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Microbiology and Biotechnology Letters

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Food Microbiology (FM)  |  Food Borne Pathogens and Food Safety

Microbiol. Biotechnol. Lett. 2024; 52(1): 24-36

https://doi.org/10.48022/mbl.2309.09012

Received: September 22, 2023; Revised: February 28, 2024; Accepted: March 7, 2024

Synergistic Effect of Essential Oils and Enterocin Produced by Enterococcus faecalis MSW5 against Foodborne Pathogens

Mansi Shukla and Shilpa Gupte*

Department of Microbiology, Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Science (ARIBAS), New Vallabh Vidyanagar, Anand 388 121, Gujarat, India

Correspondence to :
Shilpa Gupte,       shilpagupte@aribas.edu.in

This study determines the combinatorial effect of enterocin MSW5 and five essential oils (EOs-Thymus vulgaris, Cymbopogon martini, Origanum vulgare, Cinnamomum zeylanicum, and Cymbopogon citrus) against Staphylococcus aureus, Listeria monocytogenes, and Salmonella Typhimurium. The Minimum Inhibitory Concentration of each antimicrobial agent was determined. The MIC of enterocin MSW5 against test pathogens was in the following order: S. aureus (0.362 ± 0.01), S. Typhimurium (0.362 ± 0.05 mg/ml), L. monocytogenes (0.725 ± 0.08 mg/ml). Among all EOs, maximum activity was observed in the case of C. zeylanicum against S. aureus (78.12 ± 0.04 ppm), S. Typhimurium (78.12 ± 0.08 ppm), and L. monocytogenes (39.00 ± 0.05 ppm). Further, the checkerboard assay was used to determine the synergistic effect between antimicrobial agents and enterocin MSW5 in combination with C. zeylanicum has shown significant synergism with the Fraction Inhibitory Concentration index (0.372) against test pathogens. Additionally, individual EOs and enterocin MSW5 have shown anti-biofilm activity, whereas their combined use has shown more significant antibiofilm activity. The maximum anti-biofilm activity was observed with the combination of enterocin MSW5 and O. vulgares against S. aureus (92.86 ± 0.06%) and S. Typhimurium (73.63 ± 0.23%) and a combination of enterocin MSW5 and C. citrus against L. monocytogenes (87.84 ± 0.15%). Therefore, combinations of antimicrobial compounds can control the growth of foodborne pathogens better than the individual agent.

Keywords: Antimicrobial agents, biofilm assay, checkerboard assay, enterocin MSW5, Minimum Inhibitory Concentration (MIC), Fraction Inhibitory Concentration (FIC)

Graphical Abstract


In recent years, microorganisms that cause foodborne infection and food spoilage have increased mortality or morbidity by many folds. The problem of foodborne infections is precarious, affecting 10% of the global population with 33 million deaths annually [1]. Several persistent food pathogens are present in food systems such as Escherichia coli, Listeria monocytogenes, S. aureus, C. botulinum, Salmonella Typhimurium, and C. perfringens, in both the forms planktonic as well as in biofilms. Among these, L. monocytogenes is a very dangerous foodborne pathogen, responsible for 30% of mortality in population [2, 3]. It can able to grow at pH 4−10, at a 1℃ to 45℃ temperature range, in high amounts of salt, and can also survive on the surfaces of food processing instruments in industries via biofilm formation [4]. It is psychotropic and responsible for causing listeriosis, which mainly affects the immunocompromised population, and less affects the young and elderly group compared to the 64-year-old age group and pregnant women. Also, it causes meningitis, septicemia, and central nervous system infection [5]. Maximum L. monocytogenes contamination was found in fish and fishery products at about 6%, and in ready-to-eat salads approx. 4.2%, readyto- eat meat and meat products about 1.8%, soft and in semi-soft cheeses approx. 0.9%, fruits and vegetables (0.6%), and hard cheeses (0.1%) [5, 6]. Another grampositive most important food-borne pathogen of animals and humans is S. aureus. Staphylococcal enterotoxins (SEs) are produced by S. aureus when growing in foods with high cell counts [7]. Food containing SEs is responsible for causing Staphylococcal Food Poisoning (SFP) which can intoxicate humans and animals. According to The European Union One Health 2018 and Zoonoses Report in 2019, the number of SFP cases is tremendously increasing in Europe [8]. In USA, as per the report of the CDC around 2,40,000 cases in a year, resulting in 1000 hospitalizations and six deaths [9]. Also, in France SFP outbreaks are likely much higher, which is currently > 90% of all outbreaks reported [8]. Moreover, one of the major issues related to S. aureus is antimicrobial resistance, specifically, Methicillin-resistant S. aureus (MRSA), which is getting extensive attention to develop new methods for the prevention and control of S. aureus infections [7]. One of the second most common serovars responsible for causing infections in humans and animals worldwide is the gram-negative food-borne pathogen Salmonella Typhimurium, with its wider range of host tropism including food-borne infections [1, 10]. Salmonellosis mainly spreads by consuming contaminated eggs and chicken, various kinds of seafood, meats, and vegetables which show an important role in the persistent occurrence of outbreaks [11, 12]. Due to typhoid fever, every year number of people who get sick is 11−20 million, and 128,000−161,000 people die [13]. Furthermore, the marvelous rise in the range of global food distribution, with regular travel, has provoked a rise in the spreading of foodborne diseases [14]. Refrigeration is the most common way to increase the shelf life of foods, but it is unable to inhibit most of the psychrophilic foodborne pathogens. Therefore, for limiting the growth of food-borne pathogens during these processes the practice of chemical preservatives and heat treatment have proven to be effective in the past years. However, such treatments can lead to alter the organoleptic properties of food products. In addition, nowadays consumers have become more knowledgeable and interested in safe-to-eat food that is natural food with minimal process, and environmental concerns have ignited attention towards the development of more effective new natural antimicrobial agents or combination of antimicrobial agents to control and prevent foodborne infections [15]. Many natural bio-preservatives from various natural sources have been widely studied, such as microorganisms, medical plants, herbs, and animals [5, 16]. One of them is Essential Oils (EOs), which are derived from many aromatic plants. They are lipid-based, volatile, liquid, and seldom colored compounds [17]. They have antimicrobial activity by interfering in the cell membrane phospholipid bilayer or they may interfere with the genetic material like Deoxyribonucleotide (DNA) and Ribonucleotide (RNA), and enzyme systems [18]. However, they have a major drawback if they are incorporated in more amounts into the food products, the sensory impacts can change the taste of food items or beat the acceptable flavor and odor thresholds [19]. For this reason, the combinatorial treatment of antimicrobial compounds like EOs with bacteriocins can help to enhance the antimicrobial effect, with very few undesirable changes in food products [20]. Bacteriocins are ribosomally synthesized peptides or proteins produced by bacteria that can inhibit or kill other related or unrelated microorganisms. The bacteriocin's mode of action is cell permeabilization which creates pores in the cellular membrane [21]. Bacteriocins have potential application as an alternative to conventional antibiotics to control and prevent bacterial infections. They are also broadly studied for food preservation against food-spoilage bacteria as well as food-borne pathogens [22]. Furthermore, this combinatorial method acts on the pathogen’s biofilm which is not easily eradicated by an individual treatment of bacteriocin or essential oils at their minimum inhibitory concentration (MIC). Biofilm is a group of microbial cells bounded by an extracellular polymeric structure and attached to an inert or living surface at the interface or in a liquid phase [23]. Eradication of biofilm with conventional treatments is harder than their planktonic growth, so biofilm poses more challenges in food industries, where exploring new methods to eradicate biofilms is mandatory [24]. For all the above reasons, two different natural compounds were selected in the present study: enterocin MSW5 and five essential oils like Thymus vulgaris, Cymbopogon martini, Origanum vulgare, Cinnamomum zeylanicum, and Cymbopogon citrus. In the first phase of this study, the antimicrobial activity of enterocin MSW5 and five EOs was determined individually and in combination with EOs with enterocin MSW5 using a checkerboard assay against L. monocytogenes, S. aureus, and S. Typhimurium. In the second phase, the antibiofilm activity of enterocin MSW5, EOs, and their combinations was determined against L. monocytogenes, S. aureus, and S. Typhimurium at their MIC and sub-MIC value using a microtiter plate assay.

Bacterial culture conditions and media

Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 13932, and Salmonella Typhimurium ATCC 6539 test organisms were stored at -80℃ in nutrient broth (Hi-media, India) containing glycerol (20 %v/v). Before each set of experiments, stock cultures were activated after two repeated 24 h growth cycles in a nutrient broth medium at 37℃. After that, the activated cultures were centrifuged, and the pellets were washed twice in phosphate buffer saline (pH~7.4) to obtain working cultures. The optical density of each culture was then adjusted to 1.0 as per the MacFarland table for MIC determination of antimicrobials using a checkerboard test. The bacteriocin-producing strain Enterococcus faecalis MSW5 with given accession number MW672393 was isolated from a fermented watermelon sample in the lab using tryptone glucose malt extract (TGME) media.

Antimicrobial agents

Five different EOs (T. vulgaris, C. martini, O. vulgare, C. zeylanicum, and C. citrus) were purchased from the Indian local market. These EOs were selected based on their potential application as food preservatives. The purified enterocin MSW5 was prepared from our lab to isolate E. faecalis MSW5.

Preparation of enterocin MSW5

In the present study, enterocin MSW5 was obtained from the activated lab culture of E. faecalis MSW5, after incubation for 24 h at 37℃ in TGME broth. The supernatant containing the enterocin MSW5 was collected after centrifugation for 20 min at 10,000 g. The obtained supernatant was then filter-sterilized through a 0.2 μm pore-size filter (Hi-Media). Then, crude enterocin MSW5 was purified through the cold acetone precipitation method followed by ion-exchange chromatography using SP-Sepharose fast flow as a cation exchanger (Sigma Aldrich Chemicals Pvt Ltd., India). Further, for confirmation of homogeneity and determination of the rough molecular mass of purified enterocin MSW5, Tricin SDSPAGE was carried out. The purified enterocin MSW5 gel band was cut and placed on a nutrient agar plate overloaded with S. aureus (107 CFU/ml) for its activity determination. The purified enterocin MSW5 was serially diluted to obtain concentrations of 5.80, 2.90, 1.45, 0.72, 0.36, 0.18, and 0.09 mg/ml which were further used in the assay for determining the fraction inhibitory concentration (FIC) using a checkerboard assay.

Determination of Minimal inhibitory concentration (MIC) of enterocin MSW5 and essential oils

The MIC values of enterocin MSW5 and EOs (T. vulgaris, C. martini, O. vulgare, C. zeylanicum, and Lemon grass) were determined against S. aureus ATCC 6538, Listeria monocytogenes ATCC 13932, and S. Typhimurium ATCC 6539 by broth microdilution method using 96-well microplates, as per the Clinical Laboratory Standards Institute (CLSI) guidelines 2020 [25]. The test was carried out using sterile 96-well microplates (Axiva Sichem Biotech, India), each well was filled with 100 μl of nutrient broth and 20 μl of bacterial suspension, to a final optical density was adjusted 1.0 as per the MacFarland index [26, 27]. Then, 100 μl of serially diluted enterocin MSW5 were added ranging from 5.8 to 0.09 μl/ml. Similarly, five EOs were serially diluted ranging from 10,000 to 9.7 ppm in DMSO (Himedia Laboratories, India). A sterile nutrient medium was used as a negative control and incubated for 24 h at 37℃. Positive control wells consisted of nutrient broth with indicator organisms without antimicrobial agents. All the plates were incubated for 24 h at 37℃. The next day, O.D. was measured at 595 nm using an ELISA plate reader (Thermo Fisher Scientific, India). The MIC was welldefined as the least concentration of antimicrobials that inhibited the visible growth of the indicator microorganisms [28].

Determination of the synergistic effect of essential oils and enterocin MSW5 using checkerboard assay

The combined effects of essential oils and enterocin MSW5 were calculated in terms of fractional inhibitor concentration index (FIC-Index) using a checkerboard assay in a 96-well microtiter plate [5, 29]. For that, 100 μl of nutrient broth media and 20 μl of indicator microorganisms with 1.0 O.D. were added to each and every well of the microtiter assay plate. Briefly, the method allows different concentrations of two antimicrobial agents (essential oils and enterocin MSW5) added in each well of the microtiter plate in two axes (x and y) of an 8 × 8 matrix. Plates contained 25 μl of an EOs (a) in columns which were serially two-fold diluted in a medium along the x-axis. Rows of the same plates contained 25 μl of enterocin MSW5 (b) which was diluted two-fold in the medium along the y-axis. Plates were then incubated for 24 h at 37℃ and the optical density was measured at 595 nm. All microtiter assays were carried out in triplicates. The FIC-Index is calculated by determining the value of the MIC of each antimicrobial agent alone and in combinations. Calculations were carried out as follows.

FIC A = (MIC of EOs in presence of enterocin MSW5)/

FIC B = (MIC of enterocin MSW5 in presence of EO/

(MIC of enterocin MSW5 individually

FIC Index = FIC A + FICB

Essential oils and enterocin MSW5 combinations result in decreased in the MIC compared with the MICs of individual EOs and enterocin MSW5. For interpretation of the results, FIC ≤ 0.5 is assigned as a synergistic effect, whereas 0.5 ≤ FIC ≤ 1 represented an additive or non-synergistic effect, 1 ≤ FIC ≤ 4 represented no interactive effect while greater than 4 indicate antagonistic effect between two agents [29].

Determination of the anti-biofilm activity of antimicrobials alone and in combination

The effects of five EOs, enterocin MSW5, and the combination between enterocin MSW5/EOs were tested against Listeria monocytogenes ATCC 13932, S. aureus ATCC 6538, and S. Typhimurium ATCC 6539 using microtiter plate assay, as previously described with certain modifications [5]. In a sterile 96-well microtiter plate, 100 μl of sterile Tryptone Soya Broth (TSB) medium was added with 10% of activated test organisms with O.D 1.0 in each separate plate. In each well 25 μl of enterocin MSW5, EOs, and their combinations were inoculated as per their MIC and sub-MIC value. The untreated well was considered as a positive control and the plates were incubated for 24 h at 37℃. Next day, the supernatant was removed followed by fixation of the biofilm of pathogens by adding 200 μl of methanol for 5 min, and the supernatant was drained. Then, 200 μl of 0.1% of crystal violet (CV) was added to each well of plate for 5 min, the excess dye was detached by washing the plates tierce time with sterile distilled water. The bound of crystal violet was released by adding 200 μl of 33% glacial acetic acid and optical density (O.D.) was measured using an ELISA microplate reader at 595 nm. Percent Biofilm eradication was calculated as per the given below formula,

Biofilm eradication % = (Initial OD − Test ODtimes100)/(Initial OD)

(Where, Initial OD: OD of positive control; Test OD: OD of treated wells with antimicrobials)

Statistical analysis

All the experimentations were carried out in triplicate. Data were further analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test using software GraphPad Prism 8.0. Differences between two mean values were considered significant at p ≤ 0.05.

Determination of the molecular mass of purified enterocin MSW5

The molecular mass of purified enterocin MSW5 was determined using SDS-PAGE. A single band of purified enterocin MSW5 was observed at approximately 6.5 kDa (Fig. 1). This band was cut, distained and its activity was determined against S. aureus. As shown in Fig. 2 clear zone of inhibition was observed around the gel band which indicates the antimicrobial activity of the purified sample.

Figure 1.SDS-PAGE analysis of the purified enterocin MSW5.

Figure 2.Direct antimicrobial activity assay of purified enterocin MSW5 band against S. aureus.

Determination of Minimal inhibitory concentration (MIC) of EOs and enterocin MSW5

The MIC of EOs and enterocin MSW5 was determined against S. aureus, S. Typhimurium, and L. monocytogenes using the broth dilution method. No test pathogens have shown resistance to the enterocin MSW5 and the EOs. Among all EOs, maximum activity was observed in case of C. zeylanicum against S. aureus (78.12 ± 0.04 ppm), S. Typhimurium (78.12 ± 0.08 ppm), and L. monocytogenes (39.00 ± 0.05 ppm) (Table 1). Whereas T. vulgaris and O. vulgare EOs have shown the same activity against the test organisms. The average efficacy of these both EOs was in the following order: S. aureus, L. monocytogenes (78.12 ± 0.02 ppm) > S. Typhimurium for T. vulgaris (312.5 ± 0.18 ppm) and O. vulgare (156.25 ± 0.05 ppm). The C. martini and C. citrus EOs were more effective against gram-negative bacteria (S. Typhimurium) at concentrations of 78.12 ± 0.00 ppm and 39.00 ± 0.02 ppm, respectively compared to gram-positive bacteria. However, S. aureus and L. monocytogenes was affected by C. martini and C. citrus EOs at a MIC of 156.25 ppm. Lastly, the MIC of enterocin MSW5 against bacteria was in the following order: S. aureus (362 ± 0.01 ppm) and S. Typhimurium (362 ± 0.05 mg/ml ppm) < L. monocytogenes (725 ± 0.08 ppm) (Table 1).

Table 1 . Minimum Inhibitory Concentration (MIC) of antimicrobials against indicator organisms.

AntimicrobialsMIC of antimicrobials
Staphylococcus aureus ATCC 6538Salmonella typhimurium ATCC 6539Listeria monocytogenes ATCC 13932
Enterocin MSW5362±0.01 ppm362±0.05 ppm724±0.08 ppm
Thymus vulgaris78.12±0.02 ppm312.5±0.18 ppm78.12±0.00 ppm
Origanum vulgare78.12±0.01 ppm156.25±0.05 ppm78.12±0.11 ppm
Cymbopogon martini156.25±0.12 ppm78.12±0.00 ppm156.25±0.04 ppm
Cymbopogon citrus156.25±0.08 ppm39.00±0.02 ppm156.25±0.03 ppm
Cinnamomum zeylanicum78.12±0.04 ppm78.12±0.08 ppm39.00±0.05 ppm


Determination of Fractional inhibitory concentration (FIC) of EOs and enterocin MSW5

The FIC index of enterocin MSW5 in combination with the EOs against S. aureus, S. Typhimurium, and L. monocytogenes is shown in Table 2. Results indicated that the combination of enterocin MSW5 and T. vulgaris EO displayed more synergism against L. monocytogenes (0.248 FICI) followed by S. aureus (0.372 FICI) and showed an additive effect against S. Typhimurium with 0.642 FIC-index. However, the combined enterocin MSW5 and O. vulgare EO have the same synergism activity as the combination of enterocin MSW5 with T. vulgaris against S. aureus (0.372 FICI), and L. monocytogenes (0.248 FICI). This combination also had a synergistic effect against S. Typhimurium with a 0.369 FICindex. The combination of enterocin MSW5 and C. martini EO caused an inhibitory effect against S. aureus (1.121 FICI) while synergism against S. Typhimurium (0.372 FICI) and L. monocytogenes (0.372 FICI). The FIC index of enterocin MSW5 in combination with C. citrus was the lowest (0.186), which indicates the best synergism among all combinations against L. monocytogenes. However, it has an additive effect (0.671 FICI) against S. aureus. In comparison, enterocin MSW5 and C. zeylanicum have shown potential synergism with the same FIC index (0.372) against all three pathogens. Hence, our results depict that enterocin MSW5 has shown synergism with all five EOs against the foodborne pathogen L. monocytogenes followed by S. Typhimurium and then S. aureus.



Determination of anti-biofilm activity of EOs and enterocin MSW5

All three indicator strains S. aureus, S. Typhimurium, and L. monocytogenes used in the study as they are good biofilm producing bacteria. Nevertheless, all EOs and enterocin MSW5 were found effective for the removal of their biofilms (Fig. 35). The anti-biofilm activity of individual EOs and enterocin MSW5 was comparatively less, whereas the association between EOs and enterocin MSW5 was assessed as synergistic in reducing the mature biofilm’s biomass. However, enterocin MSW5 has removed the maximum biofilm of S. aureus (53.41 ± 0.24%) followed by S. Typhimurium (37.69 ± 0.36%) and L. monocytogenes (36.67 ± 0.24%) at their minimum inhibitory concentration. While enterocin MSW5 in combination with O. vulgares has shown potential biofilm eradication of S. aureus (92.87 ± 0.06%) and S. Typhimurium (73.63 ± 0.23%) and in contrast, combination of enterocin MSW5 and C. citrus (87.84 ± 0.15%) has shown maximum biofilm eradication of L. monocytogenes at their MIC value. It was noted, that when all individual enterocin MSW5 and EOs were used at their sub-MIC value, they were not able to remove significant biofilm of indicator strains. However, their combinations can be able to remove significant biofilm at their sub-MIC value. The best anti-biofilm activity against S. aureus, S. Typhimurium, and L. monocytogenes was observed when enterocin MSW5 was used with the combination of O. vulgares (90.98 ± 0.15%), C. martini (68.58 ± 0.24%) and C. citrus (79.93 ± 0.14%), respectively at their Sub-MIC value.

Figure 3.S. aureus biofilm eradication by antimicrobials. (A) enterocin MSW5 and essential oils alone at their MIC and Sub-MIC values, (B) enterocin MSW5 and essential oils in combination at their MIC and Sub-MIC values.

Figure 4.S. Typhimurium biofilm eradication of by antimicrobials. (A) enterocin MSW5 and essential oils alone at their MIC and Sub-MIC values, (B) enterocin MSW5 and essential oils in combination at their MIC and Sub-MIC values.

Figure 5.L. monocytogenes biofilm eradication of by antimicrobials. (A) enterocin MSW5 and essential oils alone at their MIC and Sub-MIC values, (B) enterocin MSW5 and essential oils in combination at their MIC and Sub-MIC values.

The higher consumption of raw or minimally processed ready-to-eat (RTE) foods has increased the frequency of diseases caused by foodborne pathogens. Nowadays, the use of synthetic additives for food safety purposes is being questioned, due to their side effects, nitrites and nitrate are responsible for colon, leukemia, bladder, and stomach cancer, sorbate and sorbic acid are responsible for urticaria as well as contact dermatitis while benzoates responsible for causing allergies, and asthma [30]. As a result, customers demand natural preservatives to increase the shelf life of food items, especially in meals consumed without thermal pretreatments such as ready-to-eat seafood [31]. As one of the solutions to these problems, EOs and bacteriocins are employed as biopreservatives because of their proven antibacterial characteristics. However, when compared with in vitro systems, the fundamental issue with EOs is that larger doses are needed to ensure their antibacterial efficacy for food preservation. In these regards, their use in food industries may be limited because a higher amount of EOs leads to changes in the textural and organoleptic properties of food items [29]. In this respect, the use of EOs in combination with bacteriocin could be a significant natural alternative. Their use can help to decrease the use of chemical preservatives as well as the intensity of thermal treatments, resulting in more natural fresh food. Many of the bacteriocins are already available in the market, but as of now, only a few studies have been carried out on a compound obtained from Lactic Acid Bacteria (LAB), a bacteria isolated from fermented fruits, as they are well adapted to growth in the food matrix compared to other sources and therefore, they are capable to compete with food pathogens in a better way than the bacteriocin of LAB isolated from other sources. On the other side, the use of essential oils, as food preservatives in food items has been already approved by the Food and Drug Administration (2001) [32]. Regarding the susceptibility of pathogens to these natural antimicrobial substances, pathogens should not develop resistance toward essential oils, which might be due to the presence of more complexity and different modes of action of active compounds present in EOs. However, it has been shown that some gram-positive bacteria are resistant to bacteriocins, which are primarily active against nisin. As a result, class II enterocin can be used as a major substitute for nisin or other lantibiotics. Additionally, some studies have indicated that gram-positive bacteria's resistance to bacteriocins may be overcome by combining them with other antimicrobial agents like essential oils and antibiotics, or by employing a variety of bacteriocins. [33]. Therefore, in the present work, we have tried essential oils in combination with enterocin MSW5 which may act synergistically to inhibit antibacterial resistance due to their capability to affect many of the target sites and perform various physicochemical interactions.

In this work, the antimicrobial activity was assayed for enterocin MSW5 and EOs (T. vulgaris, O. vulgare, C. zeylanicum, C. martini, and C. citrus) against S. aureus, S. Typhimurium, and L. monocytogenes. Among them, gram-positive organisms S. aureus and L. monocytogenes were more susceptible to T. vulgaris, O. vulgare, and C. zeylanicum while S. Typhimurium was more susceptible to C. martini and C. citrus. This variation of susceptibility to bacterial strains demonstrates that it might be due to the presence of major components being different in each EOs [17]. The main components in the structure of Origanum vulgare are carvacrol-an and thymol [34], in Thymus vulgaris, it is thymol [35], in C. zeylanicum it contains Cinnamaldehyde and eugenol [36], in C. martini, it contains Monoterpenes [37] and in C. citrus, it contains citral [38]. So, different components are present in different Eos which give different modes of action. T. vulgaris inhibits the cytoplasmic membrane of bacteria and allows the leakage of cellular materials like nucleic acids, and increased potassium permeability [35]. In the case of O. vulgare, it may inhibit efflux pumps, ATP depletion, biofilm eradication, and damage to the cytoplasmic membrane [34]. Further, C. zeylanicum also has a similar kind of mode of action against pathogens such as inhibiting cell membranes, changing the lipid profile of cells; inhibiting ATPase enzymes, stopping cell division, and anti-quorum sensing activity [36]. The C. martini EO has also shown broad-spectrum inhibitory activity with different modes of action as they contain complex mixtures of compounds in their structures such as monoterpene hydrocarbons, ketones, sesquiterpene, aldehydes, alcohols, and other chemical families like oxides, fatty acids, and sulfur derivatives. This lipophilic nature of C. martini leads to increased cell membrane permeability and leakage of cytoplasmic constitutes which can result in various alterations to pathogens via cell wall and cell membrane disturbance, inhibition of protein/peptide synthesis, DNA damage, pH disturbance, and inhibition of cell-cell communication [37]. In case of antibacterial activity of C. citratus against pathogenic bacteria including S. aureus and S. enterica might be due to the presence of oxygenated monoterpenoids geranial (α-citral) and neral (β-citral) which are responsible for altering intracellular materials of cells, like small ions, nucleic acids, and proteins [38]. As shown in the results, T. vulgaris and C. zeylanicum oils have a significant inhibitory effect. The presence of potential inhibitory activity in thyme might be due to the presence of components such as thymol and carvacrol in EO which mainly target cell membranes to make them permeable [18]. Many researchers have found that EOs are more active against gram-positive bacteria rather than gram-negative bacteria [19]. The hydrophobic components present in EOs can usually diffuse across lipid bilayers while in the case of hydrophilic agents; they are mostly passed passively through channels present in the cell membranes. The main reason for less susceptibility of antimicrobials towards gram-negative bacteria is lipopolysaccharide presence in the outer membrane which might create hindrance in the penetration of hydrophobic antimicrobial agents; therefore, more concentration of antimicrobials is needed for gramnegative bacteria rather than gram-positive bacteria [39]. However, certain exceptions of essential oils have shown significant antimicrobial activity against gramnegative bacteria. For example, in our study, C. martini and C. citrus have shown significant inhibitory activity against S. Typhimurium. This inhibitory activity might be due to the presence of monoterpene constituents in Cymbopogon essential oils like citral, limonene, elemol, 1,8 cineole, citronellal, citronellol, linalool, geraniol, methylheptenone, b-carophyllene, geranyl formate, and geranyl acetic acid derivation [40, 41]. On the other hand, enterocin MSW5 showed the same MIC for S. aureus and S. Typhimurium (0.362 ± 0.05 mg/ml) while slightly higher MIC for L. monocytogens (0.725 ± 0.08 mg/ml). Similar results were also reported for Enterocin TJUQ1 where MIC of enterocin TJUQI was higher for L. monocytogenes than for S. aureus [42]. But in our study enterocin MSW5 has also given antimicrobial activity against S. Typhimurium which indicates that it has broad-spectrum inhibitory activity. In contrast, enterocin AS-48 has shown ten times less antimicrobial activity towards gram-negative bacteria than towards grampositive bacteria [43].

Further, enterocin MSW5 and essential oils were utilized in combination against variable test organisms (S. aureus, S. Typhimurium, and L. monocytogenes). In the case of L. monocytogenes, enterocin MSW5 and all essential oils have shown synergism (FICI ≤ 0.5), in the case of S. Typhimurium, all combinations showed synergistic effects except a combination of enterocin MSW5/T. vulgaris which had an additive effect. In certain cases, no interactions were observed like a combination of enterocin MSW5 and C. martini, whereas additive effects were found for a combination of enterocin MSW5 and C. citrus against S. aureus. Similarly, combinations of enterocin MSW5 with T. vulgaris, O. vulgare, and C. zeylanicum had shown synergist effects against S. aureus. These results demonstrate that enterocin and EOs have worked together synergistically. A similar kind of observation has been reported by another group of scientists where essential oils (thyme red, thyme verbena, Spanish oregano, tea tree, ajowan, clove, and sage oils) alone had very less antimicrobial activity compared utilized with enterocin AS-48, its efficacy against L. monocytogenes was improved [44]. Likewise, enterocin A combined with Thyme EOs has shown a synergistic effect against L. monocytogenes EGDe because the MIC value of enterocin A increased five-fold from 4.57 to 0.9 μg/ml [45]. Similar research was carried out to form an edible coating, in which EOs (Salvia officinalis, Mentha piperita, Citrus limon, and Thymus vulgaris) were used in combination with bacteriocin bacLP17. Their FIC index was less than 0.5 which indicates good synergism against L. monocytogenes [46]. In the case of bacteriocins (LBB08 and LBC02) produced by E.faecium utilized with Thyme had shown an additive effect against L. monocytogenes and E. coli O157: H7. But, when utilized in combination with Oregano EO, no interaction was observed against E. coli O157: H7 and an additive effect was observed against L. monocytogenes [47]. Such kind of combinatorial study is also reported for other bacteriocins like pediocin and nisin when pediocin is applied in combination with O. vulgare, an additive effect was observed against L. monocytogenes while an antagonistic effect was observed against B. cereus, E. coli O157: H7, and L. sakei [28]. Further, when pediocin is used in combination with T. vulgaris inhibitory effect was found against E. coli O157: H7, B. cereus, L. sakei, P. putida, L. monocytogenes, S. Typhimurium, and S. aureus. Similarly, in the case of nisin, the inhibitory effect was observed against L. sakei, E. coli O157: H7, and S.aureus, while the additive effect was observed against B. cereus, and L. monocytogenes, the antagonistic effect was observed against P. putida, and synergistic effect was showed against S. Typhimurium [28]. So, in contrast to these well-reported observations, in our study, we received significant results with enterocin MSW5, its combination with O. vulgare EO had shown a significant synergistic effect against S. aureus (FICI:0.372), S. Typhimurium (FICI:0.369), and L. monocytogenes (FICI:0.278). Similarly, its combination with T. vulgaris had also shown significant synergism against S. aureus (FICI:0.372), and L. monocytogenes (FICI:0.248). Therefore, our results depict that the combination of enterocin MSW5 and EOs in the majority of cases showed synergism and their combination is effective against grampositive and gram-negative bacteria. In this regard, the combinations of enterocin MSW5 with different EOs could be a more effective natural alternative to eradicate pathogens in food items. The combination of essential oils and bacteriocins might create pores in the cell membrane, and that is responsible for altering the permeability of the cell membrane, the efflux of amino acid, the proton motive force, and the pH gradient of bacterial cells [28].

Additionally, a combination of enterocin MSW5 and EOs was analyzed for antibiofilm activity against S. aureus, S. Typhimurium, and L. monocytogenes. But biofilm is inherently more resistant to antimicrobial agents than those present in a planktonic state. Antimicrobials are required in more amounts for the removal of the complex polymeric matrix of biofilm because this matrix often creates problems in the penetration of the antimicrobial agent to the lowermost layer of the biofilm. Hence, it is mandatory to search for effective antimicrobial combinations that help to eradicate biofilm at their lower concentration. In our study as one of the solutions, a combination of EOs and enterocin MSW5 has shown significant anti-biofilm activity at their MIC and sub- MIC levels rather than being used individually. EOs have the capability of biofilm removal by the inhibition of exopolysaccharide synthesis (cellulose) and cell-to-cell signaling via decreased gene expression [36]. Bacteriocins can inhibit biofilm formation at different stages as they inhibit the adherence of bacterial cells with other cells or host surfaces at an early stage of biofilm formation. Also, they can kill the cells or detach them from already established biofilm. They generally disturb cell membrane integrity by creating pores or electrostatic force in membrane cells. Other than this, quorum sensing or other regulatory signals interfere with motility by downregulation of their genes and also interfere in matrix synthesis [48]. Similarly to our study, maximum anti-biofilm activity was found with the combination of bacLP17 and S. officinalis and bacLP17 and T. vulgaris both concerning the control and when compared to the single EOs [5]. Similarly, the nisin and linalool combination has shown antibiofilm activity against B. cereus (79.36 ± 3.78%) and S. Typhimurium (65.84 ± 5.46%) biofilms, respectively. In the case of nisin and p-coumaric acid, the combination has shown antibiofilm activity against B. cereus (91.58 ± 7.24%) and S. Typhimurium (87.4 ± 7.5%) respectively.

In conclusion, our results proved that the use of enterocin MSW5 produced by E. faecalis in combination with five different Eos had shown significant synergistic or additive effects on foodborne pathogens such as S. aureus, S. Typhimurium, and L. monocytogenes. Additionally, enterocin MSW5 and EOs have shown potential antibiofilm activity against all three pathogens when used alone or in combination with their MIC and Sub- MIC. Therefore, the combined use of enterocin and EOs serves both the purposes, of controlling food-borne pathogens in food products and minimizing undesirable organoleptic impact due to the high concentration of antimicrobials. Further, these combinations are also used as antibiofilm agents which would help to reduce economic losses in food industries, due to problems of food safety, spoilage of products, and loss of production efficiency by the formation of biofilms.

All authors have equally contributed to this manuscript. Material preparation, data collection, and analysis were performed by MS. SG analyzed these data, and necessary inputs were given for the design of the manuscript. The first draft of the manuscript was written by MS and SG commented on previous versions of the manuscript. Both authors read and approved the final manuscript.

The authors have no financial conflicts of interest to declare.

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