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JAC Advance Access originally published online on April 12, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):105-108; doi:10.1093/jac/dkn168
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© The Author 2008. 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

Original research

Interactions of antimicrobial compounds with cross-linking agents of alginate dressings

Cheong Hian Goh, Paul Wan Sia Heng, Esther Pei En Huang, Benny Kai Hui Li and Lai Wah Chan*

Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Republic of Singapore

* Corresponding author. Tel: +65-6516-3506; Fax: +65-6779-1554; E-mail: phaclw{at}nus.edu.sg

Received 27 February 2008; accepted 22 March 2008


   Abstract
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Objectives: The aim of this study was to investigate the antimicrobial activities of calcium ions and other cross-linking agents of alginate dressings, as well as their compatibility with commonly used topical antimicrobials.

Methods: The antimicrobial activities of cross-linking agents and antimicrobials (five antibiotics and four antiseptics) were evaluated by the broth dilution method. The interactions between individual cross-linking agents and antimicrobials were evaluated using the chequerboard test against common skin pathogens, Staphylococcus aureus and Pseudomonas aeruginosa.

Results: From the MIC determined, antibiotics were the most active, followed by the antiseptics and cross-linking agents. Calcium ions, which are commonly used to cross-link alginate, exhibited very weak antimicrobial activity and higher fractional inhibitory concentration than the other cross-linking agents. The use of calcium and gentamicin resulted in antagonism against S. aureus. In contrast, aluminium, zinc and copper ions exhibited higher antimicrobial activities but insignificant interactions with the antimicrobials.

Conclusions: Commonly used topical antimicrobials that are active against the skin pathogens S. aureus and P. aeruginosa could be potentially incompatible with calcium alginate dressings. Copper, zinc and aluminium ions are more suitable cross-linking agents for alginate as they do not show antagonism with the antimicrobials and could impart antimicrobial property to the resultant dressing.

Keywords: wound care , antimicrobials , interaction


   Introduction
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Wounds are avenues for microbial invasion and infection. Opportunistic infections are often causes of morbidity and mortality in wound complications.1 Topical antimicrobials may be employed with wound dressings to prevent or treat infections. Currently, only calcium alginate dressings are widely available. Our preliminary studies suggest that alginates could cross-link with polyvalent cations such as aluminium, copper and zinc. These cations are postulated to exert antimicrobial effects.2 In this study, the antimicrobial activities of individual antimicrobials and cross-linking agents, as well as their interactions with each other, were investigated against two common skin pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. The findings provide useful information on the compatibility between cross-linking agents and antimicrobials, as well as the feasibility of developing an alginate dressing with antimicrobial properties.


   Materials and methods
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Bacterial strains, antimicrobials and other materials

S. aureus (ATCC 6538P) and P. aeruginosa (ATCC 9027) in Culti-loopTM were obtained from the ATCC bacterial strain collections. The nine antimicrobials tested consisted of four antiseptics: cetrimide (BP grade), chlorhexidine diacetate, chloroxylenol (Sigma Aldrich, USA) and proflavine hemisulphate (Fluka Biochemika, Switzerland), and five antibiotics: ciprofloxacin hydrochloride, gentamicin sulphate (USP grade), erythromycin lactobionate (David Bull Laboratories, Australia), sulfadiazine sodium and tetracycline (Sigma Aldrich). Nutrient Broth No. 2 and Purified Agar purchased from Oxoid (England) were used as the culture medium. Aluminium chloride, calcium chloride, copper sulphate and zinc chloride (Merck, Germany) were used as supplied.

Preparation of test inoculum

Test organisms in Culti-loopTM were first revived in nutrient broth at 37°C for 18–24 h before subcultures were made on nutrient agar. The 4th to 10th subcultures were used in this experiment. A standardized test inoculum containing 108 cells/mL was freshly prepared using the direct colony suspension method, with the aid of the 0.5 McFarland turbidity standard.3

MIC determination

MICs were determined by the broth dilution method, in which concentrations derived from doubling dilutions of the test compounds were further serially diluted to obtain a narrower MIC range. After incubation at 37°C for 18 h, the MIC was determined as the lowest concentration showing no visible growth.3 Each MIC determination was carried out in triplicate, with inoculated and uninoculated antimicrobial-free broths as test controls.

Interaction between cross-linking agent and antimicrobial

The interaction was evaluated by the chequerboard test using agar plates of 8 x 8 configuration.4 The plates were prepared by the agar dilution method, using 2-fold serial dilutions.3 Concentrations tested ranged from 1/32x to 4x MIC of each test compound. Test inoculum used was ~4.5 x 105 cfu/spot. The plates were examined for surface growth after incubating at 37°C for 18 h. Faint hazes were regarded as an absence of growth.

The fractional inhibitory concentration (FIC) index was used to interpret the chequerboard test and was calculated as follows:



Formula 168M1

(Equation 1)

where FICC and FICA are FIC index of cross-linking agent and antimicrobial, respectively.



Formula 168M2

(Equation 2)



Formula 168M3

(Equation 3)

The interaction was synergistic if FIC ≤ 0.50, no interaction if FIC > 0.50 but ≤4.00, and antagonistic if FIC > 4.00.5


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MIC determination

MICs of the test compounds are expressed in mg/L, as well as molar concentration (Table 1). The latter gives a clearer representation of the stoichiometric interaction of antimicrobial with the target sites. The antibiotics generally exhibited significantly higher activity than the antiseptics, followed by the cross-linking agents. The antibiotics showed comparable activities against S. aureus, but notably different activities against P. aeruginosa. The antibiotics and antiseptics were more active against S. aureus. On the other hand, the cross-linking agents were active against both organisms to comparable extents. The weakest antibiotic, sulfadiazine sodium, was only slightly more active than calcium chloride. Its MIC was 10- to 70-fold higher than those of the other cross-linking agents. Among all the compounds, calcium chloride was the weakest, with MIC 40- to 300-fold higher than the other three cross-linking agents. The most active antiseptic, chlorhexidine, was comparable in activity to tetracycline.


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  		Table 1. Antimicrobial activities of cross-linking agents and antimicrobials, from highest to lowest

 
Interaction between cross-linking agent and antimicrobial

The FIC indices for all the combinations ranged from 0.53 to 4.57 (Table 2). The interactions were mostly not significant, although antagonism was observed for the combination of calcium and gentamicin against S. aureus. No synergism was identified but some had FIC close to 0.5. These include the interactions of aluminium with chlorhexidine (FIC = 0.62) or erythromycin (FIC = 0.53), zinc with sulfadiazine sodium (FIC = 0.56) and copper with cetrimide (FIC = 0.53) against P. aeruginosa, as well as copper with tetracycline (FIC = 0.62) against S. aureus.


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  		Table 2. FIC indices and categorical interpretation of cross-linking agent–antimicrobial combinations tested against P. aeruginosa and S. aureus

 

   Discussion
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Cations affect the antimicrobial activities of quinolones and tetracycline,6,7 but little is known about their effects on other antimicrobials. Cross-linking agents, such as polyvalent cations, released from alginate dressings during use could interact with the antimicrobials and significantly affect the therapeutic outcome. Hence, the type of interaction between various cross-linking agents and antimicrobials that are commonly employed in wound management was investigated. Interactions observed in this study are expected to similarly affect other antimicrobials in the same class. Since different microorganisms usually exist in wounds, two common skin pathogens with different cell wall constituents were selected.

Cell wall composition can influence the effectiveness of antimicrobials by impeding their diffusion to the sites of action. As evident from the MIC results, the outer lipid layer in Gram-negative bacteria posed greater hindrance to the entry of most antimicrobials into the bacterial cell for action. This was not observed for the cross-linking agents, probably due to their small molecular size. No marked effects of the cross-linking agents on antibiotic activities were observed. However, most combinations showed higher FIC for S. aureus than P. aeruginosa. Cations are known to form complexes with aminoglycosides, quinolones and tetracyclines.6–8 Chemically, the conjugated C=C bonds and sulphonamide functional group in sulfadiazine sodium stabilize electron distribution via the mesomeric effect and this promotes sulfadiazine sodium complexation with cations. As the molecular size of complexes is bulkier, the thicker peptidoglycan in Gram-positive bacteria would impede their diffusion to a greater extent than the thinner peptidoglycan in Gram-negative bacteria. This probably explains the higher FIC for S. aureus than P. aeruginosa.

The tetracycline–copper interaction in this study is potentially useful. In another study, a higher DNA inhibition by tetracycline was observed for copper-accumulating cells in Wilson's disease subjects, demonstrating the role of cations as co-factors in the antimicrobial activity.9 Our results suggest that the copper–tetracycline complex could break down inside the cell to liberate the cation for further action.

The MIC results suggest that antiseptics with multiple target sites, e.g. chlorhexidine, were more active. Nevertheless, cetrimide (a quaternary ammonium compound), chlorhexidine (a biguanide) and proflavine (an acridine) are all positively charged at the pH of the test medium (about pH 7), while the phenol ring of chloroxylenol (pKa ~10) remains uncharged. As such, they are unlikely to interact with the cross-linking agents. This partly accounts for the lack of antagonism between these antiseptics and the cross-linking agents which probably diffused separately into the bacterial cell. The capacity of cations as co-factors or competitors for target sites will determine the overall antimicrobial activity. In another study, zinc ions improved the activity of the quaternary ammonium antiseptic, cetylpyridinium chloride, to a greater extent than copper ions.10 The FIC of copper–cetrimide against P. aeruginosa (0.53) is close to synergism, further illustrating the potential usefulness of copper.

In summary, the antimicrobial activity is affected by a number of factors that include the nature of the compound, the composition of the bacterial cell wall and the availability of certain chemical activators. The cross-linking agents are generally compatible with the commonly used antimicrobials. Calcium ion, which is commonly employed to cross-link alginate, exhibited very weak antimicrobial activity but no antagonism with most antimicrobials. The use of calcium alginate with gentamicin is, however, discouraged due to its antagonistic effects against S. aureus. In contrast, cross-linking agents like aluminium chloride, copper sulphate and zinc chloride could impart some antimicrobial activity to the resultant dressing and are more compatible with the antimicrobials. In particular, the combinations of aluminium with chlorhexidine or erythromycin, zinc with sulfadiazine sodium, and copper with cetrimide or tetracycline are potentially useful. It is, therefore, more beneficial to develop alginate dressings using aluminium, zinc and copper salts. However, more studies on the effects of these salts on the physical properties as well as toxicity of these dressings are necessary.


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This study was funded by GEA-NUS Pharmaceutical Processing Research Laboratory and the National University of Singapore Research Scholarship.


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None to declare.


   References
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1 Edwards-Jones V, Greenwood JE. What's new in burn microbiology? James Laing Memorial Prize Essay 2000. Burns (2003) 29:15–24.[CrossRef][Web of Science][Medline]

2 Beveridge TJ, Doyle RJ. Metal ions and bacteria (1989) New York, NY: Wiley.

3 National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6 (2003) Wayne, PA, USA: NCCLS.

4 Bonapace CR, Bosso JA, Friedrich LV, et al. Comparison of methods of interpretation of checkerboard synergy testing. Diagn Microbiol Infect Dis (2002) 44:363–6.[CrossRef][Web of Science][Medline]

5 Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother (2003) 52:1.[Free Full Text]

6 Lomaestro BM, Bailie GR. Quinolone-cation interactions: a review. DICP (1991) 25:1249–58.[Abstract]

7 Quinlan GJ, Gutteridge JMC. Hydroxyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts. Free Radic Biol Med (1988) 5:341–8.[CrossRef][Web of Science][Medline]

8 Szczepanik W, Czarny A, Zaczynska E, et al. Preferences of kanamycin A towards copper(II). Effect of the resulting complexes on immunological mediators production by human leukocytes. J Inorg Biochem (2004) 98:245–53.[CrossRef][Web of Science][Medline]

9 Hartmann A, Wess D, Witte I. Enhanced cyto- and genotoxicity of tetracycline in Wilson disease fibroblasts. Mutat Res Lett (1995) 348:7–12.[CrossRef]

10 Zeelie JJ. Effects of copper and zinc ions on the germicidal properties of two popular pharmaceutical antiseptic agents, cetylpyridinium chloride and povidone-iodine. Analyst (1998) 123:503–7.[CrossRef][Medline]


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This Article
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