Impact of pH on citric acid antimicrobial activity against Gram‐negative bacteria

• Published in: Letters in applied microbiology Vol. 72; no. 3; pp. 332 - 340
• Main Authors: Burel, C., Kala, A., Purevdorj‐Gage, L.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.03.2021; John Wiley and Sons Inc
• Subjects: Aerogenes; Anti-Bacterial Agents - pharmacology; Antiinfectives and antibacterials; Antimicrobial activity; Antimicrobial agents; antimicrobial properties; antimicrobials; Biocides; Cell culture; Cell Membrane - drug effects; chelating agents; Citric acid; Citric Acid - pharmacology; E coli; Enterobacter aerogenes - drug effects; Enumeration; Escherichia coli; Escherichia coli - drug effects; Evaluation; Gram-negative bacteria; Hydrogen-Ion Concentration; K. aerogenes; Klebsiella; Klebsiella aerogenes; Membranes; Metals; Microbial Sensitivity Tests; Morphology; Original; pH effects; Surface charge; viability; Zeta potential; citric acid; pH; K. aerogenes; antimicrobials; E. coli
• ISSN: 0266-8254; 1472-765X; 1472-765X
• DOI: 10.1111/lam.13420

The antimicrobial activity of citric acid (CA) is often evaluated without pH adjustment or control and its impact on micro‐organisms is better understood in acidic conditions. However, the biocidal action of the fully ionized CA molecule, predominantly available at higher pH, has not been previously investigated. The objective of this study was to evaluate the antimicrobial effect of high (10%) and low (1%) concentrations of CA, each adjusted over a wide range of pH values (4·5, 6·5 and 9·5) relative to the controls exposed to corresponding pH levels alone (no CA). The viability and morphology of Escherichia coli and Klebsiella aerogenes were evaluated using a culture‐based enumeration assay in parallel with direct SEM imaging. Overall, the highest membrane damage and loss in viability were achieved with 10% CA at pH 9·5, which yielded at least 4·6 log10 CFU per ml (P < 0·001) reductions in both organisms. Insight into the superior efficacy of CA at high pH is proposed based on zeta potential measurements which reveal a more negatively charged bacterial surface at higher pH. This pH‐dependent increase in surface charge may have rendered the cells potentially more sensitive towards chelants such as CA3− that interact with membrane‐stabilizing divalent metals. Significance and Impact of the Study: Citric acid (CA) is an antimicrobial molecule with three ionization states that exist in an equilibrium directly governed by pH. Traditionally, non‐ionized CA was considered more antimicrobial, presumably due to the combined effects of the molecule and the acidic environment in which it occurs. By decoupling the antimicrobial impact of pH and CA on Gram‐negative bacteria, it is demonstrated, for the first time, that the fully ionized CA species alone was the most effective at destroying the bacteria. The results from SEM imaging and surface‐charge measurements provide further insight into the antimicrobial mode of action of CA against bacteria.