Microbicidal activity of N-chlorotaurine in combination with hydrogen peroxide

Abstract

N-chlorotaurine (NCT) and hydrogen peroxide are powerful endogenous antiseptics. In vivo, the reaction between hydrogen peroxide and metal ions leads to the formation of free hydroxyl radicals, which have an increased bactericidal activity. This study examined whether there is an additive antimicrobial effect of NCT combined with hydrogen peroxide. Additionally, it was tested if the additive effect is based on the formation of free radicals. We found by luminometry that, in the presence of H₂O₂, NCT caused a slow and long-lasting production of singlet oxygen in contrast to HOCl, where this burst occurred instantaneously. Both NCT and hydrogen peroxide (1.0 and 0.1%) demonstrated bactericidal and fungicidal activity. At pH 7.1 and 37 °C, hydrogen peroxide (1%, 294 mM) showed a stronger bactericidal and particularly fungicidal activity than NCT (1%, 55 mM), whereas at pH 4.0 and also in the presence of 5.0% peptone NCT revealed a stronger bactericidal activity. A combination of NCT and hydrogen peroxide led to an increased bactericidal but no increased fungicidal activity compared to both substances alone. The additive effect against bacteria was not removed in the presence of the radical scavengers NaN₃, DMSO, or peptone. As a conclusion, NCT and hydrogen peroxide used concurrently interact additive against a range of microorganisms. However, the results of this study suggest that the additive effect of NCT combined with hydrogen peroxide is rather not based on the formation of free radicals.

Keywords: Antimicrobial agent; Hydrogen peroxide; Microbicidal; N-Chlorotaurine; Oxidant; Singlet oxygen.

Fig. 1

Reaction of HOCl (a) and NCT (b) with H₂O₂. The reactions were monitored by HOCl and NCT absorbance decay. One representative experiment of 3 independent ones with similar outcome is depicted

Fig. 2

Light emission generated by the production of singlet oxygen. a HOCl and H₂O₂; b HOCl, H₂O₂ and melatonin; c NCT, H₂O₂ and melatonin. Reaction condition: HOCl and NCT 1 mM, melatonin 1 mM, H₂O₂ 1 mM in 0.1 M phosphate buffer, pH 7.1 and 37 °C. One representative experiment of 3 independent ones with similar outcome is depicted

Fig. 3

Effect of deuterium oxide in the light emission. Control reaction: NCT 1 mM, melatonin 1 mM, H₂O₂ 3 mM in 0.1 M glycine buffer pH 9.0 and 37 °C. One representative experiment of 3 independent ones with similar outcome is depicted

Fig. 4

Bactericidal activity of 1% NCT (filled square), 1% H₂O₂ (filled triangle), and 1% NCT plus 1% H₂O₂ (inverted triangle) at pH 7.1 and 37 °C. Control in phosphate buffer without additives (filled circle). Mean values ± SD of three to four independent experiments. **P < 0.01 versus all other values. BA “bactericidal activity” [log₁₀ cfu/min] as a quantitative measure for the strength of killing calculated by the integral method for the whole killing curve according to (Gottardi et al. 2015). The higher the value, the higher the microbicidal activity

Fig. 5

Bactericidal activity of 1% NCT (filled square), 1% H₂O₂ (filled triangle), and 1% NCT plus 1% H₂O₂ (inverted triangle) at pH 7.1 and 37 °C in the presence of 10 mM sodium azide (NaN₃). Control in phosphate buffer plus 10 mM NaN₃ (filled circle). Mean values ± SD of three (E. coli) to four (S. aureus) independent experiments. **P < 0.01 versus all other values. BA values calculated as in Fig. 4

Fig. 6

Bactericidal activity of 1% NCT (filled square), 1% H₂O₂ (filled triangle), and 1% NCT plus 1% H₂O₂ (inverted triangle) at pH 7.1 and 37 °C in the presence of 5% peptone. Control in phosphate buffer plus 5% peptone (filled circle). Mean values ± SD of three to four independent experiments. **P < 0.01 versus all other values. BA values calculated as in Fig. 4

Fig. 7

Fungicidal activity of 1% NCT (filled square), 1% H₂O₂ (filled triangle), and 1% NCT plus 1% H₂O₂ (inverted triangle) at pH 7.1 and 37 °C. Control in phosphate buffer without additives (filled circle). Mean values ± SD of three to four independent experiments. BA values calculated as in Fig. 4

Fig. 8

Bactericidal activity of H₂O₂ (filled triangle) and of H₂O₂ against chlorine-covered bacteria that were pretreated with 1% NCT for 1 min (inverted triangle). 1% H₂O₂ against S. aureus ATCC 6538, 0.3% H₂O₂ against E. coli and P. aeruginosa. Controls in phosphate buffer without additives, without (filled circle) or with chlorine cover (Asterisk). Mean values ± SD of three to four independent experiments. **P < 0.01 versus all other values. BA values calculated as in Fig. 4

Scheme 1

Production of singlet oxygen by NCT and H₂O₂ and its reaction with melatonin leading to light emission