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RESEARCH ARTICLE
Antibacterial activity of varying UMF-graded
Manuka honeys
Alodia Girma, Wonjae Seo, Rosemary C. SheID*
Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles,
California, United States of America
*rosemary.she@med.usc.edu
Abstract
Honey has been used as a traditional remedy for skin and soft tissue infections due to its
ability to promote wound healing. Manuka honey is recognized for its unusually abundant
content of the antibacterial compound, methylglyoxal (MGO). The Unique Manuka Factor
(UMF) grading system reflects the MGO concentration in Manuka honey sold commercially.
Our objective was to observe if UMF values correlated with the antibacterial activity of Man-
uka honey against a variety of pathogens purchased over the counter. The antibacterial
effect of Manuka honey with UMF values of 5+, 10+, and 15+ from the same manufacturer
was assessed by the broth microdilution method. Minimum inhibitory concentration (MIC)
values were determined against 128 isolates from wound cultures representing gram-posi-
tive, gram-negative, drug-susceptible, and multi-drug resistant (MDR) organisms. Lower
MICs were observed with UMF 5+ honey for staphylococci (n = 73, including 25 methicillin-
resistant S.aureus) and Pseudomonas aeruginosa (n = 22, including 10 MDR) compared to
UMF 10+ honey (p<0.05) and with UMF 10+ compared to UMF 15+ (p = 0.01). For Entero-
bacteriaceae (n = 33, including 14 MDR), MIC values were significantly lower for UMF 5+ or
UMF 10+ compared to UMF 15+ honey (p<0.01). MIC
50
for UMF 5+, UMF 10+, and UMF
15+ honey against staphylococci was 6%, 7%, and 15%, and for Enterobacteriaceae was
21%, 21%, and 27%, respectively. For Pseudomonas aeruginosa MIC
50
was 21% and
MIC
90
was 21–27% for all UMFs. Manuka honey exhibited antimicrobial activity against a
spectrum of organisms including those with multi-drug resistance, with more potent activity
overall against gram-positive than gram-negative bacteria. Manuka honey with lower UMF
values, in our limited sampling, paradoxically demonstrated increased antimicrobial activity
among the limited samples tested, presumably due to changes in MGO content of honey
over time. The UMF value by itself may not be a reliable indicator of antibacterial effect.
Background
Honey has long been used as a wound salve and has been found experimentally to stimulate
tissue regeneration, facilitate wound debridement, reduce inflammation, and exert antibacte-
rial properties [1]. Its antibacterial effects arise from its low pH, ability to dehydrate bacteria,
PLOS ONE | https://doi.org/10.1371/journal.pone.0224495 October 25, 2019 1 / 9
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OPEN ACCESS
Citation: Girma A, Seo W, She RC (2019)
Antibacterial activity of varying UMF-graded
Manuka honeys. PLoS ONE 14(10): e0224495.
https://doi.org/10.1371/journal.pone.0224495
Editor: Filippo Giarratana, University of Messina,
ITALY
Received: August 19, 2019
Accepted: October 15, 2019
Published: October 25, 2019
Copyright: ©2019 Girma et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
and phytochemical content [2]. Manuka honey, derived from flowers of the Manuka bush
(Leptospermum scoparium), in particular has been noted for its bactericidal activity. Many
types of honey contain hydrogen peroxide as the main antimicrobial mechanism, whereas the
antibacterial effects of Manuka honey are considered to be primarily from its substantial con-
tent of methylglyoxal (MGO), a compound found in only certain honeys [3,4].
MGO is a compound formed from the dehydration of dihydroxyacetone, a natural phyto-
chemical within Leptospermum flower nectar [5]. It has demonstrated selective toxicity to bac-
terial cells when applied to wounds, and has separately been shown to cause bacterial cell lysis,
inhibit flagellation, and disrupt bacterial cell division [6–8]. The concentration of MGO in
Manuka honey correlates strongly with antibacterial activity [9–11]. Additional phytochemi-
cals, such as phenolic compounds, flavonoids, and defensins likely contribute synergistically as
MGO by itself does not achieve the same level of antibacterial activity as Manuka honey of
equal MGO concentration [7,12,13]. Nonetheless, MGO is still regarded as the major antimi-
crobial constituent and various Manuka honey grading schemes for commercially sold honey
are based in large part on MGO concentrations. One grading system, termed Unique Manuka
Factor (UMF), was originally developed to express the antibacterial activity of a Manuka
honey in units equivalent to % phenol against Staphylococcus aureus in an agar well diffusion
assay [14]. With discovery of MGO and its role in antimicrobial activity in Manuka honey,
UMF grade is now primarily based on the measured level of MGO such that UMF 5+ honey
has 83 mg/kg MGO, UMF 10+ has 263 mg/kg MGO, and UMF 15+ has 514 mg/kg
MGO [15]. Manuka honey with higher UMF are presumed to have more potent antibacterial
properties and are more expensive in the consumer market [3]. Given the widespread use of
MGO content as an indicator of Manuka honey grade and the wide acceptance of MGO as the
primary antibiotic compound in Manuka honey, our objective was to observe if UMF values
correlated with the antibacterial activity of Manuka honey purchased over the counter against
a variety of clinically relevant bacterial isolates.
Materials and methods
Bacterial isolates
Isolates originated from wound cultures of clinical specimens performed in the clinical micro-
biology laboratories of Keck Medical Center of the University of Southern California and
LAC+USC Medical Center (Los Angeles, CA). Both fresh subcultures and frozen isolates were
included. From frozen glycerol stocks, organisms were subcultured two times before being
used for antimicrobial susceptibility testing [16]. Each isolate was previously identified by
matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry
(Vitek MS, bioMe
´rieux, St. Louis, MO) and undergone susceptibility testing (Vitek 2, bioMe
´r-
ieux) according to routine clinical protocol. Carbapenemase status for Enterobacteriaceae was
determined on the basis of PCR detection of carbapenemase genes (Xpert Carba-R, Cepheid,
Sunnyvale, CA). A total of 128 bacterial organisms were selected for antimicrobial susceptibil-
ity testing: 48 Staphylococcus aureus (25 methicillin-resistant S.aureus (MRSA) and 23 methi-
cillin-susceptible S.aureus (MSSA)), 25 coagulase-negative staphylococci (11 S.epidermidis, 5
S.lugdunensis, 5 S.hominis, 2 S.capitis, 1 S.warneri, and 1 S.saccharolyticus), 33 enteric gram-
negative bacilli (17 Klebsiella pneumoniae including 9 bla
KPC
carbapenemase producers, 1
carbapenem-resistant but carbapenemase-negative strain, and 1 extended-spectrum beta-lac-
tamase (ESBL) producer; 11 E.coli including 3 ESBL producers; 1 K.aerogenes, and 4 Entero-
bacter sp.), and 22 Pseudomonas aeruginosa (10 multi-drug resistant (MDR) and 12 non-
MDR). MDR status was determined using Centers for Disease Control and Prevention defini-
tions [17].
Antibacterial activity of varying UMF-graded Manuka honeys
PLOS ONE | https://doi.org/10.1371/journal.pone.0224495 October 25, 2019 2 / 9
Antimicrobial susceptibility testing
Manuka honeys graded UMF 5+, 10+, and 15+ (Comvita New Zealand LTD) were used in this
study within 6 months of purchase and prior to the expiration date. One sample of each UMF
grade was used and expiration dates were all within the same 2-month period (Sept to Nov
2020). For each UMF-graded honey, we applied the broth microdilution method following the
Clinical and Laboratory Standards Institute (CLSI) guidelines to assess minimal inhibitory
concentrations (MIC) of antibacterial agents [16]. Stock solutions were prepared prior to each
batch of testing by preparing up to 60% (w/v) honey in Mueller-Hinton broth (Remel Inc.,
Lenexa, KS). Solutions were vortexed until completely dissolved, then sterilized by serial filtra-
tion through 0.45 μm and 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore-
Sigma, Burlington, MA) to eliminate contaminating spore-forming organisms. Based on
expected MIC values from preliminary results, we tested Manuka honey concentrations (% w/
v) of 5%, 6%, 7%, 8%, 9%, 10%, and 15% for gram-positive organisms and 9%, 15%, 21%, 27%,
33%, 39%, and 45% for gram-negative organisms. The colony suspension method was used for
preparing organism inocula from blood agar media after 18–24 hr subculture. Dilutions of a
0.5 McFarland suspension of each organism were made to a final organism concentration of
~5 x 10
4
colony forming units/mL in a final test volume of 0.1 mL per well on a 96-well plate.
Each organism was tested against all three UMF-graded honeys in parallel using the same
organism preparation. MICs were read after 20–24 h incubation at 35˚C in ambient air for
bactericidal activity. Growth and sterility controls were included for each organism-honey
combination. Purity of each organism suspension was assessed by subculturing an aliquot
onto blood agar plates. Any failed controls, tests with multiple skipped wells, or mixed purity
check cultures resulted in repeat testing with a fresh subculture of the organism.
Statistical analysis
MIC results at the 50
th
percentile (MIC
50
) and the 90
th
percentile (MIC
90
) were analyzed for
each UMF and organism group. MIC values of the different UMF honeys tested against the
same organisms underwent pairwise comparisons by the two-tailed Wilcoxon signed-rank
test. MIC values for different organism groups tested by the same UMF-graded honey were
compared using the Mann-Whitney U test. Results were considered statistically significant if
p<0.05 (GraphPad Prism v8).
Results
Gram-negative organisms demonstrated distributions of MIC values that were significantly
higher than for staphylococci (p<0.01 for each UMF grade of honey). MIC
50
values for Gram-
negative organisms were 21% compared to 5–15% for staphylococci and the different UMF
honeys. Summary statistics organism groups are shown in Tables 1and 2and individual
organism results can be found in S1 Table.
Among the 73 Staphylococcus spp., MIC values were significantly lower for UMF 5+ than
UMF 10+ (p<0.01), UMF 5+ than UMF 15+ (p<0.01), and UMF 10+ than UMF 15+
(p<0.01). Statistical significance remained (p<0.01) on subset analysis of MRSA (n = 25),
MSSA (n = 23), and coagulase-negative staphylococci (n = 25) separately, for which MIC val-
ues were significantly lower for UMF 5+ than UMF 10+, UMF 5+ than UMF 15+, and UMF
10+ than UMF 15+. The 5 strains of S.lugdunensis showed similar MIC values to other coagu-
lase-negative staphylococci, with MIC ranges of 5–7% for UMF 5+ and UMF 10+ honey and
6–15% for UMF 15+ honey. There were no significant differences between MIC distributions
of MRSA versus MSSA organisms. MIC ranges, MIC
50
and MIC
90
values for each honey and
Antibacterial activity of varying UMF-graded Manuka honeys
PLOS ONE | https://doi.org/10.1371/journal.pone.0224495 October 25, 2019 3 / 9
staphylococcal group are summarized in Table 1, and example broth microdilution results are
shown (Fig 1).
For Pseudomonas aeruginosa (n = 22), MIC values were significantly lower for UMF 5+
than UMF 10+ (p<0.05), UMF 5+ than UMF 15+ (p<0.01), and UMF 10+ than UMF 15+
(p = 0.01). Among MDR P.aeruginosa (n = 10), UMF 5+ yielded lower MIC values than either
Table 1. MIC
50
, MIC
90
, and MIC ranges of Manuka honeys UMF 5+, 10+, and 15+ tested against MRSA, MSSA, and coagulase-negative staphylococci.
Organism UMF 5+ UMF 10+ UMF 15+
MRSA (n = 25) MIC
50
(% w/v) 6 7 15
MIC
90
(% w/v) 8 8 15
MIC range (% w/v) 5 to >15 5 to >15 7 to >15
MSSA (n = 23) MIC50 (% w/v) 6 7 15
MIC90 (% w/v) 7 8 15
MIC range (% w/v) 5 to 7 5 to 10 9 to >15
Coagulase-negative staphylococci (n = 25) MIC50 (% w/v) 6 7 10
MIC90 (% w/v) 7 8 15
MIC range (% w/v) 5–8 5–10 6–15
All Staphylococcus spp. (n = 73) MIC50 (% w/v) 5 6 15
MIC90 (% w/v) 7 8 15
MIC range (% w/v) 5 to >15 5 to >15 6 to >15
MIC, minimal inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible S.aureus.
https://doi.org/10.1371/journal.pone.0224495.t001
Table 2. MIC
50
, MIC
90
, and MIC ranges of Manuka honeys UMF 5+, 10+, and 15+ tested against gram-negative organisms.
Organisms UMF 5+ UMF 10+ UMF 15+
Pseudomonas aeruginosa All (n = 22) MIC
50
(% w/v) 21 21 21
MIC
90
(% w/v) 21 27 27
MIC range (% w/v) 9–27 15–27 21–33
MDR (n = 10) MIC
50
(% w/v) 15 21 21
MIC
90
(% w/v) 21 21 21
MIC range (% w/v) 9–21 15–27 21–27
Non-MDR (n = 12) MIC
50
(% w/v) 21 21 27
MIC
90
(% w/v) 21 27 33
MIC range (% w/v) 15–27 15–27 21–33
Enterobacteriaceae All (n = 33) MIC
50
(% w/v) 21 21 27
MIC
90
(% w/v) 33 33 33
MIC range (% w/v) 15–33 15–33 21–33
ESBL, CRE (n = 14) MIC
50
(% w/v) 27 33 27
MIC
90
(% w/v) 33 33 33
MIC range (% w/v) 21–33 21–33 21–33
Non-ESBL/CRE (n = 19) MIC
50
(% w/v) 21 21 27
MIC
90
(% w/v) 27 27 33
MIC range (% w/v) 15–27 15–27 21–39
All Gram-negative organisms (n = 55) MIC
50
(% w/v) 21 21 27
MIC
90
(% w/v) 33 33 33
MIC range (% w/v) 9–33 15–33 21–39
MIC, minimal inhibitory concentration; ESBL, extended-spectrum beta-lactamase; CRE, carbapenem-resistant Enterobacteriaceae; MDR, multi-drug resistant.
https://doi.org/10.1371/journal.pone.0224495.t002
Antibacterial activity of varying UMF-graded Manuka honeys
PLOS ONE | https://doi.org/10.1371/journal.pone.0224495 October 25, 2019 4 / 9
UMF 10+ honey (p<0.05) or UMF 15+ honey (p<0.05), but UMF 10+ and UMF 15+ MIC
values showed no significant difference. Among non-MDR P.aeruginosa (n = 12), MIC values
for both UMF 5+ and UMF 10+ were significantly lower than UMF 15+ honey (p<0.05).
MDR strains had significantly lower MIC values than non-MDR strains for UMF 5+ (p = 0.01)
and UMF 15+ (p = 0.01) but not UMF 10+ (p = 0.58) honey.
For Enterobacteriaceae (n = 33), MIC values were lower for UMF 5+ than for UMF 15+
honey (p<0.01) and for UMF 10+ than UMF 15+ honey (p<0.01), but not for UMF 5+
compared to UMF 10+ (p>0.05). Compared to non-ESBL/non-carbapenem-resistant
Fig 1. Representative broth microdilution results for an MRSA isolate tested against UMF 5+, 10+, and 15+
honeys. Images of the dilution series for each honey are cropped and shown for side-by-side comparison. Here, the
MIC was 6% for UMF 5+, 7% for UMF 10+, and 15% for UMF 10+.
https://doi.org/10.1371/journal.pone.0224495.g001
Antibacterial activity of varying UMF-graded Manuka honeys
PLOS ONE | https://doi.org/10.1371/journal.pone.0224495 October 25, 2019 5 / 9
Enterobacteriaceae (CRE) organisms (n = 19), ESBL and CRE organisms (n = 14) had higher
overall MIC values with UMF 5+ and UMF 10+ honeys (p<0.01), but not UMF 15+
(p = 0.81).
Discussion
The increasing incidence of multi-drug resistant bacterial infections worldwide poses new
challenges which have led to a renewed interest in Manuka honey as an alternative antibiotic
agent [3,18]. Its antibacterial mechanisms, with different target sites, are unique from those of
conventional antibiotics, thus Manuka honey could potentially be used as an alternative or
ancillary agent in MDR bacterial infections. Through multifactorial mechanisms, Manuka
honey has been shown to disrupt the metabolic processes and membrane potential of S.aureus
and E.coli and the extent of cell viability was dependent on honey concentration [19]. Tran-
scriptomic studies have shown S.aureus to produce unique expression profiles when exposed
to Manuka honey as compared to typical antibiotics [20]. There has furthermore been demon-
strated in vitro synergism between Manuka honey and conventional antibiotics, as measured
by inhibition of bacterial growth or biofilm formation [20–22]. As a topical agent, Manuka
honey may be used effectively to treat disorders like atopic dermatitis, blepharitis, rhinosinusi-
tis, and skin ulcers [23–26]. Our data corroborate the measurable antimicrobial activity of
Manuka honey against a spectrum of clinical isolates from skin and soft tissue sources, includ-
ing those with multi-drug resistance such as MRSA, ESBL producers, CRE, and MDR P.aeru-
ginosa [6,27–30]. Lower MIC values were achieved against Staphylococcus species than with
gram-negative pathogens, consistent with the overall trends of prior studies [3]. We addition-
ally demonstrated activity of Manuka honey against S.lugdunensis, a clinically important coag-
ulase-negative Staphylococcus, which to our knowledge has not yet been reported.
Contrary to our expectations, Manuka honey of lower UMF grade demonstrated equal to
significantly increased antimicrobial activity compared to higher UMF grade honey for all
organism groups tested. While unexpected, this phenomenon has occurred in several other
studies. One investigation compared Manuka honey of UMF grades between 5 and 20 against
S.aureus and E.coli organisms incorporated into tissue engineering scaffolds and found that
no significant differences in bacterial clearance regardless of the UMF grade [31]. Other
authors have also found that UMF grade did not correlate with antibacterial activity against
P.aeruguinosa, although number of isolates tested was limited [32]. We believe that these find-
ings may be explained by the dynamic nature of the chemical composition of Manuka honey.
Dihydroxyacetone (DHA) is the precursor molecule of MGO found in Leptospermum flower
nectar and by itself lacks antimicrobial activity. With maturation of the honey, a portion of
DHA will convert to MGO, thus increasing MGO concentration with time. Decreases in DHA
and increases in MGO concentrations begin to occur after Manuka honey extraction, with
changes continuing up to at least one year of storage [33,34]. The extent of DHA conversion
to MGO is not wholly predictable for a given sample, as side chemical reactions also occur and
predictions are complicated by temperature and other variables [34]. Higher DHA:MGO
ratios between 5:1 to 9:1 are observed in fresher Manuka honey compared to lower DHA:
MGO ratios approximating 2:1 in older honeys [33,35]. A major Manuka honey testing labo-
ratory found that final packed Manuka honeys of lower UMF grade tended to have higher
DHA:MGO ratios whereas higher grade UMF honeys tended to have lower such ratios and
higher content of hydroxymethylfurfural and C4 sugars, indicating honey that was older at the
time of UMF grading [35]. Therefore, MGO concentrations and antimicrobial activity at the
time of consumer use may not be accurately reflected by UMF labelling. While we did not
measure MGO or DHA concentrations of the honeys used during our study, we conjecture
Antibacterial activity of varying UMF-graded Manuka honeys
PLOS ONE | https://doi.org/10.1371/journal.pone.0224495 October 25, 2019 6 / 9
that age and storage conditions likely influenced MGO concentrations and resulting antimi-
crobial activity of the honeys tested.
Manuka honey is marketed as beneficial to health and has been publicized for its antibacte-
rial properties. There is therefore legitimate concern that honeys of higher UMF grades are
considered by consumers as higher quality and are sold at premium prices, whereas higher
UMF graded honey may not necessarily confer an increased health benefit. Future studies
could confirm the variability of in vitro antimicrobial efficacy between UMF-graded Manuka
honeys from different manufacturers and lot numbers, as our study was limited to single bot-
tles of various UMF grades. MGO and DHA concentrations should also be assessed over time
in Manuka honey sold for medicinal purposes and correlated with antibiotic activity to better
understand changes in antimicrobial efficacy over its shelf life.
While we detected statistically significant differences in the MIC values, the absolute differ-
ences in MICs between the three UMF-graded honeys would be considered small by suscepti-
bility testing standards, generally within two-fold dilutions. It is unknown whether or not
these differences in MIC would have a significant clinical impact, such as for topical treatment
of wound infections. Studies correlating antimicrobial susceptibility testing results with clinical
outcomes are lacking, but may be beneficial to developing best practices in using Manuka
honey for its antibiotic activity.
Conclusions
Manuka honey exhibited antimicrobial activity against a spectrum of MDR and non-MDR
bacterial organisms isolated from wound sites, with greater potency against staphylococcal
organisms compared to gram-negative bacteria. In a limited sampling, we also found Manuka
honey to demonstrate significantly greater antimicrobial activity at lower UMF grades when
compared to UMF 15+ honey. We conclude that UMF grade, as an indicator of MGO content
and honey quality, may be misleading to the consumer as it may not necessarily correlate with
antibacterial efficacy of the Manuka honey at the time of purchase or the time of use. Studies
investigating in vivo outcomes of Manuka honey of different UMF grades while confirming
MGO and DHA content are needed to advance our understanding of use of Manuka honey
for medicinal purposes. Despite these concerns, natural products such as Manuka honey are
promising as alternative agents in combatting MDR bacterial infections.
Supporting information
S1 Table. Minimal inhibitory concentrations of 3 different UMF grades of Manuka honey
for 128 bacterial isolates. Categories and sub-categories of organism as discussed in the text
are also indicated. MSSA, methicillin-susceptible Staphylococcus aureus; ESBL, extended-spec-
trum beta-lactamase; KPC, bla
KPC
carbapenemase producer; CRE, carbapenem-resistant
Enterobacteriaceae; MDR, multi-drug resistant.
(XLSX)
Acknowledgments
We thank Dr. Susan Butler-Wu for provision of a portion of the bacterial isolates used in this
study.
Author Contributions
Conceptualization: Alodia Girma, Rosemary C. She.
Antibacterial activity of varying UMF-graded Manuka honeys
PLOS ONE | https://doi.org/10.1371/journal.pone.0224495 October 25, 2019 7 / 9
Data curation: Alodia Girma, Rosemary C. She.
Formal analysis: Alodia Girma, Rosemary C. She.
Investigation: Wonjae Seo, Rosemary C. She.
Methodology: Alodia Girma, Wonjae Seo, Rosemary C. She.
Project administration: Rosemary C. She.
Resources: Rosemary C. She.
Supervision: Rosemary C. She.
Writing – original draft: Alodia Girma, Rosemary C. She.
Writing – review & editing: Rosemary C. She.
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