**4. Antimicrobial activity of honey: in vitro and in vivo studies**

High antimicrobial potential of honey has been confirmed in many in vitro tests, but also in vivo studies. Some important differences in the activity of honey produced from different botanical sources have been revealed. In general, molds and yeasts are less sensitive to the activity of this product, as high concentration as 30–50% is necessary to inhibit the growth of these groups of microorganisms [11]. Much higher activity has been observed in the case of bacteria, especially sensitive are Gram-positive bacteria. Taking into account mechanisms of activity, methods of application and dose of the product necessary for effective elimination of bacteria only some specific types of diseases could be treated with honey, for example, infected wounds, skin and soft tissue infections, infections located within upper respiratory tract, mucosa of digestive tract, vaginal mucosa and some specific disease, for example, stomach ulcers caused by *Helicobacter pylori*. This limitation was the main criterion for selection of bacterial species for presented below description concerning the results of research of antimicrobial activity of honey and possibilities of its application in clinical practice or prophylaxis of some disease.

#### **4.1. Antistaphylococcal activity of honey**

High, in vitro, antistaphylococcal activity of honey has been confirmed by many researchers—for details please see our previous review [19]. In fact, staphylococci belong to the most sensitive bacteria to the components of this product [19]. The growth of staphylococci is inhibited by proteinaceous components—defensin-1 and generated by glucose oxidase H2 O2 , as well as by other antimicrobial ingredients: mainly polyphenols and methylglyoxal in the case of Manuka honey. Our previous research revealed high antistaphylococcal (against *S. aureus* PCM 2051—reference strain) activity of polish honeys produced from cornflower (*Centaurea cyanus* L.), buckwheat (*Fagopyrum esculentum* Moench) and thyme (*Thymus vulgaris* L.) with MIC values of 3.12 or 6.25% (v/v); some differences of activity of different samples of honey obtained from the same botanical sources were observed [13]. The obtained results (the ranges of effective concentrations) are in agreement with the results presented by other authors who investigated honeys sourced from different geographical locations, for example, from Greece [14] or Iran [20]. High antistaphylococcal activity of honey has been also confirmed for MRSA (*Methicillin-Resistant Staphylococcus aureus*) clinical isolates. Effective inhibition of growth of MRSA isolates has been revealed in the case of Chilean honey obtained from Ulmo tree [21], Malaysian melaleuca honey [22], some Thai honeys, especially from longan flower [23], Finland [24], Ethiopia [25] and several other geographical regions. Honey is also effective in eradication of staphylococcal biofilm. Lu and coworkers [26] revealed that New Zealand Manuka-type honeys, at the concentrations they can be applied in wound dressings, are highly active in both preventing *S. aureus* biofilm formation and in their eradication and do not result in bacteria becoming resistant [26]. High efficiency in elimination of bacterial biofilm confirmed also for honeys whose activity depends mainly from hydrogen peroxide generation, for example, "Medihoney"—therapeutic honey and Norwegian Forest Honey [27]. Staphylococci are often isolated from skin and soft tissue infections; they are also important etiological factor of wound infections. The group of Blaser achieved a full healing in seven consecutive patients whose wounds were either infected or colonized with methicillin-resistant *S. aureus*. Antiseptics and antibiotics had previously failed to irradiate the clinical signs of infection [28]. Interesting results were also presented by Al-Waili [29] who used honey for treatment surgical wounds made on the dorsum of mice infected with different species of bacteria. It was found that local application of raw honey on infected wounds reduced redness, swelling, time for complete resolution of lesion and time for eradication of bacterial infection due to *S. aureus* or *Klebsiella* sp. Its potency was comparable to that of local antibiotics [29]. Because of their promising properties, the wound dressing materials containing honey (mostly Manuka honey) are already commercially available and gain popularity in treatment difficult to heal infected wounds.

### **4.2. Activity against** *Helicobacter pylori*

**4. Antimicrobial activity of honey: in vitro and in vivo studies**

of some disease.

224 Honey Analysis

**4.1. Antistaphylococcal activity of honey**

High antimicrobial potential of honey has been confirmed in many in vitro tests, but also in vivo studies. Some important differences in the activity of honey produced from different botanical sources have been revealed. In general, molds and yeasts are less sensitive to the activity of this product, as high concentration as 30–50% is necessary to inhibit the growth of these groups of microorganisms [11]. Much higher activity has been observed in the case of bacteria, especially sensitive are Gram-positive bacteria. Taking into account mechanisms of activity, methods of application and dose of the product necessary for effective elimination of bacteria only some specific types of diseases could be treated with honey, for example, infected wounds, skin and soft tissue infections, infections located within upper respiratory tract, mucosa of digestive tract, vaginal mucosa and some specific disease, for example, stomach ulcers caused by *Helicobacter pylori*. This limitation was the main criterion for selection of bacterial species for presented below description concerning the results of research of antimicrobial activity of honey and possibilities of its application in clinical practice or prophylaxis

High, in vitro, antistaphylococcal activity of honey has been confirmed by many researchers—for details please see our previous review [19]. In fact, staphylococci belong to the most sensitive bacteria to the components of this product [19]. The growth of staphylococci is inhibited by proteinaceous components—defensin-1 and generated by glucose oxidase H2

as well as by other antimicrobial ingredients: mainly polyphenols and methylglyoxal in the case of Manuka honey. Our previous research revealed high antistaphylococcal (against *S. aureus* PCM 2051—reference strain) activity of polish honeys produced from cornflower (*Centaurea cyanus* L.), buckwheat (*Fagopyrum esculentum* Moench) and thyme (*Thymus vulgaris* L.) with MIC values of 3.12 or 6.25% (v/v); some differences of activity of different samples of honey obtained from the same botanical sources were observed [13]. The obtained results (the ranges of effective concentrations) are in agreement with the results presented by other authors who investigated honeys sourced from different geographical locations, for example, from Greece [14] or Iran [20]. High antistaphylococcal activity of honey has been also confirmed for MRSA (*Methicillin-Resistant Staphylococcus aureus*) clinical isolates. Effective inhibition of growth of MRSA isolates has been revealed in the case of Chilean honey obtained from Ulmo tree [21], Malaysian melaleuca honey [22], some Thai honeys, especially from longan flower [23], Finland [24], Ethiopia [25] and several other geographical regions. Honey is also effective in eradication of staphylococcal biofilm. Lu and coworkers [26] revealed that New Zealand Manuka-type honeys, at the concentrations they can be applied in wound dressings, are highly active in both preventing *S. aureus* biofilm formation and in their eradication and do not result in bacteria becoming resistant [26]. High efficiency in elimination of bacterial biofilm confirmed also for honeys whose activity depends mainly from hydrogen peroxide generation, for example, "Medihoney"—therapeutic honey and Norwegian Forest Honey [27]. Staphylococci are often isolated from skin and soft tissue infections; they are also important etiological factor of wound infections. The group of Blaser achieved a full

O2 , In vitro anti-*H. pylori* activity of honey has been confirmed by several research groups. Using agar diffusion assay, Nzeako and Al-Namaani [30] investigated activity of eight samples of honey (four from Germany, one from Switzerland, one from Iran and two from Oman). All of them effectively inhibited the growth of *H. pylori*. The size of growth inhibition zones produced by the samples of 100 µl of undiluted honey varied from 15 mm for Blossom bee honey (Switzerland) to 29 mm for Al-Nada clove honey (Oman) [30]. Interesting results of in vitro studies of anti-*H. pylori* activity of three locally produced honeys from different regions in South Africa were presented by Manyi-Loh and coworkers [31]. The authors revealed high activity of honey but also extracts of organic, nonproteinaceous components of these products [31]. Al Somal and colleagues [32] revealed much better anti-*H. pylori* activity of Manuka honey in comparison with peroxide-dependent honey. All five isolates tested by the authors were sensitive to a 20% (v/v) solution of Manuka honey in an agar well diffusion assay, but none showed sensitivity to a 40% solution of a honey sample in which the antibacterial activity was due primarily to its content of hydrogen peroxide [32]. The observations presented by the groups of Manyi-Loh et al. [31] are especially important from the point of view of specific conditions in stomach. High concentration of HCl and low value of pH certainly affects the activity of enzymes that are present in consumed food, including glucose oxidase, which generates hydrogen peroxide and is crucial for antimicrobial activity of most types of honeys. Thus, the presence of other than H2 O2 antimicrobial components in honey is very important for possibilities of its effective application for prophylaxis and therapy of in vivo *H. pylori* infections. Recently, Sahin [33] revealed that phenolic components of chestnut and oak honeys effectively inhibited activity of two enzymes: urease and xanthine oxidase, which are important virulence factors of *H. pylori*. These results importantly confirm that regular consumption of honey (especially the products rich in polyphenols) could prevent gastric ulcers deriving from *H. pylori* [33]. Moreover, analyzing the group of 150 dyspeptic patients, Boyanova and colleagues (2015) [34] revealed that consumption of honey at least 1 day weekly significantly reduces the risk of development of infection with *H. pylori* [34]. The in vitro susceptibility of *H. pylori* to honey is well documented. In our opinion, more studies aiming in evaluation of in vivo effects of regular consumption of honey for development of *H. pylori* infection within stomach are necessary. These researches should concentrate on selection of type of honeys (probably characterized with high content of polyphenols and/or MGO), especially effective in eradication this bacterium from the tissue.

#### **4.3. Activity against** *Mycobacterium tuberculosis*

*M. tuberculosis*, being the leading member of the MTB complex (*Mycobacterium tuberculosis complex*), is the main cause of tuberculosis worldwide. Over the recent past years, resistance against antituberculous drugs has emerged rapidly, resulting in MDR (*Multi Drug Resistant*) strains. In vitro activity of Beri honey (from Pakistan) was tested against 21 clinical isolates of MDR-MTB by Hannan and coworkers (2014) [35]. The obtained results clearly demonstrate that Pakistani Beri honey exhibits significant antimycobacterial potential, and three (14%) of the isolates were susceptible at 1% (v/v) honey, while at 2% (v/v) of honey, 18 (86%) isolates were found to be susceptible. All the 21 isolates (*n* = 21) were susceptible at 3% (v/v) of honey [35]. Honey was also proposed for treatment tuberculosis by Avicenna, a known ancient Persian philosopher and physician. At the beginning of twenty-first century, this hypothesis was evaluated by the researchers from Shiraz University of Medical Sciences in Iran [36]. It was demonstrated that the growth of mycobacteria was inhibited by adding 10% honey to the growing media (Lowenstein-Jenson media and L-J media were used). *Mycobacteria* did not grow in culture media containing 10 and 20% honey, while it grew in culture media containing 5, 2.5 and 1% honey. Thus, the obtained results of in vitro tests are quite optimistic [36]. However, future research of in vivo activity of honey against *Mycobacteria* located within the lung tissue would be necessary for fully evaluation of its usefulness in the treatment of tuberculosis. According to the best of our knowledge to date, such studies have not been conducted.

#### **4.4. Activity against Gram-negative bacteria:** *P. aeruginosa* **and** *E. coli*

The most carried out to date studies revealed that Gram-negative bacteria are a bit less sensitive to the activity of honey in comparison with Gram-positive bacteria. This situation was also observed in the research carried out in our group. The collection of over 30 Polish monofloral honeys was tested, and definitely most of them were less active against *P. aeruginosa* and especially *E. coli* reference strains in comparison with *S. aureus* PCM2051. However, the activity of most of honeys against these pathogens was on satisfactory level, with MIC values in the range of concentrations from 6.25 to 25% (v/v) and from 12.5 to 25% (v/v), respectively [13]. Honey effectively eradicates biofilm formed by *P. aeruginosa* [37]. Activity of this product against this bacterium has been also confirmed in some in vivo studies. The stingless bee honey has been successfully used for treatment of *P. aeruginosa* infected conjunctivitis in Hartley guine pigs [38]. The investigation carried out by the group of Khoo (2010) revealed that Tualang honeytreated rats demonstrated a reduction in bacterial growth in *P. aeruginosa* inoculated wounds [39]. *P. aeruginosa* belongs to the important etiological factors of wound infections. Thus, activity of many potential wound dressing materials containing honey against this bacterium has been carried out. Most of them confirm high therapeutic antimicrobial potential of honey.
