Electrochemical detection of bacterial biofilms on titanium

Statement of Purpose: Periprosthetic joint infection (PJI) is a devastating complication of total joint arthroplasty. In addition to being extremely difficult to treat with antibiotics alone, PJI is also difficult to detect [1]. These problems stem from the ability of bacteria to form biofilms on the surface of implanted materials. When in the biofilm state, bacteria gain a diffusional barrier which limits the penetration of antibiotics, and causes a reduction in the metabolic activity of the resident cells. This can make detecting biofilms with traditional culture methods difficult. For this reason, new methods for the detection of biofilms on implanted orthopedic materials are needed. This work evaluated the electrochemical methods of potentiometry and electrochemical impedance spectroscopy (EIS) as diagnostic measures of bacterial biofilm formation on titanium in an in vitro model. Methods: A custom designed polycarbonate electrochemical biofilm reactor (Fig 1) was utilized to both grow biofilms and make the electrochemical measurements. Figure 1: Experimental setup of the biofilm reactor Samples were made from grade 4 commercially pure titanium (Ti). A clinical isolate of Acinetobacter baumannii (strain Ab307), a problematic Gram-negative human pathogen, was used in all experiments. Biofilms were grown by introducing a 30 mL inoculum with a concentration of ~1× 10⁷ CFU/mL to the reactor. After two hours, flow in the system was initiated at 0.02 mL/s. The reactor also functioned as a three electrode electrochemical cell utilizing the Ti as a working electrode, an Ag/AgCl reference electrode, and a graphite counter electrode. The open circuit potential (OCP) of the Ti was measured prior to inoculation of the reactor, and 24 and 48 hours after inoculation. EIS was performed using a Gamry Interface 1000 potentiostat with a 10 mV sinusoidal voltage about the OCP in the frequency range of 100 kHz-5 mHz at the same timepoints as the OCP measurements. The results of the EIS spectra were fit to a modified Randles circuit, and the polarization resistance (Rp), constant phase element (CPE) magnitude (Y0), and CPE exponent (α) were calculated. Following EIS, the Ti coupons were extracted, rinsed in phosphate buffered saline, sonicated in 0.1% saponin, and dilution plated for enumeration of CFU. Scanning electron microscopy (SEM) of the extracted Ti was also performed for qualitative assessment of bacteria on the Ti surface. Control experiments were carried out with the same experimental conditions without bacterial inoculation. A total of 9 samples used for both the biofilm and the control experiments, and a T-test was used to compare the electrochemical parameters at each time point. Results: Biofilm formation on Ti resulted in a significant cathodic shift in the OCP after 24 hours as compared to the control samples. The Rp was found to increase significantly after 24 hours of biofilm growth (Table 1). CFU enumeration and SEM imaging (Fig 2) confirm the presence of biofilms with an average of 1.1 ×10⁷ CFU/mL. Conclusions: Biofilms grown on Ti for 48 hours resulted in a statistically significant cathodic shift in the OCP of the metal at 24 and 48 hours compared to control samples. The Rp was also found to increase significantly compared to control samples after 24 hour of biofilm growth. These results show the effect of biofilm formation on the electrochemical properties of Ti. Further work will be conducted to track these electrochemical parameters as a function of biofilm formation and growth.