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.