Beehives as a Natural Source of Novel Antimicrobials

Abstract

Honey bee products have been used since ancient times as food and therapeutics. There is increasing knowledge on their content and molecular mechanism of action. Their bioactive compounds are a combination of both honey bee and plant origin. Plant immune response effectors are secondary metabolites (polyphenols, terpenes, antimicrobial peptides), and they are responsible for the antimicrobial effects of honey bee products like honey, propolis, and bee pollen. Honey bee innate immunity effectors are antimicrobial peptides, like defensin 1 and 2, apidaecins, abaecins, and hymenoptaecin, and some of them have been found in royal jelly, honey, and pollen. Plant secondary metabolites and honey bee antimicrobial peptides combine in beehive mixtures with synergistic antimicrobial activity and undoubtedly represent an interesting alternative to standard antibiotics. Further research should elucidate their interactions in honey bee products as well as their potential biotechnology applications.

Full text

373© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022
M. Rai, I. Kosalec (eds.), Promising Antimicrobials from Natural Products,
https://doi.org/10.1007/978-3-030-83504-0_15
Chapter 15
Beehives asaNatural Source ofNovel
Antimicrobials
JelenaSuran
Abstract Honey bee products have been used since ancient times as food and ther-
apeutics. There is increasing knowledge on their content and molecular mechanism
of action. Their bioactive compounds are a combination of both honey bee and plant
origin. Plant immune response effectors are secondary metabolites (polyphenols,
terpenes, antimicrobial peptides), and they are responsible for the antimicrobial
effects of honey bee products like honey, propolis, and bee pollen. Honey bee innate
immunity effectors are antimicrobial peptides, like defensin 1 and 2, apidaecins,
abaecins, and hymenoptaecin, and some of them have been found in royal jelly,
honey, and pollen. Plant secondary metabolites and honey bee antimicrobial pep-
tides combine in beehive mixtures with synergistic antimicrobial activity and
undoubtedly represent an interesting alternative to standard antibiotics. Further
research should elucidate their interactions in honey bee products as well as their
potential biotechnology applications.
Keywords Honey bees · Honey · Propolis · Royal jelly · Bee pollen · Plant
secondary metabolites · Immunity · Antimicrobial peptides
Abbreviations
10-acetooxy-2-DEA 10-Acetoxydecanoic acid
10-HDA 10-Hydroxy-2-decenoic acid
3,10-HDA 3,10-Dihydroxy-decanoic acids
AAMP Anionic antimicrobial peptides
AMPs Antimicrobial peptides
CAMP Cationic antimicrobial peptides
CAPE Caffeic acid phenethyl ester
J. Suran (*)
Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
e-mail: jelena.suran@vef.hr

375
in the past in different parts of the world to improve wound and gut healing (Zumla
and Lulat 1989). Even the Muslim prophet Mohammed and Aristotle (350BC)
recommended the use of honey for medical purposes (Molan 1999). In Ancient
Egypt, propolis was rst recognized as an adhesive for sealing cracks in wood,
while Aristotle was one of the rst to refer to it as a healing agent. In addition,
Aristotle was the rst to recognize how royal jelly promotes physical strength and
intellectual capacity (Fratini etal. 2016a).
Centuries later, with the advent of science, these products have been extensively
studied; their composition is analyzed with advanced instrumental methods, while
their biological activity is studied in different invitro and in vivo assays. As the
knowledge about their molecular mechanisms of action grows, we become more
aware of their complexities.
The beehive can be viewed as a melting pot of plant and insect defense mecha-
nisms (Fig.15.1). These defense mechanisms can be extracted in the form of bee-
hive products used as antimicrobial agents. These products are honey, propolis royal
jelly, bee pollen, beeswax, and bee venom. Each honey bee product is specic for
its content of active compounds, and many of them have a plethora of effects– from
antioxidant to antimicrobial.
The compounds vital for plant defense are plant secondary metabolites (SM),
abundant in honey bee products. Polyphenols are a huge and versatile group of SM,
and many of them can be used as representative markers of honey bee products like
propolis. Along with polyphenols, there are terpenoids and plant antimicrobial
peptides (AMP). The possible interactions among these compounds yet have to be
Fig. 15.1 The beehive as the melting-pot of honey bee and plant defense mechanisms
15 Beehives asaNatural Source ofNovel Antimicrobials

374
CNS Coagulase-negative staphylococci
CPPs Cell-penetrating peptides
CS α β Cysteine-stabilized α β motif
Cys Cysteine
Gly Glycine
HIV Human immunodeciency virus
HSV Herpes simplex virus
Imd Immune deciency pathway
Jak/STAT Janus kinase/signal transducer and activator of
transcription
JNK c-Jun N-terminal kinase
JV Junín virus
MAPK Mitogen-activated protein kinases
MIC Minimum inhibitory concentration
MRJP Major royal jelly protein
MRSA Methicillin-resistant Staphylococcus aureus
MSSA Methicillin-sensitive Staphylococcus aureus
NB-LR Nucleotide-binding leucine-rich repeat containing resis-
tance proteins
NF-κB Nuclear factor kappa B
P/MAMP Pathogen/microbe-associated molecular pattern molecules
PR Pathogenesis-related proteins
Pro Proline
PRRs Pattern recognition receptors
RJ Royal jelly
RLP/Ks Receptor-like proteins or -kinases
RNAi RNA interference
RSV Respiratory syncytial virus
SM Secondary metabolites
TMV Tobacco mosaic virus
VRE Vancomycin-resistant enterococci
VSV Vesicular stomatitis virus
VZV Varicella-zoster virus
1 Introduction
In recent decades, as antimicrobial resistance is being increasingly recognized as a
global public health threat, natural mixtures with antimicrobial effects such as products
from the honey bee Apis mellifera are being re-discovered by mainstream medicine.
Beehives have been used as a resource of food and medicines since ancient times.
The oldest evidence of humans collecting honey from wild bees dates back to
10,000years ago (Dams and Dams 1977). Beekeeping started in the early Neolithic
period (Roffet-Salque etal. 2016), while according to Crane (1999), domestication
of bees was depicted in Egyptian art from around 4500years ago. Honey was used
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376
elucidated. Honey bees’ defense is based on individual innate immunity and social,
collective immunity. Plant and animal material that honey bees integrate into honey
bee products is an essential part of the latter. Still, at the same time, these products
work through the former – by acting on the intracellular mechanisms vital for
individual innate immunity.
In this chapter, I present some of the most relevant antimicrobial compounds that
build the defense system of the beehive. These compounds are divided according to
their origin, with their role, and antimicrobial effects. Next, honey bee products are
described, followed by numerous studies of their antimicrobial efcacy.
Undoubtedly, beehives are rich resources of potent antimicrobial compounds, just
waiting to be utilized to ght against antimicrobial resistance.
2 Plant Origin ofAntimicrobial Substances intheBeehive
Using the beehive as a resource of antimicrobial compounds means considering the
immune strategies of insects like honey bees and the vast array of plant–host defense
mechanisms. These mechanisms work synergistically as plant, and insect-derived
material is combined in honeybee products. Here is where the bees, with all their
capabilities, concentrate the abundance of substances from plants and their own,
such as polyphenols (avonoids and phenolic acids), glycoproteins, and antimicrobial
peptides, in ghting and resisting various pathogens.
2.1 Plant Immune Response
Plants respond to infection using a two-branched or two-level innate immune sys-
tem (Jones and Dangl 2006) that needs to be versatile and effective, since plants
lack the mobility and a somatic adaptive immune system from animals. The rst
branch recognizes and responds to molecules common to many classes of microbes,
including non-pathogens through defense- receptor-like proteins or -kinases (RLP/
Ks) as pattern recognition receptors (PRRs), which can detect conserved pathogen/
microbe-associated molecular pattern (P/MAMP) molecules, considered to be an
early warning system for the presence of pathogens and the timely activation of
plant defense mechanisms (Jones and Dangl 2006; Dubery etal. 2012). A second
line of plant defense includes the response to pathogen virulence factors, either
directly or through their effects on host targets (Jones and Dangl 2006) via
intracellular nucleotide-binding leucine-rich repeat (NB-LR)-containing resistance
proteins, which recognize isolate-specic pathogen effectors once the cell wall has
been compromised (Dubery etal. 2012).
Proteins and peptides involved in these mechanisms can be found in plant mate-
rial collected by honey bees and integrated in honey and royal jelly products. One
of the most studied antimicrobial peptides, defensins, found in bees, honey, and
royal jelly could be partly of plant origin. Furthermore, plant polyphenols are highly
J. Suran

377
versatile secondary plant metabolites, allowing plants to respond promptly to unpre-
dictable stress agents of different origins (Wink 2008).
2.2 Plant Secondary Metabolites (SMs)
General resistance in plants is achieved by the production of secondary metabolites
(SMs), a highly diverse group of organic molecules which are not necessary for the
actual metabolism or physiology of the plants producing them. These compounds
serve as protective agents against various pathogens: bacteria, fungi, viruses, and
insects (Wink 2008). There are several different classes of SMs: phenolic compounds
(avonoids, tannins), terpenoids, N-containing compounds (non-protein amino
acids, cyanogenic glucosides alkaloids), and S-containing compounds (pathogenesis-
related (PR) proteins, phytoalexins) (Wink 2008; Jamwal etal. 2018). In nature,
these metabolites always come in complex mixtures.
Polyphenols
One of the most abundant groups of SMs in honey bee products is polyphenols.
Polyphenols can be divided into several classes: avonols, avones, avanones,
anthocyanidins, avanols, and isoavones (Daglia 2012). Polyphenols were studied
mostly because of their antioxidant effect as the basis for chronic disease prevention,
but with the increase of antimicrobial resistance, their antimicrobial potential came
into focus as well.
In general, avonoids have shown stronger antimicrobial activity than non- a-
vonoid compounds. Flavan-3-ols, avonols, and tannins were extensively studied
due to their wide spectrum and higher antimicrobial activity compared to other
polyphenols. Most of them can suppress many microbial virulence factors (such as
inhibition of biolm formation, reduction of host ligands adhesion, and neutralization
of bacterial toxins) and show synergism with antibiotics (Daglia 2012). Although
weaker than avonoids, non-avonoids such as phenolic acids (caffeic and ferulic
acids) showed activity against Gram-positive (Staphylococcus aureus, Listeria
monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas
aeruginosa) (Daglia 2012).
There are several mechanisms of polyphenol antimicrobial activity (Olchowik-
Grabarek etal. 2020): the damage of the cell membrane and cell wall (Funatogawa
etal. 2004; Yi etal. 2010; Adnan etal. 2017), inhibition of energy metabolism (Li
etal. 2017), production, secretion, structure, and activity of released toxins (Hisano
etal. 2003; Shah etal. 2008; Lee etal. 2012; Dong etal. 2013; Verhelst etal. 2013;
Wang etal. 2015; Song etal. 2016; Shimamura etal. 2015; Chang etal. 2019; Tang
etal. 2019) and biolm formation (Lin etal. 2011; Trentin etal. 2013). Polyphenols
also act at the level of target cells, increasing their resistance to toxins (Olchowik-
Grabarek etal. 2020). Regarding polyphenol interaction with cell structures, it was
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378
hypothesized that the polyphenols rich in gallate moieties might attach to the cell
surface, serve as bridges between surfaces of two neighbor cells, and initiate cell-
binding and formation of similar clusters in the membrane of the opposite cell
(Tarahovsky 2008). Phan etal. (2014) conrmed that an increase in the number of
hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the
reactivity of the polyphenols with cell membranes. Due to their polarity, they are not
able to pass the cell membrane through passive diffusion, so it is assumed that they
pass through the membranes with the help of other plant SMs (Wink 2008).
The interactions of polyphenols with proteins and peptides are interesting, not
only for a better understanding of their action on cell surfaces and signal transduction
pathways but to understand how these molecules will interact with each other in a
natural mixture like those found in the beehive. Peptides and polyphenols form
noncovalent (hydrogen, hydrophobic, and ionic bonds) and covalent bonds (between
oxidized phenolic compounds and peptides) (Sun and Udenigwe 2020).
While forming ionic bonds, negatively charged phenolate ions interact with posi-
tively charged amino acids. Depending on their size, a single polyphenol can bind
even several proteins simultaneously (Wink 2008). Bourvellec and Renard (2012)
describe how, at the same time when hydrophobic bonds form between polyphenol
aromatic rings and hydrophobic residues of amino acids, hydrogen bonds are also
formed between the hydroxyl groups of polyphenols and the acceptor site for hydro-
gen ions in the proteins (Bourvellec and Renard 2012). The primary factors affect-
ing the protein–polyphenol interaction are conformation and type of both proteins
and polyphenols. Other factors are environmental conditions, like temperature and
pH (Quan etal. 2019). It is assumed that the phenolic binding can affect protein
activity or even protect proteins from proteolytic cleavage (Wink 2008). When poly-
phenols oxidize to reactive quinones, they form covalent bonds with proteins in
honey, and this complexation can lead to decreased antioxidant, enzymatic, or anti-
microbial activity (Brudzynski and Maldonado-Alvarez 2015).
On the other hand, there is growing evidence that the formation of protein/pep-
tide conjugates results in increased antioxidant activity and stability in food (Quan
etal. 2019). Possibly, the same logic could be applied to their antimicrobial activity,
and we assume that polyphenols could increase the stability and the activity of anti-
microbial peptides.
Volatile SMs (Terpenoids)
The volatile SMs necessary for plant defense are complex mixtures of hydrocarbons
and oxygenated hydrocarbons from the isoprenoid pathways, primarily monoterpenes
and sesquiterpenes (Bankova et al. 2014). They are produced and secreted by
glandular trichomes; specialized secretory tissues diffused onto the surface of plant
organs, particularly owers and leaves (Bankova etal. 2014).
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379
Plant Antimicrobial Peptides (AMPs)
Plants produce PR proteins/peptides with numerous defense-related properties,
including antibacterial, antifungal, antiviral, antioxidative activity, chitinase, and
proteinase inhibitory activities (Tam etal. 2015). Antimicrobial peptides (AMPs)
interact with cell membrane phospholipids and cell-penetrating peptides (CPPs),
which introduce certain cargoes in the cell (Nawrot etal. 2014).
AMPs have been isolated from all parts of plants and can be divided into anionic
(AAMPs) and cationic (CAMPs) peptides. These groups have shown activity
against pathogenic microorganisms (bacteria, viruses, and fungi) and even neoplastic
cells (Montesinos 2007; Nawrot etal. 2014). Antimicrobial peptides (AMP) found
in plants are rich in Cys, enabling disulde bonds. This contributes to their stability
and resistance to enzymatic degradation. (Tam etal. 2015). According to Nawrot
etal. (2014), there are six groups of plant AMPs: thionins, defensins, lipid transfer
proteins, cyclotides, hevein-like proteins, and knottin-type proteins.
AMPs mechanism of antimicrobial action has been described through several
types of models of membrane pore formation, which leads to cell content leakage
and death. AMPs act on the microorganism cell membrane due to their negative
charge, which attracts cationic peptides. In the bacterial membrane, negatively
charged molecules, and thus main receptors of CAMPs are phospholipids. While in
fungal membranes, these are glucosylceramides and sphingolipids. In addition,
many CAMPs appear to target internal anionic cell constituents, such as DNA,
RNA, or cell wall components (Diamond etal. 2009). AMPs exhibit broad-spectrum
activity, and thus far, it appears as though bacteria do not develop resistance as
quickly as with conventional antibiotics (Diamond etal. 2009).
While the mechanisms of CAMPs are better understood, those of AAMPs are
less so. There is evidence suggesting they increase plasma membrane permeability
by binding to lipids, disrupting the envelope integrity by attaching to chitin, and
damaging intracellular structures, such as DNA.It is also proposed that AAMPs
participate in the plant innate immune response and act synergistically with CAMPs
(Prabhu et al. 2013). Prabhu et al. (2013) conclude that cyclotides are the plant
AAMPs with the greatest potential for therapeutic and biotechnical development.
Cyclotides are named after the cyclic peptide backbone and a knotted arrangement
of three conserved disulde bonds. Due to those bonds, they are relatively stable to
thermal, chemical, and enzymatic degradation and can be modied by residue
substitutions (Prabhu etal. 2013). One of the best-studied cyclotides, kalata B2, was
found to have potent antibacterial activity against Salmonella enterica, E. coli, and
S. aureus (Gran etal. 2008; Pranting et al. 2010), but also against parasites like
gastrointestinal nematodes Haemonchus contortus and Trichostrongylus
colubriformis (Colgrave etal. 2008). Other known antimicrobial cyclotides with
antibacterial activity are vaby D (Pranting et al. 2010) and cycloviolacin O24
(Ireland etal. 2006) and cycloviolacins Y1, Y4, and Y5 which exhibit anthelmintic
properties (Colgrave etal. 2008) and antiviral activity (Wang etal. 2008).
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380
The two most prominent plant CAMP families are thionins and defensins. There
are several common traits of these two CAMP families between various species
(microbes, plants, animals), and those include their amphipathic nature, positive
charge, and molecular structure. These peptides are membrane-active, while other
families of AMPs have a different mechanism of action– from enzyme inhibition to
lipid transfer. Thionins are AMPs with a small molecular weight (~5kDa) rich in
arginine, lysine, and cysteine residues (Nawrot etal. 2014). There are two groups of
thionins, α−/β- and γ-thionins (based on their structure, γ-thionins are considered to
be a part of the defensin family of peptides). They are toxic against phytopathogenic
bacteria, fungi (Ebrahimnesbat etal. 1989), and yeasts, and also some animal and
plant cells (Evans etal. 1989). They interact with the protein receptors or lipids in
membranes (Osorio e Castro and Vernon 1989; Florack and Stiekema 1994; Garcia-
Olmedo etal. 1998; Stec 2006) with their hydrophobic residues and positive surface
charge to cause cell leakage and lysis (Majewski and Stec 2001; Tam etal. 2015).
Thionins isolated from black seed (Nigella sativa) showed bactericidal and fungicidal
effects on Bacillus subtilis, S. aureus, and Candida albicans (Vasilchenko etal. 2017).
Defensins are well-known and abundant AMP in plants, vertebrates, and inverte-
brate animals (Nawrot etal. 2014; Tam etal. 2015) and fungi (Wu etal. 2014). They
are also of small molecular weight (~5kDa), cysteine rich and cationic peptides
with broad-spectrum antimicrobial activity; antibacterial, antifungal, antiviral, pro-
teinase, and insect amylase inhibitor (Nawrot etal. 2014). Their previously described
mechanisms of antimicrobial activity are based on membrane lysis. Still, there are
other processes by which they disrupt, such as interfering with cell signaling, intra-
cellular trafcking, blocking the receptor binding, and cell entry (Weber 2020).
Plant defensins are ancient and conserved; therefore, they are similar to honey bees
and vertebrate animals (Nawrot etal. 2014). They also act as immunomodulators by
attracting immune cells and modulating adaptive immune responses (Weber 2020).
Despite having only identied and isolated AMPs from honey bees and their
products, one cannot exclude the possibility that some of these peptides are of plant
origin since there is a certain amount of plant material in the beehive. One cannot
also exclude the possible relevance of these peptides, such as in the case of
polyphenols and other secondary plant metabolites that have been identied in
honey, pollen, or propolis.
3 Honey Bee Defense Mechanisms
Honey bees are social insects with a collective “social immunity” and an individual
innate immunity, which consists of humoral and cellular effectors (Evans etal. 2006).
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3.1 Honey Bee Individual Immunity
Cells involved in individual honey bee immune response are phagocytes and hemo-
cytes and humoral-induced effectors such as AMPs, thioester linkage proteins, mel-
anization, and coagulation proteins (Larsen et al. 2019). Antiviral intracellular
defense mechanisms include RNA interference (RNAi), endocytosis, melanization,
encapsulation, autophagy, and conserved immune pathways including Jak/STAT
(Janus kinase/signal transducer and activator of transcription), JNK (c-Jun
N-terminal kinase), MAPK (mitogen-activated protein kinases), and the NF-κB
mediated Toll and Imd (immune deciency) pathways (McMenamin etal. 2018).
Interestingly, RNAi is the key resistance mechanism against viruses, not only for
individual honey bees but also for the whole beehive’s immune response (Maori
etal. 2019). Similarly, Toll, Imd, Janus kinase (JAK)/STAT, and JNK are signaling
pathways induced by bacterial cell wall lipopolysaccharides or peptidoglycans
(Boutros 2002; Evans etal. 2006) and result in the release of antimicrobial effectors,
peptides, such as hymenoptaecin, defensin 1, and abaecin at the end of the cascade
(Evans etal. 2006; Gätschenberger etal. 2013). As in plants, AMPs are considered
the key component of honey bee innate immunity (Danihlík etal. 2015).
3.2 Honey Bee AMPs
Both honey bee products and antimicrobial peptides (AMPs) have been recognized
as resources of promising alternatives to conventional antibiotics. AMPs have been
described as ancient evolutionary weapons produced by many living organisms as a
part of their nonspecic immune response. Thus, they are effective against many
microorganisms (Baltzer and Brown 2011). AMPs exhibit a multimodal mechanism
of action, specically responding to various intracellular targets and binding to
lipopolysaccharides of the bacterial membrane with different, concentration-
dependent afnity (Baltzer and Brown 2011; Hughes etal. 2000; Li etal. 2012).
As plant AMPs, insect AMPs form pores on the cell membrane of bacteria in
different ways (Li etal. 2012). They can also bind to different intracellular targets
(DNA, RNA, and proteins) once inside the cell and inhibit their synthesis (Lan etal.
2010; Li etal. 2012). Moreover, insect AMPs can interfere with bacterial cytokinesis
by cell lamentation, using unique translocation mechanisms to alter the cytoplasmic
membrane septum formation (Brown and Hancock 2006; Lan et al. 2010; Li
etal. 2012).
Not only do they have broad-spectrum activity against microorganisms, but
AMPs are also able to bypass the common resistance mechanisms that render
conventional antibiotics ineffective (Wang et al. 2016). Apart from antimicrobial
activity, AMPs also modulate the immune system via cytokine activity or
angiogenesis (Li etal. 2012). Potential novel therapeutics such as AMPs could be
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382
implemented using natural mixtures that may have antimicrobial and immunomod-
ulatory activity due to their complexity and molecular synergism.
Based on their structure, insect AMPs can be divided into four categories: α-helix
(cecropin and moricin), Cys-rich (insect defensin and drosomycin), Pro-rich
(apidaecin, drosocin, and lebocin), and Gly-rich peptides (attacin) (Bulet and
Stöcklin 2005; Yi etal. 2014). Honey bees pathogens induce four families of AMPs;
apidaecins, abaecins, hymenoptaecins, and defensins. These families have a broad
spectrum of antimicrobial activity in the hemolymph (Xu etal. 2009). Besides the
active AMPS in adult honey bee lymph, inactive peptide precursors can be found in
bee larvae (Casteels etal. 1989). Apidaecins were found to be very selective and
active against human and animal Gram-negative bacteria (E. coli, Salmonella, and
Shigella species) (Casteels et al. 1989), while abaecins are more active against
Gram-positive bacteria (Casteels etal. 1990). To be more specic, in comparison to
abaecins, apidaecins showed 200-fold more activity against Agrobacterium,
Erwinia, and E. coli strains (Casteels et al. 1990). In the same study, abaecins
showed the highest specic activity against plant pathogen Xanthomonas campestris.
This was expected since honey bees are often exposed to plant-associated
microorganisms whilst gathering food, pollen, and nectar. Hymenoptaecin is active
against Gram-negative and Gram-positive bacteria, including several human
pathogens (Casteels etal. 1993). Its bactericidal effect against E. coli results from
sequential permeabilization of the outer and inner membranes (Casteels etal. 1993).
When combined in immune lymph, hymenoptaecin, and apidaecin, as the two
predominant factors, had a strong bactericidal effect against a broad spectrum of
Gram-negative (Bordetella bronchiseptica, Enterobacter cloacae, Haemophilus
inuenzae, Yersinia enterocolitica, etc.) and some Gram-positive bacteria. Defensins
killed Gram-positive bacteria (e.g., Clostridium and Streptococcus species) that
were unaffected by their combination. As Casteels etal. (1993) concluded, “it is
clear that the broad-spectrum antibacterial activity of immune lymph is the result of
an amazing complementarity.”
As in plants, defensins are the most abundant group of AMPs in insects. In gen-
eral, insect defensins have an N-terminal loop and an α-helical fragment followed
by an antiparallel β-structure, connected by two of the three disulde bridges. These
form so-called cysteine-stabilized α β (CS α β) motif (Cornet etal. 1995). Defensins
have antibacterial activity against Gram-positive bacteria, including S. aureus,
Micrococcus luteus, and Aerococcus viridans (Yi etal. 2014; Li etal. 2017). Two
types of defensins have been identied in honey bees. Defensin 1 is synthesized in
salivary glands and plays an important role in social immunity, while defensin 2 is
synthesized by cells of body fat and lymph, which is an important factor in the sys-
tem of the honey bee individual immunity (Ilyasov etal. 2013).
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383
3.3 Honey Bee Social Immunity
Honey bees use social immunity as a collective defense against pathogens
(DeGrandi-Hoffman and Chen 2015). This type of response is based on behavioral
cooperation (Evans and Spivak 2010) during small tasks that have a colony-wide
impact on reducing pathogenic invasion, for example, necrophoric and hygienic
behavior (removing the dead adults or diseased brood from the colony), or
thermoregulatory activity (workers produce high temperature) against heat-sensitive
pathogens (DeGrandi-Hoffman and Chen 2015). The previously mentioned
transmissible RNA pathway through the royal jelly and worker jelly also has an
important role in social immunity and signaling between hive members. It protects
bees against viruses and the Varroa mite (Maori etal. 2019).
Nutrition is a key factor in honey bees’ social and individual immunity (DeGrandi-
Hoffman and Chen 2015). Honey bees use plants as their food but also as a form of
their external, collective immunity. Bee pollen is a primary source of food for the
beehive, entirely of plant origin. Honey is produced partly from the sugary secretions
of plants (oral nectar). The most effective honey bee product with
immunomodulatory, antimicrobial, antioxidative activity is propolis. Propolis is a
resin derived from plants combined with animal origin substances – honey bee
saliva and beeswax– rich in polyphenols from plants (Bankova etal., 2021). These
polyphenols are used as markers of the biological activity of propolis (Fig.15.2).
As previously mentioned, to protect themselves against consumption by herbi-
vores and pathogens, plants use complex mixtures of numerous secondary com-
pounds (SM) (Wink 2008). The action of these compounds in mixtures can be
synergistic or antagonistic. Mechanisms of activity are pleiotropic and interact with
many targets at the same time. As such, these compounds have many advantages
Fig. 15.2 The typical HPLC-UV chromatogram of propolis extracts obtained in our laboratory.
Ten biomarkers are used for analysis: (1) caffeic acid, (2) p-coumaric acid, (3) ferulic acid, (4)
trans-cinnamic acid, (5) kaempferol, (6) apigenin, (7) chrysin, (8) pinocembrin, (9) CAPE, (10)
galangin
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384
over mono-target compounds (Wink 2008). Some common mechanisms include
modulation of the structure and function of proteins, interference with gene
expression, and changing membrane permeability. Most of these SMs have been
found in the beehive in honey bee products.
4 Honey Bee Products asBeehive Defense Resources
There are six main products from the beehive with antimicrobial effect described in
the scientic literature: honey, propolis, royal jelly, pollen, beeswax, and bee venom.
Of these, honey and propolis antimicrobial activities have been studied the most and
have the greatest potential in treating systemic or local infectious diseases.
4.1 Honey
The rst product from the beehive used for its antimicrobial properties (besides the
nutritional) in folk medicine was honey. Honey is the end product of nectar digestion
and is stored in honeycomb cells. In terms of content, honey is made up of a
supersaturated aqueous solution. This solution is comprised of 80% sugars, mostly
fructose, and glucose.
It is known that natural unheated honey has some broad-spectrum antibacterial
activity when tested against methicillin-resistant S. aureus (MRSA), Pseudomonas
aeruginosa, Klebsiella pneumoniae, vancomycin-resistant enterococci (VRE),
extended-spectrum β-lactamase-producing (ESBL) Proteus mirabilis, and E. coli.
There are numerous studies on the antimicrobial activity of different types of honey.
In one study, the MICs of Tualang honey ranged 8.75%–25% compared with those
of manuka honey (8.75%–20%) against the wound and enteric microorganisms:
S. pyogenes, CNS, MRSA, Streptococcus agalactiae, S. aureus, Stenotrophomonas
maltophilia, Acinetobacter baumannii, S. enterica Serovar typhi, P. aeruginosa,
P. mirabilis, Shigella exneri, E. coli, and E. cloacae (Tan etal. 2009). In time-kill
studies, antibiotic susceptible and resistant isolates of S. aureus, S. epidermidis,
Enterococcus faecium, E. coli, P. aeruginosa, E. cloacae, and Klebsiella oxytoca
were killed within 24h by 10–40% (v/v) honey (Mandal and Mandal 2011). Several
types of honey were tested against planktonic and biolm-grown bacteria and
showed 100% bactericidal efcacy against planktonic forms. The bactericidal rates
for the Sidr and two types of Manuka honey against MSSA, MRSA, and P. aeruginosa
biolms were 63–82%, 73–63%, and 91–91%, respectively (Alandejani etal. 2009).
Different types of honey also displayed specic antiviral effects. Manuka and
clover honey showed activity against varicella-zoster virus (VZV) in concentrations
ranging from 0% to 6% wt/vol (Shahzad and Cohrs 2012). In addition, a randomized
controlled trial on the efcacy of honey compared to acyclovir showed comparable
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385
success rates of topical application of medical-grade kanuka honey and 5% aciclo-
vir in the treatment of herpes labialis (Semprini etal. 2019).
These antimicrobial effects are attributed to a wide array of compounds found in
honey, such as oligosaccharides (Cornara etal. 2017), glucose oxidase, and non-
peroxide factors with antibacterial activity, like methyl syringate, methylglyoxal
(MGO), peptides from honey bees (defensin-1) (Cornara etal. 2017), and honey
glycoproteins (glps). Honey glycoproteins showed sequence identity with the major
royal jelly proteins 1 (MRJP1) precursor (Brudzynski and Sjaarda 2015), and also
the concentration-dependent antibacterial activity against Gram-positive Bacillus
subtilis and Gram-negative E. coli. These glycoproteins bind and agglutinate
bacterial cells and also cause membrane permeabilization (Brudzynski and Sjaarda
2015). Glucose oxidase is added by bees, which, by low dilution, converts glucose
into H2O2 and gluconic acid.
Active compounds of plant origin that are found in honey differ based on the
botanical origin of their néctar. Some types of honey are being marketed as specic
regarding their antimicrobial effects and so-called unique factors. What they all
have in common is supersaturation (high osmolarity, osmotic effect), low water
activity, and low pH. These factors cultivate an unfavorable environment for
microbial growth (Tan etal. 2009).
Microbiota from honey is also believed to be responsible for its antibacterial
activity. Fourteen bacterial isolates of Bacillus sp. showed antimicrobial activity
against C. albicans, E. coli, and S. aureus has been found in honey (Jia etal. 2020).
4.2 Propolis
Honey bees primarily use propolis as a construction material but also to maintain
beehive health. Propolis is also used as an important part of social immunity due to
its natural antiseptic properties (Bankova etal., 2018; Bankova etal., 2021). It is a
resinous mixture of both animal and plant origin—bees collect it from exudates and
plant buds, where it is further mixed with wax and saliva enzymes (Bankova etal.,
2021). Its chemical composition varies depending on the geographical and botanical
origin: the most common type of propolis in Europe is poplar-type, from Populus
nigra. The most prevalent types of Brazilian propolis are green due to plant
Baccharis dracunculifolia and red, from plant Dalbergia ecastophyllum. Brown
Cuban propolis, the principal type of Cuban propolis, is derived from Clusia rosea.
Each type of propolis contains about 300 bioactive compounds (Sforcin and
Bankova 2011; Pellati etal. 2013); triterpenes (50% w/w), waxes (25–30%), volatile
mono- and sesquiterpenes (8–12%) and phenolics (5–10%) (Huang etal. 2014).
Most active compounds are of plant origin and are believed to be responsible for
the antimicrobial, antioxidant, immunomodulatory, and anti-inammatory activities
of propolis (Sforcin and Bankova 2011). The antimicrobial activity of propolis was
conrmed when tested against bacteria, viruses, yeasts, and even parasites. Propolis
extracts are highly active against Gram-positive (MRSA, VRE, Streptococcus
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386
species, B. subtilis, S. aureus, Enterococcus faecalis) and less active against Gram-
negative bacteria like E. coli. However, they have bactericidal activity on
P. aeruginosa (Kosalec etal. 2005, Przybyłek and Karpin´ski 2019). Propolis is also
active against yeasts like Candida species (Kosalec etal. 2005) and many viruses
invitro and invivo (Berretta etal. 2020; Nolkemper etal. 2010; Schnitzler etal.
2010). The mechanism of action depends on inhibition of the virus’ entry into cells
and disruption of viral replication, which destroys RNA before or after its release in
the cells (Búfalo etal. 2009; Sforcin 2016). Propolis components have inhibitory
effects on the ACE2, TMPRSS2, and PAK1 signaling pathways and can potentially
interfere with the host cell invasion by SARS-CoV-2 (Berretta etal. 2020).
It is presumed that the antimicrobial activity depends on the presence of avo-
noids such as galangin, pinocembrin, rutin, quercetin, naringenin, and CAPE, since
these compounds are known to increase bacterial membrane permeability. Some of
those compounds (galangin, pinocembrin, CAPE) also inhibit bacterial RNA poly-
merase (Cornara etal. 2017). It is, therefore, clear that the antimicrobial activity of
propolis is a result of the mixture effect and synergy between the avonoid com-
pounds and that the resultant antimicrobial actions are understood so far as complex
mechanisms. Due to this complexity, propolis is active against multidrug- resistant
bacteria (Pamplona-Zomenhan etal. 2011; Przybyłek and Karpin´ski 2019).
We conrmed this synergy when we compared the MIC values of propolis
extracts with different amounts of active markers (p-coumaric acid, trans-ferulic
acid, caffeic acid, CAPE, cinnamic acid, chrysin, pinocembrin, galangin, apigenin,
kaempferol) (Fig.15.3).
Fig. 15.3 Antimicrobial susceptibility testing: minimal biolm eradication concentration (MBEC)
determination for different (separate) propolis biomarkers (a), and propolis extracts minimum
inhibitory concentrations (MICs) determination by subcultivation on agar plates (b), agar well
diffusion (c), and broth microdilution (d) method. (With courtesy of Dr. Josipa Vlainić)
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387
An interesting and completely unexpected result is that the mixture of these
active substances in small concentrations is more effective than that of much higher
concentrations of certain (pure) active substances alone (work in progress)
(Fig.15.3). It seems that the synergy effect between these compounds follows the
Goldilocks principle.
There are certainly other compounds relevant to the investigation of propolis-
mediated antimicrobial activity. These may not just be of plant, but honey bee ori-
gin, such as antimicrobial peptides found in other honey bee products. Based on the
previously posited interaction pathways between peptides and polyphenols (Wink
2008; Quan etal. 2019), peptides in propolis could exert great stability and possibly
enhanced therapeutic potential.
Surprisingly, the idea of propolis as a natural source of stabile AMPs has never
been tested before. Our preliminary and currently ongoing research conrmed
peptides like MRJP1 and some peptides related to Populus genus in raw propolis
samples. There remains a wealth of other detected peptides yet to be sequenced.
4.3 Royal Jelly asaResource ofAntimicrobials
Royal jelly (RJ) is a food for all bee larvae for the rst 3 days of their life. For the
queen bee, RJ serves as the source of all subsequent nutrition throughout her
lifespan. RJ is a white-yellow, colloidal, slightly acidic secretion produced from the
hypopharyngeal and mandibular salivary glands of young bees (nurse, aged between
5 and 14days) (Fujita etal. 2013; Fratini etal. 2016a). It consists of 60–70% water,
11%–23% carbohydrates, 9–18% proteins, 4–8% lipids, and the remaining 0.8–3%
are vitamins, minerals, and even phenolic compounds, presumably from plants
(Sabatini et al. 2009; Fratini et al. 2016a). The composition varies based on the
season and nutrition of the bees.
Bioactive peptides and proteins identied in royal jelly are the families of major
royal jelly proteins (MRJPs), royalisin, glycoproteins jelleins, apolipophorin III-
like protein, glucose oxidase (Fratini et al. 2016a), defensin, apidaecins and
hymenoptaecin (Han et al. 2014). Interesting components of royal jelly with
antibacterial activity are unsaturated fatty acids, such as 10-hydroxy-2-decenoic
(10-HDA), also known as queen-bee acid (Fratini etal. 2016a).
MRJPs have a signicant role in honey bee nutrition since they account for
82–90% of total larval jelly proteins and contain essential amino acids. There are
seven members of the MRJP family (MRJP 1–7) that have health-promoting effects
and two members without these healthful advantages (Ahmad etal. 2020). MRJP1
occurs as a monomer (mono MRJP1 or royalactin), or can also appear as an oligomer
known as apisin, when polymerized with apisimin (Ahmad etal. 2020). MRJP1 has
been shown to modulate biological function in a broad range of species and can
maintain pluripotency by activating a ground-state pluripotency-like gene network
(Wan et al., 2018). However, it seems that MRJP1 does not display specic
antimicrobial properties (Bucekova and Majtan 2016).
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Nevertheless, jelleins, peptides isolated from MRJP1, showed a broad spectrum
of activity against Gram-positive (B. subtilis, S. aureus, Paenibacillus larvae),
Gram-negative bacteria (E. coli, P. aeruginosa), and against C. albicans. The MICs
of synthetic jelleins varied between 2.5μg/ml against E. coli and 15μg/ml against
S. saprophyticus (Brudzynski and Sjaarda 2015). Jellein I and Jellein II were active
against S. aureus, Staphylococcus saprophyticus, and B. subtilis among the Gram-
positive bacteria, and E. coli, Enterobacter cloacae, K. pneumoniae, and
P. aeruginosa among the Gram-negative bacteria (Romanelli etal. 2011). Jellein III
showed a narrower spectrum of general activity (Romanelli etal. 2011) but was the
strongest in reacting against S. epidermidis (Cappareli etal. 2012).
MRJP2 and MRJP4 act as antimicrobial agents and have a wide range of activity
against bacteria (Gram-positive and Gram-negative), fungi, and yeasts (Ahmad
etal. 2020). They kill microorganisms by attaching to, and damaging, the cell wall
of fungi, yeast, and bacteria (Kim etal. 2019; Park etal. 2019).
Royalisin is strongly active against Gram-positive bacteria strains of
Bidobacterium, Clostridium, Corynebacterium, Lactobacillus, Leuconostoc,
Staphylococcus, and Streptococcus genera, with inhibitory efcacy comparable to
that of antibiotics (Fratini et al. 2016a). Apolipophorin-III-like proteins (lipid
transport proteins) and phosphorylated icarapin (venom protein-II) are the
components of royal jelly that promote immune response (Ahmad etal. 2020).
The antifungal properties of royal jelly are not limited only to their peptide prop-
erties but can also be attributed to fatty acids, such as 3,10-HDA, 10-HDA, and
10-acetooxy-2-DEA, that inhibit the growth of Candida tropicalis, C. albicans, and
Candida glabrata (Meliou and Chinou 2005).
Antiviral effects of royal jelly are not attributed to certain peptides but to the
product as a whole. Honey, royal jelly, and acyclovir have the highest inhibitory
effects on HSV-1 at concentrations of 500, 250, and 100 μg/mL, respectively
(Hashemipour etal. 2014).
4.4 Honey Bee Pollen
Honey bee pollen is used as a raw material to produce bee bread. Bee bread is the
main protein source for the bee colony and the source of nutritional and mineral
substances for royal jelly produced by worker bees (Komosinska– Vassev et al.
2015). Pollen is also important for the production and expression of antimicrobial
peptides—apidaecins and abaecin—in honey bees, not just due to its microbiota,
but possibly to certain immunomodulatory protein factors that yet have to be
determined (Danihlík etal. 2018).
Honey bee pollen composition varies depending on the botanical and geographi-
cal origin of the pollen grains. Generally, pollen consists of proteins, amino acids,
carbohydrates, lipids, fatty acids, phenolic compounds, enzymes, and coenzymes,
and vitamins and elements. There are approximately 200 substances from different
plant species found in pollen grains (Komosinska– Vassev etal. 2015). It is believed
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389
that plant SMs like avonoids and phenolic acids are responsible for pollen
antioxidant and antimicrobial activity (Bridi et al. 2019). These effects are also
possibly mediated by glucose oxidase activity, deriving from honey bee secretion
(Cornara etal. 2017).
Bee pollen extract showed antibacterial activity against Gram-positive bacteria
like Streptococcus pyogenes (Bridi etal. 2019), S. aureus, Gram-negative bacteria,
including E. coli, K. pneumoniae, Pseudomonas aeruginosa, and on fungi such as
C. albicans (Komosinska– Vassev etal. 2015).
Bee pollen is a component of honey and propolis and, as such, adds to their anti-
microbial efcacy. When compared by their pollen content, heterooral honey sam-
ples from Turkey, with pollen dominantly from Chenopodiaceae/Amaranthaceae,
Trifolium, Trigonella, Cyperaceae, Zea mays, and Anthemis taxa, had the highest
antibacterial activity against P. aeruginosa, E. coli, and S. aureus (Mercan etal.
2007). However, in our MIC study on Gram-positive and Gram- negative bacteria,
we found no bactericidal or bacteriostatic activity of Cistus pollen extracts.
4.5 Beeswax
Honey bees secrete beeswax in order to build honeycombs. Beeswax is a complex
mixture (more than 300 components) of hydrocarbons, free fatty acids, esters of
fatty acids and a fatty alcohol, diesters, and exogenous substances (Tulloch, 1980),
which are mainly residues of propolis, pollen, small pieces of oral component
factors, and pollution (Hepburn etal. 1991).
Several studies report antimicrobial activity of crude beeswax against S. aureus,
Staphylococcus epidermidis, Streptococcus pyogenes, B. subtilis, P. aeruginosa,
E. coli, S. enterica, C. albicans, and Aspergillus niger (Fratini et al. 2016b).
Similarly, beeswax methanolic and ethanolic extracts showed inhibitory activity on
L. monocytogenes, S. enterica, E. coli, A. niger, C. tropicalis, C. glabrata, and
C. albicans (Fratini etal. 2016b).
Beeswax also has good antimicrobial activity in synergy with other natural prod-
ucts, like propolis, honey, or olive oil (Fratini etal. 2016b).
4.6 Bee Venom (Apitoxin)
Honey bee venom glands secrete the venom and inject it through a stinger. Bee
venom is rich in amphipathic polycationic peptides, melittin and apamin, enzymes
such as phospholipase A2, and low-molecular-weight compounds including active
bioamines such as histamine and catecholamines (Cornara et al. 2017). This
complex mixture causes local inammation, anticoagulant effect, and immune
response in victims (Cornara etal. 2017).
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Melittin, a peptide of 26 amino acid residues, has been recognized as a peptide
with an antiviral effect. It has inhibited the viral replication of Herpes simplex virus
(HSV), human immunodeciency virus-1 (HIV-1), and Junín virus (JV), and it also
has shown to reduce the infectivity of Coxsackie virus and other enteroviruses
(Picornaviridae), Inuenza A viruses (Orthomyxoviridae), respiratory syncytial
virus (RSV; Pneumoviridae), vesicular stomatitis virus (VSV; Rhabdoviridae), and
the plant virus tobacco mosaic virus (TMV; Virgaviridae) (Memariani etal. 2020).
Melittin also showed effective antibacterial activity against Streptococcus salivarius,
Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus
sanguinis, Lactobacillus casei, and E. faecalis with MIC values ranging from 4 to
40μg/mL (Leandro etal. 2015). Although melittin has many therapeutic potentials,
the systematic administration is followed by many side effects, and its
biotechnological applications are limited to topical formulations (Moreno and
Giralt 2015).
5 Conclusion
Honey bee products result from combining the honey bee and plant-origin com-
pounds in the beehive, and as such, have been used as food and therapeutics since
ancient times. They are abundant in sugars, secondary plant metabolites, and honey
bee proteins and peptides with antimicrobial activity. With the help of powerful
modern technologies stemming from molecular biology, proteomics, and chemistry,
the evidence and mechanisms of their antimicrobial activity are being elucidated
increasingly. However, one must bear in mind the effect of the mixture and synergy
between the components in natural products.
Acknowledgments Our preliminary results mentioned in this chapter were obtained as part of the
project named Beehive as a natural resource for antibiotic alternatives nanced by private Sweden
foundation Ekhagastiftelsen, founded by Gösta Videgårds. I would like to thank them for all the
support in our research work on antibiotic alternatives.
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