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Lactic acid bacterial symbionts in honeybees - an unknown key to honey's antimicrobial and therapeutic activities: Lactic acid bacteria a key in honey production

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Could honeybees' most valuable contribution to mankind besides pollination services be alternative tools against infections? Today, due to the emerging antibiotic-resistant pathogens, we are facing a new era of searching for alternative tools against infections. Natural products such as honey have been applied against human's infections for millennia without sufficient scientific evidence. A unique lactic acid bacterial (LAB) microbiota was discovered by us, which is in symbiosis with honeybees and present in large amounts in fresh honey across the world. This work investigates if the LAB symbionts are the source to the unknown factors contributing to honey's properties. Hence, we tested the LAB against severe wound pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and vancomycin-resistant Enterococcus (VRE) among others. We demonstrate a strong antimicrobial activity from each symbiont and a synergistic effect, which counteracted all the tested pathogens. The mechanisms of action are partly shown by elucidating the production of active compounds such as proteins, fatty acids, anaesthetics, organic acids, volatiles and hydrogen peroxide. We show that the symbionts produce a myriad of active compounds that remain in variable amounts in mature honey. Further studies are now required to investigate if these symbionts have a potential in clinical applications as alternative tools against topical human and animal infections.
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International Wound Journal ISSN 1742-4801
ORIGINAL ARTICLE
Lactic acid bacterial symbionts in honeybees an unknown
key to honey’s antimicrobial and therapeutic activities
Tobias C Olofsson1, Èile Butler1, Pawel Markowicz1, Christina Lindholm2, Lennart Larsson1& Alejandra
Vásquez1
1 Medical Microbiology, Department of Laboratory Medicine Lund, Lund University, Lund, Sweden
2 Sophiahemmet University, Stockholm, Sweden
Key words
Alternative antibiotic tools; Bioactive
metabolites; Honey; Honeybees; Lactic acid
bacteria; Symbiosis; Wound management
Correspondence to
A Vásquez
Medical Microbiology
Department of Laboratory Medicine
Lund University
Sölvegatan 23
SE-223 62 Lund
Sweden
E-mail: Alejandra.Vasquez@med.lu.se
Olofsson TC, Butler È, Markowicz P, Lindholm C, Larsson L, Vásquez A. Lactic
acid bacterial symbionts in honeybees – an unknown key to honey’s antimicrobial and
therapeutic activities. Int Wound J 2014; doi: 10.1111/iwj.12345
Abstract
Could honeybees’ most valuable contribution to mankind besides pollination services
be alternative tools against infections? Today, due to the emerging antibiotic-resistant
pathogens, we are facing a new era of searching for alternative tools against infections.
Natural products such as honey have been applied against human’s infections for
millennia without sufcient scientic evidence. A unique lactic acid bacterial (LAB)
microbiota was discovered by us, which is in symbiosis with honeybees and present
in large amounts in fresh honey across the world. This work investigates if the LAB
symbionts are the source to the unknown factors contributing to honey’s properties.
Hence, we tested the LAB against severe wound pathogens such as methicillin-resistant
Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and vancomycin-resistant
Enterococcus (VRE) among others. We demonstrate a strong antimicrobial activity from
each symbiont and a synergistic effect, which counteracted all the tested pathogens.
The mechanisms of action are partly shown by elucidating the production of active
compounds such as proteins, fatty acids, anaesthetics, organic acids, volatiles and
hydrogen peroxide. We show that the symbionts produce a myriad of active compounds
that remain in variable amounts in mature honey. Further studies are now required to
investigate if these symbionts have a potential in clinical applications as alternative tools
against topical human and animal infections.
Introduction
Today, due to overuse of antibiotics and emerging
antibiotic-resistant pathogens, we are facing a new era of
searching for alternative tools against infectious diseases.
Chronic wounds infected by pathogens such as Pseudomonas
aeruginosa,Staphylococcus aureus,Klebsiella and Strepto-
coccus pyogenes are subjects for intensive research efforts
because of the bacteria’s ability to sustain antibiotic treat-
ment and maintain chronic infections by biolm production.
In a previous study (1), 70% of all patients with wounds
had critical colonisation or overt infection in their wounds.
Antibiotic-resistant bacteria in wounds caused by frequent
use of antibiotics are a threat to the Health Care Sector (2)
and, now, researchers are searching for new antimicrobial
weapons in natural products and unexplored ecological niches
for alternative tools against infections (3,4). Symbionts in an
ecological niche that are already shaped to defend their host
by producing bioactive compounds are a relatively unexploited
option (5,6).
Key Messages
for centuries, honey has been used as a folk medicine
for the treatment of upper respiratory tract infections and
wounds. Today, many of its antimicrobial characteristics
have been recognised; however, there are still unknown
substances that contribute to this action
it has been discovered that 13 LAB symbionts from the
honey stomach of honeybees are found in large concen-
trations in fresh honey as well as having a wide spectrum
of antimicrobial activity against various bee pathogens
© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd. 1
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Lactic acid bacteria a key in honey production T. C. Olofsson et al.
and bacteria and yeasts from owers. We hypothesise that
many of the unknown healing and antimicrobial proper-
ties of honey are linked with these LAB symbionts
our results show that these LAB are producing not only
common metabolites such as formic acid and lactic acid
but also a wide variety of other interesting metabolites
such as benzene and 2-heptanone. We have also identied
putative LAB proteins in different honey types, suggest-
ing their importance in honey production and antimicro-
bial activity. Interestingly, we have shown that in combi-
nation and separately these LAB symbionts have antimi-
crobial activity against a variety of severe chronic wound
pathogens
in this study, we could conrm that LAB symbionts
within honeybees are responsible for many of the antibac-
terial and therapeutic properties of honey. Our future aim
is to develop new alternative tools in wound management
against human and animal infections that are scienti-
cally proven, well dened and standardised
Less than a decade ago, we discovered a large unexplored
bacterial microbiota in symbiosis with honeybees and located
in the honey stomach (7). The novel microbiota is entirely
composed of approximately 40 lactic acid bacterial (LAB)
strains with 13 taxonomically well-dened Lactobacillus (9
spp.) (8) and Bidobacterium (4 spp.) species. LAB is a bacte-
rial group functionally related by their ability to produce lactic
acid during homo-or hetero-fermentative metabolism. In gen-
eral, certain species within LAB may produce bioactive com-
pounds such as organic acids, free fatty acids, ethanol, benzoate,
enzymes, hydrogen peroxide, antimicrobial peptides and antibi-
otics (9,10). These qualities together result in a wide inhibitory
spectrum against pathogens.
To our knowledge, this novel honeybee LAB microbiota
is one of the greatest symbiotic ora ever found in a single
organism (7,11,12). These LAB symbionts were shown to be
similarly present and active within all honeybees (Apis spp.)
and sampled stingless bees, and in their respective freshly har-
vest honey on all continents of the world (11– 13). Besides
their key role in honey production, our research shows that the
symbionts have been evolutionarily shaped to work synergisti-
cally in order to defend bees against incoming microbial threats
introduced by nectar foraging including several bacterial gen-
era and yeast by producing different metabolites, peptides and
proteins (11,14).
Honey is the best-known honeybee product and represents
the only human food source created entirely by an insect. The
medical effects of honey have been independently documented
by many cultures throughout history (15). However, while
honey has a number of applications by different cultures, it is
most well-known for its actions against upper respiratory tract
infections and in wound management (15,16). Honey’s modes
of action, besides its low pH and high osmolarity, are today
explained by the hydrogen peroxide content as an action of per-
oxidase oxidase produced by the honeybee itself, the origin of
the nectar by its different avonoids and phenolic acid contents
(17,18) and unidentied active compounds (19). Recently, other
compounds have been shown, including methylglyoxal in Lep-
tospermum scoparium (Manuka) honey (20), antibacterial pep-
tides bee defensin-1 (21) and bioactive compounds that alter the
expression of a specic protein in S. aureus (22). Furthermore,
studies have shown that honey has an anti-inammatory action
in wounds (18,23). Although clinical reports have shown posi-
tive results when using honey in wound management and recent
research has shown previously unknown bioactive compounds,
the application of honey dressings still gives a low condence
for its use in therapeutic treatment in wound management (24),
without a necessary scientic explanation of the source for
those compounds and mechanisms of action behind honey’s
properties.
Every single member of the novel LAB microbiota is
involved in varying concentrations in the process of nectar to
become honey. However, from our research we now know that
the 13 LAB species vary numerically in naturally harvested
honey depending on the nectar source, honeybee health and
presence of other microorganisms in the collected nectar
(7,11,14,25). We noticed early that the LAB symbionts react in
a synergistic matter and defend themselves by secreting a vari-
ety of active compounds that inhibit other microbial growth.
These interesting numerical variations and varying produc-
tion of active compounds appear to be a well-established
symbiosis among bees, LAB symbionts, owers’ nectars
and microbial threats that varies with season and honeybee
health (11).
The massive presence of viable LAB (108LAB/g fresh
honey) (7,11) raised the hypothesis that these novel LAB with
their active bio-products could be the reason for why honey
has been regarded as an antimicrobial agent through human
history. In this study, we explore the antimicrobial proper-
ties of LAB and their produced bioactive substances. We the-
orised that LAB substances produced during honey produc-
tion should be present in freshly harvest honey and preserved
in mature honey. Our future aim is to develop new alter-
native tools in wound management against human and ani-
mal infections that are scientically proven, well dened and
standardised.
Material and methods
Bacteria, media and honey
Lactic acid bacteria
The 13 LAB honeybee symbionts were previously isolated
from the honey stomach of the honeybee species Apis mellif-
era around the world and identied to the strain level in our
laboratory (7,8,11–13). The following bacteria were used in
this study: Lactobacillus helsingborgensis Bma5, Lactobacil-
lus kimbladii Hma2, Lactobacillus mellis Hon2, Lactobacillus
mellifer Bin4, Lactobacillus melliventris Hma8, Lactobacil-
lus apis Hma11, Lactobacillus kullabergensis Biut2, Lacto-
bacillus apinorum Fhon13, Lactobacillus kunkeei Fhon2, Bi-
dobacterium coryneforme Bma6 and Bidobacterium, Bin2,
Hma3 and Bin7. LAB strains from the honey stomach of
the dwarf honeybee Apis andreniformis, the giant honeybee
Apis laboriosa and the stingless bee Melipona beechii pre-
viously isolated by us (11) were also tested in this study.
2© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.
T. C. Olofsson et al. Lactic acid bacteria a key in honey production
All LAB strains were cultivated anaerobically for 72hours at
35C on de Man, Rogosa & Sharpe (MRS) (Oxoid, Hamp-
shire, UK) agar plates (1% agar, Oxoid) and broth supple-
mented with fructose (2%, Merck, Sollentuna, Sweden) and
-cysteine (01%, Sigma-Aldrich, Stockholm, Sweden), or in
Pollen media, freshly collected bee pollen mixed with water and
sterilised by autoclaving (26).
Human pathogens
Severe multidrug-resistant pathogens from chronic wound
infections were tested at Prof. Rose Cooper’s laboratory,
UWIC (Cardiff, Wales, UK). The used pathogens shown in
Table 1 were cultivated in nutrient broth (Oxoid) at 37Cfor
24 hours prior to test in the antagonism assays.
Dual culture overlay assay
Antimicrobial activity was measured by using dual culture over-
lay assay as previously described (11), with few modications.
LAB separately or in combinations (Table 1)were put into a l-
ter disc and placed on supplemented MRS agar plates followed
by overnight incubation at 35C. Wound pathogen cultures
were mixed with a 10-ml soft nutrient agar (08%), holding a
temperature of 42C. Each mixture of soft agar was poured as
an over layer on top of MRS plates with the overnight-cultivated
LAB. The plates were incubated at 37C for 24 hours. All the
tests were performed in triplicate. Zone diameters were mea-
sured from the centre of the disc to the zone edge.
Honey types
Stored honeys were purchased from a local Swedish
bee-keeper, which covered one summer season. These included
honeys from the following ower nectars (bloom time in paren-
thesis): rapeseed (May), raspberry (June), linden (small-leaved
lime, July), heather (August) and honeydew (pine, September).
We purchased Manuka honey (Manuka factor +10) from a
store in France (Comptoirs & Compagnies, Venelles, France).
As we know that the LAB are present in large amounts in
freshly harvest honey directly from the hive, we included
freshly harvest rapeseed honey taken directly from one colony
and stored it for 1 month 2 weeks.
Characterisation of LABs bioactive metabolites
Identication of bioactive metabolites produced by each of
the 13 LAB strains originating from honeybees (A. mellifera)
was conducted to uncover the mechanisms of action behind
antimicrobial and therapeutic characteristics.
Organic acids
Reagents and bacteria. Organic acid standards: lactic acid (L+,
98%) (Sigma, St. Louis, MO), formic acid (98%) (Sigma)
and acetic acid (100%) (Merck, Darmstadt, Germany). Milli-Q
ultrapure water (Merck) was used to dilute all standards and
stock solutions. Deionised water was used to prepare the mobile
phase. Lactobacillus Bma5, Hma2, Hon2, Bin4, Hma8, Biut2,
Fhon2 and Fhon13 were cultured in MRS broth. For Fhon2 and
Fhon13, the medium was enriched with 2% fructose (VWR,
Poole, UK). Bidobacterium Bma6, Bin2, Hma3 and Bin7 and
Lactobacillus Hma11 were cultured in Lactobacillus-carrying
medium (LCM) (27). The isolates were incubated in 15-ml
tubes under anaerobic conditions for 24 hours at 35C.
Equipment and chromatographic conditions. The HPLC
analyses were carried out on an Elite LaChrom modular
system composed of a high-pressure pump (L-2130) with
built-in degasser, a column oven (L-2300), a diode array detec-
tor (L-2455) (Hitachi, Tokyo, Japan) and a manual sample
injection valve (7725i, Rheodyne, Cotati, CA) with a 20-μl
sample loop. EZChrom Elite (Version 3.2.1) software suite
(Agilent Technologies, Kista, Sweden) was used for data
acquisition and calculations. The HPLC column used was a
Shodex RSpak KC-811 (6 μm, 300 ×80mm
2ID) (Showa
Denko K.K., Kawasaki, Japan). The mobile phase consisted of
01% phosphoric acid. Elution was carried out under isocratic
conditions with a ow rate of 10 ml/minute and a temperature
of 40C. Chromatograms of the UV absorbance were recorded
at 210 nm (from a UV-spectra of 200–400 nm). The system
was equilibrated for 30 minutes between each run.
Sample preparation. The medium containing the cultured LAB
was vortexed (MS1, IKA, Taquara, Brazil) until the pellet
had dissolved, then centrifuged for 10 minutes at 5100 g in
a Sigma 2–5 centrifuge and the supernatants were passed
through a 020-μm lter (Filtropur S, Sarstedt, Nümbrecht,
Germany). The resulting liquid was injected directly into the
HPLC without any previous dilution. Analyses were performed
in triplicate for each bacterium.
Calibrations and calculations. Standard aqueous solutions of
lactic acid (10%, v/v), formic acid (05%, v/v) and acetic acid
(10%, v/v) were used to establish the mean individual retention
time of each organic acid. Quantication was performed by the
external standard method. Multilevel calibrations [concentra-
tion (%) versus peek area] with ve loading levels in triplicates
(true average) were used to construct the calibration curves,
which were tted using linear regression. Background levels of
the corresponding medium for each of the bacterium were then
subtracted before the calculation of nal concentrations.
Free fatty acids (3-OH FAs)
The LAB species were grown in 5 ml supplemented MRS and
5 ml Pollen media until they reached their early stationary
phase, after approximately 24 hours of incubation at 35C
(14). Three millilitres of the supernatant was ltered through
a020-μm lter (Sarstedt). The ltered supernatants were
freeze-dried for 18 hours at 20C prior to the GC–MS
analysis.
The freeze-dried bacterial supernatants, and both stored
and freshly harvest honey samples (200 mg) were analysed
for 3-OH FAs. In brief, the preparations were heated in acid
methanol, extracted with heptane and puried using silica gel
columns. The hydroxy fatty acids, in the polar lipid fraction,
were then subjected to derivatisation to form trimethylsilyl
derivatives, and analysed by GC– MS/MS using an ion-trap
© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd. 3
Lactic acid bacteria a key in honey production T. C. Olofsson et al.
Ta b l e 1 Dual culture overlay assays with lactic acid bacterial (LAB) strains of bee origin against clinical isolates of pathogenic wound bacteria and yeast. The diameters of the inhibition zones are displayed
in millimetres. Antibiotics commonly used against the same pathogens are depicted as controls.
Bee species origin LAB strain
Serratia narcescens NJ19 5c
Staphylococcus areus FJ 0 2
Klebsiella aerogenes Clmp R
Citrobacter freundii CR01 5A
Staphylococcus areus 74022 PR
Staphylococcus areus CR01
Pseudomonas aeruginosa LE08
Enterobacter cloacae JSB 5B
MRSA clinical isolate 18
Klebsiella oxytoca JSB 5B
Escherichia coli V517
Candida albicans
Enterococcus faecalis E12 VRE
Acinetobacter A23 Z32524
Apis mellifera L. kunkeei Fhon2 42* ND 24* 3231* ND 3038* 33 ND 402031* 55*
L. apinorum Fhon13 8* ND 101820* ND 0 0 18 ND 12014*13
L. kimbladii Hma2 9* ND 910 0ND1059ND 7000
L. melliventris Hma8 18 ND 915 19* ND 0 17* 16 N D 120818*
L. helsingborgensis Bma5 11* ND 101813ND 0 11* 6ND 12006
L. kullabergensis Biut2 7* ND 7816ND 0 7 12* ND 100012
L. apis Hma11 0ND000ND000ND0000
L. mellifer Bin4 39* ND 26* 3222ND 29* 29* 32ND 302021 40*
L. mellis Hon2 0ND000ND000ND0000
Bifidobacterium Bin2 18* ND 15* 139* ND 1021* 0 ND 19810 15 *
Bifidobacterium Bin7 22 ND 17* 2214ND 1524* 15* ND 281213 21*
Bifidobacterium Hma3 26* ND 161815ND 1621 19 ND 20815* 22*
B. coryneforme Bma6 0 ND 0 69*ND000ND0000
Apis laboriosa All together§ 40* 42* 40* 3128 393340* 32* 36391225 32*
L. kunkeei Lahm1to13 45* ND 30* 3220ND 2738* 30 ND 461522 34*
All together§ 49* 45 30* 3124 413643* 34 32451525 43*
L. kunkeei Anhmro10 42* ND 34* 3529ND 3242* 33 ND 441529 46*
Apis andreniformis All together§ 47* 40 38* 3433393237* 31* 37401228 28*
L. kunkeei Yubipro16 45* ND 32* 3634ND 3045* 30 ND 461535* 62*
Melipona beechii All together§ 44* 40 37* 3828423742* 33 32461525 45*
Antibiotics V V F Cx Cl F V F Cn C Cn V F A A C
919292819*2919 2 9 19 3 121* 23 29* 0 19 0
MRSA, methicillin-resistant Staphylococcus aureus; ND, not determined; VRE, vancomycin-resistant Enterococcus. Used antibiotics: V, vancomycin, 30μg; F, fusidic acid, 10 μg; Cx, cefuroxime sodium,
30 μg; Cl, chloramphenicol 30 μg; Cn, gentamicin, 10 μg; A, ampicillin, 10 μg; C, ciprofloxacin, 5 μg.
*Sharp edge of inhibition zone.
Blur edge of inhibition zone.
Sporadic growth all the way in through the zone
§Repeated twice.
4© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.
T. C. Olofsson et al. Lactic acid bacteria a key in honey production
instrument (28). Some of the samples were also analysed
in scan mode using a quadrupole GC–MS instrument. The
3-hydroxy fatty acids (3-OH FAs) monitored were 3-OH C
10:0–3-OH C 22:0.
Hydrogen peroxide
Hydrogen peroxide production from each LAB was analysed
according to a previously described method (29). Shortly,
LAB were initially cultured for 3 days in supplemented MRS.
3,3,5,5-Tetramethyl-benzidine (TMB) plates were prepared
by adding solution A [25 mg of TMB (Sigma-Aldrich), dis-
solved in 6 ml methanol] and solution B [2 mg horseradish per-
oxidase, type 1, approximately 100 purpurogallin units/mg, dis-
solved in 2 ml of ddH2O] to MRS agar. The TMB plates were
then inoculated with each LAB and incubated anaerobically at
35C for 48 hours before transfer to aerobic conditions at room
temperature (RT). Blue colonies were observed after incubation
at RT for 1, 24 or 90hours.
Volatiles
All the tested LAB strains from A. mellifera were incubated
anaerobically for 7 days in Pollen media (50 ml). Bacterial
cultivations were performed separately in one anaerobic jar for
each strain. The diffusive sampler was attached onto the inner
side of the jar’s lid. A diffusive sampler having uptake rates
that fully agree with the theories behind diffusive sampling was
used for sampling and analysis of formic and acetic acids (Ferm
2001, www.diffusivesampling.ivl.se). Analysis was made using
ion chromatography with a gradient eluent generator (DIONEX
ICS 2000). Diffusive sampling of other organic vapours was
made with tube-typed sorbent tubes (PerkinElmer, Waltham,
MA). For sampling of benzene, toluene, n-octane, ethylben-
zene, m-, p-xylene, o-xylene and n-nonane, Carbopack B
(Sigma-Aldrich) was used as an adsorbent. The pollutants were
analysed via thermal desorption (ATD-400, PerkinElmer) and
gas chromatography with a ame ionisation detector (GC-FID,
Varian3800). More samplers could be analysed when Tenax
TA was used as sorbent, and analysis was made with thermal
desorption (Markes, Frankfurt, Germany) and GC– MS (Agi-
lent Technologies). Experimentally determined uptake rates
were used for the thermally desorbed hydrocarbons.
2-Heptanone
All the 13 LAB (from A. mellifera) were cultivated sepa-
rately in 10 ml (15-ml tubes, Sarstedt) supplemented MRS and
Pollen media (26) at 35C for 3 days. A viable count was per-
formed for all the LAB and their respective colony forming
unit (CFU) values are shown in Table 4. Bacterial cultures
were then cleaned by using 06 g of resin for 10 ml culture.
Bacterial samples were centrifuged at 1200 g for 10 minutes;
thereafter, 5ml of each sample supernatant was transferred
to a 10-ml glass test tube following extraction twice with
3 ml of dichloromethane (Sigma-Aldrich) containing deuter-
ated N-octanol (D17) (Cambridge Isotopes Laboratories, Inc.,
Tewksbury, MA) as an internal standard. The bottom phase was
transferred to a 1-ml GC test tube and analysed as described
below.
A Varian model 3800 gas chromatograph equipped with
a combiPAL autosampler (CTC Analytics AG, Zwin-
gen, Switzerland) and a silica capillary column (VF-5ms,
60 m ×025 mm ID, 1 μm lm thickness, Agilent Technolo-
gies) coupled to a 1200 l triple quadrupole MSMS detector
(Varian, Inc., Walnut Creek, CA) was used. Helium was used
as a carrier gas at a column ow rate of 10 ml/minute. The
column temperature was programmed to rise from 50Cto
230Cat7
C/minute, where it was held for 4 minute. The
injector temperature was 200C, the transfer line temperature
280C, the ion source temperature 200C, the electron energy
70 eV and the lament current 50μA. One microlitre injections
in the splitless mode were used.
Samples of the 13 LAB members (n=2) cultivated in
pollen medium were analysed in SCAN mode. Then, bacte-
ria that were found to produce clearly detectable amounts of
2-heptanone were re-analysed. Quantication of 2-heptanone
from these bacteria was performed using selected-ion mon-
itoring (SIM). A standard curve was obtained by injecting
15 –150 pg of 2-heptanone (Sigma-Aldrich) and 240 ng of
deuterated N-octanol (internal standard). The detection limit
of 2-heptanone was 1 ng/ml, and the extraction efciency
was 112%.
Biofilm formation
The LAB symbionts reside inside honeybees within the
honey crop in biolms (11). In order to assess if this biolm
can be formed outside the honey stomach, we investigated
biolm formation in vitro by a previously described method
(30).Shortly, each LAB symbiont was grown in supplemented
MRS and Pollen broths. LAB strains were allowed to reach
early stationary phase (108CFU/ml), and 100 μl of the culture
was inoculated into a polystyrene MicroWell plate (Nunc®,
Sigma-Aldrich) in different dilutions and incubated at 35Cfor
72 hours. The plates were then washed with sterile phosphate
buffered saline (PBS; pH 7.2). The attached bacteria were
stained by adding a crystal violet solution and rinsed with
sterile water to remove excess stain. The plates were air-dried
and the stain that bound to attached bacteria was released by
adding an ethanol/acetone solution. The optical density (OD)
was measured at 570 nm. Each LAB strain in MRS and Pollen
broths was analysed in triplicates.
Protein and peptide analyses in honey
Samples were prepared and analysed as previously described
(14) with some modications. Different types of stored honey
from the following nectars were used: heather, linden, rasp-
berry, oil rapeseed and manuka (see above) were prepared
in a 1:5 and 1:50 dilution (honey:water) and centrifuged at
3500 g for 25 minutes. Supernatant was taken from each tube
and added to 30 K Amicon ultra centrifugal lters (Millipore,
Merck, Darmstadt, Germany) and centrifuged for 10 minutes at
3500 g. Tris–HCl (02M, pH83) was added to the lter and
samples were centrifuged as mentioned before. This step was
repeated once again and 6 M urea (in 02M TrisHCl) was
added to the lter and centrifuged as mentioned before (31,32).
Samples were frozen at 20C until further use.
© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd. 5
Lactic acid bacteria a key in honey production T. C. Olofsson et al.
Ta b l e 2 Bioactive substances produced by each of the 13 LAB symbionts from honeybees (Apis mellifera)*
Genus Strain Acetic acid Formic acid Lactic H2O2Benzene Toluene Octane Ethylbenzene Xylene Nonane
Lactobacillus Fhon2 >263 >17 680 00045 0004 0000022 039 00
Lactobacillus Fhon13 >327 >28 600 00018 0008 000031 029 00068
Lactobacillus Hma11 >306 >16 500 +00005 0036 0027 00023 00127
Lactobacillus Hon2 >290 >16 770 0001 0045 0049 00004 028 002
Lactobacillus Bin4 161893600 0074 00000017 001 00
Lactobacillus Hma2 >271 >16 710 +00003 0057 0049 00025 00127
Lactobacillus Bma5 >267 >16 900 +00004 0046 0059 0004 028 00163
Lactobacillus Hma8 2064127 1060 +00008 007 0049 00005 024 002
Lactobacillus Biut2 >258 >14 950 +00006 0036 0039 00004 026 00159
Bifidobacterium Bin2 >302 >20 260 00002 0040 0369 0003 027 00147
Bifidobacterium Bin7 >297 >25 420 0009 0045 0579 0004 025 002
Bifidobacterium Hma3 >294 >20 220 00014 0040 0559 0004 026 002
Bifidobacterium Bma6 2082130 260 00005 000419 0003 001 00
Summation All 13 LAB >3451 >223 7930 0094 0427 2198 0 0695 3011 01594
*The table depicts organic acids (lactic-, acetic- and formic acids), hydrogen peroxide (H2O2) and volatiles (benzene, toluene, n-octane, ethylbenzene,
xylene and n-nonane). The depicted amounts refer to microgram per sample and ‘+‘ refers to a positive reaction.
Tris –tricine SDS–PAGE and mass spectrometry
To identify any proteins found in honey samples,
Mini-PROTEAN 10–20% Tris–Tricine precast gels (Bio-
Rad, Hercules, CA) were used as per original protocol (33).
Gel bands were prepared for mass spectrometry as outlined
in the study by Shevchenko and coworkers (34), with some
modications (14).
Peptide mass fingerprinting
The resulting mass spectra les obtained from the mass spec-
trometry analysis were searched using MASCOT against a
local database containing the predicted proteome of the 13
LAB (35). We used a cut-off ion score of 38 as a value
for determining that the protein was identied. Individual ion
scores greater than 38 indicated identity or extensive homology
(P<005) of the protein. Protein sequence similarity searches
were performed with software BLASTP in the software pack-
age BLAST 227+against a non-redundant protein database at
NCBI (36,37), Pfam (default database) (38) and InterProScan
(default databases) (39,40).
Results
Antagonism assays
The overlay assays (Table 1) show that all the tested pathogens
from clinical human wounds were inhibited by antimicrobial
compounds diffusing from each of the 13 LAB originating
from honeybees (A. mellifera) and when the 13 LAB were
grown together. The results show that the different LAB strains
produce different bioactive metabolites of varying inhibitory
effects against the pathogens. We could observe the same inhi-
bition results when L. kunkeei strains (Lahm1to13, Anhmro10
and Yubipro16) and combinations of LAB strains originating
in other bees (A. laboriosa,A. andreniformis and M. beechii)
were tested against the same pathogens. In all occasions, the
effect from the collaborating LAB was greater than from the
antibiotic discs.
Ta b l e 3 Free fatty acids, 3-OH Fas, (pmol/ml medium) in spent Pollen
medium of cultivated bacteria
Samples C 10:0 C 12:0 C 14:0 C 16:0
Blank (pollen)
Biut2 – – – –
Hon2 – – – –
Bma5 – – – –
Bma6 – – – –
Fhon2 345 369–1324
Fhon13 3079 2524 267519
Bin2 121 227– –
Bin4 – – – –
Bin7 – 150
Hma2 – – – –
Hma3 – – – –
Hma8 – – – –
Hma11 – – – –
Bioactive products from the LAB
Our results demonstrate that every single member of the
LAB microbiota of honeybees (A. mellifera) produces different
bioactive metabolites (Tables 2, 3 and 4). In general (Table 2),
organic acids were produced by all tested strains but in differ-
ent amounts. Lactic-, formic- and acetic acids were produced
by all 13 LAB. Five of the LAB strains, Hma11, Hma2, Bma5,
Hma8 and Biut2, produced hydrogen peroxide. Different toxic
volatiles were detected from every LAB. These included the fol-
lowing: benzene produced mainly by L. mellifer Bin4; toluene
by 11 of the LAB strains; octane mainly by the bidobacteria
Bin7, Bin2, Hma3 and Bma6; ethylbenzene mostly by L. apino-
rum Fhon13; xylene by 11 LAB; and nonane mostly by lacto-
bacilli Hon2 and Hma8 and Bidobacterium Bin7 and Hma3.
3-Hydroxy fatty acids
Free fatty acids (3-OH FAs) were identied from 4 of the 13
LAB strains studied; these were C 10:0, C 12:0, C 14:0 and C
16:0 (Table 3). Only results from Pollen media are shown as the
6© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.
T. C. Olofsson et al. Lactic acid bacteria a key in honey production
Ta b l e 4 Results showing 2-heptanone production by one of the 13 LAB
from honeybees (Fhon13)*
Samples ng/sample CFU
Quant1
MRS blank 117–
Fhon13 (1) 575130×107
Fhon13 (2) 6963
Fhon13 (3) 6117
Pollen (Cleaned blank) 98–
Fhon13 (1) 771330×107
Fhon13 (2) 7248
Fhon13 (3) 8758
Pollen blank 1406–
Fhon13 888280×108
Quant 2
Pollen blank 441–
Fhon13 (1) 926515×108
Fhon13 (2) 8636
kohmto18 (1) 47694×108
kohmto18 (2) 5010
kohmto18 (3) 4954
nuhmto23 (1) 565115×109
nuhmto23 (2) 5079
nuhmto23 (3) 5234
cehmto2 (1) 1172325×1010
cehmto2 (2) 13493
cehmto2 (3) 14182
*Studied L. apinorum Fhon13 strains originating in other bee species
were Lactobacillus kohmto18, Lactobacillus nuhmto23 and Lactobacillus
cehmto2 in triplicate.
results from bacteria incubated in MRS contained 3-OH FAs
in the blank. The relative amounts of the different 3-OH FAs
varied between the different strains and were most abundant
in L. apinorum Fhon13 and L. kunkeei Fhon2. Bidobacterium
Bin7 produced only C 16:0 and Bidobacterium Bin2 produced
two 3-OH FAs (C 10:0 and C 12:0), but in low amounts. In addi-
tion to the monitored 3-OH FAs compounds, both of the Fhon2
and Fhon13 strains contained a compound eluting just before
3-OH C 16:0. Its mass spectrum, as recorded by quadrupole
GC–MS, showed a peak at m/z341, strongly indicating that
the compound represents 3-OH C16:1 (data not shown).
2-Heptanone
A clear peak representing 2-heptanone (2-HE) was found in
the samples of L. apinorum Fhon13. Traces of 2-heptanone
were also found in tested L. melliventris Hma8 and L. kim-
bladii Hma2 (data not shown). Different strains of L. apinorum
Fhon13 originating in other honeybees were therefore tested
further, and results are displayed in Table 4. Pollen medium,
which was used for cultivation of the bacteria, was found to con-
tain traces of 2-heptanone, which may explain the occurrence
of the compound in small amounts in the analysed samples.
Fhon13 SIM method
SIM analyses were made of L. apinorum Fhon13 and of the
closely related strains isolated from Apis koschevnikovi (Lac-
tobacillus Kohmto 18), Apis nuluensis (Lactobacillus Nuhmto
Figure 1 Biofilm formation in vitro of the lactic acid bacterial (LAB)
strains derived from honeybees (Apis mellifera) varies between the
species. Ability to adhere and form biofilm is shown (measured by OD).
23) and Apis cerana (Lactobacillus Cehmto 2). The largest
amount of 2-heptanone per CFU was found in the samples of
L. apinorum Fhon13 cultivated in supplemented MRS medium.
The amounts found in L. apinorum Fhon13 and Kohmto18 cul-
tivated in pollen medium were similar but approximately 14
times smaller than those found in Fhon13 in MRS. Samples
of Lactobacillus Nuhmto23 and Cehmto2 strains contained the
smallest amount of 2-heptanone. Both media (supplemented
MRS and Pollen) contain traces of 2-heptanone (Table 4). The
pollen medium holds higher amounts of the analysed compound
that may be explained by the fact that the same LAB strains are
inoculated into the collected bee pollen in the production of bee
bread (honeybee larval food) (41).
Biofilm formation
Biolm formation was detected in all the 13 LAB strains
and it formed without induction or stress in vitro (Figure 1).
The biolm formation ability varied between the tested strains
and showed that all the four Bidobacterium strains (Bin2,
Bin7, Bma6 and Hma3) and L. kullabergensis Biut2 were the
ones that showed greatest ability to adhere and form biolm
in vitro independently of the growing medium used (data
not shown).
LAB metabolites found in honey
We could detect nine LAB-produced proteins in stored honeys
(Table 5). These originated in Lactobacillus Hma2, Hma8,
Hon2 and Bidobacterium Bin7, Hma3, Bma6. All the detected
proteins had different putative functions and sizes between
33 and 60 kDa. In addition, free fatty acids were detected in
varying amounts in freshly harvest honey and stored honeys
(Table 6). It appears that these substances do not disappear
with time, as they were detected in all sampled honeys. All
honeys studied here were found to contain small amounts of
most of the monitored 3-OH FAs, ranging between 01and
25 pmol/mg (Table 6). 3-OH C 10:0 and 3-OH C 12:0 were
the most common 3-OH FAs found in all stored honeys. Only
linden honey and freshly harvest honey contained 3-OH C
16:0. None of the tested honeys contained any C 14:0. Overall,
the concentrations were low (0.1– 1.3 pmol/mg). 3-OH FAs C
© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd. 7
Lactic acid bacteria a key in honey production T. C. Olofsson et al.
Ta b l e 5 Proteins produced by lactic acid bacterial (LAB) symbionts found in different stored honey types.
Honey type
Dilution factor
Identified from
Gene number
Size (kDa)
Ion score
No. of peptide matches
Putative function
Closest species ID
Accession no.
Query alignment (%)
Max ID (%)
E-value
Signal peptide
Rape seed oil 1:5 Bin7 RIAT00292 411 41 1 CRISPR family
associated
protein
Bifidobacterium
asteroides
YP_006865567.1 100 96 0 N
Linden 1:5 Bin7 RIAT00039 349 45 1 23S rRNA methyl-
transferase
Bifidobacterium
asteroides
YP_006865784.1 100 93 0 N
Linden 1:5 Hon2 RYBW01404 336 42 3 Ethanolamine
ammonia-lyase
light chain
Lactobacillus
rossiae
WP_017262946.1 99 78 600E 17 3 N
Raspberry 1:50 Hma3 RVKO00316 34 40 1 Unknown
function
Bifidobacterium
catenulatum
ZP_03324303.1 93 80 500E 143 N
Raspberry 1:5 Bma6 RLWY00667 467 45 2 NaqC family
transcriptional
repressor
Bifidobacterium
longum
YP_001955801.1 86 66 200E 174 N
Raspberry 1:5 Hma2 ROUL00302 50 44 2 Glucose 6
phosphate
isomerase
Lactobacillus
crispatus ST1
YP_003601227.1 98 87 000E +00 N
Manuka 1:5 Bin7 RIAT00292 414 53 1 CRISPR family
associated
protein
Bifidobacterium
asteroides
YP_006865567.1 99 96 000E+00 N
Heather 1:5 & 1:50 Hma8 RWLJ00689 426 41 1 Mannitol
phosphate
dehydrogenase
Enterococcus
raffinosus
WP_010747016.1 99 58 400E 15 4 N
Heather 1:50 Hon2 RYBW01023 599 40 1 Hypothetical
protein
Lactobacillus
versmoldensis
WP_010625130.1 68 29 43– 16 Y
Ta b l e 6 3-OH FAs (pmol/mg) in fresh honey and stored honeys
Sample C10C12C14C16
Fresh honey 0501–02
Two-week-old honey 0301– –
One-month rapeseed 07– – –
Rapeseed* 0401– –
Linden* 0903–09
Raspberry* 0602– –
Honey dew* 0702– –
Heather* 0502– –
Manuka* 0401– –
*Stored honeys.
18:0–C 22:0 were found in the sampled honeys, but as these
are not of LAB origin these data are not shown.
Discussion
Microorganisms are well recognised to produce bioactive
substances to defend themselves and their niche. LAB are
known producers of antimicrobial compounds; however, it is
known that the properties, qualities and substances produced
by LAB are species- and strain-dependent. Furthermore, all
LAB species neither exhibit the same antimicrobial quali-
ties nor produce the same antibacterial substances (9,10). In
this study, we demonstrate an overall inhibition of all the
human wound pathogens analysed (Table 1). The inhibitory
effect was greater than from the antibiotic discs regardless
of the antibiotic resistance among these pathogens. In some
cases, the zones of inhibition from antibiotic discs were
very vague or absent. We can hypothesise that the LAB in
this case are better than or just as effective as many of the
widely used antibiotics in wound treatment today. Combined,
the 13 LAB have another advantage over antibiotics as they
have a broad spectrum against a wide variety of pathogens
(Table 1), while as we know now, many antibiotics are active
only against certain bacteria, for example, metranidazole
and anaerobic bacteria. Antibiotics are now seldom used for
chronic wound treatment because of increase in antibiotic
resistance and their inability to penetrate the bacterial biolm
in the wound (42). It was evident that different LAB strains
produce metabolites variably active against these wound
pathogens as the inhibition zones from each member varied
(Table 1). When looking at their individual antimicrobial
effects, some are more potent than others against the tested
pathogens. L. mellifer Bin4 inhibits all encountered pathogens,
whereas L. kunkeei Fhon2 had the most potent activity
8© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.
T. C. Olofsson et al. Lactic acid bacteria a key in honey production
against the pathogens among all the LAB strains used in this
study (Table 1).
Traditionally, honey is gathered from wild honeybee colonies
by honey hunters when the wax combs contain a mixture of
both ripe honey and almost ripe honey with a total water
content between 22% and 30%. This method of harvesting fresh
honey is still used in large parts of the world and was the only
way for mankind to use honey before bee-keeping. We have
previously studied fresh honeys and the amount of viable LAB
microbiota in crops from all Apis species in the world and from
some stingless bee species. We found honey with the highest
concentration of viable LAB (108per gram honey) in Nepalese
honey of A. laboriosa and similar quantities in A. mellifera
honey from Africa (11). Eventually, the LAB die after a couple
of weeks in the harvested mature honey because of low water
content. The water content of honey in EU is not allowed to
exceed 20% with the exception of heather honey (22%). In
such honey, sold by bee-keepers and stores, harvested only
after the honey is totally ripe, with water content below 20%,
zero LAB are viable. In addition, it is a well-known narrative
in Europe that honey should not be heated as it will lose its
antimicrobial properties. It is possible that this old knowledge is
a remnant since from approximately 100 to 200 years ago when
people still were hunting honey from wild honeybee colonies
in Europe. Today honey is heated or sterilised before it can be
used in a medical setting, killing off microorganisms including
the LAB symbionts and destroying their bioactive products.
Honey collected from wild colonies of honeybees has possibly
reected a myriad of benecial effects of every specic LAB
member in the honey crop.
We now know, however, that the microbiota is also rather
consistent across Apis species (11,13). LAB diversity could be
explained by variation in nutritional content of different nectars
and pollen and also by the variation of microbes that they
encounter in, for example, owers. Transient oral microbes
trigger the growth of resident LAB microbiota in honeybees
and their production of putative antimicrobial proteins (14),
a mechanism known for LAB strains in other niches (e.g.
Lactobacillus reuteri) when producing reuterin (43,44).
In their natural environment, these LAB symbionts’ produc-
tion of active compounds is achieved when they are viable and
encounter microbial threats. The LAB symbionts are shaped to
defend their occupied niches, which are the honey stomach and
honeybee products (honey and beebread) (7,11,25,41). These
microbial threats are bacteria, yeast and moulds found in ow-
ers and surrounding environment. Microbial genera and fami-
lies that are commonly found are Pseudomonas,Enterobacte-
rioceae,Bacillus and Candida (11).Interestingly, strains from
these genera are also commonly isolated from chronic wounds
and can cause major problems in choosing the correct treatment
for the infection, as chronic wounds are usually polymicrobial
in nature (45,46) . Our hypothesis is that the LAB need to pro-
duce bioactive metabolites to defend themselves in their niches
and, therefore, their metabolites will be part of the ripe honey
as they ferment nectar and counteract microorganisms intro-
duced by foraging. We can also hypothesise that these LAB
would have the same characteristics in defending themselves in
a chronic wound environment when applied together with their
natural food, honey. In this study, we detected some of these
metabolites: 3-OH FAs in honey samples (Table 6) and extra-
cellular proteins (Table 5). However, the longevity of produced
compounds is to be associated to several factors such as storage
time, light exposure, physical conditions of honey harvesting
and so on. Another important consideration is that the LAB con-
centration varies within honeybees and their products (7,11).
If certain LAB members within honeybees are in high num-
bers, they will produce certain active compounds originating
in different LAB. When the entire microbiota work synergisti-
cally, a complex myriad of antimicrobial compounds is created,
which remains in the honey stomach and end up in honeybee
products. This could explain why that up to now the unknown
factors contributing the antimicrobial properties of honey vary
with honey type.
It has been shown that also honey from the stingless honey-
bees (Meliponinae) exhibit non-peroxide antibacterial activity
against S. aureus,Enterococcus faecalis,Escherichia coli and P.
aeruginosa (47). Yet nobody has been able to point out a con-
sensus scientic reason for those activities. Here, we showed
a clear antimicrobial activity that originates in bee’s symbi-
otic LAB microbiota including isolates from stingless bees (M.
beechii) (Table 1), which for the rst time gives a reason to
honey’s well-recognised antimicrobial effect and historical use
regardless of any specic nectar or ower. All bees possess
these LAB microbiota, but the amount present changes depend-
ing on nectar source, bee health and exposure to other microbes
(7,11). It appears to be a well-evolved defence mechanism of
the bee in order to secure their health and food.
Our results demonstrate a potential explanation for why
honey has been applied against threatening human and animal
pathogens. One of the most frequent uses of honey by humans
through history is wound management. A feasible explanation
is that the honey used in folk medicine has been freshly har-
vest honey which would contain a large amount of viable and
active LAB when applied onto wounds. Our recent results show
that the LAB produce a large quantity of putative antimicro-
bial proteins and peptides (14). The LAB sense the presence
of other threatening bacteria and start to produce substances to
defend themselves. The most common bacterial genus found in
owers is Pseudomonas, which is one of most therapy-resistant
pathogens in human chronic wounds. P. aeruginosa is a very
signicant chronic wound pathogen because of its biolm for-
mation, intrinsic multi-drug resistance and its proto cooperative
action with other microbes in the wound environment (48). The
ability of the LAB to inhibit or kill members of Pseudomonas
spp. is very pronounced for L. kunkeei Fhon2. Other commonly
found pathogens in wounds are from the family Enterobac-
teriaceae which, interestingly, also occur in nature therefore
would most likely be in contact with honeybees and the LAB
symbionts. This bacterial family contains a wide variety of sig-
nicant wound species that are showing increased antibiotic
resistance as well as being involved in biolm formation (49).
Our previous results show that L. kunkeei Fhon2 is the dom-
inant bacterial species found very frequently and in high num-
bers when sampling honeybee crops, honey, bee pollen and bee
bread, and it is always present but in varying numbers regard-
less of honeybee species, geographic location of the bees or
honey origin (7,11– 13,41). L. kunkeei type strain was primary
described as a wine spoiler because of its strong inhibition
© 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd. 9
Lactic acid bacteria a key in honey production T. C. Olofsson et al.
properties against Saccharomyces yeast involved in wine pro-
duction (50). This particular lactobacilli appears to be very
important for honeybees as it is the very rst LAB that estab-
lishes in the sterile honey stomach of an emerging bee callow
(11). L. kunkeei Fhon2 was also shown to be the most potent
LAB, potentially inhibiting both food spoiling microorganisms
following nectar and pollen to the hive when bees are foraging
and bee larval pathogens (11,25). As shown in this study, it is
also the most potent one against all human wound pathogens
tested. It produces a great variety of extracellular proteins on
microbial stress (14) and three different 3-OH FAs (C 10:0, C
12:0 and C 16:0). However, L. kunkeei Fhon2 did not produce
any bioactive volatiles (Table 2). At the moment, we do not
know the exact function of all the produced proteins (14); how-
ever, analyses of these protein’s known domains show a putative
antimicrobial action in many. It is well known that LAB produce
low-molecular-weight antifungal substances (51). Lactobacil-
lus plantarum MiLAB 14 have been reported to produce 3-OH
FAs with antifungal activity. Three of these 3-OH FAs were
C10, C12 and C14, the same 3-OH FAs found to be produced
by L. kunkeei Fhon2 and L. apinorum Fhon13 in this study. Sjö-
gren and coworkers (52) found that these fatty acids were more
active against yeast than moulds and suggested an antifungal
activity connected to the detergent-like properties of the com-
pounds that affect the cell membrane of target microorganisms.
The only pathogenic yeast tested in this study was Candida
albicans and strain Fhon2 showed an inhibition against this
wound pathogen. However, there was sporadic growth through-
out the inhibitory zone, suggesting different mechanisms of
antimicrobial action are used (Table 1). The inhibitory mech-
anism appears not to be simply linked to 3-OH FAs as the other
Lactobacilli (Fhon13) that also produced 3-OH FAs was not
active against C. albicans by itself (Table 1). Thus, the function
of bioactive compounds may need other compounds to work
synergistically is shown in the present study.
We were able to nd some proteins produced by LAB sym-
bionts in different stored honey types (Table 5). These results
show proteins of sizes above 30kDa as the lters we used had a
cut-off of 30 kDa. Larger proteins, these are more than 50 kDa,
were not detected either as larger proteins could not diffuse
through the electrophoretic gel that was used. Thus, our results
show only a small amount of extracellular LAB proteins pro-
duced, which is in agreement with our tested nectar source
hypothesis. The anti-inammatory action of honey has been
investigated by others (53,54). The mechanism by which honey
reduces inammation is not fully understood, but proteins may
be an explanation. We have found one protein with a putative
anti-inammatory function produced by one of the LAB strains
(14). However, this certain protein was neither found in any of
the honeys studied here, nor any of the other more than 143
putative LAB proteins previously detected by us (14). Conse-
quently, the fact that LAB-produced proteins vary in honeys
make an application of the viable LAB much more attractive in
future wound management to implement a standardised topical
application with a constant amount of proteins with different
functions.
Our overall results demonstrate that the LAB metabolites
in combination work synergistically (Table 1) and form a
myriad of bioactive substances. These bioactive substances
(Table 2) are the key for any future application of these LAB in
wounds and to elucidate their mechanism of action. Pathogens
in wounds are very sensitive for an acidication of the wound
environment. Production of organic acids decreases the pH and
will form a hostile environment for wound pathogens. Formic
acid is known to lower the pH of the wound environment, which
lactic acid could also do, and these are produced by all of the
LAB symbionts tested in this study. Acetic acid, which was pro-
duced by the 13 LAB strains, is known to inhibit the growth of P.
aeruginosa in wounds (55). In addition, the volatiles produced
were very interesting, as many of these compounds have known
effects in wound management (Table 2). Benzene is a toxic
volatile, but here produced mostly by L. mellifer Bin4, in minor
amounts. But still, its action may be enough to inuence the
wound environment. Benzene extracts from fruits have shown
an increased rate of wound closure and rate of epithelisation
(56). The use of nonane as a solvent may indicate an antimicro-
bial effect caused by obstruction of the bacterial membranes.
Hydrogen peroxide (H2O2) in small amounts is required for
an optimal wound healing (57). As demonstrated in this study,
ve of the tested Lactobacillus strains (Hma11, Hma2, Bma5,
Hma8 and Biut2) produced H2O2. 2-Heptanone is a known hon-
eybee pheromone that we here, for the rst time, show it is
produced by one of the honeybee LAB symbionts, L. apino-
rum Fhon13, and all tested closely related strains isolated from
other bees in the world (Table 4). It has recently been discov-
ered that 2-heptonone acts as a local anaesthetic that paralyse
Varr oa mites and wax moth larvae by the honeybeebite (58). In
a wound application, it may display the same function, which is
promising as chronic wounds cause long-term pain in patients.
Furthermore, all the 13 LAB symbionts showed the ability
to form biolms in vitro (Figure 1) and colonisation by the 13
LAB in wounds may be secured by biolm formation. We know
that the investigated LAB symbionts are highly osmotolerant,
stable and are viable much longer than other microorganisms,
including other LAB, in a honey solution consisting of no more
than 25% water content (26). The combination of osmotoler-
ance with their studied antimicrobial and therapeutic charac-
teristic and ability to form biolms make these LAB symbionts
very interesting for future wound applications. Therefore, these
LAB symbionts with its myriad of bioactive products may be
an optimal alternative in future wound management.
Present antibacterial dressings such as iodine or silver are
associated with environmental and patient-related hazards as
well as having a high cost for the patient and health sector.
An ecological, environmental-friendly wound dressing with
antimicrobial properties, as active honey dressing with viable
LAB, which is also non-toxic and promotes healing, will be
highly demanded in a near future.
Perspectives
The fact of nding new treatments in wound management is
already one of the most important tasks in today’s clinical and
biochemical research. Although a new wave of research con-
cerning honey has escalated during the last decades that may
in part be explained by the increasing antibiotic resistance, it
is assumed by many researchers that honey’s mode of action
is its osmolarity and release of hydrogen peroxide. However,
10 © 2014 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.
T. C. Olofsson et al. Lactic acid bacteria a key in honey production
we have recently discovered a unique LAB microbiota in the
honey-producing tract of the honeybee given, for the rst time,
an explanation to the before unknown factors contributing to
honey’s antimicrobial properties. In this study, we could con-
rm that LAB symbionts within honeybees are responsible for
many of the antibacterial and therapeutic properties of honey.
This is one of the most important steps forward in the under-
standing of the clinical effects of honey in wound manage-
ment. The explanation model will take honey in combination
with its viable and standardised amount of LAB into a much
wider clinical use. This has implications not least in develop-
ing countries, where fresh honey is easily available, but also
in western countries where antibiotic resistance is seriously
increasing.
Acknowledgements
This work was funded by grants from the Gyllenstierna Krappe-
rup’s Foundation, Dr P. Håkansson’s foundation, Ekhaga Foun-
dation and The Swedish Research Council Formas. We thank
Prof. Rose Cooper who welcomed TCO in her laboratory to
perform the antagonistic assays on wound pathogens.
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... Likewise, pure form of honey has proven the best against many serious pathogenic bacteria E. coli, Shigella sp., Salmonella sp., V. cholerae and other Gram -ive as well as Gram +ive microoorganisms. The observed high antibacterial potential of honey is the result of acidity, hydrogen peroxide, osmotic effect and phytochemical factors [11]. ...
... Furthermore, various authors reported LAB [16][17] and Bacillus sp. [16] residing in the HB gut producing bioactive substances with potential to be used as alternatives to antibiotics against human and animal infections [11]. ...
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... These bacteria produce different bioactive metabolites such as organic acids, lactic acids, formic acid and acetic acid, H 2 O 2 . Volatile compounds like benzene, toluene, n-octane, xylene, ethylbenzene, and n-nonane contribute to some antimicrobial actions (Table 2) against antibiotic-resistant bacteria (Olofsson et al. 2016). ...
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