In Vitro Evaluation of the Potentiating Effects of Plant-Derived Molecules on Conventional Biocides

Summary

This protocol provides a systematic framework to assess how phytochemicals can enhance the bactericidal effects of traditional biocides using bactericidal activity modeling and combinatorial interaction assays , which aim to identify synergistic mixtures that improve microbial control and reduce overall biocide usage.

Abstract

Developing innovative and efficient approaches to enhance the antimicrobial activity of conventional biocides is a promising strategy for improving microbial control for healthcare disinfection purposes. In this context, we describe a detailed protocol for evaluating the potentiating effects of plant-derived phytochemicals on synthetic biocides. These plant-based products are attractive candidates as biocide adjuvants due to their structural diversity, bioactivity, and often lower toxicity. The workflow here described allows for the determination of minimum bactericidal concentrations (MBCs) and the systematic generation of both dose-response and time-response curves for each product tested individually (biocides and phytochemicals) and for their dual or triple combinations. The resulting data allow for advanced mathematical modelling, including determining the fractional bactericidal concentration index (FBCI) and assessing the interactions between the tested products regarding synergistic, additive, or antagonistic effects using the software Combenefit. In that sense, it is possible to identify promising natural products as biocide enhancers, optimize antimicrobial efficacy, and design more sustainable and safer disinfection strategies that may reduce the concentrations required of synthetic biocides. This improves microbial control and minimizes environmental and public health risks associated with excessive biocide use.

Introduction

Healthcare-associated infections (HAIs) are among the most critical and worrisome public health challenges worldwide¹. Alarmingly, they are strongly associated with the spread of multidrug-resistant (MDR) microorganisms and the persistence of bacterial bioburden on hospital surfaces, often due to inappropriate or ineffective disinfection practices^2,3. Moreover, the cross-contamination between patients, healthcare staff, and medical equipment further amplifies this issue^4,5. In Europe, for instance, it has been estimated that nea....

Protocol

NOTE: The methodology proposed was adapted from the European Standard EN 1276⁵³.

1. Culture preparation

1. Cryopreserve the bacterial strains tested, Escherichia coli CECT 434 and Staphylococcus aureus CECT 976, by storing them in cryovials with glycerol 30% (v/v) at -80 °C.
2. For recovery, culture bacteria in plate count agar (PCA) plates and allow them to grow, for 24 h at 25 °C, by incubating the plates.
3. Prepare overnight-grown cultures of the strains under study by inoculating one bacterial colony from a streaked plate in tryptic soy broth (T....

Results

This study evaluated the bactericidal activity of all four compounds tested — BAC, PAA, SAL, and EUG — against E. coli CECT 434 and S. aureus CECT 976. As shown in Figure 1 and Figure 2, BAC, PAA, SAL, and EUG exhibited an apparent concentration-dependent effect on culturability. Table 2 presents the MBC values for the tested molecules after exposure and 24h after exposure (regrowth capacity assessment). For BAC, no CFUs were o.......

Discussion

The protocol presented in this study provides an integrated framework for assessing the bactericidal activity of two commonly used biocides – BAC and PAA – and two promising phytochemicals – EUG and SAL – individually and in combination (dual and triple combinations), against E. coli and S. aureus. Several steps within this protocol are particularly critical for ensuring reproducibility and accurate interpretation of antimicrobial interactions. These include the standardization of t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Benzalkonium Chloride	Merck, Spain	8.14363.0100	Biocide
Centrifuged Mega Star 600R	VWR, USA	VWR Mega Star 600R	-
Combenefit - Version 2.021	Cancer Research UK Cambridge Institute, Cambridge, United kingdom	-	Software for synergism assessment
Cuvettes	VWR, Germany	VWRI634-0678	1.5 mL cuvettes for optical density adjustment
Destilatted water	-	-	For media preparition
Dimethyl Sulphoxide (DMSO)	VWR, France	23500.322	Organic solvent
Disposable inoculation loops	VWR, Italy	VWRI612-9357	10 μL inoculation loops
Eppendorf tubes	VWR, China	VWRI525-1164	1.5 mL eppendorf safe-lock tubes
Escherichia coli	Spanish Type Culture Collection (CECT)	434	Reference strain used in this study
Ethanol	VWR, France	83813.36	For desisfection of work plece, material, and hands
Eugenol 99%	Sigma-Aldrich, USA	163333	Phytochemical
Falcon tubes	VWR, USA	VWRI525-1085	15 mL falcon tubes
Falcon tubes	VWR, Belgium	VWRI525-1109	50 mL falcon tubes
GraphPad Prism 9.0 for Windows 11	La Jolla, California, USA	-	Software for mathematical and statistical analysis
Lecithin	Alfa Aesar, Germany	Q04A010	-
L-histidine	Merck, USA	1.04351.0025	-
Orbital Incubator	VWR, USA	VWR 5000I Incubating shaker 230V	-
Peracetic acid 15%	AppliChem, Germany	143495-1211	Biocide
Petri dishes	VWR, Italy	VWRI391-0582	-
Pipette 1 - 10 mL	VWR, USA		-
Pipette 100 - 1000 μL	VWR, USA		-
Pipette tips 100 - 1000 μL	Frilabo, Portugal	1-202-50-0	-
Pippette 20 - 200 μL	VWR, USA		-
Pippette tips 1 - 10 mL	VWR, Belgium	vwri613-7094	-
Pippette tips 2 - 200 μL	VWR, Mexico	vwri613-6418	-
Plate Count Agar	VWR, Belgium	84608-0500	Isolation and cultivation of microorganisms
Polysorbate 80	VWR, USA	20E2756726	-
Potassium Chloride - KCl	VWR, Belgium	11D110008	-
Potassium Dihydrogen Phosphate - KH2PO4	VWR, Germany	26931.263	-
Sacilicic acid	Sigma-Aldrich, USA	84210-500G	Phytochemical
Saponin	VWR, Germany	21A124103	-
Sodium Chloride - NaCl	VWR, Belgium	27788.297	-
Sodium Hydrogen Phosphate - Na2HPO4	VWR, Belgium	28026.26	-
Sodium Thiosulphate - Na2S2O3	VWR, USA	18D2056589	-
Spectrophotometer	VWR, USA	UV-1600PC Spectrophotometer	Optical density adjustment
Staphylococcus aureus	Spanish Type Culture Collection (CECT)	976	Reference strain used in this study
Tryptic Soy Broth	Merck, Germany	1.05459.0500	Isolation and cultivation of microorganisms
Ultrapure water	-	-	For sample preparation