Incubation with a Complex Orange Essential Oil Leads to Evolved Mutants with Increased Resistance and Tolerance

Abstract

Emergence of strains with increased resistance/tolerance to natural antimicrobials was evidenced after cyclic exposure to carvacrol, citral, and (+)-limonene oxide. However, no previous studies have reported the development of resistance and tolerance to complex essential oils (EOs). This study seeks to evaluate the occurrence of Staphylococcus aureus strains resistant and tolerant to a complex orange essential oil (OEO) after prolonged cyclic treatments at low concentrations. Phenotypic characterization of evolved strains revealed an increase of minimum inhibitory and bactericidal concentration for OEO, a better growth fitness in presence of OEO, and an enhanced survival to lethal treatments, compared to wild-type strain. However, no significant differences (p > 0.05) in cross-resistance to antibiotics were observed. Mutations in hepT and accA in evolved strains highlight the important role of oxidative stress in the cell response to OEO, as well as the relevance of the cell membrane in the cell response to these natural antimicrobials. This study demonstrates the emergence of S. aureus strains that are resistant and tolerant to EO (Citrus sinensis). This phenomenon should be taken into account to assure the efficacy of natural antimicrobials in the design of food preservation strategies, in cleaning and disinfection protocols, and in clinical applications against resistant bacteria.

Phenotypic and Genotypic Comparison of Antimicrobial-Resistant Variants of Escherichia coli and Salmonella Typhimurium Isolated from Evolution Assays with Antibiotics or Commercial Products Based on Essential Oils

“…In comparison to EcWT, EcAEN 10 and EcAEN 20 showed a 25% increase in MIC value against AEN, and EcCOLIFIT 10 and EcCOLIFIT 20 showed an increase in MIC value against COLIFIT of the order of 14.3% and 28.6%, respectively. However, compared to other studies in which RVs have also emerged after exposure to complex EOs [9][10][11], the observed increase in resistance was relatively low. Apart from direct resistance, AEN-and COLIFIT-RVs (EcAEN 10 , EcAEN 20 , EcCOLIFIT 10 , and EcCOLIFIT 20 ) displayed a slight cross-resistance against AEN, COLIFIT, and cinnamaldehyde.…”

“…To determine the MIC values of the commercial EOs and the ICs, we adopted the methodology widely agreed upon in previous studies [9][10][11]. We added increasing concentrations of AEN, COLIFIT, thymol, and cinnamaldehyde (50-500 µL/L with 50 µL/L intervals) to test tubes with 5 mL of MHB.…”

“…These RVs are strains with increased resistance to the antimicrobial to which they have been exposed. The repeated application of Citrus sinensis (L.) Osbeck EO led to the selection of RVs of Staphylococcus aureus [9], while continuous exposure to Thymbra capitata (L.) EO resulted in the emergence of RVs of Listeria monocytogenes [10] and Salmonella enterica [11]. In addition, RVs in populations of E. coli [12,13], S. aureus [14], and S. enterica [15] were obtained after cyclical exposure to three different ICs (carvacrol, citral, and limonene oxide).…”

Orange essential oil in the diet of broilers: performance, organ biometrics, bone characteristics, and intestinal morphometry

“…Further, orange essential oil contains myrcene, sabinene, α-sabinene, linalool, and other compounds in minor amounts (Table 1). The antimicrobial properties of orange essential oil can be assigned to each constituent feature of isolation (Guimarães et al, 2019) and also the synergy that occurs between them (Ambrosio et al, 2019;Berdejo et al, 2020). Ambrosio et al (2019) demonstrated that limonene could collaborate with the selective activity of citrus oils when present in the gut, promoting the beneficial bacteria, while other minor compounds could act to inhibit pathogenic bacteria.…”

Progress in Alternative Strategies to Combat Antimicrobial Resistance: Focus on Antibiotics

“…It is worthwhile to highlight the demonstration of the bacteria developing resistance and tolerance towards EOs; however, cross-resistance to antibiotics was not reported [ 137 ]. Treatment of P. aeruginosa infection with a sub-inhibitory concentration of cinnamon bark oil or cinnamaldehyde as an adjunctive therapy may potentially induce expression of efflux pumps, and this needs further investigation to ascertain the use of EOs with any antagonistic effects [ 138 ].…”