Bekæmpelsesmiddelforskning fra Miljøstyrelsen, 106 – Mikrobiologiske plantebeskyttelsesmidlers skæbne i mave-tarm kanalen - Studier af Bacillus thuringiensis

Mikrobiologiske plantebeskyttelsesmidlers skæbne i mave-tarm kanalen

Summary

The bacterium Bacillus thuringiensis (Bt) is contained in plant protection products used in Denmark for e.g. the production of tomatoes, cucumbers and pot plants. Bt is toxic to insects due to the production of so-called d-endotoxins that are harmless for humans. However, the bacterium also produces enterotoxins known from the food poisoning bacterium Bacillus cereus, a causative agent of diarrhoea.

Products based on Bt have been used worldwide for decades without resulting in identified problems. However, there is an increasing demand for substituting chemical insecticides with more environmental friendly ones, and at the same time there is an increasing tendency that vegetables are consumed raw or only briefly heat-treated. It is therefore likely that consumers will experience an increased exposure to Bt in the future.

Plant protection products based on Bt contain the spore-form of these bacteria, which is an inactive and very resistant form of the bacterium. The spores germinate to active cells in the gut of the insects, but it is unknown whether the spores also germinate in the human gastrointestinal tract. Germination of spores within the gastrointestinal tract could cause a risk of diarrhoea in consumers eating vegetables treated with Bt products. The main objective of this project was to clarify this question in an animal model. This objective was met by answering the following questions: 1) does heat treatment of Bt spores prior to ingestion increase their ability to germinate in the intestine, 2) can Bt cells effect the composition of the indigenous intestinal microbial population, 3) can Bt cells in the intestine produce enterotoxins, and 4) can Bt cells transfer genetic information to other bacteria in the intestine.

To answer these questions, rats with an intestinal microbiota derived from a human source were used. Animal experiments were performed with one B. cereus strain and two Bt strains, subsp. kurstaki and subsp. israelensis, both used on the Danish market. Additionally experiments were made with rats without an intestinal microflora (germ free rats) that were fed with spores of a Bt strain, which in vegetative phase was green fluorescent. The study showed clearly that spores germinate and grow in the intestine of germ free rats without the presence of an indigenous microbiota. Also in rats with a human microflora germination and growth was observed in some animals. Heat treatment of spores prior to feeding to the rats did not increase their tendency for germination. This may indicate that heat treatment (e.g. by cooking) of vegetables sprayed with Bt does not increase the risk for germination of the bacteria in the gut of the consumer.

Even though germination was observed in the rats with a human microflora, two weeks after last dosing with Bt most of the rats did not harbour any Bt cells in their intestines. However, there was one animal fed with Bt subsp. Kurstaki in which the strain colonised the gastrointestinal tract, and in this animal penetration of the gastrointestinal barrier was observed, and the strain was recovered from the spleen. Further research is needed to clarify the risk for humans of Bt to reach other organs.

A possible effect on the indigenous bacterial population in the gut by Bt was studied using both traditional selective cultivation and a molecular biology method (Denaturing Gradient Gel Electrophoresis, DGGE). DGGE is based on variations in a certain region of the chromosome of bacteria. Those variations can be used to determine which bacteria are present in a population. Specific “fingerprints” can be obtained from each population and compared by computer analysis. The study showed that feeding of rats with Bt spores did not result in major changes in the composition of the microflora, and that the minor effects observed were merely transient.

Transfer of DNA between two isogenic Bt strains was observed using germ free rats fed with spores of the two strains. The donor strain harboured a so-called conjugative plasmid that was transferred to the recipient. This transfer supports the abovementioned results regarding germination of spores in the gastro-intestinal tract, since transfer can occur only between vegetative cells, and not between spores.

Exchange of DNA between members of the B. cereus family is a common phenomenon observed under laboratory conditions and also in the gut of insects. However, this is the first time that such transfer has been studied and observed in a mammalian gastrointestinal tract. The sequence of a Bt plasmid has shown high similarity to other plasmids from the B. cereus group and to plasmids from other Gram-positive bacteria, e.g. bacteria naturally present in a human gastrointestinal tract. This similarity indicates that exchange of DNA has happened in nature, and that one of the environments may be the gut.

Furthermore, the ability of a strain to transfer genetic information is relevant in risk assessment of products based on Bt. When dealing e.g. with a bacterium resistant to an antibiotic, it is important to know whether the resistance is intrinsic and non-transferable or acquired and transferable.

The production of cytotoxic agents (including enterotoxins) was measured by the ability of the samples to kill monkey kidney-cells (Vero cells). By testing the supernatant that the bacteria had grown in, it was clear that Bt subsp. kurstaki used on eatable crops produced less toxin that Bt subsp. israelensis used on pot plants. Furthermore subsp. kurstaki produced less toxin than a B. cereus strain that has been involved in a food poisoning case.

Intestinal samples from the animals fed with either B. cereus or one of the two Bt strains were also tested for the presence of enterotoxins. Enterotoxins were only detected in one animal out of six germ free rats fed spores of Bt subsp. israelensis, and only in small amounts. In the samples from the rats with a human derived microflora no cytotoxic effect could be detected. Not even in the animals fed the B. cereus strain previously involved in an outbreak of food poisoning. Experiments with rats are currently the number one tool when testing the human safety of new biological plant protection agents in Denmark as well as in the EU. However our results (and those of others) show that rat exposure is not the best method to reveal human relevant pathogenic traits in bacteria, and there is currently a debate in the EU for new risk assessment procedures of bacteria. By basing the risk assessment on other methods it is possible to get a better safety for the consumer as well as to spare a number of rats.

In risk assessment of plant protection products based on Bt in the future it is recommended to place more impact on the toxicity of the strains in in vitro assays (e.g. PCR analysis against toxin-genes, cell-assays), and less impact on animal experiments. Nevertheless this study shows that animal experiments are useful when studying e.g. the ability of a strain to colonise the gastrointestinal tract, the effect of a strain on the indigenous microflora, and the capacity of a strain to transfer DNA to other bacteria in the gut.

By risk assessment of Bt based products it is recommended to use a case-by-case approach, since strains of the same species can be very different, and with emphasis on the application of the products into consideration – e.g. whether they are used for vegetables or pot plants. This study showed e.g. that Bt subsp. israelensis had high cytotoxic effects in the Vero-celle assay, however since products based on this strain are only used in Denmark on pot plants, this is not a concern for the consumer – but could however be an occupational health problem.

On the whole, this study does not give rise to increased concern about the products currently on the Danish market. Even though Bt subsp. kurstaki (used on vegetables) can germinate in the gastro-intestinal tract, the strain does not have high cytotoxic effects. However the observed passage to the spleen of this strain warrants further studies of this subspecies.

Besides the consumer, also greenhouse workers spraying with Bt, could be exposed to Bt. Many of the results in this project also applies for those worker, although this project has not specific dealt with occupational health problems. This issue was investigated in an earlier study financed by Arbejdstilsynet [Jensen et al., 2001]