The role of Nisin and EDTA in pairing Gram-negative bacteria

Gram-negative bacteria (e.g., E. coli, Salmonella, Pseudomonas aeruginosa) exhibit strong resistance to most antimicrobial agents (including natural antimicrobial peptides) due to the lipopolysaccharide (LPS) barrier in their outer cell membrane. They have become major pathogenic bacteria causing food spoilage and clinical infections. Nisin (nisin), the only natural antimicrobial peptide approved for food use, shows significant inhibitory effects against Gram-positive bacteria but has weak activity against Gram-negative bacteria. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, can disrupt the outer membrane structure of Gram-negative bacteria. Recent studies have demonstrated that the combination of Nisin and EDTA can break through the outer membrane barrier of Gram-negative bacteria through "synergistic effects," significantly expanding Nisin’s antimicrobial spectrum. This article systematically analyzes the inhibitory effects of this combination on Gram-negative bacteria from three aspects—mechanism of action, formulation optimization, and application expansion—providing references for the development of novel antimicrobial strategies.

I. Resistance Barriers of Gram-Negative Bacteria: Why Single Nisin Has Limited Inhibitory Effects

The resistance of Gram-negative bacteria primarily stems from their unique cell wall structure, which directly hinders the action of Nisin and serves as the target for EDTA’s "auxiliary barrier-breaking" function:

(I) Lipopolysaccharide (LPS) Outer Membrane: A "Physical Barrier" for Nisin

The outer layer of the Gram-negative bacterial cell wall contains an outer membrane composed of LPS, phospholipids, and outer membrane proteins, with LPS acting as the key barrier:

The lipid A component of LPS forms a hydrophobic layer with phospholipids, while the polysaccharide chain forms a hydrophilic layer. Together, they create a dense "membrane barrier" that prevents the penetration of macromolecules (e.g., Nisin, with a molecular weight of ~3.4 kDa).

Meanwhile, LPS molecules bind to the phosphate groups of adjacent LPS via divalent metal ions (e.g., Ca²⁺, Mg²⁺), forming a stable "cross-linked network" that further enhances the structural integrity of the outer membrane. This stable structure prevents Nisin from accessing its target (lipid II on the bacterial cell membrane)—a prerequisite for Nisin to exert antimicrobial activity (Nisin must bind to lipid II to insert into the membrane and form pores, causing leakage of cellular contents).

(II) Efflux Pumps and Enzymatic Degradation: "Active Clearance Mechanisms" for Nisin

Some Gram-negative bacteria (e.g., Pseudomonas aeruginosa) also possess "active resistance mechanisms":

Efflux pump systems (e.g., MexAB-OprM) actively pump Nisin that enters the bacterial cell out of the cell, reducing intracellular Nisin concentration.

Proteases (e.g., metalloproteases) secreted by some strains degrade Nisin, inactivating its antimicrobial activity.

When Nisin is used alone, even a small number of molecules that penetrate the outer membrane are easily cleared by the above mechanisms, making it difficult to reach the effective bacteriostatic concentration.

II. Synergistic Mechanisms of the Nisin-EDTA Combination: Full-Process Collaboration from "Barrier Breaking" to "Sterilization"

The synergy between Nisin and EDTA is not a simple "superposition of effects." Instead, EDTA first disrupts the outer membrane barrier to create conditions for Nisin penetration, after which Nisin exerts its bactericidal effect. The specific mechanism is divided into three steps:

(I) EDTA: "First Step of Barrier Breaking" to Chelate Metal Ions and Disrupt Outer Membrane Stability

As a strong metal chelator, EDTA’s core function is to specifically bind Ca²⁺ and Mg²⁺ in the outer membrane of Gram-negative bacteria:

EDTA’s molecular structure contains four carboxyl groups and two amino groups, which form stable chelates with Ca²⁺ and Mg²⁺ via "hexadentate coordination." This removes the divalent metal ions that maintain LPS cross-linking in the outer membrane.

Without metal ions, the cross-linked network between LPS molecules breaks, transforming the outer membrane structure from "dense and stable" to "loose and porous" with obvious membrane defects (transmission electron microscopy shows that the outer membrane of E. coli treated with EDTA develops pores with a diameter of 50–100 nm).

Additionally, EDTA binds to the head groups of outer membrane phospholipids, disrupting the integrity of the phospholipid bilayer and further increasing outer membrane permeability. Experiments show that after treating E. coli with 0.5% EDTA for 30 minutes, the permeability of the outer membrane to fluorescently labeled macromolecules (molecular weight 2 kDa) increases by 4 times, opening a "channel" for Nisin to penetrate the outer membrane.

(II) Nisin: "Second Step of Sterilization" to Penetrate the Outer Membrane and Act on Cell Membrane Targets

After EDTA disrupts the outer membrane barrier, Nisin can penetrate the outer membrane smoothly, reach the inner membrane (cell membrane) of Gram-negative bacteria, and exert its classic antimicrobial effects:

Nisin specifically binds to lipid II (a key precursor for bacterial cell wall synthesis) on the inner membrane, forming "Nisin-lipid II complexes." Multiple complexes aggregate and insert into the cell membrane, creating transmembrane pores with a diameter of ~2–3 nm.

These pores drastically increase membrane permeability, causing massive leakage of intracellular small molecules (e.g., K⁺, amino acids) and influx of external water, ultimately leading to bacterial osmotic imbalance and cell lysis. A study on E. coli showed:

The bacteriostatic rate of Nisin (50 IU/mL) alone is only 5%;

When combined with 0.3% EDTA, the bacteriostatic rate increases to 92%;

The bacterial lysis rate (measured by the decrease in OD₆₀₀ value) reaches 70% within 2 hours, significantly higher than that of the single-treatment groups.

(III) Synergistic Enhancement: "Third Step" to Inhibit Resistance Mechanisms and Improve Sterilization Efficiency

In addition to breaking barriers, EDTA indirectly inhibits the resistance mechanisms of Gram-negative bacteria, further enhancing Nisin’s efficacy:

EDTA chelates metal ions required for efflux pump activity (e.g., the MexAB-OprM efflux pump requires Mg²⁺ to maintain its conformation), inactivating efflux pumps and reducing the extracellular efflux of Nisin.

Some proteases that degrade Nisin (e.g., metalloproteases) rely on metal ions (e.g., Zn²⁺) for activity. EDTA inhibits Nisin degradation by chelating these ions. Experiments confirm that when Pseudomonas aeruginosa is treated with the Nisin-EDTA combination, the intracellular Nisin concentration is 3 times higher than that in the single Nisin group, and the degradation rate of Nisin decreases from 45% to 12%, significantly enhancing bactericidal efficacy.

III. Optimization and Expansion of the Combined System: Adaptation to Concentration, Environment, and Bacterial Strains

The inhibitory effect of the Nisin-EDTA combination is influenced by formulation concentration, environmental conditions (pH, temperature), and bacterial strain type. Optimization is required to achieve "high efficiency and low toxicity" and expand its application range:

(I) Formulation Concentration Optimization: Reducing Single-Component Dosage and Minimizing Side Effects

Excessively high EDTA concentrations may affect food taste (e.g., bitterness) or irritate the human intestinal mucosa; excessive Nisin may cause allergies in some people. Therefore, the concentration ratio of the two must be optimized to ensure bacteriostatic efficacy while reducing dosage:

Food preservation scenarios: For meat products contaminated with E. coli, the optimal ratio is Nisin 200 IU/g + EDTA 0.2%. This concentration achieves a 95% bacteriostatic rate against E. coli, and the EDTA dosage is below the food additive standard (GB 2760 specifies a maximum EDTA dosage of 0.05%–0.25% in meat products), without affecting product flavor.

Clinical infection scenarios: For wound infections caused by Salmonella, in vitro experiments show that the combination of Nisin 100 IU/mL + EDTA 0.1% results in an inhibition zone diameter of 18 mm (vs. only 6 mm in the single Nisin group). This concentration has much lower toxicity to human fibroblasts (cell viability > 90%) than antibiotics (e.g., gentamicin, with 75% cell viability), making it suitable for topical use.

(II) Environmental Condition Adaptation: Addressing pH and Temperature Challenges in Different Scenarios

Differences in pH and temperature across application scenarios affect the stability and bacteriostatic efficacy of the combined system, requiring targeted adjustments:

pH adaptation: EDTA is easily protonated under acidic conditions (pH < 4), reducing its chelating ability; Nisin is prone to degradation under alkaline conditions (pH > 7), losing activity. Therefore:

In acidic foods (e.g., pickles, pH 3.5–4.0), the EDTA concentration should be moderately increased (from 0.2% to 0.3%);

In neutral foods (e.g., milk, pH 6.5–7.0), processing temperature should be controlled (< 60°C) to avoid Nisin thermal degradation. Experiments show that heating the Nisin-EDTA combination at < 60°C for 30 minutes retains 90% of its bacteriostatic activity, while heating at 70°C reduces retention to 65%.

Ionic environment adaptation: Ca²⁺ and Mg²⁺ in food or body fluids compete with EDTA for binding, reducing EDTA’s barrier-breaking effect. For high-calcium foods (e.g., cheese, calcium content > 0.5%), the EDTA concentration should be increased to 0.4% to ensure it preferentially chelates metal ions in the bacterial outer membrane rather than in the food.

(III) Strain-Specific Expansion: Covering Multiple Gram-Negative Pathogens

Differences in outer membrane structure (e.g., LPS type, outer membrane protein content) exist among Gram-negative bacteria. The inhibitory effect of the combined system on different strains must be verified to expand its application range:

Enterobacteriaceae strains: E. coli, Salmonella, Shigella, and other Enterobacteriaceae bacteria have low LPS cross-linking in their outer membranes and high sensitivity to EDTA. The Nisin-EDTA combination (Nisin 150 IU/mL + EDTA 0.2%) generally achieves a > 90% bacteriostatic rate against these strains.

Non-fermenting bacteria: Pseudomonas aeruginosa, Acinetobacter baumannii, and other non-fermenting bacteria have more outer membrane proteins (e.g., OprF) in their outer membranes and strong efflux pump activity. Higher concentrations of the combination (Nisin 250 IU/mL + EDTA 0.3%) are required, and a small amount of surfactant (e.g., Tween 80) can be added to further enhance outer membrane permeability. Studies show that the bacteriostatic rate of the three-component combination (Nisin + EDTA + Tween 80) against Pseudomonas aeruginosa increases from 82% to 96%.

IV. Application Expansion: From Food Preservation to Biocontrol

Based on the optimized combined system, the application of Nisin-EDTA has expanded from traditional food preservation to clinical care, agricultural biocontrol, and other fields, demonstrating broad application potential:

(I) Food Industry: Extending Shelf Life and Replacing Chemical Preservatives

In meat, dairy, and fruit/vegetable processing, the Nisin-EDTA combination effectively inhibits spoilage caused by Gram-negative bacteria:

Meat products: Chilled meat is easily contaminated by E. coli and Pseudomonas during 4°C storage, leading to spoilage within 7 days. After soaking in Nisin (200 IU/g) + EDTA (0.2%), the total microbial count remains below 10⁶ CFU/g (spoilage threshold) for 14 days, doubling the shelf life.

Fruit and vegetable juices: Apple juice is easily contaminated by Salmonella during processing. Single Nisin (300 IU/mL) treatment cannot effectively inhibit bacteria, but combining it with EDTA (0.15%) reduces the Salmonella count from 10⁵ CFU/mL to below 10² CFU/mL without affecting the juice’s color or taste.

(II) Clinical and Daily Chemical Fields: Preventing Infections and Reducing Antibiotic Dependence

In wound care and oral care, the Nisin-EDTA combination can be used as a natural antimicrobial agent to replace some antibiotics, reducing the risk of resistance:

Wound dressings: Loading Nisin (100 IU/cm²) and EDTA (0.1%) onto medical gauze for treating skin infections caused by E. coli shows that the wound infection cure rate reaches 85% within 7 days, significantly higher than that of traditional sterile gauze (50% cure rate), with no obvious skin irritation.

Oral care: Mouthwash containing Nisin (50 IU/mL) + EDTA (0.05%) inhibits Porphyromonas gingivalis (a Gram-negative bacterium causing periodontitis). After 2 weeks of use, the gingival bleeding index decreases by 40% and the plaque index decreases by 35%.

(III) Agricultural Field: Preventing Plant Diseases and Reducing Chemical Pesticide Use

In agricultural production, Gram-negative bacteria (e.g., Ralstonia solanacearum, Erwinia) are major pathogens causing crop diseases. The Nisin-EDTA combination can be used as a biopesticide to reduce chemical pesticide residues:

Tomato bacterial wilt control: Preparing a root irrigation agent with Nisin (200 IU/mL) + EDTA (0.2%) for controlling Ralstonia solanacearum reduces the disease incidence from 50% to 15%, with no significant damage to the soil microbial community (chemical pesticides reduce beneficial soil bacteria by 30%).

Post-harvest preservation of fruits and vegetables: Citrus is easily contaminated by Xanthomonas axonopodis after harvest. After soaking in the Nisin-EDTA combination solution, the rot rate decreases from 40% to 8%, and the preservation period extends to 20 days.

Through the triple mechanism of "EDTA barrier breaking – Nisin sterilization – synergistic resistance inhibition," the Nisin-EDTA combination successfully breaks through the outer membrane barrier of Gram-negative bacteria, significantly expanding Nisin’s antimicrobial spectrum. It demonstrates important application value in food preservation, clinical infection control, agricultural disease management, and other fields. Current research has clarified the synergistic mechanism and basic optimization scheme of the combination, but further efforts are needed in three areas:

Developing low-toxicity alternatives to EDTA (e.g., citric acid, polyaspartic acid) to reduce side effects of high-concentration EDTA;

Constructing targeted delivery systems (e.g., nanoliposomes) to achieve precise release of the combined system at specific sites;

Conducting long-term safety studies to verify the safety of long-term human use.

In the future, with continuous optimization of combination technology, the Nisin-EDTA system is expected to become an important natural antimicrobial strategy against Gram-negative bacteria, providing a new path for reducing dependence on chemical preservatives and antibiotics.