Journal of Hospital Infection 86 (2014) 68e72 Available online at www.sciencedirect.com
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
In-vitro activity of polyhexanide alone and in combination with antibiotics against Staphylococcus aureus W. Fabry a, *, H.-J. Kock b a b
Institut fu¨r Medizinische Mikrobiologie, Virologie und Krankenhaushygiene, Universita¨t Rostock, Rostock, Germany Vivantes-Humboldt Klinikum, Klinik fu¨r Unfallchirurgie und Orthopa¨die, Berlin, Germany
A R T I C L E
I N F O
Article history: Received 30 January 2013 Accepted 1 October 2013 Available online 23 October 2013 Keywords: Polyhexanide Antiseptic Antibiotic Staphylococcus aureus Meticillin-resistant Staphylococcus aureus Minimum inhibitory concentration Minimum bactericidal concentration
S U M M A R Y
Background: The resistance of Staphylococcus aureus is increasing, not only to antibiotics but also to antiseptics. Aim: To investigate the activity of the antiseptic polyhexanide and several antibiotics against clinical isolates of meticillin-susceptible and meticillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively). Polyhexanide was tested alone and in combination with oxacillin, penicillin G, ampicillin, cefazolin, cefuroxime, imipenem, gentamicin, erythromycin, doxycycline, levoflocaxin, linezolid and vancomycin. Methods: Fifty MSSA and 50 MRSA strains, including one vancomycin-intermediate (VISA) strain, were tested. All strains were typed by pulsed-field gel electrophoresis (PFGE) to exclude testing of clonal isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined using the serial broth microdilution technique according to DIN 58940. Combinations of polyhexanide and different antibiotics were investigated using the checkerboard technique. Findings: Polyhexanide MICs and MBCs in the range of 0.5e2 mg/L were found for both MSSA and MRSA, and the VISA strain had MIC and MBC values of 2 mg/L. All isolates were regarded as susceptible to polyhexanide, and no antagonism was observed between polyhexanide and the tested antibiotics. Synergism between polyhexanide and some bacteriostatic antibiotics (erythromycin, doxycycline and linezolid) was found for some strains. Conclusions: Polyhexanide appears to be suitable for the topical treatment of S. aureus alone and in combination with antibiotics. ª 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction Staphylococcus aureus is a nosocomial pathogen of increasing importance due to the emergence of meticillin* Corresponding author. Address: Institut fu ¨r Medizinische Mikrobiologie, Virologie und Krankenhaushygiene, Universita ¨t Rostock, Schillingallee 70, D-18057 Rostock, Germany. Tel.: þ49 208/631290; fax: þ49 214/374266. E-mail address: [email protected] (W. Fabry).
resistant strains (MRSA) associated with multi-drug resistance.1 Patients colonized by S. aureus are at risk for bacteraemia and severe systemic infections.2,3 The topical antibiotic mupirocin is often applied for the clearance of topical S. aureus.4 However, MRSA is developing resistance to mupirocin5,6 and also to antiseptics such as chlorhexidine7 and triclosan.8 As such, alternative agents for eradication of S. aureus are needed urgently. The toxicological safety and tissue compatibility of the antiseptic fractionated polyhexamethylene biguanide
0195-6701/$ e see front matter ª 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jhin.2013.10.002
W. Fabry, H.-J. Kock / Journal of Hospital Infection 86 (2014) 68e72 (polyhexanide) and polyethylene glycol 4000 (Lavasept, Lavasorb, Lavanid) was shown by in-vitro investigations and animal studies.9e11 Previous testing of selected strains showed antibacterial activity of polyhexanide against S. aureus.12e14 A prospective randomized controlled double-blind study including S. aureus exhibited a faster and significantly larger reduction on soft tissue wound surfaces in comparison with Ringer’s solution.15,16 For further investigations into polyhexanide activity, 100 S. aureus isolates were subjected to quantitative tests. The susceptibility of the strains to various antibiotic agents was tested alone and in combination with polyhexanide to detect potential synergistic or antagonistic effects of simultaneous administration. Prior to measurement of antiseptic activity, all strains were typed by pulsed-field gel electrophoresis (PFGE) to exclude testing of clonal isolates.
Materials and methods
69
50 mg/L) and MgCl2 (final Mg2þ concentration 25 mg/L). As such, polyhexanide was diluted in the test broth to final concentrations of 0.06e128 mg/L. The final inoculum of the tested isolates was 5 105 colony-forming units (cfu)/mL. The presence or absence of an inoculum effect was determined by repeating MIC and MBC determinations with diverse inocula (1.5 105, 1.5 106, 1.5 108 cfu/mL). The MIC was read after 24 h of incubation at 35 C as the lowest concentration of the drug preventing visible growth. For the determination of MBCs, a 10-mL aliquot from wells near the threshold for turbidity of the MIC plate was transferred in duplicate after 6 h onto blood agar plates. Incubation was performed for 24 h at 35 C. The MBC was defined as the lowest antiseptic concentration preventing visible regrowth. In addition, the MBCs of 10 MRSA and 10 MSSA were determined after incubation of the MIC plates for 3 and 24 h. MRSA type strain ATCC 33591 and MSSA type strains ATCC 25923 and ATCC 29213 were used for quality control.
Strains Antibiotic susceptibility testing One hundred clonally different isolates (50 MRSA and 50 MSSA) were collected consecutively from infected patients at the university hospitals of Essen and Rostock (Germany). The strains were identified using the Vitek2 automated system (bioMe ´rieux, Nu ¨rtlingen, Germany), the agglutination kit Slidex Staph Plus (bioMe ´rieux), and the BD DNase test agar (Becton Dickinson, Heidelberg, Germany). The Protect system (Technical Service Consultants Limited, Heywood, UK) was used for storage at 70 C. Prior to testing, an aliquot of each isolate was thawed and subcultured on sheep blood agar.
PFGE typing PFGE was performed using a modified version of Sambrook et al.’s17 method with Gene Navigator System apparatus (Pharmacia, LKB Biotechnology AB, Uppsala, Sweden), as described below. Genomic DNA was extracted from bacterial cells by lysostaphin (Sigma-Aldrich, St. Louis, MO, USA) treatment and was subsequently digested with Sma1 (Roche Diagnostics, Mannheim, Germany). The resulting DNA fragments were separated for 24 h in a 1% low melting agarose gel and a 0.05 M tris-borate EDTA buffer, pH 8.5, at 10 C and 180e185 V with a pulse time of 0.5e40 s. For documentation, gels were stained with ethidium bromide. Lambda DNA concatemers (New England Biolabs, Beverly, MA, USA) were used as molecular size standards. PFGE patterns were interpreted according to Tenover et al.18 If the patterns differed by three or more bands, isolates were considered to be genetically different.
Determination of polyhexanide activity Polyhexanide was obtained from Fresenius Kabi (Bad Homburg, Germany). The stock solution contained 200 mg polyhexanide and 10 mg polyethylene glycol (polyethylene glycol 4000) per mL. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined by serial broth microdilution tests according to DIN 5894019,20 using Mueller-Hinton broth (MHB, E. Merck, Darmstadt, Germany), supplemented with CaCl2 (final Ca2þ concentration
MICs and MBCs were determined for each drug using analytical grade powders kindly provided by their manufacturers. Antibiotic substances of defined activity were dissolved and diluted in Mueller-Hinton broth (Unipath, Wesel, Germany) supplemented with calcium (50 mg/L) and magnesium (25 mg/L). The dilution scheme covered concentrations of antibiotics as follows: oxacillin, 0.001e4 mg/L; gentamicin, erythromycin, doxycycline, levofloxacin and vancomycin, 0.007e16 mg/L; penicillin G, ampicillin and linezolid, 0.015e32 mg/L; and cefazolin, cefuroxim and imipenem, 0.031e64 mg/L. MIC and MBC determinations were performed as described for testing the antiseptic substances. For selected strains, cephalosporin, carbapenem and vancomycin MBC determination was performed with transfer after 24 h of incubation. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the following breakpoints (mg/ L) were used indicating susceptibility () and resistance (>): oxacillin, 2/2; penicillin G, 0.125/0.125; ampicillin, 2/8; cefazolin, 1/2; cefuroxime, 4/8; imipenem, 2/8; gentamicin, 1/1; erythromycin, 1/2; doxycycline, 1/2; levoflocaxin, 1/2; vancomycin, 2/2; and linezolid, 4/4.21 In the presence of resistance against penicillin G, ampicillin was also regarded as resistant. Resistance to oxacillin was regarded as resistance to all tested beta-lactams.
Synergy studies In-vitro interactions of polyhexanide with oxacillin, penicillin G, ampicillin, cefazolin, cefuroxime, imipenem, gentamicin, amikacin, erythromycin, doxycycline, levofloxacin, linezolid and vancomycin were investigated using the microdilution checkerboard technique with 96-well microtitre plates for each combination. Concentrations of antimicrobials, which were tested in combination, were at least between 1/4 MIC and 2 MIC and 1/4 MBC and 2 MBC. Growth and sterility controls were also included in each plate. The fractional inhibitory concentrations (FICs) for each isolate were calculated as follows:22,23
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W. Fabry, H.-J. Kock / Journal of Hospital Infection 86 (2014) 68e72
Table I Activity of antimicrobial agents against meticillin-susceptible Staphylococcus aureus (N ¼ 50) Agent
Polyhexanide Oxacillin Penicillin G Ampicillin Cefazolin Cefuroxime Imipenem Gentamicin Erythromycin Doxycycline Levofloxacin Linezolid Vancomycin
MIC (mg/L) and related susceptibility
MBC (mg/L) and related susceptibility
MIC50
MIC90
Range
S
I
R
MBC50
MBC90
Range
S
I
R
1 0.25 8 8 0.5 1 0.125 0.125 0.25 0.5 0.125 0.5 0.5
1 1 >32 >32 1 2 2 0.5 0.5 4 1 1 1
0.5e2 0.062e1 0.015e>32 0.015e>32 0.062e1 0.25e8 0.031e16 0.062e16 0.062e>16 0.062e32 0.062e16 0.125e4 0.25e4
50 50 11 21 50 49 48 47 46 37 27 50 50
e e 7 6 e 1 2 e 2 4 16 e e
e e 32 23 e e e 3 2 9 7 e e
1 >4 8 8 1 8 0.5 0.25 2 8 2 >32 4
2 >4 >32 >32 64 64 8 1 8 32 >16 >32 16
0.5e2 0.062e4 0.015e>32 0.015e>32 0.5e>64 1e>64 0.031e64 0.062e16 0.5e16 4e>16 0.125e16 >32 0.5e>16
50 17 10 10 27 10 48 47 7 e 21 e 27
e e 8 6 15 23 e e 25 e 4 e 3
e 33 32 34 8 17 2 3 18 50 25 50 20
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MIC50/MIC90, minimum inhibitory concentration reached by 50% or 90% of the strains, respectively; MBC50/MBC90, minimum bactericidal concentration reached by 50% or 90% of the strains, respectively; S, susceptible; I, intermediate; R, resistant.
FIC ¼
and MBC values of 2 mg/L. In 10 strains, MBC was determined after 3 and 24 h of exposure to polyhexanide. These values did not differ more than one dilution step from those obtained after 6 h of exposure in the standard MBC determinations. The results of antimicrobial testing are shown in Tables I and II. All strains were regarded as susceptible to polyhexanide. As expected for bacteriostatic antibiotics, the MBC values of bacteriostatic drugs exceeded the MIC values by three to five dilution steps for the MSSA isolates. These differences were much less pronounced for the MRSA strains. However, for the MSSA isolates, the MBC values of the cephalosporins and imipenem also exceeded the MIC values by a factor of two to three. This was observed in particular with the incubation time of 6 h. The effect was lower if it was increased to 24 h. Based on MBC values, the number of resistant strains was remarkably higher if using the same interpretative criteria as for MIC values. Several strains only exhibited bacteriostatic action against vancomycin, even if the transfer was performed after 24 h. One strain with a vancomycin MIC of 4 mg/L was regarded as a VISA strain, although it was interpreted as resistant to vancomycin using the EUCAST criteria.21
MIC of polyhexanide tested in combination with antibiotic MIC of polyhexanide MIC of antibiotic tested in combination with polyhexanide þ MIC of antibiotic
The fractional bactericidal concentrations (FBCs) were calculated accordingly. Synergy was defined as an FIC or FBC index of 0.5, indifference by an index of >0.5 to 4, and antagonism by an index of >4.
Results The PFGE profiles of the 100 strains differed in at least three bands between the individual isolates (data not shown). As a median of 15 independent determinations with polyhexanide, the MSSA type strain ATCC 29213 had MIC and MBC values of 1 mg/L, and the MRSA type strain ATCC 33591 had MIC and MBC values of 2 mg/L. The maximum deviation between tests was one dilution step. The activity of polyhexanide against MSSA and MRSA was 0.5e2 mg/L; the vancomycin-intermediate S. aureus (VISA) strain had MIC
Table II Activity of antimicrobial agents against meticillin-resistant Staphylococcus aureus (N ¼ 50) Agent
Polyhexanide Gentamicin Erythromycin Doxycycline Levofloxacin Linezolid Vancomycin
MIC (mg/L) and related susceptibility
MBC (mg/L) and related susceptibility
MIC50
MIC90
Range
S
I
R
MBC50
MBC90
Range
S
I
R
1 8 16 4 8 1 1
2 >16 >16 8 >16 4 2
0.5e2 0.25e>16 0.25e>16 2e>16 0.125e>16 0.25e4 0.5e4
50 20 15 e 3 50 49
e e 1 23 e e e
e 30 34 27 47 e 1
1 16 >16 16 16 >32 2
2 >16 >16 >16 >16 >32 >16
0.5e2 0.25e>16 4e>16 >16 0.25e>16 >32 0.5e>16
50 18 6 e 3 e 35
e e 4 e e e e
e 32 40 50 47 50 15
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MIC50/MIC90, minimum inhibitory concentration reached by 50% or 90% of the strains, respectively; MBC50/MBC90, minimum bactericidal concentration reached by 50% or 90% of the strains, respectively; S, susceptible; I, intermediate; R, resistant.
W. Fabry, H.-J. Kock / Journal of Hospital Infection 86 (2014) 68e72 Table III Checkerboard results showing synergy for the present meticillinsusceptible Staphylococcus aureus strains (N ¼ 10) according to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using different combinations of antibiotics and polyhexanide Antibiotic
Cefuroxime Erythromycin Doxycycline Linezolid
MIC
MBC
S
I
A
S
I
A
1 2 5 3
9 8 5 7
e e e e
e 4 6 4
10 6 4 6
e e e e
S, synergistic; I, indifferent; A, antagonistic.
The results of combination testing in Tables III and IV indicate no antagonistic action between polyhexanide and the tested antibiotics. Remarkably, synergy was noted between polyhexanide and some bacteriostatic antibiotics (erythromycin, doxycycline and linezolid).
Discussion MICs and MBCs of polyhexanide for MRSA and MSSA were determined in this study. All tested strains showed different PFGE profiles, and it was concluded that they were different clones. MICs and MBCs of polyhexanide for MRSA and MSSA, determined with a standardized susceptibility test, did not exceed 2 mg/L. This is significantly lower than the therapeutic preparations containing 200e400 mg/L polyhexanide, which corresponds to a 0.1e0.2% dilution of the stock solution. It is available for several applications (wound dressing, whole-body bath, oral tablets and solutions) and successful cases of MRSA eradication have been reported.24,25 A whole-body bath with a 0.1% polyhexanide solution (one to three times daily) is applied as an adjunct to nasal mupirocin or PVP-iodine in carriers for the eradication of MRSA. A preparation of 0.1% polyhexanide (Prontoderm) is also available as nasal ointment. The excess power of the concentration used clinically compared with the values determined in this study may, of course, be needed in a clinical setting where the polyhexanide solution might be further diluted by blood or other fluids, and the bacteria might be difficult to reach with the rinse solution containing polyhexanide.
Table IV Checkerboard results showing synergy for the present meticillinresistant Staphylococcus aureus strains (N ¼ 10) according to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using different combinations of antibiotics and polyhexanide Antibiotic
Erythromycin Doxycycline Linezolid
MIC
MBC
S
I
A
S
I
A
1 3 4
9 7 6
e e e
2 6 4
8 4 6
e e e
S, synergistic; I, indifferent; A, antagonistic.
71
In this study, the presence of albumin did not decrease the activity of polyhexanide, so its effectiveness was not impaired by the presence of protein. The clinical use of polyhexanide for the eradication of S. aureus in the vestibulum itself may be limited, because inactivation of polyhexanide by mucins (heavily glycosylated proteins) has been demonstrated previously.26,27 This underlines the need for further clinical trials in this field. The MBC values in this study did not differ significantly when determined after exposure of 3, 6 or 24 h. As such, the bactericidal effect of 0.5e2 mg/L polyhexanide against S. aureus is already achieved after 3 h. The higher MBC values of the cephalosporins and imipenem compared with the MIC values may be explained by the relatively short incubation time of 6 h. The MBC determination according to DIN 58940-8 does not include the use of a neutralizing agent. A possible carryover effect is minimized by spreading the inoculum of the subculture over a large surface. In a previous investigation, the efficacy of some antiseptics was compared under different standard conditions.14 The test methods included a quantitative suspension test according to DIN EN 1040, in which a neutralization step is performed.28 Polyhexanide was only found to be slightly more effective in the microdilution test than in the quantitative suspension test. This may be due to more effective neutralization in the quantitative suspension test. However, the difference between the results of the two tests did not exceed one to two dilution steps. In conclusion, the use of the microdilution test not only allows the testing of a large number of strains, but also appears to provide results comparable to standard tests designed for antiseptics. Furthermore, the activity might be affected by the development of resistance. Repeated application of a topical antibiotic over a longer period enhances the possibility of resistance development.29 For example, Brumfitt et al. showed chlorhexidine resistance in some strains of MRSA, thus demonstrating intrinsic resistance to an antiseptic.7 However, no similar data for polyhexanide have been published. No resistance against polyhexanide was detected in the present study. The application of polyhexanide to both healthy and wounded tissue appears to be safe. The application of polyhexanide led to faster closure of experimental superficial aseptic wounds in piglets than placebo or octenidine.30 Defining a biocompatibility index by comparing antibacterial activity and cytotoxicity in murine fibroblasts, polyhexanide showed good antimicrobial action against S. aureus and low toxicity compared with other antiseptics.31 In general, antibiotic therapy may lead to selection rather than eradication; therefore, the use of effective antiseptics such as polyhexanide is recommended whenever appropriate.32 Systemic antibiotic treatment is usually necessary for infected wounds if classical clinical signs or symptoms of inflammation are observed.33 In conclusion, polyhexanide appears to be suitable for the topical treatment of infections with S. aureus, including MRSA and VISA, based on its antibacterial activity and lack of inhibition of antibiotic action. Conflict of interest statement None declared. Funding source None.
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electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233e2239. Deutsches Institut fu ¨r Normung. DIN 58940-8: medical microbiology e susceptibility testing of microbial pathogens to antimicrobial agents. Part 8: microdilution method; general methodicspecific requirements. Berlin: Beuth Verlag; 2002. Deutsches Institut fu ¨r Normung. DIN 58940-7: medical microbiology e susceptibility testing of microbial pathogens to antimicrobial agents. Part 7: determination of minimum bactericidal concentration with the method of microbouillondilution. Berlin: Beuth Verlag; 2008. European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 3.0. Basel, Switzerland: European Society of Clinical Microbiology and Infectious Diseases; 2013. Moody J. Synergism testing: broth microdilution checkerboard and broth macrodilution methods. In: Isenberg HD, editor. Clinical microbiology procedures handbook. 2nd ed, Vol. 2. Washington: ASM Press; 2004. p. 5.12.1e5.12.23. Pillai S, Moellering RC, Eliopoulos G. Antimicrobial combinations. In: Lorian V, editor. Antibiotics in laboratory medicine. 5th ed. Baltimore: Lippincott Williams & Wilkins; 2005. p. 365e440. Seipp HM, Stroh A. Methicillin-resistente S. aureus (MRSA) e signifikante Reduktion von Inzidenz und Rate in einem Klinikum der Maximalversorgung (1994 bis 1999). Hygiene Medizin 1999;24:224e237. Rietko ¨tter J, Ko ¨rber A, Grabbe S, Dissemond J. Eradication of methicillin-resistant Staphylococcus aureus in a chronic wound by a new polyhexanide hydrogel. J Eur Acad Dermatol Venereol 2007;21:1416e1417. Ansorg R, Azem T, Fabry WH, Rath PM. Influence of mucin on the activity of the antiseptic Lavasept against Staphylococcus aureus. Chemotherapy 2002;48:129e133. Ansorg R, Rath PM, Fabry W. Inhibition of the anti-staphylococcal activity of the antiseptic polyhexanide by mucin. Arzneim-Forsch/ Drug Res 2003;53:368e371. Deutsches Institut fu ¨r Normung. DIN EN 1040: chemical disinfectants and antiseptics e quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics e test method and requirements (phase 1). Berlin: Beuth Verlag; 2005. Seipp HM, Stroh A. Multiresistant Staphylococcus aureus. Part 3: decontamination and prevention of recontamination. Hygiene Medizin 1997;22:285e305. Kramer A, Roth B, Mu ¨cker N. Influence of the ¨ller G, Rudolph P, Klo antiseptic agents polyhexanide 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 2004;17:141e146. Mueller G, Kramer A. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. J Antimicrob Chemother 2008;61:1281e1287. Rotter M. Staphylokokken und Desinfektion. Chemother J 2009;18:89e92. Lipinsky BA, Hoey C. Topical antimicrobial therapy for treating chronic wounds. Clin Infect Dis 2009;49:1541e1549.
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
In-vitro activity of polyhexanide alone and in combination with antibiotics against Staphylococcus aureus W. Fabry a, *, H.-J. Kock b a b
Institut fu¨r Medizinische Mikrobiologie, Virologie und Krankenhaushygiene, Universita¨t Rostock, Rostock, Germany Vivantes-Humboldt Klinikum, Klinik fu¨r Unfallchirurgie und Orthopa¨die, Berlin, Germany
A R T I C L E
I N F O
Article history: Received 30 January 2013 Accepted 1 October 2013 Available online 23 October 2013 Keywords: Polyhexanide Antiseptic Antibiotic Staphylococcus aureus Meticillin-resistant Staphylococcus aureus Minimum inhibitory concentration Minimum bactericidal concentration
S U M M A R Y
Background: The resistance of Staphylococcus aureus is increasing, not only to antibiotics but also to antiseptics. Aim: To investigate the activity of the antiseptic polyhexanide and several antibiotics against clinical isolates of meticillin-susceptible and meticillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively). Polyhexanide was tested alone and in combination with oxacillin, penicillin G, ampicillin, cefazolin, cefuroxime, imipenem, gentamicin, erythromycin, doxycycline, levoflocaxin, linezolid and vancomycin. Methods: Fifty MSSA and 50 MRSA strains, including one vancomycin-intermediate (VISA) strain, were tested. All strains were typed by pulsed-field gel electrophoresis (PFGE) to exclude testing of clonal isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined using the serial broth microdilution technique according to DIN 58940. Combinations of polyhexanide and different antibiotics were investigated using the checkerboard technique. Findings: Polyhexanide MICs and MBCs in the range of 0.5e2 mg/L were found for both MSSA and MRSA, and the VISA strain had MIC and MBC values of 2 mg/L. All isolates were regarded as susceptible to polyhexanide, and no antagonism was observed between polyhexanide and the tested antibiotics. Synergism between polyhexanide and some bacteriostatic antibiotics (erythromycin, doxycycline and linezolid) was found for some strains. Conclusions: Polyhexanide appears to be suitable for the topical treatment of S. aureus alone and in combination with antibiotics. ª 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction Staphylococcus aureus is a nosocomial pathogen of increasing importance due to the emergence of meticillin* Corresponding author. Address: Institut fu ¨r Medizinische Mikrobiologie, Virologie und Krankenhaushygiene, Universita ¨t Rostock, Schillingallee 70, D-18057 Rostock, Germany. Tel.: þ49 208/631290; fax: þ49 214/374266. E-mail address: [email protected] (W. Fabry).
resistant strains (MRSA) associated with multi-drug resistance.1 Patients colonized by S. aureus are at risk for bacteraemia and severe systemic infections.2,3 The topical antibiotic mupirocin is often applied for the clearance of topical S. aureus.4 However, MRSA is developing resistance to mupirocin5,6 and also to antiseptics such as chlorhexidine7 and triclosan.8 As such, alternative agents for eradication of S. aureus are needed urgently. The toxicological safety and tissue compatibility of the antiseptic fractionated polyhexamethylene biguanide
0195-6701/$ e see front matter ª 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jhin.2013.10.002
W. Fabry, H.-J. Kock / Journal of Hospital Infection 86 (2014) 68e72 (polyhexanide) and polyethylene glycol 4000 (Lavasept, Lavasorb, Lavanid) was shown by in-vitro investigations and animal studies.9e11 Previous testing of selected strains showed antibacterial activity of polyhexanide against S. aureus.12e14 A prospective randomized controlled double-blind study including S. aureus exhibited a faster and significantly larger reduction on soft tissue wound surfaces in comparison with Ringer’s solution.15,16 For further investigations into polyhexanide activity, 100 S. aureus isolates were subjected to quantitative tests. The susceptibility of the strains to various antibiotic agents was tested alone and in combination with polyhexanide to detect potential synergistic or antagonistic effects of simultaneous administration. Prior to measurement of antiseptic activity, all strains were typed by pulsed-field gel electrophoresis (PFGE) to exclude testing of clonal isolates.
Materials and methods
69
50 mg/L) and MgCl2 (final Mg2þ concentration 25 mg/L). As such, polyhexanide was diluted in the test broth to final concentrations of 0.06e128 mg/L. The final inoculum of the tested isolates was 5 105 colony-forming units (cfu)/mL. The presence or absence of an inoculum effect was determined by repeating MIC and MBC determinations with diverse inocula (1.5 105, 1.5 106, 1.5 108 cfu/mL). The MIC was read after 24 h of incubation at 35 C as the lowest concentration of the drug preventing visible growth. For the determination of MBCs, a 10-mL aliquot from wells near the threshold for turbidity of the MIC plate was transferred in duplicate after 6 h onto blood agar plates. Incubation was performed for 24 h at 35 C. The MBC was defined as the lowest antiseptic concentration preventing visible regrowth. In addition, the MBCs of 10 MRSA and 10 MSSA were determined after incubation of the MIC plates for 3 and 24 h. MRSA type strain ATCC 33591 and MSSA type strains ATCC 25923 and ATCC 29213 were used for quality control.
Strains Antibiotic susceptibility testing One hundred clonally different isolates (50 MRSA and 50 MSSA) were collected consecutively from infected patients at the university hospitals of Essen and Rostock (Germany). The strains were identified using the Vitek2 automated system (bioMe ´rieux, Nu ¨rtlingen, Germany), the agglutination kit Slidex Staph Plus (bioMe ´rieux), and the BD DNase test agar (Becton Dickinson, Heidelberg, Germany). The Protect system (Technical Service Consultants Limited, Heywood, UK) was used for storage at 70 C. Prior to testing, an aliquot of each isolate was thawed and subcultured on sheep blood agar.
PFGE typing PFGE was performed using a modified version of Sambrook et al.’s17 method with Gene Navigator System apparatus (Pharmacia, LKB Biotechnology AB, Uppsala, Sweden), as described below. Genomic DNA was extracted from bacterial cells by lysostaphin (Sigma-Aldrich, St. Louis, MO, USA) treatment and was subsequently digested with Sma1 (Roche Diagnostics, Mannheim, Germany). The resulting DNA fragments were separated for 24 h in a 1% low melting agarose gel and a 0.05 M tris-borate EDTA buffer, pH 8.5, at 10 C and 180e185 V with a pulse time of 0.5e40 s. For documentation, gels were stained with ethidium bromide. Lambda DNA concatemers (New England Biolabs, Beverly, MA, USA) were used as molecular size standards. PFGE patterns were interpreted according to Tenover et al.18 If the patterns differed by three or more bands, isolates were considered to be genetically different.
Determination of polyhexanide activity Polyhexanide was obtained from Fresenius Kabi (Bad Homburg, Germany). The stock solution contained 200 mg polyhexanide and 10 mg polyethylene glycol (polyethylene glycol 4000) per mL. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined by serial broth microdilution tests according to DIN 5894019,20 using Mueller-Hinton broth (MHB, E. Merck, Darmstadt, Germany), supplemented with CaCl2 (final Ca2þ concentration
MICs and MBCs were determined for each drug using analytical grade powders kindly provided by their manufacturers. Antibiotic substances of defined activity were dissolved and diluted in Mueller-Hinton broth (Unipath, Wesel, Germany) supplemented with calcium (50 mg/L) and magnesium (25 mg/L). The dilution scheme covered concentrations of antibiotics as follows: oxacillin, 0.001e4 mg/L; gentamicin, erythromycin, doxycycline, levofloxacin and vancomycin, 0.007e16 mg/L; penicillin G, ampicillin and linezolid, 0.015e32 mg/L; and cefazolin, cefuroxim and imipenem, 0.031e64 mg/L. MIC and MBC determinations were performed as described for testing the antiseptic substances. For selected strains, cephalosporin, carbapenem and vancomycin MBC determination was performed with transfer after 24 h of incubation. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the following breakpoints (mg/ L) were used indicating susceptibility () and resistance (>): oxacillin, 2/2; penicillin G, 0.125/0.125; ampicillin, 2/8; cefazolin, 1/2; cefuroxime, 4/8; imipenem, 2/8; gentamicin, 1/1; erythromycin, 1/2; doxycycline, 1/2; levoflocaxin, 1/2; vancomycin, 2/2; and linezolid, 4/4.21 In the presence of resistance against penicillin G, ampicillin was also regarded as resistant. Resistance to oxacillin was regarded as resistance to all tested beta-lactams.
Synergy studies In-vitro interactions of polyhexanide with oxacillin, penicillin G, ampicillin, cefazolin, cefuroxime, imipenem, gentamicin, amikacin, erythromycin, doxycycline, levofloxacin, linezolid and vancomycin were investigated using the microdilution checkerboard technique with 96-well microtitre plates for each combination. Concentrations of antimicrobials, which were tested in combination, were at least between 1/4 MIC and 2 MIC and 1/4 MBC and 2 MBC. Growth and sterility controls were also included in each plate. The fractional inhibitory concentrations (FICs) for each isolate were calculated as follows:22,23
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W. Fabry, H.-J. Kock / Journal of Hospital Infection 86 (2014) 68e72
Table I Activity of antimicrobial agents against meticillin-susceptible Staphylococcus aureus (N ¼ 50) Agent
Polyhexanide Oxacillin Penicillin G Ampicillin Cefazolin Cefuroxime Imipenem Gentamicin Erythromycin Doxycycline Levofloxacin Linezolid Vancomycin
MIC (mg/L) and related susceptibility
MBC (mg/L) and related susceptibility
MIC50
MIC90
Range
S
I
R
MBC50
MBC90
Range
S
I
R
1 0.25 8 8 0.5 1 0.125 0.125 0.25 0.5 0.125 0.5 0.5
1 1 >32 >32 1 2 2 0.5 0.5 4 1 1 1
0.5e2 0.062e1 0.015e>32 0.015e>32 0.062e1 0.25e8 0.031e16 0.062e16 0.062e>16 0.062e32 0.062e16 0.125e4 0.25e4
50 50 11 21 50 49 48 47 46 37 27 50 50
e e 7 6 e 1 2 e 2 4 16 e e
e e 32 23 e e e 3 2 9 7 e e
1 >4 8 8 1 8 0.5 0.25 2 8 2 >32 4
2 >4 >32 >32 64 64 8 1 8 32 >16 >32 16
0.5e2 0.062e4 0.015e>32 0.015e>32 0.5e>64 1e>64 0.031e64 0.062e16 0.5e16 4e>16 0.125e16 >32 0.5e>16
50 17 10 10 27 10 48 47 7 e 21 e 27
e e 8 6 15 23 e e 25 e 4 e 3
e 33 32 34 8 17 2 3 18 50 25 50 20
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MIC50/MIC90, minimum inhibitory concentration reached by 50% or 90% of the strains, respectively; MBC50/MBC90, minimum bactericidal concentration reached by 50% or 90% of the strains, respectively; S, susceptible; I, intermediate; R, resistant.
FIC ¼
and MBC values of 2 mg/L. In 10 strains, MBC was determined after 3 and 24 h of exposure to polyhexanide. These values did not differ more than one dilution step from those obtained after 6 h of exposure in the standard MBC determinations. The results of antimicrobial testing are shown in Tables I and II. All strains were regarded as susceptible to polyhexanide. As expected for bacteriostatic antibiotics, the MBC values of bacteriostatic drugs exceeded the MIC values by three to five dilution steps for the MSSA isolates. These differences were much less pronounced for the MRSA strains. However, for the MSSA isolates, the MBC values of the cephalosporins and imipenem also exceeded the MIC values by a factor of two to three. This was observed in particular with the incubation time of 6 h. The effect was lower if it was increased to 24 h. Based on MBC values, the number of resistant strains was remarkably higher if using the same interpretative criteria as for MIC values. Several strains only exhibited bacteriostatic action against vancomycin, even if the transfer was performed after 24 h. One strain with a vancomycin MIC of 4 mg/L was regarded as a VISA strain, although it was interpreted as resistant to vancomycin using the EUCAST criteria.21
MIC of polyhexanide tested in combination with antibiotic MIC of polyhexanide MIC of antibiotic tested in combination with polyhexanide þ MIC of antibiotic
The fractional bactericidal concentrations (FBCs) were calculated accordingly. Synergy was defined as an FIC or FBC index of 0.5, indifference by an index of >0.5 to 4, and antagonism by an index of >4.
Results The PFGE profiles of the 100 strains differed in at least three bands between the individual isolates (data not shown). As a median of 15 independent determinations with polyhexanide, the MSSA type strain ATCC 29213 had MIC and MBC values of 1 mg/L, and the MRSA type strain ATCC 33591 had MIC and MBC values of 2 mg/L. The maximum deviation between tests was one dilution step. The activity of polyhexanide against MSSA and MRSA was 0.5e2 mg/L; the vancomycin-intermediate S. aureus (VISA) strain had MIC
Table II Activity of antimicrobial agents against meticillin-resistant Staphylococcus aureus (N ¼ 50) Agent
Polyhexanide Gentamicin Erythromycin Doxycycline Levofloxacin Linezolid Vancomycin
MIC (mg/L) and related susceptibility
MBC (mg/L) and related susceptibility
MIC50
MIC90
Range
S
I
R
MBC50
MBC90
Range
S
I
R
1 8 16 4 8 1 1
2 >16 >16 8 >16 4 2
0.5e2 0.25e>16 0.25e>16 2e>16 0.125e>16 0.25e4 0.5e4
50 20 15 e 3 50 49
e e 1 23 e e e
e 30 34 27 47 e 1
1 16 >16 16 16 >32 2
2 >16 >16 >16 >16 >32 >16
0.5e2 0.25e>16 4e>16 >16 0.25e>16 >32 0.5e>16
50 18 6 e 3 e 35
e e 4 e e e e
e 32 40 50 47 50 15
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MIC50/MIC90, minimum inhibitory concentration reached by 50% or 90% of the strains, respectively; MBC50/MBC90, minimum bactericidal concentration reached by 50% or 90% of the strains, respectively; S, susceptible; I, intermediate; R, resistant.
W. Fabry, H.-J. Kock / Journal of Hospital Infection 86 (2014) 68e72 Table III Checkerboard results showing synergy for the present meticillinsusceptible Staphylococcus aureus strains (N ¼ 10) according to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using different combinations of antibiotics and polyhexanide Antibiotic
Cefuroxime Erythromycin Doxycycline Linezolid
MIC
MBC
S
I
A
S
I
A
1 2 5 3
9 8 5 7
e e e e
e 4 6 4
10 6 4 6
e e e e
S, synergistic; I, indifferent; A, antagonistic.
The results of combination testing in Tables III and IV indicate no antagonistic action between polyhexanide and the tested antibiotics. Remarkably, synergy was noted between polyhexanide and some bacteriostatic antibiotics (erythromycin, doxycycline and linezolid).
Discussion MICs and MBCs of polyhexanide for MRSA and MSSA were determined in this study. All tested strains showed different PFGE profiles, and it was concluded that they were different clones. MICs and MBCs of polyhexanide for MRSA and MSSA, determined with a standardized susceptibility test, did not exceed 2 mg/L. This is significantly lower than the therapeutic preparations containing 200e400 mg/L polyhexanide, which corresponds to a 0.1e0.2% dilution of the stock solution. It is available for several applications (wound dressing, whole-body bath, oral tablets and solutions) and successful cases of MRSA eradication have been reported.24,25 A whole-body bath with a 0.1% polyhexanide solution (one to three times daily) is applied as an adjunct to nasal mupirocin or PVP-iodine in carriers for the eradication of MRSA. A preparation of 0.1% polyhexanide (Prontoderm) is also available as nasal ointment. The excess power of the concentration used clinically compared with the values determined in this study may, of course, be needed in a clinical setting where the polyhexanide solution might be further diluted by blood or other fluids, and the bacteria might be difficult to reach with the rinse solution containing polyhexanide.
Table IV Checkerboard results showing synergy for the present meticillinresistant Staphylococcus aureus strains (N ¼ 10) according to minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using different combinations of antibiotics and polyhexanide Antibiotic
Erythromycin Doxycycline Linezolid
MIC
MBC
S
I
A
S
I
A
1 3 4
9 7 6
e e e
2 6 4
8 4 6
e e e
S, synergistic; I, indifferent; A, antagonistic.
71
In this study, the presence of albumin did not decrease the activity of polyhexanide, so its effectiveness was not impaired by the presence of protein. The clinical use of polyhexanide for the eradication of S. aureus in the vestibulum itself may be limited, because inactivation of polyhexanide by mucins (heavily glycosylated proteins) has been demonstrated previously.26,27 This underlines the need for further clinical trials in this field. The MBC values in this study did not differ significantly when determined after exposure of 3, 6 or 24 h. As such, the bactericidal effect of 0.5e2 mg/L polyhexanide against S. aureus is already achieved after 3 h. The higher MBC values of the cephalosporins and imipenem compared with the MIC values may be explained by the relatively short incubation time of 6 h. The MBC determination according to DIN 58940-8 does not include the use of a neutralizing agent. A possible carryover effect is minimized by spreading the inoculum of the subculture over a large surface. In a previous investigation, the efficacy of some antiseptics was compared under different standard conditions.14 The test methods included a quantitative suspension test according to DIN EN 1040, in which a neutralization step is performed.28 Polyhexanide was only found to be slightly more effective in the microdilution test than in the quantitative suspension test. This may be due to more effective neutralization in the quantitative suspension test. However, the difference between the results of the two tests did not exceed one to two dilution steps. In conclusion, the use of the microdilution test not only allows the testing of a large number of strains, but also appears to provide results comparable to standard tests designed for antiseptics. Furthermore, the activity might be affected by the development of resistance. Repeated application of a topical antibiotic over a longer period enhances the possibility of resistance development.29 For example, Brumfitt et al. showed chlorhexidine resistance in some strains of MRSA, thus demonstrating intrinsic resistance to an antiseptic.7 However, no similar data for polyhexanide have been published. No resistance against polyhexanide was detected in the present study. The application of polyhexanide to both healthy and wounded tissue appears to be safe. The application of polyhexanide led to faster closure of experimental superficial aseptic wounds in piglets than placebo or octenidine.30 Defining a biocompatibility index by comparing antibacterial activity and cytotoxicity in murine fibroblasts, polyhexanide showed good antimicrobial action against S. aureus and low toxicity compared with other antiseptics.31 In general, antibiotic therapy may lead to selection rather than eradication; therefore, the use of effective antiseptics such as polyhexanide is recommended whenever appropriate.32 Systemic antibiotic treatment is usually necessary for infected wounds if classical clinical signs or symptoms of inflammation are observed.33 In conclusion, polyhexanide appears to be suitable for the topical treatment of infections with S. aureus, including MRSA and VISA, based on its antibacterial activity and lack of inhibition of antibiotic action. Conflict of interest statement None declared. Funding source None.
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