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 modified plant is treated on its merits. Some proposals may be assessed as too risky and not be allowed to proceed.
Gene transfer between bacteria references:
Gast RK, Stephens JF (1986) In vivo transfer of antibiotic resistance to a strain of Salmonella arizonae. Poultry Science 65:270-279
Abstract: Newly-hatched turkey poults were dosed with a strain of Escherichia 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 diarrhea 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 persistence 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 evaluated 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 different¤ forms of plasmid DNA with an inserted kanamycin resistance gene, , 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 optimized 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 studies 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
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