Mustedanagic et al. AMB Expr (2017) 7:102 DOI 10.1186/s13568-017-0404-3 ORIGINAL ARTICLE Microbicidal activity of N-chlorotaurine in combination with hydrogen peroxide Jasmin Mustedanagic1, Valdecir Farias Ximenes2 and Markus Nagl1,3* 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 bacte- ricidal activity. This study examined whether there is an additive antimicrobial effect of NCT combined with hydro- gen 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%) demon- strated 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: N-Chlorotaurine, Hydrogen peroxide, Singlet oxygen, Microbicidal, Antimicrobial agent, Oxidant © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Introduction Human phagocytes are activated by invading microor- ganisms and produce several reactive oxygen species (ROS) by an enzymatic cascade called oxidative burst (Klebanoff 1968). Superoxide (O2.−) and hydrogen perox- ide (H2O2) are products of NADPH oxidase and super- oxide dismutase, respectively, and hypochlorous acid (HOCl) is formed from H2O2 and chloride by myelop- eroxidase (Weiss et  al. 1982; Zgliczynski et  al. 1971). HOCl immediately reacts among others with amino groups to create chloramines (R-NHCl), also designated as long-lived oxidants because of their lower reactivity. N-chlorotaurine (Cl–HN–CH2–CH2–SO3 −, NCT) is the most abundant representative of this class of compounds (Grisham et al. 1984). All these oxidants are thought to be involved in the killing of microorganisms during inflam- mation (Klebanoff et al. 2013). However, their spectrum of functions is broader, such as signal transduction by H2O2 (Kim et al. 2002; Mongkolsuk and Helmann 2002), or anti-inflammatory effects by NCT (Kim and Cha 2014; Marcinkiewicz and Kontny 2014). Moreover, chemically synthesized H2O2 and HOCl are in use as antiseptics in human medicine (Bruch 2007; Wilkins and Unverdor- ben 2013), and NCT is particularly suited for treatment of infections of sensitive body sites according to several studies (Gottardi and Nagl 2010; Nagl et al. 2003; Neher et al. 2004; Teuchner et al. 2005). As an interesting aspect, the mentioned oxidants may also react among one another, whereby further microbi- cidal ROS are formed. The reaction between HOCl and H2O2 to form singlet oxygen (1O2) (Eq. 1) was discovered by Khan and Kasha in (1963), and it was studied in details due to its physiological and pathological relevance, including its involvement in signaling and microbicidal Open Access *Correspondence: m.nagl@i-med.ac.at 3 Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstr. 41, 1st Floor, 6020 Innsbruck, Austria Full list of author information is available at the end of the article http://orcid.org/0000-0002-1225-9349 http://creativecommons.org/licenses/by/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1186/s13568-017-0404-3&domain=pdf Page 2 of 11Mustedanagic et al. AMB Expr (2017) 7:102 functions (Khan and Kasha 1963; Steinbeck et  al. 1992; Stief and Fareed 2000). The oxidation of H2O2 leading to 1O2 is not a prop- erty exclusive of HOCl (Miyamoto et al. 2014), but also shared by other halogenating species as hypobromous acid (HOBr) (Kanofsky et al. 1988), chloramines, includ- ing NCT (Stief 2003) and N-bromotaurine (De Carvalho et  al. 2016). However, although the reactivity of NCT with H2O2 leading to 1O2 has been proposed (Stief et al. 2001; Stief 2003), as far as we know, it was never inves- tigated in details. Here we identified an advantage in the use of NCT compared to HOCl regarding the production of 1O2. The aim of this study was to characterize the reaction of NCT and hydrogen peroxide with production of sin- glet oxygen and to investigate a possible additive bacteri- cidal or fungicidal effect of both compounds. Materials and methods Chemicals Taurine, melatonin, hydrogen peroxide and deuterium oxide were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Working solution of hypochlor- ous 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, Darm- stadt, 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 buff- ers and solutions. Bacto™ peptone from Becton–Dick- inson 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/ (1)H2O2 +OCl − → 1 O2 + Cl − +H2O2 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 spectrophotome- ter (Shelton, CT, USA). The reaction mixtures were com- posed 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 produc- tion of 1O2 was monitored by its dimol light emission or, indirectly, by the light emission generated by its reac- tion with melatonin using a plate luminometer (Centro Microplate Luminometer LB960, Berthold Technologies, Oak Ridge, TN, USA). The reaction mixtures were com- posed of 1.0  mM NCT or HOCl, 20  mM H2O2, in the presence or absence of 1 mM melatonin in 0.1 M phos- phate buffer, pH 7.1 and 37  °C. The reactions were trig- gered 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% blasto- conidia). 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 fil- tration (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 phos- phate 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 inves- tigated 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 Page 3 of 11Mustedanagic et al. AMB Expr (2017) 7:102 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 detec- tion limit was 100 cfu/ml, taking into account both plates and the previous 10-fold dilution in the inactivating solu- tion. 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). Con- trols, 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 cata- lase 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 pel- let 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 incuba- tion time was 1  min. Controls were treated with phos- phate 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 pre- vious paragraph. Statistics The data are presented as mean values and standard devi- ations (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 activ- ity 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 microbi- cidal activity. Moreover, the method allows an expanded statistical analysis with the tests mentioned above, par- ticularly between killing curves with small differences. Results Reaction between NCT or HOCl and  H2O2 We found that, differently of HOCl, which produces an instantaneous burst of 1O2 in the presence of H2O2, NCT caused a slow and long-lasting production of this elec- tronically excited form of molecular oxygen. Figure  1 shows the consumption of HOCl and NCT provoked by the addition of H2O2. While HOCl was almost com- pletely depleted in less than 5 s, NCT lost less than 2% of its initial concentration in 20 min. The reactions were also monitored by the weak chemiluminescence generated by dimol emission of 1O2 (Lengfelder et al. 1983). Figure 2a shows that, although of low intensity and of a short period (less than 10 s), chemiluminescence was detected by the reaction between HOCl and H2O2. On the other hand, due to its lower reactivity, light emission was not detected using NCT. Hence, to improve the efficiency, we added melatonin, which promptly reacts with 1O2 leading to light emission through the formation of an unstable diox- etane intermediate (Lu et al. 2002; Matuszak et al. 2003). The result in Fig. 2b shows that the emission of light for the reaction between HOCl and H2O2 was increased about 3 orders of magnitude in the presence of melatonin, reinforcing the involvement of 1O2. Finally, Fig.  3 shows the kinetic profile of light emission when HOCl was sub- stituted by NCT in the presence of melatonin and H2O2. Corroborating with the results obtained by monitoring the consumption of NCT, the production of 1O2 took place in more than 30  min, which is an evidence of the slow, but efficient reactivity of this chloramine with H2O2. Of note, according controls disclosed that light emission was not due to direct reaction of melatonin with H2O2 or with NCT (not showed). We also found that the reaction Page 4 of 11Mustedanagic et al. AMB Expr (2017) 7:102 rate was still lower at alkaline pH, which is consistent with the lower reactivity of NCT in this condition. Additional evidence of the formation of 1O2 was obtained by addi- tion of deuterated water in the reaction medium, which increase its lifetime (Kim et  al. 2016) and, consequently, the efficiency of the reaction (Fig. 3). Microbicidal activity of NCT and  H2O2 Against all bacterial test strains, the combination of NCT and H2O2 showed a more rapid killing than the single compounds. In phosphate buffer at pH 7.1, H2O2 had a throughout stronger activity than NCT. This is illustrated in Fig.  4. To investigate the influence of singlet oxygen formed by NCT plus H2O2, sodium azide as a scaven- ger was added to the test solutions at a concentration of 10 mM, which is a threshold that just does not kill bac- teria (Sabbahi et  al. 2008). Surprisingly, the significantly more rapid killing by the combination was still present (Fig. 5). The same was true if 5% DMSO, a hydroxyl radi- cal scavenger, was added (data not shown). Finally, we added 5% peptone as a general quencher of oxidants (Fig. 6). Of note, NCT plus H2O2 was still the highly sig- nificantly strongest bactericidal solution. In addition, the already known enhancing effect of organic matter on 0 5 10 15 20 0.2 0.4 0.6 0.8 1.0 H2O2a b Time (s) H O C l ( C /C 0) 0 500 1000 0.98 0.99 1.00 1.01 Time (s) N C T (C /C 0) Fig. 1 Reaction of HOCl (a) and NCT (b) with H2O2. The reactions were monitored by HOCl and NCT absorbance decay. One repre- sentative experiment of 3 independent ones with similar outcome is depicted 0 20 40 60 0 5.0×10 2 1.0×10 3 1.5×10 3 2.0×10 3 Time (s) Li gh t E m is si on (a .u .) 0 20 40 60 0 5.0×10 5 1.0×10 6 1.5×10 6 2.0×10 6 2.5×10 6 Time (s) Li gh t E m is si on (a .u .) 0 500 1000 1500 2000 0 5.0×10 3 1.0×10 4 1.5×10 4 Time (s) Li gh t E m is si on (a .u .) a b c 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 0 500 1000 1500 2000 0 2.0×103 4.0×103 6.0×103 8.0×103 (1) control (3) + 50% D2O (2) + 25% D2O (1) (2) (3) Time (s) Li gh t E m is si on (a .u .) Fig. 3 Effect of deuterium oxide in the light emission. Control reac- tion: 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 Page 5 of 11Mustedanagic et al. AMB Expr (2017) 7:102 NCT was seen again (Gottardi et  al. 2014), while H2O2 was weakened. Therefore, NCT turned out to kill rather more rapidly than H2O2 under these conditions. A similar result was seen at pH 4 in acetate buffer where 0.1% NCT and 0.1% H2O2 were tested. Again, the combination showed the strongest effect followed by NCT and H2O2 (BA = 0.98 ± 0.15 log10 cfu/min for NCT plus H2O2, 0.60 ± 0.07 for NCT, and 0.25 ± 0.02 for H2O2). Against C. albicans and A. fumigatus in phosphate buffer, no difference was found between NCT plus H2O2 and H2O2 alone (Fig.  7), while NCT killed fungi much slower than bacteria as expected from former work (Nagl et al. 2001). S. aureus ATCC 6538 Incubation time (min) 0 4 8 12 16 20 24 28 32 0 2 4 6 8 detection limit BA H2O2 = 0.3166 ± 0.0266 BA NCT = 0.1687 ± 0.0131 BA NCT + H2O2 = 0.4489 ± 0.0409 P < 0.01 between all BA values S. aureus ATCC 25923 Incubation time (min) 0 2 4 6 8 10 12 14 0 2 4 6 8 detection limit BA H2O2 = 0,5018 ± 0,0530 BA NCT = 0.2781 ± 0.0261 BA NCT + H2O2 = 0.6710 ± 0.0826 P < 0.01 between all BA values P. aeruginosa ATCC 27853 Incubation time (min) 0 2 4 6 8 10 12 14 0 2 4 6 8 detection limit BA H2O2 = 1.3740 ± 0.1979 BA NCT = 0.3258 ± 0.0227 BA NCT + H2O2 = 2.5892 ± 0.5681 P < 0.01 between all BA values E. coli ATCC 11229 Incubation time (min) 0 2 4 6 8 10 12 0 2 4 6 8 detection limit ** ** BA H2O2 = 1.2884 ± 0.1361 BA NCT = 0.5704 ± 0.0372 BA NCT + H2O2 = 2.2715 ± 0.4081 P < 0.01 between all BA values in phosphate buffer: NCT + H2O2 > H2O2 > NCT lo g 10 c fu /m L lo g 10 c fu /m L lo g 10 c fu /m L lo g 10 c fu /m L 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 Page 6 of 11Mustedanagic et al. AMB Expr (2017) 7:102 In some experiments, 0.005% CAT was used instead of 1% NCT against S. aureus 6538. The BA value of 1% H2O2 was 0.28 ± 0.02, that of CAT 0.55 ± 0.05 and that of CAT plus 1% H2O2 0.67 ± 0.07 log10 cfu/min. The dif- ference between CAT and CAT plus H2O2 did not reach significance but a trend after 3 independent experiments (P = 0.0676). Time‑kill assays with sequential treatment of NCT and  H2O2 When NCT-pretreated, chlorine-covered bacteria were incubated in H2O2, a differential result was gained, depending on the species used. With S. aureus, the chlo- rine cover did not enhance the susceptibility to H2O2 (Fig. 8). By contrast, E. coli and P. aeruginosa were killed significantly more rapidly by H2O2 if pretreated with NCT (Fig. 8) (P < 0.01). Discussion Both hydrogen peroxide and NCT are important low reactive components of the oxidative armament of human granulocytes and monocytes and can be used as endogenous antiseptics in human medicine (Baldry 1983; Gottardi et  al. 2013; Gottardi and Nagl 2010; Winter- bourn and Kettle 2013). For both regards, investigations of the interaction between these molecules may be of interest and contribute to elucidate biological processes. A slow and long-lasting production of singlet oxygen by NCT plus H2O2 was characterized for the first time in this study. Concentrations of the oxidants were adjusted to reveal good quantitative results. Two procedures were used to monitor the reactions and to highlight the differ- ences between NCT and its precursor HOCl. In the first one, the higher reactivity of HOCl compared to NCT was clearly demonstrated by its rate of consumption. However, the concomitant production of singlet oxygen, monitored by its phosphorescence emission, is uneasy to follow due to the low efficiency of light emission. This was the reason to add melatonin in the medium, since its reactivity with singlet oxygen is well-known and charac- terized by the efficient emission of chemiluminescence (Lu et  al. 2002). Scheme  1 depicts a mechanistic pro- posal for production of singlet oxygen and the emission of chemiluminescence through generation of a dioxetane intermediate. It is worth of note that there are other oxi- dative pathways by which melatonin could generate light emission, however, this was not the case using NCT or H2O2 alone. In addition, the amplification of the light emission provoked by deuterium oxide is an additional evidence of the intermediate singlet oxygen. S. aureus ATCC 6538 Incubation time (min) 0 5 10 15 20 25 30 0 2 4 6 8 detection limit BA H2O2 = 0.5085 ± 0.0350 BA NCT = 0.1745 ± 0.0113 BA NCT + H2O2 = 0.7063 ± 0.0524 P < 0.01 between all BA values E. coli ATCC 11229 Incubation time (min) 0 1 2 3 4 5 6 7 8 0 2 4 6 8 detection limit BA H2O2 = 1.9633 ± 0.2369 BA NCT = 0.8461 ± 0.0819 BA NCT + H2O2 = 2.7466 ± 0.4619 P < 0.01 between NCT and H2O2 P < 0.05 between H2O2 and NCT + H2O2 in 10 mM sodium azide: NCT + H2O2 > H2O2 > NCT lo g 10 c fu /m L lo g 10 c fu /m L 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 Page 7 of 11Mustedanagic et al. AMB Expr (2017) 7:102 We hypothesized that production of 1O2 would increase the microbicidal activity of NCT and H2O2 if they were used in combination. For these investigations, we applied higher, clinically applied concentrations and gained rea- sonable killing times of microorganisms. Actually, against bacteria an additive effect of both compounds was found. S . a u r e u s A T C C 6538 In c u b a tio n t im e (m in ) lo g 1 0 cf u /m L 0 4 8 1 2 1 6 2 0 0 2 4 6 8 d e te c t io n l im i t B A H 2O 2 = 0 .3 2 8 6 ± 0 .0 2 4 0 B A N C T = 0 .3 8 2 2 ± 0 .0 2 6 4 B A N C T + H 2O 2 = 1 .0 1 1 1 ± 0 .0 8 6 3 ( P < 0 .0 1 ) P = 0 .4 0 N C T v s . H 2O 2 S . a u r e u s A T C C 2 5 9 2 3 In c u b a tio n t im e (m in ) lo g 1 0 c fu /m l 0 2 4 6 8 1 0 0 2 4 6 8 d e te c t io n l im i t B A H 2O 2 = 0 .4 8 5 1 ± 0 .0 6 2 4 B A N C T = 0 .6 7 3 1 ± 0 .1 1 8 3 B A N C T + H 2O 2 = 2 .7 5 9 8 ± 0 .8 9 3 6 ( P < 0 .0 1 ) P = 0 .0 7 1 6 N C T v s . H 2O 2 P . a e r u g in o s a A T C C 2 7 8 5 3 In c u b a tio n t im e (m in ) lo g 1 0 cf u /m L 0 4 8 1 2 1 6 2 0 2 4 0 2 4 6 8 d e te c t io n l im i t B A H 2O 2 = 0 .3 6 7 3 ± 0 .0 2 3 3 B A N C T = 0 .3 4 2 5 ± 0 .0 2 0 3 B A N C T + H 2O 2 = 2 .5 1 4 3 ± 0 .5 7 3 6 ( P < 0 .0 1 ) P = 0 .1 9 2 2 N C T v s . H 2O 2 E . c o li A T C C 11229 In c u b a tio n t im e (m in ) lo g 1 0 cf u /m L 0 4 8 1 2 1 6 2 0 0 2 4 6 8 d e te c t io n l im i t B A H 2O 2 = 0 .3 5 3 4 ± 0 .0 2 2 9 B A N C T = 1 .2 3 3 6 ± 0 .1 7 2 5 B A N C T + H 2O 2 = 2 .6 5 3 6 ± 0 .4 2 2 5 P < 0 .0 1 b e tw e e n a l l in 5 % p e p to n e : N C T + H 2 O 2 > N C T ≥ H 2 O 2 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 Page 8 of 11Mustedanagic et al. AMB Expr (2017) 7:102 It was not strong, but reached high significance com- pared to the single components. This was true for single incubation time points as well as for the whole killing curves condensed by the integral method (Gottardi et al. 2015), which proved to be of high advantage for compari- son of curves. The additive effect was independent of the pH and still present at pH 4. In acidic environment, both single NCT and H2O2 showed stronger killing than at pH 7, but this was much more pronounced with NCT. There- fore, NCT exerted a stronger bactericidal effect than H2O2 at pH 4. Active chlorine compounds typically and markedly increase their microbicidal activity at acidic pH due to formation of further oxidizing species and due to loss of negative charges of the bacterial surface [for details see (Gottardi et al. 2013)]. We further hypothesized that the additive effect of NCT and H2O2 would disappear in the presence of scavengers of oxygen radicals (DMSO, sodium azide). However, this assumption turned out as not true. The combination was still stronger bactericidal, which was further confirmed in the presence of 5% peptone, a general scavenger of oxidants. These results indicate that formed singlet oxygen might not be responsible for this effect. A possible explanation is a combined attack of NCT and H2O2 on the bacterial cell wall and cell membrane. This may lead to earlier penetration into the microorganism followed by its rapid irrevers- ible inactivation. In this regard, however, we cannot completely discard the involvement of singlet oxygen, since it is possible that azide (N3 −), a charged molecule, and the other scavengers had not total access to the site of generation of singlet oxygen in the cell covers of the bacteria. Sequential application of NCT and H2O2, revealing an additive killing effect in Gram-negative bacteria but not in S. aureus, rather confirms that singlet oxygen is not responsible. Combined attack and more rapid destruc- tion of the bacterial covers followed by slightly more rapid penetration of oxidation capacity into the bacte- ria appears to be a conclusive hypothesis in our opin- ion. Detailed contribution of the multitude of single reaction partners/products are unknown, although the basic chemical reactions have been elucidated (Gottardi et  al. 2013; Peskin et  al. 2009). From previous studies it is known that the chlorine cover attached by sublethal treatment with NCT does not kill the microorganisms but removes their virulence and causes a lag of regrowth and postantibiotic effect (Gottardi and Nagl 2005; Lack- ner et  al. 2015; Nagl et  al. 1999). Therefore, we think that the damage of the surface by the chlorine cover C. albicans CBS 5982 Incubation time (min) lo g 10 c fu /m L 1 2 3 4 5 6 A. fumigatus ATCC 204305 Incubation time (min) lo g 10 c fu /m l 0 10 20 30 40 50 60 0 60 120 180 240 1 2 3 4 5 6 BA H2O2 = 0.0970 ± 0.0092 BA NCT = 0.0191 ± 0.0047 BA NCT + H 2O2 = 0.0970 ± 0.0092 P < 0.01 between NCT and H2O2 ± NCT P = 1.00 between H 2O 2 and H 2O 2 + NCT BA H 2O2 = 0.1062 ± 0.0105 BA NCT = 0.0148 ± 0.0014 BA NCT + H 2O2 = 0.0990 ± 0.0097 P < 0.01 between NCT and H2O2 ± NCT P = 0.54 between H 2O 2 and H2O2 + NCT in phosphate buffer: NCT + H 2 O2 = H 2 O 2 > NCT 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 Page 9 of 11Mustedanagic et al. AMB Expr (2017) 7:102 promotes the attack by H2O2 at least in Gram-negatives. The thicker Gram-positive cell wall seems to resist few min longer to penetration of the used oxidants than the Gram-negative one. The relatively slow killing of bacteria and fungi by mil- limolar NCT appears to approximately correlate with its penetration into the cytosol of these organisms. Slow penetration of 1 mM NCT into endothelial cells has been found (Peskin et  al. 2005), which was confirmed in our laboratory with 55 mM NCT using keratinocytes (A431), lung epithelial cells (A549), and Aspergillus fumigatus (M. Nagl, A. Windisch, unpublished results). Against fungi, no additive effect was found, at least at the tested concentration. The activity of NCT against fungi is markedly lower than that of H2O2 in phosphate buffer, probably due to slow penetration (Nagl et al. 2001, S. aureus ATCC 6538 Incubation time (min) 0 3 6 9 12 15 0 2 4 6 8 BA 1% H2O2 = 0.3578 ± 0.0305 BA chlorine cover + 1% H2O2 = 0.3443 ± 0.0243 P = 0.51 between both BA values detection limit E. coli ATCC 11229 Incubation time (min) 0 3 6 9 12 15 0 2 4 6 8 detection limit BA 0.3% H2O2 = 0.3471 ± 0.0180 BA chlorine cover + 0.3% H2O2 = 0.6348 ± 0.0476 P < 0.01 between both BA values P. aeruginosa ATCC 27853 Incubation time (min) 0 3 6 9 12 15 0 2 4 6 8 detection limit BA 0.3% H2O2 = 0.7157 ± 0.0628 BA chlorine cover + 0.3% H2O2 = 1.5589 ± 0.2133 P < 0.01 between both BA values lo g 10 c fu /m L lo g 10 c fu /m L lo g 10 c fu /m L 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 Page 10 of 11Mustedanagic et al. AMB Expr (2017) 7:102 2002). Obviously, the combined attack is not sufficiently strong to kill fungi more rapidly. Under protein load, H2O2 showed the expected decrease of activity, while NCT was markedly increased. The latter can be explained by transchlorination from NCT to amino groups of peptone, whereby among others low molecular weight chloramines are formed in equilib- rium, which have stronger microbicidal activity (Gottardi et al. 2014). This is particularly true for monochloramine (NH2Cl) because of its higher lipophilicity (Gottardi et  al. 2007). The enhancement of activity under protein load renders NCT a particularly interesting antiseptic for treatment of infections with high amounts of exudate (Gottardi et al. 2014; Gottardi and Nagl 2013). Moreover, NCT kills fungi much more rapidly in the presence of organic matter than in buffer solution (Gruber et al. 2017; Lackner et al. 2015; Nagl et al. 2001). Regarding the human defence system, the results of this study may provide evidence that single oxidants cooperate in their attack on invading microorgan- isms. Despite a high number of studies on highly reac- tive oxidants such as hypochlorite and long-lived ones such as chloramines, this aspect appears to have been underestimated up to date. Hydrogen peroxide and NCT are moderately enhanced in their bactericidal activity if applied in combination. The relatively slowly produced singlet oxygen seems not to be responsible for this effect. Consequences are not fully foreseeable presently and may comprise the understanding of the human defence system and application of oxidants as antiseptics. Abbreviations ANOVA: analysis of variance; BA: bactericidal activity; Cfu: colony forming units; DMSO: dimethylsulphoxide; NCT: N-chlorotaurine; ROS: reactive oxygen spe- cies; SD: standard deviation. Authors’ contributions JM performance of microbiological experiments, writing of the paper. MN writing of the paper, guidance of the study, statistical analysis. VX idea and concept, performance and guidance of biochemical experiments, writing of the paper. All authors read and approved the final manuscript. Author details 1 Division of Hygiene and Medical Microbiology, Medical University of Inns- bruck, Innsbruck, Austria. 2 Department of Chemistry, Faculty of Science, UNESP - São Paulo State University, Bauru, SP, Brazil. 3 Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstr. 41, 1st Floor, 6020 Innsbruck, Austria. Acknowledgements We are grateful to Andrea Windisch for excellent technical assistance. Competing interests The authors declare that they have no competing interests. Scheme 1 Production of singlet oxygen by NCT and H2O2 and its reaction with melatonin leading to light emission Page 11 of 11Mustedanagic et al. AMB Expr (2017) 7:102 Availability of data and materials All relevant data are presented in the manuscript. Funding This study was funded by the Austrian Science Fund, Grant No. KLI459-B30 and by the Brasilian Fundação de Amparo à Pesquisa do Estado de São Paulo, Grant No. 2016/20594-5, and INCT.Bio.Nat No. 2014/50926-0). 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Biochim Biophys Acta 235:419–424 http://dx.doi.org/10.1128/AAC.02527-16 Microbicidal activity of N-chlorotaurine in combination with hydrogen peroxide Abstract Introduction Materials and methods Chemicals Studies of the reaction between NCT or HOCl and H2O2 Bacteria and fungi Time-kill assays (Lackner et al. 2015; Martini et al. 2012) Time-kill assays with sequential treatment of NCT and H2O2 Statistics Results Reaction between NCT or HOCl and H2O2 Microbicidal activity of NCT and H2O2 Time-kill assays with sequential treatment of NCT and H2O2 Discussion Authors’ contributions References