ISIS Report 14/06/10
GM DNA Does Jump Species
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
was submitted to EFSA, please circulate widely, keeping the links intact
resistance genes from GM plants “unlikely” to transfer to bacteria
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  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 .
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
the scientific uncertainties, the European Commission granted approval to the GM
potato, Amflora in March 2010, in time for the planting season , more than
13 years after BASF first applied for commercial approval in the EU.
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 . 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.
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
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 ,
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  (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  (Horizontal Gene
Transfer from GMOs Does Happen, SiS 38), we drew attention to the
only feeding trial in human volunteers  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  Transgenic Lines
Unstable hence Illegal and Ineligible for Protection, SiS 38).
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 , 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 , 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 . 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
We have reported earlier in The Case
for A GM-Free Sustainable World  (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 .
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 .
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
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) . 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 . They
were able to detect transfer of plant DNA to bacteria on the surface of intact
leaves as well as on rotting, damaged leaves . 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
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 . 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 . 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 , 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.
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 . 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.
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.
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
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.
as I have long pointed out in Living with the Fluid Genome
 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  Gene Technology and Gene Ecology of Infectious
Diseases, ISIS scientific
publication) But even that is not all.
horizontal gene transfer is not the only, nor the major risk
commentators including the EFSA scientist  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.
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 . 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  Naked and Free Nucleic Acids -
Unregulated Hazards, ISIS Report).
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  (New Evidence Links
CaMV 35S Promoter to HIV Transcription, SiS 43 and ISIS scientific
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:
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.
Transfer of antibiotic resistance marker genes
in the gastrointestinal tract is particularly relevant to human and animal
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.
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
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
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
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There are 2 comments on this article so far. Add your comment
|wal Comment left 15th August 2010 14:02:34|
Then New Dehli metallo-beta-lactamase-1 : an example of successful horizontal antibiotic resistance gene transfer, most likely through the gastrointestinal tract.
|sam Comment left 30th October 2011 11:11:55|
Now I understand why our vaccines dont work.I understand why I wonder if we stop all of the genetically engineered foods from growing will we stop the holocaust we have created or will we have to live or die with the horror we have created?