Lack of detectable DNA uptake by transformation of selected recipients in mono-associated rats
© Wilcks et al; licensee BioMed Central Ltd. 2010
Received: 9 October 2009
Accepted: 1 March 2010
Published: 1 March 2010
An important concern revealed in the public discussion of the use of genetically modified (GM) plants for human consumption, is the potential transfer of DNA from these plants to bacteria present in the gastrointestinal tract. Especially, there is a concern that antibiotic resistance genes used for the construction of GM plants end up in pathogenic bacteria, eventually leading to untreatable disease.
Three different bacterial species (Escherichia coli, Bacillus subtilis, Streptococcus gordonii), all natural inhabitants of the food and intestinal tract environment were used as recipients for uptake of DNA. As source of DNA both plasmid and genomic DNA from GM plants were used in in vitro and in vivo transformation studies. Mono-associated rats, creating a worst-case scenario, did not give rise to any detectable transfer of DNA.
Although we were unable to detect any transformation events in our experiment, it cannot be ruled out that this could happen in the GI tract. However, since several steps are required before expression of plant-derived DNA in intestinal bacteria, we believe this is unlikely, and antibiotic resistance development in this environment is more in danger by the massive use of antibiotics than the consumption of GM food harbouring antibiotic resistance genes.
A major concern in relation to marketing of genetically modified (GM) plants for human consumption is the possible transfer of antibiotic resistance genes used as marker genes in GM plants to the human or animal intestinal microbiota. The uptake of these resistance genes by bacteria present in the gastrointestinal (GI) tract could potentially render pathogens resistant to antimicrobial agents currently used, thereby resulting in untreatable diseases .
Transformation is the only known gene transfer mechanism by which bacteria can take up DNA released from plants. Key factors are thus DNA persistence in the GI tract, the availability of competent bacteria, and their state of competence . Several studies indicate that DNA, and especially plant-associated DNA, is able to survive the conditions in the GI tract and be available for uptake by bacteria resident in the gastrointestinal tract [3–8].
Several of the bacteria found in the GI tract, either carried by the food or innate GI bacteria, have been found to be naturally transformable . But the question is whether these bacteria also possess or develop competence in this environment. In this work we used as recipients the naturally transformable bacteria Bacillus subtilis which is often a contaminant of food, and Escherichia coli and Streptococcus gordonii that are part of the normal gut microbiota. We used mono-associated rats that can be considered as a worst-case model, and as a biological magnifier making it possible to study one bacterial species separately and often in high number. All animal experiments were carried out under the supervision of the Danish National Agency for Protection of Experimental Animals.
Bacterial strains and plasmids
Strains or plasmids
Reference or source
DB1317 harbouring pMR1, Apr
DH5α, recA1, harbouring pMR2, Cmr
, this study
TIGR strain harbouring pMK110, Eryr
pBR322 vector, nptIIΔNco I, Apr
pACYC184 vector, nptII without promoter, Cmr
pMG36e vector, nptIIΔNco I from pMR1, Eryr
pUC19 vector, cat from pC194, Bacillus thuringiensis replicon, Apr
Solanum tuberosum cv. Apriori
Genetically modified potato containing antisense GBSS (granule bound starch synthase) and intact nptII
AVEBE, Foxhole, The Netherlands
In this study we observed low-frequency spontaneous transformation of E. coli strain DB1317 growing in LB media without addition of divalent cations or temperature shift, conditions pivotal for high frequency transformation. However, adding faecal or intestinal samples from germfree rats to the LB media let drop the number below the detection limit of 2.3 × 10-9 TF/rec. This could indicate that adding these samples either inhibited uptake of DNA, or that the DNA was degraded in the samples. A recent published paper (Nordgaard et al., 2007) observed the same inhibitory effect of intestinal content from germfree mice on the transformation process of Acinetobacter baylyi. Previous studies incubating plant DNA in GI samples ex vivo showed that the DNA was rapidly degraded in samples from the small intestine, whereas hardly any degradation was observed when incubating the DNA in samples from the lower part of the GI tract . However, DNA persistence was studied using PCR, a sensitive method needing only scarce amounts of DNA for amplification. Studies incubating plasmid DNA in faecal samples from germfree rats showed that by simple gel electrophoresis, the plasmid could not be observed after 10 minutes of incubation, whereas by PCR a strong band could be detected even after 20 min of incubation (unpublished results). Since the transformation frequency in LB media is already low, the degradation of DNA in the samples is probably lowering the transformation frequency further, so that a possible transfer event is below detection limit. The same may be true for the in vivo studies where no uptake of DNA by E. coli could be observed, in concordance with an earlier study showing that although plasmid DNA was detected in the GI tract, the concentration was very low .
An explanation of lack of transformants in the rats could be the low number of B. subtilis present in the GI tract with app. 103 cfu/g in ileum, 103 cfu/g in caecum and 105 cfu/g in colon. In vitro the highest transformation frequency observed was 10-6 TF/recipient. Therefore the likelihood of detection of a transformed B. subtilis within a faecal population of 103-105 cfu/g is very low.
Previous published studies have shown that the used strain, LTH 5597, is capable of taking up plasmid DNA and genomic plant DNA under in vitro conditions .
At day 7 to 10 of the experiment, eight animals received 1 mg DNA extracted from GM potato; this corresponds to approximately 109nptII genes. Faecal samples were taken and plated onto selective media (BHI containing 1 mg/ml kanamycin), but no transformants were detected (detection limit 10 cfu/g faeces). To select for potential transformants, samples were pooled, incubated in Brain Heart Infusion (BHI) media containing 1 mg/ml kanamycin at 37°C overnight, and plated onto selective media containing kanamycin. But again no transformants were detected (detection limit 10 cfu/ml).
At day 22 of the experiment, the test animals received an overnight culture of E. coli harbouring the plasmid pMR2, which contains a whole copy of nptII but without a promoter. The strain established well in the animals with app. 108 cfu/g faeces, but again no kanamycin resistant colonies of S. gordonii were detected (detection limit 10 cfu/g faeces). At sacrifice the total number of bacteria in the different sections of the intestine where as following: duodenum: 104 cfu/g; ileum: 106 cfu/g; caecum and colon: 108 cfu/g. And also here no transformants were detected (detection limit 10 cfu/g intestinal content). This is in agreement with another study with germ free rats mono-associated with S. gordonii and fed large amounts of plasmid DNA that also failed to show transformation in vivo.
In the present study, the three studied bacterial species were unable to take up free DNA in a germfree animal model. Using germfree mice, another study using Acinetobacter baylyi also failed to show in vivo transformation . However, there are several more transformable bacterial species that are relevant for the intestinal situation , so we cannot rule out the possibility of transformation to happen in the GI tract. Nevertheless, many steps are required before a successful transfer of an antibiotic resistance gene from plant to bacteria has occurred. The DNA has to be released from the plant material, the DNA must survive the harsh gastrointestinal environment; competent bacteria must be present and take up the DNA, and finally has the gene to be integrated into the genome in a place where it can be expressed. Therefore the development of antibiotic resistant pathogenic bacteria is much more favoured by the use of antibiotics thereby putting a selective pressure on the intestinal environment, than by the consumption of antibiotic resistance genes present in GM plants.
We want to thank Annette Christensen, Anh Pham and Amir Mujezinovic for their technical assistance, and Anne Ørngren and her co-workers for the handling of the animals. Johann de Vries and Mitra Kharazami are thanked for providing strains and plasmids. The European commission is acknowledged for financial support through the Fifth Framework Programme 1998-2002, project GMOBILITY, proposal no. QLK1-CT-1999-00527.
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