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

In vitro activities of isobavachalcone against planktonic and persister cells and biofilm of Enterococcus faecalis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vitro activities of isobavachalcone against planktonic and persister cells and biofilm of Enterococcus faecalis Lili Ouyang, Zhicao Xu, Yuanyuan Tang, Duoyun Li, Zhijian Yu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5317490/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2025 Read the published version in BMC Microbiology → Version 1 posted 4 You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction 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. Materials and methods 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 µ L 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). Results 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 (Table S1 ). 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. 1 a, 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. 1 a,b). The inhibition of the biofilm formation of E. faecalis by isobavachalcone was evaluated (Fig. 1 d,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. 2 a,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. 2 c, 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. 2 c). 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. 2 d). 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. 4 a,b) and FlgJ (Fig. 4 b,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. 4 a. Within the docking pocket, interactions between the isobavachalcone and PurH involve 9 hydrogen bonds, 5 hydrophobic interactions, and two ionic interactions (Fig. 4 b). 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. Discussion 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. 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. Declarations 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. References Kao PHN, Kline KA. Dr. Jekyll and Mr. Hide: How Enterococcus faecalis Subverts the Host Immune Response to Cause Infection. J Mol Biol. 2019;431(16):2932–45. Yadav J, Das S, Karthikeyan D, Chug R, Jyoti A, Srivastava VK, et al. Identification of Protein Drug Targets of Biofilm Formation and Quorum Sensing in Multidrug Resistant Enterococcus faecalis. Curr Protein Pept Sci. 2022;23(4):248–63. Leclercq R, Derlot E, Duval J, Courvalin P. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med. 1988;319(3):157–61. Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev. 1990;3(1):46–65. García-Solache M, Rice LB. The Enterococcus: a Model of Adaptability to Its Environment. Clin Microbiol Rev. 2019;32(2):e00058–18. Mercuro NJ, Davis SL, Zervos MJ, Herc ES. Combatting resistant enterococcal infections: a pharmacotherapy review. Expert Opin Pharmacother. 2018;19(9):979–92. Sandoe JAT, Witherden IR, Cove JH, Heritage J, Wilcox MH. Correlation between enterococcal biofilm formation in vitro and medical-device-related infection potential in vivo. J Med Microbiol. 2003;52(Pt 7):547–50. Rello J, Campogiani L, Eshwara VK. Understanding resistance in enterococcal infections. Intensive Care Med. 2020;46(2):353–6. Fisher RA, Gollan B, Helaine S. Persistent bacterial infections and persister cells. Nat Rev Microbiol. 2017;15(8):453–64. Goh HMS, Yong MHA, Chong KKL, Kline KA. Model systems for the study of Enterococcal colonization and infection. Virulence. 2017;8(8):1525–62. O’Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annu Rev Microbiol. 2000;54:49–79. Defraine V, Fauvart M, Michiels J. Fighting bacterial persistence: Current and emerging anti-persister strategies and therapeutics. Drug Resist Updat Rev Comment Antimicrob Anticancer Chemother. 2018;38:12–26. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv. 2019;37(1):177–92. de Assis LR, Theodoro RDS, Costa MBS, Nascentes JAS, Rocha MD da, de Bessa MA et al. S,. Antibacterial Activity of Isobavachalcone (IBC) Is Associated with Membrane Disruption. Membranes. 2022;12(3):269. Cui Y, Taniguchi S, Kuroda T, Hatano T. Constituents of Psoralea corylifolia Fruits and Their Effects on Methicillin-Resistant Staphylococcus aureus. Mol Basel Switz. 2015;20(7):12500–11. Chiang CC, Cheng MJ, Peng CF, Huang HY, Chen IS. A novel dimeric coumarin analog and antimycobacterial constituents from Fatoua pilosa. Chem Biodivers. 2010;7(7):1728–36. Wang M, Lin L, Lu JJ, Chen X. Pharmacological review of isobavachalcone, a naturally occurring chalcone. Pharmacol Res. 2021;165:105483. Dzoyem JP, Hamamoto H, Ngameni B, Ngadjui BT, Sekimizu K. Antimicrobial action mechanism of flavonoids from Dorstenia species. Drug Discov Ther. 2013;7(2):66–72. Cushnie TPT, Lamb AJ. Recent advances in understanding the antibacterial properties of flavonoids. Int J Antimicrob Agents. 2011;38(2):99–107. Chen Y, Hu H, Huang F, Ling Z, Chen B, Tan B, et al. Cocktail of isobavachalcone and curcumin enhance eradication of Staphylococcus aureus biofilm from orthopedic implants by gentamicin and alleviate inflammatory osteolysis. Front Microbiol. 2022;13:958132. Zheng J, Chen Z, Lin Z, Sun X, Bai B, Xu G, et al. Radezolid Is More Effective Than Linezolid Against Planktonic Cells and Inhibits Enterococcus faecalis Biofilm Formation. Front Microbiol. 2020;11:196. Zheng J, Shang Y, Wu Y, Wu J, Chen J, Wang Z, et al. Diclazuril Inhibits Biofilm Formation and Hemolysis of Staphylococcus aureus. ACS Infect Dis. 2021;7(6):1690–701. Zheng J, Shang Y, Wu Y, Zhao Y, Chen Z, Lin Z, et al. Loratadine inhibits Staphylococcus aureus virulence and biofilm formation. iScience. 2022;25(2):103731. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–9. Liu Y, Yang X, Gan J, Chen S, Xiao ZX, Cao Y. CB-Dock2: improved protein-ligand blind docking by integrating cavity detection, docking and homologous template fitting. Nucleic Acids Res. 2022;50(W1):W159–64. Xing N, Meng X, Wang S, Isobavachalcone. A comprehensive review of its plant sources, pharmacokinetics, toxicity, pharmacological activities and related molecular mechanisms. Phytother Res PTR. 2022;36(8):3120–42. de Assis LR, Theodoro RDS, Costa MBS, Nascentes JAS, Rocha MD da, de Bessa MA et al. S,. Antibacterial Activity of Isobavachalcone (IBC) Is Associated with Membrane Disruption. Membranes [Internet]. 2022;12(3). https://pubmed.ncbi.nlm.nih.gov/35323743 Sy CL, Chen PY, Cheng CW, Huang LJ, Wang CH, Chang TH, et al. Recommendations and guidelines for the treatment of infections due to multidrug resistant organisms. J Microbiol Immunol Infect Wei Mian Yu Gan Ran Za Zhi. 2022;55(3):359–86. Egan SA, Shore AC, O’Connell B, Brennan GI, Coleman DC. Linezolid resistance in Enterococcus faecium and Enterococcus faecalis from hospitalized patients in Ireland: high prevalence of the MDR genes optrA and poxtA in isolates with diverse genetic backgrounds. J Antimicrob Chemother. 2020;75(7):1704–11. Yan J, Bassler BL. Surviving as a Community: Antibiotic Tolerance and Persistence in Bacterial Biofilms. Cell Host Microbe. 2019;26(1):15–21. Defraine V, Fauvart M, Michiels J. Fighting bacterial persistence: Current and emerging anti-persister strategies and therapeutics. Drug Resist Updat Rev Comment Antimicrob Anticancer Chemother. 2018;38:12–26. Liu F, Jin P, Gong H, Sun Z, Du L, Wang D. Antibacterial and antibiofilm activities of thyme oil against foodborne multiple antibiotics-resistant Enterococcus faecalis. Poult Sci. 2020;99(10):5127–36. Liu X, Xiong Y, Shi Y, Deng X, Deng Q, Liu Y, et al. In vitro activities of licochalcone A against planktonic cells and biofilm of Enterococcus faecalis. Front Microbiol. 2022;13:970901. Harms A, Maisonneuve E, Gerdes K. Mechanisms of bacterial persistence during stress and antibiotic exposure. Science [Internet]. 2016;354(6318). https://pubmed.ncbi.nlm.nih.gov/27980159 Smith JR, Barber KE, Raut A, Rybak MJ. β-Lactams enhance daptomycin activity against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium in in vitro pharmacokinetic/pharmacodynamic models. Antimicrob Agents Chemother. 2015;59(5):2842–8. Barber KE, Shammout Z, Smith JR, Kebriaei R, Morrisette T, Rybak MJ. Biofilm Time-Kill Curves to Assess the Bactericidal Activity of Daptomycin Combinations against Biofilm-Producing Vancomycin-Resistant Enterococcus faecium and faecalis. Antibiot Basel Switz. 2021;10(8):897. Mathur H, Field D, Rea MC, Cotter PD, Hill C, Ross RP. Fighting biofilms with lantibiotics and other groups of bacteriocins. NPJ Biofilms Microbiomes. 2018;4:9. Suleyman G, Zervos MJ. Safety and efficacy of commonly used antimicrobial agents in the treatment of enterococcal infections: a review. Expert Opin Drug Saf. 2016;15(2):153–67. Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 30 Oct, 2024 Editor assigned by journal 25 Oct, 2024 Submission checks completed at journal 25 Oct, 2024 First submitted to journal 23 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5317490","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372422985,"identity":"73232459-e85b-4c8f-99b2-6a2d953003f1","order_by":0,"name":"Lili Ouyang","email":"","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Ouyang","suffix":""},{"id":372422986,"identity":"584dabbf-626b-4584-adaa-03c3265c7b8a","order_by":1,"name":"Zhicao Xu","email":"","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Zhicao","middleName":"","lastName":"Xu","suffix":""},{"id":372422987,"identity":"c8eb32e0-6ad1-46dd-99ef-1c67cf7b6b17","order_by":2,"name":"Yuanyuan Tang","email":"","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Tang","suffix":""},{"id":372422988,"identity":"6444cbc9-8d0e-4216-b1f7-16a1d98b59d7","order_by":3,"name":"Duoyun Li","email":"","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Duoyun","middleName":"","lastName":"Li","suffix":""},{"id":372422989,"identity":"05916477-4e8d-4de8-8d3c-91e9f8cd122f","order_by":4,"name":"Zhijian Yu","email":"","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Zhijian","middleName":"","lastName":"Yu","suffix":""},{"id":372422990,"identity":"a8580548-469d-4cfa-bcdf-4ca87c4e437f","order_by":5,"name":"Zewen Wen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYNACg39ybOztB0jRUnHAmI/nTAIpWs4cSJwn4WBApJOO9x7+zNt2J71NgiGB4UfFNiK0nDmXYMzb9iy3TbrxAGPPmduEtZjdyDFIzm1jzm2TOZDAzNhGjJb7bwwOA7Wks0kkGBCp5QaPYXPOmcMJxGuxP5NjzPynIs2wDRjIB4nyi2T7GeOPMwxs5OXb2w8++FFBhBYUcIBE9aNgFIyCUTAKcAEAmq0+HwWA4CwAAAAASUVORK5CYII=","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":true,"prefix":"","firstName":"Zewen","middleName":"","lastName":"Wen","suffix":""},{"id":372422991,"identity":"b1779963-bfb0-4a1a-b723-847c1ea07576","order_by":6,"name":"Haigang Zhang","email":"","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Haigang","middleName":"","lastName":"Zhang","suffix":""},{"id":372422992,"identity":"ebffd882-16d8-41f5-8bd6-78a689339def","order_by":7,"name":"Chaoqin Zhang","email":"","orcid":"","institution":"Shenzhen University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Chaoqin","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-10-23 09:08:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5317490/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5317490/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-03836-5","type":"published","date":"2025-02-28T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68328486,"identity":"7b1841b9-2bdd-4d31-9d31-3fe34d0034eb","added_by":"auto","created_at":"2024-11-06 06:36:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e16C51(a) and 16C106 (b) were treated with sub-MIC concentrations of isobavachalcone for 24 h or not, and the growth of planktonic cells was detected by optical density at 600 nm (OD600). (c,d)The effect of sub-MIC concentrations of isobavachalcone on the biofilm formation of the two \u003cem\u003eE. faecalis\u003c/em\u003e isolates. Data in panels(a,b) are the standard deviation of the mean of three replicates, and spots are representative of the mean of these independent experiments. The biofilm formation of \u003cem\u003eE. faecalis \u003c/em\u003ewas determined by crystal violet staining, measuring the absorbance at 570 nm during 24h. Data presented(c,d) are the average of three independent experiments (mean ± SEM)... Compared with control, *P \u0026lt; 0.05,**P \u0026lt; 0.01; (Student’s t-test).\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5317490/v1/531228f79d146b162172a8c1.jpg"},{"id":68329368,"identity":"4c72d4a2-57d3-4368-97e8-28c56025bdea","added_by":"auto","created_at":"2024-11-06 06:44:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":215595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e16C51(a) and 16C106(b) of time-kill curves for planktonic cells were tested with isobavachalcone of 4 × MIC in combination with some antibiotics (ampicillin, vancomycin, linezolid). The anti-persisters activity of isobavachalcone against \u003cem\u003eE.faecalis \u003c/em\u003e16C51(c) and 16C106(d) were tested with the three antibiotics and their combinations with isobavachalcone at 10 × MICs. Data panels (a,b) are the standard deviation of the mean of three replicates and spots are representative of the mean of these independent experiments. Antimicrobials were added at time point 0 and monitored until 24 h. \u0026nbsp;Amp, Ampicillin; Van, Vancomycin; Lin, Linezolid, Iso,Isobavachalcone.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5317490/v1/498ba80d767a7d8a615cbaa0.jpg"},{"id":68328345,"identity":"61b9fb7e-526c-43a9-87d4-ae4e02dc5501","added_by":"auto","created_at":"2024-11-06 06:28:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155303,"visible":true,"origin":"","legend":"\u003cp\u003eThe medication effect of isobavachalconeon the established biofilms of \u0026nbsp;4 \u003cem\u003eE. faecalis \u003c/em\u003estrains \u0026nbsp;FB-1(a), 16C51(b), 16C102 (c), 16C166 (d). The biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e at 8 × MIC was respectively shown by measuring the absorbance at 570 nm after 24h of culture. The data presented were the average of three independent experiments (mean ±SD).Compared with control, *P \u0026lt; 0.05; ***P \u0026lt; 0.001; (Student’s t-test). MIC, minimum inhibitory concentration. Amp, Ampicillin; Van, Vancomycin; Lin, Linezolid; Dap, Daptomycin; Iso, Isobavachalcone.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5317490/v1/1c2a6e9aaadf7d954d832239.jpg"},{"id":68328344,"identity":"49804de6-8a25-4cba-8a0a-4ba2eafb5e8b","added_by":"auto","created_at":"2024-11-06 06:28:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":234783,"visible":true,"origin":"","legend":"\u003cp\u003e(a,b) Interaction between isobavachalconeand PurH, FlgJ (c,d) by molecular docking. The isobavachalcone was shown with the elements in a variety of color combinations by using a ball-and-stick style (blue represents a nitrogen atom; red represents an oxygen atom; and green represents a fluorine atom).\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5317490/v1/46232d0967e6ebf2e1f301a1.jpg"},{"id":77623231,"identity":"1c6d9a39-0b62-47c7-86f6-fc0c0ea577cf","added_by":"auto","created_at":"2025-03-03 16:11:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1897718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5317490/v1/780f1b75-8933-4e43-88d3-6a4150a66400.pdf"},{"id":68328348,"identity":"78f01790-0474-4faa-abd5-085a06b9bb02","added_by":"auto","created_at":"2024-11-06 06:28:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13755,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5317490/v1/ad0a631287cc1a26f264d073.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In vitro activities of isobavachalcone against planktonic and persister cells and biofilm of Enterococcus faecalis","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eEnterococcus faecalis\u003c/em\u003e 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 \u003cem\u003eE.faecalis\u003c/em\u003e infection is gradually troubling clinicians due to the dissemination of \u003cem\u003eE.faecalis\u003c/em\u003e with intrinsic or acquired resistance towards a range of antibiotics (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In the 1970s, resistance in \u003cem\u003eEnterococci\u003c/em\u003e 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(\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). With the widespread use of antimicrobial drugs, multidrug-resistant (MDR) \u003cem\u003eE. faecalis\u003c/em\u003e, such as VRE, linezolid-resistant \u003cem\u003eenterococci\u003c/em\u003e, daptomycin-resistant \u003cem\u003eenterococci\u003c/em\u003e and \u003cem\u003eet al\u003c/em\u003e, have gradually emerged and disseminated worldwide, which often leads to clinical treatment failure(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Therefore, the rapid development of multi-drug resistance in \u003cem\u003eenterococci\u003c/em\u003e necessitated an urgent need to look for an alternative medicine with antibacterial activities against MDR \u003cem\u003eE. faecalis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. faecalis\u003c/em\u003e 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(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003cem\u003eE. faecalis\u003c/em\u003e 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(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). 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(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).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(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIsobavachalcone (Iso), isolated from plants such as \u003cem\u003eFatouapilosa\u003c/em\u003e and \u003cem\u003ePsoralea corylifolia\u003c/em\u003e 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(\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). 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(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) .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(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).In recent years, Yan Chen et al. reported that the use of isobavachalcone together with gentamicin significantly improved its antibacterial effect on \u003cem\u003eS.aureus\u003c/em\u003e biofilm(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).However, there are few reports on the antibacterial activity and anti-biofilm against \u003cem\u003eE. faecalis\u003c/em\u003e and its antibacterial mechanism. Therefore, the objective of this study was to assess the antibacterial and anti-biofilm activity of isobavachalcone against \u003cem\u003eE. faecalis\u003c/em\u003e and to identify the potential target genes of isobavachalcone A in \u003cem\u003eE. faecalis.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolates and culture conditions\u003c/h2\u003e \u003cp\u003eA total of 219 strains of \u003cem\u003eE. faecalis\u003c/em\u003e (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 (Biom\u0026eacute;rieux, Marcy l\u0026rsquo;Etoile, France) according to the manufacturer\u0026rsquo;s instructions. The \u003cem\u003eE. faecalis\u003c/em\u003e ATCC29212 and OG1RF were purchased from the American Type Culture Collection (ATCC) and used as reference strains.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eE.faecalis\u003c/em\u003e strains were stored at -80℃ in glycerol-containing (40%) Tryptic Soy Broth (TSB) medium for subsequent analysis. \u003cem\u003eE. faecalis\u003c/em\u003e isolates were cultivated in TSB) at 37\u0026deg;C with agitation at 180 rpm, unless otherwise stated. \u003cem\u003eE. faecalis\u003c/em\u003e isolates were cultivated in Cation-Adjusted Mueller-Hinton Broth (CAMHB) at 37\u0026deg;C with agitation to conduct antimicrobial susceptibility tests and time-killing tests. \u003cem\u003eE. faecalis\u003c/em\u003e isolates were cultivated in TSBG (TSB with 0.5% glucose) at 37\u0026deg;C to conduct a biofilm assay. In all experiments, CAMHB media were supplemented with 50 mg/L Ca2\u0026thinsp;+\u0026thinsp;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.)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntimicrobial susceptibility testing\u003c/h3\u003e\n\u003cp\u003eThe antimicrobial susceptibility of \u003cem\u003eE. faecalis\u003c/em\u003e 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 (Biom\u0026eacute;rieux, Marcy l\u0026rsquo;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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth curve experiment of\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwo \u003cem\u003eE. faecalis\u003c/em\u003e (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\u0026thinsp;=\u0026thinsp;0.1). Isobavachalcone was added to make the final concentrations at 1/32 \u0026times; MIC, 1/16 \u0026times; MIC, 1/8 \u0026times; MIC, 1/4 \u0026times; MIC, and 1/2 \u0026times; 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntimicrobials inhibit the biofilm formation and eradicate the established biofilms of\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwo \u003cem\u003eE. faecalis\u003c/em\u003e isolates (16C51,16C106) previously reported with positive biofilm formation were selected to evaluate the inhibition of isobavachalcone on the biofilm formation of \u003cem\u003eE.faecalis\u003c/em\u003e. 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 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e 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.\u003c/p\u003e \u003cp\u003eFour \u003cem\u003eE. faecalis\u003c/em\u003e isolates (FB-1, 16C51, 16C102, 16C166) were further used for evaluating the eradication capacity of isobavachalcone against the biofilm formation of \u003cem\u003eE.faecalis\u003c/em\u003e. The \u003cem\u003eE. faecalis\u003c/em\u003e strains were cultivated in TSB at 37℃ for 12h and then diluted in 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL 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\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e 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.\u003c/p\u003e\n\u003ch3\u003eTime-kill assay\u003c/h3\u003e\n\u003cp\u003eTwo \u003cem\u003eE. faecalis\u003c/em\u003e(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(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), 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 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003eMIC. Each experiment was repeated three times.\u003c/p\u003e\n\u003ch3\u003ePersister assay\u003c/h3\u003e\n\u003cp\u003eIn order to evaluate the additional anti-persister activity of isobavachalcone against \u003cem\u003eE.faecalis\u003c/em\u003e, two \u003cem\u003eE. faecalis\u003c/em\u003e isolates(16C51, 16C106) were selected and tested for the persister assays with isobavachalcone and some first-line antibiotics. \u003cem\u003eE.faecalis\u003c/em\u003e 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 \u0026times; 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.\u003c/p\u003e \u003cp\u003eTo 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro, induction of isobavachalcone non-sensitive\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003eisolates with possible mutations\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the possible target genes of isobavachalcone in \u003cem\u003eE.faecalis\u003c/em\u003e, the isobavachalcone non-sensitive \u003cem\u003eE. faecalis\u003c/em\u003e isolates with possible mutations were induced and screened in vitro according\u003c/p\u003e \u003cp\u003eto previous studies(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) .\u003cem\u003eE. faecalis\u003c/em\u003e isolates (16C51, 16C106) were subcultured serially in TSB containing isobavachalcone. The initial inducing concentration of isobavachalcone was 1/2 \u0026times; MIC; the concentration was then increased successively to high concentrations. \u003cem\u003eE. faecalis\u003c/em\u003e isolates\u003c/p\u003e \u003cp\u003ein each concentration of isobavachalcone were cultured for three to five passages before being inoculated and passaged to the next generation. \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e isolates were kept frozen at \u0026minus;\u0026thinsp;80◦C in glycerol containing (35%) TSB.\u003c/p\u003e\n\u003ch3\u003eGenome sequence of mutations\u003c/h3\u003e\n\u003cp\u003eThe genomic DNA of two isobavachalcone non-sensitive \u003cem\u003eE.faecalis\u003c/em\u003e isolates (16C51-T1.1 and 16C51-T1.2) was extracted, and a total amount of 1 \u0026micro;g 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 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).Coding genes, repetitive sequences, non-coding RNAs, genomics islands, transposons, prophages, and CRISPR (clustered regularly interspaced short\u003c/p\u003e \u003cp\u003epalindromic 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. \u003cb\u003eMolecular Docking\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe molecular structure pdb file of PurH and FlgJ was constructed by AlphaFoldDB(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). 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(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed by Student\u0026rsquo;s t-test. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were regarded as statistically significant. All data were analyzed in the SPSS software package (version 26.0).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eactivity of isobavachalcone against clinical\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe antibacterial activity of isobavachalcone against \u003cem\u003eE. faecalis\u003c/em\u003e was investigated by determining the MICs of isobavachalcone and other antibacterial agents against \u003cem\u003eE. faecalis\u003c/em\u003e and confirming the susceptibility results according to the CLSI-M100-S30. The MIC of isobavachalcone against the reference strains of \u003cem\u003eE. faecalis\u003c/em\u003e, ATCC29212, and OG1RF, was determined. Both strains exhibited a MIC of 12.5 \u0026micro;M (equivalent to 4.05 mg/L). Subsequently, the MICs of isobavachalcone were measured for 22 clinical isolates of \u003cem\u003eE. faecalis\u003c/em\u003e (isolated from urine, exudate, or blood), of which the MICs were12.5 \u0026micro;M for all 22 isolates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The isobavachalcone MICs in 22 clinical isolates of \u003cem\u003eE.faecalis\u003c/em\u003e, including two linezolid-resistant \u003cem\u003eE.faecalis\u003c/em\u003e and ten daptomycin-nonsusceptible strains, was also shown with 12.5\u0026micro;M(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting its excellent bactericidal activity against multi-drug resistant \u003cem\u003eE.faecalis\u003c/em\u003e. To further verify the antibacterial activity of isobavachalcone against clinical isolates of \u003cem\u003eE.faecalis\u003c/em\u003e, the MIC values of isobavachalcone against 198 additional clinical isolates of \u003cem\u003eE. faecalis\u003c/em\u003e (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 \u0026micro;M, with the MIC50 and MIC90 levels being 12.5 \u0026micro;M against the clinical isolates (Table\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGrowth curve and the anti-biofilm activity of isobavachalcone\u003c/h3\u003e\n\u003cp\u003eThis study was to investigate the effect of subinhibitory concentrations of isobavachalcone on the biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e. To this end, the effect of subinhibitory concentrations of isobavachalcone on the planktonic growth of \u003cem\u003eE. faecalis\u003c/em\u003e (16C51, 16C106) planktonic cells was determined using an automatic growth curve analyzer. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the 1/2 \u0026times; MIC of isobavachalcone demonstrated inhibitory effects on the growth of \u003cem\u003eE. faecalis\u003c/em\u003e planktonic cells during the logarithmic growth phase. In contrast, the 1/4 \u0026times;, 1/8 \u0026times;, 1/16 \u0026times;, and 1/32 \u0026times; MICs of isobavachalcone exhibited no inhibitory effects against \u003cem\u003eE.faecalis\u003c/em\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). The inhibition of the biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e by isobavachalcone was evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,c). Notably, biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e (16C51) was significantly inhibited by 1/2 \u0026times; or 1/4 \u0026times; MICs of isobavachalcone. However, against a different strain of \u003cem\u003eE. faecalis\u003c/em\u003e(16C106), there was no significant inhibition at 1/2\u0026times;,1/4 \u0026times; MIC of isobavachalcone against the planktonic growth, whereas isobavachalcone at 1/4\u0026times;MIC could inhibit biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTime-killing curve and biofilm elimination of\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003eby isobavachalcone alone or in combination\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe efficacy of the bactericidal and anti-biofilm activity of isobavachalcone alone and in combination were compared in two \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e isolates. Exposure to antimicrobial agents at 4 \u0026times;MIC resulted in a reduction in log10CFU/mL over the 0\u0026ndash;24 hour period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b), suggesting the similar bactericidal activity of isobavachalcone and the combinations with three commonly-used antibiotics in these two \u003cem\u003eE. faecalis\u003c/em\u003e. That was, time-killing curves of isobavachalcone alone in \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e 16C51 isolate was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Furthermore, isobavachalcone combination with vancomycin, linezolid or ampicillin (all used at 8 \u0026times; MIC) to treat established \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic mutations in the isobavachalcone resistant\u003c/b\u003e \u003cb\u003eE.faecalis\u003c/b\u003e \u003cb\u003eand potential targeting was identified through molecular docking\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the possible target gene of isobavachalcone in \u003cem\u003eE. faecalis\u003c/em\u003e, the isobavachalcone-resistant \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e was performed, suggesting ten nucleotide mutations in the 16C51-T1 isolate (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). 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\u003c/p\u003e \u003cp\u003ein \u003cem\u003eE. faecalis\u003c/em\u003e. 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b) and FlgJ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c) in favorable conformations, exhibiting low binding energy of \u0026minus;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;7 kcal/mol suggests a good binding force]. The optimal conformation of the isobavachalcone and PurH complex is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Within the docking pocket, interactions between the isobavachalcone and PurH involve 9 hydrogen bonds, 5 hydrophobic interactions, and two ionic interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These findings suggest that isobavachalcone has the capability to effectively bind with PurH of \u003cem\u003eE. faecalis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial susceptibilities of \u003cem\u003eE. faecalis\u003c/em\u003e determined by conventional broth microdilution\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eMIC(mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDap\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRif\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eFOS\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eIso\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOG1RF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACTCC29212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFB-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.5\u0026micro;M(4.05mg/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMIC, 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMutations in \u003cem\u003eE. faecalis\u003c/em\u003e 16C51-T1 isolate were detected by whole genome sequencing. \u003cb\u003eGenome analysis of mutations\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eref_gene_ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA mutations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAA mutations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eref_gene_product\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eref_gene_strand\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM000347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT247C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY83H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePyrroline-5-carboxylate reductase\u003c/p\u003e \u003cp\u003eAmino acid transport and metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM000484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG559A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA187T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003edTDP-4-dehydrorhamnose reductase\u003c/p\u003e \u003cp\u003eCell wall/membrane/envelope biogenesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM000718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC187T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP63S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdenine deaminase\u003c/p\u003e \u003cp\u003eNucleotide transport and metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM001010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA508G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT170A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlagellum-specific peptidoglycan hydrolase FlgJ\u003c/p\u003e \u003cp\u003eCell wall/membrane/envelope biogenesis; Cell motility ;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM001244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA688G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK230E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFatty acid/phospholipid biosynthesis enzyme\u003c/p\u003e \u003cp\u003eLipid transport and metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM001349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT938C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI313T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDNA-binding transcriptional regulator, FrmR family, Transcription\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM001679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG49A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE17K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlycerol kinase\u003c/p\u003e \u003cp\u003eEnergy production and conversion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM001760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC632A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT211K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAICAR transformylase/IMP cyclohydrolase PurH\u003c/p\u003e \u003cp\u003eNucleotide transport and metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM002262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA109G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK37E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSignal transduction histidine kinase\u003c/p\u003e \u003cp\u003eSignal transduction mechanisms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16C51T1_GM000346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA442G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI148V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhosphomannomutase\u003c/p\u003e \u003cp\u003eCarbohydrate transport and metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eE. faecalis\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIsobavachalcone is a natural flavonoid derived from a wide range of medicinal plants, including the Fabaceae, Moraceae and so on(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) .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 \u0026micro;g/mL, respectively (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). But there are no reports on the effect of isobavachalcone on \u003cem\u003eE. faecalis\u003c/em\u003e. Linezolid is a first-line treatment for VRE infections(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, with the increased use of linezolid, an increasing number of cases of linezolid-resistant \u003cem\u003eE. faecalis\u003c/em\u003e have emerged, and its propensity to form biofilms limits therapeutic options(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Biofilm formation and persister cell formation can be viewed as two types of collective bacterial behaviors (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). 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(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). It has been reported that certain small molecules and natural products exert an inhibitory effect on the growth, biofilm formation, and exopolysaccharide synthesis of \u003cem\u003eE. faecalis\u003c/em\u003e (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This study also suggested that isobavachalcone had excellent antibacterial activity against \u003cem\u003eE. faecalis\u003c/em\u003e and had a good inhibitory effect on the biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE.faecalis\u003c/em\u003e did not an increase in resistance to isobavachalcone, which shows that isobavachalcone is a promising antibacterial agent against resistant \u003cem\u003eE. faecalis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBacterial 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(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). 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(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Interestingly, our results demonstrated that isobavachalcone alone against \u003cem\u003eE. faecalis\u003c/em\u003e showed a rapid bactericidal effect, killing more planktonic cells than vancomycin, linezolid, or ampicillin. It even eradicated \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e 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(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Therefore, further studies are required to substantiate the efficacy of isobavachalcone in the removal of biofilms when combined with daptomycin.\u003c/p\u003e \u003cp\u003eTo explore the possible target site of isobavachalcone in \u003cem\u003eE. faecalis\u003c/em\u003e, the isobavachalcone non-sensitive \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e, 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(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The bactericidal effect of isobavachalcone on \u003cem\u003eE. faecalis\u003c/em\u003e 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 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eThe limitation of this study is to find the target gene of isobavachalcone in \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e through the utilization of alternative methodologies, such as CO-IP and LC-MS.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study discovered that isobavachalcone had an antibacterial effect on \u003cem\u003eE. faecalis\u003c/em\u003e, and significantly inhibited the biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e at subinhibitory concentrations. In addition, antibacterial and antibiofilm activity against clinical \u003cem\u003eE. faecalis\u003c/em\u003e isolates from China by targeting FlgJ protein.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All procedures involving human participants were performed following the ethical standards of Shenzhen Nanshan People\u0026rsquo;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\u0026rsquo;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.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis 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\u0026rsquo;s Republic of China (NS2023026, YN2022028, NS2022114; NS2022046; NS2023049; NSZD2023032; NS2024038).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eOuyang 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.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Weiguang Pan and Jie Lian (Department of Laboratory Medicine, Shenzhen Nanshan People\u0026rsquo;s Hospital, Shenzhen University Medical School,Shenzhen 518052, China) for helping identify and preserve the bacterial isolates.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKao PHN, Kline KA. Dr. Jekyll and Mr. Hide: How Enterococcus faecalis Subverts the Host Immune Response to Cause Infection. J Mol Biol. 2019;431(16):2932\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav J, Das S, Karthikeyan D, Chug R, Jyoti A, Srivastava VK, et al. Identification of Protein Drug Targets of Biofilm Formation and Quorum Sensing in Multidrug Resistant Enterococcus faecalis. Curr Protein Pept Sci. 2022;23(4):248\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeclercq R, Derlot E, Duval J, Courvalin P. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med. 1988;319(3):157\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurray BE. The life and times of the Enterococcus. Clin Microbiol Rev. 1990;3(1):46\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Solache M, Rice LB. 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Expert Opin Drug Saf. 2016;15(2):153\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Isobavachalcone, Enterococcus faecalis, Antibacterial, Persister, Biofilm","lastPublishedDoi":"10.21203/rs.3.rs-5317490/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5317490/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMounting evidences have demonstrated the extensive pharmacological activities of the natural product isobavachalcone, including antimicrobial activity, inhibition of reverse transcriptase, antitubercular and antioxidant ability \u003cem\u003eet al\u003c/em\u003e. However, the antibacterial and antibiofilm activity and its action mode of isobavachalcone against clinical \u003cem\u003eE. faecalis\u003c/em\u003e isolates remain elusive. This study aims to evaluate the in vitro antibacterial and anti-biofilm activities of isobavachalcone on clinical \u003cem\u003eE. faecalis\u003c/em\u003e isolates from China and further investigate the possible target site of isobavachalcone in \u003cem\u003eE. faecalis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur data suggested the MICs of isobavachalcone ranging from 6.25 to 12.5 \u0026micro;M against 220 \u003cem\u003eE. faecalis\u003c/em\u003e strains. The robust inhibitory effect of isobavachalcone with sub-MIC concentration ( 1/2xMIC ) against the biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e was found. The rapid bactericidal effect of isobavachalcone against \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e. Other mutations are involved in the cell wall or cell membrane biogenesis, DNA synthesis, and energy metabolism.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study discovered that isobavachalcone had an antibacterial effect on \u003cem\u003eE. faecalis\u003c/em\u003e, and significantly inhibited the biofilm formation of \u003cem\u003eE. faecalis\u003c/em\u003e at subinhibitory concentrations. In addition, antibacterial and antibiofilm activity against clinical \u003cem\u003eE. faecalis\u003c/em\u003e isolates from China by targeting FlgJ protein.\u003c/p\u003e","manuscriptTitle":"In vitro activities of isobavachalcone against planktonic and persister cells and biofilm of Enterococcus faecalis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 06:27:55","doi":"10.21203/rs.3.rs-5317490/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-30T23:08:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-25T05:13:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-25T05:09:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2024-10-23T09:01:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e7e43d13-5e29-4ce8-8856-604f2f2e208d","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T16:09:21+00:00","versionOfRecord":{"articleIdentity":"rs-5317490","link":"https://doi.org/10.1186/s12866-025-03836-5","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-02-28 15:57:23","publishedOnDateReadable":"February 28th, 2025"},"versionCreatedAt":"2024-11-06 06:27:55","video":"","vorDoi":"10.1186/s12866-025-03836-5","vorDoiUrl":"https://doi.org/10.1186/s12866-025-03836-5","workflowStages":[]},"version":"v1","identity":"rs-5317490","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5317490","identity":"rs-5317490","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}