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The antioxidant and antimicrobial activity of essential oils against Pseudomonas spp. isolated from fish

Natural products of plant origin, which include essential oils (EO) could be used as a growth inhibitor of pathogenic and spoilage microflora in food. The objective of this study was to determine the antibacterial and antioxidant activity of 21 EO against 10 Pseudomonas species isolated from freshwater fish. The chemical composition of EO was determined by gas chromatography/mass spectrometry. The disc diffusion method and detection of minimum inhibitory concentration (MIC) were used for the determination of the antimicrobial activity. All the EO tested exhibited antimicrobial activity, however, Cinnamomum zeylanicum EO was the most effective against Pseudomonas spp. both according to the disc diffusion and MIC methods. The EOs of Cymbopogon nardus, Origanum vulgare, Foeniculum vulgare and Thymus serpyllum showed the highest antioxidant activity of 93.86 μg, 83.47 μg, 76.74 μg and 74.28 μg TEAC/mL. Application of EO could be an effective tool for inhibition of growth of Pseudomonas spp. on fish.


Introduction
Essential oils (EOs) are aromatic and volatile liquids, which contain a mixture of organic compounds extracted from plant material. EOs possess a strong and generally pleasant flavour (Burt, 2004), therefore they are widely used in the cosmetic and food industry. Since the EOs exhibit antimicrobial and antioxidant properties as food additives, the research on the impact on food nutritional and microbiological properties have been intensified during the past decade. Studies on the effect of the EOs against a wide range of microorganisms, including pathogenic and food spoilage microflora, are among the most perspective for a safe food production (Trombetta et al., 2005).
The mode of action of EOs has not been completely understood yet and the main effect of EOs could be linked to the chemical compounds naturally present in EOs bearing plants (Burt, 2004;Cox and Markham, 2007). However, the antimicrobial activity of EOs depends on the composition and plant synergy showing that the chemical composition of the EOs is of great importance (Bajpai et al., 2012). The degree of antimicrobial activity exhibited by the EOs may influence their ability to penetrate through bacterial membranes and display the inhibitory activity on the functional properties of the cell (Bajpai et al., 2012;Fisher and Phillips, 2009;Guinoiseau et al., 2010). The phenolic compounds of EOs also elicit an antimicrobial response against foodborne pathogens by altering the microbial cell permeability, damaging cytoplasmic membranes, interfering with cellular energy (ATP) generation system and disruption of the proton motive force which result in the inhibition of the functional properties and the leakage of the internal cellular contents (Bajpai et al., 2012;Friedly et al., 2009). Antibacterial properties shared by the EOs allowed to identify the effect on commensal and pathogenic microorganisms as an alternative to antimicrobial agent application. The extensive use of antibiotics in intensive food animal production has resulted in the emergence of resistance among food-borne pathogens, opportunistic pathogens and commensal flora. The resistant microflora has significantly contributed to the development of antibiotic resistance in humans with the EOs to be safe for the environment and consumers and with the ability to potentially inhibit the resistant bacteria (Heuer et al., 2009).
Pseudomonas spp are a genus of Gram-negative bacteria ubiquitous in the environment. The genus consists of species with human and animal health significance, particularly Pseudomonas aeruginosa is an opportunistic human pathogen while other Pseudomonas representatives can cause an infection in plants and insects (Stead, 1992). Some species of Pseudomonas exhibit the plant growth promoting and pathogen-suppressing properties and may be considered for use in biological control and bioremediation (Keel et al., 1996). Pseudomonas spp. are metabolically versatile, and hence, they were widely isolated from the natural environment, including water. Pseudomonas species were frequently associated with fish and the bacteria have been isolated from skin, gills and intestines. Despite the bacterial flora of the fish reflect the microbial population of the aquatic habitat influenced by the bacterial load in the water and salinity, the Pseudomonas spp. can comprise a predominating part of fish microflora (Cahill, 1990). Pseudomonas could cause fish infection and contribute to the spoilage processes of freshly caught and processed fish (Tripathy et al., 2007). Studies on the effect of the EO on Pseudomonas spp. isolated from freshly caught fish from natural environment are still limited. Furthermore, Pseudomonas are inherently resistant to various antimicrobial agents (EUCAST, 2015) but the aquatic environment is a source of diverse microflora. The application of EOs for inhibition of Pseudomonas spp. growth could be an effective tool to alter bacterial growth, therefore studies on the comprehensive evaluation of the inhibitory effects of the EO on the microflora of freshwater fish are needed. The aims of the present study were (i) to determine the antioxidant activity of the EOs, and (ii) to evaluate the antimicrobial effect of 21 EOs against Pseudomonas spp. isolated from freshwater fish.

Productions of samples of the EO and analysis of their chemical compositions
A classical methodology for large-scale production of EOs was applied. The EOs were obtained with the distillation apparatus of two types specifically designed for aromatic and medicinal plants. Distillation equipment consisted of the main distillatory unit, steam condenser, steam boiler and apparatus for improving of the water quality. The used apparatus were of type HV-3000 with height and width of 5250 and 2180 mm and container for 200-250 kg of dried or 400 to 500 kg of fresh matter of plant material; and the type HV-300 with height and width of 3400 and 1300 mm and container for 40-50 kg of dry or 100-120 kg of fresh matter of plant material.

Qualitative and quantitative analysis of the EOs with GC/GC-MS
Analyses were carried out in an Agilent Technologies (Santa Clara, CA) 6890 N gas chromatograph fitted with an HP-5MS fused silica column (5% phenylmethyl polysiloxane, 30 m Â 0.25 mm i.d., film thickness 0.25 lm, Agilent Technologies), interfaced with an Agilent Technologies mass-selective detector 5975B operated by Table 1 Chemical composition of the investigated essential oils (S1-S10).
Sample concentration (% g/100 g) a Compound RI b S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 Values are given as mean value ± SD of three independent experiments. b RI-exp; S11-S. officinalis -leaves; S12-A. alba -needles; S13-C. aurantium -pericarp; S14-C. sinensis -pericarp; S15-C. nardus -leaves; S16-M. spicata -leaves; S17-T. vulgaris -herb; S18-C. carvi -fruits; S19-T. serpyllum -leaves; HP Enhanced ChemStation software (Agilent Technologies). Analytical conditions were as follows: oven temperature programmed at 50°C with an increase of 5°C/min to 280°C; injection of 1 lL (10% hexane solution); split ratio 1:50.0; carrier gas, helium at 1.0 mL/min; injector and transfer line temperatures of 250°C and 280°C, respectively; MS source temperature 230°C; MS quadruple temperature 150°C; mass scan range, 35-550 amu at 70 eV. GC analyses were performed on an Agilent model 6890 N gas chromatograph with a flame ionization detector using an HP-5MS column. The chromatographic conditions were the same as for GC/MS analyses. The constituents of the essential oils were identified by comparing their retention times with available standards, RI (retention indices) values relative to those of C 6 -C 30 n-alkanes and their mass spectral fragmentation pattern with those reported in literature (Adams, 2007) and stored in the MS library (Wiley7Nist) incorporated in the HP Enhanced ChemStation software.
In accordance to previously published procedure (Kowalski, 2008), the quantitative analysis was performed by means of the internal standard addition method (alkanes C 12 and C 19 ). Briefly, samples of essential oils were diluted one thousand times with n-hexane in order to obtain 1 mL of solutions. Then, 1 mg of n-dodecane and 1 mg of n-nonadecane were added to each sample of investigated diluted oils. Prepared samples were subjected to GC/MS and GC/FID examinations, with the fact that quantitative analysis were performed by using calibration curves for available standards within the concentration range 0.03-80%. Semiquantification: safrole from calibration curve of eugenol, trans-2-metoxycinnamaldehyde from calibration curve of transcinnamaldehyde, isomenthone from calibration curve of menthone, pulegol from calibration curve of isopulegol, c-terpinene from calibration curve of a-terpinene, b-thujone from calibration curve of a-thujone, a-caryophyllene from calibration curve of b-caryophylene, citronelal from calibration curve of citronelol.
The chemical composition of EOs is summarized in Tables 1 and  Table 2.

Origin of Pseudomonas spp.
Freshly caught freshwater fish were used for isolation of Pseudomonas spp. Pseudomonas were confirmed with the MALDI TOF MS Biotyper (Brucker, Germany) and the following species were isolated: Pseudomonas agglomerans, P. antarctica, P. brassicacearum, P. frederiksbergensis, P. koreensis, P. lundensis, P. mandelii, P. proteolytica, P. synxantha, P. veronii. Isolates were cultivated on Mueller Hinton Agar (MHA, Merck, Germany). Bacterial culture was enriched in the Mueller Hinton Broth (MHB, Merck, Germany) at 37°C for 24 h before the antimicrobial susceptibility and EOs antimicrobial activity tests.

Antibiotic susceptibility testing of Pseudomonas spp.
The antibiotic susceptibility was tested by disc diffusion method. A suspension of the Pseudomonas spp. in MHB was plated out onto MHA, then, the appropriate antimicrobial discs were placed on the agar surface. Inoculated agars were incubated at 37°C for 24 h. Pseudomonas spp. cultures were tested against ampicillin (10 mcg), gentamicin (10 mcg), imipenem (10 mcg) and meropenem (10 mcg) (Oxoid, UK). The results were interpreted according to the EUCAST, 2015.

Detection of antimicrobial activity of the EOs
Detection of antimicrobial activity of EOs was carried out with the agar disc diffusion method and detection of the minimum inhibitory concentration of EOs.
For the agar disc diffusion method, an aliquot of 0.1 mL of bacterial suspension in MHB was spread onto MHA. Then, the filter paper discs of 6 mm in diameter were impregnated with 15 mL of the EOs and placed on the MHA surface. Inoculated MHA plates were kept at 4°C for 2 h and incubated aerobically at 37°C for 24 h. The diameters of the inhibition zones were measured in mm after incubation. Each test was repeated twice.
For the detection of minimum inhibitory activity of the EO, a test oil solution was prepared in 10% aqueous dimethyl sulphoxide (DMSO, Penta, Prague, Czech Republic). Geometric dilutions from 0.75 to 100 mg/mL of the EOs in a 96-well microtitre plate were prepared. One growth control well (MHB + Tween 80) and one sterility control well (MHB + Tween 80 + test oil) were included in each assessment. The plates were incubated aerobically at 37°C for 24 h. The presence of a white ''pellet'' on the well bottom indicated on the bacterial growth.
Pseudomonas spp. growth was evaluated after incubation by measuring the well absorbance at 450 nm (Biotek EL808 with shaker, Biotek Instruments, USA). Measurements were undertaken before and after the experiment and the difference between the measurements was described as growth. Measurement error was 0.05 of values from absorbance. Each test was done in eight replicates for a higher accuracy of the MICs of used EOs.

Statistical analysis
The basic variation (disc diffusion method) was from obtained data by using the statistical programme Statgraphic and the Tukey HSD test for the comparison of the antimicrobial activity of the 21 EOs. The parameters calculated alongside with the basic variation were: average, standard deviation, minimum, maximum coefficient of variation and the frequency of size of the inhibition zones.

Antibiotic susceptibility testing
Pseudomonas antarctica, P. frederiksbergensis, P. mandelii, P. proteolytica and P. veronii were resistant to all the antimicrobial agents tested that comprised 50% of all bacterial cultures tested (Table 3). Pseudomonas synxantha was the most sensitive to application of antimicrobial agents and exhibited the sensitivity to ampicillin and imipenem, intermediate susceptibility to gentamicin and resistance to meropenem. All the Pseudomonas were resistant to meropenem (100%) while 4 out of 10 were resistant to imipenem (40%). Resistance against the ampicillin and gentamicin comprised 10% and 40%, respectively.
The present study revealed the high proportion of resistant strains among the Pseudomonas spp. isolated originated from fish. Pseudomonas spp., including P. aeruginosa, is naturally resistant to many antibiotics (Tadeu et al., 2000) with only few of antimicrobial agents were found to be effective against Pseudomonas. Fluoroquinolones, gentamicin and imipenem were described among the most effective but against all the Pseudomonas species. The high efficiency of gentamicin on Pseudomonas spp. animal isolates was confirmed. Also meropenem, imipenem, ciprofloxacin, ticarcillin and mezlocillin were described as the antimicrobials with high activity against environmental isolates of Pseudomonas spp. (Tadeu et al., 2000). The present study showed the high prevalence of the imipenem-, meropenem-, gentamicin-and ampicillinresistant strains the presence of the large proportion of antibiotic resistant Pseudomonas spp. strains in the aquatic environment.

Antimicrobial activity of EOs detected by the disc diffusion method
The results on the antibacterial activity of 21 EOs tested by the disc diffusion method varied at great extent (Table 4). The majority 2.33 ± 0.58 4.00 ± 1.00 6.00 ± 1.00 2.33 ± 0.57 1.33 ± 0.58 Thymus serpyllum L.
of the Pseudomonas spp. was sensitive to all EOs were applied. Cinnamomum zeylanicum EO was the most effective against seven Pseudomonas species, including P. agglomerans, P. antarctica, P. brassicacearum, P. koreensis, P. mandelii, P. proteolytica and P. synxantha. The most sensitive among Pseudomonas spp. to Cinnamomum zeylanicum EO was P. brassicacearum with the inhibition zone of 15.00 ± 2.00 mm. P. frederiksbergensis was the most sensitive to Abies alba Mill. EO (14.33 ± 0.58 mm) while P. veronii was the most sensitive to Pinus sylvestris L. EO (14.67 ± 0.58 mm). There were no differences between the sensitivity of P. lundensis to the EOs activity of Cinnamomum zeylanicum and Origanum vulgare L. (12.33 ± 1.53 mm, P ! 0.001). There were significant differences (P 0.001) between the antimicrobial activity of 18 EOs on Pseudomonas spp. growth for other three -Citrus sinensis, Cymbopogon nardus and Cinnamomum zeylanicum differences were not significant (P > 0.001). A broad variation in antimicrobial properties of the EOs was reported by of Mith et al. (2014). The EOs of Cinnamomum cassia, C. verum, Origanum compactum, O. heracleoticum, Thymus capitatus and T. vulgaris thymoliferum showed strong antimicrobial activity against the tested bacteria, whereas Cymbopogon flexuosus EO showed strong activity against Gram-positive bacteria only. In con-trast, Kaempferia galanga EOs did not exhibit the antimicrobial activity against any of the tested bacterial strains. In general, Gram-positive bacteria were found to be more sensitive to EOs or antibacterial compounds than Gram-negative bacteria because of the differences in cell structure, which may retain the entry of hydrophobic compounds in the cell (Burt, 2004;Cox and Markham, 2007;Dorman and Deans, 2000). Our results revealed that the EOs could be effective against Gram-negative Pseudomonas spp.

Antimicrobial activity of EOs detected by identification minimum inhibitory concentration
The best antimicrobial activity was exhibited by Cinnamomum zeylanicum EO against six Pseudomonas species, including P. agglomerans, P. brassicacearum, P. frederiksbergensis, P. lundensis, P. proteolytica and P. synxantha and our findings were in agreement with the results obtained by the disc diffusion method. The MIC of Cinnamomum zeylanicum EOs ranged from MIC50 of 3.125 and MIC90 of 6.25 to MIC50 of 6.25 and MIC90 of 12.50 ml/mL. There were no differences between the antimicrobial activity of Cinnamomum zeylanicum and Satureja hortensis on the growth  Table 5.
The present study showed that the application of EOs was effective in inhibition of Pseudomonas spp. in freshwater fish. Pseudomonas spp. are an important part of spoilage microflora, which alter the shelf-life and the quality of fish. The EOs was affective against the spoilage microflora for prolongation of the shelf-life of freshwater fish (Harpaz et al., 2003). The studies on the effect of treatment of Pseudomonas spp. with EO originated from freshwater fish are limited. However, the Pseudomonas spp. of freshwater fish were found to be the specific spoilage microorganisms and the activity of Pseudomonas spp. in fish results in rapid deterioration of the product. Therefore, the EOs activity differs from those reported from meat and another kind of products. It might be explained by different composition and percentage content of active constituents in EOs (Bozin et al., 2006), species, subspecies or variety of plants, geographical locations, harvesting, drying and extraction methods (Burt, 2004;Di Cesare et al., 2003;Hussain et al., 2008;Sarac and Ugur, 2008). Methods used to assess the antimicrobial activity, bacterial strains and their sensitivity, volume of inoculum, incubation time, and temperature may also be related to the variation in the experimental results (Burt, 2004;Bozin et al., 2006).

Antioxidant activity
The highest antioxidant activity (Table 6) was observed in Cymbopogon nardus (93.86 lg TEAC/mL), Origanum vulgare (83.47 lg TEAC/mL), Foeniculum vulgare (76.74 lg TEAC/mL) and Thymus serpyllum (74.28 lg TEAC/mL). In comparison, the antioxidant capacity of Cymbopogon citrates with DPPH in Vázquez-Briones et al. (2015) study was 44.06 ± 0.20 mg TEAC per 100 mL, equivalent to 55.57% of inhibition. The major compound of Cymbopogon oil is citral, which possesses various useful bioactivities and one of these is an anti-clastogenic effect in nickel chloride-treated mouse micronucleus system. Citral-caused inhibition of micronuclei formation and enhanced the superoxide scavenging activity were thought to be responsible for the anti-clastogenic effects of citral (Rabbani et al., 2006). Some other compounds such as geraniol and limonene have also been correlated with different types of bioactivities. Ganjewala (2009) reported that EOs from Cymbopogon spp. showed scavenging of free radicals and antiacetylcholine esterase activity proving that the EOs share strong antioxidant properties.
Strong antioxidant activity was also detected in EOs of Origanum vulgare and Thymus serpyllum. The main compounds of these EOs are thymol and carvacrol. The metabolic pathway for the carvacrol and thymol (Table 1) formation begins with the autoxidation of cterpinene to p-cymene and the subsequent hydroxylation to thymol (Alizadeh, 2013). Ruberto and Baratta (2000) confirmed that thymol and carvacrol molecules are indeed responsible for the antioxidant activity of many thymol-and carvacrol-containing EOs. Strong antioxidant activity was exhibited by the EO from Foeniculum vulgare also showed. Yoshioka and Tamada (2005) revealed that Foeniculum vulgare EO provided an inhibitory activity against platelet aggregation induced by ADP, arachidonic acid and collagen in guinea pig plasma. Similar findings were reported for aggregation of rabbit platelets. The biological activity of herbal EOs alongside with their antimicrobial activity influences the naturally occurring Pseudomonas spp. of freshwater fish, therefore the possible application of EO in aquaculture and food industry could be considered.

Conclusions
In conclusion, the EOs of EO-bearing plants used in the present study revealed a significant antimicrobial activity on the Pseudomonas spp. originated from freshwater fish with Cinnamomum zeylanicum to be the most effective. The results of the present study suggest that the EO is a potential source of natural antibacterial agents and may be used as natural compounds with antipseudomonal activity to improve the microbiological quality of freshly caught freshwater fish. The highest antioxidant activity was observed for Cymbopogon nardus, Origanum vulgare, Foeniculum vulgare and Thymus serpyllum.