Effect of Panax quinquefolius extract on Mycobacterium abscessus biofilm formation

Abstract Mycobacterium abscessus (M. abscessus) can exist either as planktonic bacteria or as a biofilm. Biofilm formation is one of the important causes of conversion to resistance to antibiotics of bacteria that were previously sensitive when in their planktonic form, resulting in infections difficult to manage. Panax quinquefolius and its active ingredient ginsenosides have the potential ability in fighting pathogenic infections. In this study, the P. quinquefolius extract (PQE) showed good antibacterial/bactericidal activity against the M. abscessus planktonic cells. The extract reduced the biomass, thickness, and number of M. abscessus in the biofilm and altered its morphological characteristics as well as the spatial distribution of dead/alive bacteria. Moreover, the ginsenoside CK monomer had a similar inhibitory effect on M. abscessus planktonic bacteria and biofilm formation. Therefore, PQE and its monomer CK might be potential novel antimicrobial agents for the clinical prevention and treatment of M. abscessus, including biofilms in chronic infections.


Introduction
Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterium (NTM). It is widely present in the natural environment, human life, and iatrogenic environments, such as soil, dust, water sources, various plumbing systems, medical catheters, and implants (Thomson et al. 2013;Falkinham 2015;Faria et al. 2015). It is an opportunistic pathogen, particularly affecting patients with immune deficiencies and underlying lung diseases, such as cystic fibrosis and bronchiectasis (Floto et al. 2016;Cowman et al. 2019;Santos-Silva et al. 2019;Brugha and Spencer 2021). The adaptive evolution of NTM has led to the acquisition of inherent resistance to most clinical antibiotics, including many anti-tuberculosis drugs (Bryant et al. 2016;Bryant et al. 2021). The current treatment of M. abscessus infections is represented by multidrug regimens, usually consisting of macrolides (azithromycin or clarithromycin), b-lactams (imipenem or cefoxitin), aminoglycosides (amikacin), and other reinforcing agents (tigecycline or clofazimine) over a lengthy period (Floto et al. 2016;Daley et al. 2020). However, long-term antibiotic therapy against M. abscessus causes not only reduced patient compliance and accumulation of toxic side effects but also adaptive resistance and acquired resistance (in addition to the intrinsic resistance), ultimately leading to an unsatisfactory clinical outcome and chronic infection (Ferro et al. 2016;Kwak et al. 2019).
It is well known that much of the persistence of infectious disease is largely associated with bacterial biofilm formation and that biofilm-harboring bacteria increase the resistance to antibiotics (approximately 50-5000 times more resistant than planktonic bacteria) and host immunity (Mu et al. 2014;Clary et al. 2018;Chakraborty et al. 2021). NTM readily forms biofilms in nutrient-poor aqueous environments and medical surfaces (Faria et al. 2015;Ghosh et al. 2017). M. abscessus biofilm has been found in the airway (Qvist et al. 2015) and lung cavity (Fennelly et al. 2016) of patients, and its formation has exacerbated the current status of the treatment.
The scarce quantity of drugs against M. abscessus urgently needs to be supplemented with new and effective candidates. Panax quinquefolius is one of the most widely used herbs in Traditional Chinese medicine, with a variety of pharmacological activities, such as anti-diabetic effect, anti-obesity, anti-ageing, anti-cancer, neuroprotective, immune-boosting, and anti-pathogenic microbial effects, as preliminarily proven by numerous studies in vitro and in vivo. P. quinquefolius contains a variety of active substances mainly consisting of ginsenosides, polysaccharides, flavonoids, and phenols (Szczuka et al. 2019;Yang et al. 2020). P. quinquefolius has good bioactivity as an anti-fungal, anti-viral, and anti-bacterial (Kachur and Suntres 2016;Kim and Yang 2018;Wang et al. 2020), showing inhibitory effects against the respiratory bacteria Pseudomonas aeruginosa and Streptococcus pneumoniae (Song et al. 2010;Iqbal and Rhee 2020;Alsayari et al. 2021). However, only a few studies are available on the antimicrobial activity of P. quinquefolius against bacteria of the genus Mycobacterium. Therefore, this study aims to investigate the effect of P. quinquefolius extract (PQE) against M. abscessus, explore its anti-biofilm formation mechanism, and search for effective active monomers.

Extract preparation, strains, media and reagents
The dried root and leaf stem of P. quinquefolius to obtain the PQE was purchased from Huilin Biotechnology Inc. (Xi'an, China). Five ginsenosides were purchased from Derick Biotechnology Inc. (Chengdu, China). A mass of 100 g of dried P. quinquefolius root and leaf stem was mixed with 2000 mL of ethanol solution (75%, v/v), stirring continuously at 25 C for 24 h. The filtrate was concentrated using a rotary evaporator (RE-3000A, Yarong, Co. Ltd., China) at 45 C and then lyophilized to obtain a dried powder. The extract was stored in a cool, dry place for subsequent use.
M. abscessus ATCC 19977 was purchased from the American type culture collection and recovered aerobically at 37 C in 7H10 agar-ADN-0.5% Tween 80. 7H9 broth-ADN-0.5% Tween 80 was used for planktonic bacteria growth. Sauton medium was used for biofilm formation and growth. ddH 2 O water was used as a solvent for PQE. DMSO was used as a solvent for ginsenosides. The PQE and ginsenosides were sterilized using 0.22 lm membrane filtration and then frozen at À20 C for subsequent use.

Susceptibility of the planktonic cells of M. abscessus to PQE
M. abscessus was cultured in 7H9 broth-ADN-0.5% Tween 80 until the OD 600 reached approximately 0.5 and it was subsequently diluted to 2Â10 5 CFU mL À1 . The antimicrobial efficacy of PQE was determined by microplate Alamar blue/resazurin assay (MABA) . Briefly, PQE was serially diluted from 200 mg mL À1 in two-fold dilution order. Then the plant extract solution and M. abscessus suspension were added to each well of a microplate. The microplate was placed in a 5% CO 2 incubator at 37 C for 1 day and then the growth well was tested by adding 1% Resazurin solution and incubated overnight to observe whether the color changed from blue to red, at which time 1% Resazurin solution was added to all the remaining wells and incubated overnight. The fluorescence was measured at 540/590 nm using a multifunctional microplate analyzer (EnVision, PerkinElmer, USA), and the growth inhibition of M. abscessus at different concentrations of the agent was calculated. The MIC 90 was defined as the concentration of P. quinquefolius that caused 90% inhibition of M. abscessus growth.
After the microdilution drug sensitivity test in a 96-well plate, the viability of M. abscessus treated with PQE was evaluated by collecting 50 lL culture from each minimum inhibition concentration (MIC 90 ), and MIC 90 above three wells (without obvious bacterial growth wells). The 10-fold dilutions of culture were plated onto a 7H10 agar plate, the colonies were counted after incubation at 37 C for 3 days. The minimum drug concentration killing 99.9% of M. abscessus was considered the minimum bactericidal concentration (MBC) of P. quinquefolius.
The CFU counting assay was also performed to investigate the effect of PQE on M. abscessus planktonic bacteria. M. abscessus suspension diluted in 7H9 broth-ADN-0.5% Tween 80 was added in a 96-well plate and the PQE concentration of 25 mg mL À1 and 50 mg mL À1 , then the plates were incubated at 37 C. The bacterial culture without PQE was used as the negative control. The CFU of the cultures was determined at 0, 2 and 4 d by plating 50 lL aliquots of 10fold dilutions onto 7H10 agar plates. After 2-4 d of incubation, the average CFU count for each group was plotted at different times.

Drug susceptibility of M. abscessus biofilms to PQE
The inhibitory effect of PQE on total biomass during biofilm formation in M. abscessus was quantified by crystal violet staining. Briefly, a saturated culture of M. abscessus was diluted using the Sauton's medium into a 96-well plate. PQE was added to each well with the final concentrations of 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 mg mL À1 . The plate was placed in a 5% CO 2 incubator at 37 C for 5 d to obtain the mature biofilm. Then, the excess medium was aspirated from the wells, which were washed once with sterile PBS to remove the planktonic bacteria. The biofilms were fixed with 4% (w/v) paraformaldehyde for 15 min and then stained with 0.01% (w/v) crystal violet solution for 5 min at room temperature. The wells were gently washed twice with sterile PBS and gently shaken with 33% (v/v) acetic acid for 30 min to dissolve the dye. Finally, 100 lL of the reaction mixture was transferred to a new 96-well plate and the absorbance was read at 575 nm.
The CFU counting assay was used to evaluate the inhibition of bacteria in M. abscessus biofilm formation by PQE in a similar way to the method used in planktonic bacteria. The suspension of M. abscessus diluted in Sauton medium was added to 96-well plates at the concentrations of PQE of 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 mg mL À1 and then incubated at 37 C. After removing the planktonic bacteria, biofilm dispersed by ultrasound energy was diluted and coated onto 7H10 agar plates at 0, 5 and 7 d and after 2-4 d of incubation, the mean CFU counts for each group were compared to the control.

Scanning electron microscopy
The morphological changes in M. abscessus biofilms under PQE treatment were observed by scanning electron microscopy (SEM) as previously described , with minor modifications. The glass slides were placed in a 24-well plate with 2 mL Sauton medium and inoculated with 1% saturated culture of M. abscessus. PQE was then added to each well to obtain the final concentrations of 0, 25 and 50 mg mL À1 . M. abscessus dilution without PQE was used as a negative control. The plate was then incubated at 37 C for 5 days. After the removal of the excess medium and loss of bacteria, the slides were fixed in 2.5% (v/v) glutaraldehyde at 4 C for 12 h. Then, graded concentrations of ethanol (30%, 50%, 70%, 80%, 90% and 100%) were added to the plates for 15 min to dehydrate the biofilm (Nguyen and Harbison 2017). The biofilm specimens were observed by SEM (Inspect F, FEI, The Netherlands) after gold spraying.

Confocal laser scanning microscopy
The three-dimensional architecture and biomass of the biofilms were visualized as previously described (Qiu et al. 2020) with minor modifications under confocal laser scanning microscopy (CLSM) (N-SIM, Nikon, Japan). Like the SEM assay, PQE and M. abscessus suspension were added to the wells containing the glass slides. M. abscessus on slides were stained using LIVE/DEAD TM BacLight TM Bacterial viability kit (Invitrogen, USA) according to the manufacturer's instructions. The floating bacteria were removed after incubation for 5 days by rinsing the biofilms twice with sterile water, and then, SYTO 9 and propidium iodide (PI) was added to the slides and incubated for 15 min at 37 C. The excess dye was discarded, and the slides were washed twice with sterile water. Finally, the specimens with the coverslip were observed under a CLSM. The images were processed using Image J COMSTAT software (NIH, USA).

LC/MS analysis
PQE was identified by a Nexera UPLC system (Shimadzu Corporation, Japan) coupled with Q-Exactive quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray ionization source (Thermo Fisher Scientific, Waltham, MA, USA). An ACQUITY UPLC HSS T3 column (1.8 lm, 2.1 Â 100 mm) was employed in both positive and negative modes. The binary gradient elution system consisted of (A) water (containing 0.1% formic acid) and (B) acetonitrile (containing 0.1% formic acid) and separation was achieved using the following gradient: 0 min, 5% B; 2 min, 5% B; 4 min, 30% B; 8 min, 50% B; 10 min, 80% B; 14 min, 100% B; 15 min, 100% B; 15.1 min, 5% and 16 min, 5% B. The flow rate was 0.35 mL min À1 and the column temperature was 45 C. All the samples were kept at 4 C during the analysis. The injection volume was 2 lL. The mass range was from m/z 125 to 1,000. The resolution was set at 70,000 for the full MS scans and 17,500 for HCD MS/MS scans. The collision energy was set at 10, 20 and 40 eV. The mass spectrometer operated as follows: spray voltage, 3,500 V (þ) and 3,500 V (-); sheath gas flow rate, 40 arbitrary units (þ) and 35 arbitrary units (-); auxiliary gas flow rate, 10 arbitrary units (þ) and 8 arbitrary units (-); capillary temperature, 320 C.

Susceptibility of M. abscessus planktonic bacteria to ginsenosides
Five ginsenosides (Rb1, Rb2, Rk1, CK and F11) were selected according to the identification results and the effective ingredients of the PQE to investigate whether ginsenosides possess antibacterial activity similar to that PQE exerted on the standard strain. Likely, MIC and MBC were determined using the same method as previously described. PQE/ASE (Angelica sinensis extract) and ginsenosides were serially diluted from 200 mg mL À1 and 0.50 mg mL À1 respectively, in twofold dilution order.

Ginsenoside CK inhibition of M. abscessus biofilm formation
Since the ginsenoside monomer CK has antibacterial activity against the standard strain, its potential similar effect to that of PQE in inhibiting the biofilm formation was evaluated using 0, 0.004, 0.008, 0.015, 0.03, 0.06, 0.12, 0.25 mg mL À1 as the final concentrations of CK against the biofilm formation of standard M. abscessus. The cultivation of the biofilm and quantification of the biofilm biomass was performed as above.

Statistical analysis
Statistical analysis and graphs were performed using GraphPad Prism. One-way ANOVA was used to compare the results of multiple groups. Results were expressed as mean ± standard deviation (SD). A value of p < 0.05 was considered statistically significant.

Results
Antimicrobial activity of PQE on M. abscessus M. abscessus collection strain was treated with different concentrations of PQE to assess quantitatively its antimicrobial activity in vitro. PQE at 50-200 mg mL À1 inhibited the growth of more than 90% of standard M. abscessus, and 25 mg mL À1 PQE inhibited the growth of more than 50% ( Figure 1A-1B). PQE at 50 mg mL À1 completely inhibited the growth of M. abscessus, while 25 mg mL À1 PQE induced less inhibition; thus, the inhibition on the bacterial growth was dose-dependent. The number of viable cells in the planktonic bacteria decreased by approximately 0.4 log CFU mL À1 and 2.4 log CFU mL À1 , respectively (compared to the drug-free control) after 2 d of 0.5/1 MIC PQE treatment, while the live bacteria decreased by approximately 1.8 log CFU mL À1 and 3.6 log CFU mL À1 , respectively, after 4 d of 0.5/1 MIC PQE treatment ( Figure 1C). In addition, the plate coating also showed that 50 mg mL À1 PQE killed more than 99.9% of the standard M. abscessus on the medium, as shown in Table 1 (compared to the colony count in the drug-free control plate). The inhibitory effect of PQE was also evaluated on seven M. abscessus clinical strains. After co-incubation with 50-100 mg mL À1 of PQE extract for 48 h, the planktonic growth of the seven isolates was reduced ( Figure S1 and Table S1, Supplementary material). This result demonstrated that PQE also exerted an antibacterial effect on clinical strains.

PQE inhibited M. abscessus biofilm formation
The macroscopic images of the biofilms of each group of M. abscessus are shown in Figure 2A. Apparently, the treatment with PQE resulted in a dose-dependent reduction in the biofilm formation of M. abscessus, and PQE !12.5 mg mL À1 resulted in almost no biofilm formation in the wells. The results of the crystal violet staining showed that a large amount of crystalline violet dye bound to the biofilm adhered to the inner wall of the control wells and only a small amount of purple was present in the wells when PQE !12.5 mg mL À1 ( Figure 2B). The quantitative results showed that 12.5-50 mg mL À1 PQE significantly reduced the total biofilm biomass of M. abscessus by 75-80% ( Figure 2C). The effect of PQE on the biofilm formation of standard M. abscessus at different time points was determined using the CFU counts at all concentrations: the viable bacteria in the biofilm decreased by approximately 5.4 log CFU mL À1 for 6.25 mg mL À1 , and 6.8 log CFU mL À1 for 50 mg mL À1 PQE after 5 d of treatment, while the live bacteria in the biofilm were reduced by approximately 5.3 log CFU mL À1 and 7.2 log CFU mL À1 after 7 d of PQE treatment, respectively ( Figure 2D). SEM images revealed visually the inhibitory effect of PQE on the biofilm formation of M. abscessus. The number of bacteria on glass coverslips and the degree of biofilm aggregation decreased with PQE treatment, which was consistent with the result of crystal violet staining. To achieve consistent inhibition of M. abscessus, both 25 mg mL À1 and 50 mg mL À1 PQE were investigated by SEM. When treated only with DMSO, M. abscessus formed a regular, dense membrane-like waxy structure. However, the structure disintegrated after the treatment with 25 mg mL À1 PQE, and a few bacteria were present in clump-like aggregates. Markedly, only a few scattered bacteria remained, with no matrix formed outside bacteria and no biofilm when 50 mg mL À1 PQE treatment was used (Figure 3). The result of crystal violet staining, SEM and CFU counts demonstrated a dose-dependent response from 25 mg mL À1 to 50 mg mL À1 PQE in the bacteria number and biofilm biomass.

PQE inhibited bacteria in the biofilm
The three-dimensional architecture and biomass of M. abscessus biofilms were observed by CLSM, and the dead and live bacteria composition in the biofilm after PQE intervention was also evaluated. The PI fluorescent dye stains the dead cells, and the SYTO 9 fluorescent dye stains the live cells. The CLSM results revealed a   significant decrease in green fluorescence intensity and no significant change in red fluorescence intensity in the 25 and 50 mg mL À1 PQE groups compared to the control group without PQE intervention ( Figure 4A). The semi-quantitative results of biofilm biomass and thickness calculated and analyzed by COMSTAT showed that PQE intervention significantly affected bacterial proliferation and biomass accumulation during biofilm formation in M. abscessus. The dose of 25 and 50 mg mL À1 PQE inhibited 88% and 93% of biofilm biomass, respectively, and inhibited 71%, and 84% of biofilm thickness, respectively, ultimately resulting in reduced biofilm formation on the slides ( Figure 4B-4C). The distribution of live and dead bacteria in each biofilm layer is shown in Figure 4D. The bacteria in the control group were accumulated in the upper part of the biofilm, approximately 6 lm below the surface; the bacteria in the 25 and 50 mg mL À1 groups were accumulated at the middle of the biofilm, approximately 5 and 2.5 lm from the surface respectively. The number of bacteria in the PQE-treated groups was relatively less than that in the control group, which was consistent with the results of SEM.

Identification of the constituent in PQE
Phytochemical identification results indicated the presence of sesterterpenoids, triterpenoids, steroidal glycosides, carbohydrates, flavans, short-chain keto acids and derivatives, cholestane steroids, stigmastanes and fatty acids in PQE. The compounds were identified by LC-MS analysis, based on the precise mass-tocharge ratio (M/z), secondary fragments, and isotopic distribution using The Human Metabolome Database, Lipidmaps (V2.3), Metlin, EMDB, PMDB, and selfbuilt databases to perform qualitative analysis. The LC chromatogram in Figure 5 showed the retention times and the peaks corresponding to the metabolites present in the extracts. The molecular structure of five identified ginsenosides is also shown in Figure 5.

Bacteriostatic activity of ginsenoside monomers on M. abscessus
The antibacterial and bactericidal activities of the five available pure ginsenosides in vitro were tested against the standard strain using the microdilution and plate  Figure 6A). The quantitative calculation based on fluorescence values showed that CK at 0.25-0.50 mg mL À1 inhibited more than 90% of the growth of the bacteria ( Figure 6D). The other four ginsenosides at 0.50 mg mL À1 scarcely inhibited M. abscessus growth ( Figure 6B-6F). The MBC results showed that CK was able to kill 99.9% of M. abscessus at 1 MIC (Table 1).

CK inhibited M. abscessus biofilm formation
The macroscopic images of M. abscessus biofilm after CK treatment are shown in Figure 7A. The drug-free control showed a well-formed biofilm at the air-liquid interface. CK at 0.015-0.25 mg mL À1 significantly reduced 70%-81% of the total biofilm biomass of M. abscessus ( Figure 7B-7C). The results of CFU counting showed that the concentration of CK ! 0.015 mg mL À1 greatly reduced the number of viable bacteria in the biofilm ( Figure 7D).

Discussion
The incidence and mortality of NTM pulmonary disease are increasing. M. abscessus is one of the most common pathogens causing NTM pulmonary disease (Mirsaeidi et al. 2014;Vinnard et al. 2016;Cowman et al. 2019;Johansen et al. 2020). The inherent widespectrum drug resistance of M. abscessus arises from the hydrophobic characteristic of the cell wall, the lack of drug activation systems, the induction of efflux pumps, and the production of a wide range of drug-modifying enzymes (Luthra et al. 2018;Lopeman et al. 2019). Existing antibiotic regimens against M. abscessus infection result in a residual infection becoming a life-threatening chronic infection and low rates of sputum conversion due to drug resistance (Ferro et al. 2016;Diel et al. 2017;Weng et al. 2020). It has become a clinical problem that cannot be ignored. Therefore, the search for new effective antimicrobial drug strategies is particularly urgent.
Biofilm formation increases virulence and antibiotic resistance in bacteria. Biofilms further exacerbate the drug-resistant phenotype since the waxy extracellular matrix of the biofilm acts as a barrier to drug penetration and the increased horizontal exchange of virulence or resistance gene ultimately leads to no efficacious cure (Faria et al. 2015;Wu et al. 2018). M. abscessus biofilms are more resistant to antibiotics and disinfectants than planktonic bacteria (Hoiby et al. 2015;Clary et al. 2018). Addressing one of the major drug resistance problems of pathogenic bacteria requires confronting the challenge of biofilm formation. The existing chain of drug development for M. abscessus is inadequate, especially regarding the anti-biofilm effect (Egorova et al. 2021).
Natural products are a treasure trove of pharmaceutical resources. In a previous study, a pharmacy type of concentrated Coptis chinensis paste inhibited the growth and biofilm formation of M. abscessus with MBC at 6 mg mL À1 and MIC at 1 mg mL À1 , and its main active ingredient berberine significantly inhibited biofilm formation of M. abscessus at 0.125 mg mL À1 (Tseng et al. 2020). In the present study, the PQE inhibited the growth of M. abscessus with MBC at 50 mg mL À1 and MIC at 50 mg mL À1 , and the MIC of ginsenoside CK was 0.25 mg mL À1 against M. abscessus. Considering different methods for determining the anti-mycobacterial effect, or different pharmacological components in different plants, we focused on the inhibition ability of herbal monomers and found that ginsenoside CK is comparable to berberine for the growth inhibition of M. abscessus. Strikingly, 0.015 mg mL À1 of CK with no anti-M. abscessus effects but significantly inhibited the biofilm formation by more than 70% of the biomass.
The P. quinquefolius is a medicinal and food homologous product. A clinical trial suggested that shortterm administration of PQE (100-3000 mg d À1 for 12 weeks) may be safe (Stavro et al. 2006). In vivo safety tests of ginsenoside component CK in animals showed that single oral administration of CK did not induce acute toxicity in rats (8 g kg À1 ) and mice (10 g kg À1 ). A 26-week oral toxicity assessment of CK showed NOAEL (no observed adverse effect levels) doses of 40 mg kg À1 and 120 mg kg À1 for female and  The biofilm production of M. abscessus with CK treatment was measured with crystal violet staining. Data were expressed as the means ± SD ( ÃÃÃÃ , p < 0.0001). Comparisons were performed using one-way ANOVA.
male mice, respectively. The NOAEL dose for Beagle dogs was 12 mg kg À1 (Gao et al. 2019;Li et al. 2019). A randomized double-blind trial from Chinese volunteers showing oral doses of 100, 200 or 400 mg CK at least nine times a week confirmed the safety of CK during the intervention (Chen et al. 2017). P. quinquefolius additional benefits, such as low toxicity as a long-term tonic and immune enhancement adaptogen, make it an excellent candidate as an antibacterial drug . The root extract of P. quinquefolius showed MIC values of 12.5-25 mg mL À1 against Pseudomonas aeruginosa. The crude extract inhibited bacterial growth and biofilm by reducing virulence expression and attenuating bacterial swarming/swimming motility (Alipour et al. 2011). Animal studies also demonstrated that PQE in combination with tobramycin significantly reduced P. aeruginosa counts in the lungs of infected rats (Alipour et al. 2013). In the present study, PQE could not only inhibit the growth of planktonic bacteria of standard M. abscessus as well as seven clinical strains but also hinder the formation of biofilm at sub-inhibitory concentrations (6.25-50 mg mL À1 ), mainly manifested as a significant decrease in the number of cells, the total biomass and the thickness of the biofilm. SEM images showed that PQE intervention prevented intact biofilm formation at the initial aggregation and adhesion and stalled biofilm at the step of forming partial microcolonies.
Previous investigations of a large group of lowpolarity ginsenosides derived from the root extract of P. quinquefolius revealed that Rk1, Rg6, and F4, among other molecules, were found to have bacteriostatic and bactericidal effects on Propionibacterium acnes, Fusobacterium nucleatum, Clostridium perfringens, and Porphyromonas gingivalis (Wang et al. 2013;Xue et al. 2017). In this study, the activity of ginsenosides (Rb1, Rb2, CK, Rk1 and F11) as responsible components of the tonic effect of ginseng was also investigated against M. abscessus survival. Among the five ginsenosides, CK was able to inhibit the growth of 90% of planktonic bacteria at 0.25 mg mL À1 . Ginsenoside CK is the final manifestation of the activity of other protopanaxadiol ginsenosides (including Rb1, and Rb2) in the intestinal tract after microbial metabolism and has higher bioavailability than its parent ginsenosides. CK has become a hot spot for pharmacological studies of ginseng active ginsenoside components, showing many health benefits including anti-diabetic, antiinflammatory, anti-ageing, and anti-cancer activity, as well as neuroprotective, hepatoprotective, and immune enhancement. These effects are confirmed by in vitro and in vivo studies, and related clinical studies and drug delivery system development are underway (Sharma and Lee 2020). The lower polarity of ginsenoside molecules leads to higher antimicrobial activity (Xue et al. 2020). Thus, the highest effectiveness of CK among the five ginsenosides tested in this study could be due to its lower polarity structure compared to the other four monomers, causing cell damage through a better affinity effect on the lipid-soluble outer membrane. Followup experiments on the investigation of the inhibitory activity of CK monomer against clinical strains of M. abscessus and their biofilms, as well as transcriptomic sequencing of M. abscessus with PQE and CK treatment are underway to discover in what form the herbal extracts and their monomers affect and block biofilm formation. In addition, the potential ability of the herbal extracts and monomers alone or in combination with clinical antibiotics to reverse drug-resistant bacteria and biofilm tolerance states should be investigated. This is extremely important in the treatment of M. abscessus infections and the prevention of biofilm formation at the lesion site.

Conclusion
PQE and the ginsenoside CK effectively inhibited the growth of planktonic M. abscessus. More importantly, they inhibited M. abscessus biofilm by disintegrating its structure. Therefore, PQE and its monomer CK are potential novel antibacterial agents in the clinical prevention and treatment of M. abscessus infections and subsequent biofilm formation in chronic infections.

Disclosure statement
All authors declare no potential conflicts of interest concerning the authorship and/or publication of this article.