Journal of Antimicrobial Chemotherapy (2005) 55, 475482 doi:10.

1093/jac/dki054 Advance Access publication 10 March 2005

JAC

Chlorine covers on living bacteria: the initial step in antimicrobial action of active chlorine compounds
Waldemar Gottardi* and Markus Nagl
Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria
Received 28 October 2004; returned 5 December 2004; revised 22 December 2004; accepted 7 January 2005
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Objectives: Although active chlorine compounds are well-known antimicrobial agents in human medicine, their initial steps of action have not been completely claried. Using N-chlorotaurine (NCT), an endogenous mild representative, we observed persisting oxidation capacity afxed to bacteria. It was the aim of this study to investigate this chlorine cover. Methods: Pathogens were incubated in NCT, which was subsequently washed off. The oxidation capacity on the bacterial surface was measured photometrically. Results: Supercial chlorination in the form of covalent N Cl bonds to Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans could be attached before killing took place. For S. aureus, 3 min incubation with NCT produced a cover of 3.3 3 10216 mol Cl1/cfu, while the cfu count was reduced by only 26%. The kind of microorganism, coating time, pH, buffer system and, basically, the chlorine compound, inuenced the cover strength. The relative cover strength on S. aureus by NCT, chloramine T, sodium dichloro-isocyanurate or N,N-dichlorotaurine was 1:15.7:38.7:0.24. Chlorine covers were surprisingly stable and could be detected for 3 h at 20 8C (> 8 h at 18C), even without a reduction of cfu. However, addition of 5% ammonium chloride caused a rapid loss of viability, explained by formation of highly bactericidal NH2Cl, an effect that resembles the ignition of a time-bomb. Conclusions: The chlorine cover can be regarded as the rst sign of interaction between chlorinating agent and microorganism, and may explain the non-lethal features of postantibiotic effect and attenuation of bacterial virulence. Furthermore, it may be a decisive step in bacterial inactivation by the myeloperoxidase-hypochlorite system in innate immunity. Keywords: chloramines, N-chlorotaurine, oxidation capacity

Introduction
One of the fascinating episodes during development of hygiene was the beating of the puerperal fever by Semmelweis,1 who reached this goal by introducing chloride of lime (bleach) as a rapid degerming agent. After this, chlorine and active chlorine compounds (bearing O Cl or N Cl functions) became indispensable in disinfection practice.2 Surprisingly, it was not until 1988, i.e. more than 120 years later, that we became aware that active chlorine compounds produce an oxidation capacity [c(Ox)] that persists on the skin surface, which was designated chlorine cover.3 6 It is not the result of adsorption, but originates from covalent N Cl bonds at the outermost layer of the horny skin. Recently, chlorine covers

on the surface of hypochlorite-sterilized rice seed and their impact on mutations on surrounding bacteria were detected.7 Chlorination of bacteria by active chlorine compounds with the aim of killing them occurs both in a variety of disinfection processes and, in vivo, in the myeloperoxidase-hypochlorite system that operates within phagolysosomes of human leucocytes.8 10 Investigations on the main long-lived oxidant produced by granulocytes and monocytes, N-chlorotaurine (NCT),11,12 revealed new insights in the consequences of the chlorination of pathogens. Incubation for a sublethal time of 1 min in 1% NCT solution caused a lag of regrowth (postantibiotic effect) of bacteria and a loss of virulence of highly encapsulated staphylococci and streptococci, demonstrated in the mouse peritonitis model.13,14 In addition, bacteria chlorinated by

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*Corresponding author. Tel: +43-512-507-3430; Fax: +43-512-507-2870; E-mail: waldemar.gottardi@uibk.ac.at

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q The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org

W. Gottardi and M. Nagl

the myeloperoxidase system lost their ability to induce nitric oxide and tumour necrosis factor-a in macrophages.15 These ndings prompted us to establish methods of detection and quantication of chlorination of bacterial surfaces and to perform the rst systematic examination of chlorine covers on Gram-positive and -negative bacteria and Candida albicans.
washed four times with saline and centrifuged at 1700 g. As shown in Figure 1, this procedure ensures a pellet virtually free from chlorinating agent. Standard procedure with NCT. It was most reasonable to suspend the pellet derived from 20 mL of an overnight culture in 4 mL of 1% NCT in 0.1 M phosphate (pH 7.0) and to incubate therein for 3 min at 20 8C. This procedure was used unless otherwise stated. For determination of bacterial counts see Regarding counted entities.

Materials and methods

Bacteria
Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Proteus mirabilis ATCC 14153, Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 27853, as well as S. aureus Smith diffuse B9 and Streptococcus pyogenes d 68, both slime producing and highly encapsulated strains (kindly provided by Dr J. Hildebrandt, Sandoz Scientic Center Vienna), and Candida albicans CBS 5982 (Centraal Bureau voor Schimmelculturen, Baarn, The Netherlands) were used. Bacterial strains deep-frozen for storage were grown overnight on tryptic soy agar (Merck, Darmstadt, Germany). Colonies from this agar were grown in tryptic soy broth (Merck) at 37 8C overnight.

Removing chlorine covers

Chlorinated bacteria were suspended for 10 min in 5 mL 0.01 M ice-cooled sodium thiosulphate. This procedure ensured a complete removal of the attached oxidation capacity.

Measurement of the bacterial chlorine cover

The c(Ox) originating in the chlorine cover was measured photometrically with the DPD and TNB methods.4,17 Procedures using the oxidation of iodide (tri-iodide method or redox potential) turned out to be imprecise, since the formed iodine was partly absorbed by the bacteria. The pellet of the chlorinated and washed bacteria was suspended in an appropriate excess volume of the reagent (according to the strength of chlorine cover volumes of 0.5 1.5 mL DPD solution or 1.0 5.0 mL TNB solution). Contrary to the statement of Thomas that the oxidation of TNB is usually complete within time of mixing,17 we found that an overall contact time of $ 8 min was necessary. The suspensions were centrifuged (2 min at 16 380 g) and the absorption of the supernatant was measured (DPD method: cell diameter 1.0 cm, l = 553 nm, 1 = 20500 L/cm/mol;4 TNB method: 0.10 cm; l = 325 and 410 nm (see above). Because oxygen in the air continuously oxidizes the reagents, care was taken to minimize the time between measuring the blank value, samples and controls. Annotation. The DPD and the TNB methods were used in parallel throughout this study. With chlorine covers up to 50 nmol/pellet both methods brought the same result, while in case of higher cover strength the DPD dye was gradually bleached, which was not the case with the TNB method, which was therefore preferred where

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Chemicals and solutions

All reagents were of the highest available purity. Sodium N-chloro4-toluenesulphonamide-sodium (chloramine T; CAT) and buffers were purchased from Merck, and dichloroisocyanuric acid sodium salt (DCI-Na) was purchased from Sigma Aldrich (Vienna, Austria). The sodium salts of NCT and N,N-dichlorotaurine (NDCT) were prepared from taurine and CAT, and DCI-Na, respectively.16 Lovibond tablets DPD 1 (containing N,N-diethyl-p-phenylenediamine; DPD) and DPD 3 (containing potassium iodide) were from BWT (Mondsee, Austria). Both tablets together were dissolved in 10 mL of water. 5,50 -Dithiobis(2-nitrobenzoic acid) (Ellmans reagent; DTNB) and 2-mercaptoethanol as starting materials for synthesizing the reagent 2-nitro-5-thiobenzoic acid (TNB) were from Sigma Aldrich. A 0.001 M aqueous solution of DTNB was reduced with the calculated amount of 2-mercaptoethanol forming TNB.17 The concentrations of TNB and DTNB were assessed photometrically using the absorptions at their peaks: DTNB, lmax = 325 nm; TNB, lmax 410 nm. The following molar absorption coefcients were found: 325 nm, 1TNB 2140 L=mol=cm, 1DTNB 17 430 L=mol=cm; 410 nm, 1TNB 13 600 L=mol=cm, 1DTNB 285 L=mol=cm.

Analysis of chlorinating agents

Since chlorinating agents release an equivalent amount of iodine in the presence of iodide, the purity of and strength of solutions were monitored by measuring their c(Ox), by iodometric titration with 0.1 M thiosulphate at pH 2 3 (acetic acid) after reaction with surplus KI using the automatic titration assembly TIM900 from Radiometer (Copenhagen, Denmark).

Attaching chlorine covers to bacteria

Portions (20 mL) of an overnight culture were centrifuged (8 min at 1700 g) and washed twice with 4.0 mL of saline. The resulting pellet was suspended in 4 mL of the buffered chlorinating solution and incubated therein for a dened time (1 20 min). In case of chlorinating agents that disproportionate at the chosen pH, e.g. NCT at pH < 8,16 the compound was directly dissolved in the suspension of the pellet in 4 mL buffer solution. After incubation, bacteria were
Figure 1. Efcacy of washing bacterial chlorine covers. S. aureus ATCC 25923 incubated for 1 min with 1% NCT. Oxidation capacity after one to seven subsequent washes, each with 4 mL saline. Supernatant and 1 mL DPD reagent (open squares); washed pellet suspended in 1 mL DPD reagent (open triangles); calculated c(Ox) of sequential dilutions of 55 mM NCT, each time 0.1 mL diluted with 4.0 mL (crosses); detection limit (dashed line, mean value of blank plus 3 S.D. ). Each point represents the mean value S.D. of three independent experiments. DPD method.

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Chlorine covers on living bacteria

high cover strength could be expected. Testing the accuracy with CAT solutions of known c(Ox) revealed an inaccuracy of 5% for both methods. potassium nitrate solution (which is isotonic to saline). Three coated pellets were used for measuring the c(Ox) of the chlorine cover with the TNB method. The other three coated pellets were reduced with thiosulphate, by which the positive chlorine was released in form of chloride:

Quantication of bacterial chlorine covers

Regarding the pellet. With the DPD method the c(Ox) of the chlorine cover, expressed in a molar scale (as positive chlorine), is: nmolCl=pellet A V 121 d21 ; where A denotes the absorption, 1 the molar absorption coefcient at 553 nm, d the diameter of the photometric cell and V the volume of the reagent (in mL). With the TNB method it is: nmol Cl=pellet 1=2{TNBC VTNB C VH2O C =VTNB C VTNB S 2 TNBS VTNB S VH2O S }; where [TNB]C and [TNB]S are the concentrations of TNB in the control and the sample, VTNB C and VTNB S the volumes of TNBreagent, and VH2O-C and VH2O-S the volumes of water (if dilution was necessary). The TNB concentrations were evaluated as described above. Regarding counted entities. Two methods were applied, relating to (i) the number of cfu and (ii) the number of discrete bacterial cells present after bringing on the chlorine cover (i.e. after chlorination and four washing steps). On a cfu basis. With the bacterial count (cfu/mL) of the suspension of the chlorinated bacteria and the volume (Vs) the pellet was made from, the chlorine cover of one cfu is: nmol Cl=cfu nmolCl=pellet=VS cfu=mL

. N Cl 2S2 O22 H2 O ! . N H S4 O22 OH2 Cl2 3 6

After 10 min the suspensions were centrifuged and the chloride concentration of the supernatant was measured with a Dionex Ion-Chromatograph. From this value the chloride concentration of controls which were treated identically was subtracted.

Evidence of release of oxidation capacity from the chlorine cover by ammonium chloride
Two pellets each of bacteria bearing a chlorine cover applied with the standard procedure were suspended in 1 mL of 5% NH4Cl solution for 1, 3 and 10 min, and centrifuged. The c(Ox) of the supernatant was assayed with the TNB method. Controls were treated with saline instead of NH4Cl.
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Statistical analyses
Students t-test was used for comparison of paired means of two groups of measurements. One-way analysis of variance and Dunnetts multiple comparison test (Graphpad Software Inc.) were applied for evaluation of more than two groups of measurements. P values of < 0.05 were considered signicant.

Results
cfu were determined by spreading aliquots of 50 mL of adequate dilutions on tryptic soy agar in duplicates by using a spiral plater (model WASP, Shipley, UK). The lowest detection limit was 100 cfu/mL taking both plates into account. Inactivation of the chlorine cover with sodium thiosulphate proved to be dispensable, since the cover did not kill, and was therefore omitted. In the killing experiments in 1% NCT solution (see Figure 4), however, aliquots were serially 10-fold diluted in 0.3% thiosulphate to ensure exact incubation times. Regarding the single bacterial cell. A pellet coated with a chlorine cover was suspended in an appropriate volume of water. A dened volume (V) was used for measuring the c(Ox) (nmol Cl+/V). Single bacteria were counted microscopically using a counting chamber including both living and dead entities (Ntot/mL). The chlorine cover of a single bacterial cell (b.c.) then is: nmol Cl=b:c: nmol Cl=V1 =Ntot =mL Finally, multiplying by Avogadros constant (NA = 6.022 1023), the number of N Cl functions bound to the surface of one bacterial entity was found.

General ndings in quantication of bacterial chlorine covers

The simplest approach for quantication of bacterial chlorine covers concerned c(Ox)/pellet, which was ideal for estimating the inuence of parameters like chlorinating agent and reaction time. The pellet size ranged from 1 to 6 1010 cfu (49 109 for S. pyogenes, and 14 109 for C. albicans, respectively). The ratio c(Ox)/cfu was determined by the varying number of bacterial cells that constitute the cluster of one cfu. It was used for relating chlorine cover strength and viability. The c(Ox) per single bacterial cell was appropriate for quantication of the chlorine cover of different bacterial strains. This quantity can be regarded as the only thoroughgoing gauge for cover strength, which, however, is subject to a dened chlorinating procedure that is ambitious to attain (see below).

Evidence of positive chlorine being the origin of the c(Ox) xed upon chlorinated bacteria
The assessed c(Ox) was 346.1 29.5 nmol Cl+/pellet (n = 3, TNB method), while the chloride values of the reduced pellets and the controls were 459.8 17.5 (n = 3) and 76.2 4.2 (n = 4) nmol Cl /pellet (Ion-Chromatograph), respectively. The amount of chloride resulting from the reduced chlorine cover was 383.6 18.3 nmol/pellet, which tted well with the c(Ox). From this nding it can be deduced that the bacterial chlorine cover is made up of covalent N Cl bonds.

Evidence of positive chlorine being the origin of the c(Ox) xed upon chlorinated bacteria
Ten identical pellets from 20 mL each of an overnight culture of S. aureus ATCC 25923 were split in two parts. Four were left uncoated and served as controls, while six were tted with a chlorine cover according to the standard procedure. Contrary to usual practice, the pellets were not washed with saline but with 0.15 M

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Quantication of bacterial chlorine covers
The standard procedure applied to S. aureus ATCC 25923 produced a chlorine cover of 3.3 1016 mol Cl+/cfu, which can be considered as a tolerable quantity. The ratio of cfu:single bacterial cell evaluated by comparison of viable counts and microscopic counts in a counting chamber was $ 1:2 in our experiments. It can be deduced that a single cell survives $ 1.6 1016 mol Cl+, which originates from 9.6 107 N Cl bonds at its surface. This strength of c(Ox) in the form of supercial NCl bonds was measured after 3 min of incubation time, and it was survived by the discrete microorganism (see Figure 4). (Figure 5b). The high value of C. albicans can be explained by its higher bulk compared with bacteria.

Stability of chlorine covers

In the absence of organic matter interacting with the NCl function, chlorine covers are rather stable dependent on the temperature. At 20 8C, 80% reduction of c(Ox) took place after 1 h, followed by a linear decrease to the detection limit of 1%, which was reached after 4 h (Figure 6a). At 1 8C (ice cooling), the c(Ox) remained stable for at least 8 h after a 30% reduction during the rst 2 h. In human serum the cover vanished comparably fast in a twophase manner. More than 60% of c(Ox) was lost within 1 min at 1 8C, while the remaining 40% decreased to the detection limit during the following 20 min (Figure 6b). At 20 8C and 37 8C the rapid loss was 80% after 1 min, and the detection limit was achieved after 12 min.
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Inuence of coating parameters on cover strength

Type of chlorine compound. The relative strength of chlorine cover per pellet afxed to S. aureus ATCC 25923 (0.055 M Cl+, pH 7, 3 min) with NCT, CAT, DCI-Na and NDCT was 1, 15.7, 38.7 and 0.24, respectively (Figure 2). The higher chlorinating potential of CAT compared with NCT is shown also in Figure 3. Coating time. As expected, prolonging the incubation time resulted in an increase of cover strength up to a range of saturation (Figure 4). Coating time appeared to be the least dened parameter because chlorination proceeded to a certain degree also during centrifugation of the pellet. pH and buffer concentration. While lowering the pH clearly increased the strength of the cover afxed by CAT (Figure 3b), the opposite was true for NCT (Figure 3a). The reason for that is the fast disproportionation of NCT at pH 5 to NDCT,16 which produced a minor chlorine cover (Figure 2). If bacteria were chlorinated in the presence of phosphate buffer instead of saline, the cover strength increased (Figure 4), which can be explained in part by the higher pH of NCT dissolved in saline (pH 8.3). Additionally, we found a positive correlation with the buffer concentration in a range of 0.050.5 M (data not shown). Type of microorganism. Under the chosen conditions the maximal cover per pellet brought about by NCT was attained with S. aureus, while it was very little with E. coli, P. aeruginosa and C. albicans (Figure 5a). A completely different result revealed the chlorine cover per individual bacterial cell, which is bettersuited to work out the real differences between microorganisms

Chlorine cover and viability

Figure 4 illustrates that killing by NCT, to a certain degree, was independent of the cover strength. Increasing the concentration of phosphate buffer raised the cover strength, while the killing

Figure 2. Inuence of chlorinating agent on cover strength. S. aureus ATCC 25923, incubated for 3 min with NCT, CAT, DCI-Na and NDCT, each containing 0.055 M Cl+ in 0.1 M phosphate pH 7.0. Mean values S.D. of three independent experiments. TNB method. P < 0.01 between all agents.

Figure 3. Inuence of pH on chlorine cover strength applied with (a) NCT and (b) CAT. S. aureus ATCC 25923 incubated for 1 min with 0.055 M Cl+ and 0.1 M buffer (citrate pH 5, phosphate pH 7, borate pH 9). Mean values S.D. of three independent experiments. TNB method. P < 0.01 between all bars.

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Chlorine covers on living bacteria

Figure 4. Inuence of incubation time in 1% NCT and of chlorinating milieu on cover strength and viability of S. aureus ATCC 25923. Chlorine strength in 0.1 M phosphate (open squares, pH 7.0), and in saline (open triangles, pH 8.2); P < 0.01 for all time points. cfu in phosphate (lled squares) and in saline (lled triangles); P > 0.05 for all time points. Mean values S.D. of three independent experiments. DPD method. Controls without NCT showed no killing and no chlorine cover (data not shown).

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Figure 6. Stability of chlorine covers (S. aureus ATCC 25923) in saline (a) and relative stability in human serum (b) at 1 8C (open squares), 20 8C (open triangles) and 37 8C (open circles). Mean values S.E.M. of three independent experiments. DPD method. P < 0.01 between 1 8C and 20 8C for all time points (a and b), and P > 0.05 between 20 8C and 37 8C (b).

Figure 5. Chlorine covers on different microorganisms, 3 min incubated with 1% NCT (0.055 M Cl+) in 0.1 M phosphate pH 7.0, (a) in nmol Cl+/pellet, (b) in mol Cl+/b.c. (b.c., bacterial cell). Mean values S.E.M. of three independent experiments. TNB method.

bond by transhalogenation. The term transhalogenation was dened for the transfer (exchange) of positive halogen between amine compounds. Contrary to the substitution of a C H bond, it occurs rather fast at room temperature, needs no catalyst and is not connected with a loss of oxidation capacity. As shown in Figure 7, an $ 5 log10 decrease of cfu took place in the presence of 5% ammonium chloride within 2 h, while there was virtually no decrease in saline and in the control of uncoated bacteria with ammonium chloride. Results described in the next paragraph revealed that the attached c(Ox) is released by ammonium chloride, most plausibly in form of NH2Cl, which is the reason for the observed bactericidal effect.

Evidence of release of oxidation capacity from the chlorine cover by ammonium chloride
The measured c(Ox) in the supernatant showed no time dependency, which suggests a mobilization completed within 1 min. The summarized result (n = 6) is 31.1 1.9 nmol Cl+/pellet released by ammonium chloride, while it was only

curve did not change. This implies that the two effects underlie different mechanisms. However, viability subsisted only in a medium like saline, which lacked components interacting with the covalent N Cl

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W. Gottardi and M. Nagl

species NCTH+, which is the real active agent executing transhalogenation.16 ClHN CH2 CH2 SO2 H2 O 3 $ ClH2 N CH2 CH2 SO2 OH2 3 NCT H2 O $ NCTH OH2 pKb NCTH aOH2 =NCT 7:9 ^ 0:2 25 8C16 2 1

Figure 7. Bactericidal action of chlorine cover in the presence of ammonium chloride. S. aureus ATCC 25923 3 min incubated with 1% NCT in 0.1 M phosphate pH 7.0. Chlorinated bacteria in 5% NH4Cl (open triangles) (P < 0.01 for all time points), chlorinated bacteria in saline (crosses), bacteria without chlorine cover in 5% NH4Cl (lled triangles). Mean values S.D. of three independent experiments. DPD method.

From Equation 2 it can be derived that the equilibrium concentration of NCTH+ increases with acidity. The ratio [NCTH+]/[NCT] comes to 126, 0.126 and 0.00126 at pH 5, pH 7 and pH 9, respectively. Therefore, transhalogenation is less probable in an alkaline milieu than at pH 7. The decrease of chlorine cover in the weak acid milieu, however, routes in disproportionation to NDCT (Equation 3), a reaction that also embodies a transhalogenation. 2ClHNCH2 CH2 SO2 H $ Cl2 NCH2 CH2 SO2 3 3

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5.9 1.2 nmol Cl+/pellet in the case of saline. It was not possible to identify the origin of the oxidation capacity in the supernatant by means of UV spectroscopy. Both with NH4Cl and NaCl, an unidentied peak was observed at 260 nm, while NH2Cl absorbs at 243 nm18 and is obviously undetectable in the bulk of proteinaceous material adhering to bacteria. However, the signicantly higher c(Ox) released by ammonium chloride plausibly suggests the formation of NH2Cl.

H3 N CH2 CH2 SO2 3 2NCT H $ NDCT taurine

Discussion
Type of chlorine compound
The observed ranking DCI-Na > CAT > NCT concerning the produced cover strength complies with their oxidative power19 and can be assigned to the different structures of NH bonds susceptible to chlorination. While the amine functions occurring in free amino acids, in N-terminals of peptides (proteins) and in basic side chains of amino acids (lysine) are easily chlorinated, the amide functions being present in form of peptide linkages need powerful agents and are not substituted by NCT. Furthermore, the attachment of a second chlorine atom to amine functions forming N,N-dichloro derivatives is not possible with NCT either. The pale yellow colour of pellets chlorinated with DCINa refers to the presence of NCl2 functions. The surprisingly low chlorinating potency of NDCT will be discussed in an upcoming specic study on this compound.

Obviously, the increasing portion of NCTH+ in the weak acid milieu promotes transhalogenation towards bacterial surfaces to a lesser degree than auto-chlorination of NCT to NDCT. Another feature concerns the promotive effect of increasing phosphate concentration. The most plausible explanation is a catalytic effect that, provoked by phosphate acting as a protondonating species,20 favours NCTH+ formation causing a faster chlorination (Equation 4).

4 A similar effect was observed by Antelo et al.21 concerning the disproportionation of NCT.

Chlorine covers on living bacteria

Since DCI-Na and CAT kill bacteria within <1 min at the applied concentration of 0.055 M Cl+, the goal to t bacteria with a chlorine cover without appreciable killing was feasible only with NCT. The conditions of 3 min incubation with 1% NCT in the presence of 0.1 M phosphate at pH 7.0 (standard procedure with NCT) represents a method that ensures a marked, well measurable chlorine cover and only little bacterial kill.

Inuence of pH
It is common knowledge that the bactericidal activity of oxidizing chlorine compounds increases with the proton activity.2 In case of CAT, the same dependence was also found concerning cover strength (Figure 3b). NCT behaved differently showing a maximum at pH 7 (Figure 3a). Both effects, the decrease in the weak acidic as well as in the weak alkaline region, seem to be a consequence of hydrolysis (Equation 1) producing the protonated

Bactericidal potential of chlorine covers

The unexpected viability of chlorinated bacteria (Figures 4 and 7) can be attributed to the nature of the attached c(Ox) that is present in form of covalent NCl bonds (the formation of O Cl bonds needs very strong chlorinating agents like hypochlorite, while in the case of NCT they can be excluded).

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Chlorine covers on living bacteria

The nearly constant c(Ox) of the pellets after two to seven washings conrms that it does not deal with an adsorption effect. In pure water or saline the only imaginable reactions are Equation 5a (hydrolytic splitting off of HOCl) and Equation 5b (comproportionation forming Cl2). Their equilibria, however, lie far on the left side so that virtually neither HOCl nor Cl2 is formed and the coated bacteria survive. M NHCl H2 O ! M NH2 HOCl M NHCl Cl2 H ! M NH2 Cl2 5a 5b cover formed in the natural environment by the myeloperoxidase hypochlorite system can exist. Nevertheless, the consequences of chlorination, basically consequences of the reactions specied under reaction (iii) (Equations 9a and 9b) have to be held responsible for the observed postantibiotic effect and attenuation of bacterial virulence.13,14,23 Subcultures of these bacteria exhibit their usual behaviour again, which conrms the transient nature of these alterations.

Approaching the mechanism of chlorine-based bacterial kill

Because of its low-level reactivity, NCT allows us to study the rst steps of the interaction between bacterium and chlorinating agent. Figure 4 shows that in the early stages ( < 10 min) of building up the chlorine cover, the killing rate of S. aureus ATCC 25923 depends on the incubation time and not on the formed cover strength. This indicates that the obviously slow penetration of NCT into the cell is the step determining the killing rate, and not the extent of the comparatively fast surface chlorination. Nevertheless, even after a sublethal incubation with NCT, intracellular changes of S. aureus have been observed by electron microscopy.14 With the highly reactive compounds CAT and chiey DCINa the high-level chlorine cover obviously exerts an immediate destructive impact on the bacterial surface. This enables rapid penetration of the quoted chlorinating molecules which, as sodium salts, are throughout charged species that are not prone to entering undamaged cells (unlike the non-charged and therefore lipophilic NH2Cl). According to the present understanding, the operation of active chlorine compounds on bacteria can be divided in a nonlethal and a lethal section. The former implies reversible chlorination of the bacterial surface. The latter is probably connected with penetration through the cell covers combined with irreversible alterations.

where M is the protein matrix of the bacterial surface. On the other hand, in the presence of reactants prone to transhalogenation such as free amino acids or, more pronounced, ammonium ions, highly bactericidal reaction products like NH2Cl emerge (Equation 6) and viability decreases.11,12,22 M NHCl NH ! M NH2 NH2 Cl H 4 6

In other words, the bactericidal potential of chlorine covers roots in a mobilization of the xed c(Ox) in form of low molecular weight bactericidal N Cl compounds. Metaphorically speaking, the chlorine cover can be compared with a time-bomb that is ignited by compounds prone for transhalogenation, particularly ammonium ions.

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The fate of chlorine covers within body uids

Since chlorine covers of bacteria exist only for a very short period within a natural environment like serum (Figure 6b), the question arises about the kind of alterations at the protein matrix of the bacterial surface that endure after reduction, i.e. the removal of the oxidizing chlorine. There exist three different mechanisms by which chlorine covers degrade:5,16 i) reaction with reducing substances: R NHCl R0 SH ! R NH2 R0 SCl ii) transhalogenation: R NHCl R0 NH2 ! R NH2 R0 NHCl iii) intra-molecular redox reactions (elimination of HCl): a R CH2 NHCl ! R CH NH !
2HCl H2 O

Conclusions
8 This rst systematic investigation of the phenomenon of bacterial chlorine cover provides the basics to understand the action of active chlorine compounds on microorganisms in general. In particular NCT enabled us to gain rst insights into the mechanism of bacterial kill and postantibiotic effect.

R CH O NH3 b NH CO CRH NHCl ! NH CO CR NH

H2 O 2HCl

9a

Acknowledgements
We thank Magdalena Hagleitner and Eva-Maria Lemberger for excellent technical assistance and Thomas Erbeznik for performing the ion-chromatographic chloride analysis. This study was supported by the Austrian Science Fund (grant no. P15240MED).

! NH CO CR O NH3 9b

Mechanisms (i) and (ii) leave behind the unchanged protein structure (Equations 7 and 8), while reaction (iii), which refers to both the basic side chain, e.g. of lysine (Equation 9a), and to the N-terminal of a peptide chain (Equation 9b) causes irreversible alterations of the protein matrix, leaving behind an aldehyde and the amide of an a-ketocarbonic acid.5 Reaction (ii) stands out in that the oxidation capacity is not destroyed but only released from the bacterial surface. Since the chlorine cover represents a manifestation of the action of active chlorine on bacteria that can be detected only in a non-reducing environment, it is debatable how long a chlorine

References
1. Semmelweis, I. P. (1861). Die Atiologie, der Begriff und die Prophylaxe des Kindbettebers, 1st edn. C.A. Hartlebens, Verlag, Wien and Leipzig, Germany. 2. Dychdala, G. R. (2001). Chlorine and chlorine compounds. In Disinfection, Sterilization and Preservation, 5th edn (Block, S. S., Ed.), pp. 135 58. Lippincott Williams & Wilkins, Philadelphia, PA, USA.

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3. Gottardi, W. (1988). Residual effects on the skin after application of disinfectants containing active halogen. Hygiene und Medizin 13, 157 61. 4. Gottardi, W. & Karl, A. (1990). Chlorine covers on skin surfaces. I. Determination of the cover strength by the DPD cuvette-method. Zentralblatt fur Hygiene und Umweltmedizin 190, 51122. 5. Gottardi, W. & Karl, A. (1991). Chlorine covers on skin surfaces. II. Parameters inuencing the cover strength. Zentralblatt fur Hygiene und Umweltmedizin 191, 478 93. 6. Gottardi, W. & Karl, A. (1991). The bactericidal effect of chlorine covers: a new model for the remanent skin disinfection. Hygiene und Medizin 16, 51 4. 7. Miche, L. & Balandreau, J. (2001). Effects of rice seed surface sterilization with hypochlorite on inoculated Burkholderia vietnamiensis. Applied and Environmental Microbiology 67, 304652. 8. Stelmaszynska, T. & Zgliczynski, J. M. (1974). Myeloperoxidase of human neutrophilic granulocytes as chlorinating enzyme. European Journal of Biochemistry 45, 305 12. 9. Thomas, E. L. (1979). Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli. Infection and Immunity 23, 522 31. 10. Weiss, S. J., Klein, R., Slivka, A. et al. (1982). Chlorination of taurine by human neutrophils. Journal of Clinical Investigation 70, 598607. 11. Nagl, M. & Gottardi, W. (1996). Enhancement of the bactericidal efcacy of N-chlorotaurine by inammation samples and selected NH compounds. Hygiene und Medizin 21, 597 605. 12. Nagl, M., Lass-Floerl, C., Neher, A. et al. (2001). Enhanced fungicidal activity of N-chlorotaurine in nasal secretion. Journal of Antimicrobial Chemotherapy 47, 8714. 13. Nagl, M., Hengster, P., Semenitz, E. et al. (1999). The postantibiotic effect of N-chlorotaurine on Staphylococcus aureus. Application in the mouse peritonitis model. Journal of Antimicrobial Chemotherapy 43, 8059. 14. Nagl, M., Hess, M., Pfaller, K. et al. (2000). Bactericidal activity of micromolar N-chlorotaurine evidence for its antimicrobial function in the human defence system. Antimicrobial Agents and Chemotherapy 44, 250713. 15. Marcinkiewicz, J., Czajkowska, B., Grabowska, A. et al. (1994). Differential effects of chlorination of bacteria on their capacity to generate NO, TNF-alpha and IL-6 in macrophages. Immunology 83, 6116. 16. Gottardi, W. & Nagl, M. (2002). Chemical properties of N-chlorotaurine sodium, a key compound in the human defence system. Archiv der Pharmazie 335, 411 21. 17. Thomas, E. L., Grisham, M. B. & Jefferson, M. M. (1986). Preparation and characterization of chloramines. Methods in Enzymology 132, 56985. 18. Snyder, M. P. & Margerum, D. W. (1982). Kinetics of chlorine transfer from chloramine to amines, amino acids, and peptides. Inorganic Chemistry 21, 2545 50. 19. Gottardi, W., Hagleitner, M. & Nagl, M. (2001). The inuence of plasma on the disinfecting activity of the new antimicrobial agent N-chlorotaurine sodium. Journal of Pharmacy and Pharmacology 53, 68997. 20. Valentine, R. L. & Jafvert, C. T. (1988). General acid catalysis of monochloramine disproportionation. Environmental Science and Technology 22, 6916. 21. Antelo, J. M., Arce, F., Calvo, P. et al. (2000). General acid-base catalysis in the reversible disproportionation reaction of N-chlorotaurine. Journal of the Chemical Society Perkin Transactions 2 2, 2109 14. 22. Nagl, M. & Gottardi, W. (1998). Rapid killing of Mycobacterium terrae by N-chlorotaurine in presence of ammonium is caused by the reaction product monochloramine. Journal of Pharmacy and Pharmacology 50, 131720. 23. Fuursted, K., Hjort, A. & Knudsen, L. (1997). Evaluation of bactericidal activity and lag of regrowth (postantibiotic effect) of ve antiseptics on nine bacterial pathogens. Journal of Antimicrobial Chemotherapy 40, 221 6.

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