In vitro activities of isobavachalcone against planktonic and persister cells and biofilm of Enterococcus faecalis

doi:10.21203/rs.3.rs-5317490/v1

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In vitro activities of isobavachalcone against planktonic and persister cells and biofilm of Enterococcus faecalis

https://doi.org/10.21203/rs.3.rs-5317490/v1

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Background

Mounting evidences have demonstrated the extensive pharmacological activities of the natural product isobavachalcone, including antimicrobial activity, inhibition of reverse transcriptase, antitubercular and antioxidant ability et al. However, the antibacterial and antibiofilm activity and its action mode of isobavachalcone against clinical E. faecalis isolates remain elusive. This study aims to evaluate the in vitro antibacterial and anti-biofilm activities of isobavachalcone on clinical E. faecalis isolates from China and further investigate the possible target site of isobavachalcone in E. faecalis.

Results

Our data suggested the MICs of isobavachalcone ranging from 6.25 to 12.5 µM against 220 E. faecalis strains. The robust inhibitory effect of isobavachalcone with sub-MIC concentration ( 1/2xMIC ) against the biofilm formation of E. faecalis was found. The rapid bactericidal effect of isobavachalcone against E. faecalis was demonstrated and more planktonic cells could be killed by isobavachalcone compared with vancomycin, linezolid, or ampicillin at 2, 4, 6, and 12h. No synergetic bactericidal activity of isobavachalcone combined with vancomycin, linezolid, or ampicillin was found. Furthermore, genetic mutation of isobavachalcone-resistant E. faecalis was compared with the parental strain by whole genome sequencing, showing that the functions of the mutated proteins were associated with the PurH and FlgJ proteins and other eight proteins involved in the cell wall or cell membrane biogenesis, DNA synthesis, and energy metabolism. Molecular docking analysis showed that FlgJ protein might serve as the potential target of isobavachalcone in E. faecalis. Other mutations are involved in the cell wall or cell membrane biogenesis, DNA synthesis, and energy metabolism.

Conclusion

This study discovered that isobavachalcone had an antibacterial effect on E. faecalis, and significantly inhibited the biofilm formation of E. faecalis at subinhibitory concentrations. In addition, antibacterial and antibiofilm activity against clinical E. faecalis isolates from China by targeting FlgJ protein.

Isobavachalcone

Enterococcus faecalis

Antibacterial

Persister

Biofilm

Enterococcus faecalis is one of the conditional pathogens causing hospital-acquired infections, such as urinary tract infections, abdominal infections, device-associated infections, etc. Recently, the increasing difficulty available for antimicrobial treatment of E.faecalis infection is gradually troubling clinicians due to the dissemination of E.faecalis with intrinsic or acquired resistance towards a range of antibiotics (1, 2). In the 1970s, resistance in Enterococci was manifested to aminoglycosides, such as gentamicin and streptomycin, and resistance to β-lactams and glycopeptides was discovered in the 1980s, for instance,vancomycin-resistant enterococci (VRE) were first described in 1986(3–5). With the widespread use of antimicrobial drugs, multidrug-resistant (MDR) E. faecalis, such as VRE, linezolid-resistant enterococci, daptomycin-resistant enterococci and et al, have gradually emerged and disseminated worldwide, which often leads to clinical treatment failure(6). Therefore, the rapid development of multi-drug resistance in enterococci necessitated an urgent need to look for an alternative medicine with antibacterial activities against MDR E. faecalis.

E. faecalis exhibit intrinsic resistance to many antibiotics because of their thick cell walls and low membrane permeability, beyond that, they have a strong biofilm-forming ability to resist unfavorable external conditions for their growth(7, 8).E. faecalis usually adopt dormant strategies to survive extreme environments under antibiotics or other stresses, and this class of dormant subclassified bacteria is known as persistent bacteria, which can respond to drug pressure by "dormancy-growth-proliferation" mode without being killed by antibiotics(9). Biofilms are complex aggregates of microorganisms in a polymeric network of polysaccharides and DNA, and the formation of biofilms often leads to decreased bacterial drug sensitivity and evasion of host immune clearance, resulting in poor therapeutic efficacy(10, 11).In other words, biofilm formation promotes the survival and persistence of infecting microbes by facilitating defense against the host immune response. Currently, the first-line antibiotics in clinical often lack both the activity to kill persistent bacteria and to remove bacterial biofilm. Thus, searching for natural plant-derived active substances with antimicrobial activity that can both kill persistent bacteria and inhibit or remove biofilms is a promising option for the development of new antimicrobial agents(12, 13).

Isobavachalcone (Iso), isolated from plants such as Fatouapilosa and Psoralea corylifolia fruits, has been reported to have significant antibacterial activity against MRSA (methicillin-resistant Staphylococcus aureus) strains tested, and the antibacterial activity of this compound is associated with membrane disruption(14–16). Isobavachalcone has been shown to have extensive pharmacological activities, including antibacterial, antifungal, anticancer, anti-reverse transcriptase, antitubercular and antioxidant. In terms of antibacterial activity, it was found to be active against Gram-positive, Gram-negative,multidrug-resistant bacteria, and mycobacteria(17) .Previous studies have found that the main antibacterial mechanisms of isobavachalcone include the enhanced membrane permeabilization and the leakage of alkaline phosphatase (AKP) due to the impairment of the cell wall and cell membrane damage, the inhibition of protein and DNA and RNA synthesis, and inhibition of energy metabolism(18, 19).In recent years, Yan Chen et al. reported that the use of isobavachalcone together with gentamicin significantly improved its antibacterial effect on S.aureus biofilm(20).However, there are few reports on the antibacterial activity and anti-biofilm against E. faecalis and its antibacterial mechanism. Therefore, the objective of this study was to assess the antibacterial and anti-biofilm activity of isobavachalcone against E. faecalis and to identify the potential target genes of isobavachalcone A in E. faecalis.

Bacterial isolates and culture conditions

A total of 219 strains of E. faecalis (non-replicative) were collected from Shenzhen Nanshan People's Hospital (a tertiary-care teaching center hospital in China with 1500 beds) over five years between 2011 and 2015. These bacteria were identified with the Vitek 2 compact system (Biomérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. The E. faecalis ATCC29212 and OG1RF were purchased from the American Type Culture Collection (ATCC) and used as reference strains.

The E.faecalis strains were stored at -80℃ in glycerol-containing (40%) Tryptic Soy Broth (TSB) medium for subsequent analysis. E. faecalis isolates were cultivated in TSB) at 37°C with agitation at 180 rpm, unless otherwise stated. E. faecalis isolates were cultivated in Cation-Adjusted Mueller-Hinton Broth (CAMHB) at 37°C with agitation to conduct antimicrobial susceptibility tests and time-killing tests. E. faecalis isolates were cultivated in TSBG (TSB with 0.5% glucose) at 37°C to conduct a biofilm assay. In all experiments, CAMHB media were supplemented with 50 mg/L Ca2 + to test the efficacy of daptomycin. (All procedures involving human participants were conducted under the ethical standards of Shenzhen University and the 1964 Helsinki Declaration and its subsequent amendments, or comparable ethical standards.)

Antimicrobial susceptibility testing

The antimicrobial susceptibility of E. faecalis isolates to isobavachalcone and the commonly used antibiotics, including ampicillin, tetracycline, doxycycline, linezolid, vancomycin, ciprofloxacin and nitrofurantoin, were tested by broth microdilution using the Vitek 2 compact system (Biomérieux, Marcy l’Etoile, France) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). Isobavachalcone and antibiotics above were purchased from the Macklin Company (Shanghai, China) and the minimum inhibitory concentrations( MICs) were determined by broth microdilution method.

Growth curve experiment of E. faecalis

Two E. faecalis (16C51, 16C106) isolates were selected and tested in the One-step growth experiments with isobavachalcone. Bacteria strains were cultured in Mueller-Hinton Broth (MHB) at 37℃ for 12h, and diluted to the optical density value at 600 nm was 0.1 (OD600 = 0.1). Isobavachalcone was added to make the final concentrations at 1/32 × MIC, 1/16 × MIC, 1/8 × MIC, 1/4 × MIC, and 1/2 × MIC, and the OD values were measured every single hour in 24h by the automatic growth curve measurement instrument. Each experiment was repeated three times.

Antimicrobials inhibit the biofilm formation and eradicate the established biofilms of E. faecalis

Two E. faecalis isolates (16C51,16C106) previously reported with positive biofilm formation were selected to evaluate the inhibition of isobavachalcone on the biofilm formation of E.faecalis. The bacteria were inoculated into 96-well polystyrene microtitre plates with TSBG containing antimicrobials (at1/2, 1/4, 1/8, 1/16, and 1/32 \(\:\times\:\) MICs). The ones with no antibiotics were used as controls. After 24 h of static incubation, the biofilm biomasses were determined by crystal violet staining. Experiments were performed in triplicate.

Four E. faecalis isolates (FB-1, 16C51, 16C102, 16C166) were further used for evaluating the eradication capacity of isobavachalcone against the biofilm formation of E.faecalis. The E. faecalis strains were cultivated in TSB at 37℃ for 12h and then diluted in 200 µL of TSBG at the volume ratio of 1:200. After 24 h of static incubation on 96-well polystyrene microtitre plates at 37℃ (mature biofilms formed), the supernatant was removed and plates were washed with 0.9% saline to remove unattached cells. Then TSBG containing antimicrobials (at 8\(\:\times\:\) MIC) was added with blank control, and incubated for 48 h of static incubation with the medium being replaced at 24 h, after which biofilm biomasses were visualized by crystal violet staining. Experiments were performed in triplicate.

Time-kill assay

Two E. faecalis(16C51, 16C106) isolates were used for the time-kill assays with isobavachalcone and some first-line antibiotics, including ampicillin, vancomycin, and linezolid as previously reported(21), Strains were cultured in MHB at 37℃ for 12h, then diluted 100 times with CAMHB and antibiotics were added to make the final concentrations at 4 \(\:\times\:\)MIC. Each experiment was repeated three times.

Persister assay

In order to evaluate the additional anti-persister activity of isobavachalcone against E.faecalis, two E. faecalis isolates(16C51, 16C106) were selected and tested for the persister assays with isobavachalcone and some first-line antibiotics. E.faecalis strains were cultured in MHB at 37℃ for 12h, then diluted 100 times with CAMHB to exponential phase, and 7 mL of the culture was exposed to various antibiotics (10 × MIC) at 37℃. After incubation with shaking at 200rpm for 12 h, samples were washed twice with 0.9% NaCl and spread on TSB plates by serial dilution. The surviving persister populations were determined by the dilution coating plate method.

To confirm whether the surviving colonies were persister cells, two colonies from each sample were re-inoculated into Luria-Bertani (LB) broth and the unchanged MIC value of isobavachalcone would be tested by the in vitro antimicrobial susceptibility testing methods.

In vitro, induction of isobavachalcone non-sensitive E. faecalis isolates with possible mutations

To explore the possible target genes of isobavachalcone in E.faecalis, the isobavachalcone non-sensitive E. faecalis isolates with possible mutations were induced and screened in vitro according

to previous studies(22) .E. faecalis isolates (16C51, 16C106) were subcultured serially in TSB containing isobavachalcone. The initial inducing concentration of isobavachalcone was 1/2 × MIC; the concentration was then increased successively to high concentrations. E. faecalis isolates

in each concentration of isobavachalcone were cultured for three to five passages before being inoculated and passaged to the next generation. E. faecalis isolates from the last passage of each concentration of isobavachalcone were collected and subcultured on tryptic soy agar plates without isobavachalcone for three passages, identified again by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IVD MALDI Biotyper, Bruker, Bremen, Germany), and the MIC of isobavachalcone was measured again. Until the 21st generation, the isobavachalcone non-sensitive E. faecalis isolates were kept frozen at − 80◦C in glycerol containing (35%) TSB.

Genome sequence of mutations

The genomic DNA of two isobavachalcone non-sensitive E.faecalis isolates (16C51-T1.1 and 16C51-T1.2) was extracted, and a total amount of 1 µg DNA per sample was used as input material for DNA sample preparations. Whole genomes were sequenced in an Illumina HiSeq2500 sequencer according to the previous study (23).Coding genes, repetitive sequences, non-coding RNAs, genomics islands, transposons, prophages, and CRISPR (clustered regularly interspaced short

palindromic repeat) sequences were predicted. Gene functions were predicted by referring to the following databases: GO(Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and Swiss-Prot. Genomic alignments between each sample genome and a reference genome (E. faecalis OG1RF, GenBank: NC_017316.1) were performed with MUMmer and LASTZ tools. Single nucleotide polymorphisms, insertions, deletions, and structural variation annotations were identified based on inter-sample genomic alignment results by MUMmer and LASTZ. Molecular Docking

The molecular structure pdb file of PurH and FlgJ was constructed by AlphaFoldDB(24). The molecular structure file for isobavachalcone was searched from Pubchem. The binding architecture between the PurH and FlgJ protein and isobavachalcone was performed by molecular docking. The best binding pocket of the PurH and FlgJ were selected by the structure-based cavity detection, and the isobavachalcone of the optimum binding site was determined by Auto Dock Vina based on the Vina score (kcal/mol) in the pocket(25).

Statistical analysis

Data were analyzed by Student’s t-test. P-values < 0.05 were regarded as statistically significant. All data were analyzed in the SPSS software package (version 26.0).

In vitro activity of isobavachalcone against clinical E. faecalis isolates

The antibacterial activity of isobavachalcone against E. faecalis was investigated by determining the MICs of isobavachalcone and other antibacterial agents against E. faecalis and confirming the susceptibility results according to the CLSI-M100-S30. The MIC of isobavachalcone against the reference strains of E. faecalis, ATCC29212, and OG1RF, was determined. Both strains exhibited a MIC of 12.5 µM (equivalent to 4.05 mg/L). Subsequently, the MICs of isobavachalcone were measured for 22 clinical isolates of E. faecalis (isolated from urine, exudate, or blood), of which the MICs were12.5 µM for all 22 isolates (Table 1). The isobavachalcone MICs in 22 clinical isolates of E.faecalis, including two linezolid-resistant E.faecalis and ten daptomycin-nonsusceptible strains, was also shown with 12.5µM(Table 1), suggesting its excellent bactericidal activity against multi-drug resistant E.faecalis. To further verify the antibacterial activity of isobavachalcone against clinical isolates of E.faecalis, the MIC values of isobavachalcone against 198 additional clinical isolates of E. faecalis (isolated from urine, exudate, blood, bile, and other sources) were determined. This investigation revealed that the MICs of isobavachalcone ranged from 6.25 to 12.5 µM, with the MIC50 and MIC90 levels being 12.5 µM against the clinical isolates (TableS1).

Growth curve and the anti-biofilm activity of isobavachalcone

This study was to investigate the effect of subinhibitory concentrations of isobavachalcone on the biofilm formation of E. faecalis. To this end, the effect of subinhibitory concentrations of isobavachalcone on the planktonic growth of E. faecalis (16C51, 16C106) planktonic cells was determined using an automatic growth curve analyzer. As illustrated in Fig. 1a, the 1/2 × MIC of isobavachalcone demonstrated inhibitory effects on the growth of E. faecalis planktonic cells during the logarithmic growth phase. In contrast, the 1/4 ×, 1/8 ×, 1/16 ×, and 1/32 × MICs of isobavachalcone exhibited no inhibitory effects against E.faecalis(Fig. 1a,b). The inhibition of the biofilm formation of E. faecalis by isobavachalcone was evaluated (Fig. 1d,c). Notably, biofilm formation of E. faecalis (16C51) was significantly inhibited by 1/2 × or 1/4 × MICs of isobavachalcone. However, against a different strain of E. faecalis(16C106), there was no significant inhibition at 1/2×,1/4 × MIC of isobavachalcone against the planktonic growth, whereas isobavachalcone at 1/4×MIC could inhibit biofilm formation of E. faecalis.

Time-killing curve and biofilm elimination of E. faecalis by isobavachalcone alone or in combination

The efficacy of the bactericidal and anti-biofilm activity of isobavachalcone alone and in combination were compared in two E. faecalis isolates (16C51, 16C106). Firstly, time-killing curve studies for isobavachalcone and three commonly used antibiotics, including linezolid, vancomycin, and ampicillin, were performed using these two E. faecalis isolates. Exposure to antimicrobial agents at 4 ×MIC resulted in a reduction in log10CFU/mL over the 0–24 hour period (Fig. 2a,b), suggesting the similar bactericidal activity of isobavachalcone and the combinations with three commonly-used antibiotics in these two E. faecalis. That was, time-killing curves of isobavachalcone alone in E. faecalis were significantly lower than those of linezolid, vancomycin, and isobavachalcone with their combinations could not further improve its bactericidal activity. Anti-persister assay of isobavachalcone in E. faecalis 16C51 isolate was shown in Fig. 2c, showing the eradication effect of isobavachalcone against 16C51. Persister cells were similar when compared with those of vancomycin or linezolid. Moreover, isobavachalcone combined with three commonly-used antibiotics was even much better than that of the individual antimicrobials(Fig. 2c). Similar results of isobavachalcone against 16C106 persister cells were found, suggesting isobavachalcone combined with these antibiotics could completely eradicate the persister cells after 48h(Fig. 2d). Furthermore, isobavachalcone combination with vancomycin, linezolid or ampicillin (all used at 8 × MIC) to treat established E. faecalis biofilms( FB-1, 16C51, 16C102, 16C166 ) were explored, indicating isobavachalcone combined with daptomycin has significant eradication effect in only 16C51, but not found in the other three E. faecalis isolates (Fig. 3).

Genetic mutations in the isobavachalcone resistant E.faecalis and potential targeting was identified through molecular docking

To investigate the possible target gene of isobavachalcone in E. faecalis, the isobavachalcone-resistant E. faecalis isolates (16C51-T1) were selected by in vitro induction of wild-type strains under the pressure of isobavachalcone and then identified. Then, the genetic mutations might be found in the possible target of isobavachalcone, and whole genome sequencing in isobavachalcone-resistant E. faecalis was performed, suggesting ten nucleotide mutations in the 16C51-T1 isolate (Table 2). Among these mutations, PurH and FlgJ protein mutations were found, and other relevant mutations were associated with cell wall or membrane biogenesis, DNA synthesis, and energy metabolism. Consequently, it was postulated that PurH and FlgJ may serve as the target for isobavachalcone

in E. faecalis. Utilizing molecular docking techniques, the potential binding affinity of PurH and FlgJ with the isobavachalcone was assessed. Remarkably, the results revealed that the isobavachalcone binds to PurH(Fig. 4a,b) and FlgJ (Fig. 4b,c) in favorable conformations, exhibiting low binding energy of − 8.6 and-7.1 kcal/mol respectively [a smaller score value (greater absolute value of the negative value) signifies a stronger binding force, and an affinity < 7 kcal/mol suggests a good binding force]. The optimal conformation of the isobavachalcone and PurH complex is depicted in Fig. 4a. Within the docking pocket, interactions between the isobavachalcone and PurH involve 9 hydrogen bonds, 5 hydrophobic interactions, and two ionic interactions (Fig. 4b). These findings suggest that isobavachalcone has the capability to effectively bind with PurH of E. faecalis.

Table 1

Antimicrobial susceptibilities of *E. faecalis* determined by conventional broth microdilution
Strains	MIC(mg/L)								MIC
	Amp	Van	Lin	Dap	Rif	Min	Gen	FOS^a	Iso
OG1RF	2	2	2	4	128	1	16	64	12.5µM(4.05mg/L)
ACTCC29212	2	2	2	4	1	4	4	8	12.5µM(4.05mg/L)
FB-1	2	1	8	4	8	1	>512	32	12.5µM(4.05mg/L)
16C35	4	1	2	8	8	16	16	64	12.5µM(4.05mg/L)
16C51	2	2	4	8	8	16	8	64	12.5µM(4.05mg/L)
16C54	2	1	2	16	16	8	>512	64	12.5µM(4.05mg/L)
16C68	4	1	2	4	4	16	>512	64	12.5µM(4.05mg/L)
16C102	4	1	8	4	8	8	32	32	12.5µM(4.05mg/L)
16C106	2	1	2	8	8	16	>512	64	12.5µM(4.05mg/L)
16C124	1	1	2	4	8	16	8	64	12.5µM(4.05mg/L)
16C125	2	1	2	4	2	16	>512	64	12.5µM(4.05mg/L)
16C137	2	0.5	2	8	32	16	>512	64	12.5µM(4.05mg/L)
16C138	2	1	2	8	8	16	>512	32	12.5µM(4.05mg/L)
16C152	2	1	2	2	4	16	>512	64	12.5µM(4.05mg/L)
16C166	2	1	2	8	4	16	16	32	12.5µM(4.05mg/L)
16C168	4	1	2	8	2	16	>512	64	12.5µM(4.05mg/L)
16C186	2	0.5	2	8	8	8	16	64	12.5µM(4.05mg/L)
16C201	2	1	2	4	8	16	>512	32	12.5µM(4.05mg/L)
16C274	4	1	2	8	16	16	16	64	12.5µM(4.05mg/L)
16C289	2	1	2	2	2	16	8	64	12.5µM(4.05mg/L)
16C350	2	1	2	4	2	16	>512	32	12.5µM(4.05mg/L)
16C353	2	1	2	16	16	16	16	32	12.5µM(4.05mg/L)
16C385	2	0.5	2	4	16	16	>512	64	12.5µM(4.05mg/L)
16C405	2	1	2	4	2	16	>512	64	12.5µM(4.05mg/L)

MIC, minimum inhibitory concentration; Amp, Ampicillin; Van, Vancomycin; Lin, Linezolid; Dap, Daptomycin; Rif, Rifampin; Min, Minocycline; Gen, Gentamicin,Fos, Fosfomycin; Iso,Isobavachalcone. a, agar dilution method.

Table 2

Mutations in *E. faecalis* 16C51-T1 isolate were detected by whole genome sequencing. **Genome analysis of mutations**
ref_gene_ID	NA mutations	AA mutations	ref_gene_product	ref_gene_strand
16C51T1_GM000347	T247C	Y83H	Pyrroline-5-carboxylate reductase Amino acid transport and metabolism	+
16C51T1_GM000484	G559A	A187T	dTDP-4-dehydrorhamnose reductase Cell wall/membrane/envelope biogenesis	+
16C51T1_GM000718	C187T	P63S	Adenine deaminase Nucleotide transport and metabolism	+
16C51T1_GM001010	A508G	T170A	Flagellum-specific peptidoglycan hydrolase FlgJ Cell wall/membrane/envelope biogenesis; Cell motility ;	-
16C51T1_GM001244	A688G	K230E	Fatty acid/phospholipid biosynthesis enzyme Lipid transport and metabolism	+
16C51T1_GM001349	T938C	I313T	DNA-binding transcriptional regulator, FrmR family, Transcription	+
16C51T1_GM001679	G49A	E17K	Glycerol kinase Energy production and conversion	-
16C51T1_GM001760	C632A	T211K	AICAR transformylase/IMP cyclohydrolase PurH Nucleotide transport and metabolism	+
16C51T1_GM002262	A109G	K37E	Signal transduction histidine kinase Signal transduction mechanisms	-
16C51T1_GM000346	A442G	I148V	Phosphomannomutase Carbohydrate transport and metabolism	+

E. faecalis strain16C51 strain was serially subcultured in TSB isobavachalcone containing Mutations in the isolate T1 from 21 generations of 16C51 strain were detected by whole genome sequencing. NA, nucleotide; AA, amino acid.ref_gene_strand, the orientation of the gene in which the SNP is located on the reference sequence.

Isobavachalcone is a natural flavonoid derived from a wide range of medicinal plants, including the Fabaceae, Moraceae and so on(26) .Previous study has shown that it was active against Gram-positive bacteria, mainly against Methicillin-Susceptible Staphylococcus aureus (MSSA) and Methicillin-Resistant Staphylococcus aureus (MRSA), with MIC values of 1.56 and 3.12 µg/mL, respectively (27). But there are no reports on the effect of isobavachalcone on E. faecalis. Linezolid is a first-line treatment for VRE infections(28). However, with the increased use of linezolid, an increasing number of cases of linezolid-resistant E. faecalis have emerged, and its propensity to form biofilms limits therapeutic options(29). Biofilm formation and persister cell formation can be viewed as two types of collective bacterial behaviors (30). It is often difficult for currently used first-line antibiotics to have both bacterial retention and bacterial biofilm scavenging activity, so screening for antibacterial compounds with both bacterial retention and biofilm scavenging activity may help to develop more effective antibacterial drugs(31). It has been reported that certain small molecules and natural products exert an inhibitory effect on the growth, biofilm formation, and exopolysaccharide synthesis of E. faecalis (32, 33). This study also suggested that isobavachalcone had excellent antibacterial activity against E. faecalis and had a good inhibitory effect on the biofilm formation of E. faecalis at sub-inhibitory concentrations. In our study, although 3 strains exhibited a lack of sensitivity to linezolid, the MICs value of 22 clinical isolates of E.faecalis did not an increase in resistance to isobavachalcone, which shows that isobavachalcone is a promising antibacterial agent against resistant E. faecalis.

Bacterial persister cells evade antimicrobial-induced killing by entering physiological dormancy and are thought to be a major cause of antimicrobial treatment failure and recurrent infections(34). Therefore, it is anticipated that a reduction in the production of bacterial persister cells will result in a reduction in chronic or relapsing infections and an improvement in the efficacy of anti-infection treatment(9). E. faecalis is frequently resistant to vancomycin and β-lactams, so combination therapy is often administered. In vitro pharmacokinetic/pharmacodynamic models and biofilm time-kill curves, β-Lactams enhance daptomycin activity against vancomycin-resistant E. faecalis(35, 36). Interestingly, our results demonstrated that isobavachalcone alone against E. faecalis showed a rapid bactericidal effect, killing more planktonic cells than vancomycin, linezolid, or ampicillin. It even eradicated E. faecalis persister cells at high concentrations, when combining it with vancomycin, linezolid or ampicillin improved its efficacy. However, isobavachalcone in combination with vancomycin, linezolid, or ampicillin was not effective in eliminating established E. faecalis biofilms but showed potential when combined with daptomycin. This is consistent with other studies which indicate that once bacterial biofilms have been established, it is challenging for antimicrobials to effectively remove them(37). Therefore, further studies are required to substantiate the efficacy of isobavachalcone in the removal of biofilms when combined with daptomycin.

To explore the possible target site of isobavachalcone in E. faecalis, the isobavachalcone non-sensitive E. faecalis clones were selected in vitro by induction of wildtype strains under the pressure of isobavachalcone, and mutations in the possible target genes were detected by whole-genome sequencing. Our study suggested that mutations in the possible target genes are associated with the PurH and FlgJ proteins, which potentially serve as targets for the isobavachalcone in E. faecalis, as determined through molecular docking analyses. Other mutations are involved in the cell wall or cell membrane biogenesis, DNA synthesis, and energy metabolism. The previous study has shown that ampicillin and vancomycin exert their antibacterial activity by interfering with the cell wall synthesis of Gram-positive bacteria, whereas linezolid exerts its antibacterial activity by interfering with the protein synthesis of Gram-positive bacteria(38). The bactericidal effect of isobavachalcone on E. faecalis planktonic cells was observed to be rapid, and it was found to be more effective than vancomycin, linezolid, and ampicillin in this study. In addition, there are studies showed that the antibacterial activity of isobavachalcone is associated with membrane disruption (27). Therefore, this study suggests that isobavachalcone may not block the cell wall or protein synthesis, but exert a greater influence on the biogenesis of the cell membrane.

The limitation of this study is to find the target gene of isobavachalcone in E. faecalis through experimental evolution or serial messages. However, this kind of experiment is more valuable to determine the propensity of a given antimicrobial to develop resistance to mutations. Consequently, further research is required to ascertain the precise target of isobavachalcone in E. faecalis through the utilization of alternative methodologies, such as CO-IP and LC-MS.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Ethics approval and consent to participate

All procedures involving human participants were performed following the ethical standards of Shenzhen Nanshan People’s Hospital and the 6th Affiliated Hospital of Shenzhen University Health Science Center and with the 1964 Helsinki Declaration and its later amendments, and this study was approved by the ethics committee of the Shenzhen Nanshan People’s Hospital and the 6th Affiliated Hospital of Shenzhen University Health Science Center. Isolates were collected as part of the routine clinical management of patients. Therefore, formal consent is not required.

Consent for publication

Not applicable.

Clinical trial number

Not applicable.

Funding

This work was supported by the following grants: National Natural Science Foundation of China (82172283); Guangdong Basic and Applied Basic Research Foundation (2022A1515110096, 2024A1515013276); Sanming Project of Medicine in Shenzhen (SMGC202305029); Shenzhen Key Medical Discipline Construction Fund (SZXK06162); Science, Technology and Innovation Commission of Shenzhen Municipality of basic research funds (JCYJ20220530141810023, JCYJ20220530141614034, JCYJ20220530142006015) and the Shenzhen Nanshan District Scientific Research Program of the People’s Republic of China (NS2023026, YN2022028, NS2022114; NS2022046; NS2023049; NSZD2023032; NS2024038).

Author Contribution

Ouyang L participated in the antimicrobial susceptibility test, growth curve experiment and drafted the manuscript. Xu Z performed the antimicrobial susceptibility test. Tang Y performed time-kill and persister assay. Li D participated in collecting E. faecalis clinical isolates and conducted statistical analysis. Wen Z conducted the genome sequence analysis and participated in the detection of PurH and FlgJ proteins. Yu Z, Zhang C, and Zhang H designed the study, participated in the data analysis, and provided critical manuscript revisions for valuable intellectual content. All authors have read and approved the manuscript.

Acknowledgement

The authors thank Weiguang Pan and Jie Lian (Department of Laboratory Medicine, Shenzhen Nanshan People’s Hospital, Shenzhen University Medical School,Shenzhen 518052, China) for helping identify and preserve the bacterial isolates.

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In vitro activities of isobavachalcone against planktonic and persister cells and biofilm of Enterococcus faecalis

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Abstract

Background

Results

Conclusion

Figures

Introduction

Materials and methods

Bacterial isolates and culture conditions

Antimicrobial susceptibility testing

Time-kill assay

Persister assay

Genome sequence of mutations

Statistical analysis

Results

Growth curve and the anti-biofilm activity of isobavachalcone

Discussion

Conclusion

Declarations

Conflict of Interest

Clinical trial number

Funding

Author Contribution

Acknowledgement

References

Additional Declarations

Supplementary Files

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