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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.
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