Minimum inhibitory (MIC) and minimum microbicidal concentration (MMC) of polihexanide and triclosan against antibiotic sensitive and resistant Staphylococcus aureus and Escherichia coli strains

dgkh000163 10.3205/dgkh000163 urn:nbn:de:0183-dgkh0001636 Research Article Minimum inhibitory (MIC) and minimum microbicidal concentration (MMC) of polihexanide and triclosan against antibiotic sensitive and resistant Staphylococcus aureus and Escherichia coli strains Minimale Hemm-Konzentration (MHK) und minimale bakterizide Konzentration (MBK) von Polihexanid und Triclosan gegen Antibiotika-empfindliche und resistente Staphylococcus aureus- und Escherichia coli-Stämme Assadian Assadian Ojan O Prof. Dr. DTMH

Clinical Institute for Hospital Hygiene, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria, Phone: +43-1-40400-1904, Fax: +43-1-40400-1907Clinical Institute for Hospital Hygiene, Medical University of Vienna, Vienna, Austria

ojan.assadian@meduniwien.ac.at author Wehse Wehse Katrin K

Institute of Hygiene and Environmental Medicine, University Medicine Greifswald, Germany

author Hübner Hübner Nils-Olaf NO

Institute of Hygiene and Environmental Medicine, University Medicine Greifswald, Germany

author Koburger Koburger Torsten T

Hygiene-North GMBH, Greifswald, Germany

author Bagel Bagel Simone S

Antiinfectives Intelligence, Clinical Microbiological Research and Communication GmbH, Campus Fachhochschule Bonn-Rhein-Sieg, Bonn, Germany

author Jethon Jethon Frank F

Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany

author Kramer Kramer Axel A

Institute of Hygiene and Environmental Medicine, University Medicine Greifswald, Germany

author German Medical Science GMS Publishing House

Düsseldorf

610 polihexanide triclosan antimicrobial inhibitory concentration microbicidal concentration Polihexanid Triclosan antimikrobiell minimale inhibitorische Konzentration MHK minimale mikrobizide Konzentration 20111215 engl 1863-5245 6 1 GMS Krankenhaushygiene Interdisziplinär GMS Krankenhaushyg Interdiszip Prevention and therapy of nosocomial infections 06 Hintergrund: In einer quantitativen in-vitro Studie wurde der antimikrobielle Effekt von Polihexanid und Triclosan unter identischen Verhältnissen gegen Referenzstämme sowie klinische Isolate von Staphylococcus aureus und Escherichia coli bestimmt.Methoden: Die minimale Hemmkonzentration (MHK) und die minimale mikrobiozide Konzentration (MMK) wurden gemäß DIN 58940-81 mittels Mikro-Verdünnungs-Essay und eines quantitativen Verdünnungstests gemäß EN 1040 bestimmt. Polihexanid wurde in einer Polyethylene-Glykol 4000 Lösung, Triclosan in wässriger Lösung angesetzt. Ergebnisse: Die MHK von Polihexanid gegen alle getesteten Stämme lag zwischen 1–2 µg/mL. Die MHK von Triclosan war stammabhängig und lag bei 0,5 µg/mL für die getesteten Referenzstämme und bei 64 µg/mL</PlainText></TextGroup> für zwei klinischen Isolate. Gegen alle Stämme erreichte Triclosan bei einer Konzentration von 0,6 µg/mL einen logRF >5 ohne und logRF >3 mit 0,2% Albuminbelastung. Als Ausnahme erwies sich <TextGroup><Mark2>S. aureus</Mark2></TextGroup>-Stamm H-5-24, für den eine Triclosan-Konzentration von <TextGroup><PlainText>0,6 µg/mL</PlainText></TextGroup> bei einer Einwirkungszeit von 1 min ohne bzw. 10 min mit Albuminbelastung erforderlich war, um dieselben logRF zu erreichen. Polihexanid erreichte bei einer Konzentration von 0,6 µg/mL einen logRF >5 ohne und logRF >3 mit Belastung bei einer Einwirkungszeit von <TextGroup><PlainText>30 s</PlainText></TextGroup>. Hier erwies sich als Ausnahme der Norddeutsche MRSA Epidemiestamm, gegen den bei selber Konzentration eine Einwirkungszeit von 5 min erforderlich war. </Pgraph><Pgraph><Mark1>Schlussfolgerungen:</Mark1> Die klinischen <Mark2>E. coli</Mark2>-Isolate erforderten höhere MHKs bei Triclosan als die untersuchten Referenzstämme. Für Polih<TextGroup><PlainText>exan</PlainText></TextGroup>id und Triclosan konnte eine stammabhängige Empfindlichkeit gezeigt werden. Beide Antiseptika werden im klinischen Einsatz jedoch regelhaft bei weit höheren Konzentrationen eingesetzt, womit die unterschiedlichen Empfindlichkeiten klinisch keine Rolle spielen sollten.</Pgraph></Abstract> <Abstract language="en" linked="yes"><Pgraph><Mark1>Background:</Mark1> An in-vitro study was conducted investigating the antim<TextGroup><PlainText>icrob</PlainText></TextGroup>ial efficacy of polihexanide and triclosan against clinical isola<TextGroup><PlainText>tes and reference</PlainText></TextGroup> laboratory strains of <Mark2>Staphylococcus aureus</Mark2> and <Mark2>E</Mark2><TextGroup><Mark2>scherich</Mark2></TextGroup><Mark2>ia coli</Mark2>.</Pgraph><Pgraph><Mark1>Methods:</Mark1> The minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC) were determined following DIN 58940-81 using a micro-dilution assay and a quantitative suspension test following EN 1040. Polihexanide was tested in polyethylene glycol 4000, triclosan in aqueous solutions.</Pgraph><Pgraph><Mark1>Results:</Mark1> Against all tested strains the MIC of polihexanide ranged between 1–2 µg/mL. For triclosan the MICs varied depending on strains ranging between 0.5 µg/mL for the reference strains and 64 µg/mL for two clinical isolates. A logRF >5 without and logRF >3 with 0.2% album<TextGroup><PlainText>in burden</PlainText></TextGroup> was achieved at 0.6 µg/mL triclosan. One exception was <TextGroup><Mark2>S. aureus</Mark2></TextGroup> strain H-5-24, where a triclosan concentration of 0.6 µg/mL required 1 minute without and 10 minutes with albumin burden to achieve the same logRFs. Polihexanide achieved a logRF >5 without and logRF >3 with albumin burden at a concentration of 0.6 µg/mL within 30 sec. The exception was the North-German epidemic MRSA strain, were an application time of 5 minutes was required. </Pgraph><Pgraph><Mark1>Conclusion:</Mark1> The clinical isolates of <Mark2>E. coli</Mark2> generally showed higher MICs against triclosan, both in the micro-dilution assay as well in the quantit<TextGroup><PlainText>ativ</PlainText></TextGroup>e suspension test than comparable reference laboratory strains. For polihexanide and triclosan strain dependant susceptibility was shown. However, both antimicrobial compounds are effective when used in concentrations common in practice.</Pgraph></Abstract> <TextBlock linked="yes" name="Introduction"> <MainHeadline>Introduction</MainHeadline><Pgraph>Due to their different chemical structure, their different use in clinical practice and their different antimicrobial capacity the matter of antibiotic resistance does not apply <TextGroup><PlainText>to antiseptics at the moment. So far, for microbicidal</PlainText></TextGroup> antiseptics no clinically relevant resistance is documented <TextLink reference="1"></TextLink>, <TextLink reference="2"></TextLink>, <TextLink reference="3"></TextLink>. For microbistatic antiseptics, however, the potential risk for development of resistance can not be excluded. Examples for microbistatic antiseptics include benzalkonium chloride <TextLink reference="4"></TextLink>, Cetylpyridinium chloride <TextLink reference="5"></TextLink>, chlorhexidine and triclosan <TextLink reference="6"></TextLink>, <TextLink reference="7"></TextLink>. For hexachlorophene <TextLink reference="1"></TextLink> and chlorhexidine <TextLink reference="8"></TextLink> a plasmid-coded resistance was shown already. </Pgraph><Pgraph>Furthermore, for some antiseptics and antibiotics, increased resistance against antiseptics was correlated with increased antibiotic resistance <TextLink reference="9"></TextLink>, <TextLink reference="10"></TextLink>; however, increased antibiotic resistance has not been associated with antiseptic resistance so far. Russel et al. <TextLink reference="11"></TextLink> could demonstrate in an in-vitro study that after <Mark2>Pseudomonas stutzeri</Mark2> was exposed to increasing concentrations of chlorhexidine after multiple passages, a stable chlorh<TextGroup><PlainText>exidin</PlainText></TextGroup>e resistance was achieved. Simultaneously resistance against benzalkonium chloride, cetylpyridinium chloride and triclosan and a number of antibiotics was increased as well. For <Mark2>Mycobacterium smegmatis</Mark2> our study group could show a parallel increase in resistance for isoniazid and triclosan <TextLink reference="7"></TextLink>.</Pgraph><Pgraph>For the majority of antiseptic compounds the first step for their antimicrobial action is penetration into the bacterial cell. If this penetration is minimized or inhibited, resistance will result. Bacteria are able to alter their cell’s permeability by changing its lipid content, the composition of the outer membrane proteins, plasmid-coded mucous production or by altering the affinity of their efflux pumps. If the efflux pump has an unspecific affinity to several compounds, resistances will result. Example for an efflux pump with a broad substrate spectrum is <Mark2>AcrAB</Mark2> of <Mark2>E</Mark2><TextGroup><Mark2>scherich</Mark2></TextGroup><Mark2>ia coli</Mark2> which is also responsible for the efflux activity of the membrane-pore-protein <Mark2>TolC</Mark2>, able of transporting tetracycline, chloramphenicol, fluoroquinolones, β-lactams, novobiocin, erythromycin, ethidium bromide, crystal violet and Acriflavin <TextLink reference="12"></TextLink>, <TextLink reference="13"></TextLink>. </Pgraph><Pgraph>In contrast to antibiotics where antibiogramms are generated in course of routine clinical microbiological diagn<TextGroup><PlainText>ostic</PlainText></TextGroup>s for a broad range of antibiotics and bacteria, such routine data is not available for antiseptics and knowledge on antimicrobial efficacy is based only on selected, most often historic, literature. In light of the possibilities for development of resistance against antiseptics it deems useful to control the antimicrobial activity of frequently used antiseptics and possible changes in regular intervals.</Pgraph><Pgraph>Therefore we conducted an in-vitro study investigating the current antimicrobial efficacy of the frequently used skin, mucous membrane and wound antiseptics polihexanide and triclosan against clinical isolates and defined laboratory strains of <Mark2>Staphylococcus aureus</Mark2> and <Mark2>E. coli</Mark2>.</Pgraph></TextBlock> <TextBlock linked="yes" name="Methods"> <MainHeadline>Methods</MainHeadline><SubHeadline>Tested antimicrobial compounds</SubHeadline><Pgraph>For polihexanide, we used the commercially available product Lavasept<Superscript>®</Superscript> (Fresenius AG, Bad Homburg, Germany) which contains 200 µg/mL polihexanide and <TextGroup><PlainText>10 µg/mL</PlainText></TextGroup> macrogolum 4000. Because triclosan as pure substance (Irgasan<Superscript>®</Superscript>DP 300; CAS No 88032-08-0; Ciba AG, Basel, Switzerland) is not solvable in water, we diluted the product in dimethylsulfoxid (DMSO) and 9.2% ethanol according to the manufacturer’s recommendation. In a separate pilot experiment, we could rule out that 9.2% ethanol itself possesses antimicrobial properties. </Pgraph><SubHeadline>Tested clinical isolates and laboratory strains</SubHeadline><Pgraph>The following bacteria were tested using the micro-dilution test: <Mark2>E. coli ATCC 25922</Mark2>; <Mark2>E. coli AG 100 wild type</Mark2> (K-12 argE3 thi-1 rpsL xyl mtl Δ(gal-uvrB) supE44) <TextLink reference="14"></TextLink>, <TextLink reference="15"></TextLink>; <TextGroup><Mark2>E. coli</Mark2></TextGroup><Mark2> AGT11</Mark2>, containing <Mark2>fabI</Mark2> mutation with resistance against triclosan; exchange of amino acid in the enoyl-reductase in position 93 <TextLink reference="16"></TextLink>; <Mark2>E. coli AGT 11K</Mark2> (AcrAB::kan, deletion of the <Mark2>E. coli</Mark2> efflux pump <Mark2>AcrAB)</Mark2> <TextLink reference="14"></TextLink>; <Mark2>E. coli AGT 11 Kan</Mark2> (AmarCORAB::kan, constructed by replacement of a chromosomal 1.24-kb BspHI fragment of the mar locus in AG100 by homologous recombination with the kanamycin resistance cassette (Kanr) from pKMN33; deletion of the regulator gene which activate the efflux pump) <TextLink reference="17"></TextLink>; <TextGroup><Mark2>S. aureus</Mark2><PlainText> ATCC 29213, </PlainText><Mark2>MRSA</Mark2><PlainText> strains H-5-18</PlainText></TextGroup>, H-5-19, H-5-20, H-5-24, H-5-26, H-5-27, and H-5-31 (University Clinic Bonn, Germany), MRSA epidemic strain North-Germany, Niedersachsen and Berlin (Robert Koch Institute, Wernigerode, Germany); <Mark2>VISA</Mark2> strains Ve 13985, Ve 1177/98, BK 13230, BK 1704/98, 18 A 026, and 20 A 063 (Robert Koch Institute, Wernigerode, Germany).</Pgraph><Pgraph>Additionally to the micro-dilution test, <Mark2>S. aureus</Mark2> ATCC 29213, <Mark2>MRSA</Mark2> H-5-24, MRSA epidemic strain North-G<TextGroup><PlainText>ermany</PlainText></TextGroup>, <Mark2>E. coli</Mark2> ATCC 25922 and <Mark2>E. coli</Mark2> AGT 11 were tested in a quantitative suspension test.</Pgraph><SubHeadline>Culture media and neutralizing agents</SubHeadline><Pgraph><TextGroup><PlainText>As polihexanide precipitates on Mueller-Hinton agar, micr</PlainText></TextGroup>o-organisms were cultured on iso-sensitest bouillon (Oxoid, Darmstadt, Germany), tryptone soya agar and broth, respectively (Oxoid, Wesel, Germany). As neutralizer for polihexanide 3% (w/v) Tween 80 (Serva, Heidelberg, Germany), 3% (w/v) saponine (Fluka, Buchs, Switzerland), 0.1 % (w/v) histidine (Serva, Heidelberg, Germany), and 0.1% (w/v) cysteine (Merck, Darmstadt, Germany) was used. As neutralization of triclosan was not possible with the usual neutralization solution, we used egg yolk (sterile egg yolk diluted to 50% by sterile distilled water) ins<TextGroup><PlainText>tead. However</PlainText></TextGroup>, only triclosan concentrations of 0.6 µg triclosan/L and less were possible. Therefore, this concentration also was the maximum tested concentration in the quantitative suspension test. </Pgraph><SubHeadline>Determination of the Minimum Inhibitory Concentration (MIC)</SubHeadline><Pgraph>The MIC was determined following DIN 58940-81 <TextLink reference="18"></TextLink> using a micro-dilution assay. Briefly, using colonies from a fresh overnight culture an inoculum of 1x10<Superscript>5</Superscript> cfu/mL in the final medium was prepared. 50 µl of the inoculum were added to 50 µl of the respective antimicrobial test-dilution and incubated at 36°C ± 1°C for 20h ± 2h.</Pgraph><SubHeadline>Determination of the Minimum Microbicidal Concentration (MMC)</SubHeadline><Pgraph>The MMC was determined using the quantitative suspension test following Pitten et al. <TextLink reference="19"></TextLink> with and without bio-burden. For each assay, 0.1 ml of the test organism (10<Superscript>8</Superscript>–10<Superscript>9</Superscript> cfu/mL) in tryptone soya broth was transferred from a fresh overnight culture into a test tube, mixed with 1 ml distilled sterile water (test without bio-burden) or, in parallel series, with 1 ml of 0.2% bovine serum albumin (test without bio-burden), and transferred in 9 ml of the test solution. At the final time of action (30 sec, 60 sec, 5 min, 10 min, 60 min, respectively) 1 ml of the test mixture was transferred into 9 ml tryptical-soy-broth with addition of the respective neutralizer. After 5 minutes of neutralisation, serial dilutions were plated on trypticase-soy-agar. Colonies were counted after 48h. The log<Subscript>10</Subscript> r<TextGroup><PlainText>eductio</PlainText></TextGroup>n factor (RF) for each application time was calc<TextGroup><PlainText>ulate</PlainText></TextGroup>d using the formula: log<Subscript>10</Subscript> (control) – log<Subscript>10</Subscript> (test sample). All experiments were performed in triplicate. <TextGroup><PlainText>Both antiseptics were tested at concentrations of 2 µg/mL</PlainText></TextGroup>, 0.2 µg/mL and 0.02 µg/mL. </Pgraph><SubHeadline>Statistical analysis</SubHeadline><Pgraph>All assays were repeated 6-fold, and number of organisms were averaged as mean cfu/ml and expressed as log<Subscript>10</Subscript>. The log<Subscript>10</Subscript> reduction factor (logRF) was calculated as log<Subscript>10</Subscript> of the pre-value minus log<Subscript>10</Subscript> of the post-value. </Pgraph></TextBlock> <TextBlock linked="yes" name="Results"> <MainHeadline>Results</MainHeadline><SubHeadline>Microbistatic activity</SubHeadline><Pgraph>For <Mark2>S. aureus</Mark2> no difference was observed in the antim<TextGroup><PlainText>icrobi</PlainText></TextGroup>al activity of polihexanide at concentrations of 1 and 2 µg/mL against ATCC reference strains, 10 MRSA isolates and 6 VISA strains (Table 1 <ImgLink imgNo="1" imgType="table"/>). Triclosan could be tested only against the ATCC strains and the epidemic North-German MRSA strain. Compared to MRSA strains the antimicrobial activity of triclosan was 512 times lower. </Pgraph><Pgraph>All <Mark2>E. coli</Mark2> ATCC reference strains and the <Mark2>E. coli</Mark2> wild type AG 100 were inhibited by triclosan concentrations ranging between 0.5 and 1 µg/mL. <Mark2>E. coli</Mark2> AGT 11 with a mutation in its enoylacyl-carrierprotein-reductase and <Mark2>E. coli</Mark2> AGT Kan11, a strain identical to <Mark2>E. coli</Mark2> AGT 11 but with addi<TextGroup><PlainText>tiona</PlainText></TextGroup>lly defect efflux pump regulation showed an increased MIC by the factor 128 against triclosan. For <TextGroup><Mark2>E. coli</Mark2></TextGroup> AGT 11K, again a strain identical to <Mark2>E. coli</Mark2> AGT 11 but additionally with switched-off efflux pump AcrAB triclosan resistance was decreased by 32 times (Table 2 <ImgLink imgNo="2" imgType="table"/>). The minimum microbistatic concentration for polihexanide was 2 µg/mL against all tested <Mark2>E. coli</Mark2> strains. </Pgraph><SubHeadline>Microbicidal activity</SubHeadline><Pgraph>In the quantitative suspension test polihexanide showed superior activity than triclosan (Table 3 <ImgLink imgNo="3" imgType="table"/>). <Mark2>S. aureus</Mark2> ATCC strains did not differ in their susceptibility against triclosan as compared to the North-German epidemic MRSA strain. The minimum requirements of a logRF >5 without and logRF >3 with 0.2% albumin burden within 5 minutes application time <TextLink reference="19"></TextLink> were achieved at a concentration of 0.6 µg/mL triclosan. The exception was <Mark2>MRSA</Mark2> strain H-5-24, where a triclosan concentration of 0.6 µg/mL was able only after 60 sec without and 10 minutes with albumin burden to achieve a logRF >5 or a logRF >3 reduction, respectively. The difference between <Mark2>E. coli</Mark2> ATCC 25922 und <Mark2>E. coli</Mark2> AGT 11 was only minute, for both strains a concentration of 0.6 µg/mL triclosan at an application time of 60 sec was needed to achieve a logRF >5 without and a logRF >3 with albumin-burden.</Pgraph><Pgraph>Polihexanide achieved a logRF >5 without and lofRF >3 with albumin burden at concentrations of 0.6 µg/mL within 30 sec, respectively, for <Mark2>S. aureus</Mark2>. The only exc<TextGroup><PlainText>epti</PlainText></TextGroup>on was the North-German epidemic MRSA strain, were an application time of 5 minutes was required. <Mark2>MRSA</Mark2> H-5-24 did not differ in its susceptibility from the ATCC reference strains. <Mark2>E. coli</Mark2> AGT 11 was only lesser susceptible to polihexanide as compared to <Mark2>E. coli</Mark2> ATCC 25922 under the presence of an albumin burden. Without albumin burden a logRF >5 was achieved with a concentration of 0.2 µg/mL and an application time of 30 sec, or 0.02 µg/mL and 60 sec, respectively. With albumin burden even an application time of 1 minute was not sufficient for a concentration of 0.02 µg/mL polihexanide to achieve a logRF >3.</Pgraph></TextBlock> <TextBlock linked="yes" name="Discussion"> <MainHeadline>Discussion</MainHeadline><Pgraph>In order to assess the efficacy of the tested antimicrobial compounds polihexanide and triclosan, our results must take their usual clinically used concentrations into account. Triclosan usually is used in liquid soap at conc<TextGroup><PlainText>entratio</PlainText></TextGroup>n of 2 to 5 µg/mL, in toothpaste starting from <TextGroup><PlainText>3 µg/mL</PlainText></TextGroup> and in hand disinfectants ranging from 2 to <TextGroup><PlainText>20 µg/mL</PlainText></TextGroup> <TextLink reference="7"></TextLink>.</Pgraph><Pgraph>Wound antiseptics containing polihexanide as their active ingredient contain concentrations of 0.2 to 1 µg/mL and sanitizers 1 to 2 µg/mL. Our results showed that the required minimum concentration for triclosan and polihexanide are 0.6 µg/mL, and therefore, concentrations used in clinical still are far above the MIC demonstrated for various clinical isolates and laboratory reference strains. The focus of our study, however, was not to assess the antimicrobial efficacy in clinical practice, but rather to explore differences in susceptibility within various antib<TextGroup><PlainText>ioti</PlainText></TextGroup>c susceptible strains of <Mark2>S. aureus</Mark2> and <Mark2>E. coli</Mark2> in order to detect possible induction of co-resistance against antiseptics. </Pgraph><Pgraph>The results of our study showed that the susceptibility of the epidemic North-German MRSA strain against triclosan was reduced as compared to antibiotic susceptible strains. Also <Mark2>MRSA</Mark2> H-5-24 showed reduced susceptibility against triclosan. As expected, triclosan was highly effective against the <Mark2>E. coli</Mark2> ATCC strain. In contrast, clinical <TextGroup><Mark2>E. coli</Mark2></TextGroup> isolates showed decreased susceptibility against triclosan. The mode of action of triclosan is based on the inhibition of the biosynthesis of lipid acid and enoylacyl-carrierprotein-reductase <TextLink reference="7"></TextLink>. For <Mark2>E. coli</Mark2> to become resistant against triclosan, a single change of 1 amino acid in the reductase enzyme is sufficient resulting in a 32- to 128-fold MIC increase.</Pgraph><Pgraph>Another reason for the increased resistance of <Mark2>E. coli</Mark2> against triclosan could also be an increased activity of the efflux pump <Mark2>AcrAB-TolC</Mark2> located in the bacterial cell membrane. <Mark2>E. coli</Mark2> mutants with increased efflux pump activity showed higher MIC values against various antib<TextGroup><PlainText>iotic</PlainText></TextGroup>s as well as triclosan than strains with normal efflux pump activity. This correlation was also observed earlier by McMurry et al. <TextLink reference="14"></TextLink> and was explained by the fact that affected compounds are substrates of the same membrane pump <Mark2>AcrAB-TolC</Mark2>. Our observation of a multiple antibiotic-resistance-phenotype combined with an increase in triclosan MIC by 1 to 2 titres in some clinical <TextGroup><Mark2>E. coli</Mark2></TextGroup> isolates confirms this possibility. </Pgraph><Pgraph>With one exception MICs of antibiotic resistant and susceptible strains against polihexanide did not differ showing that polihexanide has a different mode of action against bacteria <TextLink reference="20"></TextLink>. Furthermore, the observed low MICs for polihexanide allow the conclusion that polihexanide is not an substrate for above efflux pumps. Based on the presented in-vitro results and clinical observations <TextLink reference="21"></TextLink>, it is explainable why polihexanide is a very effective wound antiseptic. Our data show that polihexanide easily is able to kill even antibiotic resistant bacteria commonly found as the cause of wound infection. Currently, it can be concluded that unlike triclosan, polihexanide is not transported outside of the bacterial cell wall by the activity of efflux pumps, and therefore is not subject to development of possible bacterial resistance. Moreover, polihexanide is integrated into the bacterial cell membrane, which it irreversibly destroys.</Pgraph><Pgraph>However, as with all antimicrobial compounds, it seems prudent to limit its use to clinically justifiable indications. </Pgraph></TextBlock> <TextBlock linked="yes" name="Conclusion"> <MainHeadline>Conclusion</MainHeadline><Pgraph>The clinical isolates of <Mark2>E. coli</Mark2> generally showed higher MICs against triclosan, both in the micro-dilution assay as well in the quantitative suspension suspension test than comparable ATCC laboratory reference strains. For polihexanide and triclosan strain dependant susceptibility was shown. However, both antimicrobial compounds are effective when used in concentrations common in practice. </Pgraph></TextBlock> <TextBlock linked="yes" name="List of abbreviations"> <MainHeadline>List of abbreviations</MainHeadline><Pgraph>ATCC – American Type Culture Collection</Pgraph><Pgraph>logRF – log 10 Reduction factor</Pgraph><Pgraph>MIC – Minimum inhibitory concentration</Pgraph><Pgraph>MMC – Minimum microbicidal concentration</Pgraph><Pgraph>MRSA – Methicillin Resistant <Mark2>Staphylococcus aureus</Mark2></Pgraph><Pgraph>VISA – Vancomycin intermediate sensitive <Mark2>Staphylococcus aureus</Mark2></Pgraph></TextBlock> <TextBlock linked="yes" name="Notes"> <MainHeadline>Notes</MainHeadline><SubHeadline>Authors’ contribution</SubHeadline><Pgraph>AK and PR had the idea for the study, AK and PR planned and supervised the experiments, as well drafted the m<TextGroup><PlainText>anuscri</PlainText></TextGroup>pt and analyzed and interpreted the data. KW, TK, and SB participated in the technical design of the study and performed experiments. All authors analyzed and interpreted the data. NOH, OA SB, and FJ participated in the study’s design and coordination, helped to draft the manuscript, and analyzed and interpreted the data. All authors have been involved in drafting the manuscript or revising it critically for important intellectual content and have read and approved the final manuscript.</Pgraph><SubHeadline>Funding</SubHeadline><Pgraph>The study was sponsored by Fresenius Kabi GmbH, Bad Homburg, Germany. The sponsors had no participation in the study design or interpretation of the results.</Pgraph><SubHeadline>Conflicts of interest</SubHeadline><Pgraph>Dr. Frank Jethon is a paid employee of Fresenius Kabi GmbH, Bad Homburg, Germany. Prof. Dr. Assadian, <TextGroup><PlainText>Dr. Bagel</PlainText></TextGroup>, Dr. Huebner, Prof. Dr. Kramer, Dr. Rudolph and Mrs. Wehse have no financial or other conflict of interest to declare.</Pgraph></TextBlock> <References linked="yes"> <Reference refNo="1"> <RefAuthor>Lebek G</RefAuthor> <RefTitle>Genetische Grundlagen der Resistenzentwicklung von Mikroorganismen gegenüber Antiseptika bzw. Desinfektionsmitteln</RefTitle> <RefYear>1981</RefYear> <RefBookTitle>Handbuch der Antiseptik</RefBookTitle> <RefPage>170-186</RefPage> <RefTotal>Lebek G. Genetische Grundlagen der Resistenzentwicklung von Mikroorganismen gegenüber Antiseptika bzw. Desinfektionsmitteln. In: Krasilnikow AP, Kramer A, Gröschel D, Weuffen W, eds. Handbuch der Antiseptik, Vol. I/2. 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Available from: http://www.egms.de/en/journals/dgkh/2007-2/dgkh000091.shtml</RefTotal> <RefLink>http://www.egms.de/en/journals/dgkh/2007-2/dgkh000091.shtml</RefLink> </Reference> </References> <Media> <Tables> <Table format="png"> <MediaNo>1</MediaNo> <MediaID>1</MediaID> <Caption><Pgraph><Mark1>Table 1: MIC (µg/mL) of polihexanide and triclosan against </Mark1><Mark1><Mark2>S. aureus</Mark2></Mark1><Mark1> and different MRSA and VISA-strains</Mark1></Pgraph></Caption> </Table> <Table format="png"> <MediaNo>2</MediaNo> <MediaID>2</MediaID> <Caption><Pgraph><Mark1>Table 2: MIC (µg/mL) of polihexanide and triclosan against </Mark1><Mark1><Mark2>E. coli</Mark2></Mark1></Pgraph></Caption> </Table> <Table format="png"> <MediaNo>3</MediaNo> <MediaID>3</MediaID> <Caption><Pgraph><Mark1>Table 3: Time and concentration dependant bactericidal activity of polihexanide and triclosan</Mark1></Pgraph></Caption> </Table> <NoOfTables>3</NoOfTables> </Tables> <Figures> <NoOfPictures>0</NoOfPictures> </Figures> <InlineFigures> <NoOfPictures>0</NoOfPictures> </InlineFigures> <Attachments> <NoOfAttachments>0</NoOfAttachments> </Attachments> </Media> </OrigData> </GmsArticle>