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Date:

October 12, 2000

Subject:

gene transfer to gut microbes

 

Curt Hannah wrote:

> Is there any documented evidence of a microbe living in a stomach to
> take up ANY DNA from the animal's food source?
>

Dear Curt,

I don't think there is any evidence that genes can transfer from food to
microbes in the gut. Some activists will probably quote a 1999 edition of
New Scientist (MacKenzie, D. , 1999, Gut reaction. 30 Jan., p.4). I have
seen this article referenced for example on M-W.Ho's World Scientist
statement as evidence that genes transfer from plants to gut microbes. In
fact what the Dutch group referred to in this new scientist article showed
was that antibiotic genes in bacteria introduced into an artificial gut
transfer to other microbes in the gut. The author of the New Scientist
article suggests that this is the first time that it has been shown that
antibiotic genes can transfer between microbes in guts. However, my
research on this matter has found that this is incorrect. There are several
reported cases where this is known to have happened and it seems to be a
fairly common and natural phenomenon. See references below. This New
Scientist article in fact reports that antibiotic resistance genes in Flavr
Savr tomatoes DID NOT transfer to the gut microbes in this experiment. So
an article often quoted as evidence of risk actually provides evidence of
no risk.

In another issue of New Scientist (25 March 2000) an article by Andy
Coghlan "For The Moment, The Gene Genie Is Staying In Its Bottle" which
reports a paper presented at a meeting of the British Society of Animal
Science by John Heritage from the University of Leeds. The reported data
indicated that the antibiotic resistance gene in transgenic maize did not
transfer to the gut organisms of chickens fed the maize. In personal
correspondence with John Heritage I have learnt that he has tried to
publish this data but has been rejected from several journals - the usual
problem with trying to publish negative findings.

I think the reason why people can't detect transfer of chromosomal DNA from
plants to bacteria even under optimal lab conditions is because it does not
happen. Plasmid DNA on the other hand can transform bacteria under these
experimental conditions. The reason for the difference maybe the fact that
plasmids are circular and the plant DNA linear - it is well known that
linear DNA has a much much lower transformation efficiency than circular
DNA. Thus, any result which show plasmid DNAs can transform bacteria
probably have little relevance to what will happen when the same gene is
found in a plant chromosome. So when assessing the relevance of any studies
it is important to carefully examine what people are measuring. Are they
looking at plasmid DNA or plant chromosomal DNA.
This issue has been recently reviewed by Gasson.

Gasson MJ (2000) Gene transfer from genetically modified food. Curr Opin
Biotechnol 11:505-508
Abstract: The current debate about the safety of genetically modified food
includes some important scientific issues where more scientific data would
aid the robustness of safety evaluation. One example is the possibility of
gene transfer, especially from genetically modified plant material

I have been reviewing this issue too. I have been able to pull out many
references that show NO transfer from plants to bacteria even under optimal
conditions. Below is the summaries from the papers pulled from the authors
own pens. What has been found is that bacteria that already have an
antibiotic resistance gene in them can take up the same gene from
transgenic plants. The relevance of this observation to the debate is
debatable.


"These data, in combination with other published studies, argue that
horizontal gene transfer is so rare as to be essentially irrelevant to any
realistic assessment of the risk involved in release experiments involving
transgenic plants"

"Therefore, this study could not provide a valid proof that horizontal gene
transfer of plant DNA to bacteria occurs under field or microcosm conditions"

"These results suggest that chromosomal DNA released into soil rapidly
becomes unavailable for transformation of A. calcoaceticus. In addition,
strain BD413 quickly loses the ability to receive, stabilize, and/or
express exogenous DNA after introduction into soil"

"Conclusions regarding the probability of successful horizontal gene
transfer on non-homologous DNA between plant and bacteria should not be
drawn from these data"

"our results suggest that gene transfer from the plant chromosome to
bacteria might occur in soil if homologous sequences are present in
competent bacteria. However, the in situ transformation frequencies would
likely be much lower than those under laboratory conditions." The lab
frequency was measured at 1 in 6.6 billion (What this data means is
bacteria can take up antibiotic resistance genes from transgenic plants if
these bacteria already have antibiotic resistance genes at a frequency many
times lower than 1 in 6.6 billion.)

"The persistence of DNA in [the cow saliva, rumen fluid and silage]
suggests the possibility of natural transformation to antibiotic resistance
of bacteria with the microflora associated with transgenic plants that
contain antibiotic resistance marker genes. In view of the continued use of
transgenic plants with such genes, this possibility warrants further
investigation in vivo"

I note that this paper used plasmid DNA and not plant DNA. What has been
observed in the soil studies is that plasmid DNA can transform the soil
bacteria but plant DNA can not. Follow up, unpublished work, from the
authors of this paper has found that this is the also the case for gut
bacteria - plant DNA containing antibiotic R genes does not transform gut
bacteria. The work referred to in the Mackenzie New Scientist article also
indicated that this is the case.

What we have yet to see is whether gut bacteria can undergo homologous
recombination with plant DNA as has been shown for soil bacteria.

What also must be kept in mind is low frequency of these events and the "so
what" factor. The event happens at very very low frequency and the
probability of it being harmful if it did happen is also very low. So when
you multiply these two probabilities you come up with something that is
nothing to worry about. This would be different if we were talking about
something different than antibiotic resistance genes. If the probability of
harm happening was large if the rare event happened then we would be in a
different risk area. This is why each different genetically mofified plant
is treated on its merits. Some proposals may be assessed as too risky and
not be allowed to proceed.


ia coli resistant to kanamycin, tetracycline and ampicillin,
and/or a strain of Salmonella arizonae resistant to nalidixic acid and
streptomycin. Kanamycin was added to the drinking water of some poults.
Samples were collected by swabbing the rectum of the poults and by removing
segments of the intestines and livers after death. Nalidixic acid was added
to the isolation media to prevent in vitro transfer from occurring after
the samples were collected. S. arizonae resistant to nalidixic acid,
streptomycin, kenamycin, tetracycline and ampicillin was isolated from 20%
of the rectal samples taken from poults that had received both bacterial
strains. S. arizonae cells which had received resistance determinants in
vivo were also isolated from 73% of the intestinal samples and 8% of the
liver samples taken from birds inoculated with both donor E. coli and
recipient S. arizonae. Salmonella arizonae demonstrating resistance to all
five antibiotics were recovered from all intestinal samples taken from
birds given kanamycin in the drinking water immediately after the last S.
arizonae inoculation, but from only 43% of such samples taken from birds
given no kanamycin

Gyles C, Falkow S, Rollins L (1978) In vivo transfer of an Escherichia coli
enterotoxin plasmid possessing genes for drug resistance. American Journal
of Veterinary Research 39:1438-1441
Abstract: Experiments were conducted to study transfer of an enterotoxin
(Ent) plasmid from a porcine enteropathogenic Escherichia coli to an E.
coli K12 strain in the intestine of newly weaned pigs. The Ent plasmid
carried genes for resistance to tetracycline, streptomycin, and
sulfonamides, thereby permitting a selection for tetracycline-resistant
ex-conjugants in the faeces of the pigs. Transfer of the Ent plasmid
occurred when the pigs were given large oral inocula of donor and recipient
cultures, 1 hour apart. Differences in extent of transfer were not detected
in pigs given antibiotic-free feed compared with littermates on feed
containing oxytetracycline at 50 g/ton. In one experiment,
tetracycline-resistant Ent exconjugants were found which appeared to have
received an R plasmid from an enteropathogenic type of E. coli resident in
the intestine

Smith MG (1977) In vivo transfer of an R factor within the lower
gastro-intestinal tract of sheep. Journal of Hygiene 79:259-268
Abstract: The transfer of an R factor from donor E. coli introduced into
the rumen of adult sheep to the coliform microflora of the lower
gastro-intestinal tract was greatly increased when the animals were
subjected to a short period of starvation (about 24-48 h). This also
resulted in coliform organisms containing the resistance determinants of
the R factor being excreted for much longer periods, sometimes for months
afterwards. As no antibiotic treatment was given to the animals during
these experiments, possession of the R factor should have conferred no
selective advantages on the host cells, and other plasmids could possibly
be transferred similarly in sheep or other ruminants and perhaps also
within the gut of monogastric animals


Falkow S (1975) Infectious multiple drug resistance.
Abstract: After a brief historical account of transmissible drug resistance
and R factors, the early chapters give detailed consideration to
characteristics of plasmids and to the genetic properties, molecular
nature, replication, and ecology of R factors. In Chapter 9 current
knowledge on the mechanisms of R factor-mediated resistance to
chloramphenicol, penicillin, aminoglycosides, tetracyclines and
sulphonamides is reviewed. Evidence is presented that R factors existed in
the pre-antibiotic era e.g. in an E. coli strain freeze-dried in 1946.
Chapter 10 (Transfer of R factors in vivo and the public health implication
to man and domestic animals) argues that the rate of in vivo transfer of R
factors is of relatively low order, but the emergence of resistant
bacterial populations has public health implications, especially when
salmonellae are present in meat and meat products. Mention is made of the
Swann Committee Report on Antibiotics, but the author does not indicate
that any of the recommendations have been implemented. The final chapter
contains a useful review of current knowledge on the relevance of plasmids
to pathogenicity, notably the significance of the K88 plasmid in the
pathogenesis of E. coli diarrhoea in pigs

Jones FT, Langlois BE, Cromwell GL, Hays VW (1984) Effect of
chlortetracycline on the spread of R-100 plasmid-containing Escherichia
coli BEL 15R from experimentally infected pigs to uninfected pigs and
chicks. Journal of Animal Science 58:519-526
Abstract: Seven-week-old pigs from a chlortetracycline (CTC)-fed herd and
from a herd not fed antibiotics were fed diets containing 0 or 55 mg of
CTC/kg. One of five pigs in each herd-diet treatment group was infected
orally with E. coli strain BEL15R that was resistant to nalidixic acid
(NA), chloramphenicol (C), streptomycin (S), sulfamethizole (TH) and
tetracycline (TE). Effects of CTC on the quantity and duration of faecal
shedding of E. coli BEL15R and on the transmission of strain BEL15R and its
R-100 plasmid from infected pigs to uninfected pigs and chicks were
determined. Quantity and duration of shedding were greater in infected
antibiotic-herd pigs than in infected non-antibiotic-herd pigs. Feeding of
CTC increased the duration of shedding in infected pigs from both herds.
Strain BEL15R colonized and was shed in one uninfected antibiotic-herd pig
in each treatment group, but it did not colonize in any of the uninfected
nonantibiotic-herd pigs or in the uninfected chicks. In vivo transfer of
resistance to C, S, TH and TE occurred in the infected antibiotic-herd pigs
but not in the infected non-antibiotic-herd pigs. Transfer of the R-100
plasmid occurred from the infected to the uninfected antibiotic-herd pigs
and to the uninfected chicks housed near the antibiotic-herd pigs fed CTC,
but not to the chicks housed with the antibiotic-herd pigs fed the control
diet. No transfer of resistance occurred from the infected
non-antibiotic-herd pigs fed either CTC or control diet
Gene transfer between plants and bacteria references.


Gebhard F, Smalla K (1999) Monitoring field releases of genetically
modified sugar beets for persistance of transgenic plant DNA and horizontal
gene transfer. FEMS Microbiol Ecol 28:261-272
Abstract: From the Authors discussion: "Therefore, this study could not
provide a valid proof that horizontal gene transfer of plant DNA to
bacteria occurs under field or microcosm conditions"

Nielsen KM, van Weerelt MD, Berg TN, Bones AM, Hagler AN, van Elsas JD
(1997) Natural transformation and availability of transforming DNA to
Acinetobacter calcoaceticus in soil microcosms. Appl Environ Microbiol
63:1945-1952
Abstract: A small microcosm, based on optimized in vitro transformation
conditions, was used to study the ecological factors affecting the
transformation of Acinetobacter calcoaceticus BD413 in soil. The
transforming DNA used was A. calcoaceticus homologous chromosomal DNA with
an inserted gene cassette containing a kanamycin resistance gene, nptII.
The effects of soil type (silt loam or loamy sand), bacterial cell density,
time of residence of A. calcoaceticus or of DNA in soil before
transformation, transformation period, and nutrient input were
investigated. There were clear inhibitory effects of the soil matrix on
transformation and DNA availability. A. calcoaceticus cells reached
stationary phase and lost the ability to be transformed shortly after
introduction into sterile soil. The use of an initially small number of A.
calcoaceticus cells and nutrients, resulting in bacterial growth, enhanced
transformation frequencies within a limited period. The availability of
introduced DNA for transformation of A. calcoaceticus cells disappeared
within a few hours in soil. Differences in transformation frequencies
between soils were found; A. calcoaceticus cells were transformed at a
higher rate and for a longer period in a silt loam than in a loamy sand.
Physical separation of DNA and A. calcoaceticus cells had a negative effect
on transformation. Transformation was also detected in nonsterile soil
microcosms, albeit only in the presence of added nutrients and at a reduced
frequency. These results suggest that chromosomal DNA released into soil
rapidly becomes unavailable for transformation of A. calcoaceticus. In
addition, strain BD413 quickly loses the ability to receive, stabilize,
and/or express exogenous DNA after introduction into soil

Nielsen KM, Gebhard F, Smalla K, Bones AM, van Elsas JD (1997) Evaluation
of possible horizontal gene transfer from transgenic plants to the soil
bacterium Acinetobacter calcoaceticus BD413. Theor Appl Genet
Notes: Abstract The use of genetically engineered crop plants has raised
concerns about the transfer of their engineered DNA to indigenous microbes
in soil. We have evalu-ated possible horizontal gene transfer from
transgenic plants by natural transformation to the soil bacterium
Acinetobacter calcoaceticus BD413. The transformation frequencies with DNA
from two sources of transgenic plant DNA and di¤erent forms of plasmid DNA
with an inserted kanamycin resistance gene, nptII, were measured. Clear
e¤ects of homology were seen on transformation frequencies, and no
transformants wereever detected after using transgenic plant DNA. This
implied a transformation frequency of less than 10-13(transformants per
recipient) under optimised conditions, which is expected to drop even
further to a minimum of 10-16 due to soil conditions and a lowered
concentration of DNA available to cells. Previous stud-
ies have shown that chromosomal DNA released to soil is only available to
A. calcoaceticus for limited period of time and that A. calcoaceticus does
not maintain detectable competence in soil. Taken together, these results
suggest that A. calcoaceticus does not take up non-homologous plant DNA at
appreciable frequencies under natural conditions.


Schluter K, Futterer J, Potrykus I (1995) "Horizontal" gene transfer from a
transgenic potato line to a bacterial pathogen (Erwinia chrysanthemi)
occurs--if at all--at an extremely low frequency. Biotechnology (N Y )
13:1094-1098

Abstract: The frequency of possible "horizontal" gene transfer between a
plant and a tightly associated bacterial pathogen was studied in a model
system consisting of transgenic Solanum tuberosum, containing a beta-
lactamase gene linked to a pBR322 origin of replication, and Erwinia
chrysanthemi. This experimental system offers optimal conditions for the
detection of possible horizontal gene transfer events, even when they occur
at very low frequency. Horizontal gene transfer was not detected under
conditions mimicking a "natural" infection. The gradual, stepwise
alteration of artificial, positive control conditions to idealized natural
conditions, however, allowed the characterization of factors that affected
gene transfer, and revealed a gradual decrease of the gene transfer
frequency from 6.3 x 10(-2) under optimal control conditions to a
calculated 2.0 x 10(-17) under idealized natural conditions. These data, in
combination with other published studies, argue that horizontal gene
transfer is so rare as to be essentially irrelevant to any realistic
assessment of the risk involved in release experiments involving transgenic
plants
de Vries J, Wackernagel W (1998) Detection of nptII (kanamycin resistance)
genes in genomes of transgenic plants by marker-rescue transformation. Mol
Gen Genet 257:606-613
Abstract: From the authors discussion:
"Conclusions regarding the probability of successful horizontal gene
transfer on non-homologous DNA between plant and bacteria should not be
drawn from these data"

We have developed a novel system for the sensitive detection of nptII genes
(kanamycin resistance determinants) including those present in transgenic
plant genomes. The assay is based on the recombinational repair of an nptII
gene with an internal 10-bp deletion located on a plasmid downstream of a
bacterial promoter. Uptake of an nptII gene by transformation restores
kanamycin resistance. In Escherichia coli, promoterless nptII genes
provided by electroporation were rescued with high efficiency in a
RecA-dependent recombinational process. For the rescue of nptII genes
present in chromosomal plant DNA, the system was adapted to natural
transformation, which favours the uptake of linear DNA. When competent
Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the
mutant nptII gene on a plasmid were transformed with DNA from various
transgenic plants carrying nptII as a marker gene (Solanum tuberosum,
Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum),
kanamycin-resistant transformants were obtained roughly in proportion to
the concentration of nptII genes in the plant DNA. The rescue of nptII
genes occurred in the presence of a more than 6 x 10(6)-fold excess of
plant DNA. Only 18 ng of potato DNA (2.5 x 10(3) genome equivalents, each
with one copy of nptII) was required to produce one kanamycin-resistant
transformant. These experiments and others employing DNA isolated from soil
samples demonstrate that the system allows reliable and highly sensitive
monitoring of nptII genes in transgenic plant DNA and in DNA from
environmental sources, such as soil, without the need for prior DNA
amplification (e.g. by PCR)
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 Microbiol Lett 191:71-77

Abstract: From the authors discussion:"The persistence of DNA in [the cow
saliva, rumen fluid and silage] suggests the possibility of natural
transformation to antibiotic resistance of bacteria with the microflora
associated with transgenic plants that contain antibiotic resistance marker
genes. In view of the continued use of transgenic plants with such genes,
this posibility warrants further investigation in vivo"
To assess the likelihood that the bla gene present in a transgenic maize
line may transfer from plant material to the microflora associated with
animal feeds, we have examined the survival of free DNA in maize silage
effluent, ovine rumen fluid and ovine saliva. Plasmid DNA that had
previously been exposed to freshly sampled ovine saliva was capable of
transforming competent Escherichia coli cells to ampicillin resistance even
after 24 h, implying that DNA released from the diet could provide a source
of transforming DNA in the oral cavity of sheep. Although target DNA
sequences could be amplified by polymerase chain reaction from plasmid DNA
after a 30-min incubation in silage effluent and rumen contents, only short
term biological activity, lasting less than 1 min, was observed in these
environments, as shown by transformation to antibiotic resistance. These
experiments were performed under in vitro conditions; therefore further
studies are needed to elucidate the biological significance of free DNA in
the rumen and oral cavities of sheep and in silage effluent
Gebhard F, Smalla K (1998) Transformation of Acinetobacter sp. strain BD413
by transgenic sugar beet DNA. Appl Environ Microbiol 64:1550-1554
Abstract: "our results suggest that gene transfer from the plant chromosome
to bacteria migh occur in soil if homologous sequences are present in
competent bacteria. However, the in situ transformation frequencies would
likely be much lower than those under laboratory conditions.". There
transformation freq in the lab (acutally homologous recombination freq) was
1.5 x 10-10 with plant homoginate of sugar beet leaves.

The ability of Acinetobacter sp. strain BD413(pFG4 delta nptII) to take up
and integrate transgenic plant DNA based on homologous recombination was
studied under optimized laboratory conditions. Restoration of nptII,
resulting in kanamycin-resistant transformants, was observed with plasmid
DNA, plant DNA, and homogenates carrying the gene nptII. Molecular analysis
showed that some transformants not only restored the 317-bp deletion but
also obtained additional DNA

--
Opinons expressed in this posting are personal and do not reflect the
position of my employer