a Division of Microbiology, School of Biochemistry and Molecular Biology and b Centre for Animal Sciences, Leeds Institute for Plant Biotechnology and Agriculture (LIBA), University of Leeds, Leeds LS2 9JT, UK
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Abstract |
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Introduction |
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Bacterial transformation by plant-derived DNA in vitro has been demonstrated2 and may be regarded as a theoretical possibility in the animal gut. For transformation to occur, DNA must first be released from the donor organism in a form in which it can be incorporated into the genome of a putative recipient bacterium and then must survive intact for sufficient time for transformation to occur. DNA retains the ability to transform bacteria after in vitro exposure to body fluids. Mercer et al.3 reported the persistence of transforming capability of a recombinant plasmid after exposure to human saliva for 10 min. Duggan et al.4 found that plasmid DNA exposed to ovine saliva was capable of transforming Escherichia coli after 24 h, implying that DNA released from dietary components could provide a source of transforming DNA, at least in the oral cavity of sheep. DNA sequences could also be amplified by PCR from plasmid DNA after incubation in rumen contents or silage effluent for 30 min. The ability to transform E. coli to resistance, however, persisted for <1 min in these environments. Under European regulations, the potential for gene flow is considered in the safety assessment of novel foods.1 Applicants often presume that marker genes will be digested. This paper reports experimental evidence that supports this assumption.
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Materials and methods |
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On three successive days, five broiler chicks were fed 100 g of a proprietary feed, seeded with bacteria. The feed was mixed with 50 mL of water and 50 mL of an overnight culture, equivalent to c. 5 x 109 E. coli DH5(pUC18) cells. On the third day the 17-day-old birds were killed and, from each, samples of digesta were collected from the crop, stomach, duodenum, three sites along the intestine, caecum and rectum. The samples were cultured on CLED medium containing 100 mg/L ampicillin, to facilitate the isolation of ampicillin-resistant non-lactose-fermenting bacteria. Random sample colonies were examined by RFLPPCR, specific for the blaTEM ß-lactamase gene.
Maize feeding experiments
Two groups of five 8-week-old broiler chickens, which had been receiving a standard proprietary diet, were fed a diet consisting of 80% ground maize and 20% fishmeal plus 0.5% (w/w) mineral and vitamin supplement for 5 days. One group received a diet containing transgenic maize CG00526-176, which contains a pUC18-derived blaTEM; the other group received a diet containing conventional maize, lacking blaTEM. Samples of excreta were collected on the third and fourth days of feeding. On the fifth day the birds were killed and samples of digesta were collected from the crop, stomach, small intestine, large intestine, caecum and rectum of each bird. The samples were stored at 70°C before DNA extraction.
Extraction of bacterial DNA from faeces and digesta
Total bacterial DNA was extracted from faeces and digesta using the QIAamp DNA Stool Mini Kit (Qiagen Ltd, Crawley, UK) using the manufacturer's protocol. The kit has been shown to be suitable for extraction of DNA from bacteria and from plant cells. Briefly, cells in the gut samples are lysed in buffer at 70°C, PCR inhibitors present in the sample are adsorbed on InhibitEX, proteins are digested using proteinase K and DNA is purified on QIAamp spin columns. The DNA is eluted in a final volume of 100 µL, 5 µL of which is used in a PCR with a total volume of 50 µL. BSA was included in the PCR at a final concentration of 0.1 g/L, as recommended by the manufacturer, and increases the robustness of the procedure.
PCR amplification of the blaTEM gene
A fragment of blaTEM was amplified using primers described by Mabilat et al.5 E. coli DH5(pUC18) was used as a positive control, as was E. coli UB1780, which contains a wild-type blaTEM gene; E. coli UB5201 was included as a negative control, being isogenic with UB1780 except for the absence of Tn802, which carries blaTEM. A DNA fragment from nad5, a plant mitochondrial gene, was amplified using the primers described by Mannerlöf & Tenning.6 DNA was visualized on 1.2% (w/v) agarose gels in TBE buffer.
Restriction endonuclease digestion
The blaTEM amplimer was digested with PstI (Life Technologies, Paisley, UK) by mixing 5 µL with 1.5 µL of x10 restriction enzyme buffer, 1.0 µL of restriction enzyme (10 U/µL) and 7.5 µL of sterile distilled water and incubating at 37°C for 2 h. Adding loading buffer stopped the reaction. DNA was visualized on 1.5% (w/v) agarose gels in TBE buffer.
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Results |
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Discussion |
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The results presented show that blaTEM ß-lactamase genes are detectable throughout the digestive tract of chickens fed a diet of conventional maize, as well as birds fed on transgenic maize incorporating blaTEM. In birds fed on conventional maize, however, all amplimers detected had the PstI site of the wild-type gene, which is missing from blaTEM derived from pUC18. This illustrates the ease with which wild-type blaTEM genes may be detected, reflecting the widespread distribution of these genes.
When birds were deliberately fed material containing E. coli DH5(pUC18), selective culture for ampicillin-resistant non-lactose-fermenting bacteria revealed that this strain was present throughout the gastrointestinal tract and in faecal material. The results of Netherwood et al.9 support these observations. They found that microorganisms fed to chickens as probiotics could be detected in faeces during the feeding period. Once feeding of probiotics ceased, these bacteria were no longer detected.
The fate of blaTEM in bacteria fed to chickens contrasts sharply with that of blaTEM from transgenic maize. The pUC18-derived blaTEM gene in transgenic maize was not detected in digesta collected from sites lower down the alimentary tract than the stomach. In three birds of five tested, the blaTEM present in transgenic plant material was found only in the crop contents. This difference in DNA survival in the intestinal tracts of different birds can probably be attributed to the difference in the length of time between the last releases of food from the crop into the stomach before the birds were killed. In contrast to blaTEM from bacteria, blaTEM from transgenic maize does not survive the digestive processes of the stomach to pass further down the alimentary tract.
This observation is supported by the results of PCR specific for nad5, the plant mitochondrial gene. In this case, nad5 was detected in the stomach contents of all birds tested, but not lower down the intestinal tract. The apparent greater resilience of this gene is probably an experimental artefact, reflecting either the greater copy number of the mitochondrial genome or, alternatively, that wild-type blaTEM sequences greatly outnumber blaTEM sequences derived from pUC18, and so only amplimers of the former are recovered, because the commoner sequence is likely to dominate PCR amplification. Whatever the explanation of the minor difference between the detection of pUC18-derived sequences in birds fed transgenic maize and the detection of plant-specific DNA in all birds, whether fed transgenic or conventional maize, it is important to note that blaTEM from transgenic maize does not, in essence, behave differently from other plant DNA sequences and does not survive the digestive processes of the stomach. In consequence, blaTEM from transgenic maize is very unlikely to transform bacteria found in the lower gut flora of birds to ampicillin resistance. This finding provides reassurance that chickens fed this transgenic maize are unlikely to be vectors for gene flow from transgenic plant material to the gut microflora, including significant human pathogens.
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Acknowledgements |
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Notes |
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References |
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2
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Gebhart, F. & Smalla, K. (1998). Transformation of Acinetobacter sp. BD413 by transgenic sugar beet DNA. Applied and Environmental Microbiology 64, 15504.
3
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Mercer, D. K., Scott, K. P., Bruce-Johnson, W. A., Glover, L. A. & Flint, H. J. (1999). Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Applied and Environmental Microbiology 65, 610.
4 . Duggan, P. S., Chambers, P. A., Heritage, J. & Forbes, J. M. (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 Microbiology Letters 191, 717. [ISI][Medline]
5 . Mabilat, C., Goussard, S., Sougakoff, W., Spencer, R. C. & Courvalin, P. (1990). Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum ß-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae. Plasmid 23, 2734. [ISI][Medline]
6 . Mannerlöf, M. & Tenning, P. (1997). Screening of transgenic plants by multiplex PCR. Plant Molecular Biology Reporter 15, 3845. [ISI]
7 . Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strainsnucleotide sequences of the M13MP18 and pUC19 vectors. Gene 33, 10319. [ISI][Medline]
8 . Suttcliffe, J. G. (1978). Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proceedings of the National Academy of Sciences, USA 75, 373741. [Abstract]
9
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Netherwood, T., Gilbert, H. J., Parker, D. S. & O'Donnell, A. G. (1999). Probiotics shown to change bacterial community structure in the avian gastrointestinal tract. Applied and Environmental Microbiology 65, 51348.
Received 15 February 2001; returned 13 August 2001; revised 3 September 2001; accepted 23 October 2001