The Effects of Eugenol, Trans-Cinnamaldehyde, Citronellol, and Terpineol on Escherichia coli Biofilm Control as Assessed by Culture-Dependent and -Independent Methods

Abstract

Bacterial biofilms contribute to problems with preserving food hygiene, jeopardizing any conventional intervention method used by the food industry. Hence, the approach of using essential oil (EO) compounds effective in biofilm control has considerable merit and deserves in-depth research. In this study, the effect of selected EO compounds (eugenol, trans-cinnamaldehyde, citronellol, and terpineol) was assessed on Escherichia coli biofilm control by plate count, resazurin assay, and Syto® 9/PI (-/propidium iodide) staining coupled with flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). The selected EO compounds effectively inhibited the growth of planktonic E. coli at low concentrations of 3–5 mM, revealing a high antimicrobial activity. EO compounds markedly interfered with biofilms too, with trans-cinnamaldehyde causing the most prominent effects. Its antibiofilm activity was manifested by a high reduction of cell metabolic activity (>60%) and almost complete reduction in biofilm cell culturability. In addition, almost 90% of the total cells had perturbed cell membranes. Trans-cinnamaldehyde further impacted the cell morphology resulting in the filamentation and, thus, in the creation of a mesh network of cells. Citronellol scored the second in terms of the severity of the observed effects. However, most of all, it strongly prevented native microcolony formation. Eugenol and terpineol also affected the formation of a typical biofilm structure; however, small cell aggregates were still repeatedly found. Overall, eugenol caused the mildest impairment of cell membranes where 50% of the total cells showed the Syto® 9+/PI– pattern coupled with healthy cells and another 48% with injured cells (the Syto® 9+/PI+). For terpineol, despite a similar percentage of healthy cells, another 45% was shared between moderately (Syto® 9+PI+) and heavily (Syto® 9–PI+) damaged cells. The results highlight the importance of a multi-method approach for an accurate assessment of EO compounds’ action against biofilms and may help develop better strategies for their effective use in the food industry.

Biological Properties and Prospects for the Application of Eugenol—A Review

“…Eugenol, a component of essential oil, in addition to other compounds, such as trans-cinnamaldehyde, citronellol, and terpineol, has been tested for its effectiveness in eliminating bacterial biofilm [ 24 ]. Bacterial biofilm is a three-dimensional macrocolony of bacteria isolated by an extracellular matrix, produced by these microorganisms [ 25 ].…”

“…For example, in the food industry, they form on the surface of food products, contaminating the food and can causing disease development. Therefore, effective measures are being sought to solve this issue [ 24 ].…”

“…Olszewska et al [ 24 ] examined the above-mentioned compounds, which are components of essential oils, in regard to their ability to inhibit the growth of the Escherichia coli biofilm. The study was performed on the basis of the platelet count, resazurin test, and Syto ® 9/PI (-/propidium iodide) staining in combination with flow cytometry (FCM) and confocal laser scanning microscopy (CLSM).…”

Antimicrobial and Antibiofilm Effect of ε-Polylysine against Salmonella Enteritidis, Listeria monocytogenes, and Escherichia coli in Tryptic Soy Broth and Chicken Juice

“…The inhibitory and degradation effects of ε-PL on the selective pathogen biofilms were optically analyzed using CLSM ( Figure 4 ). Syto9 and PI differ in terms of the penetration capability of cell membranes [ 44 ]. Syto9 could penetrate both dead and live cells because of its high permeability, whereas PI has high molecular mass that it can only penetrate cells with damaged membranes.…”

Influence of Gallic Acid and Thai Culinary Essential Oils on Antibacterial Activity of Nisin against Streptococcus mutans

“…In addition, bacterial cells with damaged membranes often fail to attach and form biofilm structures [ 36 ]. Therefore, KLL and LG oils containing terpenes as a major constituent may be diminished the biofilm formation by these mechanisms [ 37 ]. As shown in Figures 1(b) and 1(c) , nisin at all test concentrations could not inhibit the formation of biofilm of S. mutans , whereas GA at the concentration of 8 mg/mL interfered the formation of biofilms with 88.98% inhibition.…”