Enterococcus Faecalis Biofilm Formation Research Articles

Induction of proteome changes involved in biofilm formation of Enterococcus faecalis in response to gentamicin

BackgroundEnterococcus faecalis is a significant cause of nosocomial infections and other diseases, including endocarditis, bacteremia, and urinary tract infections. This microorganism forms biofilms to overcome difficult environmental conditions, such as lack of oxygen, lack of water, and the presence of antimicrobials. These biofilms make diseases difficult by changing their proteome contents, protecting the bacterium, and increasing their pathogenicity. This study aimed to evaluate gentamicin's effect on proteome changes and biofilm formation in E. faecalis. MethodTwenty-five clinical isolates and one standard isolate were selected for the experiments. A label-free/gel-free proteomic and microtiter plate techniques were used to study proteome changes and biofilm formation, respectively. ResultsGentamicin significantly increased the biofilm formation in 62% of isolates and the rest of the isolates; no significant change was observed. The abundance of lactate utilization protein C, ribosomal RNA small subunit methyltransferase H, and protein translocase subunit SecA were increased. However, the abundances of proteins effective in cell division and metabolism, such as replication initiation protein and segregation and condensation protein A, were decreased. ConclusionThe present study's findings exhibited that antibiotics might have adverse effects on treatment and increase microorganisms' pathogenicity. It was observed in gentamicin as induction of biofilm formation through different mechanisms, particularly changes in the expression of specific proteins in E. faecalis.

Antimicrobial Ceramic Filters for Water Bio-Decontamination

Bio-contamination of water through biofouling, which involves the natural colonization of submerged surfaces by waterborne organisms, is a global socio-economic concern, allied to premature materials bio-corrosion and high human health risks. Most effective strategies release toxic and persistent disinfectant compounds into the aquatic medium, causing environmental problems and leading to more stringent legislation regarding their use. To minimize these side effects, a newly non-biocide-release coating strategy suitable for several polymeric matrices, namely polydimethylsiloxane and polyurethane (PU)-based coatings, was used to generate antimicrobial ceramic filters for water bio-decontamination. The best results, in terms of antimicrobial activity and biocide release, showed an expressed delay and a decrease of up to 66% in the population of methicillin-resistant Staphylococcus aureus bacteria on ceramic filters coated with polyurethane (PU)-based coatings containing grafted Econea biocide, and no evidence of biocide release after being submerged for 45 days in water. Biocidal PU-based surfaces were also less prone to Enterococcus faecalis biofilm formation under flow conditions with an average reduction of 60% after 48 h compared to a pristine PU-based surface. Biocidal coated filters show to be a potential eco-friendly alternative for minimizing the environmental risks associated with biofouling formation in water-based industrial systems.

Effects of ClpP protease on biofilm formation of Enterococcus faecalis.

Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Objectives Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms.Methodology In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student’s t-test or one-way analysis of variance (ANOVA).Results The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase biofilm formation in E. faecalis.Conclusion Our study shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.

Doxycycline-functionalized polymeric nanoparticles inhibit Enterococcus faecalis biofilm formation on dentine.

To evaluate in a laboratory setting the antimicrobial properties and the potential to inhibit biofilm formation of novel remineralizing polymeric nanoparticles (NPs) when applied to dentine surfaces and to ascertain the effect of the functionalization of these NPs with zinc, calcium or doxycycline. The antimicrobial activity and inhibition of biofilm formation of polymeric NPs were analysed on human dentine blocks that were infected with Enterococcus faecalis before or after application of NPs. LIVE/DEAD ® testing under Confocal Laser Scanning Microscopy and bacterial culturing were employed to analyse biofilm biovolume and bacterial viability. Field Emission Scanning Electron Microscopy was also employed to assess biofilm morphology. One-way anova with Welch's correction and post hoc comparison by the Games-Howell test were performed for comparisons between groups. The un-functionalized NPs displayed the greatest antimicrobial activity against E.faecalis biofilms as they provided the lowest biovolume (3865.7±2926.97µm3 ; P<0.001) and the highest dead/injured cells percentage (79.93±18.40%; P<0.001), followed by Dox-NPs (biovolume: 19,041.55±17,638.23µm3 , dead/injured cells: 45.53±26.50%; P<0.001). Doxycycline-loaded NPs had the largest values of inhibition of biofilm formation with the lowest biofilm biovolume (8517.65±7055.81µm3 ; P<0.001) and a high dead/injured bacterial percentage (68.68±12.50%; P<0.001). Un-functionalized NPs did not reduce biomass growth (P>0.05), but attained the largest percentage of compromised cells (93±8.23%; P<0.001), being able to disrupt biofilm formation. It also produced occlusion of dentinal tubules, potentially interfering with bacterial tubule penetration. A new generation of bioactive nano-fillers (doxycycline-functionalized polymeric NPs) had antibacterial activity and occluded dentinal tubules. Incorporating these NPs into endodontic sealers may have the potential to enhance the outcome of root canal treatment.

Involvement of Chromosomally Encoded Homologs of the RRNPP Protein Family in Enterococcus faecalis Biofilm Formation and Urinary Tract Infection Pathogenesis.

Enterococcus faecalis is an opportunistic pathogen capable of causing infections, including endocarditis and urinary tract infections (UTI). One of the well-characterized quorum-sensing pathways in E. faecalis involves coordination of the conjugal transfer of pheromone-responsive plasmids by PrgX, a member of the RRNPP protein family. Members of this protein family in various Firmicutes have also been shown to contribute to numerous cellular processes, including sporulation, competence, conjugation, nutrient sensing, biofilm formation, and virulence. As PrgX is a plasmid-encoded RRNPP family member, we surveyed the genome of the multidrug-resistant strain V583 for additional RRNPP homologs using computational searches and refined those identified hits for predicted structural similarities to known RRNPP family members. This led us to investigate the contribution of the chromosomally encoded RRNPP homologs to biofilm processes and pathogenesis in a catheter-associated urinary tract infection (CAUTI) model. In this study, we identified five such homologs and report that 3 of the 5 homologs, EF0073, EF1599, and EF1316, affect biofilm formation as well as outcomes in the CAUTI model.IMPORTANCEEnterococcus faecalis causes health care-associated infections and displays resistance to a variety of broad-spectrum antibiotics by acquisition of resistance traits as well as the ability to form biofilms. Even though a growing number of factors related to biofilm formation have been identified, mechanisms that contribute to biofilm formation are still largely unknown. Members of the RRNPP protein family regulate a diverse set of biological reactions in low-G+C Gram-positive bacteria (Firmicutes). Here, we identify three predicted structural homologs of the RRNPP family, EF0073, EF1599, and EF1316, which affect biofilm formation and CAUTI pathogenesis.

ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

BackgroundClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△clpP) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling.ResultsThe ΔclpP mutant strain showed impaired growth at 20 °C or 45 °C at 5% NaCl or 2 mM H2O2. The number of surviving ΔclpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The ΔclpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the ΔclpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the ΔclpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein (rplD), ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE), and orotidine-5′-phosphate decarboxylase (pyrF) all decreased in the ΔclpP mutant strain.ConclusionThe present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

The growth and biofilm formation of Enterococcus faecalis in ethanol extract of Citrus aurantiifolia Indonesian species

Context: Enterococcus faecalis is reported as a biofilm bacterium involved in the pathogenesis of tooth root canal infection. The ability to grow and form biofilms is the reason these bacteria are difficult to be eliminated. Aims: To analyze the antibacterial effect of Citrus aurantiifolia against the growth and biofilm formation of E. faecalis. Methods: This study used E. faecalis ATCC 29212 and ethanol extract of lime peel (Citrus aurantiifolia). The E. faecalis growth was assessed by spectrophotometry, and biofilm formation was examined with 1% violet crystals also visualized by microscope (magnification 400×). Results: Citrus aurantiifolia extract can inhibit the growth and biofilm formation of E. faecalis with varying quantities. Based on period incubations, a concentration of 6.25% had a better ability to suppress the growth of E. faecalis (&lt;300 CFU/mL). The 75% concentration was the highest among other levels to inhibit the biofilms formation of E. faecalis that assumed the higher concentration of the extract, the most significant increase in inhibition of the biofilm formation of E. faecalis. Growth and biofilm formation of E. faecalis showed linearity in line with a concentration of 6.25% (p&lt;0.05: 0.000) with a relatively stable relationship (r=0.506). Conclusions: Citrus aurantiifolia extract can inhibit the growth of E. faecalis that is in line with the inhibiton of the biofilm formation.

2604. Changes in a Fatty Acid Kinase Associated with Daptomycin (DAP) Resistance Lead to Increased Collagen Binding and Biofilm Formation in Enterococcus faecalis

BackgroundEnterococci are a major cause of healthcare-associated infections with limited treatment options. We previously identified that mutations in dak (a gene encoding a putative fatty acid kinase), ace (a collagen adhesin) and the YxdJK stress response system are associated with DAP resistance (DAP-R) in E. faecalis (Efs) in the absence of a functional LiaFSR system. Here, we examined the role of DAK in pathogenesis by examining the ability of the mutants to produce biofilm and bind to collagen, an important protein of the extracellular matrix.MethodsPreviously, the Efs strain OG1RFΔliaR (inactive LiaFSR system, DAP susceptible) was adapted to make a DAP-R derivative (mutations in yxdK, dak, and ace), and the mutant OG1RFΔliaRΔc-dak, lacking the C-terminal domain of dak, and its complement OG1RFΔliaRΔc-dak::c-dak were constructed to study the dak mutation in isolation. Biofilm formation (BF) for the above strains was assayed after growth in tryptic soy broth with glucose in 96-well plates at 37° C for 24 hours. Bacteria were fixed with Bouin’s fixative, stained with crystal violet, and biofilm was quantitated by absorbance at 570 nm. For collagen adherence, 96-well plates were coated with 10 μg/well type I collagen, with 2% bovine serum albumin (BSA) as a control. Bacteria grown at 46° C (to induce ace expression) were added at OD 600 nm of 1.0 and allowed to bind for 2 hours. Non-adherent bacteria were removed by washing, cells were fixed, stained, and quantified as above.ResultsWhen compared with OG1RFΔliaR, the DAP-R derivative exhibited a significant increase in BF (4.6 vs. 2.4, P < 0.001). This enhanced biofilm phenotype was also seen in the OG1RFΔliaRΔc-dak mutant (6.1 vs. 2.4, P < 0.001), and reverted on complementation of the full-length dak in its native chromosomal location (2.4 vs. 2.6, p = 0.72). DAK was also found to impact adherence to collagen, with OG1RFΔliaRΔc-dak showing increased binding to collagen when compared with OG1RFΔliaR (7.9 vs. 3.4, P < 0.001), a phenotype which reverted on complementation (7.9 vs. 1.2, P < 0.001).ConclusionChanges in an enzyme involved in DAP adaptation lead to biofilm formation and adherence to extracellular matrix proteins, potentially enhancing virulence in the setting of DAP-R. Disclosures All authors: No reported disclosures.

Inhibition of biofilm formation and exopolysaccharide synthesis of Enterococcus faecalis by phenyllactic acid

This study aimed to evaluate the inhibitory activity of phenyllactic acid (PLA) against the biofilm formation of Enterococcus faecalis and to explore its potential molecular mechanism. The MIC value of PLA that inhibited the growth of E. faecalis R612-Z1 in BHI broth was 5 mg/mL. PLAs at subinhibitory concentrations of 1.25 and 2.50 mg/mL were found to inhibit biofilm formation by a crystal violet staining assay. The cell swimming and swarming motilities of E. faecalis were reduced in the presence of PLA. An apparent decrease in the thickness of PLA-treated biofilms was observed through confocal laser scanning microscopy analysis. The exopolysaccharide production in E. faecalis biofilms was inhibited by EPS quantification assay and scanning electron microscopy (SEM). qRT-PCR analyses showed that PLA down-regulated the transcription of Ebp pili genes (ebpABC) and Epa polysaccharide genes (epaABE). PLA inhibited the biofilm formation by interfering with cell mobility and EPS production of E. faecalis. In addition, PLA at concentrations of 10.0 mg/mL can effectively control the bacterial cells in a three-day-old mature biofilm of E. faecalis grown on 24-well flat-bottom polystyrene plates and stainless-steel surfaces. Thus, PLA is potentially an effective agent to control E. faecalis biofilms.

Virulence factors, biofilm formation and antibiotic resistance pattern in Enterococcus faecalis and Enterococcus faecium isolated from clinical and commensal human samples in Isfahan, Iran.

The aim of this study was to determine and compare antibiotic resistance profile, biofilm formation ability and frequency of agg and ace genes in Enterococcus spp strains isolated from patients and healthy individuals. A total of 90 non-duplicate Enterococcus spp isolates were isolated from patients and healthy individuals. Antibiotic susceptibility pattern was determined by disk diffusion and E-test method. Virulence genes and two species of enterococci were determined by PCR amplification. The capacity of biofilm formation was also evaluated by microtiter plate technique. E. faecalis was the predominant species among our clinical isolates (80%). The prevalence of agg and ace genes was 37.8% and 73.3% in clinical and 8.9% and 11.1% in "healthy" samples, respectively. The rate of Multiple Drug Resistant strains was 73.3% and 11.1% in clinical and "healthy" isolates, respectively. The ability of biofilm formation was significantly higher in clinical compared to "healthy" isolates (100% vs 75.6%, P < 0.05). The frequency of ace and agg genes, antibiotic resistance and biofilm formation ability were significantly higher in clinical than in "healthy" isolates (P < 0.05). Existence of agg and ace genes, biofilm formation and antibiotic resistance among the healthy enterococci isolates has a special importance since, in case these strains spread through clinical environments or reach water sources, this issue can be considered as a risk factor for health and sanitation of societY.

Lipoteichoic acids of lactobacilli inhibit Enterococcus faecalis biofilm formation and disrupt the preformed biofilm.

Enterococcus faecalis, a Gram-positive bacterium commonly isolated in patients with refractory apical periodontitis, invades dentin tubules easily and forms biofilms. Bacteria in biofilms, which contribute to recurrent and/or chronic inflammatory diseases, are more resistant to antimicrobial agents than planktonic cells and easily avoid phagocytosis. Although Lactobacillus plantarum lipoteichoic acid (Lp.LTA) is associated with biofilm formation, the effect of Lp.LTA on biofilm formation by E. faecalis is not clearly understood. In this study, we investigated whether Lp.LTA inhibits E. faecalis biofilm formation. The degree of biofilm formation was determined by using crystal violet assay and LIVE/DEAD bacteria staining. The quantification of bacterial growth was determined by measuring the optical density at 600 nm with a spectrophotometer. Formation of biofilms on human dentin slices was observed under a scanning electron microscope. E. faecalis biofilm formation was reduced by Lp.LTA treatment in a dose-dependent manner. Lp.LTA inhibited biofilm development of E. faecalis at the early stage without affecting bacterial growth. LTA from other Lactobacillus species such as Lactobacillus acidophilus, Lactobacillus casei, or Lactobacillus rhamnosus GG also inhibited E. faecalis biofilm formation. In particular, among LTAs from various lactobacilli, Lp.LTA showed the highest inhibitory effect on biofilms formed by E. faecalis. Interestingly, LTAs from lactobacilli could remove the biofilm preformed by E. faecalis. These inhibitory effects were also observed on the surface of human dentin slices. In conclusion, Lactobacillus species LTA inhibits biofilm formation caused by E. faecalis and it could be used as an anti-biofilm agent for prevention or treatment against E. faecalis-associated diseases.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite.

This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Regulation of Enterococcus faecalis Biofilm Formation and Quorum Sensing Related Virulence Factors with Ultra-low Dose Reactive Species Produced by Plasma Activated Water

Enterococcus faecalis (E. faecalis) is the species which can cause life-threatening infections in human beings and exhibit highly antibacterial resistance in the nosocomial environment. The purpose of this study was to evaluate the inhibition effect and the mechanisms of ultra-low dose reactive oxygen species produced by plasma activated water (PAW) to the E. faecalis biofilm. The bactericidal effect of E. faecalis planktonic growth was evaluated to find out the maximum processing time. The level of intracellular reactive oxygen species (ROS) and extracellular hydrogen peroxide (H2O2) produced by the PAW without significant killing effect were defined as ultra-low. Then the pre-treated bacteria suspension by the PAW were incubated with brain heart infusion (BHI) broth for 6 h, 12 h, 24 h and 48 h. The biofilm development was evaluated with 96-well polystyrene method (OD570) and confocal laser scanning microscopy (CLSM). Meanwhile, total organic carbon (TOC) was measured with a Shimadzu TOC-L analyzer. The quorum sensing related virulence genes, cylR1, cylA, gelE and sprE, were evaluated with real-time PCR. The PAW did not have significant killing effect within 30 s and the maximum corresponding fluroscence intensity of ROS is under 57.33 ± 0.58 and the concentration of H2O2 is 19.87 ± 2.01 μmol/L. The OD570, CLSM results and TOC results indicated that the biofilm development was effectively inhibited with PAW treatment. Four quorum sensing related virulence genes were all down-regulated. In conclusion, the PAW treatment has potential application to control E. faecalis biofilm development and reduce quorum sensing related virulence genes expression in vitro.

Quorum sensing LuxS/autoinducer-2 inhibits Enterococcus faecalis biofilm formation ability.

Objective:To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2.Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions.Results:LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05).Conclusions:Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.

Presence of virulence factor genes (gelE and esp) and biofilm formation in clinical Enterococcus faecalis and Enterococcus faecium isolated from urinary tract infection in Isfahan, Iran

BackgroundThe aim of present study was to determine the prevalence of two major virulence genes (gelE and esp) and biofilm-forming ability in Enterococcus faecalis and E. faecium isolated from patients with urinary tract infections (UTIs), and the correlation of selected virulence factors with biofilm formation. MethodsOne hundred Enterococcus isolates were collected from urine of patients with urinary tract infections (UTIs) attending to Alzahra and Gharazi hospitals affiliated to Isfahan University of Medical Sciences. The isolates were confirmed by phenotypic and genotypic methods. Biofilm formation was determined by microtiter plate method and the frequency of gelE and esp genes was evaluated using PCR assay. ResultsOf the 100 confirmed isolates, 83% and 17% were E. faecalis and E. faecium recpectively. The capacity of biofilm formation was 91.5% and 58.7% in E. faecalis and E. faecium respectively. The prevalence of gel E and esp genes was 81.9% and 79.5% in E. faecalis and 76.5% and 58.8% in E. faecium, respectively. There was a significant correlation between biofilm formation and presence of both virulence genes (P < 0.05). ConclusionsIn conclusion, we observed a high frequency of biofilm formation, esp and gelE genes in Enterococcoci strains isolated from UTIs. As an important result was a significant association between the presence of these two genes and biofilm production. Therefore, the new and more effective strategies should be applied to prevent and spread of these strains.

Inhibition of Enterococcus faecalis Growth and Biofilm Formation by Molecule Targeting Cyclic di-AMP Synthetase Activity

IntroductionEnterococcus faecalis is correlated with oral diseases including recurrent root canal treatment failure because of its biofilm formation ability and various virulence factors. Cyclic di-AMP (c-di-AMP) is an omnipresent second messenger involved in many crucial cellular physiological processes, including biofilm formation. ST056083 is a small molecule working as an inhibitor of the c-di-AMP synthetase DNA integrity scanning protein (DisA) in vitro. In this study, the impact of ST056083 on E. faecalis DisA activity, bacterial growth, and biofilm formation was tested. MethodsThe binding affinity between the protein and ligand was evaluated using the Amber score, and the binding mode was analyzed and visualized using UCSF Chimera (Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco, San Francisco, CA). The effect of ST056083 on E. faecalis DisA was evaluated using the coralyne assay. The effect of ST056083 on E. faecalis biofilm formation was determined by the biofilm quantification assay, scanning electron microscopic examination, and 3-dimensional confocal laser scanning microscopic assay. The effect of ST056083 on E. faecalis exopolysaccharide synthesis was measured by the anthrone-sulfuric method. ResultsWe expressed and purified E. faecalis DisA in vitro and confirmed the inhibitory effect of ST056083 on its biological activity. In addition, we showed the inhibitory effect of ST056083 on E. faecalis growth, biofilm formation, and exopolysaccharide synthesis. ConclusionsOur findings enhance the understanding of the physiological role of c-di-AMP in E. faecalis and represent a preliminary study on the ST056083 inhibitory effect and mechanism.

Effects of three γ-alkylidene-γ-lactams on the formation of multispecies biofilms.

This study evaluated the inhibitory effects of lactams on Streptococcus mutans, Enterococcus faecalis, and Candida glabrata multispecies biofilm formation. γ-Alkylidene-γ-lactams 1, 2, and 3 [solubilized in 3.5% dimethyl sulfoxide (DMSO)] were tested. Glass coverslips were conditioned with either the lactams or 3.5% DMSO (control) for 1 h, inoculated with microbial cultures, and incubated for 48 h. To assess the effect of the lactams on biofilm formation, the following parameters were determined: the biofilm biomass (by both crystal violet staining and protein determination); the amount of insoluble polysaccharides of the extracellular matrix; and the number of viable and total cells [by both colony-forming unit counting and quantitative real-time PCR (qPCR)]. Data were analysed using one-way anova and post-hoc Tukey tests. Lactams 1, 2, and 3 promoted a statistically significant reduction in the amount of biofilm biomass, but only lactam 3 resulted in a statistically significant reduction in the number of attached viable E. faecalis. Both total protein content and the amount of extracellular polysaccharides decreased significantly. The effects of γ-alkylidene-γ-lactams 1, 2, and 3 on the inhibition of multispecies biofilm formation were evident by their ability to reduce the amount of protein and extracellular polysaccharides.

Effect of DNase Treatment on Adhesion and Early Biofilm Formation of Enterococcus Faecalis.

Objective:Extracellular DNA (eDNA) has been shown to be important for biofilm stability of the endodontic pathogen Enterococcus faecalis. In this study, we hypothesized that treatment with DNase prevents adhesion and disperses young E. faecalis biofilms in 96-well plates and root canals of extracted teeth.Methods:E. faecalis eDNA in 96-well plates was visualized with TOTO-1®. The effect of DNase treatment was assessed in 96-well plates and in extracted single-rooted premolars (n=37) using a two-phase crossover design. E. faecalis was treated with DNase (50 Kunitz/mL) or heat-inactivated DNase for 1 h during adhesion or after 24 h of biofilm formation. In 96-well plates, adhering cells were quantified using confocal microscopy and digital image analysis. In root canals, the number of adhering cells was determined in dentine samples based on colony forming unit counts. Data from the 96-well plate were analyzed using one-tailed t-tests, and data from extracted teeth were analyzed using mixed-effect Poisson regressions.Results:eDNA was present in wells colonized by E. faecalis after 1 h of adhesion and 24 h of biofilm formation; it was removed by DNase treatment, as evidenced by TOTO®-1 staining. DNase treatment reduced the area covered by cells in 96-well plates after 1 h (P<0.05), but not after 24 h (P=0.96). No significant differences in the number of adhering cells were observed in extracted teeth after 1 (P=0.14) and 24 h (P=0.98).Conclusion:DNase treatment does not disperse endodontic E. faecalis biofilms. The sole use of DNase as an anti-biofilm agent in root canal treatments is not recommendable.

EFFICACY OF PROPOLIS FLUORIDE AND NANO SILVER FLUORIDE FOR INHIBITION OF STREPTOCOCCUS MUTANS AND ENTEROCOCCUS FAECALIS BIOFILM FORMATION

Objective: The objective of this study is to investigate the potency of propolis fluoride (PPF) and nano silver fluoride (NSF) as fluoride-based varnishes for inhibiting Streptococcus mutans and Enterococcus faecalis biofilm formation. In this study, both varnishes were compared to silver diamine fluoride (SDF) varnish, the gold standard for anticariogenic agents.Methods: The suspensions of S. mutans and E. faecalis were cultured and then plated into 96 - well plates and combined with SDF (38%), NSF (3.16, 3.66, and 4.16%), or PPF (3, 6, and 10%). E. faecalis was incubated in an anaerobic environment for 24 h, and the same protocol was used for S. mutans. The amount of biofilm inhibition was evaluated by optical density measurements at 570 nm using a microplate reader. Data were analyzed using one-way ANOVA.Results: The minimum inhibitory concentration (MIC) of PPF for S. mutans was 3%, and minimum bactericidal concentration (MBC) was 10%. The MIC of PPF for E. faecalis was at 6%, but no MBC was established. The MIC of NSF for S. mutans was 3.16%, and the MBC was 4.16%. The MIC of NSF for E. faecalis was 3.16%, while the MBC was 4.16%. Biofilm formation was inhibited dose-dependently by both NSF and PPF.Conclusion: NSF and PPF fluoride-based varnishes show clear antibacterial effects that are comparable to those obtained with SDF fluoride-based varnish.

Interplay between Antibiotic Efficacy and Drug-Induced Lysis Underlies Enhanced Biofilm Formation at Subinhibitory Drug Concentrations.

ABSTRACTSubinhibitory concentrations of antibiotics have been shown to enhance biofilm formation in multiple bacterial species. While antibiotic exposure has been associated with modulated expression of many biofilm-related genes, the mechanisms of drug-induced biofilm formation remain a focus of ongoing research efforts and may vary significantly across species. In this work, we investigate antibiotic-induced biofilm formation in Enterococcus faecalis, a leading cause of nosocomial infections. We show that biofilm formation is enhanced by subinhibitory concentrations of cell wall synthesis inhibitors but not by inhibitors of protein, DNA, folic acid, or RNA synthesis. Furthermore, enhanced biofilm is associated with increased cell lysis, increases in extracellular DNA (eDNA) levels, and increases in the density of living cells in the biofilm. In addition, we observe similar enhancement of biofilm formation when cells are treated with nonantibiotic surfactants that induce cell lysis. These findings suggest that antibiotic-induced biofilm formation is governed by a trade-off between drug toxicity and the beneficial effects of cell lysis. To understand this trade-off, we developed a simple mathematical model that predicts changes in antibiotic-induced biofilm formation due to external perturbations, and we verified these predictions experimentally. Specifically, we demonstrate that perturbations that reduce eDNA (DNase treatment) or decrease the number of living cells in the planktonic phase (a second antibiotic) decrease biofilm induction, while chemical inhibitors of cell lysis increase relative biofilm induction and shift the peak to higher antibiotic concentrations. Overall, our results offer experimental evidence linking cell wall synthesis inhibitors, cell lysis, increased eDNA levels, and biofilm formation in E. faecalis while also providing a predictive quantitative model that sheds light on the interplay between cell lysis and antibiotic efficacy in developing biofilms.