Propolis Extract: A Possible Antiseptic Oral Care against Multidrug-Resistant Non-Fermenting Bacteria Isolated from Non-Ventilator Hospital-Acquired Pneumonia

Non-ventilator Hospital-acquired Pneumonia (NV-HAP) is a significant burden in acute care hospitals and poses a risk to nonelderly, non-intensive care unit (ICU) patients, which have been increasing worldwide. In addition, poor oral hygiene has been associated to significant increases in the number of cases of NV-HAP. Unfortunately, preventive options are limited. Thus, there is a need for oral antiseptics, similar to those of natural products or plant sources. The aim of this study was to assess the antibacterial activity of various bee products (BPs); for example, honey, propolis, and bee venom against multidrugresistant (MDR) non-fermenting bacteria (e.g., Pseudomonas and Acinetobacter), which were collected from NV-HAP patients to investigate its use as a possible antiseptic oral care. Bacterial susceptibility to different antibiotics were performed. The antimicrobial activity of BPs against non-fermenting bacteria, the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC) were assessed. Eighteen Pseudomonas aeruginosa isolates and five Acinetobacter baumannii isolates were identified. P. aeruginosa isolates displayed high resistance to the antibiotics: meropenem and imipenem (55.6% and 77.8% respectively), whereas A. baumannii isolates were 100% resistant to meropenem and imipenem. All isolates remained sensitive to colistin. Propolis showed the best antibacterial activity (p<0.001) in comparison to honey and bee venom against P. aeruginosa (13 36 mm, MIC =1.4-22.5%, and MBC=2.8-45%) and A. baumannii (7-20 mm, MIC=5.6-22.5%, and MBC=11.3 -22.5%). While bee venom expressed the least antibacterial activity against all isolates with a zone diameter ranging from 0-12 mm, propolis, which is a non-toxic, natural, and inexpensive, had antibacterial activity towards the MDR bacteria: P. aeruginosa and A. baumannii collected from pneumonic patients. Additionally, we confirmed that propolis could be used as a potential antiseptic oral care product.


INTRODUCTION
Non-ventilator Hospital-acquired Pneumonia (NV-HAP) is one of the most common hospital-acquired infections (HAIs). Even though it conveys similar mortality rates as Ventilator-Associated Pneumonia (VAP), its incidence rates are higher; thus, the accompanying charges and deaths could be more considerable 1 .
Three fundamental factors increase a patient's susceptibility to HAP: alterations in oral microorganisms (virulent gram-negative organisms), microaspiration (tiny droplets being aspirated), and a debilitated host. These factors generate a perfect environment for microorganisms to invade the respiratory area. In addition, neglected nursing care increased the likelihood of contracting NV-HAP triggered by an enlarged load of microbes 2 . Fortunately, one's risk in contracting pneumonia can be reduced by simple care procedures; for example, evaluating the patient's aspiration hazard, regular mobilization, and uplifting the head off the bed. Additionally, the decontamination of the respiratory tract by topical antiseptic agents is an applicant protective interference 3 .
Non-fermenting Gram-negative (GN) species, particularly P. aeruginosa and A. baumannii have long been documented as common and recurrent pathogens that cause HAP. It has been observed that P. aeruginosa is the second leading cause of HAP, right behind Staphylococcus aureus, which may account for 15%-20% of patients infected with HAP 4 . Additionally, A. baumannii has developed a substantial presence for HAP in intensive care units 5 . A. baumannii has been associated with a high mortality rate, which is approximately 35% to 70% of cases. 6 Currently, clinicians are faced with bacterial strains, such as P. aeruginosa and Acinetobacter species that are resistant to the majority of antibiotics, resulting in HAIs, with only a few available options 7 . Therefore, the development of new, naturally complementary, and alternative medicinal products against MDR GN pathogens is of the utmost importance in the field of HAIs 8 .
Bees are insects belonging to the genus Apis. Bees are nature's developers and accumulators of honey, which is their primary product. However, bees also produce beeswax, royal jelly, bee venom, and propolis, which are derived from the excretion glands of the honeybee. It is worth mentioning that these products have been used for medicinal purposes for millennia 9 .
Honey comprises approximately 200 celluar molecules, such as, vitamins, amino acids, enzymes, and minerals. However, honey is primarily composed of water and sugar (i.e., glucose and fructose). The antibacterial agents in honey provide inhibitory properties to Gram positive (GP) and GN bacteria. Honey has low mean pH of 4.4, which is lethal to microorganisms. Even when honey is heavily diluted, the hydrogen peroxide activity of honey can still affect microbes by inhibiting their growth 10 .
Propolis is a resin-like material made by bees, produced through mixing beeswax and secretions of salivary glands with exudate collected from plants. Propolis is sometimes referred to as "bee penicillin" 11 . A beehive is a very sterile environment found in nature. Propolis not only acts as glue but is also used to clean and disinfect the honeycombs. Ancient Egyptians used it for preserving bodies during mummification. Recently, Egyptian propolis has become a topic of interest and is receiving increasing attention from chemists and biologists 12 .
Propolis is indicated in treating tuberculosis and upper respiratory tract infections, Human immunodeficiency Virus, H1N1 "swine" flu, and the common cold. Propolis contains phenolic substances, including flavonoids, with antimicrobial potential 13 .
Bee venom (BV) called apitoxin includes a combination of active enzymes, peptides, and amines. It is a colorless liquid with a pH of 5.0-5.5 14 . The antibacterial effect of BV has been recognized to be effective against Salmonella spp, Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Coagulase-Negative Staphylococcus, and S. aureus 10 .
This study sought to assess the antibacterial effects of Egyptian Bee products (BPs): honey, propolis, and BV against MDR nonfermenting bacterial isolates collected from NV-HAP patients as a possible antiseptic oral care.

MATERIAL AND METHODS Preparation of Bee products (BPs)
The study was carried out in El-Zawia village at EL-FAYOUM Governorate, a city near middle Egypt. Ten colonies were used for honey, propolis, and bee venom collection. Honeycombs were taken from these colonies where they were weighed, followed by honey-extraction. A stock honey solution of 50% (w/v) was prepared by liquefying 25 g of honey in 50 mL Muller-Hinton broth (Thermo Fisher Scientific Remel product, Lenexa, KS, USA) in a 50 mL flask. The solution was diluted for further analysis. Concentrations of honey were expressed as weight/volume percent (w/v %). Propolis was gathered by collecting the hive scrapings while washing the honey supers throughout the honey harvest. The scrapings were covered with 5.08 to 7.62 cm of water and placed in an oven at 93.3°C. The contents of the container were heated for approximately 2 h and were then moved. The liquefied wax is attached to pieces of wood, where it hangs to the surface of the water while the propolis attaches to the container bottom. After the container is detached from the oven and well-ventilated, the waxy layer on the surface of the water is detached, and the water is then cautiously poured off to reveal the colored propolis bulk underneath it. The cleaned propolis pieces are spread out on a piece of paper to dry before being stored. In total, 100 mg of the propolis powder sample was homogenized with 10 ml of 70% alcohol for 30 min. The alcoholic extract was separated by centrifugation at 6000-7000 rpm followed by evaporation. The electric shock method with the bee venom collector device was employed to gather the bee venom. The device was put vertically between the hive combs. Next, the collector device was used for 15 min/colony/ week. The deposited venom between the two glass sheets was dried, then scraped off by a razor blade. Finally, the bee venom was collected and placed into dark bottles in a powder form 14 .
Next, the physicochemical properties for honey, bee venom, and propolis were determined.

Antimicrobial activity of bee products by disc diffusion test
T h e i s o l ate s we re t ra n s p o r te d in a brain-heart infusion broth media to the research laboratory of Medical Microbiology and Immunology Department at the Fayoum University (Faculty of Medicine). For the detection of antibacterial activities of the BPs, the agar disc diffusion method was performed. The suspension of the tested bacteria (10 6 CFU/μl) was spread on Mueller Hinton agar (Oxoid Ltd., Basingstoke, England). Then, bacterial strains were incubated for 20 min at 37°C. The agar was cut into discs of 5 mm in diameter, and wells were filled with 100μL containing 10%, 20%, 50%, and 90% concentrations of BPs. They were incubated at 37°C for 24 h. The inhibition zone diameters were detected in millimeters. The negative control was performed using distilled water. From triplicate measurements, the results were displayed as mean values 34 .

Minimum inhibitory concentration determination (MIC) for Honey and Propolis
The Minimum Inhibitory Concentrations (MICs) of honey and propolis were assessed by a microdilution technique as both honey and propolis showing the highest antimicrobial activity through the agar disc diffusion method. The tests were performed in 96-well plates (Corning  Costar  Ltd., Corning, NY, USA) according to the CLSI guidelines 19 . Given that no diameter inhibitions were found at concentrations of 10%, 20%, and 50% of BPs, a volume of 100μl of 90% BPs concentration (w/v) was distributed in wells. Positive and negative controls (broth) were tested. Subsequently, wells were seeded with an aliquot of 10 μl of approximately 10 6 CFU/mL of overnight bacterial culture and incubated for 24 h aerobically at 37 °C. The concentrations of BPs obtained in the well were between 45% and 0.01%.
To determine the minimum bactericidal concentration determination (MBC) of honey and propolis, 10μl of content from the MIC wells were spread out without visible turbidity onto Mueller Hinton agar and incubating (aerobically) as required for evaluation. Positive and negative control wells were included. MBCs were determined on three separate occasions 35 .

Ethical considerations
This study was approved by the Faculty of Medicine Research Ethical Committee and Fayoum University Hospitals director. The study was conducted after explaining the study's aims.

Statistical analysis
Using Statistical Package for Social Science (SPSS version 16, Chicago IL, USA), data were collected, coded, and analyzed. Data were quantified by calculating the means, median, standard deviation (SD), and interquartile range (IQR) for numerical data. Frequencies and percent were completed for qualitative data. A comparison of quantitative variables was made using the Mann-Whitney U test between two groups and Kruskal-Wallis H tests (i.e., one-way ANOVA on ranks) for more than two groups. P ≤ 0. 05 was considered statistically significant.

RESULTS
A total of twenty-three isolates were collected from NV-HAP patients. Eighteen isolates were P. aeruginosa, and five isolates were A. baumannii. P. aeruginosa isolates revealed a high resistance rate to meropenem and imipenem (55.6% and 77.8%, respectively). Additionally, all P. aeruginosa isolates were susceptible to polymyxin and colistin (Table 1). A. baumannii isolates were found to be resistant to all tested antibiotics except for colistin and polymyxin.

Antimicrobial activity of Bee Products
At the concentration of 90% (w/v), the inhibitory effect of BPs on bacterial growth was apparent. Significant differences in inhibition zone values, MIC, and MBC values for propolis in comparison to honey and bee venom (p<0.001) were found (Table 3, 4). The best antibacterial  Table 5.   Thus, research and investigation into potential antibacterials are currently of great interest. In recent times, public awareness in the therapeutic use of bee products has dramatically increased, especially since they have no reported record of adverse side effects on tissues 22 .
In the current work, among 18 isolates of P. aeruginosa, high resistance rates to meropenem and imipenem (55.6% and 77.8% respectively) were found. Additionally, five A. baumannii isolates were found to be resistant to all the antibiotics used in this study. These results were in agreement with the results reported by Liu et al. who reported the rates of resistance of P. aeruginosa to meropenem and imipenem (48.8% and 70.7%, respectively), and high resistance rates of A. baumannii to carbapenems (more than 70%) were found 23 . Moreover, Chung et al. found P. aeruginosa with a resistance rate to imipenem (56.9%), and Acinetobacter spp. presented a high rate of resistance to imipenem (67.3%) 24 . All isolates in our study persisted in being sensitive to colistin. However, in this report: the resistance rate to colistin was observed to be 0.8 %.
Several scientific studies showed propolis having antibacterial activity (with active constituents including flavonoids like galanin and hydroxyl-cinnamic acids) 25 . The synergistic activity between various active constituents has been attributed as the chief reason for getting the complex antibacterial effect of propolis 26 . Many researchers have shown that Acinetobacter spp exhibited either low sensitivity or total absence of sensitivity against propolis. 12,31,32 These variations in the antibacterial activity of propolis among different studies could be due to the differences in origin.
In the present study, the antimicrobial activity of honey against MDR non-fermenter bacteria were less than propolis, which the diameter zone of inhibition of honey for P. aeruginosa was 9-28 mm with MIC=5.6-45% and MBC=11.3->45%, and for A. baumannii was 0-16 mm with MIC=11.3-45%, and MBC= 22.5->45%. Those results were higher than the results reported by Henriques et al. who found the values of MIC and MBC of manuka honey against P. aeruginosa (9.5% and 12%, respectively) 33 . However, they were lower than the results revealed by Moussa et al. who observed the diameter zone of inhibition of honey for P.  40 . It has been revealed that honey might have antibacterial action from less than 3% to 50% or higher concentrations 36 .
These results from different studies approved that honey from diverse regions and countries could have vast differences in their antibacterial activity. Our results demonstrated little activity of bee venom against all isolates with zone diameter from 0-12 mm with mean: 4.7. Zolfagharian et al. agreed with our results, who evaluated the antimicrobial effect of bee venom against six GP and GN bacteria and found bee venom had no noticeable effect on P. aeruginosa 14 . Also, Fennell et al. reported that the bee venom is more active against GP than GN bacteria 41 .
The findings of our study showed that propolis had good antimicrobial activity against MDR non-fermenting bacteria, which helps to be used as a possible oral antiseptic in pneumonic patients.

CONCLUSION
Given the promising antimicrobial activity of propolis, low cost, and safety, we suggest its potential application as an oral antiseptic to reduce the NV-HAP incidence. Further investigation is warranted to confirm these promising findings, and additional studies on varied kinds of GP and GN bacteria are needed, and in vivo activity of propolis for the pneumonic patients would be essential for the assessment of its efficacy in pneumonia prevention.