A Novel Method to Determine the Longitudinal Antibacterial Activity of Drug-Eluting Materials

This protocol involves testing the real-time antibacterial efficacy of antibiotics eluted from a polymeric device, which can aid in accurately determining effective formulations increasing their translational value. This method enables real-time antibacterial efficacy testing capability to monitor the longitudinal activity of drug-eluting materials, and to understand the range of antibacterial activity for a given implant formulation. This polymeric material is used in over 90%of total joint replacements as a bearing surface.

And these antibiotic-eluting formulations of it are developed as possible treatments for periprosthetic joint infections. This method can be used with any drug-eluting device, such as porous metals, degradable and non-degradable polymers, gels, and possibly particulate materials with material specific protocol modifications. To begin, culture the bacteria overnight in one milliliter of tryptic soy broth.

Then dilute the overnight grown bacterial suspension in sterile Mueller Hinton Broth, or MHB, and verify the viable bacterial count using the luminescence units before the start of the experiment as described in the manuscript. Place the virgin and drug loaded UHMWPE strips within a three-milliliter syringe. Then draw MHB-containing bacterial suspension into the syringe through the attached needle up to the 1.5 milliliter mark.

Place the syringe setup on a shaking incubator until the indicated time points of six hours, and then every day until day seven. After each indicated time point, take out the syringe setup and dispense the media in a two-milliliter tube. Perform a real-time microbial viability assay as described in the manuscript using 100 microliters of bacterial suspension.

After determining the viable bacteria count, calculate the colony forming units per milliliter from the luminescence units using the corresponding standard curve. To verify the absence of viable bacteria in the samples that showed luminescence values below the limit of detection, spread the culture on tryptic soy agar plates. Incubate the plates at 35 degrees Celsius overnight.

Then check the presence of colonies the next day. Centrifuge the remaining bacterial suspension. Gently aspirate the spent media.

Leave 100 microliters of supernatant in the tubes with unseen pellets. Resuspend the pelleted bacteria in fresh MHB. Vortex for 10 seconds to ensure complete resuspension.

Then draw the bacterial suspension into the same syringe setup through the attached needle. Begin by retrieving virgin and drug-loaded UHMWPE surfaces from the syringe setup after the completion of the study on day seven. Then transfer the surfaces to 1.5-milliliter tubes and rinse with one milliliter of sterile PBS three times.

Sonicate the surface for 40 minutes in one milliliter of sterile PBS. Determine adherent bacteria viability by performing a luminesce assay on 100 microliters of the sonicated sample. The drug release from vancomycin and gentamicin-loaded UHMWPE demonstrated a burst release at six hours followed by a steady release rate with a concentration greater than the minimum inhibitory concentration until seven days.

UHMWPE with vancomycin and gentamicin demonstrated more than three-log reduction for susceptible ATCC 12600 starting at six hours and complete eradication was observed at day three. For the gentamicin-susceptible and vancomycin-intermediate strain, L1163, both drug-eluting materials caused more than three-log reduction at six hours and no colony growth was observed on day one. Gentamicin elution from UHMWPE did not affect the bacterial viability of the gentamicin-resistant and vancomycin-intermediate strain, L1101.

On the contrary, vancomycin elution from UHMWPE significantly reduced bacterial viability at six hours. The surfaces of both, gentamicin and vancomycin-eluting UHMWPE showed no viable adherent bacteria when exposed to susceptible and intermediate resistance strains after day seven. However, some viable bacteria were present on gentamicin-eluting UHMWPE exposed to gentamicin-resistant L1101.

The surface of control virgin polyethylene showed viable adherent bacteria when exposed to each strain. The separation of spent media at each time point facilitating carryover of the microbial population is crucial to simulate the sustained exposure to the antibiotics. While an improvement on static methods, this semi-static simulation method can be followed up with a continuous setup to understand further the activity kinetics of novel drug formulations against infections.

The protocol has enabled us to capture the effect of drug elution on strain-dependent bacterial dynamics. It enhances the tools for the efficacy evaluation of sustained antibiotic delivery devices.