The fate of antibiotic resistance marker genes in transgenic plant feed material fed to chickens

Philip A. Chambersa, Paula S. Duggana, John Heritagea,* and J. Michael Forbesb

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We have examined the fate of an antibiotic resistance marker, incorporated into transgenic maize when fed to chicks. Plant-derived markers were found in the crops of five birds fed transgenic maize and in the stomach contents of two birds. The plant-derived marker gene was not found in the intestines. The survival of the antibiotic resistance marker gene mirrored that of plant DNA targets, demonstrating that it survives no better than other DNA and indicating that it is very unlikely that bacteria in the gut of chickens will be transformed to ampicillin resistance when the birds are fed transgenic maize.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
There has been increasing concern regarding the problem of bacterial antibiotic resistance genes.1 These are used as markers in the production of transgenic plants, a use that has the potential to increase their dissemination by providing novel situations for gene transfer. In some transgenic plants, resistance genes are engineered to express in eukaryotic cells to enable selection of transformed plant cells. In others, resistance genes are present through incorporation of vector DNA from bacterial constructs. In these cases it is unlikely that the resistance gene will be expressed in the plant cell but it may be expressed were it to return to a bacterial host.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Feeding experiments using bacteria containing plasmid pUC18

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{alpha}(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 RFLP–PCR, 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{alpha}(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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
When birds were given conventional feed material with E. coli DH5{alpha}(pUC18) added, these bacteria were detected at all sites sampled and RFLP–PCR analysis showed amplimers that lacked a PstI site, establishing that RFLP–PCR can differentiate blaTEM associated with pUC18 from the wild-type blaTEM gene in samples derived from the chicken gastrointestinal tract (data not shown). The fate of the pUC18-derived blaTEM gene in transgenic maize fed to chickens was then examined. PCR amplification demonstrated that blaTEM sequences were detected at all sites tested in all birds, whether fed conventional or transgenic maize. From the five birds fed on conventional maize, all blaTEM amplimers were digested with PstI (Figure 1Go), indicating that these were not derived from plasmid pUC18. In contrast, from the five birds fed transgenic maize, all crop samples yielded amplimers that characteristically lacked the PstI site (Figure 2Go). Given that wild-type blaTEM is easily detected in birds fed conventional maize and that the predicted PCR products are of the same size, the appearance of the maize-derived blaTEM restriction pattern indicates that it is present in considerable excess over the wild-type blaTEM gene in birds fed transgenic material. In two of the five birds fed transgenic maize, the maize-derived blaTEM was also detected in the contents of the stomach. In the other three birds, the amplimers carried the PstI site characteristic of wild-type blaTEM. All blaTEM sequences detected from lower in the intestine and from faecal material were of the wild type, indicating that blaTEM characteristic of the transgenic maize did not persist throughout the intestinal tract in detectable amounts.



View larger version (81K):
[in this window]
[in a new window]
 
Figure 1. PCR–RFLP of blaTEM using targets derived from along the gastrointestinal tract of chickens fed conventional maize. Lanes 1 and 10, 100 bp ladders; lane 2, E. coli UB1780, a strain carrying a single chromosomal copy of blaTEM; lane 3, E. coli DH5{alpha}(pUC18); lane 4, crop material; lane 5, stomach contents; lane 6, small intestine; lane 7, colon; lane 8, caecum; lane 9, rectum.

 


View larger version (73K):
[in this window]
[in a new window]
 
Figure 2. PCR–RFLP of blaTEM using targets derived from the gastrointestinal tract of chickens fed transgenic maize. Lanes 1 and 10, 100 bp ladders; lane 2, E. coli UB1780, a strain carrying a single chromosomal copy of blaTEM; lane 3, E. coli DH5{alpha}(pUC18); lane 4, crop material; lane 5, stomach contents; lane 6, small intestine; lane 7, colon; lane 8, caecum; lane 9, rectum.

 
The persistence of plant-derived DNA was examined by amplification of nad5, a maize mitochondrial DNA sequence. Since mitochondrial DNA is present in excess copies compared with chromosomal DNA, this target offers a sensitive indicator of plant DNA. In all birds tested, whether fed on conventional or transgenic maize, plant-specific DNA was detected in samples from the crop and stomach only (data not shown). This indicates that blaTEM from transgenic maize survives no better than other DNA derived from the animal feed.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Broiler chickens were fed bacteria and transgenic plant material, both of which contained blaTEM associated with pUC18. RFLP–PCR differentiates this blaTEM from the wild-type blaTEM found in the microflora of the avian alimentary tract. The absence of the characteristic PstI site in pUC18-derived blaTEM7 found in wild-type blaTEM8 makes differentiation of blaTEM from transgenic plant material a relatively simple process.

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{alpha}(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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Novartis, formerly Ciba-Geigy, for providing the genetically modified maize seeds used in this study. This work was funded by a grant from the Food Standards Agency.


    Notes
 
* Corresponding author. Tel: +44-113-233-5592; Fax: +44-113-233-5638; E-mail: j.heritage{at}leeds.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Commission of the European Communities (1997). Commission Recommendation of 29 July, 1997, concerning the scientific aspects and the presentation of information necessary to support applications for the placing on the market of novel foods and novel food ingredients and the preparation of initial assessment reports under Regulation (EC) No. 258/97 of the European Parliament and of the Council. Official Journal of the European Communities 40, L253, 1–36.

2 . Gebhart, F. & Smalla, K. (1998). Transformation of Acinetobacter sp. BD413 by transgenic sugar beet DNA. Applied and Environmental Microbiology 64, 1550–4. [Abstract/Free Full Text]

3 . 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, 6–10. [Abstract/Free Full Text]

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, 71–7. [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, 27–34. [ISI][Medline]

6 . Mannerlöf, M. & Tenning, P. (1997). Screening of transgenic plants by multiplex PCR. Plant Molecular Biology Reporter 15, 38–45. [ISI]

7 . Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains—nucleotide sequences of the M13MP18 and pUC19 vectors. Gene 33, 103–19. [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, 3737–41. [Abstract]

9 . 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, 5134–8. [Abstract/Free Full Text]

Received 15 February 2001; returned 13 August 2001; revised 3 September 2001; accepted 23 October 2001





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (13)
Disclaimer
Request Permissions
Google Scholar
Articles by Chambers, P. A.
Articles by Forbes, J. M.
PubMed
PubMed Citation
Articles by Chambers, P. A.
Articles by Forbes, J. M.