Investigation of the susceptibility of clinical infection loads to nitric oxide antibacterial treatment

Abstract

The persistent infection of medical devices by opportunistic pathogens has led to the development of antimicrobial medical device polymers. Nitric oxide (NO) is an endogenous antimicrobial molecule that is released through the degradation of synthetic donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP) embedded into polymer membranes. It is hypothesized that the clinical success of these polymers is enhanced by the physiological release of NO and the consequent prevention of infection. However, such NO-releasing materials have never been evaluated against microbial loads that are commensurate with clinical infection levels. This study aimed to develop a standardized polymer film impregnated with SNAP that consistently releases NO and evaluates its efficacy against bacterial loads that represent clinical infection parameters. Microbial loads of 10³, 10⁵, and 10⁸ (colony-forming units) CFU mL^-1 were exposed to the NO-releasing polymer, corresponding to bloodstream infections, catheter-associated urinary tract infections, and standard laboratory exposure levels that have been reported in the scientific literature. By 24 h, SNAP films led to >1 log reduction of adhered and viable E. coli at all tested microbial loads compared to control polydimethylsiloxane (PDMS). Further, SNAP films displayed no viable adhered S. aureus at the 10³ microbial level for the entire study and showed total planktonic killing by 8 h. NO localization within bacterial cells adhering to the films was evaluated, revealing higher NO uptake and consequent bacterial killing by SNAP samples. This unique study shows that NO-releasing polymers not only kill bacteria adhered to the polymer surface, but localized delivery leads to environmental planktonic bacterial killing that prevents adhesion from occurring. Furthermore, the promising findings of NO-releasing polymers in scientific research indicate their potential for successful application in clinical settings to prevent infections.

Keywords: Antibacterial; Clinical infection; Medical device; Nitric oxide; S-nitroso-N-Acetylpenicillamine (SNAP).

Figure 1.

(A) Fabrication of NO-releasing PDMS followed a well-established solvent swelling method. (B) SEM images of the surfaces of the films revealed no significant morphological changes, (C) though the SNAP-PDMS films displayed the green hue characteristic of the SNAP molecule. (D) Contact angle measurements on the surfaces of the films showed no significant change in surface hydrophobicity following SNAP inclusion.

Figure 2.

(A) NO is released from RSNOs following reactions with heat, light, and catalysis by metal ions. (B) SNAP leaching from films was quantified over 24 h. (C) Release of NO from SNAP films was also measured over 24 h, showing no change in NO flux dependent on the absence or presence of FBS in the PBS.

Figure 3.

(A) Bacterial counts (Log CFU cm⁻²) of E. coli adhered to the surface of PDMS and SNAP-PDMS over 24 h was quantified and (B) percent reduction of adhered bacteria was calculated for SNAP-PDMS films compared to control PDMS films. (C) Viable planktonic bacteria were also quantified and (D) percent reductions of SNAP samples compared to control PDMS were calculated. Time points where CFUs for control samples were below the detection limit are denoted by ‘#.’ Statistical analysis is denoted by * p < 0.05, + p< 0.01, and § p < 0.0001.

Figure 4.

(A) Bacterial counts (Log CFU cm⁻²) of S. aureus adhered to the surface of PDMS and SNAP-PDMS over 24 h was quantified and (B) percent reduction of adhered bacteria was calculated for SNAP-PDMS films compared to control PDMS films. (C) Viable planktonic bacteria were also quantified and (D) percent reductions of SNAP samples compared to control PDMS were calculated. Time points where CFUs for control samples were below the detection limit are denoted by ‘#.’ Statistical analysis is denoted by * p < 0.05, + p< 0.01, and § p < 0.0001.

Figure 5.

(A) Fluorescent quantification of NO (green) localized within adhered E. coli bacterial cells and Eth-DIII (red), signaling dead adhered bacteria after 4 h and 24 h. (B) Representative images of adhered E. coli display fluorescent differences after 24 h treatment of control PDMS and SNAP samples. (C) Planktonic E. coli bacteria were also stained for NO localization in the treatment well at 4 h and 24 h and (D) representative images at 24 h demonstrate the enhanced NO localization within SNAP-PDMS-treated bacteria. White scale bars represent 5 µm. Statistical analysis is denoted by § p < 0.0001 when comparing PDMS to SNAP samples at each time point and for each dye.

Figure 6.

(A) Fluorescent quantification of NO (green) localized within adhered S. aureus bacterial cells and Eth-DIII (red), signaling dead adhered bacteria after 4 h and 24 h. (B) Representative images of adhered S. aureus at 24 h reveal greater fluorescence intensity for NO localization and killing for SNAP samples. (C) Planktonic S. aureus were also stained for NO localization and dead cell were quantified at 4 h and 24 h. (D) Representative images of planktonic S. aureus at 24 h display enhanced bacterial killing by NO-releasing samples. White scale bars represent 5 µm. Statistical analysis is denoted by § p < 0.0001 when comparing PDMS to SNAP samples at each time point and for each dye. Mean fluorescence intensity (a.u.) of zero is denoted by # across tested sample types.