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Antimicrobial Activity of Topical Skin Pharmaceuticals – an in Vitro Study

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Antimicrobial Activity of Topical Skin Pharmaceuticals – an in Vitro Study Acta Derm Venereol 2010; 90: 239–245 INVESTIGATIVE REPORT Antimicrobial Activity of Topical Skin Pharmaceuticals – An In vitro Study Mikael ALSTERHOLM1, Nahid KARAMI1,2 and Jan FAERGEMANN1 Departments of 1Dermatology and Venereology and 2Clinical Bacteriology, Sahlgrenska University Hospital, Göteborg, Sweden The aim of this study was to investigate the antimicro- yeast infections have been prescribed extensively with bial activity of currently available topical skin phar- less development of resistance (4, 5). maceuticals against Candida albicans, Escherichia coli, Biocides are broad-spectrum chemical agents that Staphylococcus aureus, Staphylococcus epidermidis and inactivate microbes. When they are used on living tissue Streptococcus pyogenes. The agar dilution assay was used they are usually referred to as antiseptics. These agents, to determine the minimal inhibitory concentration for for instance chlorhexidine and triclosan, represent an- cream formulations and their active substances. Corti- other group of topical antimicrobials that are used in costeroid formulations with the antiseptics clioquinol or modern healthcare and in consumer products. Antisep- halquinol were active against all microbes. The hydro- tics have a broader spectrum of activity than antibiotics gen peroxide formulation was primarily active against and often act on multiple cellular targets (6). staphylococci. Clotrimazole, miconazole and econazole Resistance to antibiotics used in the treatment and showed an effect against staphylococci in addition to prevention of infectious disease in humans and animals their effect on C. albicans. In contrast, terbinafine had is a rapidly increasing global problem. In the past two no antibacterial effect. Fusidic acid was active against decades there has been a large number of reports of staphylococci, with slightly weaker activity against S. multi-resistant bacteria causing considerable morbidity pyogenes and no activity against C. albicans or E. coli. In and mortality. Antibiotic overuse in humans and animals summary, some topical skin pharmaceuticals have broad and dissemination of bacteria through the food chain, antimicrobial activity in vitro, clioquinol and halquinol trade and human migration has lead to the current, being the most diverse. In limited superficial skin infec- highly worrying, situation. In dermatology, the resis- tion topical treatment can be an alternative to systemic tance pattern of Staphylococcus aureus, one of the most antibiotics and should be considered. With the global important skin pathogens, has been of special interest. threat of multi-resistant bacteria there is a need for new, Methicillin-resistant S. aureus (MRSA) have resisted topical, non-resistance-promoting, antimicrobial prepa- eradication attempts and have spread successfully from rations for the treatment of skin infections. Key words: hospitals into the community. In the USA and Europe azoles; clioquinol; fusidic acid; halquinol; hydrogen per- community-associated MRSA (CA-MRSA), first recog- oxide; skin infection; terbinafine. nized in the mid-1990s, are causing necrotic SSTIs in dermatology out-patients (7–9). (Accepted December 10, 2009.) In the late 1990s outbreaks of bullous impetigo among Acta Derm Venereol 2010; 90: 239–245. children were observed in Sweden and Norway (10, 11). These outbreaks were caused by a fusidic acid-resistant Mikael Alsterholm, Department of Dermatology and Vene- clone of S. aureus (FRSA) and its emergence was as- reology, Sahlgrenska University Hospital, SE-413 45 Göte- sociated with an increase in the use of topical fusidic borg, Sweden. E-mail: [email protected] acid. The FRSA has since flourished in several European countries and there are some reports of high frequencies of FRSA in patients with atopic dermatitis (12–16). Skin and soft tissue infections (SSTIs) are common Microbes are believed to trigger, exacerbate or sustain conditions causing considerable morbidity. Microbes inflammatory skin disease, such as atopic dermatitis, such as bacteria and yeasts often co-contribute to these seborrhoeic dermatitis and psoriasis (17). Due to this, infections. An appealing way of treating superficial skin treatment of skin disease often requires antimicrobial infections is through the use of topical antimicrobials. therapy. At the same time it is vital, whenever possible, Topical treatment delivers a high concentration of a drug to limit and carefully target the use of antibiotics. Faced to the desired area with limited or no systemic effects. with this dilemma, dermatologists, general practitio- Unfortunately, some topical antibiotics, such as fusidic ners and other healthcare professionals dealing with acid and mupirocin, have proved to be promoters of re- superficial skin infections need to be aware and make sistance and restrictive use is advised (1–3). In contrast, use of the anti-microbial potential of already existing topical azoles and allylamines against dermatophyte and topical skin pharmaceuticals. For instance, it is known © 2010 The Authors. doi: 10.2340/00015555-0840 Acta Derm Venereol 90 Journal Compilation © 2010 Acta Dermato-Venereologica. ISSN 0001-5555 240 M. Alsterholm et al. that some of the azoles, originally developed as anti- aureus (CCUG 17621), Staphylococcus epidermidis (CCUG fungals, also have activity against certain Gram-positive 39508) and Streptococcus pyogenes (CCUG 4207). bacteria (18). It can also be hypothesized that diols, preservatives and other additives in skin creams may Agar dilution assay have an antimicrobial effect of their own. Topical skin MICs of the formulations and substances were determined by pharmaceuticals with broad, non-resistance-promoting agar dilution assay. Dilution series for each formulation/sub- activity against staphylococci, streptococci, dermato- stance (individual plates for each dilution step) were prepared in blood agar medium (Columbia agar (Oxoid, Basingstoke, phytes or yeasts can be of great use in dermatology UK) + 5% defibrinated horse blood) for S. pyogenes and in where infections are often mixed. Diagnostic Sensitivity Test (DST) agar (Oxoid) for C. albicans, The aim of this study was to investigate the anti- E. coli, S. aureus and S. epidermidis. All dilution series were microbial properties of currently available antibacterial, made with freshly prepared and autoclaved agar that had been antifungal and anti-inflammatory topical formulations. allowed to cool to a temperature of 48–50°C in an oven before a formulation or substance was added. We have adopted an unconventional approach whereby Formulations were weighed and dissolved directly in agar we test the formulations in their marketed form and not for the concentrations 20% (10 g cream in a final volume of merely the designated active substance of each formu- 50 ml agar, weight/volume (w/v)), 10% (w/v), 5% (w/v) and lation. With the agar dilution assay we have determined 1% (w/v). For the lower concentrations a stock solution of 1% the minimal inhibitory concentration (MIC) against (0.5 g cream in a final volume of 50 ml agar) was prepared and further diluted with agar to obtain concentrations 0.5–0.0001% common skin pathogens for a panel of formulations (w/v). A pre-warmed oven with a constant temperature of 50°C and substances. kept the stock solution at an appropriate temperature during experimental procedures. Final concentration ranges were; Betnovat® 1–20% (w/v), Betnovat® with chinoform 0.005–20% MATERIALS AND METHODS (w/v), Emovat® 1–20% (w/v), Dermovat® 1–20% (w/v), Ca- nesten® 1% 0.001–20% (w/v), Daktar® 2% 0.001–5% (w/v), Topical skin pharmaceuticals Daktacort® 0.001–20% (w/v), Fucidin® 2% 0.0001%–20% (w/v), Hydrokortison CCS® 1% 1–20% (w/v), Kenacutan® The following skin pharmaceuticals were tested. All formula- 0.005–20% (w/v), Lamisil® 1% 0.001–20% (w/v) and Microcid® tions were creams. 1% 0.001–20% (w/v). ® Antibiotic formulation. Fucidin 2% (fusidic acid) (Leo Pharma, Substances were dissolved in dimethylformamide (DMF) Malmö, Sweden). to prepare a stock solution of 10 mg/ml and this solution was Antifungal formulations. Canesten® 1% (clotrimazole) (Bayer, further diluted and mixed with agar. When testing S. pyogenes, Solna, Sweden), Daktar® 2% (miconazole) (Janssen-Cilag, the stock solution was instead prepared with ethanol 99.5% Sollentuna, Sweden) and Lamisil® 1% (terbinafine) (Novartis, (Kemetyl AB, Haninge, Sweden) because DMF was no longer Täby, Sweden). recommended for laboratory use. Final concentration ranges Antifungal formulation with corticosteroid. Daktacort® (mico- were: clioquinol 0.125–100 µg/ml, econazole nitrate salt 1.0–50 nazole 20 mg/g + hydrocortisone 10 mg/g) (Janssen-Cilag, µg/ml, fusidic acid 0.03125–25 µg/ml, ketoconazole 0.125–100 Sollentuna, Sweden). µg/ml, miconazole nitrate salt 0.125–100 µg/ml and terbinafine Antiseptic formulation. Microcid® 1% (hydrogen peroxide) hydrochloride 0.5–50 µg/ml. The MICs against all microbes (Bioglan Pharma, Malmö, Sweden). for DMF (range tested 0.625–10% (volume/volume = v/v)) or Corticosteroid formulation. Betnovat® (betamethasone), Emo- ethanol (range tested 0.625–10% (v/v)) were also determined vat® 0.05% (clobetasone butyrate) and Dermovat® 0.05% to ensure that antimicrobial effect could not be attributed to the (clobetasol), all from GlaxoSmithKline, Solna, Sweden and solvent for the active substances. Hydrokortison CCS® 1% (hydrocortisone) (Clear Chemical Standard Petri dishes (92 × 16 mm (diameter × height)) were Sweden, Borlänge, Sweden). used and the agar
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