Synergistic use of anti-inflammatory ketorolac and gentamicin to target staphylococcal biofilms

Abstract

Background While antibiotics remain our primary tools against microbial infection, increasing antibiotic resistance (inherent and acquired) is a major detriment to their efficacy. A practical approach to maintaining or reversing the efficacy of antibiotics is the use of other commonly used therapeutics, which show synergistic antibacterial action with antibiotics. Here, we investigated the extent of antibacterial synergy between the antibiotic gentamicin and the anti-inflammatory ketorolac regarding the dynamics of biofilm growth, the rate of acquired resistance, and the possible mechanism of synergy. Methods Control (ATCC 12600, ATCC 35984) and clinical strains (L1101, L1116) of Staphylococcus aureus and Staphylococcus epidermidis with varying antibiotic susceptibility profiles were used in this study to simulate implant-material associated low-risk and high-risk biofilms in vitro. The synergistic action of gentamicin sulfate (GS) and ketorolac tromethamine (KT), against planktonic staphylococcal strains were determined using the fractional inhibitory concentration measurement assay. Nascent (6 h) and established (24 h) biofilms were grown on 316L stainless steel plates and the synergistic biofilm eradication activity was determined and characterized using adherent bacteria count, minimum biofilm eradication concentration (MBEC) measurement for GS, visualization by live/dead imaging, scanning electron microscopy, gene expression of biofilm-associated genes, and bacterial membrane fluidity assessment. Results Gentamicin-ketorolac (GS-KT) combination demonstrated synergistic antibacterial action against planktonic Staphylococci. Control and clinical strains showed distinct biofilm growth dynamics and an increase in biofilm maturity was shown to confer further resistance to gentamicin for both ‘low-risk’ and ‘high-risk’ biofilms. The addition of ketorolac enhanced the antibiofilm activity of gentamicin against acquired resistance in staphylococcal biofilms. Mechanistic studies revealed that the synergistic action of gentamicin–ketorolac interferes with biofilm morphology and subverts bacterial stress response altering bacterial physiology, membrane dynamics, and biofilm properties. Conclusion The results of this study have a significant impact on the local administration of antibiotics and other therapeutic agents commonly used in the prevention and treatment of orthopaedic infections. Further, these results warrant the study of synergy for the concurrent or sequential administration of non-antibiotic drugs for antimicrobial effect.

Investigating the translational value of Periprosthetic Joint Infection (PJI) models to determine the risk and severity of Staphylococcal biofilms

“…Antibiotic-loaded implant materials that have been developed to locally release high antibiotic concentrations in a sustained manner could be best suited for this application[60–62]. For inherently resistant infections, it is advisable to use stronger tools such as the synergistic use of antibiotic and non-antibiotic compounds to enhance the antibacterial activity as early as possible to achieve effective eradication[36,63,64]. The study highlights the importance of diagnostics for characterizing the infecting bacteria in situ and developing more nuanced situation-specific and environment-specific guidelines for antibacterial treatment.…”

“…Antibiotic-loaded implant materials that have been developed to locally release high antibiotic concentrations in a sustained manner could be best suited for this application [60][61][62]. For inherently resistant infections, it is advisable to use stronger tools such as the synergistic use of antibiotic and non-antibiotic compounds to enhance the antibacterial activity as early as possible to achieve effective eradication [36,63,64].…”

“…Scanning electron microscopy was performed on the implant materials retrieved from rats on POD3 and from in-vitro experiments [36]. The implant materials with adherent bacteria were fixed using 2.5% glutaraldehyde in 0.1M PBS for 48 hours.…”