Anti-biofilm forming and anti-quorum sensing activity of selected essential oils and their main components on food-related micro-organisms

Authors


Correspondence

Erika-Beáta Kerekes, Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Közép fasor 52., Hungary.

E-mail: kerekeserika88@gmail.com

Abstract

Aims

The aim of this study was to investigate the effect of clary sage, juniper, lemon and marjoram essential oils (EOs) and their major components on the formation of bacterial and yeast biofilms and on the inhibition of AHL-mediated quorum sensing (QS).

Methods and Results

Biofilm formation was measured by crystal violet and resazurin staining, and QS inhibition was detected by paper disc diffusion assay.

Marjoram EO inhibited Bacillus cereus, Pichia anomala, Pseudomonas putida and mixed-culture biofilm formation of Ps. putida and Escherichia coli and showed the best QS inhibitor effect on Chromobacterium violaceum.

For B. cereus, all components showed better antibiofilm capacity than the parent EOs. Lemon EO inhibited E. coli and mixed-culture biofilms, and cinnamon was effective against the mixed forms. Scanning electron microscopy showed the loss of three-dimensional structures of biofilms.

Conclusions

The EOs and components used seem to be good candidates for prevention of biofilm formation and inhibition of the AHL-mediated QS mechanism.

Significance and Impact of the Study

Biofilm formation on foods and food industrial equipment is a serious problem causing food spoilage and emergence of foodborne diseases. This article highlights the importance of studying EOs as potential disinfectants and food preservatives.

Introduction

The continuously expanding market and the reluctance of consumers towards artificial food components and sanitization products motivated researchers to start testing natural, more eco-friendly materials for their antimicrobial effect. Contaminated foods are a substantial problem in health care, with severe acute diseases often resulting from food poisoning. Conventional preservatives can often produce unpleasant by-products, so to find efficient natural antimicrobial agents to at least partially replace them has become necessary (Burt 2004).

It is of high relevance that the majority of these organisms are capable of forming biofilms. Costerton et al. (1999) determine biofilms as a functional consortium of micro-organisms attached to a surface and embedded in a hydrated polymeric matrix of their own synthesis. Bacteria develop these three-dimensional structures for the same reason as other organisms: for protection from predators and other negative factors, for better availability of nutrients and for increased genetic diversity (Johnson 2007). Bacteria growing as biofilms are tough to remove from surfaces and are highly resistant to common antibacterial agents (Høiby et al. 2010). They can contain both spoilage and pathogenic microflora, and they will be formed easily on food surfaces and in processing environment, representing a considerable problem of postprocessing contamination and cross-contamination (Kumar and Anand 1998). Outbreaks of serious diseases caused by Salmonella sp. and Escherichia coli O157H7 were associated with biofilms formed on cantaloupe, lettuce and spinach (Annous et al. 2009). Several mechanisms can account for the increased antibiotic resistance in biofilms including the physical barrier formed by extracellular polymeric substance (EPS) (Costerton et al. 1999; Lynch and Robertson 2008), the slow-growing, dormant bacteria in the interior of these structures that are inert towards antibiotics (Costerton et al. 1999; Mah and O'Toole 2001) and resistance genes that are uniquely expressed in biofilms (Mah et al. 2003; Nguyen et al. 2011). These bacterial features create resistance to antimicrobial agents and drive the need for novel strategies that will eradicate bacterial biofilms. Pathogenic yeasts such as Candida albicans can also adhere to surfaces, for example medical devices, and form drug-resistant biofilms (Hawser and Douglas 1994).

The biofilm-forming bacterial cells are able to communicate by the density-dependent cell-to-cell communication mechanism which is called quorum sensing (QS). This plays an important role in biofilm development, resistance, virulence and the production of EPS. Researchers believe that by controlling QS mechanism and biofilm formation, the resistance of these structures could be influenced (Wang et al. 2007). Anti-QS agents are of interest as these affect only the virulence factors but not the growth of the pathogens, so the development of resistance is much less likely (Bakkiyaraj et al. 2012). It is worth noting that this is dependent on the concentration of the substance used. Gram et al. (2002) reported the widespread occurrence of N-acyl homoserine lactones (AHLs) in fish products, poultry and vacuum-packed meat. AHL production is a widespread phenomenon in food-spoiling bacteria, and QS antagonist might be useful to inhibit growth or biofilm formation of food spoilage bacteria (Annous et al. 2009).

The antiseptic qualities of aromatic and medicinal plants and their extracts have been recognized since antiquity. Essential oils (EOs) are volatile liquids obtained from herbs, spices and different plants mainly by steam distillation. They can have more than 50 components in different ratios (Bakkali et al. 2008).

There seems to be a growing interest in using EOs by the food industry as natural preservatives against food spoilage and foodborne pathogenic microbes (Burt 2004).

Several studies have reported inhibitory effects of several oils and their components on human pathogens (Ahmad and Beg 2001; Bagamboula et al. 2004), foodborne microbes and food spoilage pathogens (Niu and Gilbert 2004; Rahman and Kang 2009; Angienda et al. 2010). EOs can be used as disinfectant agents in washing solutions for leafy vegetables and might be suitable alternatives to chlorine (Gündüz et al. 2010; Karagözlü et al. 2011). EOs containing edible coatings on foods of plant or animal origin have been proved to be a good solution against food spoilage and pathogenic microbes (Min and Oh 2009; Gómez-Estaca et al. 2010; Vu et al. 2011). The combined treatment of two Cymbopogon sp. EOs against Listeria monocytogenes biofilms on stainless steel eliminated 100% of the surface-adhered cells in a 240 h old biofilm showing an excellent disinfection effect (de Oliveira et al. 2010).

EOs in most cases confer antimicrobial activity by damaging the cell wall and membrane, leading to cell lysis and leakage of cell contents (Burt 2004).

Furthermore, many EOs are relatively easy to obtain, have low mammalian toxicity and degrade quickly in water and soil, so they are relatively environmentally friendly (Isman 2000).

According to these facts, it is assumed that these oils can help prevent the formation of biofilms and could be used in sanitization and food preservation. In some cases, EOs and their components resulted to be good inhibitors of biofilm formation (Hendry et al. 2009) or even better than some antibiotics against these structures (Kavanaugh and Ribbeck 2012).

Even so, their use in foods as preservatives is often limited due to flavour considerations, as effective antimicrobial doses may exceed organoleptically acceptable levels (Lambert et al. 2001).

In the present study, we investigated the effect of clary sage, juniper, lemon and marjoram EOs and their major components (α-pinene, limonene, linalool and terpinene-4-ol) on the formation of bacterial and yeast biofilms and on N-acylhomoserine lactone (AHL)-derived bacterial QS.

Materials and methods

Bacterial strains

All the strains used were from the Szeged Microbiological Collection (SZMC; WDCM 987) maintained by the Department of Microbiology of the University of Szeged, Hungary. One Gram-positive (Bacillus cereus var. mycoides 0042) and two Gram-negative (E. coli 0582, Pseudomonas putida 291T) bacteria and a yeast (Pichia anomala 8061Mo) strain were used to test the inhibition of biofilm formation. The model organism Chromobacterium violaceum 6269 was used for the anti-QS tests. B. cereus and S. marcescens were grown in supplemented meat medium (MEE; 4 g beef extract, 4 g peptone (OXOID, United Kingdom), 10 g glucose (BDH PROLAB, Belgium), 1 g yeast extract for 1000 ml), Ps. putida on TGE broth (TGE; 10 g glucose, 5 g peptone, 2·5 g yeast extract (Alfa Aesar GmbH and Co KG, Germany), for 1000 ml), E. coli on LB medium (LB; 10 g peptone from casein, 10 g NaCl, 5 g yeast extract, for 1000 ml) and the yeast in malt extract medium (MEA; 50 ml malt extract, 5 g yeast extract, 5 g glucose for 1000 ml). All ingredients were purchased from Merck, Hungary. B. cereus and P. anomala were incubated at 30°C, E. coli at 37°C and Ps. putida at 25°C. Selective media for quantifying bacteria present in the mixed culture were Chromocult for E. coli and Cetrimide agar for Pseudomonas putida, both obtained from Merck, Hungary.

For C. violaceum, a special medium was used with the following composition: 1 g of yeast extract, 10 g of NaCl, 1 g of K2HPO4, 0·3 g of MgSO4 + 7H2O, 36 mg of Na-EDTA, 10 mg of ammonium iron (III) citrate and 10 g of triptone for 1000 ml. All ingredients were purchased from Sigma-Aldrich, Hungary. Cultures were grown at room temperature (22–25°C).

Essential oils

The EOs of clary sage (Salvia sclarea), juniper (Juniperus communis), lemon (Citrus lemon) and marjoram (Origanum majorana) and their main components α-pinene, limonene, linalool and terpinene-4-ol were tested for their inhibitory effect on biofilm formation. The EOs were purchased from Aromax Natural Products Zrt. (Budapest, Hungary) and the components from Sigma-Aldrich (Hungary). Selection of the EOs was based on the lack of phenolic compounds and effectiveness showed in previous studies. Components of the used EOs were determined by the producer (Aromax Zrt) using GC-MS analysis, and the main components except for clary sage were chosen for further investigations (Tserennadmid et al. 2011). Cinnamon EO was used to inhibit mixed biofilm formation.

Determination of MIC values

The dilution of the EOs and their components was made in liquid culture media in combination with Tween 40 (1%). Concentration of EOs and their components ranged from 0·5 up to 16 μl ml−1. Hundred microlitres of cell suspension (105 CFU ml−1) in liquid culture medium was added to the wells, followed by 100 μl of the diluted EO or the component. Positive controls contained the inoculated growth medium without any EOs or components, and negative controls contained EOs or components in sterile medium. After 24 h incubation at corresponding temperatures, absorbance was measured at 600 nm (ASYS Jupiter HD microplate reader). Decreases in the absorbance of lower than 10% of the positive control samples were considered the MIC values. Measurements were made in triplicates.

Biofilm formation and treatment

Ninety-six-well microtitre plates were inoculated with 200 μl of 24 h old liquid culture containing approx. 108 CFU ml−1. Following 4 h of cell adhesion at corresponding temperatures, the supernatant was removed from each well, and the plates were rinsed with physiological saline. Subsequently, 200 μl of fresh medium containing the EO or the component to be examined was added in MIC/2 concentration (μl/ml) to each well, and the plates were further incubated for 24 h. Positive controls contained only the inoculated growth medium and negative controls contained EOs or components in growth medium (Peeters et al. 2008). Experiments were repeated at least two times, and six parallel measurements were made each time.

Biofilm formation of mixed cultures

Escherichia coli and Ps. putida liquid cultures containing approx. 108 CFU ml−1 were mixed in 1 : 1 ratio in TGE broth. The cultures were incubated at 30°C. (This was the maximum temperature on which Ps. putida could grow.) The biofilm formation procedure was as described above. In this experiment, lemon, marjoram and cinnamon oils were used in different concentrations. To find out which species dominate the biofilm under normal and EOs-treated conditions, we investigated the cell number of 24-h-old pure culture of E. coli, Ps. putida and mixed-culture biofilm using TGE agar and selective media. The biofilm-forming capacity of E. coli was tested in monoculture at 30°C as well.

Crystal violet staining

Inhibition of biofilm formation by bacteria was detected by the crystal violet staining method. After 24 h treatment, the supernatant was removed, and the wells were rinsed with physiological saline. For fixation of the biofilms, methanol was added, and the supernatant was removed again. Then, 0·1% crystal violet (CV) solution was added to all wells, and after 20 minutes, the excess dye was removed by washing the plates under running tap water. Finally, bound crystal violet was released by adding 33% acetic acid. The absorbance was measured at 590 nm (Peeters et al. 2008).

Crystal violet staining was performed also on yeast biofilms. As, however, it gave no statistically significant results, it was not used for the evaluation of biofilm formation.

Staining with CellTiter-Blue Cell Viability assay

In case of food spoilage by yeast P. anomala, the inhibition of biofilm formation was measured by CellTiter-Blue Cell Viability assay. This provides a reproducible fluorometric method for estimating the number of viable cells present in multiwell plates. It uses the indicator dye resazurin to measure the metabolic capacity of cells – an indicator of cell viability. Viable cells retain the ability to reduce resazurin into resorufin, which is highly fluorescent. Nonviable cells rapidly lose metabolic capacity, do not reduce the indicator dye and thus do not generate a fluorescent signal. After 24 h treatment, the supernatant was removed, and the wells were rinsed with physiological saline. Hundred microlitres of physiological saline and 20 μl of resazurin were added to each well. After 1 h incubation at 30°C, fluorescence was measured at 590 nm with a fluorescence plate reader (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany).

Scanning Electron Microscopy (SEM)

Scanning electron microscopy was used to investigate the structural modifications of biofilms after treatment with EOs. For biofilm formation, 5 ml of overnight P. anomala culture and a mixed culture of Ps. putida and E. coli were added to 6-well macrotitre plates. Sterile coverslips were placed in the wells and served as the attaching surface for the cells. The plates were incubated for 4 h at 30°C, then the supernatant was removed, and plates were rinsed with physiological saline. For treatment of P. anomala biofilms, 5 ml of juniper or α-pinene solution of MIC/2 concentration was added, and a third well was used as the untreated control. The mixed cultures of Ps. putida and E. coli were treated with lemon and cinnamon oils in the same concentration. Control samples contained only liquid culture media. The macrotitre plates were incubated for 24 and 48 h. After incubation, the supernatant was removed, and the wells were washed with physiological saline. The preparation of the samples for electron microscopy was performed as follows: soaking of the sample with 2·5% glutaraldehyde in 0·05 mol l−1 cacodylate buffer (pH = 7·5), for 2 h at room temperature, followed by dehydration using different ethanol concentrations: 50, 70, 80, 90, 95 and 98%. Each ethanol treatment lasted for 2 × 15 min at room temperature. The next step was dehydration with t-butyl–100% ethanol solution in 1 : 2, 1 : 1 and 2 : 1 ratios. For each ratio, the dehydration lasted for 1 h at room temperature, then dehydrated with absolute t-butyl alcohol for 2 × 1 h at room temperature; and changed to new t-butyl. The sample was stored at 4°C for 1 h and freeze–dried overnight. The sample was coated with a gold membrane and observed with Hitachi S4700 scanning electron microscope.

Detection of QS inhibition

The synthesis of violacein in Cviolaceum is under QS regulation, and this strain is an AHL (N-acylhomoserine lactone) biosensor. The literature describes this bacterium as a good model organism for screening of compounds that inhibit AHL-mediated QS inhibition (Adonizio et al. 2006; Szabó et al. 2010). The culture was reactivated from glycerol stock culture kept at −80°C. It was cultured in Petri plates on a specific C. violaceum broth (see 2·1) for 24–48 h at room temperature. Sterile paper discs (6–8 mm diameter) were saturated with the investigated EOs, and their main components (1, 2 and 3 μl per disc) and were placed in the centre of Petri plates initially spread with C. violaceum (10CFU ml−1). Quorum sensing inhibition was determined by measuring the colourless halo developed behind the inhibition zone around the paper discs. Paper discs saturated with distilled water served as a negative control. Growth inhibition zones have also been measured in both cases.

Statistical analysis

Statistical analysis was conducted using a one-way anova followed by Tukey's HSD testing (R Works 2.8.0, The R Foundation for Statistical Computing, Vienna, Austria). Significance was considered at P < 0·05.

Results

Effect of EOs on biofilm formation

The MIC values obtained for each EO and their component are presented in Table 1. In most cases, components had major MIC values than the EOs, indicating the role of minor components in the growth-reducing effect of parent EOs.

Table 1. The MIC values (μl ml−1) of essential oils and components
EOs/components Bacilius cereus Escherichia coli Pichia anomala Pseudomonas putida
  1. –, the effect of the component was not tested.

Clary sage0·521>30
Juniper120·5>30
Lemon 140·7525
Marjoram0·250·50·52
α-Pinene611
Limonene461
Linalool631
Terpinen-4-ol6214

Only marjoram EO showed significant reduction in B. cereus biofilm formation (Fig. 1).

Figure 1.

Effect of essential oils and components in MIC/2 concentration on the biofilm formation of Bacilius cereus. Biofilms were stained with crystal violet. Different letters represent significant changes.

The majority of the EOs and components had inhibitory effect on biofilm formation of E. coli. Marjoram did not differ significantly from the control sample. Besides limonene, all the components proved to be better inhibitors than the parent oils (Fig. 2a).

Figure 2.

Effect of essential oils and components in MIC/2 concentration on the biofilm formation of Escherichia coli (a) and Pseudomonas putida (b). Biofilms were stained with crystal violet. Different letters represent significant changes.

Regarding Ps. putida biofilms, the MIC values for clary sage, juniper and lemon were too high, so no further analysis was performed with these EOs. On the other hand, marjoram and its component had inhibitory effect on the formation of these structures (Fig. 2b).

Pichia anomala biofilm formation was significantly inhibited by all the oils, α-pinene and terpinene-4-ol as well. We did not measure any reduction in the case of the other two components tested. In contrast with bacterial biofilms, the yeast biofilms were more susceptible to the EOs than to components (Fig. 3). Tween 40 used for better disperse of the EOs did not have any effect on the growth of the microbes in the used concentration (0·5% in the medium).

Figure 3.

Effect of essential oils and components in MIC/2 concentration on the biofilm formation of Pichia anomala. Biofilms were stained with resazurin. Different letters represent significant changes.

Effect of EOs on mixed-culture biofilms

Lemon oil inhibited biofilm formation, but the inhibition was not concentration dependent. Cinnamon and marjoram oils had a strong inhibitory effect on the formation of mixed-culture biofilms; the lowest concentrations tested already inhibited the formation of these structures (Fig. 4).

Figure 4.

Effect of essential oils in different concentrations (μl ml−1) on the biofilm formation of mixed-culture Escherichia coli and Pseudomonas putida. Biofilms were stained with crystal violet.

Before mixed-culture experiments, the biofilm-forming capacity of E. coli at 30°C was tested. Biofilm formation was reduced by 14% compared with biofilm at 37°C.

In the 24 h old control of mixed-culture biofilms, E. coli overgrew Ps. putida in 99 : 1 ratio. Treatment with lemon EO resulted in an inverse picture: E. coli became more sensitive, the cell number was reduced by 2 logs, but no change could be seen in P. putida cell number. Marjoram inhibited E. coli and Ps. putida cells as well; the cell number was reduced by 2 logs in both cases. No bacterial colonies could be seen after treatment with cinnamon EO (data not shown).

Investigation of biofilm structure with SEM

In case of P. anomala, the images of the control samples captured the characteristic morphological elements of a mature, three-dimensional biofilm (Fig. 5 a,b). Juniper and α-pinene had an inhibitory effect on the formation of biofilms, the cells attached to the surface, but they did not form biofilm-specific structures (Fig. 5 c,d).

Figure 5.

Scanning electron microscopic images of Pichia anomala biofilms: 24 h (a) and 48 h (b)-old control samples and treated with juniper oil (c) and α-pinene (d). Images of Escherichia coli and Pseudomonas putida mixed biofilms: 24 h old control samples (e, f) and treated samples with lemon (g) and cinnamon oil (h). Essential oils and components were used in MIC/2 concentration.

The control sample of the mixed cultures showed the formation of microcolonies, which is the next step in biofilm formation after attachment (Fig. 5 e,f). Treatment with lemon oil had little inhibitory effect on the mixed cultures in contrast with cinnamon oil, which significantly inhibited the formation of these structures (Fig. 5 g,h).

QS inhibition

The results regarding QS and growth inhibition are shown in Table 2. Lemon and limonene oils had no or minimal inhibitory effect on the production of violacein of C. violaceum. Juniper and α-pinene and also clary sage and linalool had a quantity-dependent effect, inhibiting production at 3 μl. The amount of α-pinene used did not influence the halo diameter. Marjoram and terpinene-4-ol had the best anti-QS effect at 2 μl. (Fig. S1). In all cases, the oils proved to be better QS inhibitors than their major components.

Table 2. Growth inhibition zones and colourless halo diameters of the anti-quorum sensing (QS) effect of investigated essential oils (EOs) and components (mm)
EOs/componentsGrowth inhibition halo (mm)Anti-QS halo (mm)
1 μl2 μl3 μl1 μl2 μl3 μl
Clary sage444102025
Linalool553101520
Lemon 122·500·51
Limonene476·5000
Marjoram133101520
Terpinen-4-ol12201520
Juniper23301020
α-Pinene555111

Discussion

The increasing resistance of micro-organisms towards antibiotics and products of sanitization encourage researchers to look for new, natural antimicrobial agents that are effective but do not lead to the development of resistant strains. The ability of micro-organisms to develop biofilms makes them even harder to eradicate as these structures are more resistant than the planktonic forms (Lewis 2001). The present study focuses on the antibiofilm-forming and anti-QS effect of some essential oils and their major components.

In the case of the Gram-positive bacterium, B. cereus marjoram inhibited biofilm formation, and all the components used had better inhibitory effect than the parent EOs. It has been reported that EOs containing phenolic compounds such as carvacrol, eugenol or thymol have the strongest antimicrobial activity (Burt 2004). The EOs used in these experiments have terpenoids as main constituents: cyclic terpenes and terpene alcohols. It seems that in our case, monoterpenes (α-pinene and limonene) inhibited biofilm formation in a higher degree than terpene alcohols (terpinene-4-ol and linalool). The main target of these components is the cell wall and cytoplasmic membrane or proteins embedded in the membrane (Dorman and Deans 2000). Cell membrane lost its integrity, and leakage of cell compounds can lead to death of the cell. It is also possible that the damaged wall lost its ability to attach to the surface and form biofilms (Burt 2004).

Gram-negative bacteria are known to be more resistant to essential oils than the Gram-positive ones (Burt 2004). Pseudomonas putida resulted to have a very high MIC value for lemon, juniper and clary sage EO and their components. This means that the use of these oils in food preservation would be difficult because they may exceed organoleptically acceptable levels. For this reason, their antibiofilm effect was not tested. On the other hand, marjoram and its component significantly inhibited the formation of Ps. putida biofilms. This was not the case with E. coli biofilms, and they resulted to be less susceptible to treatment with marjoram, but the other oils and components inhibited the formation process. In most environments, bacteria form mixed-culture biofilms (Hall-Stoodley et al. 2004). Mixing E. coli and Ps. putida, we obtained a biofilm in which, based on the plating on selective media, E. coli overgrew Ps. putida in the control samples, but showed higher susceptibility to lemon EO treatment. Marjoram and cinnamon had an inhibitory effect on both strains. These findings suggest that EOs might be able to act against the cell wall of Gram-negative strains. They can also act against mixed forms, which occur most commonly in the environment.

Previous studies have shown that the antimicrobial effect of essential oils is due to the interaction between all the components present and not due to an individual component (Lis-Balchin and Deans 1997; Mourey and Canillac 2002). In this study, the use of a single component to treat a biofilm resulted to be sufficient in some cases (B. cereus and E. coli) to inhibit biofilm formation. This finding is in contrast with the study of Sandasi et al. (2008) where Listeria monocytogenes biofilm growth was enhanced by α-pinene, limonene and linalool components.

For P. anomala biofilms, marjoram was the best inhibitor, and the other oils and components did not have this effect. Marjoram EO showed also a strong anti-QS effect on C. violaceum, the indicator strain for AHL-mediated QS. In case of Ps. putida, the AHL system is responsible for the QS mechanism, which has been implicated in the regulation of biofilm formation (Bertani and Venturi 2004). We are going to confirm in further experiments whether there is a link between the anti-QS effect of marjoram EO and the reduction in Ps. putida biofilm formation.

We can conclude that marjoram EO was one of the best inhibitor of bacterial and yeast biofilm formation and AHL-mediated QS in the indicator strain. Surprisingly, some of the EOs did not have inhibitory effect on biofilm formation of Gram-positive bacteria, but their main components significantly inhibited the process. Gram-negative bacterial biofilms were inhibited by the EOs, but the components were more effective, suggesting that only the main components could be enough for sanitization processes. Yeast biofilms were resistant to components but showed a high susceptibility for EOs. These results showed that the individual susceptibility of microbes is very different and plays a crucial role in the effectiveness of EOs and EO components.

There is an ongoing need for foodborne illness investigation and prevention but also for the use of natural sanitizing products. Our results revealed that the investigated EOs and their main components may be suitable for food-preserving industrial methods, so further analyses are needed referring to their applicability in the food industry.

Acknowledgements

This work was supported by a Bilateral S&T Cooperation Project (MN-1/2009) and a personal grant of Institute Balassi for E.B. Kerekes (2011 and 2012).

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