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Antimicrobial Activity of Different Finnish Monofloral Honeys Against Human Pathogenic Bacteria
Antimicrobial Activity of Different Finnish Monofloral Honeys Against Human Pathogenic Bacteria
DOI 10.1111/apm.12039
used in wound management, Revamil® and was obtained from Northern Finland at 65° N
Manuka honeys (19), have additional antimi- (Oulu), and cloudberry honey (R. chamaemorus)
crobial mechanisms. The main active compo- was collected at 68° N (Sodankyl€ a) during the year
2008. Identification of the floral source of honey
nent in Manuka honey is methylglyoxal (20) was based on organoleptic characteristics of the
and an antimicrobial peptide, bee defensin-1, honey and it was supported by pollen analysis.
has been identified from Revamil® honey (12). Honey samples were diluted in phosphate buffered
Many studies on antimicrobial activity of saline (PBS) (Gibco, Paisley, UK) containing Todd-
honey have been conducted in non-European Hewitt broth supplemented with yeast extract
countries (21), and especially in southern hemi- (THY) (Becton Dickinson, Le Pont de Claix,
sphere (9, 10, 22, 23). New Zealand Manuka France). The concentration of THY in PBS was
0.05%. The concentrations of the studied honeys
honey is widely studied and used clinically. are expressed as the percentage of honey weight per
However, it has been found that other honeys total reaction volume used in the antimicrobial
with different floral backgrounds exhibit equiv- assay (w/v). The tested honey concentrations were
alent inhibitory activity (9). It is thus reason- 60%, 40%, and 20% (w/v).
able to search for new antimicrobial honey
candidates from different parts of the world.
In this study, we tested the antimicrobial Phenolic standards
activity from different Finnish monofloral hon- The phenolic compounds: p-hydroxybenzoic acid
eys against important human pathogens: S. (H5376), vanillic acid (V2250), gallic acid (G8647),
pneumoniae, S. pyogenes, S. aureus, and caffeic acid (C0625), ferulic acid (F3500), p-couma-
MRSA. The tested honeys were buckwheat ric acid (C9008), 3,4-hydroxybenzoic acid (P5630),
honey (Fagopyrum esculentum), cloudberry (+)-catechin (C1251), ( )-epicatechin (E1753) were
honey (Rubus chamaemorus), heather honey purchased from Sigma Chemical Co. (St Louis,
MO, USA) and quercetin 3-glucoside (1327S) from
(Calluna vulgaris), lingonberry honey (Vaccini- Extrasynthese (Genay Cedex, France).
um vitis-idaea), and willow herb honey (Epilo-
bium angustifolium). Phenolic compounds in
honeys were analyzed by HPLC-DAD Microbroth dilution assay
method. A modification of a microtiter broth microdilution
assay described by Amsterdam 2005 (25) was chosen
MATERIALS AND METHODS for testing the antibacterial activity of different
honey samples. The antimicrobial assay was per-
formed as described before (24). Shortly: bacteria
Bacterial strains and culture conditions were cultured overnight on sheep agar plates and
Streptococcus pyogenes (ATCC 8184), S. aureus one colony of each bacterial strain was collected
(ATCC 25923), and MRSA (ATCC 43300) were into 5 mL of THY broth (S. pneumoniae and S. py-
from ATCC. S. pneumoniae clinical strain SB 53845 ogenes) or brain heart infusion (BHI) broth (Oxoid,
(isolated from lung) was received from Sauli Ha- Cambridge, UK) (S. aureus and MRSA) for subcul-
ataja (University of Turku, Finland). S. pyogenes, S. ture. Then the culture was carried out at 37 °C for
aureus, and MRSA were cultured at 37 °C on sheep 3–4 h or overnight depending on the growth of the
blood agar plates (Becton Dickinson, Franklin bacterial strain. The cultures were followed by mea-
Lakes, NJ, USA). S. pneumoniae was cultured at suring A600 absorbance values. Each culture inocu-
37 °C in CO2 atmosphere on sheep blood agar lum was individually standardized. The densities of
plates (Labema Inc., Kerava, Finland) as previously the bacterial suspensions were adjusted to the A600
described (24). values of 0.4–0.7 to achieve colony-forming units
(CFUs)/mL approximately of 109–1013 depending
on the bacterial strain. Then appropriate dilutions
Preparation of the honey samples were made in PBS to bring the inoculum density to
the range of 103–104 CFU/mL depending on the
Monofloral honey samples were obtained from bacterial strain. The diluted bacteria were then inoc-
Finnish Beekeepers Association. Heather (C. vulga- ulated to the test and control samples. Because of
ris) and lingonberry honeys (V. vitis-idaea) were har- the differences in growth between the bacterial
vested from Western Finland at 63° N (Kaustinen pathogens, inhibition induced by honey samples was
and Pihtipudas, respectively), buckwheat honey (F. always compared to the respective bacterial control
esculentum) was collected from Eastern Finland at prepared as the samples, but without honey addi-
62° N (Kitee), willow herb honey (E. angustifolium) tion. The controls were made each time for the stud-
ied strain in question and carried in the experiment increase of the organic phase from 5% to 30% dur-
at the same time with the sample. Different concen- ing 25 min (acetonitrile and methanol, ACN:
trations of the honey samples (80 lL) were then MeOH, 85:15, v/v) in the 1% formic acid water
mixed with the diluted bacteria (20 lL) and the bac- phase. Separation was followed by rising the organic
teria-honey mixtures were incubated in microtiter phase to 90% during 10 min, returning to initial
plates (Falcon Flexible Plate; Becton Dickinson conditions during 5 min and then by re-equilibra-
Labware) at 37 °C for 2 h. The controls were pre- tion of the column for 10 min. The flow rate was
pared without adding honey to the reaction mixture 1.0 mL/min.
or by adding ampicillin as described before (24, 26). HPLC combined with diode array detection was
Shortly: a quantity of 80 lL of 0.05% THY or BHI used for UV–Vis spectral analysis and quantifica-
broth with yeast extract in PBS was mixed with the tion. Identification of the polyphenols in the chro-
diluted bacterial suspension of 20 lL (bacteria- matograms was based on retention times and
broth/PBS control) and incubated in microtiter plate comparison of their UV–Vis spectral shape, and
wells as described for the bacteria-honey samples. wavelengths of maximum absorption, and wave-
The ampicillin control was prepared as the bacteria- lengths of shoulders (sh) of representative standards.
broth/PBS controls with ampicillin addition to the The representative standards were analyzed near
final concentration of 80 lg/mL. To measure the their wavelengths of maximum absorption: benzoic
antibacterial activity of the honeys, all the bacteria- acid and hydroxybenzoic acids (vanillic acid,
honey samples and the bacteria-broth/PBS controls p-hydroxybenzoic acid, gallic acid, and 3,4-hydroxy-
were plated on sheep blood agar plates in triplicate benzoic acid) at 260 nm, hydroxycinnamic acids
and the numbers of the colonies were counted next (caffeic acid, ferulic acid, and p-coumaric acid,) at
day. Bacterial survival was calculated by comparing 320 nm, flavonol glycosides of flavonoids (quercetin
the CFU numbers of the bacteria-honey samples 3-O-glucoside) at 360 nm. In honey samples typical
with CFU numbers of the bacteria-broth/PBS con- spectra neither of ellagic acid, anthocyanins nor fla-
trols of the respective bacterial strain. Two indepen- van-3-ols were detected (27, 28). Identified individ-
dent antimicrobial assays were done for each of the ual compounds were quantified within the linear
studied bacterial strain. range using standard curves of representative stan-
dards. The response factors were determined from
freshly prepared solutions in the concentration
Extraction of polyphenols ranges 2–250 mg/L.
The honey samples (5 g) were weighed in centrifuge
tubes and deionized water was added to equilibrate Statistical analysis
the content of soluble solids to 20 g/100 g in all
honey samples. Rare flavonol, morin (1 mg/mL Results for antimicrobial tests were reported as
methanol, 0.1 mL) was added as an internal stan- means standard deviation. Two-tailed, unpaired
dard, to follow extractability of polyphenols. Student’s t-test (Microsoft Excel 2007, Microsoft
Extractions were performed with ethyl acetate Corp., Santa Rosa, CA, USA) was used to calculate
(4 9 10 mL) by shaking the samples vigorously the statistical significance of the differences between
several times followed by centrifugation. The whole CFUs from plated bacteria-broth/PBS controls and
40-mL portion of the ethyl acetate extract was evap- plated bacteria-honey mixtures. Significance was
orated to dryness with a rotary evaporator, dis- defined as a value of p of <0.01.
solved in 1 mL of methanol and analyzed with
HPLC-DAD.
RESULTS
HPLC-analysis of honey samples
Antimicrobial activity of honey
All samples were filtered through a 0.45-lm syringe
filter before injection into the HPLC. The HPLC- Antimicrobial activities of five Finnish monofl-
DAD apparatus consisted of a Hewlett-Packard oral honeys (buckwheat honey, cloudberry
instrument with a 1100 series quaternary pump, an honey, heather honey, lingonberry honey, and
autosampler, and a diode array detector linked to willow herb honey) were studied against
an HP-ChemStation data handling system (Wald- human pathogens S. pneumoniae, S. pyogenes,
bronn Analytical Division, Waldbronn, Germany). S. aureus and MRSA. In this study we found
HPLC separation was achieved on a (150 9 4.6 mm that all the tested bacteria: S. pneumoniae,
i.d., 5 lm) Phenomenex Gemini reversed-phase (RP)
column (RP-18; Merck, Darmstadt, Germany) pro- S. pyogenes, S.aureus and MRSA were suscep-
tected with a guard column of the same material tible (p < 0.01) to the tested willow herb,
(4 9 3 mm). The linear gradient was based on heather and buckwheat honeys (Fig. 1).
A B
C D
Fig. 1. Antimicrobial activity of buckwheat, cloudberry, heather, lingonberry, and willow herb honeys (60%,
40%, and 20%) concentrations (w/v) against Streptococcus pneumoniae (A), Streptococcus pyogenes (B),
Staphylococcus aureus (C), and methicillin-resistant S. aureus (D). The values represent the mean and standard
deviation calculated from two individual experiments. Bacterial survival was compared to control. Standard
deviation, n = 6, *p < 0.01 against the bacterial control.
S. pneumoniae, S. pyogenes, and S. aureus, but buckwheat, and lingonberry honeys had anti-
not MRSA were also susceptible to the lingon- microbial effect (p < 0.01). The corresponding
berry honey. The studied cloudberry honey bacterial survivals for the heather honey were
was active only against S. pneumoniae. The 18% and 32%, respectively, for the buckwheat
detected antimicrobial effect was mostly honey 33% and 43%, respectively, and for the
growth inhibiting (results not shown). In the lingonberry honey the bacterial survivals were
presence of ampicillin (80 µg/mL) there was 50% and 45%, respectively, The studied
no bacterial growth, except in MRSA cultures cloudberry honey had no antimicrobial effect
(8% compared to broth/PBS control). against S. pyogenes.
S. pneumoniae was significantly (p < 0.01) The growth of S. aureus was inhibited by
sensitive to all the tested honeys compared to the buckwheat and the heather honeys in all
the control at the honey concentrations of 60% the tested concentrations of 60%, 40%, and
and 40% (Fig. 1A). Best activities were obtained 20% (p < 0.01) (Fig. 1C). The bacterial sur-
with the willow herb honey. Pneumococcal sur- vival was 10% for the 60% buckwheat honey,
vival was 8% when 60% concentration of the 6% survival for the 40% buckwheat honey
willow herb honey was used and 16% with 40% and 28% survival for the 20% buckwheat
willow herb honey. For the heather honey the honey. In the presence of the heather honey
respective survivals were 17% and 15%, for the the survival of S. aureus was 26%, 27%, and
buckwheat honey the respective survivals were 38% vs the 60%, 40%, and 20% heather hon-
17% and 43%, for the lingonberry honey the eys, respectively. The willow herb honey of
survivals were 23% and 24%, respectively, and 60% and 40% had significant antimicrobial
for the 60% and 40% cloudberry honey the activity as well (p < 0.01) with the respective
respective survivals were 22% and 30%. bacterial survivals of 28% and 33%. The 60%
The growth of S. pyogenes was inhibited sig- lingonberry honey had significant antimicro-
nificantly (p < 0.01) by all the studied concen- bial activity (p < 0.01) with the bacterial sur-
trations (60%, 40%, and 20%) of the willow vival of 56%. The tested cloudberry honey
herb honey, when bacterial survival was 11%, was ineffective against S. aureus.
27%, and 62%, respectively (Fig. 1B). Also There was no significant difference between
60% and 40% concentrations of the heather, antibiotic-resistant and antibiotic-sensitive
and buckwheat honeys, did not affect their anti- honeys against all the studied bacteria, willow
microbial activity (results not shown). Neither herb, and buckwheat honeys, were very low,
did heating reduce the bioactivity of the willow 0.65 and 2.91 mg/100 g of honey, respectively,
herb and heather honeys against S. pyogenes and thus phenolic compounds do not explain
nor the bioactivity of the buckwheat honey their antimicrobial activity. Characterization of
against S. aureus or MRSA (results not shown). the unknown active components in Finnish
High osmolarity, low pH, and hydrogen perox- honeys remains to be carried out in future.
ide are the main antimicrobial factors in honeys Against S. pyogenes, S. aureus and MRSA
(15–17). Because heating reduced some, but not several honeys with different floral backgrounds
all the bioactivity of the tested honeys, it gives a have been reported to possess antimicrobial
clue that hydrogen peroxide may have a role, activity (9, 20, 21, 30). Parallel to our results
but there are also other contributing antimicro- (Fig. 1C) against S. aureus, heather, and buck-
bial factors in these honeys. wheat honeys from different geographical ori-
In our study, we found that the 20% con- gins have been previously reported to be active
centrations of willow herb, heather, and buck- (8, 22). There are honeys that do not possess
wheat honeys had significant antimicrobial any antimicrobial activity against certain bacte-
activity at neutral pH. This refers to other ria. Here we show that the studied cloudberry
contributing antimicrobial factors than high honey was inactive against S. pyogenes, S. aur-
osmolarity or pH in buckwheat, heather, and eus, and MRSA strains, but was antimicrobial
willow herb honeys. Cloudberry honey was against S. pneumoniae. This demonstrates
not active against S. pyogenes, S. aureus or higher sensitivity of S. pneumoniae to varying
MRSA at the 60% concentration. This further levels of antibacterial compounds present in
supports the finding that the antimicrobial honey samples. Variations in antimicrobial
activity against these bacteria was not due to activity and active components between differ-
high osmolarity of the studied honeys. ent honeys may result from different geographi-
Phenolic content varies between different cal locations and floral sources, as well as
honeys, and phenolic compounds may contrib- differences in harvesting, processing, storage
ute to antimicrobial activities in honeys (18). In conditions, and bee-related factors (23, 33).
the studied Finnish honeys only very small Honey is a natural, nontoxic, and inexpen-
amounts of phenolic acids, benzoic acid, and sive product for the need of novel therapies
flavonoids were present. The highest concentra- against bacterial infections. The clinical use of
tion of phenolic compounds was detected in honey has an enormous potential, especially in
heather honey, almost 9 mg/100 g honey, which the fight against antibiotic-resistant strains.
composed mostly of benzoic acid. Heather Here we show antimicrobial activity and anti-
honey was active against all the studied bacte- infective potential of Finnish monofloral wil-
ria, and thus benzoic acid may possibly have an low herb, heather, buckwheat, lingonberry,
additional role as an antimicrobial agent in the and cloudberry honeys for the first time
studied honeys. However, for antimicrobial against important human pathogen S. pneumo-
activity of berry phenolic compounds, at least niae. In this study we also show that against
100 times higher concentrations were needed for S. pyogenes, S. aureus, and MRSA bacteria
antimicrobial activity against S. pneumoniae, the tested willow herb, heather, and buckwheat
when the same assay methodology was used honeys had significant antimicrobial activity.
(24). In the studied honeys, benzoic, hydroxy- Future studies are needed both to reveal the
benzoic, and hydroxycinnamic acids were found active components and to clinically prove
in free forms and flavonoids were detected as the efficacy of these Northern honeys against
aglycones and glycosides. In berries benzoic, hy- the tested pathogens.
droxybenzoic sand hydroxycinnamic acids
occur predominantly as sugar esters and glyco-
sides (27, 28). This difference may have an effect We thank Finnish Beekeepers Association for pro-
on the antimicrobial activity of phenolic com- viding the honey samples, and Jukka Hyt€ onen and
pounds in honeys. The total concentration of Sauli Haataja for bacterial strains. We also thank
the phenolic compounds in the two other active Sari Ukkonen and Eeva-Liisa Palkisp€ a€
a for skilful
technical assistance. This study was funded by the tance to medical-grade manuka honey. Eur J
European Regional Development Fund and the Clin Microbiol Infect Dis 2010;29:1237–41.
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Fazer Makeiset Oy (Vantaa, Finland), Kiantama honey, as hydrogen peroxide and its origin in
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