Lack of detectable DNA uptake by bacterial gut isolates grownin vitroand byAcinetobacter baylyicolonizing rodentsin vivo

Abstract

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 10 3 −10 5 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 10 3 −10 5 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.

Scientific Opinion on applications (EFSA-GMO-UK-2008-57 and EFSA-GMO-RX-MON15985) for the placing on the market of insect-resistant genetically modified cotton MON 15985 for food and feed uses, import and processing, and for the renewal of authorisation o

“…Several bacterial species with the potential to develop competence belong to the common gut microbial community (EFSA, 2009b;Rizzi et al, 2012). However, actual competence development and transformation of such bacteria by genomic DNA of plants has not yet been observed in the lower gastrointestinal tract even with optimised model systems providing a selective advantage (Nordgård et al, 2007;EFSA, 2009b;Rizzi et al, 2012). In contrast, some studies have shown that introduced bacteria can be naturally transformed in the oral cavity of humans and animals (Mercer et al, 1999a(Mercer et al, , b, 2001Duggan et al, 2000Duggan et al, , 2003.…”

Scientific Opinion on an application from Pioneer Hi‐Bred International and Dow AgroSciences LLC (EFSA‐GMO‐NL‐2005‐23) for placing on the market of genetically modified maize 59122 for food and feed uses, import, processing and cultivation under Regulation (EC) No 1829/2003

“…However, competence development and transformation of such bacteria with genomic DNA of plants has not been observed in the lower gastrointestinal tract, even with optimised model systems providing a selective advantage (Nordgård et al, 2007;EFSA, 2009c;Rizzi et al, 2012). In contrast, some studies have shown that introduced bacteria can be naturally transformed in the oral cavity of humans and animals (Duggan et al, 2000(Duggan et al, , 2003Mercer et al, 1999aMercer et al, ,b, 2001Rizzi et al, 2012).…”

Acinetobacter baylyi ADP1: Transforming the choice of model organism

“…The spread of transgenic plant DNA has been observed from decaying leaves, by simulated rainwater, in field plots, from roots, and via pollen (21,22). While efforts to detect HGT to A. baylyi in the gastrointestinal tracts of insects and animals were unsuccessful, digested transgenic plant tissue in insect fecal matter remains capable of transforming A. baylyi (23)(24)(25)(26). These studies inform continued assessment of HGT and provide insights into ways to design genetically modified organisms to decrease the risk of transgene dissemination.…”