Octenidine's Efficacy: A Matter of Interpretation or the Influence of Experimental Setups?

Abstract

With its broad antimicrobial spectrum and non-specific mode of action via membrane disruption, any resistance to octenidine (OCT) seems unlikely and has not been observed in clinical settings so far. In this study, we aimed to investigate the efficacy of OCT against Escherichia coli and mutants lacking specific lipid head groups which, due to altered membrane properties, might be the root cause for resistance development of membrane-active compounds. Furthermore, we aimed to test its efficacy under different experimental conditions including different solvents for OCT, bacterial concentration and methods for analysis. Our primary goal was to estimate how many OCT molecules are needed to kill one bacterium. We performed susceptibility assays by observing bacterial growth behavior, using a Bioscreen in an analogous manner for every condition. The growth curves were recorded for 20 h at 420-580 nm in presence of different OCT concentrations and were used to assess the inhibitory concentrations (IC_100%) for OCT. Bacterial concentrations given in cell numbers were determined, followed by Bioscreen measurement by manual colony counting on agar plates and QUANTOM^TM cell staining. This indicated a significant variance between both methods, which influenced IC_100% of OCT, especially when used at low doses. The binding capacity of OCT to E. coli was investigated by measuring UV-absorbance of OCT exposed to bacteria and a common thermodynamic framework based on Bioscreen measurements. Results showed that OCT's antimicrobial activity in E. coli is not affected by changes at the membrane level but strongly dependent on experimental settings in respect to solvents and applied bacterial counts. More OCT was required when the active was dissolved in phosphate or Hepes buffers instead of water and when higher bacterial concentration was used. Furthermore, binding studies revealed that 10⁷-10⁸ OCT molecules bind to bacteria, which is necessary for the saturation of the bacterial surface to initiate the killing cascade. Our results clearly demonstrate that in vitro data, depending on the applied materials and the methods for determination of IC_100%, can easily be misinterpreted as reduced bacterial susceptibility towards OCT.

Keywords: Bioscreen; E. coli; MIC; QUANTOMTM; adaption; binding to bacteria; cell density; inoculum effect; lipid mutants; mechanism of action; multidrug resistance; octenidine; reduced susceptibility; resistance; tolerance.

Figure 1

Inhibition of E. coli growth is strongly influenced by OCT solvent and bacterial cell counts. (A) Growth curves of 1 × 10⁶ CFU/mL E. coli wildtype recorded in presence and absence of the indicated OCT concentrations over 20 h by Bioscreen at 420–560 nm. OCT was dissolved in water (upper panel), PBS (middle panel) or Hepes buffer (lower panel) before being added to the initial bacterial cell suspension. (B) Growth of E. coli treated with 1 mg/L OCT dissolved in water (dotted line), PBS (black line) and Hepes buffer (grey line). MIC values represent the lowest concentration of OCT that prevented the growth of 1 × 10⁶ E. coli. (C) Total inhibitory concentration (IC_100%) for OCT dissolved in the indicated buffer systems against E. coli of different concentrations ranging from 1 × 10⁶–2.5 × 10⁸ CFU/mL, as determined by Bioscreen at 420–560 nm. All results are representative data of at least three independent experiments.

Figure 2

Inhibition of E. coli wildtype growth is strongly influenced by OCT_water concentration and initial bacterial load. Growth curves of inoculum of 1 × 10⁷–1 × 10⁸ cells/mL or CFU/mL as measured directly by QUANTOM^TM technology (a–c) or optical density following the recalculation of CFU/mL by colony counting on agar plates (d–f), respectively. Bacterial growth in the absence (a,d) and presence of indicated OCT concentrations (b,c,e,f) over 20 h as recorded by Bioscreen at 420–560 nm. The results are representative data of at least three independent experiments.

Figure 3

Estimating the number of OCT molecules binding to an E. coli cell. (A). The unbound fraction of OCT_water as estimated by 281 nm after exposure to 1 × 10⁶ and 1 × 10⁷ cells/mL dissolved in PBS. OCT was applied at concentrations in the range where it exerts antimicrobial activity against E. coli and above the IC_100% (IC_100% for 1 × 10⁶ CFU/mL and 1 × 10⁷ CFU/mL in PBS buffer is 2 mg/L). The insert shows absorbance spectra of OCT at 281 nm. The lowest spectra correspond to the lowest OCT concentrations and their absorbance intensity increased with increasing OCT concentrations. In the presence of bacterial cells, the slightly lower absorbance in all spectra was observed indicating association of OCT with bacteria. Measurements were performed at least three times. To obtain the number of molecules per cell, the bound (partitioned) fraction is calculated by subtracting the unbound (free) fraction from the overall OCT concentration. In this case, the bound fraction is proportional to bacterial concentration, and results in an estimate of 1–2 × 10⁸ OCT molecules per single cell at IC_100%. (B) IC_100% values as a function of cell concentration. MIC or IC_100% corresponding to OCT´s concentration where no bacterial growth was observed, were calculated from growth curves recorded in MHB for OCT_water in presence of different bacterial concentrations (1 × 10⁶, 1 × 10⁷, 1 × 10⁸ and 2.5 × 10⁸ CFU/mL). To obtain the number of molecules per cell, the overall OCT concentration is divided in bound and unbound fractions. Free OCT is independent of cell concentration and is revealed at low bacterial presence (about less than 2 mM [1.2 mg/L]). The steep increase of IC_100% instead is due to the bound OCT fraction. It is linear with cell concentration and enables the extraction of OCT molecules bound on a single cell (it is around 1 × 10⁷). Solid lines are the best data fits, and dashed lines enclose the area accounting for the level of confidence.