Recombinant Enterococcus faecalis L-rhamnose isomerase (rhaA)

What is the biochemical function of E. faecalis L-rhamnose isomerase?

E. faecalis L-rhamnose isomerase (L-RhI) catalyzes the reversible isomerization between L-rhamnose (6-deoxy-L-mannose) and L-rhamnulose (6-deoxy-L-fructose), which constitutes the first step in L-rhamnose catabolism in bacteria. This enzyme belongs to the rhamnose isomerase family and plays a crucial role in the microbial metabolism of L-rhamnose, a deoxy monosaccharide widely distributed in bacteria and plants . The enzyme can also catalyze isomerization reactions with various other aldoses and ketoses, exhibiting broad substrate specificity that has attracted research interest for rare sugar production .

What is the genetic organization of the rhaA gene in E. faecalis?

The rhaA gene in E. faecalis strain ATCC 700802/V583 encodes a protein of 428 amino acids with a molecular mass of approximately 48.9 kDa . The gene is part of the L-rhamnose utilization pathway. Unlike some other bacteria where rhamnose metabolism genes are organized in clusters, comparative genomic analysis of rhamnose utilization pathways across bacterial lineages has shown variations in genetic organization . The transcription of rhaA in E. faecalis can be regulated by growth conditions and may be influenced by the presence of L-rhamnose in the environment .

How conserved is rhaA across E. faecalis strains?

Genomic diversity studies of E. faecalis strains using comparative genomic hybridization have shown that metabolic genes, including those involved in sugar utilization, may display variation across strains . While specific data on rhaA conservation was not directly provided in the search results, the enzyme belongs to the rhamnose isomerase family which is broadly distributed across bacterial species. Research examining multiple E. faecalis strains would be required to fully characterize the conservation and potential sequence variations of rhaA across clinical and environmental isolates.

What are the key structural features of E. faecalis L-rhamnose isomerase?

Based on the amino acid sequence and comparison with characterized L-rhamnose isomerases, E. faecalis L-RhI is likely to adopt a structure similar to other members of this enzyme family. While the specific crystal structure of E. faecalis L-RhI has not been directly reported in the search results, related L-RhIs from other bacteria, such as Bacillus halodurans, have been crystallized and characterized. The B. halodurans L-RhI crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution . It's likely that E. faecalis L-RhI adopts a similar fold and quaternary structure.

What are the optimal conditions for E. faecalis L-rhamnose isomerase activity?

While the search results don't specifically detail the optimal conditions for E. faecalis L-RhI, we can draw comparisons with well-characterized L-RhIs from other bacterial sources. For example, B. halodurans L-RhI has an optimal pH and temperature of 7 and 70°C, respectively, with a kcat of 8,971 min⁻¹ and a kcat/Km of 17 min⁻¹mM⁻¹ for L-rhamnose . Other thermostable L-RhI variants, such as one identified from a hot spring metagenome, maintain substantial activity (80% or more) across a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80°C) ranges . Researchers working with recombinant E. faecalis L-RhI should conduct enzyme characterization experiments to determine the specific optimal conditions for this enzyme.

How does the quaternary structure of E. faecalis L-rhamnose isomerase affect its function?

Based on studies of L-RhIs from other bacterial sources, E. faecalis L-RhI likely forms a homodimeric or homotetrameric structure. For instance, the B. halodurans L-RhI was estimated to be ~48 kDa by SDS-PAGE and 121 kDa by gel filtration chromatography, suggesting a homodimeric structure . The quaternary structure is important for the enzyme's stability and catalytic activity. The arrangement of subunits creates the proper conformation of the active site, including metal binding residues that are essential for the isomerization reaction. To study the quaternary structure-function relationship, researchers could employ techniques such as size-exclusion chromatography, analytical ultracentrifugation, and site-directed mutagenesis of interface residues, followed by activity assays.

What expression systems are most effective for producing recombinant E. faecalis L-rhamnose isomerase?

Escherichia coli is the most commonly used expression system for recombinant L-rhamnose isomerases, including those from E. faecalis. Based on the search results and typical approaches for similar enzymes, an effective methodology would include:

• PCR amplification of the rhaA gene from E. faecalis genomic DNA

• Cloning into an expression vector (e.g., pET28a) with an N-terminal or C-terminal His-tag

• Transformation into an appropriate E. coli strain (e.g., BL21(DE3))

• Induction with IPTG (typically 0.5 mM) at optimal temperature (often 16-37°C)

For example, in the case of B. halodurans L-RhI, the gene was amplified with primers incorporating restriction sites (BamHI and HindIII), cloned into pQE-80L vector under the control of the T5 promoter, and expressed as a His-tagged protein . Similar approaches would be applicable to E. faecalis rhaA.

What purification strategy yields the highest purity and activity for recombinant E. faecalis L-rhamnose isomerase?

A recommended purification strategy based on protocols for similar enzymes would include:

• Cell lysis by sonication in an appropriate buffer (e.g., 50 mM HEPES pH 7.0, 300 mM NaCl)

• Clarification of the lysate by centrifugation (typically 10,000 RPM, 45 min, 4°C)

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

• Washing with increasing concentrations of imidazole (e.g., 10 mM for equilibration, 40 mM for washing)

• Elution with high imidazole concentration (e.g., 300 mM)

• Buffer exchange to remove imidazole via dialysis or gel filtration

This approach has been successfully applied to similar enzymes such as the L-rhamnose isomerase from metagenomic samples, yielding pure and active enzyme . The purified protein can be concentrated using an Amicon filter with an appropriate molecular weight cutoff (e.g., 30 kDa) and quantified by Bradford assay.

How can researchers overcome solubility and stability issues when working with recombinant E. faecalis L-rhamnose isomerase?

To address solubility and stability challenges:

• Optimization of expression conditions:

  • Lower induction temperature (16-20°C)

  • Reduced IPTG concentration (0.1-0.5 mM)

  • Co-expression with chaperones (e.g., GroEL/ES, DnaK)

• Buffer optimization:

  • Screen various buffers (HEPES, Tris, phosphate) at different pH values

  • Include stabilizing agents (glycerol 5-10%, trehalose, sucrose)

  • Add metal ions that may serve as cofactors (Mn²⁺, Mg²⁺)

• Storage conditions:

  • Flash-freeze aliquots in liquid nitrogen

  • Add protease inhibitors if degradation is observed

  • Store at -80°C for long-term stability

• Protein engineering approaches:

  • Surface entropy reduction

  • Introduction of disulfide bonds

  • Fusion to solubility-enhancing tags (MBP, SUMO)

These approaches have been successful with thermostable enzymes and would likely be applicable to E. faecalis L-rhamnose isomerase .

What are the most reliable methods for measuring E. faecalis L-rhamnose isomerase activity?

Several complementary approaches can be used to reliably measure L-rhamnose isomerase activity:

• Spectrophotometric assays:

  • Cysteine-carbazole method for ketose detection

  • Coupled enzyme assays with L-rhamnulose kinase and NADH-dependent enzymes

  • Monitoring UV absorbance changes associated with the keto-enol tautomerization

• Chromatographic methods:

  • HPLC analysis with refractive index detection

  • Ion-exchange chromatography to separate substrate and product

  • Liquid chromatography-mass spectrometry (LC-MS) for precise quantification

• Nuclear Magnetic Resonance (NMR):

  • Real-time monitoring of the isomerization reaction

  • Quantification of both substrate and product simultaneously

  • Structural verification of the reaction products

Standard reaction conditions would typically include substrate (L-rhamnose, 10-50 mM), buffer (50 mM HEPES or phosphate, pH 7.0-8.0), temperature (37-70°C depending on enzyme stability), and reaction times of 5-60 minutes followed by appropriate detection methods.

How can researchers accurately determine the kinetic parameters of E. faecalis L-rhamnose isomerase?

To determine kinetic parameters of E. faecalis L-rhamnose isomerase:

• Initial velocity measurements:

  • Measure reaction rates at varying substrate concentrations (typically 0.1-10× Km)

  • Ensure <10% substrate conversion to maintain initial velocity conditions

  • Maintain constant enzyme concentration, temperature, and pH

• Data analysis:

  • Use Michaelis-Menten equation to fit data: v = Vmax[S]/(Km + [S])

  • Employ linearization methods (Lineweaver-Burk, Eadie-Hofstee) for verification

  • Utilize non-linear regression software (GraphPad Prism, Origin) for accurate fitting

• Determine kcat:

  • Calculate kcat = Vmax/[E] where [E] is the molar enzyme concentration

  • Ensure accurate enzyme concentration determination (Bradford assay, absorbance at 280 nm)

• Temperature and pH effects:

  • Repeat kinetic measurements at different temperatures to determine activation energy (Ea)

  • Investigate pH-rate profiles to identify catalytically important ionizable groups

For B. halodurans L-RhI, kinetic parameters included a kcat of 8,971 min⁻¹ and a kcat/Km of 17 min⁻¹mM⁻¹ for L-rhamnose . Similar methodologies would be applicable to E. faecalis L-RhI.

How does substrate specificity of E. faecalis L-rhamnose isomerase compare to other bacterial L-rhamnose isomerases?

To investigate substrate specificity:

• Substrate screening methodology:

  • Test activity on various aldoses and ketoses (L-rhamnose, L-mannose, D-allose, D-ribose, etc.)

  • Maintain equivalent substrate concentrations and reaction conditions

  • Employ appropriate detection methods for different sugar products

• Comparative analysis:

  • Calculate relative activity (%) with L-rhamnose as the reference (100%)

  • Determine kinetic parameters (Km, kcat, kcat/Km) for each viable substrate

  • Create a substrate specificity profile

• Structural basis of specificity:

  • Compare with known L-RhIs (e.g., from B. halodurans, thermophilic metagenomes)

  • Identify key active site residues through sequence alignment and homology modeling

  • Consider structural aspects of substrate recognition

L-rhamnose isomerases generally exhibit broad substrate specificity and can catalyze reactions with various sugars, making them valuable for rare sugar production. For example, some L-RhI variants have been shown to convert D-allose to D-allulose . A comprehensive substrate specificity analysis would provide insights into the unique properties of E. faecalis L-RhI compared to other bacterial enzymes.

How can E. faecalis L-rhamnose isomerase be optimized for rare sugar production?

To optimize E. faecalis L-RhI for rare sugar production:

• Protein engineering approaches:

  • Rational design based on structural information and mechanism

  • Site-directed mutagenesis of active site residues to alter substrate specificity

  • Directed evolution through error-prone PCR or DNA shuffling

  • High-throughput screening for variants with improved catalytic properties

• Reaction condition optimization:

  • Determine optimal temperature, pH, and metal ion requirements

  • Investigate substrate concentration effects on reaction equilibrium

  • Develop systems for continuous product removal to shift equilibrium

• Immobilization strategies:

  • Covalent attachment to solid supports

  • Entrapment in polymeric matrices

  • Cross-linked enzyme aggregates (CLEAs)

  • Evaluation of activity, stability, and reusability of immobilized enzyme

• Process integration:

  • Coupling with other enzymes in multi-enzyme cascades

  • Development of continuous flow reactors

  • Scale-up considerations and process economics

These strategies have been employed with other L-RhIs to improve their application in rare sugar production and could be adapted for E. faecalis L-RhI .

What role might E. faecalis L-rhamnose isomerase play in understanding bacterial pathogenesis?

E. faecalis is an opportunistic pathogen causing healthcare-associated infections, and its metabolic capabilities contribute to survival in host environments. The potential roles of L-rhamnose isomerase in pathogenesis include:

• Contribution to metabolic versatility:

  • Enables utilization of L-rhamnose as a carbon source

  • May provide metabolic advantages in specific host niches

  • Could contribute to survival during nutrient limitation

• Relationship to virulence-associated structures:

  • L-rhamnose is a component of the Enterococcal Polysaccharide Antigen (EPA), which is essential for biofilm formation, antibiotic resistance, and pathogenesis

  • EPA consists of a rhamnan backbone made of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains

  • Understanding rhamnose metabolism may provide insights into EPA synthesis regulation

• Potential as a therapeutic target:

  • Inhibiting rhamnose metabolism could affect EPA production

  • May represent a novel approach to attenuate virulence

  • Could be explored in combination with conventional antibiotics

Experimental approaches to investigate this connection could include:

• Creation of rhaA knockout mutants and evaluation in infection models

• Transcriptional analysis of rhaA under host-relevant conditions

• Investigation of EPA production in the presence of rhamnose metabolism inhibitors

How does E. faecalis L-rhamnose isomerase compare with other enzymes in the rhamnose metabolic pathway?

To compare E. faecalis L-rhamnose isomerase with other enzymes in the pathway:

• Pathway organization and comparison:

  • L-rhamnose isomerase (RhaA): Converts L-rhamnose to L-rhamnulose

  • L-rhamnulose kinase (RhaB): Phosphorylates L-rhamnulose to L-rhamnulose-1-phosphate

  • L-rhamnulose-1-phosphate aldolase (RhaD): Cleaves L-rhamnulose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde

  • L-lactaldehyde dehydrogenase: Converts L-lactaldehyde to L-lactate

• Comparative analysis approaches:

  • Determine rate-limiting steps through pathway flux analysis

  • Compare kinetic parameters and regulation of all pathway enzymes

  • Evaluate expression levels and coordination under various conditions

• Novel pathway variants:

  • Some bacteria possess a bifunctional enzyme, L-rhamnulose-phosphate aldolase (RhaE) fused to L-lactaldehyde dehydrogenase (RhaW)

  • Others have an alternative L-lactaldehyde processing enzyme, L-lactaldehyde reductase (RhaZ)

  • Comparative genomics approaches can identify these variations across bacterial lineages

• Regulatory integration:

  • Examine transcriptional regulation by specific regulators like RhaR

  • Investigate metabolic integration with other sugar utilization pathways

  • Study catabolite repression effects on pathway expression

This comparative approach would provide insights into the evolutionary and functional relationships between E. faecalis L-rhamnose isomerase and other enzymes in the rhamnose metabolic pathway.

How do post-translational modifications affect E. faecalis L-rhamnose isomerase activity and stability?

To investigate post-translational modifications (PTMs) of E. faecalis L-rhamnose isomerase:

• Identification of potential PTMs:

  • Mass spectrometry-based proteomics approaches

  • Western blot with specific antibodies (phosphorylation, glycosylation)

  • Chemical labeling methods for specific modifications

• Functional impact assessment:

  • Site-directed mutagenesis of modified residues

  • Comparison of enzymatic properties before and after modification

  • In vitro modification systems to generate specifically modified forms

• Environmental regulation of PTMs:

  • Examine PTM patterns under different growth conditions

  • Investigate stress responses and their impact on PTMs

  • Study the enzymes responsible for installing or removing PTMs

• Structural basis of PTM effects:

  • Crystallography or cryo-EM of modified and unmodified forms

  • Molecular dynamics simulations to assess conformational changes

  • Hydrogen-deuterium exchange mass spectrometry for conformational impact

While specific information on PTMs of E. faecalis L-rhamnose isomerase was not present in the search results, these approaches would be valuable for understanding potential regulatory mechanisms affecting enzyme function.

What are the molecular determinants of thermostability in E. faecalis L-rhamnose isomerase compared to thermophilic homologs?

To investigate molecular determinants of thermostability:

• Comparative sequence and structure analysis:

  • Multiple sequence alignment with thermostable homologs (e.g., B. halodurans L-RhI, thermophilic metagenomic L-RhI)

  • Identification of conserved and divergent residues

  • Structural modeling to locate these residues in the 3D structure

• Key features to analyze:

  • Salt bridge and hydrogen bond networks

  • Hydrophobic core composition

  • Surface charge distribution

  • Presence of proline residues in loops

  • Disulfide bond formation potential

• Experimental approaches:

  • Thermal shift assays (Thermofluor) to determine melting temperatures

  • Circular dichroism for secondary structure stability

  • Half-life measurements at elevated temperatures

  • Engineering approaches to introduce stabilizing features

• Specific examples to consider:

  • B. halodurans L-RhI shows high thermal stability with 100% activity for 10 hours at 60°C

  • The metagenomic L-RhIM has a half-life of about 12 days at 65°C and 5 days at 70°C

  • Comparative analysis with these thermostable L-RhIs could provide insights into stabilizing features that might be introduced into E. faecalis L-RhI

How does the metal cofactor coordination in E. faecalis L-rhamnose isomerase influence its catalytic mechanism?

To explore metal cofactor coordination and catalytic mechanism:

• Metal cofactor identification and characterization:

  • Inductively coupled plasma mass spectrometry (ICP-MS) for metal content

  • Activity assays with various metal ions (Mn²⁺, Mg²⁺, Co²⁺, etc.)

  • Metal chelation experiments to establish dependence

  • X-ray absorption spectroscopy for coordination geometry

• Structural analysis of metal binding:

  • Crystallography in the presence of different metals

  • Identification of coordinating residues

  • Comparison with metal-binding sites in related isomerases

• Mechanistic studies:

  • Site-directed mutagenesis of metal-coordinating residues

  • pH-rate profiles to establish ionization states

  • Solvent kinetic isotope effects to probe proton transfer

  • Substrate analogs and transition state inhibitors

• Computational approaches:

  • Quantum mechanics/molecular mechanics (QM/MM) simulations

  • Density functional theory (DFT) calculations of transition states

  • Free energy calculations for reaction pathway

This integrated approach would provide detailed insights into how metal cofactors influence the catalytic mechanism of E. faecalis L-rhamnose isomerase, which is crucial for understanding its function and for rational enzyme engineering efforts.

How does expression of rhaA correlate with E. faecalis growth on different carbon sources?

To investigate the physiological regulation of rhaA expression:

• Growth and expression analysis methodology:

  • Cultivate E. faecalis in defined media with different carbon sources (glucose, rhamnose, other sugars)

  • Monitor growth rates and yield on each carbon source

  • Quantify rhaA transcript levels using RT-qPCR

  • Measure L-rhamnose isomerase protein levels using Western blotting or targeted proteomics

• Regulatory elements investigation:

  • Identify potential regulatory elements in the rhaA promoter region

  • Construct transcriptional reporter fusions (e.g., rhaA promoter-GFP)

  • Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the rhaA promoter

• Metabolic context analysis:

  • Examine co-regulation with other rhamnose metabolic genes

  • Investigate carbon catabolite repression effects

  • Study induction kinetics upon exposure to L-rhamnose

Based on studies in other bacteria, rhaA expression is likely induced by L-rhamnose and may be subject to carbon catabolite repression in the presence of preferred carbon sources like glucose . Similar approaches to those used for studying transcriptional regulation in other sugar utilization pathways could be applied to E. faecalis rhaA.

What is the significance of L-rhamnose metabolism in E. faecalis biofilm formation and pathogenesis?

To explore the connection between L-rhamnose metabolism and E. faecalis virulence:

• Biofilm formation analysis:

  • Compare biofilm formation between wild-type and rhaA knockout strains

  • Examine biofilm architecture using confocal microscopy

  • Quantify extracellular polymeric substances (EPS) production

  • Investigate EPA composition and structure in the rhaA mutant

• Virulence phenotype characterization:

  • Perform infection studies in appropriate animal models (e.g., Caenorhabditis elegans, mouse models)

  • Examine host colonization efficiency

  • Study persistence in specific host niches

  • Evaluate antimicrobial resistance phenotypes

• Molecular connections to explore:

  • EPA contains a rhamnan backbone essential for biofilm formation and virulence

  • L-rhamnose metabolism may influence EPA synthesis through substrate availability

  • Metabolic adaptation to host environments may involve L-rhamnose utilization

E. faecalis biofilm formation plays a key role in its virulence and drug resistance attributes . The production of EPA, which contains L-rhamnose, is required for biofilm formation, antibiotic resistance, and pathogenesis . Understanding how L-rhamnose metabolism interfaces with these processes could provide insights into E. faecalis pathophysiology and potential therapeutic targets.

How does the evolutionary conservation of rhaA in E. faecalis relate to its ecological niche adaptations?

To investigate evolutionary aspects of L-rhamnose metabolism in E. faecalis:

• Comparative genomic analysis:

  • Compare rhaA sequences across diverse E. faecalis strains (clinical, commensal, environmental)

  • Analyze synteny of rhamnose utilization genes across strains

  • Examine presence/absence patterns of pathway components

• Phylogenetic approaches:

  • Construct phylogenetic trees of rhaA sequences

  • Compare with species phylogeny to identify potential horizontal gene transfer events

  • Analyze selection pressures (dN/dS ratios) on rhaA

• Ecological correlations:

  • Correlate rhaA presence/sequence variants with isolation sources

  • Compare L-rhamnose utilization capacity across strains from different niches

  • Investigate potential correlations with virulence or colonization capacity

• Functional implications:

  • Examine expression patterns in different ecological contexts

  • Compare substrate specificity and catalytic efficiency across variants

  • Evaluate temperature and pH optima in relation to niche adaptations

L-rhamnose is a component of plant cell walls and bacterial exopolysaccharides, suggesting that the ability to metabolize L-rhamnose could provide advantages in specific environmental niches. Comparative genomic approaches, as used in studies of other sugar utilization pathways , would be valuable for understanding the evolutionary significance of L-rhamnose metabolism in E. faecalis.

How does E. faecalis L-rhamnose metabolism interact with host glycan structures during infection?

To investigate interactions between E. faecalis L-rhamnose metabolism and host glycans:

• Host glycan analysis:

  • Characterize glycan structures at sites commonly colonized by E. faecalis

  • Identify potential L-rhamnose-containing structures in host tissues

  • Examine changes in host glycosylation during infection

• Bacterial adaptation studies:

  • Monitor expression of rhaA and other rhamnose metabolism genes during host colonization

  • Compare growth on host-derived glycans between wild-type and rhaA mutants

  • Investigate potential glycosidase activities that might release L-rhamnose from host structures

• Interaction with immune system:

  • Study the impact of L-rhamnose metabolism on innate immune evasion

  • Examine recognition of EPA by pattern recognition receptors

  • Investigate the role of L-rhamnose-containing structures in modulating inflammatory responses

Research has shown that EPA plays a role in immune evasion, and mutations affecting its decorations can alter interactions with the host immune system . The glycosaminoglycan (GAG) degradation capability of E. faecalis, which involves enzymes like hyaluronidases, contributes to tissue damage and immune evasion , suggesting complex interactions between bacterial metabolism and host glycobiology.

Can L-rhamnose isomerase activity be leveraged as a diagnostic marker for E. faecalis infections?

To evaluate the potential of L-rhamnose isomerase as a diagnostic marker:

• Specificity assessment:

  • Compare L-rhamnose isomerase sequences and activities across Enterococcus species and other bacteria

  • Identify unique features of E. faecalis L-RhI that could be targeted

  • Develop species-specific antibodies or activity-based probes

• Detection method development:

  • Enzymatic activity-based assays for L-RhI in clinical samples

  • Immunological detection methods (ELISA, lateral flow)

  • PCR-based detection of rhaA gene sequences

  • Mass spectrometry-based proteomic identification

• Clinical validation:

  • Test detection methods on clinical samples from various infection types

  • Determine sensitivity and specificity compared to standard diagnostic methods

  • Evaluate correlation between L-RhI levels and infection severity or prognosis

• Practical implementation considerations:

  • Sample preparation requirements

  • Time to result and point-of-care potential

  • Cost-effectiveness compared to existing methods

While not directly addressed in the search results, the unique properties of E. faecalis L-rhamnose isomerase could potentially be exploited for diagnostic purposes, particularly if species-specific features can be identified and targeted.

How might inhibitors of E. faecalis L-rhamnose isomerase be designed and evaluated for antimicrobial potential?

To develop potential inhibitors targeting E. faecalis L-rhamnose isomerase:

• Rational inhibitor design strategy:

  • Structure-based drug design using crystal structures or homology models

  • Targeting the active site or allosteric sites

  • Virtual screening of compound libraries

  • Fragment-based drug discovery approaches

• Inhibitor classes to consider:

  • Substrate analogs and transition state mimics

  • Metal-chelating compounds (if metal-dependent)

  • Allosteric inhibitors targeting protein dynamics

  • Covalent inhibitors targeting specific residues

• In vitro evaluation methods:

  • Enzyme inhibition assays with purified recombinant protein

  • Determination of inhibition constants (Ki) and mechanisms

  • Thermal shift assays to assess binding

  • X-ray crystallography to confirm binding modes

• Cellular and in vivo assessment:

  • Growth inhibition of E. faecalis cultures

  • Biofilm formation assays

  • Cytotoxicity evaluation against mammalian cells

  • Efficacy in infection models

  • Pharmacokinetic and pharmacodynamic studies