Drug-resistant bacteria, especially those belonging to the Enterobacteriaceae family, have become increasingly problematic in the nosocomial setting. However, a solution may be to exploit bacteria’s ability to produce inhibitory proteins, like bacteriocins, to suppress competitors and synergistically pair these proteins with antibiotics. Our lab has discovered a potentially novel plasmid-mediated antimicrobial protein produced by as specific strain of Klebsiella pneumoniae. To verify the genetic elements of this plasmid necessary to produce the antimicrobial, a gene interruption plasmid library was generated by transposon mutagenesis using the EZ-TN5TM system. These transposon plasmids were then electroporated into competent E. coli. The resulting E. coli were then plated and screened on agar containing kanamycin to ensure successful plasmid uptake and were now able to secrete the antimicrobial protein. The transposon’s unique sequence allowed primer- binding sites, which were used to sequence the plasmid. Four different sequences were analyzed by NCBI BLAST comparisons and matched with high similarity to: 1) a predicted colicin; 2) an uncharacterized Klebsiella protein, 3) a TraM recognition domain containing protein. The K. pneumoniae antimicrobial protein has been shown, when spotted on lawns of Citrobacter freundii, Enterobacter cloacae and Enterobacter aerogenes to inhibit their growth. It has been additionally shown to inhibit the growth of closely related strains including Klebsiella pnuemoniae strain 9997 when spotted on a lawn. When the protein was synergistically paired with subinhibitory levels of common antibiotics, there was an increase in the effectiveness of the antibiotic, it was paired with. The optical density, MTT, and CFUs demonstrate that when the K. pneumoniae protein is paired with Streptomycin or Kanamycin, growth is inhibited greater than the antibiotic alone. These results demonstrate the importance of studying polymicrobial interactions as a means to combat drug resistance and discover novel antimicrobial derived proteins for new therapeutics.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:asrf-2030 |
| Date | 25 April 2023 |
| Creators | Fowler, Donald, Becker, Ethan, Fox, Sean, PhD |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Source | Appalachian Student Research Forum |
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