The Effect of Honeybee Propolis on Escherichia coli Biofilm Formation in vitro

Colibacillosis is an infectious disease that affects livestock such as cattle, pigs, sheep, horses, goats and poultry caused by the bacterium Escherichia coli (Singh et al., 2023). E. coli is a gram-negative bacterium belonging to the order Enterobacterales (Sora et al., 2021). E. coli can cause infectious diseases in livestock such as cattle, pigs, goats, sheep, horses and poultry which is called colibacillosis. Colibacillosis in pigs causes diarrhea, dehydration, acidosis and even death (Castro et al., 2022; Li et al., 2021). Colibacillosis infection in chickens and poultry can be divided into two types, localized and systemic infections (Thakre et al., 2017). In poultry, the infection is characterized by coliform cellulitis, hemorrhagic septicemia, swollen head syndrome, colisepticemia, omphalitis, peritonitis, salpingitis, coligranuloma, orchitis, osteomyelitis or synovitis, panophthalmitis and enteritis (Kabir, 2010). In horses, E. coli is the main cause of urinary tract infections, reproductive organ infections, respiratory diseases and other tissue infections (Nielsen et al., 2022).
       
E. coli is a rod-shaped bacterium with virulence genes fim, krl, csg and agn43, allowing it to form biofilms during its growth phase. Biofilm formation occurs when environmental conditions are unfavorable (Basavaraju and Gunashree, 2021; Zhou et al., 2022; Rabins et al., 2018). A biofilm is a collection of bacterial cells that adhere to surfaces and bind with secreted exopolymer matrix (Zhao et al., 2023). According to Zhou et al., (2022), five steps are typically involved in the creation of E. coli biofilms: colony formation, biofilm maturation, dispersion, reversible adhesion and irreversible adhesion. Biofilm formation in E. coli makes these bacteria more resistant to antibiotics than when they are not in a biofilm state.
       
As antibiotics lose their ability to effectively stop bacterial development, microorganisms develop the ability to withstand the effects of antimicrobial drugs (Reygaert, 2018; Chakraborty et al., 2020). According to Wang et al., (2023), E. coli has been reported to exhibit resistance to 17 types of antibiotics. Antimicrobial agents diffusing through a limited biofilm matrix, communication between the biofilm matrix (polymers and cells) and the antimicrobial agents, enzyme-mediated resistance, levels of metabolic activity within the biofilm, genetic adaptation, efflux pumps and outer membrane structure are the causes of antibiotic resistance in E. coli (Singh et al., 2017).
       
The use of antibiotics for the treatment of colibacillosis has been widely practiced. Indonesia is one of the countries with the highest antibiotic usage, with a percentage of 202% (Dewi et al., 2023). Due to indiscriminate antibiotic use, various antibiotic resistance issues arise, rendering the treatment of colibacillosis ineffective. This issue needs to be addressed through research on how to inhibit biofilm formation and development with the emergence of antibiofilm activity. However, research on testing honeybee propolis extract as an antibiofilm against E. coli has not been conducted. Honeybee propolis is a safe natural and readily available substance. Honeybee propolis extract is known to inhibit biofilm formation in Staphylococcus bacteria. This occurs because honeybee propolis contains flavonoid, saponin, tannin, alkaloid, terpenoid, phenol and glycoside that can act as antibiofilm agents (Almuhayawi, 2020; Aziz et al., 2021).

Study area and sample collection
 
The research was conducted over a period of July 2023 to November 2023 at the Bacteriology and Mycology Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga. The reading of OD value was conducted in the Molecular Biology Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga. The materials used in this research include aluminium foil, the ATCC 25922 E. coli culture from the Bacteriology and Mycology Laboratory of the Faculty of Veterinary Medicine of Universitas Airlangga, Noble Broth (NB) MERCK media, MacConkey Agar (MCA) media, honeybee propolis from Melia, Phosphate Buffer Saline Solution (PBS), Paraformaldehyde (PFA), Crystal Violet 0.1% and Acetic Acid.
 
Preparation of noble broth
 
First, Noble broth media for the cultivation of E. coli bacteria and the dilution of honeybee propolis extract was created. The medium was dissolved in 100 mL of distilled water to make 0.8 grams of Noble broth. Second, the dissolved medium in distilled water was slowly stirred around over a flame on the stove. Third, the medium was then transferred into wide-mouth laboratory bottles type I Borosilicate 100 mL. Forth, the bottles were sealed and covered with aluminum foil. Fifth, the medium was autoclaved for 15 minutes at 121°C and 1-2 atm of pressure to sterlize it.
 
Preparation and microdilution of honeybee propolis
 
The honeybee propolis used in this research is a product of Melia Propolis purchased from a pharmacy in Surabaya. The propolis underwent microdilution in 7 microtubes that had been labeled 1 to 7. Microtube 1 was filled with 1000 μl of honeybee propolis, while microtubes 2 to 7 were initially filled with 500 μl of culture. Dilution was performed by transferring 500 μl of honeybee propolis from microtube 1 to microtube 2. This process was sequentially repeated for the solutions in microtubes 2 through 7 to achieve appropriate dilution results.
       
The concentration value of the result of the dilution was determined through the modification and the application of the Minimal Inhibitory Concentration (MIC), recommended by the NCCLS, while maintaining sterile conditions throughout the experiment (Clinical and Laboratory Standards Institute, 2015). The lowest concentration of an antibacterial drug expressed in mg/L (μg/mL) is known as the MIC. The concentration values of propolis were determined based on the previous research, namely 0.39 mg/mL, 0.78 mg/mL, 1.56 mg/mL, 3.125 mg/mL and 6.25 mg/mL (Kaligis and Mokosuli, 2022).
 
Preparation of E. coli inoculum
 
Inoculation of E. coli bacteria into Noble broth media was performed by retrieving bacteria from the bacterial culture on MacConkey agar using a needle, then transferring it into the Noble Broth media. The mixture was then incubated for one day at a temperature of 37°C. Successful bacterial inoculation was indicated by a change in the media color to cloudy yellow.
 
Biofilm inhibition assay
 
This testing included positive and negative control, which were prepared in planktonic form. The testing was conducted in triplicate as described by Kayanadath et al., (2019) with modifications, comprising positive control, negative control and treatment groups. The positive control contained 100 μl of NB media and 100 μl of E. coli bacteria, the negative control contained 200 μl of NB media and the treatment group contained 100 μl of E. coli bacteria and 100 μl of various concentrations of propolis (0.39 mg/mL, 0.78 mg/mL, 1.56 mg/mL, 3.125 mg/mL and 6.25 mg/mL).
 
Wells in the positive control and treatment group were inoculated with E. coli bacteria. Then, wells in the treatment group were provided with honey propolis according to the concentration of each treatment. Wells filled with bacteria and honey propolis were subsequently shaken using an orbital shaker. The microtiter plate was then incubated for 3 days at a temperature of 30°C.
 
Biofilm quatification of crystal violet assay
 
After the incubation period, planktonic cells were removed by carefully aspirating the supernatant. Biofilm fixation was done at room temperature with 150 μl per well of 4% paraformaldehyde (PFA) diluted in phosphate buffer saline (PBS) for 30 minutes. Subsequently, the PFA was removed and the wells were rinsed twice with 200 μl per well of PBS. Evaluation of biofilm formation required staining with 175 μl per well of 0.1% crystal violet pipetted into the wells and incubated for 10 minutes (O’Toole, 2011). Unbound staining was removed and the wells were washed with 200 μl per well of PBS. The microtiter plate was air-dried at room temperature for approximately 3 hours. Remaining color residues in the wells were dissolved by adding 200 μl per well of 30% acetic acid, then 100 μl of the solution was transferred to a new microtiter plate for analysis.
 
Reading of optical density values using an ELISA reader
 
The microtiter plate was subjected to optical density (OD) readings at a wavelength of 595 nm using a microplate spectrophotometer or ELISA Reader (Wahyudi and Wael, 2021).
 
Data analysis
 
To identify significant differences among mean pair, the collected data were subjected to analysis using the Post Hoc Dunn Test.

This research employs honeybee propolis extract as an antibiofilm agent against E. coli in vitro. The decrease in absorbance at 595 nm in the presence of the inhibitor confirms the inhibitory effect of honeybee propolis. It has been proven that biofilm formation by some bacteria is a crucial defense strategy against xenobiotics. There are three antibiotic resistance mechanisms associated with biofilm-forming bacterial strains: the biofilm matrix reduces antibiotic penetration, the chemical composition of the biofilm protects bacterial cells and biofilm-forming bacteria can grow and form spores (Zhao et al., 2023). The bacterium E. coli was treated with concentrations of honeybee propolis extract and incubated for 3 days. Then, the wells were washed to remove planktonic cells and residue. Propolis is a compound known to exhibit antibiofilm activity against Gram-negative bacteria. This is because propolis contains high levels of flavonoids. This study focuses on E. coli, which is a pathogen capable of expressing various virulence factors. Honeybee propolis extract was able to prevent biofilm formation, as indicated by the low optical density values compared to the control group.
       
The average OD values, which vary across each research group, are presented in the following Table 1 and Fig 1. The results presented in Table 1 indicate that the OD values and the average OD values for each treatment and control group. The treatments with concentrations of 0.39 mg/mL, 0.78 mg/mL, 1.56 mg/mL, 3.125 mg/mL and 6.25 mg/mL yielded average values of 0.1715, 0.142375, 0.115, 0.099625 and 0.078857, respectively. The positive control and negative control exhibited average values of 0.205125 and 0.079375, respectively.
 

 

       
Based on the data in Table 1, it is evident that the growth media utilized in this study lacks antibiofilm activity. This is indicated by the absence of a decrease in optical density values. While, in the group treated with honeybee propolis, antibiofilm activity is observed as demonstrated by the decreasing optical density values compared to the control group.The data were then analyzed using post hoc dunn test. The results of the post hoc dunn test in Table 1 indicate that there were no significant differences between the negative control, the concentration of 3.125 mg/mL and the concentration of 6.25 mg/mL (p>0.05). The concentrations of 1.56 mg/mL and 3.125 mg/mL in the treatment group did not change significantly (p>0.05). There were no discernible variations (p>0.05) between the concentrations of 1.56 mg/mL and 0.78 mg/mL. The concentration of 0.39 mg/mL and the positive control showed significant differences compared to all treatments (p<0.05).
       
Honey propolis at concentrations of 3.125 and 6.25 mg/mL which represents the minimum concentrations in the group is capable of inhibiting E. coli biofilm formation in vitro. This is indicated by OD values that do not significantly differ when compared to the negative control. Based on Fig 1, it can be concluded that the higher the concentration of extract added to the medium, the greater the ability of the antibiofilm from the extract. This aligns with the findings of De Marco et al., (2017), which suggest that the higher the concentration of propolis used, the higher the antibiofilm activity. At a concentration of 100 μg/mL, the biofilm mass was lower than at a concentration of 50 μg/mL. Meanwhile, at a concentration of 50 μg/mL, the biofilm mass was lower than at a concentration of 10 μg/mL.
       
The antibiofilm activity of honeybee propolis extract is caused by its high flavonoid content. Flavonoids are active ingredients with antibiofilm ability. The presence of flavonoid compounds in honeybee propolis extract disrupts the adhesion and polymerization processes of biofilm-associated proteins (ALaerjani et al., 2022). Flavonoids also inhibit the icaA and icaD genes responsible for EPS formation. According to Roy et al., (2018), the antibiofilm mechanism involves disrupting bacterial adhesion to surfaces, inhibiting quorum sensing and inhibiting EPS growth. The flavonoids contained in honeybee propolis inhibit the formation of DnaK. DnaK is a protein crucial for biofilm formation, as it enhances the expression of CsgA and CsgB. CsgA and CsgB function to aggregate curli fibers, which is one of extracellular matrix components in biofilm formation (Tursi and Tükel, 2018). According to Buchmann et al., (2023), By stopping the production of amyloid structures CsgA, CsgB and CsgD, flavonoids can stop E. coli and other Gram-negative bacteria from forming biofilms. CsgD is a gene that functions as a connector for adhesion factor production, transportation, metabolism and gene regulation in biofilm formation (Gualdi et al., 2007). CsgA and CsgB are genes involved in forming curli fimbriae. Curli fibers play a critical role in the initial stages of biofilm formation, particularly in the attachment phase. Disruption of curli fiber formation hampers bacterial attachment to surfaces. If the formation of CsgA and CsgB is inhibited, biofilm formation in E. coli cannot occur.
       
According to Bhargava et al., (2021), honeybee propolis extract is capable of inhibiting the motility of Gram-negative bacteria. This occurs due to the reciprocal relationship between curli and flagella that mutually inhibit each other. When cells transition to a non-motile state to form biofilms, the intracellular c-GMP levels increase and activate CsgD, which functions to activate the expression of curli genes. CsgD suppresses flagellar gene operons, thereby reducing flagella synthesis. Decreased bacterial motility can increase the force of repulsion to the surface, making adhesion unstable. To achieve stable attachment, bacteria must suppress or decrease their motility after contact with the surface.

Based on the conducted research, it can be concluded that honeybee propolis extract holds potential as an antibiofilm agent against E. coli in vitro. The concentrations of 3.125 mg/mL and 6.25 mg/mL are the minimum concentrations of honeybee propolis extract capable of inhibiting E. coli biofilm formation in vitro.

This study was supported by the Faculty of Veterinary Medicine, Universitas Airlangga and Ministry of Education, Culture Research and Technology, Republic of Indonesia.

The authors declare that there is no conflict of interest.