Plasmid Elimination from Klebsiella pneumoniae using Anti-plasmid Compounds

Multidrug resistant Klebsiella pneumoniae are responsible for a variety of infections that include pneumonia, bloodstream, wound and surgical infections. Plasmids containing antibiotic resistance genes (R-plasmid), which are mostly transferable, are responsible for enabling bacteria to become resistant to a large number of antibiotics. Therefore, a successful elimination of R-plasmids from pathogenic bacteria can reveal more about the biology of the plasmid. The hypothesis of this study was: elimination of R-plasmid from K. pneumoniae (cured strain) increases susceptibility of bacteria to antibiotics. The strains used in this study were: K. pneumoniae SW2957R carrying two plasmids (191 kb and 157 kb), Escherichia coli SW2957R-MDR+AZI carrying K. pneumoniae R-plasmid (191 kb), and E. coli J53 (negative control). A plasmid curing experiment was done by exposing the cells with sub-minimum inhibitory concentration (sub-MIC) of acridine orange, amitriptyline, and thioridazine. Plasmid cured strains were screened using replica plating on TSA only, TSA supplemented with trimethoprim (20 mg/L) or tetracycline (30 mg/L). Plasmid isolation, antibiotic disk diffusion assay, and gel electrophoresis were performed on SW2957R, SW2957R-MDR+AZI, plasmid cured strains, and J53. A 191 kb R-plasmid was eliminated from SW2957R and SW2957R-MDR+AZI isolates after treatment with 1/2 MIC of acridine orange, 100 mg/L and 50 mg/L, respectively. The percentage of plasmid curing efficiency of acridine orange was 2% and 4% for SW2957R and SW2957R-MDR+AZI, respectively. Amitriptyline and thioridazine did not eliminate the plasmids on either strain. K. pneumoniae SW2957R was resistant to seven antibiotic classes: 1. Beta-lactams (ampicillin, cefotaxime), 2. Fluoroquinolone (ciprofloxacin), 3. Aminoglycosides (kanamycin, neomycin, gentamicin streptomycin, tobramycin), 4. Tetracyclines (tetracycline, oxytetracycline, doxycycline, minocycline), 5. Sulfonamide (sulfisoxazole), 6. Chloramphenicol, and 7. Trimethoprim. Plasmid cured SW2957R (without 191 kb R-plasmid) became susceptible to kanamycin, gentamicin, neomycin, tobramycin, ciprofloxacin, chloramphenicol, minocycline and trimethoprim. Our control, E. coli SW2957R-MDR+AZI carrying a 191 kb K. pneumoniae plasmid, was resistant to 6 antibiotic classes: 1. Beta-lactams (ampicillin, cefotaxime), 2. Aminoglycosides (kanamycin, neomycin, gentamicin, and streptomycin), 3. Tetracyclines (tetracycline, oxytetracycline), 4. Chloramphenicol, 5. Sulfonamide (sulfisoxazole), and 6. Trimethoprim. Based on the antibiotics that this strain became susceptible to, it is expected to find at least six different antibiotic resistance genes in this plasmid. Sequencing of this plasmid would be the next step that would offer more information on the biology of this plasmid.