Antibacterial and anti-biofilm activity of cinnamon essential oil and eugenol

Biofilms are responsible for most of the interference caused by microorganisms in food processing. The aim of this study was to evaluate the cinnamon (Cinnamomum zeylanicum) essential oil and eugenol sanitizer and anti-biofilm activity against biofilms. Concentrations used of essential oil were 0.0% (control) 0.12%; 0.48%; 0.96% and 1.92%; the amount of eugenol was 0.76%. Concentrations were determined from other published studies. Number of viable cells and quantification the bacterial biomass were determined. Anti-biofilm treatment was effective in preventing the formation of biofilms. The 1.92% concentration was the most satisfactorily with Escherichia coli reduction of 5.91log CFUcm-2 and Staphylococcus aureus reduction of 5.17log CFUcm-2 (P<0.05) biomass of the two bacteria. Sanitizing treatment was not effective in reducing biomass. Seen this, the cinnamon and eugenol essential oil may be promising alternatives for controlling biofilms.


INTRODUTION
A biofilm is an assemblage of microbial cells that is irreversibly associated (not removed by gentle rinsing) with a surface and enclosed in a matrix of primarily polysaccharide material. Noncellular materials such as mineral crystals, corrosion particles, clay or silt particles, or blood components, depending on the environment in which the biofilm has developed, may also be reported in the biofilm matrix. Biofilm-associated organisms also differ from their planktonic (freely suspended) counter parts with respect to the genes that are transcribed (DONLAN, 2002). Biofilm formation in equipment and food processing surfaces causes several problems, including the corrosion of metal surfaces and crosscontamination of food products (MENON, 2016;DIAS et al., 2018). Among the pathogenic microorganisms S. aureus and E.coli are able to form biofilms, which are complex structures consisting of surface attached bacteria surrounded by a selfproduced extracellular polymer matrix (MILLEZI et al., 2016;FROZIET al., 2017).
Escherichia coli is one of the most versatile microorganisms reported in nature. Due to frequent precarious hygienic sanitary food production, it is common to observe them contaminating E. coli, as an aggravating factor, the bacteria form biofilms with ease in stainless steel surface (FROZI et al., 2017), polystyrene (MILLEZI et al., 2016) and polipropilene Millezi et al. (MILLEZI et al., 2012). Staphylococcus aureus is a Gram-positive organism, mesophyll; however, studies showed that it can grow in a wide range of temperatures. S. aureus is able to form biofilms, which are complex structures consisting of surface-attached bacteria surrounded by a selfproduced extracellular polymer matrix. Biofilms of staphylococci on surfaces in the food industry pose a serious risk of food contamination. Additionally, S. aureus biofilm formation contributes to its pathogenesis in a number of conditions, such as damaged skin infections, chronic and recurrent airway infections, osteomyelitis and mastitis (KAWARAI et al., 2009;ROGERS et al., 2010).
The essential oils (EOs) of aromatic plants and their components have a wide range of applications in ethno-medicine, preservation, food flavoring and fragrances and in the perfume industries. Therefore, considerable attention has been focused on the various biological effects of naturally occurring agents (BAKKALIA et al., 2008;MILLEZI et al., 2013;SZCZEPANSKI & LIPSKI, 2014;XIAOFENG et al., 2018). Among them are cinnamon EO and its constituents, which are known to possess various biological activities (WANG & YANG, 2009).
Cinnamomum zeylanicum bark is widely used as a spice. It is principally employed in cooking as a condiment and flavoring material, being largely used in the preparation of some desserts, chocolate, spicy candies, tea, hot cocoa, and liqueurs. In medicine, it acts like other volatile oils and was once used as a cure for colds. It has also been used to treat diarrhea and other problems of the digestive system; C. zeylanicum bark is high in antioxidant activity (OUSSALAH et al., 2007).
Given the public health problem that these bacterial species represent, studies have been performed to identify new compounds with antibacterial activity for use in the food industry. In the current study, the C. zeylanicum EO and eugenol sanitizer and anti-biofilm activity was evaluated against E. coli and S. aureus. Biofilms on a stainless steel surface after single cultivation. Stainless steel was chosen for being one of the most utilized materials in the food industries.

Experiment execution sites
The experiment was carried in the Universidade de Minho (Braga, Portugal), in Applied Microbiology Laboratory.

Essential oils and concentrations
Antibacterial solutions were prepared by homogenization of the EO or its constituent in sterile distilled water containing 0.5% (v/v) Tween 80. The solutions were prepared in test tubes and agitated vortexing for 2min. The EO of C. Zeylanicum leaves was purchased from Ferquima Indústria e Comércio Ltda (Vargem Grande Paulista, São Paulo, Brazil). The chemical characterization of the EO, as specified by the supplier, were: color yellow, free of impurities, density (20 o C): 1.045, refractive index (20 o C): 1.533, main component eugenol (about 90%). Eugenol (>93%) was purchased from Sigma Aldrich. The EO concentrations used were 0.12%, 0.48%, 0.96% and 1.92%. The amount of eugenol in the antibacterial solutions was 0.76%. The concentrations were selected based on a study already performed by MILLEZI et al 2016.

Bacterial species, standardization and inoculum preparation
The microorganisms used were Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 24922 obtained from the Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. The standardization of the number of cells was determined by growth curve. Throughout the experiment, the strain was stored under refrigeration in freezing culture medium (15mL glycerol, 0.5g bacteriological peptone, 0.3 of yeast extract and 0.5g NaCl, per 100mL of distilled water, with the final pH 7.4) and stored at -80ºC. For strain reactivation and use, an aliquot of the freezing culture medium was transferred to test tubes containing Trypticase Soy Broth (TSB, Merck, Portugal), with two subcultures at 37°C for 24h. The culture was striated in Trypticase Soy Agar (TSA, Merck, Portugal) added to Petri dishes and incubated at 37°C for 24h. Of the colonies formed on the TSA surface, some were removed and transferred into an Erlenmeyer flask containing 150mL of TSB, which was incubated at 37°C until approximately 10 8 UFCml -1 (MILLEZI et al., 2013 with modifications).

Biofilm formation, activity anti-biofilm and sanitizing
The experimental model of biofilm formation elaborated based on a system first used by MILLEZI et al. (2013) with modifications. In the present study, the experimental model consisted of 20 stainless steel coupons AISI 304 (1x10x20mm) in erlenmeyer containing TSB. AISI 304 was chosen because it is one of the most used stainless steel.
In anti-biofilm activity, were mounted systems containing coupons, flasks, solution with essential oil, eugenol and TSB. The coupons were hung with stainless steel wire and immersed in a total volume of 40mL. The solution was composed of standardized bacterial suspension to 10 8 CFUml -1 , sanitizing solution (essential oil, DMSO, water saline Tween 80) and TSB. For each concentration solution with essential oil and eugenol prepared an individual system. The systems were incubated for 24 hours 37°C and 120rpm and proceeded to quantification of cultivable cells in biofilms and quantification of biomass.
For activity sanitizing, biofilms were formed in systems containing coupons, flasks and TSB. The coupons were hung with stainless steel wire and immersed in TSB total volume of 40mL. Systems were incubated for 24 hours 37°C and 120rpm. After, the coupons were removed, washed two times in sterile peptone water were placed in 12 well plates. Biofilms were exposed to the sanitizing action for 30 minutes.

Quantification of cultivable cells in biofilm
After 24 hours of cultivation, the cells adhered on stainless steel coupons were removed using ultrasonic bath. Each coupon was placed in a well of 12-well sterile plates (Orange Scientific Braine-l'Alleud, Belgium) was added 1 ml of peptone water in each well and proceeded for 6 minutes ultrasonic bath. After this procedure, was performed a serial dilution and aliquots of 0.1mL were removed and the number of viable cells determined by Eosin Methylene Blue Agar (EMB) to count E. coli and mannitol agar for S. aureus, using the technique of microdrop. The drop plate (DP) method was used to determine the number of viable suspended bacteria, less time and effort are required to dispense the drops onto an agar plate than to spread an equivalent total sample volume into the agar (HERIGSTAD et al., 2001). Dishes were incubated at 37°C/24 hours. After this period, took place on plate count and the values were expressed in total number of colony forming units (CFUs) per unit area (log CFUcm -2 ). All assays were performed in three separate occasions.

Biomass quantification by crystal violet staining
Biomass of biofilms were quantified by Crystal Violet (CV) (STEPANOVIC´et al., 2000). For fixation of the adhered cells and biofilms the coupons stainless steel coupons were added in sterile 12-well plates (Orange Scientific, Brainel'Alleud, Belgium), 2mL of 99% methanol (Vaz Pereira, Portugal) was added to each well, after 15min the methanol was removed and the coupons were allowed to dry about 25°C. Then, 2mL of crystal violet stain (CV; 1% v/v) (Merck, Portugal) were added to all wells. After 5min, excess of CV was removed and the coupons were washed in water. Finally, 1mL of acetic acid (33% v/v) (Pronalab, Portugal) were added to all wells to dissolve the CV stain and the absorbance was measured at 570nm. The classification follows the parameters according to the values of Optical Density (OD): OD control <OD≤2OD control, weakly adherent/ weak biofilm producer; 2xODcontrol<OD≤4xOD control, moderately adherent/moderate biofilm producer; 4xODc<OD, strongly adherent/strong biofilm producer; where OD is optical density of the negative control and OD control is the cut-off OD value defined as three standard deviation values above the mean OD of the negative control. All assays were performed in triplicate and on three separate occasions.

Statistical analysis
Data were analyzed with GraphPad Prism ® One-way ANOVA (Bonferroni) tests were performed and P<0.05 was considered significant.

RESULTS AND DISCUSSION
According to the parameters used to classify the formation of biofilms, by biomass analysis (STAPANOVIC et al., 2000), S. aureus and E. coli adhered moderately in 24 hours of culture; however, PARIZZI et al. (2004) considered biofilm, when the number of adhered cells oscillates between 10 4 and 10 5 UFC/cm 2 in the present study, results oscillated between 10 6 and 10 7 UFC/cm 2 , being higher than this parameter.
Treatments anti-biofilm eugenol and C. zeylanicum EO treatments anti-biofilm were effective in preventing the formation of bacterial communities on the surface of stainless steel coupons. Significant reductions (P<0.05) were achieved with the eugenol and all EO concentrations. Eugenol reduced E. coli in 3.65log CFUcm -2 (56.85%), the concentration of 1.92% was the most satisfactorily with a reduction Millezi et al. of 5.91 log CFUcm -2 (92.0% ) ( Figure 1A). The Staphylococcus aureus was susceptible to the action of eugenol and OE, all treatments were significant (P<0.05) ( Figure 1A). Eugenol reduced 2.2log CFUcm -2 ; however, the concentration 1.92% was the best result (reduction of 5.17log CFUcm -2 ) corresponding to 87.0% ( Figure 1A).
The effect of compound eugenol on the formation of biofilm bacterial biomass was satisfactory (P<0.05). Escherichia coli was more susceptible at a concentration of 1.92% with 97.45% reduction in biomass ( Figure 1B). At the concentration of 0.12%, there was 93.0% reduction of the formation of biomass S. aureus ( Figure 1B). There was no concentration-dependent effect on the activity of the EO on biomass produced by S. aureus and E. coli, increasing concentrations did not reduce the formation of biomass.
Eugenol and EO agents were effective as sanitizers against biofilms formed by E. coli and S. aureus in log CFU cm -2 reduction (P<0.05). Both bacteria were less susceptible to treatment with eugenol, a reduction of 1.85 log CFUcm -2 for E. coli and 2.11 log CFU cm -2 for S. aureus (Figure 2A) .The greatest reduction of E. coli concentration was 1.92% of EO, 3.85 log CFUcm -2 (64.27%) (Figure 2). Simultaneously, we observed concentration-dependent effect in which, the higher the concentration used, the greater was the reduction of log CFUcm -2 of cells of E. coli. However, the treatment against S. aureus no effect concentration dependent, the greatest reduction was in the concentration of 0.48% of EO, of 3.46 log CFUcm -2 (61.34%). For biomass formed, all treatments showed poor removal capacity without significant results (P>0.05) ( Figure 2B).
Biofilms are communities on the surfaces of harmful food industries. Thus, there is a high probability that the irreversibly adhered cells will remain even in the surfaces after sanitation. This is one of the main reasons for biofilm formation on surfaces in contact with food. The C. zeylanicum EO and its major constituent eugenol are effective antimicrobials with broad-spectrum anti-biofilm and sanitizer activity.
In the food industry, considerable number of surfaces such as stainless steel, glass, low density polyethylene, cast iron, rubber, polycarbonate and polypropylene, are susceptible to microbial adhesion. However, the surface characteristics, such as electric charge, water retention capacity, free energy and topography play an important role in the accession process. In a study the authors mentioned that the cells adhere better on hydrophilic surfaces (stainless steel, glass) than on hydrophobic surfaces (rubber and plastics) (STEPANOVIC´et al., 2000). Several other bacteria readily form biofilms on stainless steel surface (MILLEZI et al., 2012). In the present study, it was found that 24 hours was sufficient for the formation of biofilms of E. coli and S. aureus on stainless steel coupons.
In studies that evaluate the antibacterial potential of different agents, it is common for authors to use both gram-negative and grampositive bacterial strains, which usually have different susceptibility profiles. Normally, gram-negative bacteria, such as E. coli, are more resistant than gram-positive bacteria because their outer membrane serves as an additional barrier making the diffusion of the constituents of the EOs into the bacterial cell difficult (BURT, 2004). The nucleus and the O antigen in the lipopolysaccharide of the outer membrane of gram-negative bacteria are hydrophilic, which prevents the accumulation of EOs in the cytoplasmic membrane (PSALTIS et al., 2007). In this study, E. coli was more sensitive to the action of the antibacterial activity anti-biofilm; however, the activity sanitizer; E. coli was more resistant than S. aureus. This demonstrated that it is important to conduct tests in different situations or stages of biofilm because the bacterial behavior against stress caused by sanitizing agents is versatile.
Disinfectants effectiveness is frequently determined by the number of surface-adhered cells they are capable to reduce, obtained by standard plate count. In present study, beyond the standard plate count was performed to quantify biomass biofilm. Results of the activity of anti-biofilm all treatments were unsatisfactory (P>0.05), however, when eugenol and EO were used as sanitizers, already formed biofilm by 24 hours, there were significant biomass reductions. Once formed, biofilms showed greater power of resistance to treatments.
In the research BURT (2004) examined the anti-biofilm activity of EOs some of their major constituents on biofilms formed by S. aureus and E. coli on the surface of medical biomaterials. Melaleuca alternifolia and Melissa officinales OEs, as well as constituents alpha-terpineol and terpinen-4-ol, showed high activity anti-biofilm. According to these same authors, the death rate of biofilm formed by S. aureus within 24 hours, treated with EOs and its constituents, said partial reduction (50%) of the biomass metabolism. Concentrationdependent effect was observed for E. coli biofilm; however, was more susceptible to the action of the treatments that biofilms of S. aureus. Results of this study are similar to those obtained (BURT, 2004), there was a high biomass reduction of both biofilms. Furthermore, E. coli was also more susceptible to anti-biofilm activity and decreased concentration dependent EO treatment. Satisfactory results were obtained with reduction of log CFU in treated and biomass EO C. zeylanicum and its major compound isolated eugenol. However, the highest concentrations of EO (0.96% and 1.92%) logarithmic reduction was more significant (P>0.05). Previous studies have shown that the antimicrobial effect of essential oils is due to the interaction between all the essential oil components present and not due to an individual component (NEU et al., 2010). In research SIMIC et al. (2004) reported the use of a single EOs majority components to treat a biofilm in this study was not sufficient to inhibit biofilm growth. However, in this study, eugenol was effective except as a sanitizing agent in removal of biofilms formed by biomass.
Mechanism of action against bacteria is yet not fully understood, but it is speculated to involve membrane disruption through lipophilic products. (MILLEZI et al., 2016). Phenolic compounds, such as eugenol, can cause the disruption of energy production due to enzyme inhibition by the oxidized products, through reaction with sulfhydyl groups or through more nonspecific interactions with proteins (MENDOZA et al.,1997).
In this research, we obtained important findings, we found that the use of EO C. zeylanicum and eugenol may be a promising alternative, especially as anti-biofilm agent to prevent or reduce the formation of biofilms of E. coli and S. aureus on surfaces of stainless steel AISI 304. However, the effectiveness of treatments against biofilms does not always lead to its total destruction and eradication.

DECLARATION OF CONFLICTING INTERESTS
There is no conflict of interest.