Dissemination of antibiotic resistance genes from antibiotic producers to pathogens

Abstract

It has been hypothesized that some antibiotic resistance genes (ARGs) found in pathogenic bacteria derive from antibiotic-producing actinobacteria. Here we provide bioinformatic and experimental evidence supporting this hypothesis. We identify genes in proteobacteria, including some pathogens, that appear to be closely related to actinobacterial ARGs known to confer resistance against clinically important antibiotics. Furthermore, we identify two potential examples of recent horizontal transfer of actinobacterial ARGs to proteobacterial pathogens. Based on this bioinformatic evidence, we propose and experimentally test a 'carry-back' mechanism for the transfer, involving conjugative transfer of a carrier sequence from proteobacteria to actinobacteria, recombination of the carrier sequence with the actinobacterial ARG, followed by natural transformation of proteobacteria with the carrier-sandwiched ARG. Our results support the existence of ancient and, possibly, recent transfers of ARGs from antibiotic-producing actinobacteria to proteobacteria, and provide evidence for a defined mechanism.

Figure 1. Proteobacterial proteins related to Streptomyces ARG proteins.

Relationships between all experimentally validated Streptomyces ARG proteins and their most similar homologues in proteobacteria were analysed by phylogenetic analysis (Supplementary Fig. 1). Selected pairs that might be connected via interphylum HGT are summarized here, with the proposed transfer direction indicated by an arrow. Protein sequence identity was determined by BLASTP. ^aThe proteins were BLASTed against pathogen genome database PATRIC to see if they have close homologues (sequence identity threshold of 90%) in pathogens.

Figure 2. A tanglegram between the phylogeny of the genus representative hits of cmx and the host phylogeny of their corresponding 16S rRNA sequences.

The red labels correspond to Gram-positive actinobacteria, while the blue labels correspond to Gram-negative proteobacteria. Genomes were selected if they have BLASTP hits against the Cmx protein (WP_005297378.1). The best hit per genus is taken as the representative hit. Hits with over 99% identity to WP_005297378.1 are framed. These cmx genes are located in transposons together with a transposase gene tnp45. Triangles represent collapsed branches. The full trees are provided in Supplementary Fig. 4.

Figure 3. Transfer of a resistance gene from actinobacteria to proteobacteria by the ‘carry-back' model.

Actinobacterial and proteobacterial cells are marked in red and blue, respectively. DNA sequences of actinobacterial and proteobacterial origins are likewise labelled in red and blue. Dashed line represents DNA that can be either a fragment of a chromosome or a plasmid. The terminal inverted repeats of cmx transposon are represented by red bars. (a) The proposed ‘carry-back' interphylum gene transfer mechanism. (b) Examples of actual sequences found in different bacteria that resemble the proposed intermediates: (1) carrier sequence in proteobacteria; (2) carrier sequence in actinobacteria; (3) sandwich structure with cmx transposon flanked by carrier sequences in actinobacteria; (4) cmx transposon incorporated into genome in proteobacteria. Double-slash indicates additional resistance genes and mobile elements inserted into the proposed intermediate structures. NCBI accession numbers of these intermediate sequences are provided in Supplementary Data 4. (c) Experimental reconstruction of the interphylum gene transfer of cmx from actinobacteria to proteobacteria. C. resistens DSM 45100 naturally carries the sandwich structure. Plasmid pXJ83 is constructed by cloning the carrier sequence from P. aeruginosa BM4530 onto pUCP24 vector. *Kanamycin (kan) instead of chloramphenicol was used for the selection plates. Kanamycin resistance is coded by Tn5393e inserted in the cmx transposon. (d) Efficiency of natural transformation using A. baylyi ADP1/pXJ83 as recipient strain (error bar: s.d.; n=3). A. baylyi ADP1/pUCP24 was used as a negative control. The detection limit is one transformant per 10⁹  colony-forming unit (c.f.u.) of recipient cells. ND, not detected. (e) Colony PCR of transformants using primers that target cmx. C. resistens DSM 45100 was used as a positive control. A. baylyi ADP1/pXJ83 was used as a negative control.