Assessing the Incidence of Plasmid-borne Resistance to Clinically-Significant Antibiotics in Stream Sediments

Plasmids in agriculturally-impacted bodies of water may play a significant role in the dissemination of antibiotic resistance (AR). High bacterial loads in stream sediment and selective pressures introduced by agricultural practices may facilitate the exchange and recombination of genetic material, creating reservoirs of AR genes that can potentially be accessed by fecal and other animal and human pathogens.

  Transmissible plasmids were captured “exogenously” from stream sediment samples by conjugating sediment cells with a rifampicin-resistant strain of Escherichia coli. Transconjugants were tested for decreased antibiotic susceptibility using a modified Stokes disk diffusion method.  Twenty-three of thirty captured plasmids conferred decreased susceptibility to multiple antibiotics in addition to tetracycline.

  One plasmid, pEG1-1, conferred resistance to tetracycline, tobramycin, kanamycin, ticarcilin, piperacillin, piperacillin-tazobactam, and cefepime. A method to sequence multi-drug resistance plasmids using both Oxford Nanopore MinION and Ion Torrent Personal Genome Machine sequencers was developed to sequence plasmid pEG1-1. A hybrid assembly generated a single 73,320 bp contig. Analysis of the genome revealed pEG1-1 to be an IncP-1β plasmid with two mobile genetic elements – a tn21-related transposon and an in104 complex integron – both of which carry multiple antibiotic resistance genes.

  These findings suggest that plasmids in stream sediment are prone to the incorporation of mobile genetic elements that introduce a broad range of antibiotic resistance genes into their genome. This could cause serious risk to human health since IncP-1β plasmids are capable of transferring into nearly all Gram-negative bacteria, including fecal pathogens that get introduced to stream sediment.