Integron Gene Cassettes Harboring Novel Variants of D-Alanine-D-Alanine Ligase Confer High-level Resistance to D-Cycloserine

During a PCR-based screen of antibiotic resistance genes (ARGs) associated with integrons in saliva-derived metagenomic DNA of healthy human volunteers, two novel variants of genes encoding a D-alanine-D-alanine ligase (ddl6 and ddl7) located within gene cassettes in the first position of a reverse integron were identified. Treponema denticola was identified as the likely host of the ddl cassettes. Both ddl6 and ddl7 conferred high level resistance to D-cycloserine when expressed in Escherichia coli with ddl7 conferring four-fold higher resistance to D-cycloserine compared to ddl6. A SNP was found to be responsible for this difference in resistance phenotype between both ddl variants. Molecular dynamics simulations were used to explain the mechanism of this phenotypic change at the atomic scale. A hypothesis for the evolutionary selection of ddl containing integron gene cassettes is proposed, based on molecular docking of plant metabolites within the ATP and D-cycloserine binding pockets of Ddl. Significance StatementThe drivers of antimicrobial resistance are varied and numerous. One such hypothesis is that compounds not normally considered to be antibiotics may be able to select for genes that can provide resistance to conventional antibiotics. Here we provide evidence that dietary flavonoids are likely to provide the selective pressure for two novel variants of the ddl gene, encoding D-alanine-D-alanine ligases, to be maintained as the first gene cassette of a reverse integron detectable in the human oral cavities from both the UK and Bangladesh. We show that Ddl is functional, able to confer resistance to D-cycloserine and that the dietary flavonoids quercetin and apigenin are likely able to compete with both ATP and D-cycloserine within their Ddl binding sites.