Enhanced Killing of Biliary Bacterial Biofilm Using Electroporation: An In Vitro Evaluation

Abstract

Background: Bacterial biofilm is a known cause of chronic device related infections requiring removal of implanted device such as a biliary stent. Biofilm bacteria are resistant to antibiotic therapy requiring 100 to 1000 times minimum inhibitory concentration (MIC) for complete eradication. Electroporation (EP) - a pulsed electrical field induces cell membrane permeability and enhances penetration of drugs into bacterial cells. Aim: To determine effects of EP and antibiotics therapy in eradication of biofilm bacteria. Method: Mixed biofilm were formed on micro TFA filters (Applied Biosystems, CA) by incubation with E. coli (E) and Enterococcus (En) in ox bile for 72 hours. The established biofilm was tested against Ciprofloxacin at 100xMIC for E. coli (Cipro) or 4% tetra sodium EDTA (TE) with or without EP. The filters were inserted into cuvettes and electroporated (ECM 630, Harvard Apparatus, MA) with constant voltage, capacitance, pulse duration, frequency and number of pulses for 1 minute. The experiments were divided into 2 groups with 10 or 15 EP pulses and exposure to individual antimicrobials for 4 hours. Experiments included: 1. Control; 2. 10 pulses of EP at 1500 volts; 3. 4% TE; 4. Cipro at 100xMIC; 5. EP plus Cipro; 6. EP plus TE. Experiments were repeated using 15 EP pulses. The filters were rinsed and then sonicated in 5 ml sterile PBS to release the bacteria. Bacterial counts in colony forming units (CFU/cm2) were determined by serial dilution and viable plate count method. The experiments were repeated and the mean bacterial count (n = 2) was calculated and expressed as mean (log10 CFU/cm2) ± SD. Results: Mean control bacterial counts were (E 5.6-5.8, En 4.8-4.4). Compared with controls, EP at 10 pulses or Cipro alone produced a 100-fold (2 log) reduction in E. coli but no significant effects on Enterococcus; 2. EP plus Cipro produced a 100-fold decrease in E. coli and 10-fold decrease in Enterococcus. 3. TE alone killed all E. coli but not Enterococcus; 4. EP plus 4% TE produced a 100-fold decrease in Enterococcus. With 15 EP pulses, all E. coli were killed by Cipro. A combination of TE and 15 EP pulses killed ALL bacteria in the biofilm. The differences were statistically significant (Box plot, p < 0.001). Conclusion: Electroporation enhanced the killing effects of antibiotics/antimicrobials against established biofilm bacteria and may offer an alternative to prevent biliary biofilm and stent blockage. Background: Bacterial biofilm is a known cause of chronic device related infections requiring removal of implanted device such as a biliary stent. Biofilm bacteria are resistant to antibiotic therapy requiring 100 to 1000 times minimum inhibitory concentration (MIC) for complete eradication. Electroporation (EP) - a pulsed electrical field induces cell membrane permeability and enhances penetration of drugs into bacterial cells. Aim: To determine effects of EP and antibiotics therapy in eradication of biofilm bacteria. Method: Mixed biofilm were formed on micro TFA filters (Applied Biosystems, CA) by incubation with E. coli (E) and Enterococcus (En) in ox bile for 72 hours. The established biofilm was tested against Ciprofloxacin at 100xMIC for E. coli (Cipro) or 4% tetra sodium EDTA (TE) with or without EP. The filters were inserted into cuvettes and electroporated (ECM 630, Harvard Apparatus, MA) with constant voltage, capacitance, pulse duration, frequency and number of pulses for 1 minute. The experiments were divided into 2 groups with 10 or 15 EP pulses and exposure to individual antimicrobials for 4 hours. Experiments included: 1. Control; 2. 10 pulses of EP at 1500 volts; 3. 4% TE; 4. Cipro at 100xMIC; 5. EP plus Cipro; 6. EP plus TE. Experiments were repeated using 15 EP pulses. The filters were rinsed and then sonicated in 5 ml sterile PBS to release the bacteria. Bacterial counts in colony forming units (CFU/cm2) were determined by serial dilution and viable plate count method. The experiments were repeated and the mean bacterial count (n = 2) was calculated and expressed as mean (log10 CFU/cm2) ± SD. Results: Mean control bacterial counts were (E 5.6-5.8, En 4.8-4.4). Compared with controls, EP at 10 pulses or Cipro alone produced a 100-fold (2 log) reduction in E. coli but no significant effects on Enterococcus; 2. EP plus Cipro produced a 100-fold decrease in E. coli and 10-fold decrease in Enterococcus. 3. TE alone killed all E. coli but not Enterococcus; 4. EP plus 4% TE produced a 100-fold decrease in Enterococcus. With 15 EP pulses, all E. coli were killed by Cipro. A combination of TE and 15 EP pulses killed ALL bacteria in the biofilm. The differences were statistically significant (Box plot, p < 0.001). Conclusion: Electroporation enhanced the killing effects of antibiotics/antimicrobials against established biofilm bacteria and may offer an alternative to prevent biliary biofilm and stent blockage.