Essential Oils in the Control of Listeria monocytogenes: History
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Listeria monocytogenes is a foodborne pathogen, the causative agent of listeriosis. Infections typically occur through consumption of foods, such as meats, fisheries, milk, vegetables, and fruits. Chemical preservatives are used in foods; however, due to their effects on human health, attention is increasingly turning to natural decontamination practices. One option is the application of essential oils (EOs) with antibacterial features, since EOs are considered by many authorities as being safe. In this review, we aimed to summarize the results of recent research focusing on EOs with antilisterial activity.

  • essential oil
  • efficacy
  • food
  • method
  • antibacterial
  • preservation

1. Methods to Reveal Antilisterial Activity of Essential Oils and Their Active Components

A wide range of methods are available to study the antilisterial activities of EOs. The most effective for activity screening is the simple drop plate or paper filter-based disc diffusion method [60], which is used on the lawn of the test organism. Other studies preferred using the agar diffusion assay [61]. To define the lowest EO concentration which inhibits proliferation and which kills L. monocytogenes, the terms minimal inhibitory and minimal bactericidal concentration (MIC and MBC) are used. These values can be determined using macro- or microdilution methods, in glass reagent tubes [62,63,64] or in 96-well microdilution plates [65,66], respectively. Due to solubility problems of the hydrophobic EOs and their compounds, the usage of detergents (e.g., Tween-20 and Tween-80) is sometimes required [67]. Transmission and scanning electron microscopy (TEM and SEM) are adequate techniques to visualize the morphological changes accompanying antimicrobial effects [31,47,62,68,69]. To identify active compounds responsible for the antilisterial activity of an EO, bioautography is a proper method. This is based on thin-layer chromatography (TLC) performed on silica gel [70]. Active compounds are identified on the basis of their Rf values, while nonidentifiable active volatile compounds can be cut out from the silica gel and analyzed using the headspace solid-phase microextraction method coupled to gas chromatography–mass spectrometry (HS-SPME/GC-MS) [71]. With this, the purity or percentage composition of antimicrobial active compounds from silica gel can be determined.
For the biofilm-inhibitory or biofilm-degrading capacity of EOs or active compounds, the classical crystal violet staining assay, performed in 96-well microplate format, is most commonly used [66,72,73,74]; however, other methods, such as the TEMPO system (bioMérieux), VIDAS system (bioMérieux), or the discrete element method (DEM), have also been suggested [75]. Polystyrene, polypropylene, polyethylene, glass, and stainless steel are typically tested abiotic surface materials [31,45,72]. SEM and Confocal laser scanning electron microscopy (CLSM) are used to analyze changes in the biofilm integrity as a result of treatment [76].
The molecular changes accompanying the antilisterial activities of EOs and their compounds uncovered using the above methods can be further analyzed with molecular biological tools. Changes in protein profiles are often studied with 1D [47,62] or with the more detailed 2D polyacrylamide gel electrophoresis (PAGE). A fast alternative of 1D PAGE is capillary electrophoresis, in which expression differences between treated and control samples can be detected in a couple of minutes. A similar quantitative analysis uses liquid chromatography–mass spectrometry (LC–MS/MS) [69].
Further analysis of affected proteins, isolated with 2D PAGE, requires cutting and extraction from the acrylamide gel, followed by separation with liquid chromatography–mass spectrometry (LC–MS/MS) [69,77].
Today, high-throughput molecular biological methods are effectively used to uncover the underlying molecular events of bacterial metabolism in the presence of EOs or their active compounds. Whole-transcriptome analysis (WTA) is a proper approach to get a global view of the level of RNA synthesis [78], while the involvement of individual target genes, such as virulence-associated genes, can be further analyzed more precisely using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) [47,79,80].
Certain enzymatic assays are also preferably used to gain insight into which part of the metabolism is affected on an enzymatic level. Measurement of the level of β-galactosidase and ATPase gives feedback about the energy metabolism of L. monocytogenes, while the appearance of alkaline phosphatase outside the cell suggests weakened cell-wall integrity [47,62]. Membrane integrity can also be studied by quantifying the appearance of extracellular DNA in the medium [80].

2. Essential Oils with Antilisterial Activities

A broad range of EOs have been investigated for their antilisterial activity in the last two decades. Results of these tests are summarized in Table 1. Most of the represented articles focused on the activity of single EOs, but some also investigated synergistic effects when different EOs were combined [81,82]. The significance of this is that most EOs have a strain-dependent effect on L. monocytogenes [44,82]; therefore, a combination of different EOs could be an adequate approach against this foodborne pathogen in practice.
Results indicate that members of the Lamiaceae family, involving different Thymus and Oregano spp., showed the most extended antilisterial activity. Additionally, Cinnamomun spp. was proven to be effective. Typically, the major compounds were responsible for the antilisterial activities, especially if they belonged to the groups of mono- and sesquiterpenes. For the antilisterial effects, in most cases, compounds such as carvacrol, thymol, p-cymene, alpha-pinene, terpinene, or citral were responsible [44,83].
Most of the investigated EOs were extracted by steam distillation and originated from different countries and different producers. Especially in earlier studies, the compound composition (determined by gas chromatography) of the investigated EOs was not presented, which is a shortcoming that compromises the comparability of the different studies. This is an important issue as the compound composition of EOs is determined by geographical localization, weather, and time of harvest [84,85], which can influence the test results. A good example is that the compound composition of oregano EO in three studies differed significantly [44,86,87]; in the study of Gottardo, carvacrol content was 91%, whereas, in the studies of Maggio and Pesavento, these values were 68% and 71.8%, respectively. This was also the case with thyme showing differences in major compound content, albeit without influencing the antilisterial activity [44,45,88].
Another problem in the comparability of the results is that the bacterial cell numbers applied in different studies, either for the simple drop plate method or for MIC and MBC determinations, showed discrepancies. Furthermore, during tests, different media were used, e.g., Luria–Bertani (LB), Mueller–Hinton Broth (MHB), Brain Heart Infusion (BHI), and Peptone Yeast glucose (PYG) [60,62,63]. In most cases, in vitro tests were performed at 37 °C, whereas tests were only rarely conducted at lower temperatures under refrigerated conditions, which are mostly applied in food systems. Certainly, it would be a mistake to overstate the importance of the above factors. Moreover, since growth conditions influence gene regulation and, thus, phenotypic heterogeneity in bacteria [89], these factors could also influence the sensitivity of L. monocytogenes to EOs.
The antilisterial effect of EOs, summarized in Table 1, was mostly investigated using the standard disc diffusion technique [90], as this is the simplest screening method. In positive cases, the diameter of an inhibition zone around the EO spot or disc was typically between 20 and 30 mm, as demonstrated in several cases: black seed oil (31.50 mm) [90], broccoli sprout extract (17.84 ± 0.34 mm) [91], Citrus medica L. var. sarcodactylis Swingle citron oil (23.45 ± 1.23 mm) [78], Ceratonia siliqua EO (17 ± 0.3 mm) [65], Hibiscus surattensis L. calyces EO (25.26  ±  1.53 mm) [63], and Melaleuca alternifolia EO (30 ± 8.8 mm). In addition to this method, the agar diffusion assay [44,72,78], disc volatilization method [92], and plate colony counting [62] in pure, nanoliposome [91], nanocapsule [91], nanoemulsion [60,93,94], and liposome [95] systems were used. The advantage of the use of nanoemulsions is that this formulation is able to increase the biological activity and stability of EOs [96]. Nanoemulsion systems consist of three components: EO, water, and a nonionic surfactant, e.g., Tween-80 [60,94]. These three components are mixed, and then the particle size can be decreased using a sonicator [60].
Considering the tests, we have to emphasize again that the effect of EOs can be strain-dependent. A good example demonstrating this was a recent study in which the EO of Melissa officinalis was tested on three strains of L. monocytogenes (LMG 13305, 16779, and 16780), and three different inhibition zone diameters were found: 38.4 ± 4.2 mm, 54.6 ± 1.3, mm and 48.6 ± 1.7 mm, respectively [74]. In the case of Schinus terebinthifolius Raddi, EOs were produced from both ripe and unripe fruits. The inhibition zone of the latter was 35.22 ± 0.79 mm, while that of the former was 40.86 ± 0.31 mm [97].
Another aspect to be considered is which part of the plant and which procedure were used for the extraction. In a recent study, the authors revealed that the antilisterial effect of thyme was the strongest if acetone extract from the leaves was used, while ethanolic extract from the seeds exhibited the lowest antilisterial activity [98].
In most of the recent studies, kinetic assays were also performed in order to reveal the course of the antilisterial effect [68,99,100]. Kinetic curves are necessary if molecular changes on genomic and proteomic levels, accompanying the antilisterial effect, are intended to be investigated [78]. Through these analyses, the antibacterial mode of action of certain EOs can be revealed.
Since L. monocytogenes is able to form biofilms, a number of experiments focused on the antibiofilm capacity of EOs [45,67]. In such experiments, the biofilm-forming capacities of L. monocytogenes strains were hindered by Cinnamomun zeylanicum or Eugenia caryophyllata EOs [101]. Furthermore, it was also investigated whether EOs have the ability to destroy the already established biofilm on certain surfaces. They found that, within 2 h, clove could already drastically weaken the established biofilm. Such capacity is not typical for all EOs, as demonstrated by Guo et al. After establishing a firm biofilm in 72 h, they treated it with Citrus Changshan-huyou EO for 24 h, but the formed biofilm remained intact after treatment [31].
Revealing the antilisterial effect in in vitro studies is inevitable before considering practical uses; from this perspective, the results of screening and exploring the antimicrobial mode of action are all relevant issues, but the real challenge is always how a certain EO with potential antilisterial effects performs under harsh conditions if applied in different food systems.

This entry is adapted from the peer-reviewed paper 10.3390/microorganisms11061364

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