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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Abstract |
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Methods: Pathogens were incubated in NCT, which was subsequently washed off. The oxidation capacity on the bacterial surface was measured photometrically.
Results: Superficial chlorination in the form of covalent NCl 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 x 1016 mol Cl+/cfu, while the cfu count was reduced by only 26%. The kind of microorganism, coating time, pH, buffer system and, basically, the chlorine compound, influenced 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 °C (>8 h at 1°C), 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 first 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
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Introduction |
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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.36 It is not the result of adsorption, but originates from covalent NCl 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.810
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 the myeloperoxidase system lost their ability to induce nitric oxide and tumour necrosis factor- in macrophages.15
These findings prompted us to establish methods of detection and quantification of chlorination of bacterial surfaces and to perform the first systematic examination of chlorine covers on Gram-positive and -negative bacteria and Candida albicans.
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Materials and methods |
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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 Scientific 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 °C overnight.
Chemicals and solutions
All reagents were of the highest available purity. Sodium N-chloro-4-toluenesulphonamide-sodium (chloramine T; CAT) and buffers were purchased from Merck, and dichloroisocyanuric acid sodium salt (DCI-Na) was purchased from SigmaAldrich (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.
5,5'-Dithiobis(2-nitrobenzoic acid) (Ellman's reagent; DTNB) and 2-mercaptoethanol as starting materials for synthesizing the reagent 2-nitro-5-thiobenzoic acid (TNB) were from SigmaAldrich. 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, max=325 nm; TNB,
. The following molar absorption coefficients were found: 325 nm,
,
; 410 nm,
,
.
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 23 (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 defined time (120 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 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.
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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 0.51.5 mL DPD solution or 1.05.0 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,
=553 nm,
=20500 L/cm/mol;4
TNB method: 0.10 cm;
=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 high cover strength could be expected. Testing the accuracy with CAT solutions of known c(Ox) revealed an inaccuracy of ± 5% for both methods.
Quantification 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:
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With the TNB method it is:
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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:
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Evidence of positive chlorine being the origin of the c(Ox) fixed 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 fitted 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 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:
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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.
Statistical analyses
Student's t-test was used for comparison of paired means of two groups of measurements. One-way analysis of variance and Dunnett's multiple comparison test (Graphpad Software Inc.) were applied for evaluation of more than two groups of measurements. P values of < 0.05 were considered significant.
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Results |
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The simplest approach for quantification of bacterial chlorine covers concerned c(Ox)/pellet, which was ideal for estimating the influence of parameters like chlorinating agent and reaction time. The pellet size ranged from 1 to 6 x 1010 cfu (49 x 109 for S. pyogenes, and 14 x 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 quantification 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 defined chlorinating procedure that is ambitious to attain (see below).
Evidence of positive chlorine being the origin of the c(Ox) fixed 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 fitted well with the c(Ox). From this finding it can be deduced that the bacterial chlorine cover is made up of covalent NCl bonds.
Quantification of bacterial chlorine covers
The standard procedure applied to S. aureus ATCC 25923 produced a chlorine cover of 3.3 x 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 x 1016 mol Cl+, which originates from 9.6 x 107 NCl bonds at its surface. This strength of c(Ox) in the form of superficial NCl bonds was measured after 3 min of incubation time, and it was survived by the discrete microorganism (see Figure 4).
Influence of coating parameters on cover strength
Type of chlorine compound. The relative strength of chlorine cover per pellet affixed 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.
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pH and buffer concentration. While lowering the pH clearly increased the strength of the cover affixed 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 better-suited to work out the real differences between microorganisms (Figure 5b). The high value of C. albicans can be explained by its higher bulk compared with bacteria.
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In the absence of organic matter interacting with the NCl function, chlorine covers are rather stable dependent on the temperature. At 20 °C, 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 °C (ice cooling), the c(Ox) remained stable for at least 8 h after a 30% reduction during the first 2 h.
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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 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 NCl bond by transhalogenation. The term transhalogenation was defined for the transfer (exchange) of positive halogen between amine compounds. Contrary to the substitution of a CH 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.
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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 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 unidentified 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 significantly higher c(Ox) released by ammonium chloride plausibly suggests the formation of NH2Cl.
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Discussion |
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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 DCI-Na refers to the presence of NCl2 functions. The surprisingly low chlorinating potency of NDCT will be discussed in an upcoming specific study on this compound.
Influence 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 species NCTH+, which is the real active agent executing transhalogenation.16
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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 proton-donating species,20
favours NCTH+ formation causing a faster chlorination (Equation 4).
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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 fit 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.
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 OCl bonds needs very strong chlorinating agents like hypochlorite, while in the case of NCT they can be excluded). The nearly constant c(Ox) of the pellets after two to seven washings confirms 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.
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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
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The fate of chlorine covers within body fluids
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
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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 -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 cover formed in the natural environment by the myeloperoxidasehypochlorite system can exist. Nevertheless, the consequences of chlorination, basically consequences of the reactions specified 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 confirms 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 first 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 chiefly DCI-Na 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 non-lethal 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.
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Conclusions |
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Acknowledgements |
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References |
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