Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes

Abstract

Tetracyclines are broad-spectrum antibiotics used to prevent or treat a variety of bacterial infections. Resistance is often mediated through mobile resistance genes, which encode one of the three main mechanisms: active efflux, ribosomal target protection or enzymatic degradation. In the last few decades, a large number of new tetracycline-resistance genes have been discovered in clinical settings. These genes are hypothesized to originate from environmental and commensal bacteria, but the diversity of tetracycline-resistance determinants that have not yet been mobilized into pathogens is unknown. In this study, we aimed to characterize the potential tetracycline resistome by screening genomic and metagenomic data for novel resistance genes. By using probabilistic models, we predicted 1254 unique putative tetracycline resistance genes, representing 195 gene families (<70 % amino acid sequence identity), whereof 164 families had not been described previously. Out of 17 predicted genes selected for experimental verification, 7 induced a resistance phenotype in an Escherichia coli host. Several of the predicted genes were located on mobile genetic elements or in regions that indicated mobility, suggesting that they easily can be shared between bacteria. Furthermore, phylogenetic analysis indicated several events of horizontal gene transfer between bacterial phyla. Our results also suggested that acquired efflux pumps originate from proteobacterial species, while ribosomal protection genes have been mobilized from Firmicutes and Actinobacteria. This study significantly expands the knowledge of known and putatively novel tetracycline resistance genes, their mobility and evolutionary history. The study also provides insights into the unknown resistome and genes that may be encountered in clinical settings in the future.

Keywords: antibiotic resistance; hidden Markov model; metagenomics; microbiome; resistome; tetracycline resistance.

Fig. 1.

Phylum analysis of genomes carrying tetracycline resistance genes. The analysis was based on genomes present in NCBI RefSeq and HMP genomic databases. The significance of the ratios was assessed using Fisher’s exact test and results with P <0.001 are marked by asterisks. RPG, Ribosomal protection genes.

Fig. 2.

Number of reconstructed genes Gb⁻¹ for each metagenomic dataset. A reconstructed gene was classified as novel if it had a sequence identity of <70 % to any previously known tetracycline resistance gene. RPG, Ribosomal protection genes.

Fig. 3.

Phylogenetic tree of the enzymatic tetracycline-resistance genes predicted in this study. The tree was recreated from all previously known enzymatic tetracycline-resistance gene families, together with gene families predicted in this study. Each gene family contains genes with >70 % amino acid sequence identity, and the number of unique genes in each gene family is presented within the square brackets. The gene functional in E. coli is indicated by an asterisk and the gene families for which the tested genes did not function in E. coli are indicated by hash signs. The scale bar indicates number of substitutions per site.

Fig. 4.

Phylogenetic tree of the ribosomal protection genes predicted in this study. The tree was recreated from all previously known ribosomal protection gene families, together with gene families predicted in this study. Each gene family contains genes with >70 % amino acid sequence identity, and the number of unique genes in each family is presented within the square brackets. The gene families functional in E. coli are indicated by asterisks and the gene families for which the tested genes did not function in E. coli are indicated by hash signs. The scale bar indicates number of substitutions per site.

Fig. 5.

Phylogenetic tree of the efflux pump genes predicted in this study. The tree describes the previously known efflux gene families of MFS group 1, together with gene families predicted in this study. Each gene family contains genes with >70 % amino acid sequence identity, and the number of unique genes in each group is presented within the square brackets. The gene families functional in E. coli are indicated by asterisks and the gene families for which the tested genes did not function in E. coli are indicated by hash signs. The scale bar indicates number of substitutions per site.