Reproducibility and relative validity of a newly developed web-based food-frequency questionnaire for assessment of preconception diet

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An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats

Background: Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes. Results: Plasmid DNA with two resistance genes (nptII and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, where constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. Conclusions: The analyses showed that extensive ingestion of DNA (100 \ub5g plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit <1 transformant per 1.1 x 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed

Survival and uptake of feed-derived DNA in the mammalian intestinal tract

Lise Nordgård har i sin avhandling fra Universitetet i Tromsø og GenØk-Senter for biosikkerhet studert hva som skjer med introduksjon av transgene genkonstrukter som inntar en organsime via fòr. Blir DNA brutt ned når det passerer tarmkanalen eller overlever det? Kan dette DNAet passere tarmbarrierer og videre tas opp i blod og transporteres rundt i organsimen for å bli tatt opp av celler i vev? Hva med bakteriene i tarmen, kan disse ta opp fremmed DNA og utrykke det? I arbeidet sitt har Lise Nordgård gjennomført ulike foringsforsøk hvor rotter har blitt foret med transgene genkonstrukter. I studiene ble eventuelt opptak av DNA via tarmkanalen og transport til ulike organer ble undersøkt, herunder blodbane, lever, lymfeknuter milt, etc. undersøkt. Fragmenter av de transgene konstruktene har blitt gjenfunnet i flere indre organer som milt lever, lymfevev og bukspyttkjertel hos unge, aktivt voksende rotter. I et annet forsøk har hun sett på bakteriens evne i tarmsystemet til å ta opp genkonstrukter ved en opptaksmekanisme kalt naturlig transformasjon. Antibiotikaresistensgener brukes ofte som seleksjonsmarkører ved genmodifisering og markørgenene finnes derfor i mange GMOer på det internasjonale markedet. Det har derfor vært reist spørsmål om bakterier i tarmen hos levende organismer kan ta opp disse genene og på den måten bli resistente mot antibiotika. I innhold fra mage, tynntarm, cecum og tykktarm hos rotter foret med transgene genkonstrukter har ulike størrelser fragmenter av de transgene konstruktene blitt gjenfunnet. Ulike modellsystemer med sterile rotter ble etablert for å kunne studere kontrollert opptak av fremmed DNA hos bakterier i tarmsystemet. I disse studiene viste det seg at innholdet fra de ulike tarmdelene virket hemmende eller hindret eksponering av DNA fragmenter til Acinetobacter baylyi, som er en kjent og ofte brukt modell bakterie for naturlig transformasjon. Deler av dette studiet ble utført i samarbeid med Karolinska Instituttet, Stockholm, Sverige. I tillegg ble flere kjente humane tarmbakteriers evne til å ta opp nakent DNA inn i cellen via naturlig transformasjon undersøkt

Lack of detectable DNA uptake by bacterial gut isolates grown

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

Reproducibility and relative validity of a newly developed web-based food-frequency questionnaire for assessment of preconception diet

publishedVersio