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1999: A detailed assessment of the potential risks from the occurrence of the aad gene in the two Monsanto cottonseed lines.
The ACNFP was consulted to see if there were any concerns associated with the use of these GM crops when used as animal feed that might have an impact on human health. The detailed assessment below of the potential risks from the occurrence of the aad gene in the two Monsanto cottonseed lines was prepared on behalf of the ACNFP by one of its members in discussion with others. This position was subsequently endorsed by the whole Committee (by correspondence) and forwarded to ACRE.
Monsanto, both in correspondence and in a recent meeting with ACNFP experts, made a good case in support of their position that the occurrence of this gene in the cottonseeds is not of significant risk. While the risk is small, it does, however, give rise to serious concerns at the potential, in some cases life-threatening, implications should human pathogens, in particular Neisseria gonorrhoeae, acquire the aad gene, which confers resistance to streptomycin and spectinomycin.
The company have concentrated on the probability of gene transfer occurring in the human gut. It is implied but never stated explicitly that the only concern is with the bowel flora, an important source of endogenous infection. Scant regard is taken of the oral flora or of environmental bacteria, which could act as potential gene reservoirs for other pathogens. The company have indicated they consider transformation of the gut flora "is effectively zero". This assumption is based, amongst other things, on unpublished experiments using an ampicillin marker. Extrapolation from these data is unwise. The report by Basker correctly states that streptomycin resistance conferred by the aad gene is associated with integrons; ampicillin resistance is not. Also, recent work by Flint at the Rowett Institute indicates that rumen bacteria are indeed amenable to transformation, with the uptake and expression of naked DNA by bacterial cells. He has recently published work on the transformation of an oral streptococcus with free plasmid DNA (Applied and Environmental Microbiology, 1999, 65: 6-10). This work is considered to be of particular significance for a product that will be used as an animal feed. Furthermore, in a recent paper Gebhard and Smalla (Applied and Environmental Microbiology, 1998, 64: 1550-4) have demonstrated transformation of a soil bacterium Acinetobacter sp. BD413 by DNA from transgenic sugar beet in a marker rescue for the npt gene.
It is implied in information from the company that gonococci, the principal target for spectinomycin, are unlikely to acquire resistance genes from gut flora because they cause genital infection. There are reservations on this position. Penicillinase-producing Neisseria gonorrhoeae acquired a plasmid conferring ampicillin resistance at about the same time as that plasmid was first described in the respiratory pathogen Haemophilus influenzae. It may be reasonable to believe that a transfer occurred during a co-infection of the throat. Likewise, tetracycline-resistant Neisseria gonorrhoeae that carry a tet(M) gene acquired at this time when the gene was rapidly spreading through bacteria, both Gram-positive and Gram-negative, that constitute the human bowel flora and the flora of the female genital tract. It is possible that gonococci acquired this resistance during a rectal infection. Current sexual practices encourage infection not simply of the genitalia as acquisition of these resistances may illustrate. Spectinomycin is used to treat certain gonococcal infections during pregnancy. Treatment failure here has the potential to lead to neonatal infection with a resistant gonococcus.
In the production of novel foods or the exploitation of novel processes, we open opportunities for microbial evolution that would not otherwise exist. The production of large numbers of crop plants increases enormously the biomass of resistance genes. We cannot predict what the effect of such amplification will be, but the situation is not dissimilar to the first use of antibiotics in clinical practice. Microbiologists did not foresee the problems of antibiotic resistance in the bacteria they were targeting. It took only a few weeks, however, for resistant bacteria to emerge in the hospitals where the antibiotics were being used. The genes for antibiotic resistance predate the clinical use of these drugs; it was the intensive use of antibiotics that provided the selective pressure leading to widespread occurrence of these genes.
It is considered that the growth and use of transgenic plants containing the aad gene will increase significantly the number of resistance genes in the environment and could create new opportunities for this gene to spread to microbes that would not normally encounter this resistance determinant. A major concern is what will happen when large quantities of naked DNA are released from transgenic plants, for example when ingested and digested by animals. Recent unpublished work at the University of Leeds, shows that plant DNA is really quite resilient and can survive mild processing (Prof. Forbes: personal communication). Work carried out at TNO in Holland (New Scientist, 30 January 1999; "Gut reaction") appear to add weight to these concerns. This suggests that there is a significant risk of environmental bacteria acquiring these resistance genes and that these microbes could then act as a gene pool for pathogens.
In the light of this sort of evidence, the assurances that transformation is unlikely to occur in the gut may not be safe. Gut bacteria have evolved not to leak their DNA in their natural environment. In contrast, the DNA from plant material will be released as a result of the natural digestive processes. Until we test the survival of DNA from digested plant material, we are unable to estimate the likely risk of naturally competent bacteria acquiring and expressing bacterial genes from transgenic plants.
It is evident that resistance to streptomycin and spectinomycin is widespread amongst the Enterobacteriaceae and that the gene encoding this resistance is located on a transposable element. I do, however, take issue with the company's assertion that this makes the marker safe to use. The principal pathogens treated with streptomycin and spectinomycin are resistant strains of Mycobacterium tuberculosis and Neisseria gonorrhoeae. Of these, my particular concerns are with the treatment of infections caused by Neisseria gonorrhoeae. This bacterium could potentially acquire the aad gene from a transgenic plant during infection of the alimentary canal. The consequences of this would be severe.
I have discussed this issue with acknowledged experts in the field of medical microbiology, one of whom has a long standing interest in infections caused by antibiotic resistant Neisseria gonorrhoeae. The other is an authority on gonococcal infections.
Currently, extended spectrum cephalosporins can be used to treat complicated gonococcal infections. I have researched extended-spectrum beta-lactamases. These enzymes confer resistance to the "third-generation" cephalosporins, including those used in gonococcal therapy. An important class of these beta-lactamases, the TEM-type extended-spectrum beta-lactamases, are derived by point mutation from the blaTEM-1 gene, which confers narrow-spectrum penicillin resistance. Penicillinase-producing Neisseria gonorrhoeae first appeared in the mid-1970's and have persisted ever since. We both expect that the blaTEM-1 gene will ultimately mutate in this pathogen. Given the rate at which extended-spectrum mutants are appearing, this may not be too far into the future. Ciprofloxacin is an alternative drug used in gonococcal therapy but resistance is easily selected in this bacterium. Furthermore, ciprofloxacin cannot be safely used during pregnancy. Spectinomycin is currently available for the treatment of resistant strains of Neisseria gonorrhoeae. If a gene conferring spectinomycin resistance were to arise in resistant strains of Neisseria gonorrhoeae, then this pathogen would effectively become untreatable. The diseases that this organism causes are not all trivial and include septic arthritis, endocarditis and pelvic inflammatory disease, as well as serious infections of the neonate. In view of the very serious consequences arising if a spectinomycin resistant bacterium were to evolve, it is our opinion that it would be very unwise to allow increased opportunity for such an event to happen by the introduction of plants containing this resistance gene. It is accepted that the risk of such an event is small and cannot be quantified. The clinical consequences of such an evolutionary step would, however, be grave. It is therefore entirely appropriate in this instance to adopt a precautionary stance.
The principal use for streptomycin is as a second-line drug for the treatment of tuberculosis. I am not as concerned about the risk of Mycobacterium tuberculosis acquiring the aad gene as I am about the case of Neisseria gonorrhoeae. Resistance to streptomycin in clinical isolates of Mycobacterium tuberculosis results from alterations in the ribosome structure rather than from the action of aminoglycoside modifying enzymes. I do, however, take issue with the company's claim that Mycobacterium tuberculosis does not acquire DNA from other organisms. This observation is at odds with the most recent studies taken from bacterial genome sequence analyses. Cole et al. in a paper in Nature on 11 June 1998 (vol 393, pp 537-544) report the occurrence of prophage DNA in Mycobacterium tuberculosis that can also be found in anonymous mycobacteria and streptomycetes. This indicates that a major human pathogen is able to share genetic information with environmental bacteria. Again, the latest information in this area leads me to believe that we should adopt a precautionary approach to the use of this resistance marker in transgenic plants.
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