Taurine, melatonin, hydrogen peroxide and deuterium oxide were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Working solution of hypochlorous acid (HOCl) 100 mM was prepared by diluting a 5% commercial solution in water. The concentration of HOCl was determined spectrophotometrically after diluting the working solution in 0.01 M NaOH, pH 12 (λmax = 292 nm, ε = 350/M cm). N-Chlorotaurine (NCT) (molecular weight 181.57 g/mol, lot 2015-02-05) was prepared as crystalline sodium salt in our laboratory (M. Nagl, Innsbruck) at pharmaceutical grade, as reported (Gottardi and Nagl 2002), stored at minus 20 °C, and freshly dissolved in sterile 0.1 mM sodium phosphate buffer at pH 7.1 to a concentration of 55 mM (1%), 5.5 mM (0.1%), 1 mM (0.018%) or 0.25 mM (0.0045%) for each experiment.
Hydrogen peroxide (H2O2) 100 mM was prepared by dilution of a 30% commercial solution (Merck, Darmstadt, Germany) in water. The concentration of H2O2 was determined spectrophotometrically by its absorbance (λmax = 240 nm, ε = 43.6/M cm). The chemicals used for preparation of phosphate buffer and solutions were of analytical grade. Ultrapure Milli-Q water (Millipore, Belford, MA, USA) was used for the preparation of buffers and solutions. Bacto™ peptone from Becton–Dickinson and Company (NJ, USA) was dissolved in distilled water to a 10% stock solution and autoclaved. Sodium azide (NaN3) was dissolved in distilled water to a 0.65% (100 mM) stock, dimethyl sulfoxide (DMSO) to a 10% stock (1.28 M) in 0.1 M phosphate buffer. Catalase from Micrococcus lysodeikticus containing 65,000–150,000 U/ml was from Sigma-Aldrich (Germany). Chloramine T from Merck was dissolved in 0.1 M phosphate buffer to 0.005% (0.178 mM).
Studies of the reaction between NCT or HOCl and H2O2
Consumption of NCT and HOCl were monitored by their absorbances at 252 and 290 nm, respectively, using a Perkin Elmer Lambda 25 UV–visible spectrophotometer (Shelton, CT, USA). The reaction mixtures were composed of 0.25 mM NCT or HOCl, and 0.5 mM H2O2 in 0.1 M phosphate buffer, pH 7.1 and 37 °C. The production of 1O2 was monitored by its dimol light emission or, indirectly, by the light emission generated by its reaction with melatonin using a plate luminometer (Centro Microplate Luminometer LB960, Berthold Technologies, Oak Ridge, TN, USA). The reaction mixtures were composed of 1.0 mM NCT or HOCl, 20 mM H2O2, in the presence or absence of 1 mM melatonin in 0.1 M phosphate buffer, pH 7.1 and 37 °C. The reactions were triggered by the addition of H2O2.
Bacteria and fungi
Bacteria and yeasts deep frozen for storage were grown on Mueller–Hinton agar plates (Oxoid, Hampshire, UK) and subcultivated overnight in tryptic soy broth (Merck) at 37 °C. Subsequently, they were washed twice in 0.9% saline before use. Strains used were Staphylococcus aureus ATCC 25923 and 6538, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 11229, and Candida albicans CBS 5982 (60% pseudohyphae and 40% blastoconidia). Aspergillus fumigatus ATCC 204305 was grown on Sabouraud agar (Becton & Dickinson, Heidelberg, Germany) for 72 h. Suspensions of conidia were gained by harvesting them from the agar plates with 5.0 ml of 0.9% saline plus 0.01% Tween 20, followed by 10-µm filtration (CellTrics; Partec GmbH, Görlitz, Germany) to gain a pure conidia suspension without hyphae and three washing steps in phosphate-buffered saline (Lackner et al. 2015).
Time-kill assays (Lackner et al. 2015; Martini et al. 2012)
All experiments were done at 37 °C in a water bath. NCT (1.98 ml) was mixed with H2O2 (1.98 ml) in 0.1 M phosphate buffer (pH 7.1) or 0.1 M sodium acetate buffer (pH 4.0) to final concentrations of 1% (equals 55 mM NCT and 294 mM H2O2) or 0.1% each. In parallel, 3.96 ml of 1 or 0.1% NCT and 3.96 ml of 1 or 0.1% H2O2 were investigated in each experiment. A control in phosphate or acetate buffer without additives was done in parallel. In separate experiments, single radical scavengers were added to all test tubes. Thereby, respective volumes of the stock solutions were mixed immediately before the start of the test, for instance 1.98 ml of 2% NCT plus 2% H2O2 with 1.98 ml 10% peptone. Final concentrations were 5% peptone, 5% DMSO, or 10 mM sodium azide. To 3.96 ml of the test solutions, 40 µl of the respective suspension containing bacteria or fungi were added at time zero and vortexed. Final starting concentrations of microorganisms were 1.6–6.6 × 106 colony forming units (cfu)/ml for S. aureus ATCC 25923, 1.1–2.3 × 107 cfu/ml for S. aureus ATCC 6538, 1.1–3.1 × 107 cfu/ml for P. aeruginosa and E. coli, 2.0–4.4 × 105 cfu/ml for C. albicans, 4.4–1.0 × 106 cfu/ml for A. fumigatus. Incubation times ranged between 1 and 240 min and are indicated in the figures. At the end of each incubation time, aliquots of 100 µl were diluted in 900 µl NCT-inactivating solution consisting of 890 µl of 3% sodium thiosulphate plus 10 µl (approximately 1000 U) catalase in distilled water. Aliquots of 50 µl of this solution were spread on Mueller–Hinton agar plates in duplicate using an automatic spiral plater (model WASP 2, Don Whitley, Shipley, United Kingdom). The detection limit was 100 cfu/ml, taking into account both plates and the previous 10-fold dilution in the inactivating solution. Plates were grown for 48 h (bacteria) to 72 h (fungi) at 37 °C, and the number of cfu was counted. Plates with no growth or only a low cfu count were grown for up to 5 days (bacteria, Candida) or ten days (Aspergillus). Controls, i.e. plain 0.1 M phosphate buffer with and without scavengers (peptone, DMSO, NaN3) were performed in parallel. Inactivation controls, where NCT and H2O2 were mixed with their inactivators thiosulphate plus catalase immediately before addition of pathogens at low cfu counts, showed full survival of bacteria and fungi. This proved rapid and sufficient inactivation.
Time-kill assays with sequential treatment of NCT and H2O2
To investigate if there is an additive bactericidal effect after sequential incubation in both compounds, the pellet of washed bacteria (S. aureus ATCC 6538, E. coli, P. aeruginosa) was resuspended in the slower acting agent, 1% NCT, in 0.1 M phosphate buffer (pH 7.1) to 2–5 × 109 cfu/ml at room temperature first. The incubation time was 1 min. Controls were treated with phosphate buffer without NCT. After that time, the cfu count is not influenced, but the surface of the bacteria becomes chlorinated (“chlorine cover”) (Gottardi and Nagl 2005). Then, the bacteria were centrifuged at 4000×g for 5 min, washed in 0.9% NaCl, centrifuged again, and resuspended in saline. Subsequently, 40 µl of the bacterial suspension was added to 3.96 ml 1% H2O2 (0.3% H2O2 for tests with E. coli and P. aeruginosa) in 0.1 M phosphate buffer (pH 7.1), and incubated at 37 °C (controls in buffer without H2O2). Quantitative cultures from aliquots after different incubation times were performed as described in the previous paragraph.
The data are presented as mean values and standard deviations (SD) of at least three independent experiments. Student’s unpaired t test in case of two groups or one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test in case of more than two groups were used to test for a difference between the test and control groups. A P value of <0.05 was considered significant for all tests. Calculations were done with the GraphPad Prism 6.01 software (GraphPad, Inc., La Jolla, CA, USA).
To gain an improved survey on the microbicidal activity of NCT against the different strains, the recently introduced Integral Method was used, which transforms the whole killing curve (log10 cfu/ml versus time) into one value of “bactericidal activity (BA)” (Gottardi et al. 2015). The higher the value, the stronger is the microbicidal activity. Moreover, the method allows an expanded statistical analysis with the tests mentioned above, particularly between killing curves with small differences.