  Citation: Coimbra, A.; Miguel, S.; Ribeiro, M.; Coutinho, P.; Silva, L.; Duarte, A.P.; Ferreira, S. Thymus zygis Essential Oil: Phytochemical Characterization, Bioactivity Evaluation and Synergistic Effect with Antibiotics against Staphylococcus aureus. Antibiotics 2022, 11, 146. https://doi.org/10.3390/ antibiotics11020146 Academic Editors: Noura Dosoky and William N. Setzer Received: 22 December 2021 Accepted: 20 January 2022 Published: 24 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 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 Thymus zygis Essential Oil: Phytochemical Characterization, Bioactivity Evaluation and Synergistic Effect with Antibiotics against Staphylococcus aureus Alexandra Coimbra 1 ,Sónia Miguel 1,2 , Maximiano Ribeiro 1,2 , Paula Coutinho 1,2 ,Lúcia Silva 3 , Ana Paula Duarte 1 and Susana Ferreira 1, * 1 CICS-UBI—Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; alexandra.coimbra@hotmail.com (A.C.); spmiguel@ipg.pt (S.M.); mribeiro@ipg.pt (M.R.);coutinho@ipg.pt (P.C.); apcd@ubi.pt (A.P.D.) 2 Center of Potential and Innovation of Natural Resources, Polytechnic Institute of Guarda, 6300-559 Guarda, Portugal 3 FibEnTech—Department of Chemistry, FibrousMaterials and Environmental Technologies, University of Beira Interior, Rua Marquês d’Ávila e Bolama, 6201-001 Covilhã, Portugal; mlas@ubi.pt * Correspondence: susana.ferreira@fcsaude.ubi.pt Abstract: Staphylococcus aureus is a nosocomial bacterium causing different infectious diseases, ranging from skin and soft-tissue infections to more serious and life-threatening infections such as sepsis, meningitis and endocarditis, which may be exacerbated by antibiotic resistance. Plant products may be seen as an alternative as antibacterial agents, namely, against S. aureus. Thus, the aim of this work was to characterize the chemical composition and evaluate the bioactive properties of the T. zygis essential oil (EO), with a focus on antimicrobial activity against S. aureus. Gas chromatography coupled with mass spectrometry was used to assess the chemical composition of the T. zygis EO, and the antioxidant activity was evaluated using the DPPH method and β-carotene-bleaching assay. The antimicrobial activity against S. aureus strains, the interaction with different antibiotics and the attenuation of this bacterium’s virulence were evaluated. The T. zygis EO showed antioxidant activity acting through two different mechanisms and antibacterial activity against S. aureus, with antibiofilm and antihaemolytic properties. This EO also demonstrated synergistic or additive interactions in combination with ampicillin, ciprofloxacin or vancomycin against S. aureus strains and, in some cases, changed the antibiotic-resistance phenotype from resistant to susceptible. Therefore, the present work demonstrates the good bioactive properties of the EO of T. zygis, mainly the antimicrobial activity against S. aureus, revealing its potential to be used as an antibacterial agent. Keywords: Thymus zygis; essential oil; antioxidant activity; antimicrobial agent; Staphylococcus aureus; interaction with antibiotics 1. Introduction Antibiotics are used as the primary weapon against infections; while, at first, antibiotics were highly effective, their inappropriate use and high selective pressure have led to the emergence and spread of antibiotic-resistant bacteria [1]. In fact, antibiotic resistance has increased dramatically in recent decades and is now considered one of the greatest global health threats [2,3]. Staphylococcus aureus is a Gram-positive facultative anaerobic human pathogen of both nosocomial and community-acquired infections worldwide [4,5]. S. aureus is a com- mensal bacterium located on the skin and mucous membranes, but also a virulent bac- terial pathogen associated with high morbidity and mortality [6–9]. This opportunistic pathogen can cause numerous acute and chronic infections [5,10], such as moderately severe skin infections, fatal pneumonia, sepsis, meningitis, endocarditis, or toxic-shock syndrome [7,11,12]. The higher rates of colonization, augmented use of surgical implants, Antibiotics 2022, 11, 146. https://doi.org/10.3390/antibiotics11020146 https://www.mdpi.com/journal/antibiotics