Bacterial strains
L. plantarum CGMCC 1.12974 was purchased from the China General Microbial Strain Collection Management Center. The probiotic was seeded in De Man, Rogosa, and Sharpe (MRS) broth and incubated for 24 h at 37°C in parthenogenic anaerobic condition. P. aeruginosa strain PAO1, S. aureus and E. coli isolated from clinical specimens were selected as indicator and grown in Luria–Bertani (LB) broth overnight at 37°C with 200 rpm shaking before use.
In vitro antibacterial activity of honey and L. plantarum
The vitro antibacterial assay was carried out to evaluate the antimicrobial activity of honey and L. plantarum. Briefly, a single colony of L. plantarum was grown in MRS broth at 37°C for 24-48 h. An appropriate amount of bacterial culture was centrifuged at 12000 rpm for 5 min, and the pellet was washed three times and re-suspended with PBS buffer. The concentration of bacteria in the solution was approximately 1 × 109 CFU/mL when the OD600 was 0.7. In addition, a S. aureus single colony were grown in LB broth overnight at 37°C with 200 rpm shaking. There are approximately 1 × 108 CFU/mL of bacteria in the fluid when the culture's OD600 value is 0.1. Subsequently, S. aureus (1×108 CFU/mL), L. plantarum (1×108 CFU/mL), and honey (20% v/v) were mixed together in LB broth (HL), and a negative control (S, S. aureus), L. plantarum control (L, S. aureus + L. plantarum), and honey control (H, S. aureus + honey) were prepared. Each mixture was added to 24-well plates, incubated at 37℃ for 12h. To determine bacterial loads, the sample was serially diluted to an appropriate concentration with PBS buffer before being seeded on a LB agar plate and cultivated at 37 °C for 24 h. Finally, the colonies were counted and CFU/mL was calculated.
Optimal antibacterial formulation of honey and L. plantarum
A multilevel experimental design was used to optimize the honey-L. plantarum formulation. In brief, honey content (X1) and L. plantarum concentration (X2) were set as two independent variables and three levels of each variable according to the results of the preliminary pre-experiment (Table 1). When different ratios of honey and L. plantarum acted together with S. aureus (1×108 CFU/mL) for 12 h, the viable count of S. aureus (Y) was set as the dependent variable. Experimental trials were performed under all nine possible combinations, with three replicate experiments done simultaneously for each combination. Additionally, a blank control (containing only S. aureus, without honey and L. plantarum) was also set. The results were analyzed by SPSS statistics to select the best antibacterial formulation for honey and L. plantarum.
Table 1 3×3 factorial design levels and factors
Factors
|
Level 1
|
Level 2
|
Level 3
|
X1(Honey ratio, %,v/v)
|
10
|
20
|
30
|
X2 (L. plantarum, CFU/mL)
|
107
|
108
|
109
|
Inhibition of S. aureus, P. aeruginosa and E. coli by honey-L. plantarum formulation
The best formulation of honey and L. plantarum selected by the previous step was evaluated for inhibition of S. aureus, P. aeruginosa and E. coli by referring to step “In vitro antibacterial activity of honey and L. plantarum”. In brief, the honey-L. plantarum group (HL, pathogens + honey + L. plantarum), negative control (C, Pathogens), L. plantarum control (L, pathogens + L. plantarum), and honey control (H, pathogens + honey) were configured separately. First, all three bacteria were cultured overnight at 37°C with 200 rpm shaking. Then, the value of OD600 was adjusted to 0.1(1×108 CFU/mL). Next, the mixed formulations with each bacterial, were added to 24-well polyethylene plates at 37°C for stationary culture. At 6 h, 12 h, 18h and 24 h, the cultures were collected and diluted to a suitable concentration with PBS before being seeded on the LB agar plate. Finally, the CFU of bacterial on the LB agar plate was counted after being cultured for 24 h at 37 °C.
Inhibition of biofilm by honey-L. plantarum formulations
The detection of biofilm production was conducted using crystal violet (CV) assay methods[25].The honey-L. plantarum formulation was mixed with S. aureus (5×105 CFU/mL) and P. aeruginosa (5×105 CFU/mL) in LB broth (containing 0.1% glucose), respectively. Separate settings were set to place negative control (S, S. aureus; P, P. aeruginosa), L. plantarum control (L, S. aureus / P. aeruginosa + L. plantarum), and honey control (H, S. aureus / P. aeruginosa +honey). Each bacterial solution was added to a 24-well microplate and incubated at 37°C for 24 h. After incubation, the wells were rinsed twice by 1 × PBS to remove planktonic and non-adhering cells. Biofilms that formed in wells were fixed for 10 min with ethanol (75 %) and stained with CV (0.1% (w/v)). The wells were rinsed with PBS three times. After that, 30% (v/v) of acetic acid was added to every well and kept for 10 min to solubilize adhered biofilm. The optical density (OD) in every well was estimated at 595 nm by Enzyme Markers.
Antibacterial mechanism of honey-L. plantarum formulation
Changes in the growth of L. plantarum in the formulation
In this part, changes in the growth of L. plantarum were evaluated to understand the antimicrobial mechanism of the formulation. The honey-L. plantarum group (HL) and L. plantarum group (L) from step “Inhibition of S. aureus, P. aeruginosa and E. coli by honey-L. plantarum formulation” were incubated for 24h and then the bacterial solution was diluted with PBS. Next, the diluted solution was coated with MRS agar plates, and the number of L. plantarum was counted after incubation at 37°C for 24h. On the other hand, 1×109CFU/mL L. plantarum (L) and 10% honey-L. plantarum (HL) were added to LB broth medium, and the growth of L. plantarum in it was detected on a fully automated microbial growth curve instrument.
Antibacterial effect of honey-L. plantarum culture supernatant
To further investigate the antibacterial effect of honey-L. plantarum formulation, we tested the growth inhibition of S. aureus and P. aeruginosa by the supernatant of honey-L. plantarum cultures and the effect of honey-L. plantarum on the pH value of the medium. Firstly, 10% honey and 1×107 CFU/mL L. plantarum were incubated in MRS medium for 24 h. The cultures were then centrifuged to extract the supernatant and the pH of the medium was tested. After filtering the supernatant through a sterile membrane, it was (10% - 20%, v/v) added to LB (HL) containing 1×107 CFU/mL of S. aureus or P. aeruginosa and mixed well. Meanwhile, S. aureus or P. aeruginosa alone cultured in LB broth were also set as negative controls (C). Finally, the effect of the supernatant on the growth curves of the two bacteria was incubated and measured at 37°C for 24h with a fully automated microbial growth curve analyzer.
In vivo animal experiment
Wound infection model
Eight-week-old male Sprague-Dawley (SD) rats weighing 200 ± 20g were bought from the Chongqing Medical University’s Animal Experiment Center with license number SCXK2018-003. Rats were housed under standard conditions with food and water ad libitum. The experimental animal handling methods conformed to animal ethics standards and were approved by the Experimental Animal Ethics Committee of Chongqing Medical University.
Twelve adult male Sprague-Dawley rats were randomly divided into two groups, six in each group: the control group (S. a) and the medicated group (HL). After one week of adaptation, the experiment started following previously reported methods with modifications [26-30]. After being anesthetized with 10% chloral hydrate (300 mg / kg), the back of each rat was shaved, depilated, and washed with 75% of ethanol.[31, 32]. Following that, a circular wound with a diameter of 10 mm was created on the back of each rat, which was subsequently infected with 30 μL S. aureus (1 × 108 CFU / mL). Blank control (PBS) and honey-L. plantarum (HL) were applied to the wounds of the control group (S. a) and the medicated group (HL) of rats separately after an hour of drying. Afterward, the wounds were covered with commercially available transparent film dressings (3 M Tegaderm) and secured with medical tape. On the 0, first, third, and fifth days, the formulation was changed once a day, and the wound healing was measured and photographed. The rats were euthanized after five days of medicating changes.
Evaluation of the antibacterial effect of honey-L. plantarum formulation on the wounds
The rats were euthanized on the 1st and 5th days after treatment to assess the formulation's antibacterial effect, and the skin tissue along the wound edge was collected and homogenized in 1 ml of PBS with a homogenizer. From that, the sample solution was diluted to the optimal concentration, and 100 μL of the diluted solution was placed on the Baird-Parker ager plate. The number of colonies on the Baird-Parker agar plates were counted after 24 h of incubation at 37°C.
Histological analysis
For histological examination, excised wound skin tissue on days 1, 3, and 5 was fixed in 4% paraformaldehyde for at least 24 h, dehydrated in a graded series of ethanol, followed by xylene, and embedded in paraffin. Tissue sections were obtained from the center of the excised skin tissue and cut into 5 μm thick sections. Skin sections were stained with hematoxylin and eosin (H-E) to assess granulation tissue formation and wound maturity. Finally, the skin sections were observed and photographed using an optical microscope equipped with an imaging system.
Statistical Analysis
All Data were analyzed and graphed using BIM SPSS statistics 25 and Graph Pad Prim 7 software, with quantitative results expressed as mean ± standard deviation (SD). Statistical comparisons were performed using t-test and one-way ANOVA, followed by Tukey’s Multiple Comparison test as the post-hoc test. A significant difference is marked as * (p< 0.05), ** (p < 0.01), *** (p < 0.001) and **** (p < 0.0001).