Recombinant Escherichia coli O127:H6 Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase (arnC)

What is the role of arnC in Escherichia coli O127:H6?

ArnC in E. coli O127:H6 functions as an integral membrane glycosyltransferase that attaches a formylated form of aminoarabinose (L-Ara4FN) to undecaprenyl phosphate (UndP). This enzymatic reaction is a critical step in the lipopolysaccharide (LPS) modification pathway that contributes to bacterial antimicrobial resistance mechanisms. In Gram-negative bacteria like E. coli O127:H6, this modification ultimately leads to the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A portion of LPS, which reduces the negative charge of the bacterial outer membrane and decreases its affinity for cationic antimicrobial peptides and polymyxin antibiotics .

How does arnC fit into the broader context of E. coli O127:H6 pathogenicity?

E. coli O127:H6 strain E2348/69 is a prototype enteropathogenic E. coli (EPEC) strain that has been extensively studied worldwide for its biology, genetics, and virulence mechanisms. While this strain is primarily known for its type III secretion system (T3SS) that facilitates attaching and effacing lesion formation on intestinal epithelial cells, the arnC gene plays a complementary role in pathogenicity by contributing to antimicrobial resistance .

The arnC gene is part of the broader bacterial defense system against host immune responses and therapeutic interventions. By enabling modifications to LPS structure, arnC indirectly supports the bacterium's ability to colonize host tissues and persist during infection. The complete genomic sequence of E2348/69 has revealed that many virulence factors, including those related to membrane modifications, are carried on mobile genetic elements, facilitating potential transfer of resistance mechanisms to other bacteria .

What is the relationship between undecaprenyl phosphate metabolism and arnC function?

Undecaprenyl phosphate (UP) serves as an essential lipid carrier in the biosynthesis of various bacterial extracellular polysaccharides, including peptidoglycan, O-antigen, and teichoic acid . In E. coli O127:H6, UP acts as the acceptor substrate for the arnC-catalyzed attachment of L-Ara4FN.

The metabolism of UP involves two primary pathways:

• De novo synthesis through dephosphorylation of undecaprenyl diphosphate (UPP)

• Recycling pathways that regenerate UP after polysaccharide biosynthesis

ArnC specifically utilizes UP from these metabolic pools as its substrate. The availability of UP is therefore a rate-limiting factor in the arnC-mediated LPS modification pathway. Experimental designs targeting arnC function must consider the broader UP metabolic context to accurately interpret results, as perturbations in UP availability can indirectly affect arnC activity independent of direct modulation of the enzyme itself .

What do we know about the three-dimensional structure of arnC and how does it inform function?

Recent structural studies using cryo-electron microscopy have revealed critical insights into ArnC's structure-function relationship, though these studies were conducted with the S. enterica homolog rather than E. coli O127:H6 specifically. The enzyme adopts distinct conformations in its apo (unbound) and nucleotide-bound states .

Key structural features include:

• A GT-A glycosyltransferase domain that contains the catalytic site

• A transmembrane domain (TMD) that anchors the protein in the bacterial inner membrane

• Juxtamembrane (JM) helices that create a passage for the undecaprenyl phosphate substrate to access the active site

Upon binding of UDP (a partial donor substrate), ArnC undergoes a significant conformational change characterized by a clamshell-like motion that brings the GT-A domain closer to the juxtamembrane helices. This structural rearrangement is essential for properly positioning the catalytic residues relative to both donor and acceptor substrates .

The catalytic mechanism involves the first aspartate of the DXD motif, which does not coordinate the metal ion but instead functions as a catalytic base to abstract a proton from UndP, activating it for nucleophilic attack on the C1 carbon of the sugar substrate .

How do divalent metal ions affect arnC enzymatic activity?

ArnC belongs to the metal-dependent polyprenyl phosphate glycosyltransferase family, and its activity is significantly modulated by divalent metal ions. Microscale thermophoresis (MST) experiments have demonstrated that manganese ions (Mn²⁺) significantly enhance the affinity of ArnC for its partial donor substrate UDP .

The metal ion coordination occurs through the DXD motif, where the second aspartate participates in metal binding. This coordination is critical for properly orienting the sugar donor substrate and facilitating the glycosyl transfer reaction. Researchers studying ArnC should carefully control the metal ion composition in experimental buffers, as variations can dramatically affect enzyme kinetics and potentially lead to inconsistent results across different experimental conditions .

A comparison of metal ion effects on ArnC activity:

What are the critical residues in the active site of arnC and how can they be experimentally probed?

Based on structural and simulation data from related enzymes, several critical residues have been identified in the ArnC active site:

• The DXD motif (typically D92 and D94 in homologous proteins) involved in metal coordination and catalysis

• Positively charged residues that interact with the phosphate groups of the UDP-sugar donor

• Hydrophobic residues that form the binding pocket for the undecaprenyl chain

These residues can be experimentally probed through:

Site-directed mutagenesis approach:

• Generate point mutations at suspected catalytic residues (e.g., D→N substitutions in the DXD motif)

• Express and purify the mutant proteins

• Conduct comparative enzyme kinetics assays with wild-type and mutant enzymes

• Perform thermal stability assays to distinguish between mutations that affect catalysis versus those that disrupt protein folding

Chemical modification strategies:

• Use group-specific reagents to modify suspected catalytic residues

• Determine the effect of modification on enzyme activity

• Attempt to protect the active site through substrate binding before chemical modification

When designing such experiments, researchers should consider that membrane proteins like ArnC require special handling protocols to maintain their native conformation and activity after extraction from the membrane environment .

What are the optimal conditions for expressing recombinant arnC from E. coli O127:H6?

Expressing functional recombinant ArnC presents several challenges due to its nature as an integral membrane protein. Based on successful expression strategies for similar glycosyltransferases, the following approach is recommended:

Expression system selection:

• E. coli BL21(DE3) or C41(DE3) strains are preferred for membrane protein expression

• C41(DE3) is particularly useful as it has adaptations for toxic membrane protein expression

Vector design considerations:

• Include a C-terminal His₆ or His₁₀ tag for purification

• Consider using a fusion partner like MBP to enhance solubility

• Include a precision protease cleavage site between the tag and arnC sequence

Induction protocol:

• Grow cultures at 37°C to OD₆₀₀ of 0.6-0.8

• Shift temperature to 18-20°C before induction

• Induce with a low IPTG concentration (0.1-0.5 mM)

• Express for 16-20 hours at the reduced temperature

Buffer considerations during cell lysis:

• Use buffers containing 20 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol

• Include protease inhibitors (PMSF, leupeptin, pepstatin)

• Add 5 mM MgCl₂ or MnCl₂ to stabilize the enzyme

This protocol typically yields 1-3 mg of purified protein per liter of culture when optimized. The protein should be stored in buffer containing 0.03-0.05% DDM or LMNG detergent to maintain stability if not reconstituted into nanodiscs or liposomes immediately .

How can researchers establish a reliable in vitro assay for arnC activity?

Developing a robust in vitro assay for ArnC activity requires careful consideration of substrate preparation, reaction conditions, and detection methods. The following methodology provides a framework for establishing such an assay:

Substrate preparation:

• UDP-L-Ara4FN (donor substrate):

  • Chemical synthesis or enzymatic preparation using the ArnA and ArnB proteins

  • Typical purity requirement: >95% by HPLC

• Undecaprenyl phosphate (acceptor substrate):

  • Commercial sources or extraction from bacterial membranes

  • Solubilization in suitable detergent (e.g., Triton X-100 at 0.1%)

Reaction buffer composition:

• 50 mM HEPES pH 7.5

• 100 mM NaCl

• 5 mM MnCl₂ (preferred metal cofactor)

• 0.1% Triton X-100 or 0.05% DDM

• 1 mM DTT

Assay conditions:

• Mix 50 μM UDP-L-Ara4FN with 30 μM undecaprenyl phosphate

• Add purified ArnC (1-5 μg)

• Incubate at 30°C for 30-60 minutes

• Terminate reaction with equal volume of chloroform:methanol (2:1)

Detection methods:

• Radiochemical assay using ¹⁴C or ³H-labeled UDP-L-Ara4FN:

  • Extract lipid phase and quantify by scintillation counting

  • Provides high sensitivity (detection limit ~0.1 nmol)

• HPLC-based assay:

  • Monitor UDP release or undecaprenyl phosphate-L-Ara4FN formation

  • Use reverse-phase C18 column with appropriate gradient

• Coupled enzymatic assay:

  • Link UDP release to NADH oxidation through pyruvate kinase and lactate dehydrogenase

  • Monitor decrease in absorbance at 340 nm

A well-optimized assay should yield linear kinetics for at least 15-20 minutes and maintain proportionality with enzyme concentration .

What methods are available for studying the integration of arnC into the bacterial membrane?

Investigating ArnC membrane integration requires specialized techniques that can provide insights into topology, orientation, and interactions within the lipid bilayer. The following methodologies are particularly useful:

1. Cysteine accessibility methods:

• Strategic introduction of cysteine residues throughout the protein sequence

• Selective labeling with membrane-permeable and impermeable thiol-reactive reagents

• Analysis of labeling patterns to determine membrane-embedded regions

2. Green fluorescent protein (GFP) fusion analysis:

• Creation of truncated ArnC-GFP fusions

• Fluorescence patterns indicate correct folding and membrane insertion

• Particularly useful for identifying the boundaries of transmembrane segments

3. Proteoliposome reconstitution:

• Purification of ArnC in detergent

• Reconstitution into liposomes of defined lipid composition

• Assessment of orientation using protease protection assays

4. Molecular dynamics simulations:
The successful application of coarse-grained (CG) and atomistic simulations has revealed critical insights about ArnC-membrane interactions. These computational approaches have identified:

• Cardiolipin preferentially binds to a groove on the periplasmic transmembrane domain face

• Spontaneous and stable binding of undecaprenyl phosphate (UndP) within the GT-A domain

• Two different coordination positions for UndP (P1 "standby" position and P2 "catalysis" position)

These methods are complementary and should ideally be used in combination to build a comprehensive understanding of ArnC membrane integration.

How does arnC contribute to polymyxin resistance, and what experimental approaches can quantify this relationship?

ArnC plays a critical role in polymyxin resistance by catalyzing a key step in the pathway that ultimately modifies lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N). This modification reduces the negative charge of the bacterial outer membrane, decreasing its affinity for positively charged polymyxins .

Quantitative experimental approaches:

1. Minimum inhibitory concentration (MIC) determination:

• Compare polymyxin MICs between wild-type and arnC deletion mutants

• Complement arnC deletion with plasmid-expressed arnC to confirm specificity

• Test across a range of polymyxin concentrations (0.125-64 μg/ml)

A typical data pattern from such experiments:

2. Mass spectrometry analysis of LPS modifications:

• Extract LPS from wild-type and arnC mutant strains

• Perform mild acid hydrolysis to release lipid A

• Analyze by MALDI-TOF or LC-MS/MS to quantify L-Ara4N-modified lipid A

3. Radioactive labeling of L-Ara4N incorporation:

• Grow bacteria in media containing ¹⁴C-labeled arabinose

• Extract LPS and quantify incorporation into lipid A

• Compare incorporation levels between wild-type and arnC mutants

4. Real-time PCR analysis of the complete arn operon:

• Monitor expression of all arn genes under polymyxin exposure

• Determine if arnC is rate-limiting in the pathway

• Assess potential compensatory mechanisms in response to arnC deletion

These approaches collectively provide a comprehensive assessment of ArnC's contribution to polymyxin resistance at both the molecular and phenotypic levels .

What is known about the interaction between arnC and the undecaprenyl phosphate substrate, and how can these interactions be experimentally investigated?

The interaction between ArnC and undecaprenyl phosphate (UndP) involves a specific binding mechanism where the lipid molecule threads between the juxtamembrane (JM) helices to reach the catalytic GT-A domain. Molecular dynamics simulations have revealed detailed insights into this process .

Key features of the interaction:

• UndP binds within a groove formed by the JM helices

• The phosphate group of UndP extends beyond the plane of the membrane

• UndP assumes two coordination positions within the GT-A domain:

  • P1: A "standby" position before catalysis

  • P2: The "catalysis" position that enables nucleophilic attack

Experimental approaches to investigate these interactions:

1. Binding affinity measurements:

• Isothermal titration calorimetry (ITC) with detergent-solubilized ArnC and UndP

• Microscale thermophoresis (MST) using fluorescently labeled UndP

• Surface plasmon resonance (SPR) with immobilized ArnC

2. Photo-crosslinking studies:

• Incorporate photo-reactive analogs of UndP

• UV-activate crosslinking to ArnC

• Identify crosslinked residues by mass spectrometry

3. Structural investigations:

• Cryo-EM of ArnC in nanodiscs with bound UndP

• Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• Solid-state NMR of reconstituted ArnC with labeled UndP

4. Mutagenesis of putative UndP-binding residues:

• Target residues identified in the juxtamembrane helices

• Create alanine substitutions and assess impact on UndP binding

• Evaluate effects on catalytic efficiency using kinetic assays

These approaches provide complementary information about the ArnC-UndP interaction, from binding thermodynamics to structural details of the complex .

How can researchers distinguish between the roles of arnC and other transferases in the L-Ara4N modification pathway?

The L-Ara4N modification pathway involves multiple enzymes, including ArnA, ArnB, ArnC, and ArnT. Distinguishing the specific role of ArnC from these other transferases requires strategic experimental approaches:

1. Targeted gene deletion and complementation:

• Generate single and combinatorial deletions of arn genes

• Evaluate the accumulation of pathway intermediates in each mutant

• Complement deletions with plasmid-expressed genes under controlled promoters

A non-polar deletion of arnC would not impede the formation of UDP-Ara4FN (which requires ArnA and ArnB) but would preclude the synthesis of endogenous BP-Ara4N and LPS-Ara4N. This specific pattern of intermediate accumulation helps distinguish ArnC's role from other transferases in the pathway .

2. In vitro reconstitution of the complete pathway:

• Purify all Arn enzymes individually

• Establish assays for each enzymatic step

• Combine enzymes sequentially to track substrate conversion

3. Metabolic labeling with substrate analogs:

• Use synthetic analogs of pathway intermediates

• Track their incorporation and modification by different Arn enzymes

• Apply click chemistry to visualize specific intermediates in situ

4. Protein-protein interaction studies:

• Investigate potential physical interactions between Arn proteins

• Use bacterial two-hybrid or co-immunoprecipitation approaches

• Determine if ArnC functions independently or as part of a larger complex

5. Substrate competition assays:

• Introduce competing substrates for each enzyme

• Determine substrate preferences and specificity

• Identify unique substrates that distinguish ArnC activity

These approaches collectively allow researchers to delineate the specific contributions of ArnC within the broader L-Ara4N modification pathway .

What are common challenges in purifying functional recombinant arnC and how can they be addressed?

Purifying functional recombinant ArnC presents several technical challenges due to its nature as an integral membrane protein. Here are the most common issues and their solutions:

1. Low expression levels:

• Problem: Typical yields of membrane proteins are often 10-100 fold lower than soluble proteins.

• Solution: Optimize codon usage for E. coli, reduce induction temperature to 16-18°C, and extend induction time to 16-20 hours. Consider using specialized expression strains like C41(DE3) or Lemo21(DE3) specifically designed for membrane proteins.

2. Protein aggregation during extraction:

• Problem: Inappropriate detergent selection can cause aggregation and loss of function.

• Solution: Screen multiple detergents (DDM, LMNG, UDM) and detergent concentrations. Consider adding lipids (0.1-0.5 mg/ml) during solubilization to stabilize the native structure.

3. Loss of activity during purification:

• Problem: Metal cofactors may be stripped during purification.

• Solution: Include 1-5 mM MnCl₂ in all purification buffers. Avoid chelating agents like EDTA.

4. Heterogeneity in purified preparations:

• Problem: Mixed populations of properly folded and misfolded protein.

• Solution: Include a size exclusion chromatography step as the final purification stage. Consider thermal stability assays to identify buffer conditions that maximize the proportion of correctly folded protein.

5. Verification of functional state:

• Problem: Difficult to confirm that purified protein retains native activity.

• Solution: Develop a simplified activity assay that can be performed directly after purification, such as binding of fluorescent substrate analogs or detection of metal cofactor coordination using intrinsic tryptophan fluorescence.

Using these approaches, researchers can typically achieve 70-80% purity with 30-50% retention of native activity, which is sufficient for most biochemical and structural studies .

How should researchers address experimental variability when working with membrane-associated enzymes like arnC?

Working with membrane-associated enzymes like ArnC introduces several sources of variability that must be carefully controlled:

1. Membrane mimetic environment:

• Issue: Different detergents or lipid compositions can dramatically alter enzyme behavior.

• Recommendation: Once a functional reconstitution system is established, maintain strict consistency in detergent type, concentration, and lipid composition across experiments. Consider using nanodiscs with defined lipid composition for highest reproducibility.

2. Batch-to-batch variation in substrate preparation:

• Issue: Lipid substrates like undecaprenyl phosphate are difficult to prepare consistently.

• Recommendation: Prepare large batches of substrate that can be aliquoted and stored at -80°C. Verify substrate quality by TLC or mass spectrometry before each experimental series.

3. Time-dependent activity loss:

• Issue: Membrane proteins often lose activity over time, even when stored appropriately.

• Recommendation: Include standard control reactions with each experiment to normalize for activity changes. Consider fresh protein preparation for critical experiments rather than relying on stored samples.

4. Temperature sensitivity:

• Issue: Membrane fluidity and enzyme activity are highly temperature-dependent.

• Recommendation: Use temperature-controlled reaction vessels and equilibrate all components to the reaction temperature before mixing. Document and control the laboratory ambient temperature during experiments.

5. Statistical approach to variability:

• Issue: Higher inherent variability requires robust statistical analysis.

• Recommendation: Increase technical and biological replicates (minimum n=5 for technical replicates, n=3 for biological replicates). Apply appropriate statistical tests that account for non-normal distributions, which are common with membrane enzyme data.

By implementing these controls, coefficient of variation can typically be reduced from >30% to ~10-15%, which is acceptable for membrane enzyme systems .

What are promising strategies for developing inhibitors of arnC as potential antimicrobial adjuvants?

Given ArnC's role in antimicrobial resistance, its inhibition represents a promising strategy for developing adjuvants that could restore the efficacy of polymyxins against resistant bacteria. Several approaches show particular promise:

1. Structure-based design of competitive inhibitors:

• Target the UDP-L-Ara4FN binding site based on the recently determined structures

• Design non-hydrolyzable analogs of UDP-L-Ara4FN that compete with the natural substrate

• Focus on compounds that maintain the critical interactions with metal ions and catalytic residues

2. Allosteric inhibitors targeting protein dynamics:

• Exploit the conformational changes observed upon nucleotide binding

• Identify small molecules that stabilize the "open" conformation, preventing the clamshell motion required for catalysis

• Screen for compounds that bind at the interface between the GT-A domain and the juxtamembrane helices

3. Disruption of UndP binding:

• Design compounds that mimic the phosphate head group but contain bulky substituents that prevent threading through the JM helices

• Target the specific lipid-binding groove identified in molecular dynamics simulations

• Focus on compounds with appropriate lipophilicity to penetrate the bacterial membrane

4. Metal chelation approach:

• Develop chelators with specificity for the DXD motif coordination environment

• Design molecules that can compete with Mn²⁺ binding while maintaining membrane permeability

• Create pro-drug approaches to deliver chelators specifically to the periplasmic space

5. Peptide-based inhibitors:

• Design peptides that mimic the juxtamembrane helices to disrupt UndP binding

• Incorporate non-natural amino acids to enhance stability and membrane penetration

• Use cyclic peptides to constrain them in bioactive conformations

These strategies should be pursued with consideration of bacterial penetration, target selectivity, and potential for resistance development. High-throughput screening approaches combined with rational design offer the most promising path forward .

How might genetic variation in arnC across different bacterial species impact antimicrobial resistance profiles?

The arnC gene shows varying degrees of conservation across different bacterial species, with important implications for antimicrobial resistance profiles. Understanding these variations offers insights into species-specific resistance mechanisms and potential therapeutic vulnerabilities:

1. Conservation analysis across bacterial species:

• ArnC homologs show 40-90% sequence identity across different Gram-negative species

• The DXD motif and metal-coordinating residues show highest conservation

• Membrane-spanning regions display greater variability than catalytic domains

2. Species-specific polymorphisms and their functional impacts:

• Variations in the juxtamembrane helices may affect UndP binding efficiency

• Polymorphisms in the GT-A domain could alter substrate specificity

• Regulatory region differences potentially affect expression levels under stress conditions

3. Correlation with intrinsic polymyxin resistance:

• Species with naturally higher polymyxin MICs often contain ArnC variants with enhanced catalytic efficiency

• Structural differences in ArnC may contribute to the varying degrees of L-Ara4N modification observed across species

• Some highly resistant species may have evolved additional stabilizing interactions in the ArnC active site

4. Experimental approaches to investigate variation:

• Complementation studies using arnC genes from different species in a standard host

• Site-directed mutagenesis to introduce species-specific polymorphisms

• Biochemical characterization of recombinant ArnC variants from diverse sources

5. Implications for inhibitor development:

• Inhibitors may show variable efficacy against different bacterial species based on ArnC sequence variations

• Broad-spectrum inhibitors should target the most conserved regions of the enzyme

• Species-specific approaches may be required for pathogens with highly divergent ArnC variants

This comparative analysis approach provides a foundation for understanding the evolutionary adaptations in LPS modification systems and their impact on antimicrobial resistance profiles across the bacterial kingdom .

How can systems biology approaches be applied to understand the regulation of arnC expression in response to environmental signals?

The expression of arnC is regulated as part of the broader bacterial response to environmental stresses, particularly those affecting membrane integrity. Systems biology approaches offer powerful tools to understand this complex regulatory network:

1. Transcriptomic profiling under varying conditions:

• RNA-seq analysis of bacteria exposed to different antimicrobial peptides

• Time-course experiments to capture the dynamics of arnC regulation

• Comparative transcriptomics across multiple strains with varying resistance levels

2. ChIP-seq identification of transcription factor binding:

• Map binding sites of known regulators like PmrA/PmrB and PhoP/PhoQ

• Identify new transcription factors involved in arnC regulation

• Characterize the composition of regulatory complexes at the arnC promoter

3. Metabolomic integration:

• Correlate changes in cellular metabolites with arnC expression levels

• Identify potential metabolic signals that influence the Ara4N modification pathway

• Map interactions between central metabolism and LPS modification systems

4. Network modeling of regulatory circuits:

• Construct mathematical models of the signaling pathways controlling arnC

• Perform sensitivity analysis to identify the most influential network components

• Predict system responses to novel perturbations and validate experimentally

5. Single-cell analysis techniques:

• Use fluorescent reporters to monitor arnC expression at single-cell resolution

• Characterize population heterogeneity in response to antimicrobial stress

• Identify potential bet-hedging strategies in antimicrobial resistance development

These approaches collectively provide a comprehensive understanding of how bacteria regulate arnC expression as part of their adaptive response to environmental challenges, offering potential targets for disrupting resistance mechanisms .

What is the relationship between arnC function and biofilm formation in E. coli O127:H6?

The relationship between ArnC function and biofilm formation represents an important but understudied aspect of E. coli O127:H6 biology. LPS modifications are known to affect surface properties that influence bacterial adhesion and community formation:

1. Impact of LPS modifications on initial attachment:

• L-Ara4N addition alters surface charge and hydrophobicity

• These physicochemical changes may affect bacterial adhesion to abiotic surfaces

• ArnC activity potentially influences the initial stages of biofilm development

2. Experimental approaches to investigate this relationship:

• Compare biofilm formation between wild-type and arnC mutants using crystal violet staining

• Analyze biofilm architecture with confocal microscopy and 3D reconstruction

• Measure surface properties using zeta potential and contact angle measurements

3. Gene expression patterns in biofilms:

• Examine arnC expression at different stages of biofilm development

• Compare planktonic vs. biofilm expression patterns

• Investigate co-regulation with known biofilm-related genes

4. Extracellular matrix interactions:

• Study how L-Ara4N-modified LPS interacts with biofilm matrix components

• Characterize potential differences in extracellular DNA binding

• Examine polysaccharide distribution in wild-type vs. arnC mutant biofilms

5. Susceptibility of biofilms to antimicrobials:

• Test whether arnC mutations affect biofilm tolerance to polymyxins

• Compare diffusion rates of antimicrobials through wild-type and mutant biofilms

• Evaluate potential synergy between biofilm disruptors and polymyxins in arnC mutants

This research direction has significant implications for understanding persistent infections and developing strategies to combat biofilm-associated antimicrobial resistance in E. coli O127:H6 .