**2. Antimicrobial activity of bee products**

#### **2.1. Honey**

colonization with *S. aureus* is observed in approximately 20% of human population, about 30% carry these microorganisms transiently and 50% are non-carriers [3–5]. *S. aureus* similarly as several other species of the genus *Staphylococcus* are classified as commensal Grampositive bacteria. Like other commensal microorganisms, staphylococci have the ability to cause disease under certain conditions. It should be noted, however, that *S. aureus* is definitely one of the most dangerous commensal bacteria which colonize any part of the human body. Its high pathogenicity is based on production of a wide array of virulence factors such as protein A, coagulase, collagenase, hyaluronidase, hemolysins, lipases, multiple toxins, adhesive proteins and also proteins involved in biofilm formation. The ability to express these virulence factors has been confirmed not only for clinical isolates of *S. aureus*, but also for strains isolated from animal sources, e.g. bovine mastitis [6, 7] and food [8, 9]). All these isolates are potentially dangerous human pathogens. *S. aureus* has also developed several mechanisms that enable them escape from protective immune responses of infected humans or animals. Among them, protein A (SpA), staphylokinase and staphylococcal binder of immunoglobulin are the most important and the best characterized [10]. Another crucial characteristic for this group of bacteria is that they can easily acquire mechanisms of antibiotic resistance for a plethora of antibiotics currently in use for human and animal therapies. Especially important problem is rapidly growing number of isolation of strains resistant to methicillin, designated as *Methicillin Resistant Staphylococcus aureus* (MRSA). MRSA isolates are resistant to all β-lactam antibiotics. Moreover, some of them are classified as multidrug resistant (MDR)—not susceptible to the antibiotics that belong to various chemical groups. The MDR phenotype is usually associated with strains recovered from medical environment—*Healthcare Associated* MRSA (HA-MRSA). The drugs of choice for treatment of MRSA infections are glycopeptides, mainly vancomycin. Unfortunately, staphylococci developed also resistance to this group of antibiotics, and prevalence of isolation of vancomycin-intermediate *S. aureus* (VISA) and vancomycin-resistant *S. aureus* (VRSA) strains is constantly growing. The number of other antibiotics, which could be used for eradication of HA-MRSA infections, is very limited. In fact, only three antibiotics recently introduced to routine medicine procedures are effective in treatment of HA-MRSA infections: from the oxazolidinone group—linezolid; streptogramin group—quinupristin/dalfopristin, and from the glycylcycline group—tigecycline [11–13]. HA-MRSA are also usually susceptible to rifampicin, fusidic acid and co-trimoxazole; however, the last one does not give results good enough in treatment of acute infections, and in the case of two other drugs, resistance is easily acquired by treated bacteria [12–14]. The problem regarding therapy of staphylococci-related infections is additionally enhanced by the staphylococcal ability to form biofilm. Some of the previous studies proved that the biofilm-forming bacteria (including *S. aureus*) may be in fact 10–1000 times more resistant to antimicrobial agents than the same cells growing in a

Staphylococci also belong to the most important animal pathogens. Infections caused by these bacteria leads to huge economic losses in agriculture and food industry. Contamination of food products by these bacteria is a serious issue due to their completely lack of susceptibility to lysozyme and very low susceptibility to nisin, two important agents used as preservatives

planktonic manner [15, 16].

204 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

in food industry [17, 18].

#### *2.1.1. Basic information and mechanisms of antimicrobial activity*

From ancient times, honey was a very valued component of the diet. This is mainly due to its characteristic sweet taste. The main uses of honey are as follows: as a spread on bread, a sweetener for tea, milk and coffee, for preparing desserts and cakes. Recently, it has been also proposed to use honey as a component of healthy, high energetic, beverages. The sweet taste of honey is apparent from its chemical composition. Sugars, mainly fructose and glucose, and minor amounts of oligosaccharides account for about 80% of its weight. The centuries of observations have also shown that regular consumption of honey is beneficial to the health of consumer. Therefore, this product was widely used as drug in traditional medicine, one of the most important except of herbs. The research carried out during several last decades finally confirmed that honey (and also other bee products) is a beneficial agent in treatment of a wide spectrum of human diseases, such as some cardiovascular and gastrointestinal tract disorders, infections within upper respiratory tract including cough, as well as in healing of infected wounds [19]. While folk medicine was based only on tradition and experience, the achievements of modern science and medicine have found confirmation of the therapeutic potential of honey, as the consequence of its chemical composition. It has been revealed that crucial for therapeutic properties of honey are its non-sugar components such as: enzymes, peptides, free amino acids, vitamins, organic acids, flavonoids, phenolic acids and other phytochemicals, minerals [20]. Especially interesting subject seems to be its antimicrobial activity. As it was mentioned above, this product was successfully used in treatment of skin and soft tissue infections and for elimination of pathogens infecting mucosal of respiratory tract. In all these situations, physical contact of honey (its components) with infected tissue (microorganisms infecting the tissue) is possible. The antimicrobial action of honey is based on several mechanisms: the acidity (low pH, usually in the range from 3.4 to 6.1), osmotic pressure of sugars present in honey and the presence of bacteriostatic and bactericidal substances such as H2 O2 , antioxidants, lysozyme, polyphenols, phenolic acids, flavonoids, methylglyoxal and bee peptides [21–24]. The crucial component that is responsible for the antimicrobial activity of majority of the honey types is hydrogen peroxide, which is formed as a side product of the oxidation of glucose by glucose oxidase—the enzyme which is introduced to the honey from the salivary glands of bees. Interestingly glucose oxidase is inactive in non-diluted honey [25]. The inhibition of growth of bacteria in honey, at the original concentration of this product, is mainly caused by high concentration of sugars (high osmotic pressure) coupled with high acidity [21, 22]. Therefore, it can be stored for long period of time (at least 2 years) without any additional treatment or supplementation with preservatives. When honey is diluted to certain extent, its antibacterial activity is shifted from osmotic- and pH-dependent to peroxide-dependent mechanism of action based on the generation of H2 O2 [26]. This scenario takes place when honey is used for treatment of infections, e.g. infected wound. On the basis of current state of knowledge, it can be said that other mentioned above components of honey, mainly phytochemicals, seem to only support the antibacterial effect of generated H<sup>2</sup> O2 . The dominant role of the enzyme in the antimicrobial activity of several polish unifloral honeys was recently confirmed in the researches that were carried out in our group. Preincubation of honeys solutions in 80°C for only 10 min resulted in complete loss of antibacterial activity of all tested honeys. The same effect was observed when suspensions of tested bacterial cells in solutions of honeys were supplemented with catalase. In both cases the observed, complete losses of activity of honeys were the consequence of lack of possibilities for H2 O2 generation. Heat treatment resulted in denaturation of the enzyme, and in the presence of catalase, the generated hydrogen peroxide was immediately decomposed [27]. On the other hand, antimicrobial activity of honey also depends on the botanical source which was used by bees to collect the nectar. Buckwheat, thyme and cornflower honeys usually exhibit high antimicrobial activity, whilst produced in Poland in large amounts rape honey do not affect the growth of neither Gram-positive nor Gram-negative bacteria [27, 28]. Thus, the types of phytochemicals as well their concentration is important for final antimicrobial potential of honey. The issue of role of phytochemicals in the antimicrobial potential of honey is still not clear and is a subject of many interesting research. Hydrogen peroxide is also a known cytostatic agent; however, its concentration in honey is on a very low level which is safe for humans and animals. According to Lusby et al. [29], the concentration of H2 O2 in honey is thousand times lower than in the common 3% antiseptic solution available in pharmacies; however, its constant production causes prolonged activity which can be considered as an advantage. The concentration of hydrogen peroxide on a non-toxic and stable level is probably regulated by antioxidants and pollen-derived catalase which destroy excess amounts of H2 O2 [30].

#### *2.1.2. Honey as a potential antistaphylococcal agent*

of the most important except of herbs. The research carried out during several last decades finally confirmed that honey (and also other bee products) is a beneficial agent in treatment of a wide spectrum of human diseases, such as some cardiovascular and gastrointestinal tract disorders, infections within upper respiratory tract including cough, as well as in healing of infected wounds [19]. While folk medicine was based only on tradition and experience, the achievements of modern science and medicine have found confirmation of the therapeutic potential of honey, as the consequence of its chemical composition. It has been revealed that crucial for therapeutic properties of honey are its non-sugar components such as: enzymes, peptides, free amino acids, vitamins, organic acids, flavonoids, phenolic acids and other phytochemicals, minerals [20]. Especially interesting subject seems to be its antimicrobial activity. As it was mentioned above, this product was successfully used in treatment of skin and soft tissue infections and for elimination of pathogens infecting mucosal of respiratory tract. In all these situations, physical contact of honey (its components) with infected tissue (microorganisms infecting the tissue) is possible. The antimicrobial action of honey is based on several mechanisms: the acidity (low pH, usually in the range from 3.4 to 6.1), osmotic pressure of sugars present in honey and the presence of bacteriostatic and bactericidal substances such

, antioxidants, lysozyme, polyphenols, phenolic acids, flavonoids, methylglyoxal and

O2

[26]. This scenario takes

O2 . The

O2

bee peptides [21–24]. The crucial component that is responsible for the antimicrobial activity of majority of the honey types is hydrogen peroxide, which is formed as a side product of the oxidation of glucose by glucose oxidase—the enzyme which is introduced to the honey from the salivary glands of bees. Interestingly glucose oxidase is inactive in non-diluted honey [25]. The inhibition of growth of bacteria in honey, at the original concentration of this product, is mainly caused by high concentration of sugars (high osmotic pressure) coupled with high acidity [21, 22]. Therefore, it can be stored for long period of time (at least 2 years) without any additional treatment or supplementation with preservatives. When honey is diluted to certain extent, its antibacterial activity is shifted from osmotic- and pH-dependent to perox-

place when honey is used for treatment of infections, e.g. infected wound. On the basis of current state of knowledge, it can be said that other mentioned above components of honey,

dominant role of the enzyme in the antimicrobial activity of several polish unifloral honeys was recently confirmed in the researches that were carried out in our group. Preincubation of honeys solutions in 80°C for only 10 min resulted in complete loss of antibacterial activity of all tested honeys. The same effect was observed when suspensions of tested bacterial cells in solutions of honeys were supplemented with catalase. In both cases the observed, complete losses of activity of honeys were the consequence of lack of possibilities for H2

generation. Heat treatment resulted in denaturation of the enzyme, and in the presence of catalase, the generated hydrogen peroxide was immediately decomposed [27]. On the other hand, antimicrobial activity of honey also depends on the botanical source which was used by bees to collect the nectar. Buckwheat, thyme and cornflower honeys usually exhibit high antimicrobial activity, whilst produced in Poland in large amounts rape honey do not affect the growth of neither Gram-positive nor Gram-negative bacteria [27, 28]. Thus, the types of phytochemicals as well their concentration is important for final antimicrobial potential of honey. The issue of role of phytochemicals in the antimicrobial potential of honey is still not clear

mainly phytochemicals, seem to only support the antibacterial effect of generated H<sup>2</sup>

ide-dependent mechanism of action based on the generation of H2

as H2 O2

206 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

The results of many investigations, carried out in different geographical regions of the world, revealed especially high efficiency of honey in treatment of infections caused by Grampositive microorganisms, e.g. staphylococci. The mentioned above investigation carried out in our group revealed that honeys obtained from some species of plants, namely cornflower (*Centaurea cyanus* L.), buckwheat (*Fagopyrum esculentum* Moench) and thyme (*Thymus vulgaris* L.) were able to inhibit the growth of reference strain *S. aureus* PCM 2051 at the concentration of 3.12 or 6.25% (v/v). Satisfactory activity (minimal inhibitory concentration—MIC ≥6.25%) was observed for honeys obtained from linden tree (*Tilia* spp.), heather (*Calluna vulgaris* L.), savory (*Satureja hortensis* L.) and coriander (*Coriandrum sativum* L.). Other tested bacteria *Staphylococcus epidermidis* PCM 2118 and especially *Pseudomonas aeruginosa* ATCC 27853 and *Escherichia coli* K12 were less susceptible [27]. Quite similar results were obtained in the research carried out by the other polish group that investigates antimicrobial potential of bee products. Buckwheat, linden tree and heather honeys were found very active. Moreover, the authors also revealed high activity of goldenrod honey (*Solidago* spp.)—that was not observed in our research and honeydew honey—not investigated in our research. The latter is a specific kind of honey produced by bees, which collect the honeydew, the sweet secretions of aphids or other plant sap-sucking insects [28]. The observed in our studies activity against *S. aureus* was similar to the activity exerted by Slovenian chestnut, fir honeydew and forest honey (MIC = 2.5%, v/v) reported by Kuncic and coworkers [31] as well as Chilean Ulmo tree honey (MIC = 3.1%, v/v) reported by group of Sherlock [32]. The MIC values (against *S. aureus*) in the range of concentrations from 3.12 to 12.5% (v/v) were also observed by Anthimidou and Mossialos, who investigated a collection of 31 Greek and Cypriot honeys, they also revealed that Gram-negative bacteria *P. aeruginosa* revealed a bit higher resistance with MICs values in the range from 6.25 to 25% (v/v) [33]. Lallam and coworkers investigated antibacterial potential of 32 samples of honey (14 monofloral and 18 multifloral) collected from the Algerian Sahara Desert against four bacteria; *Bacillus subtilis, Clostridium perfringens, Escherichia coli* and *S. aureus*. The research confirmed high antimicrobial activity of honeys collected from this region; however, only disc diffusion method was used by the authors. All floral origins of honey showed antimicrobial activity against *S. aureus* but with rather similar reactions (9–10.5 mm), except with *P. persica*-based honey, whose activity was only 6 mm [34]. High anti-staphylococcal potential of honeys was also confirmed for clinical isolates of these bacteria, including MRSA strains. Effective inhibition of growth of MRSA isolates has been revealed in the case of mentioned above Chilean honey obtained from Ulmo tree [32], Malaysian melaleuca honey [35], some Thai honeys, especially from longan flower [36], Finland [37] and also many other geographical regions. Moreover, some authors revealed high activity of honey in eradication MRSA infections using *in vivo* models [38]. Considering honey as a therapeutic, antimicrobial agent the honey produced from the manuka bush (*Leptospermum scoparium*) indigenous to New Zealand and Australia deserves special attention. In contrast to majority of other nectar and honeydew honeys, the crucial factor responsible for the bactericidal activity of manuka honey is high concentration of 1, 2-dicarbonyl compound—methylglyoxal (MGO) in this product. High acidity and sugar concentration as well as hydrogen peroxide generation play in this case supporting roles [39, 40]. As a consequence, antimicrobial activity of this honey is not affected by heat treatment, catalase or proteolytic enzymes (hydrolysing glucose oxidase) [37, 40]. Several investigations *in vitro* confirmed high bactericidal, including antistaphylococcal activity of this honey [e.g. 27, 33]. Jenkins and Cooper revealed synergistic action between manuka honey and some antibiotics against MRSA and *P. aeruginosa* strains isolated from wounds [41]. Moreover, manuka honey has been successfully used for treatment of chronic wound infections caused by MRSA [42].

#### *2.1.3. Perspectives and limitations of treatment staphylococcal infections with honey*

From the point of view of possibilities of exploiting of the therapeutic potential of honey especially promising, and most realistic, seem to be application of this product as a component of wound dressing materials. Using honey to eliminate pathogens from infected wounds has a long tradition, which can be counted in hundred or even thousands of years. Currently, several companies specialize in production of dressings containing honey. However, many technical problems still have to be solved: (1) elimination of indigenous flora of honey, especially spores, without thermal or chemical treatment—results in deactivation of glucose oxidase and loss of antimicrobial activity, (2) inhibition of natural process of crystallization of honey, (3) large diversity of antimicrobial activity of honey—only the honeys with certified antibacterial activities should be used for medical applications. These problems could be partly solved by using manuka honey. However, research of many investigators revealed that many "classical honeys"—which activity is based mainly on the generation of hydrogen peroxide, exhibit even higher activity in comparison to manuka honey with a high content of methylglyoxal—550 mg/L [27, 28, 33]. Thus, they are also good candidates to be used in clinical practice as a component of dressings, ointments and creams, or direct application on the surface of infected skin, sores, diabetic foot, or mucous, e.g. in the oral cavity or genital tract.

#### **2.2. Propolis**

#### *2.2.1. Basic information*

The bees collect four products: nectar and honeydew for production of honey (source of carbohydrates), pollen (source of proteins) and propolis. Propolis is not a component of bees' diet; however, it is absolutely necessary for the proper development of bee colonies. This product, which is also called bee glue, is a natural resinous substance produced from plants' buds and exudates, modified by addition of bees' salivary secretions and wax. Similarly, as in the case of honey, propolis is a product of complex chemical composition. Some of its ingredients mainly polyphenols and flavonoids exhibit high antimicrobial activity. As a consequence, it is used as a hive disinfectant. Bees use propolis for elimination of pathogenic microorganisms from the walls of hive and cells of honeycomb, in which larvae develop (nest wells) and honey is stored [43]. It is important for prevention of development of such dangerous pathogens as *Paenibacillus larvae*, the bacteria responsible for American foulbrood, one of the most important diseases of bees [44]. Some authors also suggest that components of propolis, at least partially, affect the growth of *Varroa destructor*—most damaging parasite affecting honeybee colonies [45]. Because of its antimicrobial activity, propolis is also used by bees for mummification of larger pests such as mice invading the hive, which were killed as a result of the sting. Mummification prevents the decomposition of the body of the pest—development of pathogenic flora as well as generation of unpleasant odour. Due to its physical and chemical properties (high viscosity, low water solubility), propolis is also used as an important building material for sealing the hive or natural habitats of these insects (protection against wind or rain, stabilization of construction), which is also important for safety of bees' colonies.

#### *2.2.2. Possibilities of application of propolis in therapy of bacterial infections*

many other geographical regions. Moreover, some authors revealed high activity of honey in eradication MRSA infections using *in vivo* models [38]. Considering honey as a therapeutic, antimicrobial agent the honey produced from the manuka bush (*Leptospermum scoparium*) indigenous to New Zealand and Australia deserves special attention. In contrast to majority of other nectar and honeydew honeys, the crucial factor responsible for the bactericidal activity of manuka honey is high concentration of 1, 2-dicarbonyl compound—methylglyoxal (MGO) in this product. High acidity and sugar concentration as well as hydrogen peroxide generation play in this case supporting roles [39, 40]. As a consequence, antimicrobial activity of this honey is not affected by heat treatment, catalase or proteolytic enzymes (hydrolysing glucose oxidase) [37, 40]. Several investigations *in vitro* confirmed high bactericidal, including antistaphylococcal activity of this honey [e.g. 27, 33]. Jenkins and Cooper revealed synergistic action between manuka honey and some antibiotics against MRSA and *P. aeruginosa* strains isolated from wounds [41]. Moreover, manuka honey has been successfully used for treat-

ment of chronic wound infections caused by MRSA [42].

**2.2. Propolis**

*2.2.1. Basic information*

208 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

*2.1.3. Perspectives and limitations of treatment staphylococcal infections with honey*

infected skin, sores, diabetic foot, or mucous, e.g. in the oral cavity or genital tract.

The bees collect four products: nectar and honeydew for production of honey (source of carbohydrates), pollen (source of proteins) and propolis. Propolis is not a component of bees' diet; however, it is absolutely necessary for the proper development of bee colonies. This product, which is also called bee glue, is a natural resinous substance produced from plants' buds and exudates, modified by addition of bees' salivary secretions and wax. Similarly, as in the case of honey, propolis is a product of complex chemical composition. Some of its ingredients mainly polyphenols and flavonoids exhibit high antimicrobial activity. As a consequence, it

From the point of view of possibilities of exploiting of the therapeutic potential of honey especially promising, and most realistic, seem to be application of this product as a component of wound dressing materials. Using honey to eliminate pathogens from infected wounds has a long tradition, which can be counted in hundred or even thousands of years. Currently, several companies specialize in production of dressings containing honey. However, many technical problems still have to be solved: (1) elimination of indigenous flora of honey, especially spores, without thermal or chemical treatment—results in deactivation of glucose oxidase and loss of antimicrobial activity, (2) inhibition of natural process of crystallization of honey, (3) large diversity of antimicrobial activity of honey—only the honeys with certified antibacterial activities should be used for medical applications. These problems could be partly solved by using manuka honey. However, research of many investigators revealed that many "classical honeys"—which activity is based mainly on the generation of hydrogen peroxide, exhibit even higher activity in comparison to manuka honey with a high content of methylglyoxal—550 mg/L [27, 28, 33]. Thus, they are also good candidates to be used in clinical practice as a component of dressings, ointments and creams, or direct application on the surface of Similarly to honey, propolis was widely used in traditional medicine. The detailed history of using propolis in medicine and a discussion of perspectives of its future application have been recently presented by Silva-Carvalho and coworkers [46]. The chemical composition of this product depends on many factors: the geographical region where it was collected (the species of plants which were available for bees), season, weather conditions and many other. Some significant correlations were found primarily in the case of chemical composition of propolis and place of its isolation. On this basis, several different types of propolis have been proposed, e.g. poplar propolis, birch, green, red, "Pacific" and "Canarian". This classification still evaluates and new, different types of propolis are being recognized, e.g. Mediterranean or Portuguese [46]. At least, 13 different types of propolis have been identified in Brazil [47]. Because of differences in chemical composition the biological, including antibacterial, activity of different propolis samples may vary significantly. However, the carried out to date studies revealed that staphylococci and other Gram-positive bacteria are usually highly sensitive to this product collected in many different geographical locations. Propolis remains especially popular in the non-conventional medicine in Brazil as well as in other tropical countries, thus the product obtained in this region is well-characterized. The global market size of propolis was about 2300 tons in 2015 (it is established that it will increase to 2900 tons in 2021), and Brazil is the largest production and exporting country of this product (https://www.whatech.com/market-research/materials-chemicals/125806 world-propolis-industry-trends-share-size-2021-forecast-report). High anti-staphylococcal activity of propolis sourced from State of Paraná, in Brazil, was observed by Pamplona-Zomenhan and coworkers, the MIC50 and MIC90 for the 210 strains (162 MSSA and 48 MRSA) were both 1420 μg/mL [48]. The results of investigations performed by the groups of Fidoralisi [49] and Santana [50] indicated that propolis extracts might be effective against mastitis-causing *S. aureus*. The group of Fidoralisi observed reduction in *S. aureus* growth on average, 1.5 and 4 log10 times at concentration of propolis 200 and 500 μg/mL, respectively. At concentrations of 1000 μg/mL, all tested propolis samples reduced bacterial growth to zero [49]. The same effect was observed by Santana and coworkers, but only in BHI (Brain Hearth Infusion) medium. The authors noticed that in milk the bactericidal dose was at least 20-fold greater [50]. Interesting results were also published by Suleman and colleagues [51]. Some of 39 South Africa propolis samples exhibited much higher antistaphylococcal activity in comparison to three tested samples of Brazilian propolis with MIC and MBC values of only 6 μg/mL [51]. Al-Waili and coworkers revealed that ethanolic extracts of propolis collected from Saudi Arabia (EEPS) and from Egypt (EEPE) effectively inhibited the growth of antibiotic resistant *E. coli, S. aureus* and *C. albicans* in single and polymicrobial cultures [52]. Strong antioxidant and antibacterial activity of propolis sourced from three different areas of Sonoran Desert in northwestern Mexico were confirmed in the research of Velazquez group [53]. The MIC against *S. aureus* of the most active sample (coming from Ures) was 100 μg/mL [53]. An antimicrobial effect of propolis harvested from honeybees in subtropical eastern Australia was investigated by Massaro and coworkers. The two tested propolis crude, ethanolic extracts showed bactericidal effects against *S. aureus* ATCC 25923 reference strain at the concentrations of 0.37–2.04 mg/mL [54]. Propolis produced in many Asian and European apiaries is also effective in elimination of staphylococci. The number of publications presenting results of biological properties of propolis sourced from this region has evidentially increased during the last decade. Because of limited size of this chapter only selected investigations can be described herein. Some promising results regarding antistaphylococcal activity of ethanolic extract of Polish propolis (EEPP) against methicillin-sensitive *S. aureus* (MSSA) and methicillin-resistant *S. aureus* (MRSA) clinical isolates have been recently revealed by Wojtyczka and coworkers. The investigated EEPP displayed varying effectiveness against twelve *S. aureus* strains, with MIC in the range from 0.39 to 0.78 mg/mL, determined by broth microdilution method, and minimal bactericidal concentration (MBC) of the EEPP ranged from 0.78 to 3.13 mg/mL. The disk diffusion assay revealed also that EEPP-enhanced antistaphylococcal activity of eight classical antimicrobial antibiotics, namely: cefoxitin, clindamycin, tetracycline, tobramycin, linezolid, trimethoprim+sulfamethoxazole, penicillin and erythromycin [55]. The same authors also investigated EEPP activity against biofilm-forming coagulase-negative *Staphylococcus* strains. The biofilm formation ability of all tested *S. epidermidis* strains was inhibited at EEPP concentrations ranging from 0.39 to 1.56 mg/mL [56].

Due to its health-promoting properties, propolis is widely used as a component of cosmetics, some food and beverages. In our opinion, these applications are not adequate to the biological properties of this product. The results of many presented above studies clearly indicate that propolis is a promising antimicrobial agent. Moreover, many other biological activities of this product have been described, including antioxidant, antiviral, antitumor, antifungal and immunomodulatory properties [46]. Large diversity of its chemical composition and consequently its biological activity eliminate propolis from clinical applications. Nevertheless, this limitation should be considered in therapy of serious, life-threatening infections (or other diseases), where the exact amount of biological active agent has to be used. *Staphylococci* are often responsible for many, not really serious, but bothersome infections. We would suggest more frequent use of propolis for treatment of this type of diseases. Currently, most of these infections are treated with antibiotics, which should be rather reserved for serious infections. It leads to the overuse of antibiotics and development of resistance phenomenon. In contrast to antibiotics, propolis is a mixture of many antimicrobial components, thus development of resistance against this product (parallel resistance to all active constituents) is not possible. We also agree with other authors that it is necessary to continue research on correlation of biological activity and chemical composition of this product. Identification of crucial ingredients or their compositions could be used also for treatment serious, life-threatening diseases.

#### *2.2.3. Other bee products: pollen and royal jelly*

in BHI (Brain Hearth Infusion) medium. The authors noticed that in milk the bactericidal dose was at least 20-fold greater [50]. Interesting results were also published by Suleman and colleagues [51]. Some of 39 South Africa propolis samples exhibited much higher antistaphylococcal activity in comparison to three tested samples of Brazilian propolis with MIC and MBC values of only 6 μg/mL [51]. Al-Waili and coworkers revealed that ethanolic extracts of propolis collected from Saudi Arabia (EEPS) and from Egypt (EEPE) effectively inhibited the growth of antibiotic resistant *E. coli, S. aureus* and *C. albicans* in single and polymicrobial cultures [52]. Strong antioxidant and antibacterial activity of propolis sourced from three different areas of Sonoran Desert in northwestern Mexico were confirmed in the research of Velazquez group [53]. The MIC against *S. aureus* of the most active sample (coming from Ures) was 100 μg/mL [53]. An antimicrobial effect of propolis harvested from honeybees in subtropical eastern Australia was investigated by Massaro and coworkers. The two tested propolis crude, ethanolic extracts showed bactericidal effects against *S. aureus* ATCC 25923 reference strain at the concentrations of 0.37–2.04 mg/mL [54]. Propolis produced in many Asian and European apiaries is also effective in elimination of staphylococci. The number of publications presenting results of biological properties of propolis sourced from this region has evidentially increased during the last decade. Because of limited size of this chapter only selected investigations can be described herein. Some promising results regarding antistaphylococcal activity of ethanolic extract of Polish propolis (EEPP) against methicillin-sensitive *S. aureus* (MSSA) and methicillin-resistant *S. aureus* (MRSA) clinical isolates have been recently revealed by Wojtyczka and coworkers. The investigated EEPP displayed varying effectiveness against twelve *S. aureus* strains, with MIC in the range from 0.39 to 0.78 mg/mL, determined by broth microdilution method, and minimal bactericidal concentration (MBC) of the EEPP ranged from 0.78 to 3.13 mg/mL. The disk diffusion assay revealed also that EEPP-enhanced antistaphylococcal activity of eight classical antimicrobial antibiotics, namely: cefoxitin, clindamycin, tetracycline, tobramycin, linezolid, trimethoprim+sulfamethoxazole, penicillin and erythromycin [55]. The same authors also investigated EEPP activity against biofilm-forming coagulase-negative *Staphylococcus* strains. The biofilm formation ability of all tested *S. epidermidis* strains was

210 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

inhibited at EEPP concentrations ranging from 0.39 to 1.56 mg/mL [56].

Due to its health-promoting properties, propolis is widely used as a component of cosmetics, some food and beverages. In our opinion, these applications are not adequate to the biological properties of this product. The results of many presented above studies clearly indicate that propolis is a promising antimicrobial agent. Moreover, many other biological activities of this product have been described, including antioxidant, antiviral, antitumor, antifungal and immunomodulatory properties [46]. Large diversity of its chemical composition and consequently its biological activity eliminate propolis from clinical applications. Nevertheless, this limitation should be considered in therapy of serious, life-threatening infections (or other diseases), where the exact amount of biological active agent has to be used. *Staphylococci* are often responsible for many, not really serious, but bothersome infections. We would suggest more frequent use of propolis for treatment of this type of diseases. Currently, most of these infections are treated with antibiotics, which should be rather reserved for serious infections. It leads to the overuse of antibiotics and development of resistance phenomenon. In contrast Investigation of antimicrobial activity of pollen, fermented pollen (bee bread) and royal jelly is not as popular as in the case of honey and propolis. However, several authors confirmed that these products also reveal some antimicrobial potential. Boukraa and coworkers revealed that MIC value for royal jelly against *S. aureus* is about 2% (v/v) [57], and the group of Gunaldi [58] when using this product obtained promising results in the preservation of implant-related infection in rats. The activity of royal jelly is probably a consequence of presence within composition of this product some peptides that inhibit growth of bacteria including members of the genus *Staphylococcus* [59].

Antimicrobial, antimutagenic, antioxidant and even anti-inflammatory activity of bee pollen (collected in Portugal and Spain) has been revealed in the research of Pascola and colleagues [60], and *S. aureus* was found as the most sensitive microorganism to the activity of these products. High antimicrobial activity of methanolic and ethanolic extracts of several monofloral Slovakian bee pollens was observed by the group of Fatracova-Sramkowa. *S. aureus* was found as most sensitive to the poppy pollen ethanolic extract. The most sensitive bacteria of rape bee pollen methanolic extract and sunflower ethanolic extract was *Salmonella enterica* [61].

#### *2.2.4. Honey and other bee products as a source of bacteriocinogenic bacteria with bactericidal antistaphylococcal activity*

However, bacteriocins are usually active against bacteria closely related to producing strain, but there are also many exceptions to this general rule, e.g. nisin, which is active against broad spectrum of Gram-positive bacteria [62]. Recently carried out research of the group of Prof. Worobo from Cornell University revealed that honey should be considered as a potential source of microorganisms producing promising antimicrobial compounds, especially bacteriocins [63]. The mentioned authors analyzed two Manuka honeys from New Zealand and six domestic honeys from US. The 2217 isolates out of 2398 strains (92.5%) exhibited activity at least against one of the tested microorganisms. Among all the bacterial indicator strains, *Listeria monocytogenes* had highest susceptibility (69%) to the various antimicrobial compounds produced by all active bacterial isolates (1655 out of 2398), whereas *S. aureus* showed the second highest susceptibility of all indicator microorganisms tested. Growth of this bacterium was inhibited by 66.9% of all the isolates (1605 out of 2398) [63]. The bacterial isolate TH13 was found as an efficient producer of peptide compound with high activity against *P. larvae* spp. *larvae*, which are etiological agents of American Foulbrood. It is a disease of honeybees that results in the annihilation of the honeybee colony [64]. Another isolate, identified as *Bacillus thuringiensis* SF316, was shown to efficiently produced thuricin H, a bacteriocin which strongly inhibits the growth of *Bacillus cereus* [65]. Thus, it was shown that antimicrobial activity of honey is attributed not only to hydrogen peroxide (formed by glucose oxidase originating from honeybees), antioxidant components or dimethyloglyoxal (in the case of Manuka honey), but also to microorganisms which are present in this product.

However, results of some successful investigations have been published the trials of isolation of bacteriocinogenic bacterial strains from honey and other than honeybee products were rarely carried out to date. In our opinion, especially, promising source of bacteria producing interesting bacteriocins could be fermented pollen—it is pollen which is collected by bees for the winter and early spring. The high antimicrobial activity of fermented pollen is the consequence of lactic acid bacteria (LAB) presence in this product, and the products of LAB metabolism—lactic acid, as well as bacteriocins.
