Lack of detectable DNA uptake by transformation of selected recipients in mono-associated rats

Abstract

Background: An important concern revealed in the public discussion of the use of genetically modified (GM) plants for human consumption, is the potential transfer of DNA from these plants to bacteria present in the gastrointestinal tract. Especially, there is a concern that antibiotic resistance genes used for the construction of GM plants end up in pathogenic bacteria, eventually leading to untreatable disease.

Findings: Three different bacterial species (Escherichia coli, Bacillus subtilis, Streptococcus gordonii), all natural inhabitants of the food and intestinal tract environment were used as recipients for uptake of DNA. As source of DNA both plasmid and genomic DNA from GM plants were used in in vitro and in vivo transformation studies. Mono-associated rats, creating a worst-case scenario, did not give rise to any detectable transfer of DNA.

Conclusion: Although we were unable to detect any transformation events in our experiment, it cannot be ruled out that this could happen in the GI tract. However, since several steps are required before expression of plant-derived DNA in intestinal bacteria, we believe this is unlikely, and antibiotic resistance development in this environment is more in danger by the massive use of antibiotics than the consumption of GM food harbouring antibiotic resistance genes.

Figure 1

Colonization of four mono-associated rats with E. coli strain MS15978. Faecal samples were plated on both BHI (◆) and BHI including ampicillin (■). From day 10 all rats were dosed with 1000 μg ampicillin each day to restore the population of pMR1 containing E. coli cells. The detection limit for detection of transformants was 100 cfu/g faeces. Vertical bars represent standard error of the mean (SEM).

Figure 2

In vitro transformation frequencies (transformants per recipient) of B. subtilis 168. An overnight culture was diluted in MS media to OD₄₅₀ = 0.25. The culture was grown under aerobic conditions at 37°C. Every hour, one ml was taken out and different amounts of plasmid (0.1 μg/ml [Δ], 1 μg/ml [◆], and 10 μg/ml [■] were added. The cultures were incubated for further 1 hr before plating on selective media for counting of recipients (LB) and transformants (LB + 5 μg/ml chloramphenicol).

Figure 3

Faecal concentration (cfu/g faeces) of B. subtilis recipients (■) in eight mono-associated rats. The concentration of the dosage culture (cfu/ml) given during the three weeks is also indicated (◆). The detection limit for detecting transformants was 10 cfu/g faeces. Vertical bars represent SEM.

Figure 4

Colonization of eight animals with S. gordonii LTH 5597. Faecal samples were plated on both BHI (◆) and BHI including erythromycin (■). From day eight, all rats were dosed with 100 μg erythromycin each day to restore the population of pMK110 containing cells. The detection limit for detection of transformants was 10 cfu/g faeces. Vertical bars represent SEM.