Sub-Inhibitory Doses of Individual Constituents of Essential Oils Can Select for Staphylococcus aureus Resistant Mutants.

Daniel Berdejo,Rafael Pagán,Beatriz Chueca,Adriana Renzoni,Elisa Pagán,William Kelley,Diego Garcia-Gonzalo

doi:10.3390/molecules24010170

Abstract

Increased bacterial resistance to food preservation technologies represents a risk for food safety and shelf-life. The use of natural antimicrobials, such as essential oils (EOs) and their individual constituents (ICs), has been proposed to avoid the generation of antimicrobial resistance. However, prolonged application of ICs might conceivably lead to the emergence of resistant strains. Hence, this study was aimed toward applying sub-inhibitory doses of the ICs carvacrol, citral, and (+)-limonene oxide to Staphylococcus aureus USA300, in order to evaluate the emergence of resistant strains and to identify the genetic modifications responsible for their increased resistance. Three stable-resistant strains, CAR (from cultures with carvacrol), CIT (from cultures with citral), and OXLIM (from cultures with (+)-limonene oxide) were isolated, showing an increased resistance against the ICs and a higher tolerance to lethal treatments by ICs or heat. Whole-genome sequencing revealed in CAR a large deletion in a region that contained genes encoding transcriptional regulators and metabolic enzymes. CIT showed a single missense mutation in aroC (N187K), which encodes for chorismate synthase; and in OXLIM a missense mutation was detected in rpoB (A862V), which encodes for RNA polymerase subunit beta. This study provides a first detailed insight into the mechanisms of action and S. aureus resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide.

Highlights

Emergence of stable resistant strains during food processing can compromise food safety and shelf-life
In order to carry out the selection of stable-resistant strains by exposing S. aureus USA 300 to sub-inhibitory concentrations of individual constituents (ICs) during bacterial growth, the minimum inhibitory concentration (MIC) of wild type (WT) strain against carvacrol, citral, and (+)-limonene oxide was first determined (Table 1)
Hereinafter, the strains isolated after carvacrol exposure were referred to as CAR, those isolated after citral exposure as CIT, and after (+)-limonene oxide as OXLIM

Summary

Introduction

Emergence of stable resistant strains during food processing can compromise food safety and shelf-life. Development of stable bacterial resistance is based on genetic modifications caused by mutations (i.e., genotypic resistance) [1] that produces variations in cellular responses to stress and damage [2]. Mutated microorganisms with an elevated resistance and/or tolerance might survive food preservation treatments that were previously considered sufficient as a control measure for spoiling. In recent years many efforts have been carried out in the search for new food preservatives capable of avoiding the generation of antimicrobial resistance. Many authors have proposed the use of natural compounds, such as essential oils (EOs) and their individual constituents (ICs) as food preservatives. Citral, and (+)-limonene oxide are monoterpenes generally recognized as safe (GRAS), whose antimicrobial properties have been

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