Jensen Gert B, Larsen Preben, Jacobsen Bodil L, Madsen Bodil, Wilcks Andrea, Smidt Lasse, Andrup Lars
Department of Chemical Working Environment, National Institute of Occupational Health, Copenhagen, Denmark.
Int Arch Occup Environ Health. 2002 Mar;75(3):191-6. doi: 10.1007/s00420-001-0293-1.
Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B. thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD).
Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis.
Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product.
The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces. The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.
自20世纪初发现苏云金芽孢杆菌的杀虫活性以来，这种细菌已越来越多地用于防治各种害虫。尽管苏云金芽孢杆菌被广泛使用，但仅发表了零星的临床病例报告。近年来，已证实苏云金芽孢杆菌与人类病原体蜡样芽孢杆菌关系密切。实际上，只有苏云金芽孢杆菌的杀虫活性能区分这两个物种。然而，这两个物种都由数千个分离株组成，它们对人类产生不良反应的可能性各不相同。本研究的目的是通过测定包括质粒图谱和随机扩增多态性DNA（RAPD）在内的特定遗传信息，采用分子生物学方法评估职业性接触苏云金芽孢杆菌基生物农药的情况。
收集了12名在丹麦温室工作的人员的粪便样本进行微生物分析。其中7人使用苏云金芽孢杆菌基杀虫剂，而另外5人在未使用苏云金芽孢杆菌的温室工作。细菌在蜡样芽孢杆菌特异性固体培养基上分离，菌落进一步通过聚合酶链反应（PCR）进行鉴定。PCR方法用于鉴定肠毒素基因HblA和BceT。用两种商业血清学试剂盒检测肠毒素的表达。用于16S - 23S间隔区的特异性引物用于将细菌鉴定为蜡样芽孢杆菌属的成员。使用了几种针对杀虫基因的引物，以便进一步将分离株鉴定为苏云金芽孢杆菌的亚种。
来自接触苏云金芽孢杆菌的温室工人的两份粪便样本中，蜡样芽孢杆菌样细菌呈阳性。一个分离株显示出苏云金芽孢杆菌特有的细胞内晶体包涵体，产生蜡样芽孢杆菌肠毒素并含有相关基因，并且对一组杀虫毒素引物呈PCR阳性。粪便分离株的RAPD图谱与从一种商业产品中分离出的菌株相同。
所应用的方法证实粪便分离株与在所用生物农药中发现的苏云金芽孢杆菌分离株相同。这是首次报道从人类粪便中分离出细菌生物农药的案例。该生物农药被证明含有并表达肠毒素基因。然而，没有证据表明这对分离出这些细菌的人造成了任何不良影响。
