Abstract
BackgroundHorizontal 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.ResultsPlasmid DNA with two resistance genes (nptI 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, were 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. No transformants were identified among 441 tested isolates.ConclusionsThe analyses showed that extensive ingestion of DNA (100 μg 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 × 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.
Highlights
Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described
To address the question whether the plasmids are sufficient for natural transformation studies of phylogenetically distant bacteria, natural transformation assays were performed with pM1, pM2 and pM3 using ~30 different marine isolates as potential recipients
These studies revealed the ability of different Gammaproteobacteria and Actinobacteria to be naturally transformed with the three DNA constructs, even in the presence of significant differences in 16S rRNA identity (Table 1)
Summary
Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Few studies have examined the occurrence of horizontal gene transfer (HGT) by natural transformation in the gastrointestinal tract (GIT) of mammals under in vivo conditions [1,2,3,4,5]. It is unclear if the lack of observable competence among bacteria in the GIT is due to experimental limitations, limited occurrence in the few model bacteria examined, or due to true lack of conditions conductive for the development of competence among members of the GIT microbiota of various mammals. The lower part of the GIT has been suggested to be the most active site for HGT processes due to abundance of nutrients, high bacterial density and slower degradation rates of DNA [11,12]
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