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Liam J Reynolds, Adam P Roberts and Muna F Anjum
Frontiers in Microbiology
A human saliva metagenomic library was created using E. coli as a host. A total of 27,000 clones from this library were screened for tetracycline resistance by culturing the clones on agar containing inhibitory concentrations of tetracycline. A tetracycline resistant clone was characterised genetically and phenotypically using subcloning and mutagenesis techniques.
One of the tetracycline resistant clones identified also exhibited resistance to tigecycline but to none of the other antibiotic classes tested. The clone was found to contain a 7,765 bp insert. Sequencing revealed the insert to encode five genes including two novel half-ABC (ATP binding cassette) transporter genes. Subcloning of both of these genes was required for the resistance phenotype. Subsequent mutagenesis studies showed that deletions within the ATP binding domain of either gene resulted in a loss of tetracycline and tigecycline resistance providing evidence that the genes encoded a functional ABC transporter that hydrolysed ATP to pump these antibiotics from the cell. As these genes conferred resistance only to tetracycline antibiotics they were designated tetAB(60).
Further work is required to determine how prevalent and abundant these genes are in the human saliva microbiome and other environments. It would also be of interest to determine if tetAB(60) are associated with mobile genetic elements which would aid in the dissemination of these resistance genes. Probing the human saliva metagenomic DNA using a tetAB(60) hybridising DNA probe could be used in this respect. It would potentially allow for the isolation of larger fragments of DNA containing these genes which would provide us with greater genomic context.
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