Nanopore Sequencing of Transmissible Tetracyline Plasmids Captured without Cultivation from Stream Sediment Reveals Linked Genes Encoding Resistance to Multiple Human Clinical Antibiotics

Background. Transmissible plasmids in environmental ecosystems may facilitate the exchange of antibiotic resistance genes between introduced fecal and native bacterial populations. Bacteria in aquatic and soil habitats may thus act as incubators and sites for recombination for genes subsequently transferred to human pathogens.

Methods. Transmissible plasmids were captured “exogenously” from uncultivated stream bacteria by conjugation with E. coli. After electroporation, plasmids were tested for decreased antibiotic susceptibility using a modified Stokes disk diffusion method. In addition to tetracycline, antibiotics tested were chosen based upon their use in human clinical settings. Two multiresistance plasmids were sequenced using an Oxford Nanopore MinION DNA sequencer, assembled using the PBcR pipeline of the Celera assembler, and annotated using Prokka. Both plasmids were also sequenced on an Ion Torrent Personal Genome Machine.

Results. Twenty-three of 30 plasmids conferred decreased susceptibility to multiple antibiotics in addition to tetracycline. The most common phenotypic resistance profiles were tetR/kanamycinR/ticarcillinR/piperacillinR, and tetR/kanR/ticR/pipR/cefipimeR. One plasmid conferred decreased susceptibility to seven of 12 tested antibiotics (tet, kan, tic, tzp, pip, fep, and tobramycin). Nanopore sequencing and assembly of this plasmid resulted in two ca. 15-kb contigs. Contigs were screened for resistance genes against the ResFinder database. The following genes were identified with >93% identify: sul1 (2X) – suggesting the presence of one or more Class 1 integrons – tetC, tetG, aadA9, aadA2, sfloR, aph(3')-Ic, strB, and blaCARB-2. A second plasmid exhibiting decreased susceptibility to tet, kan, tic, pip, and ciprofloxacin was also sequenced.

Conclusions. The presence of genes encoding resistance to multiple human clinical antibiotics on transmissible plasmids selected using tetracycline suggests that there may be a significant reservoir of such genes in stream sediments, that they can be selected by tetracycline use, and that they may be capable of transmission to pathogenic Enterobacteriaceae.