ISIS Report 14/06/10

GM DNA Does Jump Species

Antibiotic Resistance not the Only Risk

Dr. Mae Wan Ho corrects some misconceptions about the risks of horizontal gene transfer from GMOs and calls for an urgent review

This report was submitted to EFSA, please circulate widely, keeping the links intact

Antibiotic resistance genes from GM plants “unlikely” to transfer to bacteria

Horizontal gene transfer – DNA being taken up and integrating into the genome of cells – came under scrutiny by the European food Safety Authority (EFSA)  in relation the safety of  antibiotic resistance marker genes in genetically modified (GM) crops grown commercially or entering the market. EFSA failed to reach a unanimous opinion. The published Statement [1] acknowledged scientific uncertainties, but claims it is “unlikely” that antibiotic resistance genes in GM crops pose health and environment risks.

However, two senior scientists on EFSA’s biohazard panel, which carried out the assessment jointly with the GMO panel, did not agree with the conclusion and issued a minority opinion included in an annex to the Statement. The key issue is the probability that the antibiotic resistance genes could transfer from plant to bacteria. The two scientists stated that the adverse effects cannot be assessed, and that the probability of gene transfer from plants to bacteria ranges widely “from unlikely to high.”

EFSA had already given a positive opinion to Germany chemical company BASF’s GM potato that has an antibiotic resistance marker gene, but was asked by the European Commission to re-examine the risks of antibiotic resistance, after failing to address persisting legal and health concerns [2]. An EU law from 2001 requires antibiotic resistance genes that may have adverse effects on human health and the environment to be phased out by the end of 2004, while the World Health Organisation considers the antibiotics inactivated by the resistance gene in the GM potato vital for treating serious infections such as tuberculosis.

Despite the scientific uncertainties, the European Commission granted approval to the GM potato, Amflora in March 2010, in time for the planting season [3], more than 13 years after BASF first applied for commercial approval in the EU.

The importance of kanamycin/neomycin

The gene nptII contained in the BASF GM potato codes for resistance against the antibiotics kanamycin and neomycin. The WHO considers kanamycin and neomycin vital in the treatment of serious diseases caused by multiple drug resistant pathogens, such as tuberculosis, which are not yet resistant to these antibiotics. If the nptII gene spreads widely, the important “second line” defence provided by kanamycin and neomycin against life-threatening infections will be lost.

One major justification for the continued use of certain antibiotic resistance marker genes in GMOs is that the genes are already common in the environment. For example, the beta-lactamase gene bla, present in a large number of GMOs commercially grown, appears to be widely distributed; occurring to varying degrees in fields growing GM and non-GM crops, even in prairies that were not disturbed by agricultural practices [1]. However, the same is not true of the nptII gene. While the gene was found to be quite abundant and functional in manure, sewage and water samples, both in and outside hospitals, it was scarce in the soil, where the potential for horizontal transfer from GM plants is greatest.

In addition, animals and humans eating GM plant material containing the nptII gene could also acquire the gene through microorganisms resident in the gastrointestinal tract, known to be a hotspot for horizontal gene transfer. This would severely compromise their chances of surviving a multidrug resistant infection.

Does DNA transfer from plant to bacteria?

So, is there evidence that genes can transfer from plants to bacteria? Yes, plenty of evidence in the laboratory, for all kinds of transgenes, as noted by EFSA [1], as well as evidence that GM DNA can persist in debris and residues in the soil long after the GM crops have been cultivated. But EFSA insists that horizontal gene transfer can only be demonstrated under “optimised conditions” in the laboratory, and there is no evidence it can happen in the field. This is not strictly true. Circumstantial evidence of horizontal transfer of GM DNA from plants to bacteria came from the very first field monitoring study carried out on GM sugar beet [4] (Horizontal Gene Transfer Happens, ISIS News 5). It is notoriously difficult to detect horizontal transfer of plant DNA to bacteria resident in the soil, as more than 90 percent of the bacteria species cannot be cultured; and the soil is a very complex, spatially structured environment.

We have reviewed horizontal gene transfer from GMOs on numerous occasions since then, as evidence continued to accumulate, despite a paucity of dedicated research. In the most recent review [5] (Horizontal Gene Transfer from GMOs Does Happen, SiS 38), we drew attention to the only feeding trial in human volunteers [6] involving a single meal containing GM soya with about 3 x 10¹² copies of the soya genome. Researchers found that the complete 2 266 bp of the epsps transgene for glyphosate tolerance was recovered from the colostomy bag in six out of seven ileostomy subjects, though at highly variable levels, ranging from 10¹¹ copies (3.7 percent) in one subject to 105 copies in another. This is a strong indication that DNA is not rapidly broken down in the gastrointestinal tract, confirming earlier results from the same research group. Significantly, in three of the seven subjects, about 1 to 3 per million bacteria cultured from the contents of the colostomy bag were positive for the GM soya transgene, indicating that horizontal transfer of transgenic DNA had occurred from plant to bacteria in the gut. This must have happened either before the experiment, as the researchers claimed, or else as the result of the single GM soya meal, a possibility that cannot be ruled out. Also significantly, no bacteria were found to have taken up non-transgenic soya DNA, suggesting that transgenic DNA may be more successfully transferred as I have highlighted time and again. The reason is that transgenic DNA is designed to jump into genomes, a design feature that also makes them more likely to jump again and insert in another location in the same genome causing rearrangement, or else transfer horizontally into the genome or another cell. That is a major cause of genetic instability of GMOs (see [7] Transgenic Lines Unstable hence Illegal and Ineligible for Protection, SiS 38).

The gastrointestinal tract is a hotspot for horizontal gene transfer

The transfer of transgenic DNA demonstrated in the single human trial is just the tip of the iceberg, it shows how readily transgenic DNA, including antibiotic resistance genes, can transfer to bacteria, especially in the gastrointestinal tract. The gastrointestinal tract is a hotspot for horizontal gene transfer as successive reviews make clear [8, 9].

Gene transfer from a GM probiotic in the avian GI tract is much higher than the rates observed by culturing them on a petri-dish [8], basically because the latter depends on the bacteria being able to grow in culture at the same time. Furthermore, anaerobic bacteria make up 99 percent of human gut flora, and these would not grow in ordinary culture. Organisms residing in the gastrointestinal tract are thought to be reservoirs of antibiotic resistance and virulence genes. Studies using simulated ileum of the pig gut provided clear evidence that antibiotic resistance could be transmitted between resident and pathogenic members of the Enterobacteriaceae passing through the gut [9], and by implication, transfers could also take place from the pathogen to the resident organisms.

Gene transfer in the colon has been found in Bacteriodes species. Frequently, the environment of the gut is exposed to low levels of antibiotics used as therapeutic agents, growth promoters, or as contaminants in food. Antibiotics have been shown to stimulate the transfer of mobile genetic elements such as conjugative transposons (jumping genes involved in conjugation, a process whereby bacteria exchange genes via cell contact) and genomes of bacterial viruses. The mouse gastrointestinal tract enabled a shiga toxin 1(Stx1)-encoding phage (bacteria virus) to be transmitted between two E. coli strains and produce infectious virions capable of infecting yet other E. coli strains in the gut.

The rumen is the first ‘stomach’ of cattle, sheep and goats, where high-fibre plant materials are digested by a mixture of micro-organisms, both prokaryotes and eukaryotes, providing a great opportunity for horizontal gene transfer [9]. Transfer of antibiotic resistance in the rumen was first documented in sheep in the 1970s, and since then indirect evidence has mounted for rumen transfer events, with the protozoa in the rumen playing an important role in facilitating gene transfer between bacteria inhabiting the rumen [10].

We have reported earlier in The Case for A GM-Free Sustainable World  [11] (Independent Science Panel Report, ISIS publication) on how free DNA survives for a considerable period of time in saliva and was able to transfer to Streptococcus gordonii, a natural inhabitant of the mouth; so horizontal gene transfer is likely to start right away in the mouth [12].

All the more so, as foods such as ultra heat treated milk, cacao drink and tomato juice have been reported to support horizontal gene transfer when external DNA was added along with the bacterial strains [9]. The highest transformation frequencies of E. coli occurred in milk, soy drink, tomato and orange juice, and DNA was released and taken up by E. coli under food processing conditions.

Biofilms - biologically active matrices consisting of cells and extracellular substrates, have become an important issue with respect to food hygiene. Biofilms are formed on any submerged surface in any environment where bacteria are present and can form on food products or food product contact surfaces, such as pipes and rubber seals. Many pathogens have been shown to persist in biofilms. Bacteria present in biofilms exhibit increased resistance to antimicrobial agents, and decreased susceptibility to a range of antibiotics. Biofilms are also hotspots for horizontal gene transfer.

Inadequate conceal the true extent of horizontal gene transfer

The failure to demonstrate horizontal gene transfer in many experiments cited by EFSA and other regulatory bodies is due to inadequate methodologies that either underestimate the frequencies, or are insufficiently sensitive, or downright misleading.

Researchers at Cardiff University in the UK were able to detect horizontal transfer of plant DNA to bacteria in both sterile and non-sterile soil down to a frequency of 5.5 per 100 billion recipient cells (5.5 x 10^-11) [13]. The rhizosphere (zone surrounding the roots in soil) is an acknowledged hotspot for horizontal gene transfer

Using direct visualization methods that restores function to a green fluorescent protein transgene and without the need to culture and select for transformants with antibiotics, researchers at Cardiff University and their colleagues in other institutions confirmed that investigations based on culture methods underestimate transformation frequencies [14]. They were able to detect transfer of plant DNA to bacteria on the surface of intact leaves as well as on rotting, damaged leaves [15]. Rotting and damaged leaves release nutrients that promote bacterial growth, and bacterial that can take up foreign DNA are at their most receptive (competent) state for horizontal gene transfer during exponential growth. In particular, the researchers have identified ‘opportunistic’ hotspots for transfer of plant DNA to bacteria in plant material infected with pathogens.

Misleading experiment cited by EFSA and other regulatory bodies

One downright misleading recent experiment that failed to detect any horizontal gene transfer in the gastrointestinal tract was carried out by researchers or the National Food Institute at the Technical university of Denmark [16]. It involves infecting ‘germ-free’ rats with single strains of bacteria to create ‘mono-associated rats’, which, the researchers claim, is a worst case scenario [for horizontal gene transfer].

In fact, the germ-free mono-associated model is so unphysiological and abnormal that it can tell you nothing about horizontal gene transfer in the typical gastrointestinal tract. Germ-free rodents are indeed abnormal in many respects, especially in their intestinal tract [17]. There are between 500 to 1 000 species of bacteria living in the human gut, more than 90 percent cannot be cultured, in addition to a number of archaebacteria and fungi [18], which interact with one another and with the host in a complex web of synergistic and antagonistic relationships. But there were also other problems with the study.

The researchers investigated three bacteria that normally live in the gut - E. coli, Bacillus subtilis, and Streptococcus gordonii - all shown to be transformed to antibiotic resistance with a plasmid carrying chloramphenicol resistance on the petri dish [16]. But no transformation could be detected for E. coli when faeces or intestinal contents from germfree rats were added. There was obviously an inhibitor of transformation present in the faeces, if not in the intestinal contents, but that was not pursued further.

For the in vivo experiment with E. coli, they chose a strain carrying a plasmid with an incomplete kanamycin-resistance gene and force-fed them to four germ-free rats daily for 17 days. On day 8-17, three of the rats were dosed additionally with a plasmid carrying the complete kanamycin-resistance gene but without a promoter, and also a chloramphenicol-resistance gene. At the end of the experiment, they checked to see if the resident bacterial strain has taken up the plasmid and therefore acquired chloramphicol-resistance, and if there has been recombination between the plasmid to restore kanamycin-resistance. The results were negative. They could find no horizontal transfer of plasmid or recombination in the faeces, or intestinal samples. However, the densities of bacteria cells in the samples were very low, simply too low for detecting any horizontal gene transfer event, given the detection limit of their method is 2.3 per billion recipient cells.

Bacillus subtilis transformation in vitro was not affected by addition of 10 percent intestinal contents. However, the in vivo results were still negative, because the colonisation of germfree rats by this bacterium is even less successful than E. coli.

Previous published studies showed that S. gordonii is capable of taking up plasmid DNA and genomic plant DNA under in vitro conditions. The bacterium did not have problems getting established in the germfree rat, but needed antibiotic to maintain its plasmid. On day 7 to 10, eight animals received 1 mg DNA extracted from GM potato, corresponding to approximately 10⁹ nptII genes. Unsurprisingly, no horizontal gene transfer was detected in the faeces. The possibility of an inhibitor of transformation in faeces was again not considered.

One feature that may appear to work against horizontal gene transfer to bacteria is that integration of DNA into bacterial genomes depends largely on base sequence similarity (homology) between the foreign DNA and host genome DNA. That is why, as the EFSA report rightly points out, there is little evidence of plant to bacteria transfer in the past history of evolution, as revealed by comparing the many bacteria and plant genomes that have been sequenced so far.

However, as I have long pointed out in Living with the Fluid Genome [19] and elsewhere, GM DNA typically contains a mosaic of sequences, many homologous to bacteria and viruses widespread in the environment, which would facilitate horizontal gene transfer to numerous species of potential pathogens.

Moreover, contrary to the impression given, successful horizontal gene transfer of antibiotic resistance is by no means the only hazard from horizontal transfer of GM DNA, as we have repeatedly emphasized [4, 5]. Another hazard is the creation of new pathogens by transferring genes for virulence, or simply recombining genes that cause diseases (see [20]  Gene Technology and Gene Ecology of Infectious Diseases, ISIS scientific publication)  But even that is not all.

“Successful” horizontal gene transfer is not the only, nor the major risk

Many commentators including the EFSA scientist [1] give the impression that the only risk from horizontal transfer of GM DNA is the successful transfer and establishment of antibiotic resistance marker genes. That is not the case. One reason that detectable horizontal gene transfer is so low is because the process is highly damaging for the recipient cell. So the actual transfer frequency is at least two to three orders of magnitude higher. This can be gauged from typical transformation experiments in making GMOs, where even the apparent successes later fail to get established.

From the point of view of biosafety, the unsuccessful horizontal gene transfer events are far more significant, especially for human beings and animals. That is because genomes of higher organisms (eukaryotes) take up DNA much more readily than bacterial (prokaryote) genomes, and do not depend on homology (similarity) of DNA sequences [5]. Most of the integration events will result in cell death, because the GM DNA is known to disrupt genes and scramble genomes during and after the transformation process. Some of the events may also lead to activation of dormant viruses, a large number of them having transferred horizontally into the human genome in our evolutionary past. Some will have the potential to trigger cancer. “Insertion carcinogenesis” is a well-known phenomenon. We have reviewed this subject in detail and alerted our regulators ten years ago (see [21] Naked and Free Nucleic Acids - Unregulated Hazards, ISIS Report).

In addition, we have called attention to hazards from specific components of the GM DNA, such as the cauliflower mosaic virus (CaMV) 35S promoter, widely used in transgenic plants, often in multiple copies and enhanced synthetic forms. The promoter functions promiscuously in species across the living kingdoms including humans, and is recently found to promote the replication of viral sequences that cause diseases, including HIV [22] (New Evidence Links CaMV 35S Promoter to HIV Transcription, SiS 43 and ISIS scientific publication)  

Conclusion

EFSA’s claim that it is “unlikely” for antibiotic resistance genes in GM crops to pose health and environment risks is based on failure to detect horizontal gene transfer events in the field is  unjustified, in view of evidence from studies that conflict with those on which it has based its conclusion, in particular:

1. Transfer of GM DNA from plant to bacteria has been found both in the field and in the gastrointestinal tract, despite the paucity of dedicated research.

2. Transfer of antibiotic resistance marker genes in the gastrointestinal tract is particularly relevant to human and animal health

3. Recent research based on non-selective direct visualisation of horizontal gene transfer events suggests that previous methods underestimate the true extent of horizontal gene transfer.

4. The antibiotic resistance marker gene nptII is not common in the environment and its release in GM crops can severely compromise antibiotics vital to treating multidrug life-threatening infections

5. EFSA has failed to consider the damage from unsuccessful horizontal gene transfer, which is at least a hundred to a thousand fold that of successful horizontal gene transfer, especially for human and animal cells

6. It has failed to consider evidence suggesting that GM DNA is more likely to transfer horizontally than natural DNA

EFSA needs to consider the full hazards of horizontal gene transfer as a matter of urgency before it considers further environmental releases of GMOs.

References

1. Statement of EFSA. EFSA-Q-2009-00589 and EFSA-Q-2009-00593. Consolidated presentation of the joint Scientific Opinions of the GMO and BIOHAZ panels on “Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants” and the Scientific Opinion of the GMO Panel on “Consequences of the Opinion on the Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants on Previous EFSA Assessments of Individual GM Plants” Prepared by GMO and BIOHAZ Units. The EFSA Journal 2009, 1108, 1-8.

2. “Disagreement in EFSA opinion puts future of B ASF GM potato in doubt”, Greenpeace European Unit Press Release, 11 June 2009, http://www.greenpeace.org/eu-unit/press-centre/press-releases2/EFSA-opinion-GM-potato-09-06-11

3. “European Commission approves Amflora starch potato”. BASF Corporate Website, accessed 10 May 2010, http://www.basf.com/group/pressrelease/P-10-179

4. Ho MW. Horizontal gene transfer happens. A practical exercise in applying the precautionary principle. i-sis news5, July 2000, http://www.i-sis.org.uk/isisnews/i-sisnews5.php#hori

5. Ho MW. Horizontal gene transfer from GMOs does happen. Science in Society 39, 22-24, 2008.

6. Netherwood T, Martin-Orue SM, O-Donnell AG, Gockling S, Graham J, Mathers JC and Gilbert JH. Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nature biotechnology 2004; 22: 204-209.

7. Ho MW. Transgenic lines unstable hence illegal and ineligible for protection. Science in Society 39, 28-29, 2008.

8. Netherwood, T., Bowden, R., Harrison, P., O’Donnell, A.G., Parker, D.S., Gilbert, H.J., 1999. Gene transfer in the gastrointestinal tract. Applied and Environmental

Microbiology 65, 5139–5141.

9. Kelly BG, Vespermann A, Bolton DJ Gene transfer events and their occurrence in selected environments. Food Chem Toxicol 2009, 47, 978–83.

10. McCuddin, Z., Carlson, S.A., Rasmussen, M.A., Franklin, S.K., 2006. Klebsiella to Salmonella gene transfer within rumen protozoa: implications for antibiotic resistance and rumen defaunation. Veterinary Microbiology 2005, 114, 275–84.

11. Ho MW, Lim LC et al. The Case for a GM-Free Sustainable World, Independent Science Panel Report, ISIS, London, 15 June 2003.

12. Mercer DK, Scott KP, Bruce-Johnson WA, Glover LA and Flint HJ. Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL by plasmid DNA in human saliva. Applied and Environmental Microbiology 1999, 65, 6-10.

13. Simpson DJ, Fry JC, Rogers HJ and Day MJ. Thematic issue on horizontal gene transfer. Transformation of Acinetobacter baylyi in non-sterile soil using recombinant plant nuclear DNA. Environ Biosafety Res 2007, 6, 101-12.

14. Rizzi A, Pontiroli A, Brusetti L, Borin S, Solini C, Abruzzese A, Sacchi GA, Vogel TM, Simonet P, Bazzicalupo M, Nielsen KM, Monier J-M and Daffoncho D. Strategy for in situ detection of natural transformation-based horizontal gene transfer events. Applied and Environmental Microbiology 2008, 74, 1250-4.

15. Pontiroli A, Rizzi A, Simonet P, Daffonchio D, Vogel TM and Monier JM. Visual evidence of horizontal gene transfer between plants and bacteria in the phytosphere of transplastomic tobacco. Appl Environ Microbio 2009, 75, 3314-22.

16. Wicks A and Jacobsen B Bl. Lack of detectable DNA uptake by transformation of selected recipients in monoassociated rats. Wicks and Jacobsen BMC Research Notes 2010, 3, 49.

17. Schaedler RW, Dubos R and Costello R. Association of germfree mice with bacteria isolated from normal mice. 1965, Jem.rupress.org.]

18. Human flora. Wikipedia, 13 May 2010, http://en.wikipedia.org/wiki/Human_flora

19. Ho MW. Living with the Fluid Genome, ISIS/TWN, London/Penang, 2003. http://www.i-sis.org.uk/fluidGenome.php

20. Ho MW, Traavik T, Olsvik R, Tappeser B, Howard CV, von Weizsacker C and McGavin GC. Gene technology and gene ecology of infectious diseases. Microbial Ecology in Health and Disease 1998, 19, 33-59.

21. Ho MW, Ryan A, Cummins J and Traavik T. Slipping Through the Regulatory Net: Naked and Free Nucleic Acids, TWN Biotechnology and Biosafety Series no. 5, 2001, http://www.i-sis.org.uk/onlinestore/books.php

22. Ho MW and Cummins J. New evidence links CaMV 35S promoter to HIV transcription. Science in Society 43, 26-27, 2009; and Microbial Ecology in Health and Disease 2009, 21, 172-4.

© 1999-2010 The Institute of Science in Society

Contact the Institute of Science in Society