(279b) Prevention of Select Eskape Pathogens from Attaching to Titanium Using Cathodic Voltage Controlled Electrical Stimulation Combined with Antibiotic Therapy | AIChE

(279b) Prevention of Select Eskape Pathogens from Attaching to Titanium Using Cathodic Voltage Controlled Electrical Stimulation Combined with Antibiotic Therapy

Authors 

Canty, M. - Presenter, University at Buffalo
Luke-Marshall, N., University at Buffalo
Campagnari, A., University at Buffalo
Ehrensberger, M., University at Buffalo
INTRODUCTION: Infection following total joint arthroplasty (TJA) has been associated with longer patient healing times, prolonged hospital stays and increased costs to the healthcare system. ESKAPE pathogens are a concerning class of multidrug resistant bacteria in the medical setting that are contributing to these infections. In a previous study, we revealed that cathodic voltage-controlled electrical stimulation (CVCES) could prevent coupon-associated and planktonic colony forming units (CFU) of Gram-positive methicillin resistant Staphylococcus aureus and Gram-negative Acinetobacter baumannii from reaching detectable levels in a magnitude and time-dependent manner [1]. In this study we evaluated if application of CVCES at -1.0V, -1.5V, or -1.8V vs. Ag/AgCl, in conjunction with prophylactic antibiotics, is an effective method to prevent attachment of Staphylococcus epidermidis (S. epidermidis), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), and Pseudomonas aeruginosa (P. aeruginosa) to the surface of commercially pure titanium (cpTi).

METHODS: cpTi coupons were wet sanded to a 600-grit finish, sonicated in deionized water for 10mins, and sterilized under ultraviolet light for 30mins. Experiments were conducted with blood isolates of Gram-positive S. epidermidis, strain SE94-309-0594, and Gram-negative A. baumannii, strain Ab307-029, K. pneumoniae, strain cKP1, and P. aeruginosa, ATCC(T) strain PA27853. Fresh cultures were inoculated into 5 mL of either tryptic soy broth supplemented with 0.25% glucose (TSBG, for SE94-309-0594) or Mueller-Hinton (MH) broth (Ab307-0294, cKP1, PA27853) at a 10:1 flask to volume ratio, adjusted to an OD600 of 0.1 and placed in a 37°C shaking water bath at 180 rpm. Cultures were grown to mid-log phase, diluted back to an OD600 of 0.1 and then further diluted 1:10,000 in the appropriate fresh media for a starting inoculum of approximately 103 CFU/mL. Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) [18]. The MIC for cefazolin against SE94-309-0594, Ab307-0294, and cKP1 was determined to be 1 μg/mL, 400 μg/mL, and 2 μg/mL, respectively. MIC of gentamicin was tested against PA27853 and found to be 3 μg/mL. Titanium was incubated for 24-hours at open circuit potential (OCP), OCP+drug, CVCES alone, or CVCES+drug. The stimulation chamber uses a standard three-electrode system with cpTi as the working electrode, a graphite counter electrode and an Ag/AgCl reference electrode [2]. With respect to Ag/AgCl, voltages of -1.0V, -1.5V, and/or -1.8V for 24 hours were applied to the working electrode at room temperature using a potentiostat (Interface 1000, Gamry Instruments). After incubation, cpTi coupons were washed in sterile PBS, sonicated and dilution plated for CFU. The media was collected for pH measurement and dilution plated for CFU. Four independent samples were evaluated in all conditions. A one-way ANOVA followed by Tukey post-hoc compared log-transformed CFU across stimulation conditions. If the Levene’s test was significant, a Welch’s test followed by a Games-Howell post-hoc test was performed. Student’s t-tests were used to assess differences between independent stimulation conditions with and without antibiotic treatment.

RESULTS: Remarkably, CVCES of P. aeruginosa at -1.0V+gentamicin and -1.5V+gentamicin showed an 8-log reduction, to below detection, from controls and CVCES alone. -1.0V+cefazolin significantly reduced coupon-associated A. baumannii from control (p<0.001), and -1.5V resulted in no detectable coupon-associated CFU and reduced planktonic CFU to below 101 CFU/mL. Compared to controls -1.8V and -1.8V+cefazolin prevented all K. pneumoniae from reaching detectable levels (p<0.001), but had no effect at -1.5V and -1.5V+cefazolin. -1.0V+cefazolin to S. epidermidis reduced planktonic CFU by 93.2% and resulted in no detectable coupon-associated CFU. -1.5V alone significantly reduced S, epidermidis coupon-associated and planktonic CFU from controls (p<0.005), and prevented all CFU at -1.5V+cefazolin (data not shown). Increasingly cathodic CVCES had a higher average cathodic current density and cumulative charge transfer. CVCES can prevent coupon-associated and planktonic CFU of select ESKAPE pathogens from reaching detectable levels on cpTi, and antibiotics synergistically enhance these effects.

DISCUSSION: Implant associated infection following orthopedic intervention is a devastating clinical complication associated with increased patient morbidity, extended treatment times, and increased costs to the health care system. The purpose of this in vitro study was to evaluate if CVCES of titanium in conjunction with antibiotics could synergistically prevent initial attachment and/or reduce the viability and subsequent bacterial biofilm formation of select ESKAPE pathogens. The outcomes of this study revealed that CVCES could prevent coupon-associated CFU and planktonic CFU of select ESKAPE pathogens from reaching detectable levels in a magnitude-dependent manner. In general, increasingly cathodic potentials had more robust antimicrobial effects and this effect was enhanced in the presence of antibiotic treatment. Bactericidal activity of stimulation corresponded with an alkaline shift in pH, which is thought to be the causative link between stimulation and bacterial cell death. In general, the addition of clinically relevant antibiotics enhanced the efficacy of CVCES to have a more robust preventative effect and these effects were dependent on the magnitude, antibiotic, and species being tested.

SIGNIFICANCE: Current antibiotic prophylactic treatment methods for joint revision surgeries are not effective against all pathogens; however alternative methods, such as CVCES in conjunction with antibiotics, shows promise to be an effective broad-spectrum prevention method for Gram-positive and Gram-negative pathogens.

REFERENCES: [1] Canty M, et al. Acta Biomaterialia: 2017;48:451-460 [2] Ehrensberger MT et al. Biomaterials 2015;41:97-105.