Antibiotics Enhance Prevention and Eradication Efficacy of Cathodic-Voltage-Controlled Electrical Stimulation against Titanium-Associated Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa Biofilms

Abstract

Periprosthetic joint infection (PJI) develops clinically, even with antibiotic treatment, and methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are predominant causes of these infections. Due to biofilm formation, antibiotic treatment for patients with PJI can perpetuate resistance, further complicating the use of noninvasive treatments. This study evaluated cathodic-voltage-controlled electrical stimulation (CVCES) of titanium, in combination with a clinically relevant antibiotic, to synergistically prevent MRSA and P. aeruginosa PJIs by inhibiting bacterial adherence or as a treatment for eradicating established biofilms. CVCES of -1.0 V, -1.5 V, or -1.8 V (versus Ag/AgCl), with or without vancomycin for MRSA or gentamicin for P. aeruginosa, was applied to sterile titanium incubated with cultures to evaluate prevention of attachment or eradication of preestablished biofilms. Treatments were 24 h long and included open-circuit potential controls, antibiotic alone, CVCES, and CVCES plus antibiotic. Biofilm-associated and planktonic CFU were enumerated. In general, CVCES at -1.8 V alone or with antibiotic completely eradicated biofilm-associated CFU for both strains, and these parameters were also highly effective against planktonic bacteria, resulting in a >6-log reduction in MRSA and no detectable planktonic P. aeruginosa All CFU were reduced ∼3 to 5 logs from controls for prevention CVCES plus antibiotics at -1.0 V and -1.5 V against MRSA. Remarkably, there were no detectable P. aeruginosa CFU following prevention CVCES at -1.0 V or -1.5 V with gentamicin. Our results suggest that CVCES in combination with antibiotics may be an effective approach for prevention and treatment of PJI.IMPORTANCE Periprosthetic joint infections (PJIs) develop clinically in the presence of antibiotic therapies and are responsible for increased patient morbidity and rising health care costs. Many of these infections involve bacterial biofilm formation on orthopedic hardware, and it has been well established that these biofilms are refractory to most antibiotic treatments. Recent studies have focused on novel methods to prevent and eradicate infection. Cathodic-voltage-controlled electrical stimulation (CVCES) has previously been shown to be effective as a method for prevention and eradication of Gram-positive and Gram-negative infections. The present study revealed that the utility of CVCES for prevention and eradication of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa is enhanced in the presence of clinically relevant antibiotics. The synergistic effects of CVCES and antibiotics are effective in a magnitude-dependent manner. The results of this study indicate a promising alternative method to current PJI mitigation techniques.

Keywords: bacteria; biofilms; orthopedic infection; prevention; treatment.

FIG 1

Eradication of established MRSA strain NRS70 biofilms on cpTi after 24 h of CVCES in the presence or absence of vancomycin. Experiments were conducted at −1.0 V (A), −1.5 V (B), and −1.8 V (C). The conditions reported are for incubations with no stimulation (control), vancomycin alone (drug), CVCES alone (−1.0 V, −1.5 V, or −1.8 V), or CVCES with vancomycin (−1.0 V, −1.5 V, or −1.8 V plus drug). ø indicates that no detectable CFU were enumerated. Bar plots show mean values ± 1 standard deviation calculated from the four independent samples under each condition. * indicates statistically significant differences between the groups as specified. The limit of detection is denoted with a dashed line.

FIG 2

Eradication of established P. aeruginosa strain PA27853 biofilms on cpTi after 24 h of CVCES in the presence or absence of gentamicin. Experiments were conducted with CVCES at −1.0 V (A), −1.5 V (B), and −1.8 V (C). The conditions reported are for incubations with no stimulation (control), gentamicin alone (drug), CVCES alone (−1.0 V, −1.5 V, or −1.8V), or CVCES with gentamicin (−1.0 V, −1.5 V, or −1.8 V plus drug). ø indicates that no detectable CFU were enumerated under the test condition. Bar plots show mean values ± 1 standard deviation calculated from the four independent samples under each condition. * indicates a statistically significant differences between the groups as specified. The limit of detection is denoted with a dashed line.

FIG 3

Prevention of MRSA strain NRS70 growth and biofilm formation on cpTi after 24 h of CVCES in the presence or absence of vancomycin. Experiments were conducted with CVCES at −1.0 V (A) or −1.5 V (B). The conditions reported are for incubations with no stimulation (control), vancomycin alone (drug), CVCES alone (−1.0 V or −1.5 V), or CVCES with vancomycin (−1.0 V or −1.5 V plus drug). ø indicates that no detectable CFU were enumerated under the test condition. Bar plots show mean values ±1 standard deviation calculated from the four independent samples under each condition. * indicates statistically significant differences between the groups as specified. The limit of detection is denoted with a dashed line.

FIG 4

Prevention of P. aeruginosa strain PA27853 growth and biofilm formation on cpTi after 24 h of CVCES in the presence or absence of gentamicin. Experiments were conducted with CVCES at −1.0 V (A) or −1.5 V (B). The conditions reported are for incubations with no stimulation (control), gentamicin alone (drug), CVCES alone (−1.0 V or −1.5 V), or CVCES with gentamicin (−1.0 V or −1.5 V plus drug). ø indicates that no detectable CFU were enumerated under the test condition. Bar plots show mean values ± 1 standard deviation calculated from the four independent samples under each condition. * indicates statistically significant differences between the groups as specified. The limit of detection is denoted with a dashed line.

FIG 5

Survival of MRSA strain NRS70 and P. aeruginosa strain PA27853 at alkaline pH after 24 h of exposure. Shown are CFU for NRS70 biofilm (A) and planktonic cultures (B) and PA27853 biofilm (C) and planktonic cultures (D) following exposure to medium adjusted to a pH ranging from 7 to 12 for 24 h. ø indicates that no detectable CFU were enumerated under the test condition. A minimum of 3 independent replicates were performed under each condition. The limit of detection is denoted with a dashed line. * indicates statistically significant differences between the groups specified and the pH 7 condition.

FIG 6

Schematic representing the custom-designed three-electrode potentiostatic electrochemical stimulation chamber. An eradication CVCES setup is depicted with a preformed biofilm (green) on the cpTi working electrode. (Reprinted from reference with permission.)