Effects of Paeonol Sub-inhibitory Concentration on Streptococcus suis Biofilm and Expression of Virulence Genes

Streptococcus suis (S. suis) is a vital gram-positive zoonotic pathogen, that can stick to each other, adhere to the host cell to form a biofilm shape and cause persistent infection, which is related to arthritis, septicaemia, endocarditis, bronchopneumonia, meningitis and other diseases in swine (Tang et al., 2006). The bacterial carrying rate of swines is close to 100% and the mortality rate is close to 20% (Cloutier et al., 2003), which cause huge property damage to the global swine industry (Guo et al., 2009).
       
Bacterial Biofilm (BF) is a membrane-like structure formed by bacteria that combine together by secreting polysaccharide complex and polysacchari, de matrix in order to adapt to harsh environment (Fuente et al., 2013; Wang et al., 2014). The biological activities of bacteria under the biofilm are changed (Wang et al., 2011; Wang et al., 2018; Li et al., 2021). In addition, studies have shown that biofilms are related to the production of bacterial resistance and the expression of virulence factors (Penesyan et al., 2015; Qi et al., 2016).
       
Antibiotics are commonly used to prevent and treat infections caused by S. suis, but the overuse of antibiotics has led to strains showing multiple resistance (Devi et al., 2017; Liu et al., 2020). Chinese herbal medicine has become a hot spot in the research and development of new bacteriostatic agents because of its green, natural and non-toxic advantages (Abdallah et al., 2018; Raju et al., 2018; Millar et al., 2021). Many studies have shown that Chinese herbal medicine has an inhibitory effect on bacterial (Szliszka et al., 2009; Chen et al., 2016; Ding et al., 2017; Acharya et al., 2020). Paeonol, also known as paonol or paeoniflorin, is an active ingredient extracted from the dry root bark of the belladonna vitex (Xu et al., 2005). Studies have shown that paeonol has such effects as sedation, cooling blood and clearing heat, bacteriostasis and anti-tumor (Li et al., 2010; Zhang et al., 2019). Recent studies have also shown that paeonol can inhibit bacterial adhesion and thus affect their biofilms (Qian et al., 2021). Based on this, paeonol was selected in this study to explore its effects on S. suis biofilm and the expression of virulence genes.

This experiment was conducted in Luoyang Key Laboratory of Animal Molecular Pathogens and Immunology, Henan University of Science and Technology from November 2020 to August 2021 to study the effects of paeonol on S. suis biofilm and the expression of related virulence factors.
 
Bacterial strains and paeonol susceptibility testing
 
HA9801 (HA) was isolated from swine infected with S. suis in Jiangsu Province which identified as a virulent strain (Wang et al., 2014). Add 100 mg paeonol to 10 ml sterile distilled water which containing 10% Tween-80 (Papandreou et al., 2002), the final concentration of paeonol mother liquor was 10 mg/ml. HA strains were grown in Tryptone soya broth (TSB) (Thermo Fisher Scientific, Shanghai, China). The MIC of Paeonol to HA was determined according to the double dilution method stipulated by CLSI.
 
Effects of paeonol on biofilm formation under sub-inhibitory concentration
 
Crystal violet staining method was used to determine the effect of paeonol on the biofilm formation. A single colony was placed into 3 ml of TSB and incubated density to 10⁶ CFU/mL (Li et al., 2021). HA suspension was added to the 96-well plate (Costar, Corning, Shanghai Aiyan Biological Technology Co., LTD, Shanghai, china), followed by adding 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC paeonol solutions respectively. After 48 h of culture, the sample wells were washed lightly twice with 200 μl Phosphate buffered saline (PBS) and immobilized with 95% methanol (w/w) at 37°C for 20 min. Then stained the biofilm with 0.1% crystal violet for 15 min and dissolved with 95% ethanol for 30 min, the OD value was measured at OD₅₉₅ nm using Automatic microplate reader (Infinite M Nano, Tecan Trading Co., Ltd., Shanghai, China). All assays were set 6 replicate controls and repeated 3 times.
 
Field emission scanning electron microscopy (FE-SEM)
 
The structure of Streptococcus suis biofilm were observed by Field emission scanning electron microscopy (FE-SEM). 1 ml of HA with the final concentration of 10⁶ CFU/ml was added to the 24-well plate of cell culture plate (Corning, Beijing Yaanda Biotechnology Co., Ltd. Beijing, China) and 1 ml of TSB containing 1/2 MIC and 1/4 MIC paeonol were added respectively and cultured at 37°C for 48 h. The slides were washed with PBS lightly, adding 2.5% pentaglycol and immobilized at 4°C overnight and washed with PBS for 3 times, the slides were dehydrated in gradients of 30%, 50%, 70%, 80% and 90% ethanol (w/w). After being added for 15 min each time, the slides were finally washed twice with 100% ethanol (Yi et al., 2020). After drying, the gold-plated film is sputtered and the air is pumped, then the scanning electron microscope is used to observe the biofilm structure at 5.0 KV (JSM-7800F; Japan Electronics Co. LTD, Tokyo, Japan).
 
Effects of paeonol on mature biofilm under Sub-inhibitory concentration
 
A single colony was placed into 3 ml of TSB and incubated density to 10⁶ CFU/mL After multiple dilution, HA was added to 96-well plates with 6 replicates in each group. Culture 48 h, suspension was absorbed, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC paeonol solutions were added respectively. Incubate for another 8 h, the detection method was carried out in accordance with the method for the effect of paeonol on the formation of biofilm.
 
Quantitative RT-PCR
 
Detection of HA virulence genes expression level by qRT-PCR. In brief, cultivate a single colony in TSB overnight, transfer to TSB containing 1/4 MIC or 1/8 MIC paeonol according to 1:100 and cultivate for 8 h. The total RNA of HA was extracted by TRIzon (Shenggong Biological Engineering Co., Ltd, Shanghai, china) method and the cDNA obtained by reverse-transcription was used PrimeScriptTM reagent kit (CoWin Biosciences Co., Ltd, Biejing, china). qRT-PCR was used for detecting the transcription levels of virulence genes ef, gapdh, srtA, fbps, gdh, mrp and 16S rRNA were calculated by using the 2^-ΔΔCT method, the 16S rRNA gene as an internal control. Primer information is shown in Table 1.
 

 
Data analysis
 
Statistical analysis was performed using SPSS 24.0. Biofilm formation and mature biofilm results are based on one-way analysis of variance (ANOVA), qRT-PCR results are based on two-way analysis of variance. The data presented represent three independent experiments and are represented as mean±SE. Control group without paeonol. *p<0.05 was considered statistically significant.

Paeonol susceptibility testing
 
The results showed the MIC of paeonol to HA was 640 μg/mL. The tween-80 which used to dissolve paeonol had no significant effect on the life activities of HA, so we excluded the interference caused by tween-80 to the experiment.
 
Sub-inhibitory concentration paeonol inhibited biofilm formation
 
Sub-MIC drug was selected to rule out the possibility that paeonol could weaken the biofilm by inhibiting the growth of HA. The results (Fig 1) showed that in the range of 1/2 MIC to 1/8 MIC, the number of biofilms showed a downward trend as the concentration of paeonol changed. 1/4 MIC and 1/8 MIC Paeonol significantly affected the ability of HA biofilm formation. Then as the concentration of paeonol decreased, the ability of HA to form biofilm gradually recovered.
 

 
Morphology of biofilm under field emission scanning electron microscope (FE-SEM)
 
The structural variation difference of HA biofilm was observed under scanning electron microscope after paeonol treatment in control group (A), 1/4 MIC paeonol (B) and 1/8 MIC paeonol (C). The FE-SEM image shows a three-dimensional structure by the adhesion of HA on the cell slipper. The magnification of electron microscope is 5000 ×. From the electron microscope (Fig 2), It was observed that the biofilm of the control group was compact and HA was tightly folded. After the sub-MIC (1/4 MIC, 1/8 MIC) paeonol action, the HA quantity was reduced and the membrane structure became dispersed and fragmental. In addition, the biofilm structure under the effect of 1/8 MIC paeonol was looser than that under the effect of 1/4 MIC paeonol, indicating that the effect of 1/8 MIC paeonol on the biofilm formation was greater than that under the effect of 1/4 MIC paeonol. This is also consistent with our previous conclusion that paeonol has a significant effect on the biofilm formation at 1/4 MIC and 1/8 MIC.
 

 
Sub-inhibitory concentration paeonol inhibited mature biofilm
 
The biofilm was placed in automatic microplate reader to determine the value of OD₅₉₅ nm. Compared with the control group (Fig 3), the paeonol in the experimental concentration range has a certain degree of influence on the biofilm of HA. But the effect of paeonol on the formation of biofilms is much greater than that on mature biofilms.
 

 
Regulation of paeonol on virulence factors
 
1/4 MIC and 1/8 MIC paeonol had the most significant effect on the biofilm. Therefore, the 1/4 MIC and 1/8 MIC paeonol were selected in this experiment to explore the effects on the expression levels of HA-related virulence genes. The results (Fig 4) showed that 1/4 MIC paeonol down-regulated the transcription levels of ef, gapdh and srtA virulence genes in HA, while 1/8 MIC paeonol up-regulated the expression levels of ef, gapdh, srtA, fbps, gdh and mrp virulence genes in HA, with the most significant effect on mrp. Up-regulation of mrp virulence gene expression can inhibit bacterial adhesion, so as to inhibit the formation of HA biofilm (Wang et al., 2011). Studies have also shown that mrp is related to bacterial biofilm and the expression of virulence and the up-regulation of mrp is also inextricably linked to the decrease of biofilm (Wang et al., 2011; Wang et al., 2014).
 

       
In the presence of sub-MIC tetracycline, the biofilm formation ability and the number of viable bacteria under the envelope increase in Streptococcus pneumoniae, which may be regulated by the LuxS/AI-2 density sensing system (Ahmed et al., 2009). Sub-MIC amoxicillin can induce the production of extracellular genes of Staphylococcus aureus, thereby stimulating the formation of biofilms (Mlynek et al., 2020), Azithromycin with sub-MIC could weaken the formation of biofilm and increase the content of capsular polysaccharide (Yang et al., 2016), Paeoniflorin inhibits the AI-2 signaling molecules in Streptococcus suis through mass effect, thereby attenuating the formation of Streptococcus suis biofilm (Li et al., 2021), Paeonol down-regulated genes related to density sensing in Pseudomonas aeruginosa and attenuated the virulence of the pathogen (Yang et al., 2021). Therefore, the effect of paeonol with low inhibitory concentration on HA biofilm may also be realized by affecting the density sensing system or the synthesis and secretion of exopolysaccharide and the specific regulatory mechanism needs to be further studied.

Paeonol sub-MICs affects biofilm formation and virulence gene expression in S. suis. These findings suggested that investigating the effect of Chinese herbal medicine paeonol on bacterial biofilms may lead to the development of natural substance treatments.

We are grateful to Chenlong Mao, Mengxia Gao and thank all the researchers at the Key Laboratory of Animal and Poultry Molecular Etiology and Immunology of Henan University of Science and Technology for their assistance.

JG contributed to the execution of the trial and the writing of the initial draft. PL and QX performed the design of the experiment. Material preparation, data collection and analysis were conducted by YF and LZ. The funds were collected by YW and YW. All authors read and approved the final manuscript.

The datasets generated during and analyzed during the study are available from the corresponding author on reasonable request.

Not applicable.

This work was supported by the National Natural Science Foundation of China (32172852, 31772761), Excellent Youth Foundation of He’nan Scientific Committee (222300420005) Student Research Training Program (SRTP) of Henan University of Science and Technology (2020360, 2021386).

Declaration of competing interest
 
The author declares that there is no conflict of interest between the publication of this paper and other persons or organizations.

None.