G01010: Assessment of the risks of transferring antibiotic resistance determinants from transgenic plants to micro-organisms

Study Duration: May 1998 to April 2001

Contractor: Centre for Animal Sciences / Institute for Plant Biotechnology and Agriculture, University Of Leeds

In the production of genetically modified (GM) plants, genes conferring 'desirable' traits are identified and are then assembled in bacteria before delivery into the target plants. The assembly of genes in bacteria is made simple by including antibiotic resistance marker genes. The presence of a gene that codes for resistance to an antibiotic in a single bacterial cell means that, by adding the appropriate antibiotic to a culture, the single cell can be isolated from millions or even billions of bacteria lacking the resistance gene present in the same culture. By linking a gene that confers antibiotic resistance on its host bacterium to the genes that are to be inserted into plants, sufficient quantities of DNA can be harvested from a bacterial culture to allow the novel DNA to be delivered easily to the plant. One marker gene commonly used in the early days of GM crop development, called bla_TEM, confers resistance to antibiotics such as ampicillin and amoxycillin. This gene causes the production of an enzyme, known as a beta-lactamase, which breaks down the structure of these antibiotics, rendering them ineffective against bacteria. Although the bacteria that live in animal and human guts frequently carry bla_TEM, which occurs naturally at a high frequency, this project is designed to examine the risk that bla_TEM from GM maize can pass from the modified plant tissues into microbes in animals that are fed GM maize.

In essence, this project sought evidence for the occurrence of a very rare event. In order to tackle this problem, a critical step analysis was undertaken. This project was designed to address six main areas of concern. The research approaches identified at the the start of the project are summarised below:

1) What is the risk that the bla gene will "loop out" from transgenic plant cells?
The DNA cassette inserted into the maize plant should be stably incorporated. If, however, it can "loop-out" from the plant DNA, its chances of spreading will be enhanced.

2) Do animals fed on transgenic maize release the bla gene from plants?
In order to transfer from GM plant tissues into microbes, it is necessary that the digestion processes release DNA in a form that can be taken up and also be expressed in bacteria. Sheep were used as the model ruminant animal; chickens were used as the non-ruminant. Both chickens and sheep may be fed significant quantities of maize or maize products.

3) With what efficiency can gut organisms take up and express the bla gene?
The demonstration that the antibiotic resistance marker gene is digested in the same way as other plant DNA, and that it is thus unavailable for uptake and expression by bacteria in the lower intestine, rendered this question of lesser importance than at the outset of the project.

4) Can the bla gene be mobilised from bacteria within an animal gut?
The transfer of bla_TEM from GM plant material to microbes in the animal gut, were it to happen, is the first step in spreading this gene to new hosts. In theory, once one bacterium has acquired bla_TEM, it may be transferable to other bacteria in the gut environment.

5) Does ensilage of transgenic maize result in the release of the bla gene?
Much of the maize feed in the United Kingdom is presented in the form of silage. To make silage, fresh plant material is fermented in the absence of air. Microbes associated with the fresh crop cause the production of organic acids, which preserve the plant material in a form that animals find desirable. The bacteria responsible for the later stages of silage production are fed to animals as part of the silage product. These have the potential to take up and express resistance genes, such as bla_TEM found in the maize that is the subject of this study.

6) To what extent does the bla gene undergo mutation to extended spectrum activity in the gut environment?
Over the past 15 years, hospitals have seen the emergence of bacteria that are resistant to important first-line antibiotics. This resistance may arise from the mutation of bla_TEM, causing substitution of different amino acids in the enzyme that confers resistance to antibiotics. About 90 such mutations have now been described in bla_TEM. Many of these allow the enzyme to confer resistance on a much broader spectrum of antibiotics than the original enzyme.

1. Outcome/key results obtained

There is no evidence that the antibiotic resistance gene, bla_TEM, can "loop-out" of the maize plant DNA.

The survival of bla_TEM DNA was then studied in a series of experiments that modelled various environments. Sheep saliva, sheep rumen fluid and the fluid that runs off from silage production, effluent, were collected and survival of DNA was monitored under laboratory conditions. It was discovered that in rumen fluid and in silage effluent, bla_TEM very rapidly lost its biological activity. In contrast, bla_TEM survived in sheep saliva and in this environment it retained its ability to cause susceptible bacteria to become resistant to ampicillin.

Feeding experiments with chickens demonstrated that although the plant-associated bla_TEM could be found in the crops of all birds fed on GM maize, it could only be detected in the stomach contents of five birds and was undetectable in any samples taken from below the stomach. This experiment further showed that the plant-associated bla_TEM was digested in the same way as other plant DNA.

Parallel experiments feeding GM plant material to sheep indicated that bla_TEM DNA survives very poorly in the rumen of animals that are fed transgenic maize or silage made from transgenic maize (Duggan et al., 2003). In contrast, free DNA in the sheep oral cavity retains the ability to transform susceptible bacteria to resistance for up to eight minutes, implying that DNA released within the mouth from GM plants in the diet may retain sufficient biological activity for genes to transfer from plant material to susceptible oral bacteria.

Culture experiments, selecting for ampicillin resistant bacteria, showed no evidence that the bacteria involved in silage formation could take up and express GM plant-associated bla_TEM. Data from molecular studies suggest, however, that bacteria in silage may take up the bla_TEM sequence but once taken up, it remains silent.

2. What it means and why it's important

It can be concluded that the antibiotic resistance marker is stably incorporated into the GM maize that was the subject of this study. Thus, the chances that it will escape easily and transfer to microbes associated with the GM plant or animals feeding on GM material are slight. DNA, however, is released from plants during digestion, and may be available to transfer to microbes. The oral cavity receives a large number of incoming bacteria with the diet and other bacteria that are normally resident at this site. This project indicates that although biologically active DNA from GM plants (and non-GM plants) can survive in the oral cavity for long enough to transfer to microbes in this site the risk of this occurring is very low. However, even this small possibility makes it essential, in the achievement of maximum risk reduction, for the regulatory process to consider each GM crop as a individual entity with its own potential risks. Similarly, the bacteria involved in silage production may acquire bla_TEM from GM maize but nevertheless silage bacteria do not express the gene and are not resistant to antibiotics such as ampicillin.

The final report for this project has been published.

Duggan PS, Chambers PA, Heritage J, Forbes JM (2000). Survival of free DNA encoding antibiotic resistance from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and silage effluent. FEMS Microbiology Letters, 191, 71-77.

Chambers PA, Duggan PS, Forbes JM, Heritage J (2001). A rapid, reliable method for the extraction from avian faeces of total bacterial DNA to be used as a template for the detection of antibiotic resistance genes. Journal of Antimicrobial Chemotherapy, 47, 241-243.

Chambers PA, Duggan PS, Forbes JM, Heritage J (2001). The fate of antibiotic marker genes in transgenic plant feed material fed to chickens. Journal of Antimicrobial Chemotherapy, 49, 161-164.

Duggan PS, Chambers PA, Heritage J, Forbes JM (2003). Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. British Journal of Nutrition, 89, 159-166.

Contact: For any enquiries concerning this research project, please contact the relevant Programme contact or email science@foodstandards.gsi.gov.uk