Citation: Sateriale, D.; Forgione, G.; De Cristofaro, G.A.; Pagliuca, C.; Colicchio, R.; Salvatore, P.; Paolucci, M.; Pagliarulo, C. Antibacterial and Antibiofilm Efficacy of Thyme (Thymus vulgaris L.) Essential Oil against Foodborne Illness Pathogens, Salmonella enterica subsp. enterica Serovar Typhimurium and Bacillus cereus. Antibiotics 2023, 12, 485. https://doi.org/10.3390/ antibiotics12030485 Academic Editors: Joanna Kozlowska, Anna Duda-Madej, Filippo Maggi and Nicholas Dixon Received: 19 December 2022 Revised: 18 January 2023 Accepted: 27 February 2023 Published: 28 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antibiotics Article Antibacterial and Antibiofilm Efficacy of Thyme (Thymus vulgaris L.) Essential Oil against Foodborne Illness Pathogens, Salmonella enterica subsp. enterica Serovar Typhimurium and Bacillus cereus Daniela Sateriale 1 , Giuseppina Forgione 1 , Giuseppa Anna De Cristofaro 1 , Chiara Pagliuca 2 , Roberta Colicchio 2 , Paola Salvatore 2,3 , Marina Paolucci 1 and Caterina Pagliarulo 1, * 1 Department of Science and Technology, University of Sannio, Via F. De Sanctis snc, 82100 Benevento, Italy 2 Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy 3 CEINGE-Biotecnologie Avanzate s.c.ar.l., Via G. Salvatore 486, 80145 Naples, Italy * Correspondence: caterina.pagliarulo@unisannio.it; Tel.: +39-0824-305141 Abstract: Nowadays, the wide spread of foodborne illness and the growing concerns about the use of synthetic food additives have shifted the focus of researchers towards essential oils (EOs) as possible antimicrobials and preservatives of natural origin. Thanks to their antimicrobial properties against pathogenic and food spoilage microorganisms, EOs have shown good potential for use as alternative food additives, also to counteract biofilm-forming bacterial strains, the spread of which is considered to be among the main causes of the increase in foodborne illness outbreaks. In this context, the aim of this study has been to define the antibacterial and antibiofilm profile of thyme (Thymus vulgaris L.) essential oil (TEO) against widespread foodborne pathogens, Salmonella enterica subsp. enterica serovar Typhimurium and Bacillus cereus. TEO chemical composition was analyzed through gas chromatography-mass spectrometry (GC-MS). Preliminary in vitro antibacterial tests allowed to qualitatively verify TEO efficacy against the tested foodborne pathogens. The subsequent determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values allowed to quantitatively define the bacteriostatic and bactericidal effects of TEO. To evaluate the ability of essential oils to inhibit biofilm formation, a microplate assay was performed for the bacterial biofilm biomass measurement. Results suggest that TEO, rich in bioactive compounds, is able to inhibit the growth of tested foodborne bacteria. In addition, the highlighted in vitro anti- biofilm properties of TEO suggest the use of this natural agent as a promising food preservative to counteract biofilm-related infections in the food industry. Keywords: thyme essential oil; antibacterial agent; antibiofilm activity; natural food preservatives; foodborne illness pathogens 1. Introduction Foodborne illness encompasses a wide spectrum of illnesses defined by the WHO’s Department of Food Safety, Zoonoses and Foodborne Diseases (FOS) as a growing public health problem worldwide [1]. In recent years, most of the foodborne outbreaks in Eu- rope and the United States were related to the consumption of contaminated meat and meat products; fresh processed products of pig meat were the most frequently involved category, along with chicken meat [2]. Meat and meat products contain essential amino acids, B-group vitamins, minerals and other nutrients ideal both for human nutrition and microbial growth [3]. Fresh and transformed meat are rich substrates that strongly support significant microbial growth, since they have nutrients, pH values and water contents generally compatible with the growth of a large number and variety of microorganisms [4]. Antibiotics 2023, 12, 485. https://doi.org/10.3390/antibiotics12030485 https://www.mdpi.com/journal/antibiotics