JAC Advance Access originally published online on March 25, 2008
Journal of Antimicrobial Chemotherapy 2008 61(6):1281-1287; doi:10.1093/jac/dkn125
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity
Institute of Hygiene and Environmental Medicine, University of Greifswald, W.-Rathenau-Str. 49a, D-17487 Greifswald, Germany
* Corresponding author. Tel: +49-3834-515542; Fax: +49-3834-515541; E-mail: kramer{at}uni-greifswald.de
Received 16 October 2007; returned 28 November 2008; revised 12 February 2008; accepted 29 February 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Objectives: To assess the suitability of an antiseptic agent, both the microbicidal activity and the cytotoxic effect must be taken into consideration to derive biocompatible antibacterial agents.
Methods: We defined the biocompatibility index (BI) by measuring the antibacterial activity against the test organisms Escherichia coli and Staphylococcus aureus and, in parallel, the cytotoxicity on cultured murine fibroblasts. The antiseptic agents tested were benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), chlorhexidine digluconate (CHX), mild silver protein (MSP), octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), povidone iodine in solution [PVP-I(s)], povidone iodine in ointment [PVP-I(o)], silver nitrate (AgNO3), silver (I) sulfadiazine (SSD) and triclosan (TRI). Assays were carried out for 30 min of exposure at 37°C in the presence of cell culture medium containing 10% fetal bovine serum. The resulting dimensionless BI was defined as the ratio of the concentration at which 50% of the murine fibroblasts are damaged and the microbicidal effect producing at least 3 log10 (99.9%) reduction.
Results: The resulting rank ordering of BI for the ratio of fibroblast cytotoxicity to E. coli toxicity was OCT > PHMB > CHX > PVP-I(o) > PVP-I(s) > BAC > CPC > TRI > MSP and that to S. aureus was OCT > PHMB > CHX > CPC > PVP-I(o) > BAC > PVP(s) > TRI > MSP. OCT and PHMB were the most suitable agents with a BI greater than 1.
Conclusions: The BI presented may be a useful tool to evaluate antiseptic agents for use in clinical practice.
Keywords: antibacterial effectiveness , rank ordering , in vitro
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Antiseptics are anti-infective substances that, after topical administration, destroy or inhibit the growth of microorganisms in or on living tissue (skin, mucous membrane and wound).1,2 Antiseptics are applied externally and, to prevent the development of biocide resistance, they are used at concentrations considerably higher than minimal bactericidal concentrations (MBCs).3 Ideally, antiseptics should have a broad microbicidal spectrum and potent germicidal activity with rapid onset and long-lasting effect. Antiseptic preparations should not be toxic to host tissues/cells and in line with the concept of biocompatibility of medical products, as far as possible, they should not impair healing processes.4 Attempts to characterize both cellular and bacterial toxicities of topical antimicrobials have been carried out to derive biocompatible antimicrobials.5–8 However, in these studies, the in vitro tests for the evaluation of cytotoxic and microbicidal effects of antimicrobial agents were carried out in different media. Although methods of assessing the antibacterial activity of these agents have been standardized using suspension-based methods, broth dilution, agar dilution or agar disc diffusion,9 experiments for evaluating the cytotoxicity of antiseptics have mostly been carried out in cell culture medium containing 10% fetal bovine serum (FBS). For this reason, we have used cell culture medium containing 10% FBS (which is similar to the composition of artificial wound fluid)10 for the evaluation of both the cytotoxic and the microbicidal effects of selected antiseptic agents. Additionally, we defined a biocompatibility index (BI) for comparing the antiseptic active substances, which may more realistically mimic antiseptic action in vivo than do previous indices. The BI takes into account both the results of the in vitro cytotoxicity, i.e. the concentration at which 50% of the murine fibroblasts are damaged, and the microbicidal effect, i.e. the concentration at which the baseline burden of the test microorganisms (Staphylococcus aureus and Escherichia coli) is reduced by at least 3 log10 (99.9%). This BI is a dimensionless number. A BI greater than 1 represents an antiseptic substance with an effective microbicidal activity combined with a relatively low cytotoxicity, whereas a BI less than 1 indicates an antimicrobial agent with a relatively high cytotoxicity in a defined medium.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
L929 cell line
L929 cells (ATCC CCL 1), derived from an immortalized mouse fibroblast cell line and routinely used in in vitro cytotoxicity assessments, were grown in the presence of the cell culture medium containing 10% FBS, which is similar to the composition of the artificial wound fluid.10 Murine fibroblasts were purchased from the Collection of Cell Lines in Veterinary Medicine, FLI, Isle of Riems, Germany. Stock cultures of L929 cells were routinely propagated in the cell culture minimal essential medium Eagle (MEM) containing 10% FBS (see Cell culture medium section).
MEM with Earle's salts and L-glutamine (PAA Laboratories, Germany) was supplemented with 10% FBS (Gibco). The cell culture medium was used exclusively without antibiotics.
The following substances and commercially available antiseptics were tested.
Betaisodona® solution [PVP-I(s), Mundipharma, Limburg, Germany]: 100 mL of the solution contains 10 g of poly(1-vinyl-2-pyrrolidone-)iodine-complex, with a content of 11% available iodine.
Betaisodona® ointment [PVP-I(o), Mundipharma]: 100 g of the ointment contains 10 g of poly(1-vinyl-2-pyrrolidone-)iodine-complex, with a content of 10% available iodine.
Chlorhexidine digluconate (CHX, Sigma) was used in a 20% (w/v) stock solution in water.
Lavasept® concentrate (Fresenius AG, Bad Homburg, Germany) contains 20 g of polyhexamethylene biguanide (PHMB) with an average molecular weight of 2800 and 1 g of polyethylene glycol 4000 in 100 mL of aqueous solution.
Octenisept® (Schülke & Mayr, Norderstedt, Germany) contains 0.1 g of octenidine dihydrochloride (OCT) and 2 g of phenoxyethanol in 100 mL of aqueous solution.
Mild silver protein (MSP) containing 20% (w/w) silver (Lot S14841-266) and silver (I) sulfadiazine (SSD) were purchased from Aldrich.
Silver nitrate (AgNO3, Sigma), benzalkonium chloride (BAC, Fluka, Buchs, Switzerland), cetylpyridinium chloride (CPC, Fluka) and triclosan (TRI, Irgasan, Fluka) were used at the highest available purity.
Stock solutions/dispersions of 10% (w/v) MSP, 10% (w/v) SSD, 1% (v/v) PVP-I and 0.01% (w/v) OCT were prepared directly in the culture medium, 10% (w/v) AgNO3, 1% (w/v) PHMB, 0.25% (w/v) BAC, 0.25% CHX and 0.25% CPC in water for injection and 1% (w/v) TRI in dimethyl sulfoxide (DMSO). Stock solutions in water and DMSO were diluted at least 1 : 10 in the cell culture medium to get the highest starting concentration for the tests. Additionally, six to eight graded dilutions were prepared in the cell culture medium.
Stocks and dilutions were prepared under sterile conditions and used within 2 h. This is the shortest period required for preparation, application of the reagent and incubation in the two parallel assays. To reduce the time involved in setting up the assays to an absolute minimum, the agents were assayed one by one, rather than in a single large experiment. Each antiseptic agent was tested in parallel in one experiment for both microbicidal and cytotoxic activities to ensure that the dwell time of the active agent in the culture medium was identical in both assays.
The neutral red (NR) assay and the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] test have been standardized for L929 cells (a recommended cell line for cytotoxicity testing in accordance with EN ISO 10993-5)11 and correlated with the cell number in separate experiments, essentially as described for human cancer cells.12 Procedures for the NR assay13–15 and the MTT test15–17 have been described in detail elsewhere. Briefly, L929 cells were seeded onto 96-well cell culture plates, 0.1 mL/well, at a density of 1 x 106 cells/mL to reach about >80% confluence after 24 h. At 24 h after seeding, 0.1 mL of fresh medium and graded concentrations of the antiseptic agent were added. Six replicate wells per concentration were tested. After 30 min of incubation in a humidified atmosphere of 5% CO2/95% air at 37°C, the medium was removed and the wells were washed twice for 2–3 min with 0.2 mL of the fresh medium. Thereafter, 0.2 mL of the fresh medium containing 50 mg/L NR (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) dye, which was pre-incubated at 37°C at least for 3 h and sterile-filtered before use, was added for 3 h at 37°C. The NR dye is incorporated into the lysosomes of viable cells.
In the MTT assay, 0.1 mL of the fresh medium containing 0.5 mg of MTT was added for 4 h at 37°C. The yellow tetrazolium salt is reduced by mitochondrial enzymes in viable cells to an insoluble blue formazan product.
After incubation, the media were completely removed, and only cells with the NR medium were washed carefully with two washes of 0.25 mL of warm PBS for 3 min.
Incorporated NR dye was extracted by adding 0.2 mL of 1% (v/v) acetic acid/50% (v/v) ethanol, and the blue formazan product was solubilized in 0.2 mL of 0.04 M HCl in 2-propanol. The plates were agitated on an orbital shaker for at least 1 h in order to ensure quantitative extraction and solubilization of the dyes.
The optical density of each well was measured spectrophotometrically using an automated plate reader (Bio-Rad, Benchmark) with a 540 nm test wavelength and a 655 nm reference wavelength. The results are expressed as a percentage of the control (unexposed cells). The inferred percentage viability based on the MTT and NR reading for each concentration was used to establish log-dose–response curves. IC50 value was defined as the concentration allowing 50% survival of cells and was determined graphically after log dose–response transformation.
The cytotoxicity tests were separately repeated three times using the mean of IC50 of both the MTT assay and NR test for the calculation of BI.
The investigation was carried out with S. aureus (ATCC 6538) and E. coli (ATCC 11229).
Biguanides, ammonium and pyridinium compounds and triclosan were inactivated as recommended18,19 using TSHC [3% (w/v) Tween 80 (Serva, Heidelberg, Germany), 3% (w/v) saponin (Fluka), 0.1% (w/v) histidine (Serva) and 0.1% (w/v) cysteine (Merck, Darmstadt, Germany)]. TLA-thio18,19 [a mixture of 3% (w/v) Tween 80 (Serva), 0.3% lecithin from soy bean (Wasserfuhr GmbH, Bonn, Germany), 0.1% (w/v) histidine (Serva) and 0.5% (w/v) sodium thiosulphate (Merck)] was used for the inactivation of test combinations containing iodine and silver.
Either alone or in combination with the active agent, TSHC was suited for the inactivation of BAC, CPC, CHX, OCT, PHMB and TRI and TLA-thio for MSP, PVP-I, AgNO3 and SSD in the investigated concentration range without any inhibitory effect on bacterial growth, as judged by separate experiments on inactivation combinations (data not shown).
The quantitative suspension tests were done in accordance with the guidelines for testing disinfectants and antiseptics of the European Committee for Standardization, technical Committee 216.18 The bacteria broth culture contained 108–109 cfu/mL as judged by separate experiments. A 1 mL aliquot of this inoculum was transferred into 9 mL of the cell culture medium with and without antiseptic agent. Incubation was for 30 min at 37°C. The antiseptic agent was eliminated by transfer of 1 mL of the incubated test combination into 9 mL of TSHC or TLA-thio depending on the substance. After 30 min of inactivation, serial dilutions were prepared in Trypticase soy broth; 0.1 mL of each dilution was plated in triplicate on Trypticase soy agar. The cfu of the test microorganisms were counted after 48 h of incubation at 37°C. The log10-reduction factor (rf) for each contact time was calculated according to the formula:
|
|
where nc is the number of viable cells (cfu) in the inoculum in the presence of PBS and nd is the number of viable cells (cfu) in the inoculum after contact with the antiseptic agent (test combination).
All experiments were repeated three times.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
The tested antiseptic agents demonstrated a different cytotoxic effect on murine fibroblasts based on both the mass and molecular weight (Table 1). The rank order of cytotoxicity in relation to their (w/v) concentration was AgNO3 > OCT > SSD > BAC >CPC > CHX > TRI > PHMB > MSP > PVP-I(s) > PVP-I(o). The most toxic substances were AgNO3 and OCT, and the best tolerated were povidone iodine-containing formulations. The rank ordering of cytotoxicity changes on the basis of molecular weight was: PHMB > OCT > CHX > AgNO3 > SSD >BAC > CPC > TRI > MSP > PVP-I(s) > PVP-I(o).
|
The concentration at which the baseline burden of the test microorganisms S. aureus and E. coli was reduced by at least 3 log10, the minimum efficacy in the presence of organic matter,19 is presented in Table 2. Surprisingly, no concentration of AgNO3 and SSD, tested at concentrations of 2.5–10 mg/mL, produced at least 3 log10 reduction. A maximum of 1.5–2 log10 reduction was found for E. coli (Gram-negative) after 30 min of exposure, but there was no microbicidal activity against S. aureus (data not shown). MSP was more effective against E. coli, but not against S. aureus. However, the cytotoxicity of MSP was more pronounced than its microbicidal activity, demonstrated by a very low BI of 0.22 and 0.11, respectively.
|
The resulting BIs of the investigated antiseptic agents, which was defined as the ratio of the mean values of IC50 on L929 cells and of the concentration producing 3 log10 reduction in microbial cfu after 30 min of exposure at 37°C in the cell culture medium, are presented for each test microorganism in Table 2. The resulting rank ordering for biocompatibility of selected antiseptic agents on the basis of cytotoxicity on murine fibroblast and microbicidal effect on E. coli was OCT > PHMB > CHX > PVP-I(o) > PVP-I(s) > BAC > CPC > TRI > MSP. The BIs of AgNO3 and SSD were not calculable, but must be much lower than 0.002 and 0.006, respectively. The resulting rank ordering for biocompatibility of selected antiseptic agents on the basis of cytotoxicity on murine fibroblast and microbicidal effect on S. aureus was similar to that for E. coli as follows: OCT > PHMB > CHX >CPC > PVP-I(o) > BAC > PVP(s) > TRI > MSP (Table 2). The antiseptic agents OCT and PHMB were the only substances yielding BI values of greater than 1. Additionally, the antiseptic agent OCT was very effective on S. aureus within 30 min of exposure. Despite its high cytotoxicity on L929 cells on the basis of both the mass and molecular concentration, the calculated BI was greater than 2 (Table 2).
The surfactants BAC and CPC, the bisguanide CHX, the bispyridine OCT, the diphenylether TRI, and the ointment formulation containing povidone iodine were more effective on S. aureus (Gram-positive) after 30 min of exposure. MSP and TRI were not suited to be antiseptic agents, as the BI is less than 0.5 (Table 2).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
This study employed two techniques to assess the biocompatibility of antiseptic agents in vitro: a cytotoxicity assay using murine fibroblasts and a quantitative suspension test using the Gram-negative test microorganism E. coli and the Gram-positive test microorganism S. aureus.
Cytotoxic effects of topical antimicrobials and antiseptic agents on fibroblasts and/or keratinocytes have been investigated in many other studies to compare toxicity of these compounds.5–7,20–27 In most of these studies, the resulting dose–response cytotoxicity curves of the antimicrobial agents were presented in the form of mass concentration–cell vitality curves. This is understandable from a practical point of view, because, in practice, concentrations based on mass are used. However, our data of IC50 (Table 1) show that the rank ordering of cytotoxicity of the investigated antiseptic agents based on mass concentration differs considerably from that based on molar concentration, especially for a compound with a relative high molecular weight. For example, from the results shown in Table 1, PHMB and TRI have similar IC50 values on the basis of mass concentration and this is commonly interpreted as showing that these substances have comparable cytotoxicity. However, on the basis of the molecular weight, PHMB has an approximately 10 times higher cytotoxic effect. For the comparison of only cytotoxic or only microbicidal effect of substances, it is recommended to use molar concentrations that best describe the effect of the reagents at the molecular level.
As shown in Table 1, the resulting mean of IC50 of the NR assay is in good agreement with that of the MTT assay, suggesting that for this kind of toxicity screening, the different in vitro assays used are interchangeable. Additionally, the resulting IC50 of the two cytotoxicity tests can be combined to one mean, which is then used for further calculations. The somewhat higher toxicity in the NR assay resulting in a lower mean IC50 may be due to the preferred initial interaction of the active agent, such as BAC, CHX, CPC, OCT and PHMB, with phospholipids in the cell membrane or other membrane components. This is especially acute with silver compounds.28,29
PVP-I and MSP might appear to be the best-tolerated compounds, albeit with low bactericidal activity (Tables 1 and 2). However, these results have to be treated with caution. First, the concentration of the active compounds (free molecular iodine and silver, respectively) in the medium used was unknown so that the molar concentrations of the active agents could not be calculated exactly. Secondly, the antiseptics lose activity through reaction with components of the cell culture medium containing 10% FBS.
Ionic silver is considered to be effective against a broad range of microorganisms at low concentrations and is therefore used in clinical practice.30–32 In our experimental design using culture medium with 10% FBS, we could not demonstrate a reduction of 99.9% of the baseline burden of either E. coli or S. aureus after 30 min of exposure with concentrations of between 0.25% and 1% (w/v) AgNO3 or SSD. Only MSP at concentrations of >0.25% produces on E. coli and concentrations of >0.5% on S. aureus, a reduction in bacterial counts of 103 after 30 min of action (Table 2).
Silver has been described also as being oligodynamic because of its ability to give rise to a bactericidal effect at minute concentrations.29,31 Additionally, we investigated concentrations of AgNO3 and SSD of 0.25–1 and 25–100 mg/L for microbicidal effect after 30 min of exposure in the cell culture medium, in water for injection and in hard water. No microbicidal activity was demonstrated after 30 min in any of these three diluents (data not shown). Therefore, a microbicidal effect of silver may be evident only after a prolonged exposure, at least after 3 h33 or after an even longer exposure time.31,34–36 However, the IC50s of AgNO3 and SSD of 18 mg/L (0.11 µmol/mL) and 60 mg/L (0.17 µmol/mL), respectively, demonstrate that after 30 min of exposure, these silver compounds damage cells more than they do the test microorganisms. This is in line with the work by others.37,38 Unfortunately, experiments for the detection of antibacterial activity of silver were carried out with the wet disc antimicrobial solution assay,29,31 using a medium different from that used for cell culture.
Similar experimental designs as presented here have been used in other studies using both the cytotoxic effect and the microbicidal activity of antimicrobial and antiseptic agents.5–8,20 Boyce and Holder5 determined the cytotoxicity of the antiseptic CHX and other antimicrobial substances on human keratinocytes and fibroblasts, and simultaneously their microbicidal effect utilizing the wet disc assay using different media. The tested CHX was uniformly effective against six strains of common burn microorganisms, but was disqualified for clinical use because it was also highly cytotoxic. Lineaweaver et al.6 compared the toxic effect of dilutions of povidone iodine, sodium hypochlorite, acetic acid and hydrogen peroxide in saline on suspensions of the same densities of both human fibroblasts and S. aureus. After 15 min of exposure, the percentage of living fibroblasts and colonies of microorganisms were compared with controls. They found that dilutions of povidone iodine (0.001%) and sodium hypochlorite (0.005%) remained bactericidal while no longer damaging fibroblasts. Others8 investigated the suitability of the topical antiseptic agent sodium hypochlorite using three different cytotoxicity assays on human dermal fibroblasts in the presence of 2% to 10% FBS, and the antimicrobial activity was assessed using a macrodilution broth technique using appropriate culture medium for four different bacterial strains isolated from the wounds of burn patients. They found that cytotoxicity of NaOCl was reduced with increasing concentration of FBS due to chlorine consumption, but this result was not transferred to the antimicrobial activity test. Sanchez et al.20 investigated the effectiveness of a 30 min exposure to dilutions of CHX and povidone iodine in PBS on canine embryonic fibroblasts utilizing the Trypan Blue technique and on cultures of S. aureus utilizing the broth dilution technique. They found that both the antiseptics tested were lethal to canine fibroblasts at bactericidal concentrations in vitro.
Only two papers presented a toxicity index for the assessment of antimicrobial and antiseptic agents. Damour et al.25 utilized MIC for antibiotic agents and MBC for antiseptic agents in relation to a CD50 value, which was equal to the IC50 value in the present study, using the MTT assay. Human fibroblasts and keratinocytes were exposed to dilutions of the antimicrobial agents for 15 min. They derived the ratio of CD50/MIC and CD50/MBC from their results and generated a rank order of cytotoxicity for the tested antiseptics: BAC > PVP-I > CHX > CHX/BAC. In another report, Wilson et al.39 presented a toxicity index for 20 commercial skin and wound cleansers. They characterized the dilution in PBS required in a 30 min exposure to leave 85% of the human fibroblasts viable in comparison with the medium controls. Hydrogen peroxide, sodium hypochlorite and PVP-I were found to be more toxic in PBS than in media containing FBS, which can be explained by consumption of activity by the FBS proteins.
In the present study, experiments to determine cytotoxic and microbicidal effects were performed in the cell culture medium with 10% FBS, which more realistically reflects the composition of wound fluids.10 The described BI, which is a dimensionless value, derived from the ratio of IC50 on fibroblasts to the concentration for 99.9% microbicidal reduction on test microorganisms, may be a useful tool to assess the biocompatibility of antiseptic agents. Because of the different effectiveness of antiseptic agents on Gram-positive and Gram-negative microorganisms, it is recommended to present the individual BI on the basis of the used microorganism. In this study, a BI greater than 1 is achieved only by the antiseptic agents OCT and PHMB, meaning that these substances are more toxic to both the test microorganisms than to murine fibroblasts. This could be of great benefit using OCT and PHMB in different antiseptic treatments, especially if considerably lower concentrations are used, as described for OCT. In a rat peritonitis model, irrigating the peritoneal cavity with 0.05% OCT caused an inflammatory reaction, resulting in peritoneal adhesions. This did not occur when 0.01% OCT was used; however, this concentration was still effective as an antiseptic against intraperitoneally injected E. coli.40 Moreover, OCT seems to be superior to CHX, and this conclusion is supported by other studies.41–45
The BI greater than 1 corresponds with the results on artificial wounds on piglets. In that study, the endpoint of wound healing did not differ between OCT and Ringer solution (control), whereas PHMB even significantly stimulated the wound healing.45 A wound dressing with 0.3% PHMB does not appear to result in cytotoxicity, although it retains antiseptic efficacy.46 Moreover, concentrations of PHMB in the range of 0.2–2 mg/L stimulate proliferation of human keratinocytes and fibroblasts in vitro.47 Additionally, in a controlled, prospective, randomized double-blind study on wounds contaminated with bacteria, the tissue compatibility of 0.04% (w/v) PHMB was significantly better than that for Ringer solution.48 On mesh grafts, PHMB proved clinically and histologically superior to povidone iodine and silver nitrate. Second-degree burn wounds treated with polyhexanide epithelized without any further debridement with a remarkable freedom from pain. Compared with the silver nitrate treatment, no fibrin film was observed on the wound. From that clinical study, PHMB is recommended for the treatment of second-degree burns, because in addition to its antiseptic efficacy it does not inhibit the re-epithelization process.49
Compared with OCT and PHMB, only a minor antibacterial effect of silver compounds was shown against the Gram-negative organism E. coli, but no activity against the Gram-positive S. aureus. Using this study design, silver-containing compounds are shown to exert a profound cytotoxic effect on murine fibroblasts with little efficacy against the test microorganisms. Our results of a BI much less than 1 were also supported by others.50,51 These authors indicated that SSD delays the wound-healing process and that silver may have serious cytotoxic effects on various host cells. Additionally, results of a recently published Cochrane database analysis failed to provide evidence, which would not support a recommendation for the use of topical silver agents for the treatment of infected or contaminated chronic wounds.52 Moreover, a delayed effectiveness of silver compounds could be accompanied by a possible development of bacterial resistance.32,53,54
In animal studies, 0.05% to 0.5% CHX significantly inhibited both wound healing and granulation.55,56 Moreover, in a prospective monocentric randomized clinical study using mesh split-thickness skin grafts, 0.5% CHX induced significantly delayed neo-epithelization.57 All these clinical studies support the BI data presented here, which were obtained in vitro.
The combined assessment of cellular cytotoxicity and antimicrobial activity of antiseptic agents in the form of a BI may be useful for the development and application of a suitable biocompatible dilution of a defined antiseptic agent, which is effective against bacteria within a short exposure time (<30 min) without harming host cells/tissues. In addition, we used a cell culture medium containing 10% FBS, which more realistically reflects the composition of wound fluid than does PBS. Additional experiments are required to assess the effect on the BI of different organic challenges, such as mucin or blood, which may alter the ranking of the antiseptics. Were this to be the case, then the choice of the most suitable antiseptic would depend on the nature of the clinical situation in which it is to be applied.
Furthermore, some of the test substances lost a significant part of activity during the preparation of the test solutions and dilutions by reacting with constituents of the cell culture medium. These reactions may result in products that themselves can considerably influence the BI. To avoid spurious results arising from reaction of the test antiseptics with the medium, an assessment of cytotoxicity and bactericidal capacity should be performed additionally in a simple buffer solution, such as PBS, or in media that mimic the particular clinical situation of interest. The IC50 values determined in this study are considerably lower than concentrations of the respective antiseptics generally applied in clinical practice. Human tissue tolerates exposure to antiseptics better than tissue culture cells do. Therefore, the IC50 values have to be considered to be a relative measure rather than an absolute measure. Moreover, the apparent toxicity of antiseptics might well depend on the cell type used. Nevertheless, for substances with a high BI, a dose reduction may be considered to minimize cytotoxicity and possibly promote wound healing without loss of bactericidal effectiveness. Naturally, this has to be verified in clinical studies.
The BI may thus be a useful measure in evaluating new antiseptic products, although it must be borne in mind that its interpretation will be affected by the chemical structure of the test substance, the composition of the test media used and the nature of the proposed clinical application sites.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
No benefit in any form has been received or will be received from a commercial party related directly or indirectly to the subject of this article. This study was supported exclusively by the University of Greifswald, Germany.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
We thank Margret Schultz for competent technical assistance. We thank Prof. Dr R. Jack (Institute of Immunology, University of Greifswald) for critical reading and correcting of the manuscript.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
1 Block SS. Definition of terms. In: Disinfection, Sterilization, and Preservation—Block SS, ed. (2001) Philadelphia: Lippincott Williams & Wilkins. 19–28.
2
McDonell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev (1999) 12:147–79.
3 Russell AD, McDonnell G. Concentration: a major factor in studying bicidal action. J Hosp Infect (2000) 44:1–3.[CrossRef][Web of Science][Medline]
4 Schulmann G. A review of the concept of biocompatibility. Kidney Int (1993) 43(Suppl 41):S209–12.[Web of Science]
5 Boyce ST, Holder IA. Selection of topical antimicrobial agents for cultured skin for burns by combined assessment of cellular cytotoxicity and antimicrobial activity. Plast Reconstr Surg (1993) 92:493–500.[Web of Science][Medline]
6 Lineaweaver W, McMorris S, Soucy D, et al. Cellular and bacterial toxicities of topical antimicrobials. Plast Reconstr Surg (1985) 75:394–6.[Web of Science][Medline]
7 Alacam T, Ömürlü H, Özkul A, et al. Cytotoxicity versus antibacterial activity of some antiseptics. J Nihon Univ Sch Dent (1993) 35:22–7.[Medline]
8 Hidalgo E, Bartolome R, Dominguez C. Cytotoxicity mechanisms of sodium hypochlorite in cultured dermal fibroblasts and its bactericidal effectiveness. Chem Biol Interact (2002) 139:265–82.[CrossRef][Web of Science][Medline]
9 Hobson DW, Bolsen K. Methods of testing oral and topical antiseptics and antimicrobials. In: Disinfection, Sterilization, and Preservation—Block SS, ed. (2001) Philadelphia: Lippincott Williams & Wilkins. 1329–59.
10 Campbell KE, Keast D, Woodbury G, et al. Wear time in two hydrocolloid dressings using a novel in-vivo model. Wounds (2003) 15:40–8.
11 DIN EN ISO 10993-5. Biological evaluation of medical devices—part 5. Tests for cytotoxicity: in vitro methods. (1999) German version EN ISO 10993-5.
12 Fiennes AGTW, Walton J, Winterbourne D, et al. Quantitative correlation of neutral red uptake with cell number in human cancer cell cultures. Cell Biol Int Rep (1987) 11:373–8.[CrossRef][Web of Science][Medline]
13 Borenfreund E, Puerner J. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Cult Methods (1984) 9:7–9.[CrossRef]
14 Borenfreund E, Puerner J. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett (1985) 24:119–24.[CrossRef][Web of Science][Medline]
15 Wilson AP. Cytotoxicity and viability assays. In: Animal Cell Culture—Masters JRW, ed. (2000) Oxford: Oxford University Press. 175–219.
16 Edmondson JM, Armstrong LS, Martinez AO. A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer cultures. J Tissue Cult Methods (1988) 11:15–7.[CrossRef]
17 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Meth (1983) 65:55–63.[CrossRef][Web of Science][Medline]
18 DIN EN 1040. Chemical disinfectants and antiseptics—quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics—test method and requirements (phase 1). (2005) German version EN 1040.
19 Pitten F-A, Werner H-P, Kramer A. A standardized test to assess the impact of different organic challenges on the antimicrobial activity of antiseptics. J Hosp Infect (2003) 55:108–15.[CrossRef][Web of Science][Medline]
20 Sanchez IR, Nusbaum KE, Swaim SF, et al. Chlorhexidine diacetate and povidone-iodine cytotoxicity to canine embryonic fibroblasts and Staphylococcus aureus. Vet Surg (1988) 17:182–5.[CrossRef][Web of Science][Medline]
21 McCauley RL, Linares HA, Pelligrini V, et al. In vitro toxicity of topical antimicrobial agents to human fibroblasts. J Surg Res (1989) 46:267–74.[Web of Science][Medline]
22 Ponec M, Haverkort M, Soei YL, et al. Use of human keratinocyte and fibroblast cultures for toxicity studies of topically applied compounds. J Pharm Sci (1990) 79:312–6.[CrossRef][Web of Science][Medline]
23 Cooper ML, Boyce ST, Hansbrough JF, et al. Cytotoxicity to cultured human keratinocytes of topical antimicrobial agents. J Surg Res (1990) 48:190–5.[CrossRef][Web of Science][Medline]
24 Cooper ML, Laxer JA, Hansbrough JF. The cytotoxic effects of commonly used topical antimicrobial agents on human fibroblasts and keratinocytes. J Trauma (1991) 31:775–84.[Web of Science][Medline]
25 Damour O, Hua SZ, Lasne F, et al. Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes. Burns (1992) 18:479–85.[CrossRef][Web of Science][Medline]
26 Teepe RGC, Koebrugge EJ, Löwik CWGM, et al. Cytotoxic effects of topical antimicrobial and antiseptic agents on human keratinocytes in vitro. J Trauma (1993) 35:8–19.[Web of Science][Medline]
27 Boyce ST, Warden GD, Holder IA. Cytotoxicity testing of topical antimicrobial agents on human keratinocytes and fibroblasts for cultured skin grafts. J Burn Care Rehabil (1995) 16:97–103.[CrossRef][Medline]
28 Gilbert P, Moore LE. Cationic antiseptics: diversity of action under a common epithet. J Appl Microbiol (2005) 99:703–15.[CrossRef][Medline]
29 Clement JL, Jarrett PS. Antibacterial silver. Met Based Drugs (1994) 1:467–82.[CrossRef]
30 Russell AD, Hugo WB. Antimicrobial activity and action of silver. Prog Med Chem (1994) 31:351–70.[Medline]
31 Hidalgo E, Bartolome R, Barroso C, et al. Silver nitrate: antimicrobial activity related to cytotoxicity in cultured human fibroblasts. Skin Pharmacol Appl Skin Physiol (1998) 11:140–51.[CrossRef][Web of Science][Medline]
32 Lansdown ABG. Silver I: its antibacterial properties mechanism of action. J Wound Care (2002) 11:125–30.[Medline]
33 Müller G, Winkler Y, Kramer A. Antibacterial activity and endotoxin-binding capacity of Actisorb® Silver 220. J Hosp Infect (2003) 53:211–4.[CrossRef][Web of Science][Medline]
34 Lansdown ABG. Silver absorption and antibacterial efficacy of silver dressings. J Wound Care (2005) 14:155–60.[Medline]
35 Thomas S, McCubbin P. A comparison of the antimicrobial effects of four silver-containing dressings on three organisms. J Wound Care (2003) 12:101–7.[Medline]
36 Thomas S, McCubbin P. An in vitro analysis of the antimicrobial properties of 10 silver-containing dressings. J Wound Care (2003) 12:305–8.[Medline]
37 Poon VKM, Burd A. In vitro cytotoxicity of silver: implications for clinical wound care. Burns (2004) 30:140–7.[CrossRef][Web of Science][Medline]
38 Hollinger MA. Toxicological aspects of topical silver pharmaceuticals. Crit Rev Toxicol (1996) 26:255–60.[Web of Science][Medline]
39 Wilson JR, Mills JG, Prather ID, et al. A toxicity index of skin and wound cleansers used in vitro fibroblasts and keratinocytes. Adv Skin Wound Care (2005) 18:373–8.[CrossRef][Medline]
40 Güzelsagaltici N, Girgin S, Gedik E, et al. Intraperitoneal octenidindihydro-chloride—phenoxyethanol solution to prevent peritoneal adhesion formation in a rat peritonitis model. Acta Obstet Gynecol (2007) 86:395–400.[CrossRef]
41 Emilson CG, Bowen WH, Robrish SA, et al. Effect of the antibacterial agents octenidine and chlorhexidine on the plaque flora in primates. Scand J Dent Res (1981) 89:384–92.[Web of Science][Medline]
42 Robrish SA, Emilson C-G, Kemp CW, et al. A comparison of viable counts and adenine nucleotide analysis to determine the effect of antimicrobial agents on dental plaque. Curr Microbiol (1981) 5:343–7.[CrossRef][Web of Science]
43
Slee AM, O'Connor JR. In vitro antiplaque activity of octenidine dihydrochloride (WIN 41464-2) against preformed plaques of selected oral plaque-forming microorganisms. Antimicrob Agents Chemother (1983) 23:379–84.
44 Kramer A, Höppe H, Krull B, et al. Antiseptic efficacy and acceptance of Octenisept® compared to common antiseptic mouthwash solutions. Int J Hyg Environ Med (1998) 200:443–56.
45 Kramer A, Roth B, Müller G, et al. Influence of the antiseptic agents polihexanide and octenidine on FL-cells and on healing of experimental superficial aseptic wounds in piglets. A double-blind, randomised, stratified controlled, parallel-group study. Skin Pharmacol Physiol (2004) 17:141–6.[CrossRef][Web of Science][Medline]
46 Mulder GD, Cavorsi JP, Lee DK. Feature: polyhexamethylene biguanide (PHMB): an addendum to current topical antimicrobials. Wounds (2007) 19:173–82.
47 Wiegand C, Abel M, Kramer A, et al. Stimulation of proliferation and biocompatibility of polihexanide. GMS Krankenhaushyg Interdiszip (2007) 2. Doc43 (20071228).
48 Schmit-Neuerburg KP, Bettag C, Schlickewei W, et al. Effectiveness of an improved antiseptic in treatment of contaminated soft tissue wounds. Chirurg (2001) 72:61–71.[CrossRef][Web of Science][Medline]
49 Daeschlein G, Assadian O, Bruck JC, et al. Feasibility and clinical applicability of polihexanide for treatment of second-degree burn wounds. Skin Pharmacol Physiol (2007) 20:292–6.[CrossRef][Web of Science][Medline]
50 Innes ME. The use of silver coated dressings on donor site wounds: a prospective, controlled matched pair study. Burns (2001) 27:621–7.[CrossRef][Web of Science][Medline]
51 Atiyeh BS, Costagliola M, Hayek SN, et al. Effect of silver on burn wound infection control and healing: review of the literature. Burns (2007) 33:139–48.[CrossRef][Web of Science][Medline]
52 Vermeulen H, van Hattem JM, Storm-Versloot MN, et al. Topical silver for treating infected wounds. Cochrane Database Syst Rev (2007) 1. CD005486.
53 Silver S. Bacterial silver resistance: molecular biology and misuses of silver compounds. FEMS Microbiol Rev (2003) 27:341–53.[CrossRef][Web of Science][Medline]
54 Percival SL, Bowler PG, Russell D. Bacterial resistance to silver in wound care. J Hosp Infect (2005) 60:1–7.[CrossRef][Web of Science][Medline]
55 Bassetti C, Kallenberger A. Influence of chlorhexidine rinsing on the healing of oral mucosa and osseous lesions. J Clin Periodontol (1980) 7:443–56.[CrossRef][Web of Science][Medline]
56 Paunio KU, Knuuttila M, Miellityinen H. The effect of chlorhexidine gluconate on the formation of experimental granulation tissue. J Periodontol (1978) 49:92–5.[Web of Science][Medline]
57 Reimer K, Vogt PM, Broegmann B, et al. An innovative topical drug formulation for wound healing and infection treatment: in vitro and in vivo investigations of a povidone-iodine liposome hydrogel. Dermatology (2000) 201:235–41.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||