Journal of Antimicrobial Chemotherapy

JAC Advance Access originally published online on April 17, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1114-1122; doi:10.1093/jac/dkm094

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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Evaluation of amino acids as mediators for the antibacterial activity of iodine-lithium--dextrin in vitro and in vivo

Tigran K. Davtyan^1,*, Isabelle S. Hakobyan¹, Rafael E. Muradyan², Hrachya G. Hovhannisyan³ and Emil S. Gabrielyan²

¹ Laboratory of Immunology and Virology, Armenicum Research Center, Yerevan, Republic of Armenia ² Drug and Medical Technology Agency of Ministry of Health, Yerevan, Republic of Armenia ³ Institute of Microbiology of Armenian National Academy of Sciences, Abovyan, Republic of Armenia

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^* Corresponding author. Tel: +374-1-74-14-31; Fax: +374-1-54-80-13; E-mail: tigdav{at}excite.com

	Abstract

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Objectives: The systemic therapeutic application of iodophores has not yet been accepted due to limited availability of safe and effective ionized iodine preparations. Here we evaluated the antibacterial activity of iodine-lithium--dextrin (ILD) both in vitro and in vivo.

Methods: The MIC values of ILD against 189 bacterial isolates in various growth media and in vivo toxicity and protective efficacy of ILD in preventing mortality of rats infected with Staphylococcus aureus were determined. The intracellular killing of S. aureus by neutrophils in the presence of ILD and myeloperoxidase (MPO)-catalysed oxidation of iodide was also determined.

Results: The MIC values of ILD against 189 Gram-positive cocci and Gram-negative bacilli ranged between 124–512 mg/L in growth media and 6.2–12.5 mg/L in buffer solution, and were highly variable in the presence of amino acids. We observed protection of S. aureus-infected rats from death with significant reduction of bacterial growth in organs upon intravenous administration of ILD at doses that are 4–12 times lower than maximal in vivo tolerability dose. Intracellular killing of S. aureus by neutrophils increased in the presence of ILD probably due to MPO-catalysed oxidation of iodide into hypoiodous acid. The pattern of ILD reaction with amino acids at different pH or halide ion content determined both the generation of long-lived secondary oxidants and antibacterial activity.

Conclusions: Systemic application of ILD proved to be successful in the rat infection model by promoting host defence. Probable mechanisms are increased intracellular killing of bacteria by production of hypoiodous acid and iodamines as well as anti-inflammatory activity.

Keywords: intracellular killing , iodophores , myeloperoxidase-catalysed oxidation of iodide , microbiocides

	Introduction

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Microbicides destroy viruses and bacteria. Iodophores such as povidone–iodine are broad-spectrum microbicides with activity against bacteria, viruses, fungi and protozoa.¹ They consist of elementary or ionized iodine bound to polymer carriers (such as poly-1-vinyl-2-pyrrolidine and dextrins), which increase solubility and provide a reservoir of iodine. Due to the oxidizing effects of free iodine on key groups of proteins, nucleotides, fatty acids and the subsequent non-specific mechanism of cell killing, iodophores are used as potent microbicides. They do not lead to the development of microbial resistance allowing for the repeated antimicrobial usage of such substances in topical formulations.^2–4 However, the systemic therapeutic application of iodine has not yet been accepted and suitable iodine preparations have not been investigated to date.

The polysaccharide chain of single-helical V-amylose (as in cyclodextrins or -dextrins), consisting exclusively of (1-4)-linked glucose residues, is folded into a left-handed screw helix that contains six glucoses per turn with an 8 Å pitch.^5–7 In the complex with the polyiodide, they form a channel-type structure, occupied by disordered (I₂I₃^–)_n depending on the counterion Li.^8–10 Water-solubility of lithium-iodine--dextrin (ILD) was achieved by polymerization with polyvinyl alcohol, known to be able to form an inclusion complex with iodine and increasing the surface-active properties of polyiodide screwed polysaccharides.^11,12

Recently we have shown that ILD diluted in saline solution displayed marked bactericidal activity against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Streptococcus pyogenes and Yersinia enterocolitica during 0.5–30 min exposures. The bactericidal efficacy of ILD was fast and proportional to the concentration of iodine.¹³ Although ILD exhibited pronounced bactericidal activity against S. aureus and S. pyogenes in higher dilutions in which iodine concentration varies over a relatively low range (30–50 mg/L), the bactericidal efficacy was highly variable, depending on growth media composition. The quantitative suspension test showed that the bactericidal efficacy of ILD against S. aureus and S. pyogenes decreased to 3.6 and 2.6 log₁₀ when diluted in nutrient broth, whereas the bactericidal activity of ILD diluted in saline solution increased more than 10-fold and 8.0 log₁₀ reductions of S. aureus and S. pyogenes were observed.¹⁴

Here we studied the in vitro antibacterial efficacy of ILD against Gram-positive and Gram-negative bacteria in various growth media and the antibacterial activity of ILD in the presence of amino acids. We also examined the in vivo toxicity and protective efficacy of ILD in preventing mortality and reducing the number of viable bacteria in organ homogenates and the blood of rats infected with S. aureus. The intracellular killing of S. aureus by neutrophils in the presence of ILD and MPO-catalysed oxidation of iodide as well as in the presence of different stimulators, amino acids and pH or halide ion contents was also determined.

	Materials and methods

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Study material, reagents and media

ILD ‘Armenicum^TM’ obtained from ‘Armenicum’ CJSC (Yerevan, Armenia) is an aqueous solution of iodine–lithium inclusion complex with low molecular weight -dextrin and polyvinyl alcohol. ILD contains 0.16 g of iodine, 0.24 g of potassium iodide, 0.004 g of lithium chloride, 0.06 g of polyvinyl alcohol, 20 g of -dextrin and 0.114 g of sodium chloride in a total volume of 20 mL.¹¹ Working dilutions of ILD were prepared after vigorous shaking and quick v/v dilution in 0.15 M PBS or in M9 minimal salt medium. N-formyl-Met-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA) and amino acids were purchased from Sigma Chemical Co. (St Louis, MO, USA). The liquid media used were nutrient broth (NB), Muller-Hinton broth (MHB; Sigma Chemical Co.) and the solid media were nutrient agar and desoxycholate citrate agar (Oxoid, UK).

Bacterial isolates

The clinical isolates of E. coli (22), S. aureus (36), S. typhimurium (32), Salmonella typhi (29), S. pyogenes (20), Klebsiella pneumoniae (8), Proteus vulgaris (11) and Pseudomonas aeruginosa (31) were derived from the Bacteriology Dept of the Armenian National Institute of Health. All isolates were identified by conventional biochemical profiles according to the CLSI (formerly NCCLS) criteria.¹⁵ In addition, reference strains S. aureus 209D and E. coli K-12 () derived from Culture Collection of Institute of Microbiology of National Academy of Sciences (Abovyan, Armenia) were used.

In vitro antibacterial activity testing

MICs for ILD were determined by the broth dilution method according to the CLSI.¹⁶ Serial 2-fold dilutions of ILD were prepared in 1 mL volumes of MHB. Each bacterial isolate was washed three times and suspended in 5 mL of sterile saline solution, adjusted to a turbidity equivalent to a 0.5 McFarland standard. Bacterial suspension was diluted 1:100 with broth with a final inoculum of 5 x 10⁵ or 5 x 10⁷ cfu/mL. After 24 h of incubation at 37°C, the MIC value was recorded as the lowest iodine equivalent concentration of ILD that inhibited visible growth compared with the control growth tube. To detect the variations in antibacterial efficacy of ILD in various growth media, the same procedure was repeated using PBS for initial dilution of ILD. In addition, gentamicin sulphate (Sigma Chemical Co.) was used as a control compound, the MIC values of which against E. coli and Staphylococcus spp. were found to be 0.25–2 mg/L either diluted in PBS or in MHB.

The time–killing studies against E. coli K-12 and S. aureus 209D were performed with ILD at double the MIC doses. The mid-logarithmic phase preparations were diluted in ILD-containing NB or M9 (PBS) test tubes (2 mL) to achieve a final inoculum of 2.5–3 x 10⁸ cfu/mL. The tubes were incubated at 37°C for 18 h and aliquots of 10 µL were removed after 1 h and every following 2 h and spread on agar plates. cfu were counted after 24 h of incubation at 37°C. The detection limit was 100 cfu/mL.

Antimicrobial activity of ILD against E. coli K-12 in the presence of amino acids was carried out in 1 mL M9, containing 1:100 v/v dilution of ILD (80 mg/L final iodine concentration) and 0.1 mL E. coli K-12 suspension in NB. Stock solutions of individual amino acids at final concentration 20 mg/mL were diluted 2-fold in test tubes containing diluted ILD and the number of bacteria was adjusted in each individual test tube to 3 x 10⁸ cfu/mL. Each series of experiments contained duplicates of test tubes and appropriate controls. After 24 h of incubation at 37°C, the MIC value was recorded.

In vivo tests

All animal studies were carried out in accordance with the Code of Practice for the Housing and Care of Animals used in Scientific Procedures 1989 after approval by the Pharmacological Committee of the Drug and Medical Technology Agency of the Ministry of Health of Armenia.

S. aureus 209D was passaged several times through Wistar strains of rats to enhance its virulence. The 50% minimum lethal dose (MLD₅₀) of untreated rats was found to be 1.2 x 10⁹ cfu per rat by the intravenous route of challenge. To determine the toxicity and lethal doses of ILD that kill 50% and 100% animals (LD₅₀ and LD₁₀₀), 150 male rats (4–6 weeks old, weighting range 50–100 g) were included in the test and 50 rats were included in control groups. Rats were intravenously (iv) administered with the ILD, diluted in sterile 0.9% NaCl solution at 0.8–100 mg final iodine concentration per kg and were kept under observation for up to 30 days.

Eighty rats (each rat weighing 70 g) were iv challenged with 1 MLD₅₀ of S. aureus 209D and divided into four groups. After 48 h of infection, the experimental animals (20 rats in each group) were one-time iv administered with the ILD, diluted in sterile 0.9% NaCl solution at either 1.6, 3.2 or 4.8 mg/kg final iodine concentration and control animals (n = 20) were injected iv with 0.9% NaCl solution. The protective capacity of ILD was determined by recording the mortality and results were compared statistically by ² test. Six animals from control and each test group were euthanized 24 h after ILD treatment in order to assess tissue burden of organs. Their blood was collected aseptically via cardiac puncture and their liver, spleen, lung and kidney were removed aseptically and homogenized in tissue homogenizers, and cfu counts of the individual organs were determined and statistically analysed by Student's t-test.

Intracellular killing of bacteria

Human peripheral blood neutrophils were isolated as previously described.¹⁷ Purified cells were suspended at 1 x 10⁷ cells/mL in Hanks balanced salt solution (HBSS), containing 10% human AB serum. The overnight culture of S. aureus 209D (1 x 10⁷ cfu/mL) was opsonized by incubation with 0.1% gelatin and 10% human AB serum in HBSS and a 1 mL suspension of opsonized bacteria (1 x 10⁷ cfu/mL) was added to 100 µL of HBSS (1 x 10⁷ cells/mL).¹⁸ Cells were incubated with bacteria for 10 min at 37°C with continuous rotation to promote phagocytosis and non-ingested bacteria were discarded by differential centrifugation for 5 min at 1200 rpm. Cells containing ingested bacteria were cultured for the next 45 min at 37°C with slow rotation in the presence of ILD, diluted in HBSS at 20 mg/L and 40 mg/L final iodine concentration.¹⁴ Killing was stopped by placing the cells on ice after adding 1 mL of distilled water containing 0.01% BSA. Cells were disrupted by vigorous vortexing, and the number of viable bacteria was determined by plating 10-fold serial dilutions. The percentage of killing was calculated as [1-(number of viable bacteria at 45 min/number of viable bacteria at 0 min)] x 100 in the presence or absence of ILD. In order to detect a possible contribution of Cl^– ions to ILD-mediated intracellular killing of bacteria, the same procedure was conducted using a Cl^–-free medium.

Oxidation of ILD-derived iodide by neutrophils

MPO-catalysed oxidation of ILD-derived iodide was studied using isolated human neutrophils according to Thomas and Fishman.¹⁹ Neutrophils (4 x 10⁶) either in 0.5 mL Cl^– (HBSS) or Cl^–-free (0.1 M Na₂SO₄, 1 mM MgSO₄, 1 mM glucose, pH 7.4) media were incubated for 30 min at 37°C with 100 ng/mL fMLP or 20 ng/mL PMA or 3 mM amino acids or 0.03 mM H₂O₂ in the presence or absence of different doses of ILD. Cells were washed and suspended in media with or without Cl^–, containing 10 mM taurine, and were incubated for an additional 1 h at 37°C. The reaction was stopped by the addition of 50 mg/L catalase. Oxidant concentration (µM) in cell-free supernatant was calculated by adding 5-thio-2-nitrobenzoic acid (TNB) and the decrease in A₄₁₂ was measured (₄₁₂ 14 100/M · cm).²⁰ Formation of oxidants in vitro was studied in media with or without Cl^– at pH 7.4 and 6.0 in reactions started by adding 10 mM taurine, 150 nM MPO and different doses of ILD. Reactions were run at room temperature for 10 min and the concentration of oxidant present in the reaction system was then measured by determining its ability to bleach TNB.²¹ Dose–response curves were generated using Graph Pad Prism v4.01 software.

	Results

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In vitro antibacterial activity of ILD

The MIC values of ILD diluted both in MHB and in PBS were determined for clinical isolates with a final inoculum of 5 x 10⁵ or 5 x 10⁷ cfu/mL (Table 1). The MIC values of ILD diluted in MHB for S. aureus, S. pyogenes, K. pneumoniae and P. vulgaris isolates ranged between 124 and 256 mg/L and for S. typhimurium, S. typhi, P. aeruginosa and E. coli isolates ranged between 256 and 512 mg/L. MIC values of ILD diluted in PBS for these isolates ranged between 6.2 and 12.5 mg/L. Thus, the in vitro antibacterial activity of ILD was dependent on the growth media composition, bacterial inoculum and studied strain.

View this table: [in this window] [in a new window]   Table 1.. MIC values of ILD diluted in MHB and in PBS against clinical isolates to a final inoculum of 5 x 105 and 5 x 107 cfu/mL

 
ILD diluted in MHB loses carrier-bound iodine-dependent bactericidal activity by interacting with nutrients i.e. amino acids, peptides, proteins etc. existing in the broth. The MIC of ILD diluted in M9 (or PBS) and NB against E. coli K-12 () and S. aureus 209D was found to be 6.2 and 200 mg/L, respectively. At the logarithmic growth phase of culture (cfu 2.5–3 x 10⁸), 12.5 mg/L and 400 mg/L (double the MIC) of ILD were added to each culture. Subsequently, the cfu counts of the cultures were determined (Figure 1). For E. coli K-12 and S. aureus 209D more than 6.0 log₁₀ reduction was observed within 60 min by ILD diluted in M9/PBS, whereas only 3–4 log₁₀ reduction was observed within 4–18 h exposure when ILD was diluted in NB. Thus, ILD diluted in M9/PBS had higher antibacterial activity. Next, we studied the effect of amino acids on antibacterial activity of ILD against E. coli K-12 (Table 2). Some of amino acids tested i.e. Met, Trp, Ile and Lys completely abolished the antibacterial activity of ILD. While His, Tyr, Thr, Ser, Arg and Cys as well as human serum albumin caused a 2–8-fold decrease in ILD antibacterial activity. However, in the presence of Val, Leu and Pro, the antibacterial activity of ILD increased more than 2-fold. No changes of antibacterial activity of ILD were observed in the presence of other amino acids. Thus, amino acids containing two aliphatic methyl groups (Val and Leu) and pyrrolidine derivates (Pro) increased the antibacterial efficacy of ILD, while other amino acids decreased or did not influence the in vitro antibacterial activity of ILD. These results suggest that antibacterial efficacy of ILD is highly variable, depending upon interactions with amino acids or proteins, and therefore in vivo it could possess toxicity rather than antibacterial activity.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1.. Time–killing curves of ILD against E. coli K-12 () (continuous line) and S. aureus 209D (broken line) at double the MIC doses diluted in M9 or PBS (12.5 mg/L) and NB (400 mg/L). Bacteria were incubated without ILD in M9/PBS (growth control). Data points are means ± SEM of six experiments.

 

View this table: [in this window] [in a new window]   Table 2.. Influence of amino acids on in vitro antibacterial activity of ILD against E. coli K-12

 
In vivo antibacterial activity of ILD

We tested whether iv administration of ILD is safe and could protect animals in vivo from experimental S. aureus infection. First, we studied the in vivo toxicity of ILD in rats. The LD₅₀ and LD₁₀₀ were found to be 42.4 (36.6–49.2 range) and 80.0 (75.8–91.4 range) mg of final iodine concentration per kg for rats, respectively. The corresponding maximum tolerability dose for iv route of ILD administration was found to be 20 mg/kg. The main toxic symptoms preceding the death of animals after administering lethal doses of ILD were an acceleration of respiration, immobility, inhibition of behavioural reactions, tremor and exopththalmia. Death usually ensued within the first 5–10 minutes following the iv administration of lethal doses of ILD. The symptoms of intoxication (transiently occurring phlebitis at the site of injection, general weakness and loss of appetite) noted in the surviving animals were transitory in nature. At the end of the experiment, the animals' general condition and body weight did not differ from the initial indices and control group. ILD did not exert an influence on the animals' behaviour, body weight, temperature and food and water consumption. Haematological, biochemical, morphological and histological investigations revealed no abnormalities (data not shown).

Next, we studied the in vivo protective effect of ILD at 1.6, 3.2 and 4.8 mg/kg doses, which were 4–12.5 times lower than the maximum tolerability dose for iv route of administration. S. aureus when delivered by the systemic route caused fatal disease within 5 days in non-treated, infected control rats (Figure 2). Survival rates were significantly higher in animals treated after 48 h of infection with any of the three doses of ILD (P < 0.001, ² test) compared with controls. However, there were no significant differences in the survival rates when different doses were compared with each other (P > 0.05, ² test). We observed, respectively, 83.3%, 85.7% and 87.5% surviving rats for 1.6, 3.2 and 4.8 mg/kg doses of ILD on day 12 (Figure 2). Table 3 demonstrates that all three doses of ILD significantly reduced viable bacterial count from heart blood, liver, spleen, lung and kidney of rats at 24 h after challenge, compared with untreated control rats. Interestingly, ILD reduced the number of viable bacteria in a dose-dependent manner in the blood and spleen, whereas in the liver, lung and kidney the dose-effect relation was not statistically significant. Thus, ILD in vivo protects S. aureus infected rats from death and reduces bacterial growth in blood and organs, which in contrast to in vitro antibacterial efficacy mainly is not dose-dependent. Therefore, we hypothesized that in addition to its direct antimicrobial action, ILD in vivo could possess the potential to modulate microbial cell ingestion and subsequently enhance their intracellular killing by innate immune cells due to the oxidation of iodide.

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2.. Survival of rats challenged with S. aureus 209D intravenously and treated after 48 h of infection with 1.6, 3.2 and 4.8 mg/kg doses of ILD. Animals were iv challenged with 1 MLD50 of S. aureus and after 48 h of infection, experimental animals (20 rats in each group) were iv administered with the ILD, diluted in sterile 0.9% NaCl solution at 1.6, 3.2 or 4.8 mg/kg concentration. Control untreated animals (n = 20) were injected iv with 0.9% NaCl solution.

 

View this table: [in this window] [in a new window]   Table 3.. Reduction of cfu of S. aureus 209D at 24 h following treatment with ILD in heart blood and organ homogenates of rats

 
ILD effect on antibacterial activity of neutrophils

The ILD effect on bactericidal activity of human peripheral blood neutrophils against S. aureus and possible contribution of halides to the ILD-mediated intracellular killing of bacteria were also studied. In the presence of both two doses (20 and 40 mg/L) of ILD, the killing of S. aureus by neutrophils significantly increased in media with and without Cl^– (compared with non-treated cells: P = 0.04), however the best effect was observed for media without Cl^– compared with HBSS (P = 0.02) (Figure 3a). In the case of non-treated cells (controls), killing of S. aureus in HBSS was more effective than in media without Cl^– (P = 0.01). There were no significant differences in the intracellular killing of bacteria when different doses of ILD were compared in both media (with and without Cl^–). Thus, ILD possesses pronounced capability to stimulate intracellular killing of bacteria in a dose-independent manner, especially in media without Cl^–.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3.. ILD effect on intracellular killing of S. aureus (a) and oxidation of ILD-derived iodide by human peripheral blood neutrophils (b). Neutrophils either in media with or without Cl– were incubated with 100 ng/mL fMLP or 20 ng/mL PMA in the presence of 40 mg/L ILD and intracellular killing of bacteria and hypohalous acid formation were measured as described in the Materials and methods section. All data represent means ± SEM (error bars) of six independent experiments.

 
Next, we studied whether the ILD-induced activation of intracellular killing is dependent on MPO-catalysed oxidation of iodide (I^–) as one of key compounds of ILD. Neutrophils either in media with or without Cl^– were activated by fMLP or PMA in the presence or absence of ILD. MPO-catalysed oxidation of I^– or Cl^– into hypohalous acids (HOCl, HOI) was determined by scavenging with taurine (converted into membrane-impermeable taurine iodamine or taurine chloramine capable of bleaching TNB).^19,20 As shown in Figure 3(b), ILD at 40 mg/L iodine equivalent concentration in media without Cl^– caused high accumulation of oxidants (16.5 ± 2.1 µM) in extracellular medium, while a low yield of oxidants was obtained at the same concentration of ILD, diluted in Cl^–-containing media (3.9 ± 1.1 µM; P = 0.04). A high yield was obtained with 20 ng/mL of PMA and a yield similar to that with ILD was obtained with 100 ng/mL of fMLP as the stimulus in both Cl^– and Cl^–-free media. However, adding ILD significantly increased the yield of oxidants by PMA- and fMLP-stimulated neutrophils in comparison with PMA or fMLP alone (P = 0.02 and P = 0.04, respectively) and these effects were observed either in media with or without Cl^–. In order to test how these effects of ILD are dose- and stimulus-dependent, neutrophils in media without Cl^– were activated by fMLP, PMA or incubated with amino acids Met, Pro and Cys or H₂O₂ in the presence of different doses of ILD. Dose–response curves of oxidant accumulation in the presence of both neutrophil stimulators, amino acids or H₂O₂ starts at the bottom of 2.5–10 mg/L iodine equivalent concentration of ILD and goes to plateau at the top 20–40 mg/L concentration of ILD (Figure 4a and b). In the absence of ILD, there were no oxidants obtained by neutrophils in the presence of 0.03 mM amino acids and H₂O₂ and low yield (3.7 ± 1.9 µM) was obtained in the presence of either PMA or fMLP, at 10 times lower than their optimal concentration (Figure 4a). However, adding 2.5–40 mg/L ILD increased the oxidant yield up to 92 µM in the presence of PMA, 80–85 µM in the presence of H₂O₂ and fMLP, and surprisingly up to 75 µM in the presence of Met. A low yield of oxidants (20 µM) was obtained with Cys and Pro in the presence of 2.5–40 mg/L ILD (Figure 4b). Thus, ILD significantly increased the yield of taurine iodamine by neutrophils in Cl^–-free media and enhanced the accumulation of hypohalous acids by PMA- and fMLP-stimulated neutrophils either in Cl^– or Cl^–-free media.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4.. Dose- and stimulus-dependent oxidation of ILD-derived iodide by neutrophils in media without Cl–. Cells were activated by fMLP or PMA (a) or incubated with amino acids Met, Pro and Cys or H2O2 (b) in the presence of different doses of ILD. Dose–response curves were generated using Graph Pad Prism v4.01 software.

 
Although ILD-activated neutrophils in the presence of stimuli and amino acids generate various amounts of hypoiodous acids in a saturation dose–response manner, they are highly unstable reactive oxidants capable of generating long-lived secondary oxidants. We tested how ILD alone in the absence of neutrophils (MPO-halide system) could interact with amino acids. ILD differentially oxidizes taurine in media with or without Cl^–, depending on pH milieu (Figure 5). In media without Cl^–, ILD oxidizes taurine and leads to accumulation of oxidants, which is linearly dependent on the dose of ILD and independent of pH of reaction media. However, saturation dose–response curves were obtained for ILD-mediated taurine iodination in media with Cl^–, which is highly dependent on pH. In media with Cl^–, the maximal yield of oxidants by ILD as well as enhancement of antibacterial activity against E. coli K-12 were found at nearly acidic (pH 6) milieu; whereas in neutral pH, antibacterial activity and taurine iodination were decreased 2-fold. In contrast, both the taurine iodination and antibacterial activity of ILD were found to be similar in Cl^–-free media at pH 6 and pH 7.4. Similarly, we observed that interaction of ILD with Pro and Cys and subsequent oxidant yield is also dependent on pH and the presence of Cl^– ions (data not shown). Thus, the pattern of ILD interaction with amino acids at different pH or halide ions content determined both the generation of long-lived secondary oxidants and antibacterial activity.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5.. Interaction of ILD with taurine in media with or without Cl– at different pH values. Formation of oxidants was assayed in reactions started by adding different doses of ILD to 150 nM MPO/10 mM taurine in Cl– (grey lines) and Cl–-free (black lines) media at pH 6.0 (continuous lines) and pH 7.4 (broken lines) and the concentration of oxidant (left axis) was then measured by determining its ability to bleach TNB. The MIC values (right axis) of ILD, diluted in Cl– and Cl–-free media at different pH values, against E. coli K-12 () were measured in the presence of 10 mM taurine as described in the Materials and methods section.

 

	Discussion

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At least seven iodine species (I₂, HOI, [H₂O I]⁺, I₃^–, I^–, OI^– and IO₃^–) are present in the solution as a complex equilibrium system with molecular and oxidized iodine being responsible for antimicrobial efficacy. Although aqueous or alcoholic solutions of iodine have been used for more than 150 years as antimicrobials, they are generally unstable in solution and associated with irritation and cytotoxicity.² These problems were overcome by the development of iodophores i.e. iodine-carriers, which are complexes of iodine and serve as a solubilizing agent and a reservoir of the iodine.^1,22 Due to its excellent antimicrobial properties, iodophores such as povidone–iodine are used in numerous topical formulations as well as for control of nosocomial infection.^23–27 Here we described the simple iodine–lithium inclusion complex with low molecular weight -dextrin and polyvinyl alcohol which is a safe and powerful antibacterial agent capable of preventing mortality and reducing bacterial growth in the organs and blood of infected rats.

The MIC values of ILD against 189 bacterial isolates in MHB and saline solution demonstrate that in vitro antibacterial activity of ILD was unstable in nutrient-rich growth media. These results coincide with the reported in vitro antibacterial activity of povidone–iodine in saline solution and in the presence of organic substances.^3,25 Moreover, the antibacterial efficacy of ILD is highly variable, depending upon interaction with amino acids. These results suggest that ILD interacts with amino acids due to oxidizing effects of iodine on key functional (amino, hydroxyl or thiol) groups yielding compounds which are bacteriostatic rather than bactericidal. The hypothesis that the interaction of ILD with amino acids or proteins could lead to either high toxicity or a decrease in ILD antibacterial efficacy in vivo was not confirmed. Here, we provided evidence that iv route of ILD administration is associated with both low in vivo toxicity and prevention of mortality with reduction of the number of viable bacteria in the organs and blood of rats infected with S. aureus.

The maximal tolerated dose of ILD in the in vivo experiments was 2–3 times higher than the MIC of the compound in saline solution and 6–25 times less than the MIC in MHB, and even at LD₅₀ dose its concentration was 3–12 times lower than the MIC value of ILD in MHB. The iv route of ILD administration caused prevention of mortality and reduction in the number of viable bacteria in organs and blood of rats infected with S. aureus at doses that are 1.9–3.9 times lower than the MIC in saline solution and 38.8–155 times lower than the MIC value of ILD against S. aureus in MHB. These results together with the data we obtained on instability of ILD antibacterial activity in the presence of organic substances suggested that ILD at the maximal tolerated dose in vivo appears to be neither bactericidal nor sufficient for direct killing of bacteria in the blood. However, ILD at the maximal tolerated dose was sufficient for increasing the intracellular killing of bacteria by neutrophils, depending on MPO-mediated oxidation of iodide into hypoiodous acid. The increasing of intracellular killing of bacteria by neutrophils in the presence of ILD was not a result of direct attenuation or killing of bacteria by the ILD, because the compound was added to cells containing ingested bacteria.

The most intriguing feature of ILD is the dose-independent mode of its in vivo action on the survival of lethally infected rats. There was no significant difference in the survival rates when various doses of ILD were compared with each other (Figure 2). In addition, ILD in blood and, to lesser extent, in spleen reduced the number of viable bacteria in a dose-dependent manner, whereas in liver, lung and kidney the dose-effect relation was not significant. We proposed three possible mechanisms for explanation of the dose-independent mode of in vivo antimicrobial action of ILD: (i) in addition to its direct antimicrobial action, ILD in vivo could possess the potential to modulate intracellular killing of bacteria due to the oxidation of iodide by innate immune cells in a saturation dose–response manner; (ii) ILD alone or in the presence of intracellular MPO-halide system could generate long-lived secondary oxidants such as mono-, di-haloamines, amino acids and thiol derivates, the in vivo antimicrobial action of which could depend on different factors, including pH, available halide ions, endogenous pro- or antioxidants etc.; (iii) ILD could enhance the survival of infected rats not only by promoting antimicrobial host defence; it could also serve as a potent inhibitor of infection-mediated inflammation.

The first proposed mechanism of ILD action in vivo was confirmed by studying the intracellular killing of S. aureus by neutrophils in media with and without Cl^–, where it was shown that ILD possesses pronounced capability to stimulate intracellular killing of bacteria in a dose-independent manner, particularly in media without Cl^–. It is well known that neutrophils utilize respiratory burst-derived H₂O₂ in combination with MPO and halides to generate reactive oxidants that can kill bacterial, fungal and viral pathogens.^28,29 The overall reaction is: H₂O₂+X^–+H⁺ HOX + H₂O₂ where X^– = Cl^–, Br^–, SCN^– and I^–, and HOX is the corresponding hypohalous acid.^30,31 Moreover, when the pH is lowered, as may occur in the phagosome, HOX predominates and could react with excess halides to form chlorine (Cl₂), bromine (Br₂) or iodine (I₂).³² These products are highly reactive and short-lived oxidizing agents that can attack pathogens at a variety of chemical sites.^33–35 Cl^– is generally accepted to be a physiological halide, as it is present at high concentrations in plasma (100–140 µM).^30,31 I^– is the most effective halide on molar basis; however, the concentration of free I^– in biological fluids is very low (<1 µM).^36,37 Br^– and SCN^– are intermediate between Cl^– and I^– in effectiveness and concentration (20–120 µM in plasma). Despite this, when I^– is the halide, iodination is detected in the phagosome and iodination appears to involve neutrophil constituents, extracellular proteins and ingested bacteria.^38–40 ILD at the in vivo maximal tolerated dose (corresponding to 100 µM equivalent iodine concentration) which is sufficient for both increasing the intracellular killing of bacteria by neutrophils and MPO-mediated oxidation of iodide into hypoiodous acid (Figures 3 and 4) in a molar basis is 100 times in excess of the physiological concentration of I^–. Moreover, the iv route of ILD administration at the doses that are in a molar basis 8–32 times in excess of the physiological concentration of I^– caused prevention of mortality and a reduction of the number of viable bacteria in the organs and blood of rats infected with S. aureus. These data suggested that ILD at either the therapeutic or tolerated doses is high enough to provide a sufficient amount of iodide for neutrophils to form considerable amounts of hypoiodous acid. Using dihydrorhodamine-123 as a probe that reacts with the pool of reactive oxygen species (HO^•, H₂O₂ and HOX), we have previously shown that the ILD effect on phagocytic cell oxidative burst is similar to the respiratory burst low stimulant fMLP effect.¹⁴ In the present study, we used another approach to address how the ILD-induced activation of intracellular killing of bacteria is dependent on MPO-catalysed oxidation of halides into hypohalous acids by determining the conversion of taurine into taurine iodamine (chloramine) in activated neutrophils. We showed that ILD significantly increased the yield of taurine iodamine by neutrophils in Cl^–-free media and enhanced the accumulation of hypohalous acids by PMA- and fMLP-stimulated neutrophils either in media with or without Cl^–. The enhancement of taurine chloramine formation upon neutrophil stimulation by PMA, H₂O₂ and fMLP in the presence of physiological concentrations of Cl^– is well known.^19,20 However, the observed increase in oxidant yield by ILD in the presence of amino acids is surprising. It is known that both Met and Cys can react with hypochlorous acids and cause the neutralization of the latter's oxidizing potential.²¹ How these results reflected the ability of ILD to generate the long-lived secondary sulphur-centred oxidants is presently unknown; however our preliminary results suggested that ILD in the presence of sodium thiosulphate caused up-regulation of human neutrophil oxidative burst.⁴¹ Therefore, we suggest that an additional mechanism of ILD-mediated intracellular killing of bacteria could be a generation of long-lived secondary oxidants.

We speculated that ILD per se could possess the MPO-like activity even in the absence of neutrophils. We observed a linear dose-dependent generation of taurine iodamine and in vitro pH-independent antimicrobial activity of ILD only in media without Cl^–. In media with Cl^– both the formation of taurine iod(chlor)amine and in vitro antimicrobial activity were shown to be pH- and saturable dose-dependent. Interestingly, the optimal pH for in vitro antimicrobial activity of ILD was found to be acidic as it was shown earlier for both taurine brom- and chloramine.^42,43 Thus, essentially, any oxidizable chemical group on host or pathogen molecules e.g. sulphydryl, amino, hydroxyl, sulphur-ether, hems groups, iron–sulphur centres and unsaturated fatty acids can be oxidized by ILD and some of them, particularly iodamines, retain oxidizing activity. As taurine is the most abundant free amino acid in leucocyte cytosol,⁴⁴ the major haloamines generated by activated neutrophils are taurine haloamines including chloramine^35,45 and taurine bromamine.⁴⁶ These compounds are less toxic than corresponding HOXs, long-lived oxidants with well-documented in vitro microbicidal activity.⁴⁷ Moreover, data from several laboratories demonstrate that both taurine chloramine and bromamine are powerful regulators of inflammation, exerting anti-inflammatory activity in vitro and in vivo.^43,48,49 Recently we have shown that ILD is a potent inhibitor of IL-1ß, IL-12 and TNF- production by activated human lymphocytes and monocytes in vitro.⁵⁰ ILD down-regulates CD14 receptor surface expression on monocytes and CD11a/CD18 integrin surface expression on neutrophils, monocytes and lymphocytes and like dexamethasone and colchicine possesses anti-inflammatory activity in vitro.⁵¹ In addition, we have shown that ILD exhibited potent anti-endotoxin activity by inhibition of E. coli lipopolysaccharide induced pro-inflammatory activation of monocytes and endotoxin-tolerance induction.^51,52

In conclusion, ILD reaction with amino acids may support hypohalous acids and taurine haloamines in the regulation of inflammatory response and in killing of bacteria by neutrophils via in vivo formation of long-lived secondary oxidants including taurine iodamine.

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None to declare.

	Acknowledgements

 
We thank Honorary Doctor of NAS RA Levon A. Gevorkyan for his support to this study in the Armenicum Research Center. We also thank Professor Gayane Martirosian MD, PhD, Department of Medical Microbiology, Medical University of Silesia, for her critical reading of the manuscript. This study was supported by the ‘Armenicum+’ CJSC.

	References

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