Surface charge modification decreases Pseudomonas aeruginosa adherence in vitro and bacterial persistence in an in vivo implant model

Abstract

Objective: Chronic, persistent infections complicate otologic procedures utilizing implantable devices such as cochlear implants or tympanostomy tubes. These infections are thought to be due to the establishment of microbial biofilms on implant surfaces. To address this issue, we hypothesized that surface charge modification may inhibit the formation of Pseudomonas aeruginosa biofilms on implant surfaces in vitro and in vivo.

Study design: We evaluated the effect of surface charge modification on bacterial biofilm formation by assessing the effect of the surface charge on bacterial adhesion in vitro and bacterial persistence in vivo.

Methods: To study the effect of surface charge in vitro, the surface wells in culture plates were modified using a layer-by-layer polyelectrolyte assembly method. Bacterial adherence was measured at 30-, 60-, and 120-minute intervals. To study the effect of surface charge modification in vivo, the surface of titanium microscrews was similarly modified and then surgically implanted into the dorsal calvaria of adult rats and inoculated with bacteria. Two weeks after implantation and inoculation, the number of bacteria remaining in vivo was evaluated.

Results: Surface charge modification results in a significant decrease in adherence of bacteria in vitro. Surface charge modification of titanium microscrew implants also resulted in a significant decrease in P. aeruginosa recovered 2 weeks after surgical implantation.

Conclusion: Charge modification decreases the number of bacteria adherent to a surface in vitro and decreases the risk and severity of implant infection in an in vivo rat infection model. These results have promising biomedical applications.

Level of evidence: NA. Laryngoscope, 127:1655-1661, 2017.

Keywords: P. aeruginosa; Surface modification; biofilm; infection; persistence.

Figure 1. Charge modification of 24 well plates and titanium screws

24 well polystyrene plates and titanium microscrews were modified using the layer-by-layer polyelectrolyte assembly method. To confirm a charge on plates, they were exposed to negatively charged gold nanorods and examined by UV-vis spectrophotometry. To confirm a charge on the titanium screws, they were exposed to negatively charged hydrazide salt, which appears blue when bound. A) UV-vis absorption spectra of plates exposed to negatively charged gold nanorods. Results for negatively charged plates are indicated by the red line and positively charged plates by the blue line. The gold nanorods have an absorption spectra with peak wavelength between 509-550 nm and will exhibit this on spectrometry. B) An unmodified titanium screw treated with negatively charged hydrazide salt has a low baseline level of binding. C) A positively charged titanium screw bound with negatively charged hydrazide salt (blue) exhibits binding. D) A negatively charged titanium screw treated with negatively charged hydrazide salt has a minimal level of binding.

Figure 2. Adherence of P. aeruginosa strains to unmodified and charge modified surfaces

Wild type strains of Pseudomonas aeruginosa (PAO1 and PA14) and otopathogenic strains of P. aeruginosa (OPPA 8, 13, 15) were bound to unmodified, positive and negative charge modified plates. The plates were incubated for 30, 60, and 120 minutes, washed and the bacteria were enumerated by microscopy (bacteria/hpf). P-values represent statistical difference between unmodified and modified samples.

Figure 3. PAO1 biofilm formation on charge modified surfaces

Wild type P. aeruginosa strain PAO1 biofilms were developed on unmodified and charge modified plates. Plates were incubated for 48 hours, washed, stained with crystal violet and the absorbance at OD595 was measured. P-values comparing unmodified versus modified plates were measured.

Figure 4. Testing of charge modification on titanium screws during an infection in vivo

Rats were implanted with sterile unmodified or charge modified titanium screws. Prior to closure, a drop of P. aeruginosa was placed on top of the screw, the incision was closed, and the animals were allowed to recover for two weeks. After euthanasia, the skin was removed above the implant and photographs were taken. Photographs are shown for an infected unmodified implant (A), a negatively charged implant (B) and a positively charged implant. The results shown are representative of multiple animals done in three independent experiments.

Figure 5. Distribution of bacteria recovered from unmodified and charge modified implants following infection

Following 2 weeks of infection, the tissue overlying the screws was harvested, homogenized in Luria Bertani broth, and plated onto LB-agar plates to allow enumeration of colony forming units. Median colony forming units are depicted by the horizontal bar in within each column and error bars indicated the standard deviation. The star around 1000 CFU/mg in the positive group represents an outlier. The results shown are representative of multiple animals done in three independent experiments.