicity of the 2 samples were 247 mouse unit （MU） and 270 MU / 100g tissue （wet weight）, respectively. TTX was detected in 9 strains among the 14 strains of bacteria isolated from the snail samples and their habitat. TTX content in the toxic strains was very low, which ranged from 15 ng/g to 96 ng/g. Partial of the 16S ribosomal DNA （rDNA） of the toxic strains were then sequenced after PCR amplification, and the toxic strains of bacteria were tentatively identified based on the alignment of these sequences with published data. Toxic strains were closely affiliated with Vibrio, Shewanella, Planococcus, Marinomonas, Photobacterium. [Conclusion] These findings suggested that TTX-producing bacteria may play an important role in TTX production or accumulation in nassariid gastropod.