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Lack of detectable DNA uptake by bacterial gut isolates grown in vitro and by Acinetobacter baylyi colonizing rodents in vivo

Published online by Cambridge University Press:  26 October 2007

Lise Nordgård ,
Thuy Nguyen ,
Tore Midtvedt ,
Yoshimi Benno ,
Terje Traavik  and
Kaare M. Nielsen
Lise Nordgård
Affiliation:
Norwegian Institute of Gene Ecology, Science Park, 9294 Tromsø, Norway
Thuy Nguyen
Affiliation:
Norwegian Institute of Gene Ecology, Science Park, 9294 Tromsø, Norway Microbe Division, Japan Collection of Microorganisms, RIKEN Bioresource Center, Wako, Saitama 351-0198, Japan
Tore Midtvedt
Affiliation:
Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden
Yoshimi Benno
Affiliation:
Microbe Division, Japan Collection of Microorganisms, RIKEN Bioresource Center, Wako, Saitama 351-0198, Japan
Terje Traavik
Affiliation:
Norwegian Institute of Gene Ecology, Science Park, 9294 Tromsø, Norway Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway
Kaare M. Nielsen
Affiliation:
Norwegian Institute of Gene Ecology, Science Park, 9294 Tromsø, Norway Department of Pharmacy, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway

Abstract

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Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is presented that were designed to determine if genetic transformation of the naturally competent bacterium Acinetobacter baylyi BD413 occurs in the GIT of mice and rats, with feed-introduced bacterial DNA containing a kanamycin resistance gene (nptII). Strain BD413 was found in various gut locations in germ-free mice at 103-105 CFU per gram GIT content 24–48 h after administration. However, subsequent DNA exposure of the colonized mice did not result in detectable bacterial transformants, with a detection limit of 1 transformant per 103-105 bacteria. Further attempts to increase the likelihood of detection by introducing weak positive selection with kanamycin of putative transformants arising in vivo during a 4-week-long feeding experiment (where the mice received DNA and the recipient cells regularly) did not yield transformants either. Moreover, the in vitro exposure of actively growing A. baylyi cells to gut contents from the stomach, small intestine, cecum or colon contents of rats (with a normal microbiota) fed either purified DNA (50 µg) or bacterial cell lysates did not produce bacterial transformants. The presence of gut content of germfree mice was also highly inhibitory to transformation of A. baylyi, indicating that microbially-produced nucleases are not responsible for the sharp 500- to 1 000 000-fold reduction of transformation frequencies seen. Finally, a range of isolates from the genera Enterococcus, Streptococcus and Bifidobacterium spp. was examined for competence expression in vitro, without yielding any transformants. In conclusion, model choice and methodological constraints severely limit the sample size and, hence, transfer frequencies that can be measured experimentally in the GIT. Our observations suggest the contents of the GIT shield or adsorb DNA, preventing detectable exposure of feed-derived DNA fragments to competent bacteria.

Keywords

horizontal gene transfernatural transformationgastrointestinal tractgnotobioticDNA persistence
Type
Research Article
Information
Environmental Biosafety Research , Volume 6 , Issue 1-2: Thematic Issue on Horizontal Gene Transfer , January 2007 , pp. 149 - 160
Copyright
© ISBR, EDP Sciences, 2007

References

Bensasson, D, Boore, JL, Nielsen, KM (2004) Genes without frontiers. Heredity 92: 483–489 CrossRef
Chambers PA, Duggan PS, Heritage J, Forbes MJ (2002) The fate of antibiotic resistance marker genes in transgenic plant feed material fed to chickens. J. Antimicrob. Chemother. 49: 161–164
Chowdhury EH, Mikami O, Nakajima Y, Hino A, Kuribara H, Suga K, Hanazumi M, Yomemochi C (2003a) Detection of genetically modified maize DNA fragments in the intestinal contents of pigs fed StarLinkTMCBH351. Vet. Hum. Toxicol. 45: 95–96
Chowdhury, EH, Kuribara, H, Hino, A, Sultana, P, Mikami, O, Shimada, N, Guruge, KS, Saito, M, Nakajama, Y (2003b) Detection of corn intrinsic and recombinant DNA fragments and cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11. J. Anim. Sci. 81: 2546–2551 CrossRef
Chowdhury, EH, Mikami, O, Murata, H, Sultana, P, Shimada, N, Yoshioka, M, Guruge, KS, Yamamoto, S, Miyazaki, S, Yamanaka, N, Nakajima, Y (2004) Fate of intrinsic and recombinant genes in calves fed genetically modified maize Bt11. J. Food Protection 67: 365–370 CrossRef
Deaville, ER, Maddison, BC (2005) Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. J. Agric. Food Chem. 53: 10268–10275 CrossRef
Doerfler W (2000) Foreign DNA in mammalian systems. Wiley-VCH, Weinheim, Germany
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 CrossRef
Duggan, PS, Chambers, PA, Heritage, J, Forbes, MJ (2003) Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. Br. J. Nutr. 89: 159–166 CrossRef
EFSA - European Food Safety Authority (2004) Opinion of the scientific panel on genetically modified organisms on the use of antibiotic resistance genes as marker genes in genetically modified plants. EFSA J. 48: 1–18
Einspanier, R, Klotz, A, Kraft, J, Aulrich, K, Poser, R, Schwägele, F, Jahreis, G, Flachowsky, G (2001) The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material. Eur. Food Res. Technol. 212: 129–134 CrossRef
Forsman, A, Ushameckis, D, Bindra, A, Yun, Z, Blomberg, J (2003) Uptake of amplifiable fragments of retrotransposon DNA from the human alimentary tract. Mol. Gen. Genomics 270: 362–368 CrossRef
Hohlweg, U, Doerfler, W (2001) On the fate of plant or other foreign genes upon the uptake in food or after intramuscular injection in mice. Mol. Gen. Genomics 265: 225–233 CrossRef
Jennings, JC, Albee, LD, Kolwyck, DC, Surber, JB, Taylor, ML, Hartnell, GF, Lirette, RP, Glenn, KC (2002) Attempts to detect transgenic and endogenous plant DNA and transgenic protein in muscle from broilers fed YieldGard corn borer corn. Poultry Sci. 82: 371–380 CrossRef
Kharazmi, M, Sczesny, S, Blaut, M, Hammes, WP, Hertel, C (2003) Marker rescue studies on the transfer of recombinant DNA to Streptococcus gordonii in vitro, in foods and gnotobiotic rats. Appl. Environ. Microbiol. 69: 6121–6127 CrossRef
Klotz, A, Mayer, J, Einspanier, R (2002) Degradation and possible carry over of feed DNA monitored in pigs and poultry. Eur. Food Res. Technol. 214: 271–275 CrossRef
Lorenz MG, Wackernagel W (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58: 563–602
Martín-Orúe, SM, O'Donnell, AG, Ariño, J, Netherwood, T, Gilbert, HJ, Mathers, JC (2002) Degradation of transgenic DNA from genetically modified soya and maize in human intestinal simulation. Br. J. Nutr. 87: 533–542 CrossRef
Maturin, L, Curtiss, R (1977) Degradation of DNA by nucleases in the intestinal tract of rats. Science 196: 216–218 CrossRef
Mercer DK, Melville CM, Scott KP, Flint HJ (1999a) Natural genetic transformation in the rumen bacterium Streptococcus bovis JB1. FEMS Microbiol. Lett. 179: 485–490
Mercer DK, Scott KP, Bruce-Johnson WA, Glover AL, Flint HJ (1999b) Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl. Environ. Microbiol. 65: 6–10
Mercer DK, Scott KP, Melville CM, Glover LA, Flint HJ (2001) Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva. FEMS Microbiol. Lett. 200: 163–167
Netherwood, T, Bowden, R, Harrison, P, O'Donnell, AG, Parker, DS, Gilbert, HJ (1999) Gene transfer in the gastrointetinal tract. Appl. Environ. Microbiol. 65: 5139–5141
Netherwood, T, Martín-Orúe, SM, O'Donnell, AG, Gockling, S, Graham, J, Mathers, JC, Gilbert, HJ (2004) Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nat. Biotechnol. 22: 204–209 CrossRef
Nielsen KM, Daffonchio D (2007) Unintended horizontal transfer of recombinant DNA. In Traavik T, Lim LC, eds, Biosafety first: holistic approaches to risk and uncertainty in genetic engineering and genetically modified organisms, Tapir Academic Press, Trondheim, Norway, pp 221–237
Nielsen KM, Townsend JP (2004) Monitoring and modeling horizontal gene transfer. Nat. Biotechnol. 22: 1110–1114
Nielsen KM, Bones AM, van Elsas JD (1997) Induced natural transformation of Acinetobacter calcoaceticus in soil microcosms. Appl. Environ. Microbiol. 63: 3972–77
Nielsen, KM, Smalla, K, van Elsas, JD (2000) Natural transformation of Acinetobacter sp. strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens and Burkholderia cepacia in soil microcosms. Appl. Environ. Microbiol. 66: 206–212 CrossRef
Nielsen KM, Berdal KG, Kruse H, Sundsfjord A, Mikalsen A, Yazdankhah S, Nes I (2005) An assessment of potential long-term health effects caused by antibiotic resistance marker genes in genetically modified organisms based on antibiotic usage and resistance patterns in Norway, VKM-Report, Norwegian Scientific Committee for Food Safety, Oslo, Norway
Nordgård L, Traavik T, Nielsen KM (2005) Nucleic acid isolation from ecological samples – vertebrate gut flora. In Methods in Enzymology, Academic Press, Vol. 224, pp 38–48
O'Hara, AM, Shanahan, F (2006) The gut flora as a forgotten organ. EMBO Rep. 7: 688–693 CrossRef
Palka-Santini, M, Schwarz-Herzke, B, Hösel, M, Renz, D, Auerochs, S, Brondke, H, Doerfler, W (2003) The gastrointestinal tract as a portal of entry for foreign macromolecules: fate of DNA and proteins. Mol. Gen. Genomics. 270: 201–215 CrossRef
Palmen, R, Hellingwerf, KJ (1997) Uptake and processing of DNA by Acinetobacter calcoaceticus: A review. Gene 192: 179–90 CrossRef
Pettersen, AK, Primicero, R, Bøhn, T, Nielsen, KM (2005) Modeling suggest frequency estimates are not informative for predicting the long-term effect of horizontal gene transfer in bacteria. Environ. Biosafety Res. 4: 222–233
Ray JL, Nielsen KM (2005) Experimental methods for assaying natural transformation and inferring horizontal gene transfer. In Methods in Enzymology, Academic Press, Vol. 224, pp 491–520
Schubbert, T, Lettmann, C, Doerfler, W (1994) Ingested foreign DNA (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice. Mol. Gen. Genet. 242: 495–504 CrossRef
Schubbert, R, Renz, D, Schmitz, B, Doerfler, W (1997) Foreign (M13) DNA ingested by mice reaches peripheral leucocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. USA 94: 961–966 CrossRef
Schubbert, R, Hohlweg, U, Renz, D, Doerfler, W (1998) On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus. Mol. Gen. Genet. 259: 569–576 CrossRef
Sharma, R, Alexander, TW, John, SJ, Forster, RJ, McAllister, TA (2004) Relative stability of transgene DNA fragments from GM rapeseed in mixed ruminal cultures. Br. J. Nutr. 91: 673–681 CrossRef
Sharma, R, Damgaard, D, Alexander, TW, Dugan, MER, Aalhus, JL, Stanford, K, McAllister, TA (2006) Detection of transgenic and endogenous plant DNA in digesta and tissues of sheep and pigs fed Roundup Ready Canola Meal. J. Agric. Food Chem. 54: 1699–1709 CrossRef
Sun, D, Zhang, Y, Mei, Y, Jiang, H, Xie, Z, Liu, Z, Chen, X, Shen P (2006) Escherichia coli is naturally transformable in a novel transformation system. FEMS Microbiol. Lett. 256: 249–255 CrossRef
Thomas, CM, Nielsen, KM (2005) Mechanisms and barriers to horizontal gene transfer between bacteria. Nat. Rev. Microbiol. 3: 711–721 CrossRef
Tony, MA, Butschke, A, Broll, H, Grohmann, L, Zagon, J, Halle, I, Dänike, Schauzu, M, Hafez, HM, Flachowsky, G (2003) Safety assessment of Bt176 maize in broiler nutrition: degradation of maize-DNA and its metabolic fate. Arch. Tierernahr. 57: 235–52
Wilcks, A, Hoek, AHAM, Joosten, RG, Jacobsen, BBL, Aarts, HJM (2004) Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation. Food Chem. Toxicol. 42: 493–502 CrossRef