(279g) Electrochemical Detection of Bacterial Biofilms on Titanium

Electrochemical Detection of Bacterial
Biofilms on Titanium

Caelen M. Clark and Mark T. Ehrensberger

State University of New York at Buffalo, Buffalo NY,
USA

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.

Table 1: Electrochemical measurement
results

 Figure
2:
SEM micrograph of Ab307 biofilm

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.

References: [1] Sendi P, Zimmerli W. International
Journal of Artificial Organs. 2012;35:913-22.