What Are the Effects of Irreversible Electroporation on a Staphylococcus aureus Rabbit Model of Osteomyelitis?
Authors: Muñoz Nina M. , Minhaj Adeeb A. , Dupuis Crystal J. , Ensor Joe E. , Golardi Natalia , Jaso Jesse M. , Dixon Katherine A. , Figueira Tomas Appleton , Galloway-Peña Jessica R. , Hill Lori , Shelburne Samuel A. , Tam Alda L. 
Published: August 2019

Abstract

Background
The treatment of osteomyelitis can be challenging because of poor antibiotic penetration into the infected bone and toxicities associated with prolonged antibiotic regimens to control infection. Irreversible electroporation (IRE), a percutaneous image-guided ablation technology in which the targeted delivery of high-voltage electrical pulses permanently damages the cell membrane, has been shown to effectively control bacterial growth in various settings. However, IRE for the management of bone infections has yet to be evaluated.

Questions/purposes
We aimed to evaluate IRE for treating osteomyelitis by assessing (1) the efficacy of IRE to suppress the in vitro growth of a clinical isolate of S. aureus, alone or combined with cefazolin; and (2) the effects of IRE on the in vivo treatment of a rabbit model of osteomyelitis.

Methods

S. aureus strain UAMS-1 expanded in vitro to the log phase was subjected to an electric field of 2700 V/cm, which was delivered in increasing numbers of pulses. Immediately after electroporation, bacteria were plated on agar plates with or without cefazolin. The number of colony-forming units (CFUs) was scored the following day. ANOVA tests were used to analyze in vitro data. In a rabbit osteomyelitis model, we inoculated the same bacterial strain into the radius of adult male New Zealand White rabbits. Three weeks after inoculation, all animals (n = 32) underwent irrigation and débridement, as well as wound culture of the infected forelimb. Then, they were randomly assigned to one of four treatment groups (n = eight per group): untreated control, cefazolin only, IRE only, or combined IRE + cefazolin. Serial radiography was performed to assess disease progression using a semiquantitative grading scale. Bone and soft-tissue specimens from the infected and contralateral forelimbs were collected at 4 weeks after treatment for bacterial isolation and histologic assessment using a semiquantitative scale.

Results
The in vitro growth of S. aureus UAMS-1 was impaired by IRE in a pulse-dependent fashion; the number of CFUs/mL was different among seven pulse levels, namely 0, 10, 30, 60, 90, 120, and 150 pulses. With the number of CFUs/mL observed in untreated controls set as 100%, 10 pulses rendered a median of 50.2% (range 47.1% to 58.2%), 30 pulses rendered a median of 2.7% (range 2.5% to 2.8%), 60 pulses rendered a median of 0.014% (range 0.012% to 0.015%), 90 pulses rendered a median of 0.004% (range 0.002% to 0.004%), 120 pulses rendered a median of 0.001% (range 0.001% to 0.001%), and 150 pulses rendered a median of 0.001% (range 0.000% to 0.001%) (Kruskal-Wallis test: p = 0.003). There was an interaction between the effect of the number of pulses and the concentration of cefazolin (two-way ANOVA: F [8, 30] = 17.24; p < 0.001), indicating that combining IRE with cefazolin is more effective than either treatment alone at suppressing the growth of S. aureus UAMS-1. Likewise, the clinical response in the rabbit model (the percentage of animals without detectable residual bacteria in the bone and surrounding soft tissue after treatment) was better in the combination group than in the other groups: control, 12.5% (one of eight animals); IRE only, 12.5% (one of eight animals); cefazolin only, 25% (two of eight animals); and IRE + cefazolin, 75% (six of eight animals) (two-sided Fisher’s exact test: p = 0.030).

Conclusions
IRE effectively suppressed the growth of S. aureus UAMS-1 and enhanced the antibacterial effect of cefazolin in in vitro studies. When translated to a rabbit osteomyelitis model, the addition of IRE to conventional parenteral antibiotic treatment produced the strongest response, which supports the in vitro findings.

Clinical Relevance
Our results show that IRE may improve the results of standard parenteral antibiotic treatment, thus setting the stage for models with larger animals and perhaps trials in humans for validation.