Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
ORIGINAL RESEARCH Antibacterial Honey (Medihoney™): in-vitro Activity Against Clinical Isolates of MRSA, VRE, and Other Multiresistant Gram-negative Organisms Including Pseudomonas aeruginosa Narelle May George;1 Keith F. Cutting2,3 WOUNDS 2007;19(9):231–236 From 1Queensland Health Pathology Service, Royal Brisbane Hospitals Campus, Herston, Queensland, Australia; 2Tissue Viability Specialist, Harefield Hospital, Middlesex; 3Principal Lecturer–Tissue Viability, Buckinghamshire Chilterns University College, Chalfont St. Giles, United Kingdom Address correspondence to: Keith F. Cutting Tissue Viability Specialist Harefield Hospital Hill End Road Middlesex UB9 6JH United Kingdom E-mail: [email protected] Disclosure: Mr. Cutting received a research and consultancy grant from Medihoney Pty LTD, and Derma Sciences Inc. Abstract: The clinical use of honey has received increasing interest in recent years, particularly its use as a topical antibacterial dressing. Results thus far are extremely encouraging, and demonstrate that honey is effective against a broad range of microorganisms, including multiresistant strains. This in-vitro study complements the work of others and focuses on the impact that a standardized honey can have on multiresistant bacteria that are regularly found in wounds and are responsible for increased morbidity. he media regularly reminds both the public and healthcare professionals of the dangers infection poses to good health, in particular the difficulties in successfully treating infection caused by multiresistant microorganisms.The development of bacterial resistance to antibiotic therapy is understood to be a natural occurrence. The emergence of resistant strains of bacteria and the ensuing management challenges are compounded by the fact that the development of new antibiotics has decreased in recent years.1 This situation prompts revisiting traditional approaches to infection management and use of those antimicrobials where the emergence of resistant strains has not been demonstrated and is highly unlikely to occur. More recently, interest in honey as a therapeutic agent has undergone a renaissance. Molan,2 in a review article on honey used as a wound dressing, eloquently presented an array of supportive evidence ranging from case studies to randomized controlled trials that clearly indicates the value of honey in wound care—particularly its antibacterial activity. Molan concludes that the antibacterial activity of honey “rapidly clears infection and protects wounds from becoming infected.”2 This statement brings into sharp belief the antibacterial potency of honey and its value as a therapeutic agent in wound care. This notion is supported in a 2005 report the Australian government commissioned that states “honey has been successfully used on infections not responding to standard antiseptic and antibiotic therapy” and that the full potential of honey will be recognized as the number of antibiotic resistant bacteria increases.3 T Vol. 19, No. 9 September 2007 231 George and Cutting The antibacterial activity of honey has been related to 4 properties (Figure 1). Methods A challenge set of 130 clinical isolates with multiple antibiotic resistances was prepared (Figure 2). High sugar content/low water activity: osmotic action Acid pH 3.2–4.5: inhibits bacterial growth Glucose oxidase enzyme: production of hydrogen peroxide Plant-derived factors: present in some honeys and not specifically identified Figure information adapted from Lusby4 and Blair5 11 strains multiresistant Acinetobacter baumannii 6 strains Enterobacter cloacae 3 strains of vancomycin susceptible Enterococcus faecalis 10 strains ESBL producing isolates of Escherichia coli 12 strains ESBL positive Klebsiella pneumoniae 20 strains of Pseudomonas aeruginosa Figure 1. The antibacterial activity of honey has been related to 4 properties. Honey appears to offer distinct advantages over “traditional” antibiotic therapy. Nonetheless, it is important to remember that although natural honey from the comb is antibacterial, it is not medical grade and should not be used in wound care. Medical grade honey is filtered, gamma irradiated, and produced under exacting standards of hygiene. All honeys are not the same and do not possess the same therapeutic advantages; therefore, honey should not be considered as a generic term.6 Medihoney™ Antibacterial Honey (Medihoney™ Pty LTD, Richlands, Australia) is a standardized medical honey that is available in many countries including Australia, United Kingdom, Finland, Germany, Austria, and Turkey. It is selected for its antibacterial activity and predominantly sourced from Leptospermum species. Sterility of products is validated against international standards and products are manufactured to meet international quality system requirements. The antibacterial activity of Medihoney is validated for the shelf life of the product, complying with the European Medical Device Directive. The Maori (Polynesian settlers of New Zealand) vernacular name for Leptospermum honey is manuka, the name by which it is more popularly known. Although the antibacterial activity of honey is recognized, potency varies between types.7 Relevant microbiological data is required in order to better understand the antibacterial activity of specific types of medical honey—particularly its impact on resistant bacterial strains. Aim. An in-vitro study was initiated in order to gain insight into the antibacterial activity of Medihoney antibacterial honey against a range of multiresistant organisms. 232 WOUNDS A Compendium of Clinical Research and Practice 48 strains Staphylococcus aureus (multiresistant and non-multiresistant) 20 strains VRE (VanA, VanB, VanC) Figure 2. Organisms tested. The clinical source and antimicrobial phenotype of test strains is shown in the Appendix. Test strains were nonreplicate, nonclonal clinical isolates that were cultured from diagnostic specimens submitted to a large tertiary referral hospital in Australia over a 14-year period (1990–2004).All organisms were identified using standard methods in accordance with those outlined in the Manual of Clinical Microbiology.8 Antimicrobial susceptibility profiles for all staphylococcal and gram-negative organisms were determined using the automated Vitek™ system version R07.1 (bioMerieux, Marcy l'Étoile, France). Enterococcal resistance profiles were determined using CLSI agar dilution protocol.9 Vancomycin-resistant phenotypes were confirmed using genotyping and the species identification confirmed using polymerase chain reaction (PCR) of specific Ddl ligases.10 Clonality of test strains was assessed from pulsed field gel electrophoresis profiles obtained using the GenePath™ system (Bio-Rad Laboratories, Hercules, Calif). Three ATCC type strains were also tested— Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212. Plate preparation and inoculum. The antibacterial honey was serially diluted from 1%–20% v/v in MuellerHinton agar (BBL211438). Control plates containing only Mueller-Hinton agar were also prepared. Plates were inoculated on the same day as preparation. Test organisms were subcultured from the -70˚C freezer onto 5% horse blood Columbia agar base (Oxoid CM 331) and George and Cutting incubated at 35˚C in ambient air for 18 h.After this time, a second subculture onto horse blood Columbia agar was performed. After 18 h of incubation at 35˚C, 4–5 colonies of each test isolate were inoculated in 2 mL Tryptone Soy Broth (Oxoid CM 129) and incubated at 35˚C in ambient air for 2 h. Following incubation, the turbidity of each culture was adjusted to a 0.5 McFarland standard.Ten microliters of the adjusted suspension was then added to 500 uL of sterile physiological saline in a multipoint inoculator. Mueller-Hinton agar plates containing serial dilutions of Antibacterial honey were inoculated using a multipoint inoculation device that delivered an inoculum per plate of approximately 104 cfu/mL.Agar plates were incubated at 35˚C in ambient air for 18 h.After this time, each plate was examined for the presence of bacterial growth. Complete inhibition of bacterial growth was recorded as “no growth.” Media showing partial inhibition (shadowing) or 1–2 colonies of test isolates were reported as “positive for growth.” Definitions of Terms ATCC: American Type Culture Collection—an international standard culture collection BORSA: mecA neg—strain with high production of beta lactamase ESBL: extended spectrum ß-lactamases confer resistance to cephalosporins and are a globally important cause of multidrug resistance in gram-negative bacteria. Minimum inhibitory concentration (MIC): the minimum concentration of antibacterial that inhibits visible growth of bacteria (colony forming units) in a culture medium. It is a culture dependent definition. MIC50: MIC for 50% of isolates MIC90: MIC for 90% of isolates VRE: vancomycin-resistant enterococci Results All test isolates grew on the control Mueller-Hinton (control) agar plates that contained no antibacterial honey.The MIC data for the 3 ATCC type strains were: 4% v/v for S aureus ATCC25923, 8% v/v for P aeruginosa ATCC 27853, and 8% v/v for E faecalis ATCC 29212. All test strains of MRSA were inhibited at a concentration of 4% v/v irrespective of antimicrobial phenotype. With the gram-negative organisms, 8/10 ESBL producing strains of Escherichia coli (80%) were inhibited at 6% v/v. The remaining 2 strains (20%) were inhibited at 8% v/v. Similarly for Klebsiella species, 11/12 strains (91%) were inhibited at 6% v/v, while the remaining strain was only inhibited at 8% v/v. For Enterobacter species, all 6 test strains (100%) were inhibited at 6% v/v. For Acinetobacter baumannii, concentrations of 8% were required to inhibit 9/11 (82%) of test isolates. All 5 panresistant strains of A baumannii were inhibited at 8% concentrations of the antibacterial honey. Vancomycin-resistant enterococci, the more resistant VanA/B strains of E faecalis and Enterococcus faecium required 8% for inhibition compared to 6% for the less resistant isolates. Two strains of vancomycin-resistant E faecalis and E faecium were inhibited at 6%, while 4 and 9 strains required a higher concentration of 8% v/v.All 3 strains expressing the VanC phenotype were inhibited at 6% v/v as were 2/3 strains of vancomycin susceptible E faecalis. Clinical isolates of P aeruginosa were more resistant to the antibacterial honey than other bacterial species. Concentrations of 14% v/v were required to inhibit 17/20 (85%) of test isolates.The remaining 3 strains were inhibited at a lower concentration of 12%. The above results and comparative MIC50 and MIC90 values for the different organism types are listed in Table 1. Table 1. Comparative MIC values by organism type. Organism (No. strains tested) S aureus BORSA mecA neg (2) Multiresistant mecA neg (2) MRSA Multiresistant (18) Non-multiresistant (26) Acinetobacter baumannii (11) E faecalis Vancomycin susceptible (3) Vancomycin resistant (7) E faecium Vancomycin resistant (11) Enterococcus Vancomycin resistant (VanC) (3) ESBL-positive strains E coli (10) Klebsiella pneumoniae (12) Enterobacter cloacae (6) P aeruginosa (20) ATCC strain S aureus ATCC 25923 P aeruginosa ATCC 27853 E faecalis ATCC 29212 MIC MIC50 MIC90 (%v/v) (%v/v) (%v/v) 4 4 4 4 4 4 4 4 4 4 4 4 6–8 8 8 6–8 6–8 6 8 8 8 6–8 8 8 6 6 6 6–8 6–8 6 12–14 6 6 6 12 8 6 6 14 4% 8% 8% N/A N/A N/A N/A N/A N/A N/A = not applicable Vol. 19, No. 9 September 2007 233 George and Cutting Discussion The findings of the present study add to the body of evidence and clearly demonstrate that honey has a valuable therapeutic role to play in wound care, often where modern approaches have failed.2 There are a number of in-vitro studies that have shown the effectiveness of medical honeys on antibiotic resistant organisms, such as MRSA and P aeruginosa.5,11 Although there is variability between MICs reported for similar organisms using nonstandardized honeys with different bacterial potencies, there was generally good correlation between the present findings and those of other researchers11,12 for gram-positive organisms (MRSA, VRE). In the present study, all strains of MRSA, including both resistant phenotypes of MRSA as well as sensitive strains of S aureus, were inhibited at low antibacterial honey concentrations (4% v/v). These data compare favorably with the earlier data from George et al13 comparing medical honeys obtained from New Zealand and Australia Leptospermum sources and those of Cooper et al14 (3% v/v), Blair5 (4.3% v/v), and Allen et al12 (3%–7% v/v). For VRE, the inhibition profiles of antibacterial honey varied between 6%–8% v/v depending on the species. Favorable comparisons can again be made with 3.8%–10% v/v ranges reported by Cooper et al11 and Allen et al.12 There is a paucity of good comparative data for invitro assessment of antibacterial activity of medical honeys against gram-negative organisms other than P aeruginosa. Gram-positive aerobic cocci, (eg, beta-hemolytic streptococci and S aureus) may be considered as primary pathogens often infecting chronic wounds in the earlier stages of wound formation.With delayed healing, wounds are more likely to be colonized by gram-negative coliforms, Pseudomonas species, and anaerobic bacteria. Infections in wounds of longer duration may be considered to be polymicrobial (aerobes and anaerobes). For the multiresistant gram-negative organisms other than P aeruginosa investigated in the present study, the majority of the 3 different species tested were inhibited at antibacterial honey concentrations of 6% v/v with all strains inhibited by 8% v/v. Likewise, antibacterial honey concentrations of 8% v/v were required to inhibit the more resistant Acinetobacter strains. Given the high levels of antibiotic resistance demonstrated in this group of organisms, the low MIC90 values clearly suggest that antibacterial honey has the potential to be an effective alternative antibacterial agent in vivo even with up to a 10234 WOUNDS A Compendium of Clinical Research and Practice fold dilution of the preparation. Surprisingly, inhibitory concentrations in the order of 12%–14% v/v for P aeruginosa with antibacterial honey were found. This contrasts with the earlier work of George et al13 where Australian and New Zealand Leptospermum honeys were shown to effectively inhibit the growth of more than 100 clinical strains of this organism at concentrations of 5%–6% v/v. Similar potency against P aeruginosa (4%–9% v/v) has been reported by Cooper et al.14 The findings of the present study highlight the variability of the antibacterial potency of different honeys. Medihoney combines honeys of differing antibacterial actions. The wide range of MICs reported when comparing different honeys against the same class of microorganism illustrate the differences in antibacterial potency that may be encountered between honeys.15 This underlines the value of using a standardized medical grade honey preparation that demonstrates consistent antibacterial activity against a broad range of microorganisms. In an in-vitro study comparing the antibacterial activity of 13 honeys including 3 commercial honeys of manuka, Medihoney Antibacterial Honey, and Rewarewa against E coli and P aeruginosa, only Medihoney and one beekeeper honey demonstrated inhibition of both organisms at 2.5% wt/v dilution.16 Cooper et al11 investigated the sensitivity of gram-positive cocci to honey. Eighteen strains of MRSA, 7 strains of vancomycin sensitive enterococci isolated from wounds, and 17 strains of vancomycin resistant enterococci were isolated from hospital environmental surfaces. The mean MIC values of manuka and pasture honey (with peroxide activity) were 3.0% and 3.1%, respectively, for MRSA strains. Mean MICs of honey for vancomycin-sensitive enterococci isolated from infected wounds were 4.9% (manuka) and 9.7% (pasture). Similar values were recorded for vancomycin-resistant enterococci—mean MICs for manuka and pasture were 4.6% and 8.3% (v/v), respectively. Conclusion The findings of the present study in respect to the antibacterial activity of honey are not unique as they complement previous findings. Although variations in bacterial potency are recognized, the presence of MRSA and other multiresistant bacteria that may be found in wounds causes much concern and leads to an increased consumption of available resources.The results are clear and provide additional in-vitro evidence that Medihoney George and Cutting Antibacterial Honey is an effective antibacterial agent, the implication being that Medihoney provides a valuable opportunity to manage wound infection caused by a range of multiresistant strains of bacteria. In-vitro activity requires corroborative evidence from rigorously conducted studies if claims are to be substantiated. The recent clinical findings of Johnson et al17 and Simon et al18 Appendix. Phenotypic antimicrobial resistance profiles of clinical isolates screened for susceptibility to Medihoney Antibacterial Honey. Organism MRSA Multiresistant Non-multiresistant S aureus BORSA mecA neg Multiresistant mecA neg ESBL positive strains E coli No. Phenotypic antimicrobial strains resistance profile 18 26 2 2 PenR, OxaR PenR, EryR, GenR 2 AmpR, SXTR, GenR, TobR, TimR, CipR AmpR, SXTR, GenR, TobR, TimR AmpR, SXTR, TimR AmpR, GentR, TimR 3 2 3 Klebsiella pneumoniae 5 3 4 Enterobacter cloacae 1 2 3 Acinetobacter baumannii 3 3 4 1 P aeruginosa PenR, OxR, EryR, GenR PenR, OxaR, EryR 15 1 1 2 1 AmpR, SXTR, GenR, TobR, TimR, CipR AmpR, GenR, TimR AmpR, SXTR, GenR, TimR, CipR AmpR, SXTR, GenR, TobR, TimR, CipR AmpR, SXTR, GenR, TobR, TimR AmpR, SXTR, GenR, TimR, CipR AmpR, GenR, TobR, AkR, TimR, CipR, MemR AmpR, TimR, MemR AmpR, GenR, TobR, TimR, CipR, MemR AmpR, GenR, TimR, CipR, MemR Fully susceptible CazR, TimR GenR TimR CipR provide much encouragement that these claims will be confirmed. The Johnson et al17 study also indicates that Medihoney may have an important role to play in infection prophylaxis as it demonstrated that Medihoney was equally effective as mupirocin in the prevention of catheter associated infections. Given that mupirocin can reduce infection rates by at least 7–13 fold,19,20 the prospect that Medihoney will prove to be an effective prophylactic is extremely hopeful. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Charles PG, Grayson ML. The dearth of new antibiotic development: why we should be worried and what we can do about it. Med J Aust. 2004;181(10):549–553. Molan PC.The evidence supporting the use of honey as a wound dressing. Int J Low Extrem Wounds. 2006;5(1):40–54. Davis C. The use of Australian honey in moist wound management. A report for the Rural Industries Research and Development Corporation. Kingston, Australia. October 2005. Publication No. W05/159. Project No. DAQ-232A. Lusby PE, Coombes A, Wilkinson JM. Honey: a potent agent for wound healing? J Wound Ostomy Continence Nurs. 2002;29(6):295–300. Blair S. Honey and drug resistant pathogens. Presented at: Joint Scientific Meeting of The Australian Society for Microbiology; July 2000; Cairns,Australia. Molan P. Not all honeys are the same for wound healing. European Tissue Repair Society Bulletin. 2002;9(1):5–6. Allen KL, Molan PC, Reid GM.A survey of the antibacterial activity of some New Zealand honeys. J Pharm Pharmacol. 1991;43(12):817–822. Murray PR, Baron EJ, Jorgensen JH, Pfaller MA,Yolken RH. Manual of Clinical Microbiology. 8th ed. Washington, DC:ASM Press; 2003. National Committee for Clinical Laboratory Standards. Methods for dilution: antimicrobial susceptibility tests for bacteria that grow aerobically. 5th ed. Wayne, Pa: Approved Standard M7-A5, NCCLS; 2000. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol. 1995;33(1):24–27. Cooper RA, Molan PC, Harding KG. The sensitivity to honey of gram-positive cocci of clinical significance isolated from wounds. J Appl Microbiol. 2002;93(5):857–863. Allen KL, Hutchinson G, Molan PC.The potential for using Vol. 19, No. 9 September 2007 235 George and Cutting 13. 14. 15. 16. 17. 18. 19. 20. 236 honey to treat wounds infected with MRSA and VRE. Presented at: First World Wound Healing Congress; September 10–13, 2000; Melbourne,Australia. George N, Sturgess R, Faoagali J, Davis C. Antimicrobial agents au naturel. Presented at: The Royal Brisbane Hospital Healthcare Symposium, 1999. Cooper RA, Halas E, Molan PC. The efficacy of honey in inhibiting strains of Pseudomonas aeruginosa from infected burns. J Burn Care Rehabil. 2002;23(6):366–370. Lusby PE, Coombes AL, Wilkinson JM. Bactericidal activity of different honeys against pathogenic bacteria. Arch Med Res. 2005;36(5):464–467. Wilkinson JM, Cavanagh HM. Antibacterial activity of 13 honeys against Escherichia coli and Pseudomonas aeruginosa. J Med Food. 2005;8(1):100–103. Johnson DW, van Eps C, Mudge DW, et al. Randomized, controlled trial of topical exit-site application of honey (Medihoney) versus mupirocin for the prevention of catheter-associated infections in hemodialysis patients. J Am Soc Nephrol. 2005;16(5):1456–1462. Simon A, Sofka K, Wiszniewsky G, Blaser G, Bode U, Fleischhack G. Wound care with antibacterial honey (Medihoney) in pediatric hematology-oncology. Support Care Cancer. 2006;14(1):91–97. Sesso R, Barbosa D, Leme IL, et al. Staphylococcus aureus prophylaxis in hemodialysis patients using central venous catheter: effect of mupirocin ointment. J Am Soc Nephrol. 1998;9(6):1085–1092. Johnson DW, MacGinley R, Kay TD, et al. A randomized controlled trial of topical exit site mupirocin application in patients with tunnelled, cuffed haemodialysis Dial Transplant. Nephrol catheters. 2002;17(10):1802–1807. WOUNDS A Compendium of Clinical Research and Practice