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. 2020 Sep 9;13(9):239.
doi: 10.3390/ph13090239.

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

Daniel Berdejo  1 , Elisa Pagán  1 , Natalia Merino  1 , Rafael Pagán  1 , Diego García-Gonzalo  1
Affiliations

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

Daniel Berdejo et al. Pharmaceuticals (Basel). .

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.

Keywords: Staphylococcus aureus; antibiotic susceptibility; complex orange essential oil; genotypic resistance; growth kinetics; minimum inhibitory and bactericidal concentrations; mutagenesis frequency; whole genome sequencing.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Growth curves of Staphylococcus aureus USA300 (A; SaWT) and evolved strain (B; SaROEO) in the absence (▬) and presence of 250 (▬), 500 (▬), 750 (▬), 1000 (▬), 1250 (▬), 1500 (▬), 2000 (▬), 5000 (▬) of orange essential oil (OEO), modelled using the modified Gompertz equation (Equation (1)).
Figure 2
Figure 2
Survival curves of Staphylococcus aureus USA300 (●; SaWT) and evolved strain (●; SaROEO), after 2000 µL/L orange essential oil (OEO) treatment at pH 7.0 (A) and pH 4.0 (B) at 37 °C. Data are means ± standard deviations (error bars) obtained from at least 3 independents experiments. Dashed line represents the detection limit (−5.0 log10).
Figure 3
Figure 3
Mutagenesis frequency in S. aureus USA300 grown in broth (control, ■) and with orange essential oil (OEO; 750 µL/L; ■), carvacrol (50 µL/L, ■) and rifampicin (0.01 mg/L, ■). Mutagenesis frequency was expressed as rifampicin-resistant cells in the total microbial population. Data are means ± standard deviations (error bars) obtained from five independent experiments. ns: no statistically significant differences (p > 0.05); ***: statistically significant differences (p ≤ 0.001), in comparison with control.
Figure 4
Figure 4
Genomic map of evolved strain (SaROEO) in comparison with Staphylococcus aureus USA300 (SaWT).
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