The effect of essential oils and their combinations on bacteria from the surface of fresh vegetables

Abstract During the study, we determined the antimicrobial activity of different selected essential oils (thyme, lemongrass, juniper, oregano, sage, fennel, rosemary, mint, rosehips, dill) on some pathogenic and spoilage bacteria isolated from the surface of various fresh vegetables. At the same time, in the case of some volatile oil combinations we followed the phenomena of synergism and antagonism. The identification of the isolated bacterial strains was made using 16S rDNA gene sequence analysis. The most resistant isolates appeared to be Curtobacterium herbarum, Achromobacter xylosoxidans, and Enterobacter ludwigii, while Pseudomonas hibiscicola was the most sensitive. Of the chosen plant essential oils, the most pronounced antimicrobial effect was detected in the case of oregano. The essential oils of thyme and mint also showed elevated antimicrobial activity. A synergistic effect was observed in case of five combinations of essential oil. Based on the results, we find that some individual essential oils and mixture compositions (due to synergic effect) could be good candidates for the preservation of fresh vegetables. These preliminary findings suggest that essential oils from locally grown spices could contribute to decreasing the health risk and also to the suppression of emergence of antibiotic resistance.

Minimally processed vegetables are one of the largest rising sectors in food industry. Ongoing advancement in natural food preservatives has led to the use of essential oils or their components in food packaging, and they represent an alternative to the synthetic additives (Patrignani, Siroli, Serrazanetti, Gardini, & Lanciotti, 2015).
Essential oils have exhibited antagonistic properties against different pathogenic or spoilage microorganisms. In vitro studies have shown antibacterial activity of essential oils against Bacillus cereus, Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus with concentration ranging from 0.2 to 10 μl/ ml (Scollard, Francis, & O'Beirne, 2013). Some essential oils such as Citrus spp., cinnamon, oregano, and thyme have been utilized as natural antimicrobials in different food preparations, while uncommon, plant-derived essential oils have received minimal attention. For example, Thymbra capitata essential oil, containing mainly carvacrol (93.31%) as oxygenated monoterpenes, exhibited strong antimicrobial activity. It was assayed as a potential sanitizing solution in food industry for food contact surfaces (Falcó, Verdeguer, Aznar, Sánchez, & Randazzo, 2019). Essential oils of Azadirachta indica and Litsea cubeba were recognized as promising and successful new antimicrobials with significant antimicrobial activity against the Staphylococcus aureus and Escherichia coli, concerning possible applicability in food (Thielmann, Muranyi, & Kazman, 2019).
Subinhibitory concentrations of Origanum vulgare and Rosmarinus officinale combined essential oils effectively inhibited the growth and survival of different pathogenic (Listeria monocytogenes, Yersinia enterocolitica, Aeromonas hydrophila) and spoilage (Pseudomonas fluorescens) bacteria related to minimally processed vegetables. Sensory evaluation showed that consumers find it acceptable for the different essential oils to be used together at subinhibitory concentrations as vegetable sanitizer. These forms of essential oils may contribute to the extension of the storage and shelf-life of vegetables (Azeredo et al., 2011). The effectiveness of essential oil combination is associated with the increased membrane damages showing a stronger antimicrobial effect (Yuan, Teo, & Yuk, 2018). A recent study proposes the use of essential oils as natural antibacterial inhibitors in nanoemulsion formulations against foodborne pathogens isolated from fresh fruits and vegetables (Amrutha, Sundar, & Shetty, 2017).
The microbial quality and safety of minimally processed vegetables have been remarkably improved with the application of essential oil-based nanoemulsions. These formulations were used as washing disinfectant or incorporated into edible coatings on packaging (Dvir et al., 2019) or product surface (Prakash et al., 2018). A new application is small concentrations of cinnamon essential oil, which are capable of reducing the attachment of Salmonella strains to the lettuce surface during refrigerated storage, with a remarkably positive effect on food safety (Rossi et al., 2019).
For the prevention of foodborne disease occurrence, it could be useful to know the specific pattern of pathogenic strains in different geographic regions. Susceptibility of these bacteria to essential oils could be a good candidate for combating them using antimicrobial packaging, without risk of the emergence of antibiotic resistance.
In this research, we determined the antimicrobial effect of different essential oils and certain combinations of essential oils, with the agar diffusion method on pathogenic as well as spoilage bacteria isolated on selective culture media and identified, from the surface of various fresh vegetables.

| MATERIAL S AND ME THODS
The first step of our research was to examine the microbial contamination of the surface of various fresh vegetables (cucumber, lettuce, tomatoes, peppers, hot peppers, cabbage, radishes, broccoli, and onions) with cultivation methods on different selective media. The vegetables were purchased from the local market. The bacterial strains occurring at highest amount on selective media were identified by molecular biological method. Subsequently, the antibacterial activity of a variety of individual herbal essential oils and certain combinations of essential oils were studied by agar diffusion method in the case of nine isolated and identified bacterial species: Pseudomonas hibiscicola, Brevibacillus agri, Enterobacter ludwigii, Curtobacterium herbarum, Acinetobacter beijerinckii, Acinetobacter calcoaceticus, Achromobacter xylosoxidans, Staphylococcus succinus, and Staphylococcus sciuri. Ten commercially available essential oils were used in this study: thyme, lemongrass, juniper, oregano, sage, fennel, rosemary, mint, rosehips, and dill. These oils were selected based on their herbal use in traditional culinary practices. These oils were produced by different companies (Solaris Plant, Adams Vision, Herbavit, Aromax, Fares Bio Vital, and Hofigal Export-Import) and extracted from different parts of the herbs.
The identification of the isolated bacterial strains was obtained using 16S rDNA sequence analysis. AccuPrep ® Genomic DNA Extraction Kit from Bioneer was used for Genomic DNA isolation.
Isolations were performed according to the manufacturer's protocol. For the amplification of one part of the bacterial 16S rDNA gene, universal oligonucleotides were used 27f and 1492r (5' AGAGTTTGATCMTGGCTCAG 3', 5' TACGGYTACCTTGTTACGACTT 3'). The PCR program for amplification was as follows: an initial denaturation at 94°C for 5 min, which was followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 7 min. Sequencing was performed by Biomi KFT (Hungary). The sequences were edited and aligned using Chromas (Technelysium Pty. Ltd., South Brisbane, Australia); Molecular Evolutionary Genetics Analysis 4 system was used for the phylogenetic analyses. The isolates were identified through comparison of the sequences using the EzTaxon server on the basis of 16SrDNA sequence data (Laslo, György, Ábrahám, & Mara, 2017).
In the agar diffusion method, 20 ml nutrient agar medium was poured in a sterilized Petri dish. After solidification, the medium was inoculated on the surface with a 0.1 ml suspension of bacteria (10 8 CFU/ml) taken in study. In the center of all of the inoculated media, an 8 mm diameter hole was cut with the help of a sterile testtube. In this hole 0.05 ml of essential oil was dropped. The incubation was carried out at the temperature of 30ºC, 48 hr. In the case of the essential oil combinations, in the holes made in the inoculated media, 0.025 ml essential oils were added. After incubation, the results were read and expressed in accordance with the size of the inhibition zone (György, Laslo, & András, 2015).

| RE SULTS AND D ISCUSS I ON
Among the studied vegetables, in the case of the lettuce, cucumber, radishes, and onions, the contamination was higher compared to the other vegetables. The results show that the microbiota occurring on the surface of fresh vegetables is highly variable and the molecular biological studies have identified a variety of pathogenic, saprophytic, and food spoilage bacteria (Table 1). It is important to highlight that the majority of the isolated and identificated bacteria during our research are not commonly found on the vegetable surfaces. It has been shown that different factors contribute to the diversity and composition of bacterial communities associated with the surfaces of fresh vegetables (Leff & Fierer, 2013).  (Anzai, Kim, Park, Wakabayashi, & Oyaizu, 2000). Bacterial strain designated SD8 was isolated from sea muds of the Qinhuangdao sea area in China. This marine bacterial strain was able to produce alkaline protease approximatively at a low yield. The bacterial strain was identified as

TA B L E
Pseudomonas hibiscicola based on 16S rDNA sequence analysis and morphological, physiological, and biochemical characterization (Cui, Yang, Wang, & Xian, 2015). Pseudomonas hibiscicola was isolated from ticks (Murrell et al., 2003). This bacterium is naturally occurring on the surface of roots, in the soil, and is an opportunistic plant pathogen. Other isolated Pseudomonas species were as follows: Pseudomonas oryzihabitans, P. geniculata, P. putida, and P. monteilii.
Other isolated bacteria with highest occurrence on the surface of fresh vegetables were as follows:  The essential oils of thyme and mint also show elevated antimicrobial activity ( should be treated as potential pathogen. In humans, after surgery or trauma, pathogenic bacteria should also be considered (Tena, Martínez, Losa, & Solís, 2014).
The sensitivity of bacterial strains isolated from the surface of fresh-cut vegetables relative to other selected essential oils was low or even shows lack of effects. The weakest activity on bacteria was shown by rosehips and sage essential oils (Tables 2 and 3).
In case of the dill essential oil, total inactivation was observed for Acinetobacter beijerinckii. Acinetobacters are present naturally in soil and water and occur in sewage. They have been detected in different foods such as raw, washed, and frozen vegetables, and they have also occurred in fresh, frozen, and stored fish products, as well as in spoiled animal-origin foods such as meat, milk, and cheese (Brenner, Krieg, & Staley, 2005). Different strains of Acinetobacter beijerinckii were isolated from human clinical samples (Nemec et al., 2009), from ready to eat fruit and lettuce (Carvalheira, Silva, & Teixeira, 2017).

The most sensitive bacterium against essential oils was
Pseudomonas hibiscicola, and the most resistant was Curtobacterium herbarum. Strains belonging to Curtobacterium have been isolated from different plants and rice, and C. herbarum was isolated from grass. Curtobacterium strains are rarely isolated from clinical specimens. It is recommended for clinical microbiologists to be aware of the possible occurrence of these bacteria in human samples.

TA B L E 3
The effect of the essential oils on growth of the studied bacteria II. (Inhibition zone in mm, average ± S.D., n = 10) Because of the everyday exposure of people to Curtobacterium their pathogenicity is considered rather low (Funke, Aravena-Roman, & Frodl, 2005).
Based on the results, the most resistant strain against the essential oil combinations was the Curtobacterium herbarum. The essential oils exert only a small antimicrobial effect on them (the most effective was the oregano essential oil), and in 7 cases, a lack of inhibition was observed (Tables 4, 5, 6 and 7). Similar results were obtained for the strain Achromobacter xylosoxidans, 7 essential oil combinations did not inhibit them, and generally a slight antibacterial effect was observed. The Enterobacter ludwigii strain shows resistance to essential oils, since in most cases, minimal inhibition was detectable (likewise, the oregano was the most effective). However, in this last two cases, the combination of thyme and dill essential oils showed synergism.
The most susceptible to single essential oil inhibition was the bacteria Pseudomonas hibiscicola, alongside the essential oil combinations.
In case of five combinations of essential oil, synergistic effect was observed (i.e., a larger inhibition zone was observed for combinations in comparison with the effect of the individual essential oils).
The synergistic combinations were as follows: lemongrass-rosemary, sage-rosemary, dill-rosemary, juniper-cumin, and cumin-dill. For the bacterial strain Acinetobacter calcoaceticus juniper-cumin essential TA B L E 5 The effect of the combination of essential oils on the studied bacteria II. (Inhibition zone in mm, average ± S.D., n = 10)     Using essential oil as antimicrobial agent (e.g., in food preservation and packaging) may decrease the risk of foodborne infections and could decrease the overuse of antibiotics, especially the emergence of antimicrobial resistance (AR). Essential oil compounds affect the bacterial cells by different mechanisms in comparison with traditional antibiotics. A synergic effect between these two compound classes may even occur. Another important strategy in the antibacterial fight is the reversing the antibiotic resistance (Kristiansen, Thomsen, Martins, Viveiros, & Amaral, 2010). The general principle of this strategy is given by the concept of collateral sensitivity, namely that microbial populations adapted to one class of antibiotics will have low fitness for one other class of antimicrobial compounds (Pál, Papp, & Lázár, 2015). A main AR mechanism is the increased activity of efflux pumps. The decreased activity of these pumps make bacteria vulnerable (Lázár et al., 2013), and active blocking of the efflux pump function or cell wall disruption is an effective way to counteract the AR (Langeveld, Veldhuizen, & Burt, 2014;Mouwakeh, Telbisz, Spengler, Mohácsi-Farkas, & Kiskó, 2018). As these two mechanisms are very common for essential oil compounds, this could be the key for AR reversing activity of the EOs (Yap, Yiap, Ping, & Lim, 2014). The efficiency of this strategy was confirmed experimentally for Gram-negative multidrug-resistant strains (Lorenzi et al., 2009;Yap, Lim, Hu, & Yiap, 2013). In case of multidrug resistance, blocking the efflux pumps with essential oil compounds may prevent the ejecting of multiple drugs from microbial cells, which have lost their efficiency due by AR. Previously, the strategy of regaining antibiotic efficiency by incorporation of nonantibiotic compounds (e.g., clavulanic acid, a β-lactamase inhibitor) was effective in the fight with AR in the case of penicillin-resistant strains (Cheesman, Ilanko, Blonk, & Cock, 2017). Until now, only several EOs possess a demonstrated strong individual antimicrobial effectiveness and more research is needed. Even though the antimicrobial effect of the majority of EO compounds is significantly weaker compared to antibiotics, their low toxicity level, as well as their natural origin, makes them attractive in both the food and cosmetic industries (Wińska et al., 2019).

| CON CLUS ION
The present study has shown that different pathogenic and food spoilage bacteria occur on the surface of fresh vegetables. The antimicrobial activity of studied essential oils of locally grown spices on the growth of identified bacteria shows differences in function of bacterial strains. The highest efficiency was observed for oregano essential oil. Some of the essential oil used in our experiments contains thymol and carvacrol (oregano and thyme) as main active component, with demonstrated efflux pump inhibitor effects, that are good candidate in prevention or even reversal of antibiotic resistance. Based on the results, it can be concluded that some individual essential oils and mixture compositions have the potential for practical antimicrobial application. These could be good candidates for the preservation of fresh vegetables, decreasing the health risk of foodborne infections, and also could contribute to the suppression of antibiotic resistance.

ACK N OWLED G M ENTS
We would like to thank the Sapientia Foundation-Institute for Scientific Research for financial support.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.