Inhibition of quorum sensing in drug-resistant Staphylococcus epidermidis by flavaspidic acid BB

Background: To investigate the effect of flavaspidic acid BB (BB) on biofilms and quorum sensing-related genes of drug-resistant Staphylococcus epidermidis (SE) and to provide a theoretical basis for the development of BB as a new type of quorum sensing inhibitor. Methods: The microdilution method was applied to screen 15 clinical isolates of S. epidermidis for drug-resistant S. epidermidis to be used as the test strain. The effects of BB on the biofilms of drug-resistant S. epidermidis at different growth stages were studied using the cell counting kit-8 assay and scanning electron microscopy. The gene expression of sigB, sarA, and luxS, regulators involved in quorum sensing of biofilm formation, was measured by PCR. Results: The minimum inhibitory concentrations of BB against 15 clinical strains of S. epidermidis ranged 11.67–66.67 µg/mL. BB significantly inhibited biofilm formation by drug-resistant SE06 in the aggregation and maturation stages, with an activity superior to those of mupirocin and fusidic acid ( P < 0.05). At 40 µg/mL, BB reduced the secretion of extracellular polymers and promoted the disruption of biofilm structure. At 80 µg/mL, BB specifically interfered with the quorum sensing system of drug-resistant SE06 by downregulating sarA and luxS, thereby inhibiting this microbe’s biofilm formation. Conclusion: BB exhibited strong inhibitory activity against SE06 by suppressing biofilm formation and downregulating quorum sensing-related genes and was identified as a new potential quorum sensing inhibitor against S. epidermidis .


Background
Skin and soft tissue infections are defined as bacterial invasions of the epidermis, dermis, and subcutaneous tissues [1].Regarded as a widely distributed and important opportunistic pathogen, S. epidermidis is normally found on the human epidermis and mucosa and is one of the main pathogens causing skin and soft tissue infections [2,3].Common symptoms of clinical S. epidermidis infection in the skin and soft tissues include venous ulcers, traumatic ulcers, diabetic ulcers, and other chronic wounds; the formation of biofilms has a significant impact on wound healing and the development of drug resistance [4,5].Improper treatment can lead to the necrosis of blood vessels and nerve tissues, which can be life-threatening in serious cases [5].
Quorum sensing regulatory components of S. epidermidis biofilm formation consists of the accessory gene regulator (Agr) system and the LuxS/AI-2-mediated system.SigB, sarA, and luxS are important regulatory genes in the quorum sensing system of S. epidermidis.SarA specifically binds to the P2 and P3 promoters of the AIP-mediated quorum sensing Agr operon and activates the transcription of RNA II and III [6].Deletion of sigB in S. epidermidis leads to the upregulation of icaR and a reduction in biofilm formation [7].The major global regulator sarA and the alternative sigma factor sigB promote the expression of the Agr operon, regulate the ica system, and upregulate poly N-acetyl glucosamine, which induces the synthesis of polysaccharide intercellular adhesin (PIA), an outer membrane protein essential for the bacterial extracellular matrix, thus promoting surface adhesion and biofilm formation [8,9].In the LuxS/AI-2-mediated interspecies quorum sensing system, the AI-2 signalling factor, a universal signalling molecule among bacterial species, is synthesised by the LuxS enzyme encoded by the luxS gene and is secreted from the bacterial cell.AI-2 secretion affects the capacity for biofilm formation by regulating the ica operon and transcription of genes encoding biofilm-associated proteins.LuxS synthase is highly conserved and has the same biosynthetic pathway and molecular structure as that of other strains [10].
A variety of inhibitors of quorum sensing signalling molecules capable of inhibiting bacterial biofilm formation exist in nature [11].Dryopteris fragrans is a herb commonly used in folk medicine formulations for the treatment of skin diseases and is especially effective in treating bacterial and fungal infections, such as dermatophytosis and rashes [12,13].The main active ingredients of Dryopteris fragrans are phloroglucinols, and the in vitro antibacterial activity of BB is the most pronounced.This compound exhibits desirable inhibitory effects on gram-positive bacteria [14,15]; however, its inhibitory effect on the quorum sensing-related genes of drug-resistant S. epidermidis has not been reported.
In this study, we hypothesised that flavaspidic acid BB (BB) has a significant impact on the formation of bacterial biofilms and functions as a quorum sensing inhibitor (QSI) in drug-resistant S. epidermidis.Our objective was to investigate the effects of BB on biofilm structure and formation, cell viability, and the expression of quorum sensing-related genes at different stages of drug-resistant S. epidermidis growth.These data should help evaluate the potential of BB for the development of a QSI acting on S. epidermidis and provide a theoretical basis for such a development.

Strains, media, and culture conditions
Fifteen clinical isolates of S. epidermidis were identified and provided by Guangdong Lewwin Pharmaceutical Research Institute Co., Ltd.(Guangdong, China) (Supplementary Figure S1).The quality control (QC) strain Staphylococcus aureus (ATCC 29213) was provided by the Microbial Culture Collection Center of Guangdong (Guangdong, China).All the strains were cultured in test tubes containing nutrient agar at 37 °C for 24 h and thereafter incubated in Cation-adjusted Mueller-Hinton broth or tryptone soy broth (TSB) (Guangdong Huankai Microbial Technology Co., Ltd., Guangzhou, China).The concentration was adjusted to 1 × 10 6 CFU/mL using the 0.5 McFarland standard.

Antibacterial activity of selected compounds against S. epidermidis
The clinical isolates were screened for drug-resistant S. epidermidis using a microdilution assay according to CLSI M07-A9 guidelines.Briefly, the drug stock solution was diluted in 96-well plates (Cellstar ® ; Greiner Bio-One Co., Ltd., Frickenhausen, Germany) with Cation-adjusted Mueller-Hinton broth by two-fold serial dilution to a final volume of 100 μL in the concentration range of 2,560 to 10 µg/mL.Next, 1 × 10 6 CFU/mL of the prepared bacterial suspension was added to each well of a 96-well plate.The plates were incubated at 37 °C for 24 h.The lowest concentration of the agent that inhibited the growth of S. epidermidis was considered the minimum inhibitory concentration (MIC).Minimum bactericidal concentrations (MBCs) of the drugs were determined by adding 50 μL of the suspensions without bacterial turbidity on the surface of the agar, and the lowest concentration without colony formation was considered the MBC.Drug-resistant S. epidermidis strains were screened according to CLSI M100-S22 guidelines and the relevant literature as test microorganisms for subsequent experiments [19].Based on the MIC of BB against drug-resistant S. epidermidis, 10, 20, 40, and 80 µg/mL were selected as the test concentrations of BB for the subsequent experiments.
S. aureus (ATCC 29213) was used as the QC strain, and cefazolin was used as the QC drug.The assay was considered valid and reliable if the MIC of the QC strain was within a range of 0.25-1 μg/mL under the conditions of parallel operation.

Determination of the biofilm inhibitory effect of BB Determination of biofilm biomass of S. epidermidis by cell counting kit-8 (CCK-8) assay.
The effect of drugs on biofilm formation by drug-resistant S. epidermidis was evaluated by measuring biofilm biomass using CCK-8 assay [20].TSB was used to prepare a suspension of drug-resistant S. epidermidis at a concentration of 1 × 10 6 CFU/mL.Next, 200 μL of the bacterial suspension was added into each well of 96-well culture plates, and the plates were incubated at 37 °C for different durations (3, 8, and 24 h) without agitation.At the end of the incubation period, the cultures were removed, and the wells were washed three times with sterile PBS to remove non-adherent cells.After that, drugs were added into the wells at different final concentrations (10,20,40, and 80 µg/mL) and incubated for 24 h.After adding 10 μL of the CCK-8 solution into each well, the plates were incubated for 1 h.The change in the biofilm adhesion rate due to drug administration was calculated by measuring the A 450nm nm using an ELISA plate reader to indirectly determine the number of viable cells according to Equation (1): Observation of biofilm microstructure of S. epidermidis by scanning electron microscopy (SEM).The effects of BB and E on the biofilm formation of drug-resistant S. epidermidis were visualised using SEM [21].Biofilms of drug-resistant S. epidermidis at different growth stages were established on glass coverslips in 24-well plates containing TSB with or without the drug and incubated at 37 °C for 3, 8, and 24 h.The glass coverslips with growing biofilm were washed with PBS, and the samples were transferred to 2.5% glutaraldehyde and soaked overnight at 4 °C.The samples were dehydrated in graded ethanol solutions, placed in a desiccator, coated with gold-palladium alloy, and examined under a JEM-2100 scanning electron microscope (JEOL, Tokyo, Japan).

Quantification of sigB, luxS, and sarA mRNA expression
PCR was performed to determine the effects of BB on the expression of sarA, sigB, and luxS in drug-resistant S. epidermidis at the transcriptome level.Drug-resistant SE06 was treated with BB (20, 40, or 80 µg/mL) for 24 h.Total RNA was extracted from drug-resistant S. epidermidis using the TRIzol kit (Invitrogen, Takara Bio Inc., Beijing, China) according to the manufacturer's instructions.cDNA was synthesised using reverse transcriptase.The samples were placed in a thermocycler programmed as follows: 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min.The PCR products were subjected to agarose gel electrophoresis, and two bands of different sizes corresponding to the target fragments of the exogenous and endogenous genes were visualised using a gel imaging system.The mean gray value (MGV) of the bands was quantified using ImageJ software [22].Sequences of genes sigB, sarA, and luxS (ATCC 12228, CP043845.1)were retrieved from the NCBI nucleic acid database and are listed in Table 1.The 16S rRNA gene was used as the internal reference.The relative expression of the target genes was calculated according to Equation (2):

Statistical analysis
All experiments were conducted in triplicate in a single multi-well plate on three different days.Mean values were calculated for biofilm formation and percent biofilm inhibition, and the means were compared using ANOVA and Tukey's multiple comparison test (with significance set to P < 0.05) using the SPSS software.

Determination of MIC and MBC of BB
The MIC and MBC of the drug against the 15 clinical isolates of S. epidermidis were determined using the broth microdilution method (Table 2).The MIC of BB against 15 clinical strains of S. epidermidis was 11.67-66.67µg/mL.The MICs of the positive control drugs against SE06 were as follows: E (213.33 µg/mL) > MUP and FD (2,133.33µg/mL).The MICs and MBCs of the positive controls met the drug resistance criteria (as prescribed in CLSI M100-A2 guidelines).An MBC/MIC ≥ 32 indicates that the microorganism is resistant to the tested drug [19].The results showed that four strains -SE06, SE09, SE10, and SE11 -were resistant to the three positive control drugs.BB had the lowest MIC against SE06 among the MICs against the four strains mentioned above, indicating that SE06 was more sensitive to BB than were the other three strains.Therefore, SE06, which was resistant to the three positive drugs and sensitive to BB, was selected as the test strain for the analysis of biofilm inhibition and expression of genes related to biofilm formation and quorum sensing.
The MIC of cefazolin against the S. aureus QC strain was 0.71 µg/mL, which was within the range of 0.25-1 μg/mL, indicating that the results of this experiment were reliable.

Effects of BB on biofilm adhesion
The biofilm-inhibitory activity of BB was confirmed at different stages of biofilm formation.As shown in Figure 2, minimal biofilm eradication concentration of BB against SE06 was 40 μg/mL at all aggregation stages.Figure 2A shows that drug resistance in the initial adhesion stage was relatively low, and the inhibitory effects were ranked in descending order as follows: E > BB > FD > MUP.The biofilm adhesion rates were 3.83% and 9.94% in the presence of 10 µg/mL E and 40 µg/mL BB, respectively.
Figure 2B shows that the resistance of biofilms to the drugs increased as biofilm maturation progressed during the aggregation stage.The adhesion rates were 97.31% and 98.36% in the presence of 80 µg/mL MUP FD, respectively.The biofilm adhesion rate was 8.19% after treatment with 40 µg/mL E. The biofilm adhesion rate when treated with 40 µg/mL BB was only 3.14%, indicating the superior anti-adhesive effect of BB compared to the effect of other drugs.
As presented in Figure 2C, the biofilms in the maturation stage formed a complete three-dimensional reticular mesh structure with strong drug resistance.No inhibitory activity was observed with 80 µg/mL MUP or FD.The adhesion rate of biofilms treated with 40 µg/mL E was 38.65%, whereas that of biofilms treated with the same concentration of BB was 32.04%.Thus, 40 µg/mL BB exerted significant anti-adhesive effect (P < 0.05) on the biofilm of SE06 at all growth stages.

Effects of BB on biofilm structure
The structural features of the biofilms of drug-resistant S. epidermidis were examined before and after treatment with BB, with E as the positive control (Figures 3, 4).In the absence of drug treatment, the bacteria tended to form large agglomerates and aggregated into distinct clusters with visible dispersion of extracellular polymers among the clusters.After treatment with BB, the biofilms appeared to swell and rupture in the initial adhesion stage and dispersed more during the aggregation and maturation stages, with a reduction in bacterial aggregation.The dispersion of bacterial cells on the surface of BB-treated biofilm was greater than that on the surface of biofilms treated with E at the same concentration, and some fields of view showed reduced secretion of extracellular polymers and well-pronounced intercellular gaps; however, no swelling, rupture, or complete disintegration of the biofilm was observed.

Expression of genes of the quorum sensing system
The influence of BB on the key genes of the quorum sensing system in drug-resistant S. epidermidis was examined using PCR and ImageJ analysis.The relative changes in the expression of the tested key genes are shown in Table 3.Compared with their expressions in a blank control, sarA and luxS in the biofilm were significantly downregulated 2.06 and 2.19 times, respectively, after 80 µg/mL BB treatment (P < 0.05).SigB was also downregulated by 1.89-fold after 80 µg/mL BB treatment, but the result was not statistically significant (P > 0.05).

Discussion
Quorum sensing system is closely associated with bacterial biofilm formation, and QSIs can suppress biofilm formation and biofilm-associated infections by interfering with the regulation of bacterial quorum sensing [23].
In the present study, we screened 15 clinical isolates and used the drug-resistant strain SE06 for further analysis; BB showed a significant inhibitory effect on SE06 biofilm formation.SEM revealed that 40 µg/mL BB promoted the dispersion or swelling of cells during the initial adhesion stage; in the aggregation stage and maturation stage, 40 µg/mL BB effectively reduced the secretion of extracellular polymeric substance, inhibited its ability to wrap bacterial extracellular vesicles, and promoted the dispersion of bacterial cells in the biofilms.Moreover, 40 µg/mL BB inhibited the biofilm of drug-resistant S. epidermidis in the initial adhesion, aggregation, and maturation stages by more than 50%.Therefore, we hypothesised that the inhibitory effect of BB on gram-positive bacterial biofilms is based on its influence on the system regulating population behaviour that plays a key role in microbial cell differentiation and biofilm structure formation, that is, the quorum sensing system [8,24].
To investigate the effect of BB on the quorum sensing system of drug-resistant S. epidermidis, we tested the relative expression levels of several key genes of the quorum sensing system that may be potentially regulated by SE06.The mRNA expression levels of sigB, sarA, and luxS were examined using PCR and ImageJ densitometry.The results showed that 80 µg/mL BB significantly repressed sarA, a key gene of the AIP-mediated quorum sensing system (P < 0.05).Tao et al. found that sarA knockout resulted in defective biofilm formation by S. epidermidis and reduced S. epidermidis resistance to E and kanamycin [25].In S. epidermidis, S. aureus, and methicillin-resistant S. aureus, mutations in the sarA gene hampered the production of PIA, a major component of biofilms, thereby impairing biofilm formation [6,26].Thus, the downregulation of sarA by BB probably directly interferes with the synthesis of PIA in Staphylococcus spp., thus affecting the normal development of biofilms and reducing the pathogenicity and drug resistance of these bacteria.SigB is an upstream gene of sarA that positively regulates sarA and promotes PIA production and the transcription of cell wall adhesion factor-related genes.In addition to sigB, the MgrA protein, ArlS-ArlR two-component system, and other factors influence sarA expression [27,28].Here, 80 µg/mL of BB was proven to downregulate sigB, but the finding was not statistically significant (P > 0.05).This suggests that BB affects the normal expression and regulatory role of sarA by inhibiting pathways other than those of sigB.
In addition, the expression of luxS, a key gene in the LuxS/AI-2-mediated quorum sensing system, was significantly inhibited by 80 µg/mL BB (P < 0.05).The LuxS protein encoded by luxS is a key enzyme in the synthesis of AI-2, a "universal signal for intercellular communication", and hence, the LuxS/AI-2 system is known as a universal interspecies quorum sensing system [29].By exogenously adding AI-2 signalling molecules, Xue et al. demonstrated that AI-2 enhanced the transcription of the ica operon and bhp, a gene encoding a biofilm-associated protein, thus significantly improving the formation of S. epidermidis biofilms [30].LuxS is an essential factor in the activation of methyl cycle pathways, and luxS mutations lead to metabolic dysfunction in bacteria, resulting in strain growth defects [31,32].Therefore, we hypothesised that the function of BB is the same as that of the currently identified inhibitors of the LuxS/AI-2-mediated quorum sensing system, that is, downregulation of LuxS.On the one hand, LuxS inhibits the synthesis of AI-2 signalling molecules and interferes with the regulation of biofilm formation via LuxS/AI-2-mediated quorum sensing, and on the other hand, it hinders the activation of methyl cycle pathways and metabolic homeostasis, affecting the normal growth and development of various bacterial species and their biofilms.
In summary, as a QSI, BB exhibited a strong suppressive effect on the biofilms of drug-resistant S. epidermidis at different growth stages and caused significant repression of sarA and luxS.It can be inferred that BB inhibits biofilm formation in drug-resistant S. epidermidis by suppressing the quorum sensing system, thereby regulating the population behaviour of this bacteria.The results reveal the mechanism of action of BB on bacterial biofilms through the quorum sensing system and highlight the potential of BB as a novel QSI, providing a theoretical basis for its development.

Conclusion
The results reveal the mechanism of action of BB on bacterial biofilms through the quorum sensing system and highlight the potential of BB as a novel QSI, providing a theoretical basis for its development.

Figure 2
Figure 2 Biomass reductions in biofilm after exposure to antimicrobials.(A) 3 h, initial adhesion stage.(B) 8 h, aggregation stage.(C) 24 h, maturity stages.Each group was tested in triplicate and data presented as mean values of biofilm densities in OD 450nm ; * Means statistically significant values (P < 0.05), according to a t-test.CG, control group; MUP, mupirocin; E, erythromycin; FD, fusidic acid; BB, flavaspidic acid BB.

Figure 3 Figure 4
Figure 3 Effect of SE06 biofilm following treatment with BB for different stages by SEM.Scale bar is 1 μm (×5,000).BB, flavaspidic acid BB.

Table 1 Nucleotide sequences of primer used for PCR in this study
a manuscript: https://www.tmrjournals.com/tmr

Table 2 In vitro antimicrobial susceptibilities of S. epidermidis clinical isolates
a ND, not detected, because of out of range MIC or MBC value (> 2,560.00μg/mL).MUP, mupirocin; E, erythromycin; FD, fusidic acid; BB, flavaspidic acid BB.

Table 3 Changes in relative expression of key genes
a Means statistically significant values (P < 0.05) in comparison with control group, according to a t-test.BB, flavaspidic acid BB.