Antimicrobial Activity and Chemical Composition of Essential Oils against Pathogenic Microorganisms of Freshwater Fish

Antimicrobials are widely applied in aquaculture for treatment of infectious diseases in fish. The increased antimicrobial resistance of fish pathogens to conventional antimicrobial treatment highlights the need for research on the antibacterial properties of natural products—in this case, essential oils (EOs). The aim of the present study was to detect the antimicrobial activity of the essential oils on pathogenic microorganisms found in freshwater fish. Freshwater fish isolates of Aerococcus spp., Aeromonas spp., Enterococcus spp., Escherichia spp., Pseudomonas spp., Shewanella spp., Yersinia spp., and Vagococcus spp. were tested for antimicrobial resistance and antimicrobial activity against 14 commercially available essential oils. Antimicrobial resistance was identified in Pseudomonas spp. isolates against cefepime and ciprofloxacin; while all Aeromonas, Enterococcus, and Yersinia isolates were fully susceptible. All tested EOs revealed antimicrobial activity against the tested freshwater fish isolates at different extents. Cinnamomum camphora exhibited strong antimicrobial activity against Aeromonas spp. (3.12 μL/mL), Enterococcus spp. (0.78–1.56 μL/mL), and Pseudomonas spp. with the MIC method. EOs of Gaultheria procumbens and Litsea cubeba showed strong antibacterial activity against Yersinia spp. and Vacococcus spp. (6.25 μL/mL). The study shows the antimicrobial activity of EOs against the most relevant freshwater fish pathogens and indicates the application opportunities in aquaculture.


Introduction
Essential oils are present in different parts of plants and consist of aromatic and volatile compounds. The primary role of EOs is the protection of plants against pathogens, which is attributed to the antimicrobial activity that EOs have shown [1]. The effects of EOs are created by their chemical composition, and an amount of a single compound from different plants, sharing different chemotypes affects the chemical composition of EOs [2]. Application of EOs may lead to alterations in the cell structure, e.g., the degradation of the cytoplasmic membranes and cell wall, and the synthesis of membrane lipids. EOs have been described as regulating the quorum sensing systems by the formation of biofilms and expression of virulence factors [3].
EOs are widely applied in cosmetics, perfumes, and food production because of their strong smells and flavors, and microbial growth inhibiting properties [4]. Different features of EOs have been revealed to have antimicrobial properties, which have been intensively investigated to find out their possible applications for replacements of existing antimicrobial treatments. The antibacterial, antiviral, antifungal, and insecticidal properties of EOs have been demonstrated, which allow us to consider EOs as an alternative of the current antimicrobials used for humans and animals [5][6][7].
Antimicrobial resistance is a global threat to animal and public health that affects the availability of antimicrobials for treatment of bacterial infections in animal and humans [8]. Fish pathogens may cause outbreaks with high mortality, leading to significant economic damage [9]. In aquaculture, the elevated stress, increased fish density, and deteriorated animal welfare may facilitate the rapid spread of fish diseases. Treatment with antimicrobials is needed to address this condition with application of antimicrobials to water and feed, which may result in ineffective therapeutic concentration and treatment of uninfected fish, which in turn may increase antimicrobial resistance [5,10].
Reduction in consumption of antimicrobials in human and veterinary medicine may minimize the problems related to the spread of antimicrobial resistance. Previous reports showed the antimicrobial activity of EOs against different microorganisms, including human and animal pathogens, from the perspective of applications in aquaculture [6,7,11,12]. EOs could be a promising agent in fish health, since their antimicrobial and immunomodulating properties have been described. Antimicrobial effects against such different fish pathogens and contaminants as Aeromonas spp., Enterobacter spp., Pseudomonas pp., Vibrio spp., etc. have been recorded with therapeutic application of EOs as recommended previously [11][12][13][14]. However, the diversity of fish microbiota and chemical composition of Eos, as well as the different antimicrobial effects of the EOs tested indicates the need for further research into these antimicrobial activities. Therefore, the aim of the present study was to investigate the antimicrobial activities of 14 commercially available EOs on the pathogens of freshwater fish.

Antimicrobial Resistance in Fish Isolates
Antimicrobial resistance in P. fluorescens, P. frederiksbergensis, and P. gessardii against cefepime and ciprofloxacine was found, while P. lundensis showed antimicrobial resistance only against cefepime and P. proteolitica only against ciprofloxacin. Antimicrobial resistance in Aeromonas spp., Enterococcus spp., and Yersinia spp. was not identified ( Table 2).

Discussion
Among the chemical compositions of the EOs in the present study, several compounds, including α-pinene, β-pinene, α-limonene, α-terpineol, and 1,8-cineole have been reported previously. Studies on the antimicrobial activities of the isomers and enantiomers of pinene showed that α-pinene and β-pinene had antibacterial activity against Cryptococcus neoformans, Candida albicans, Rhizopus oryzae, and MRSA [15]. Limonene was found to have high antibacterial activity against Gram-positive and Gram-negative foodborne pathogens-E. coli, S. enterica, and S. aureus [16]. One of the major compounds in the leaf essential oils, α-terpineol, possessed the strongest antibacterial activities compared with the other components [17].
The antimicrobial effect of the EO of Cinnamomum spp. is attributed to the chemical composition of the plant. In the present study, 1,8-cineole and a-terpineol were found to be the main constituents of the EO, in agreement with Bottoni et al. [18]. Excellent antibacterial activities of 1,8-cineole was reposted in S. aureus and E. coli, associated with damage with cell compounds confirmed with electron microscopy [17].
In the present study, Aeromonas spp. was susceptible to all antimicrobials, while Pseudomonas spp. exhibited resistance to cefepime and ciprofloxacin. High rates of antimicrobial resistance in Pseudomonas spp. and low in Aeromonas spp. were in agreement with previous studies [19,20]. Susceptibility of Enterococcus spp. to antimicrobials was in line with Ellis-Iversen et al. [21], who found that the majority of E. faecalis and E. faecium were fully susceptible against antibiotics, while multiresistant Enterococcus was isolated from the Mediterranean aquaculture site and the fish-rearing ponds in Bangladesh without history of enterococcal infection [22,23]. Yersinia spp. found in pigs exhibited resistance to chloramphenicol, ciprofloxacin, and cephalosporins, and the ability of Y. ruckeri to develop antimicrobial resistance to quinolones was identified [24,25]. In general, the rates of antimicrobial resistance were low in the present study and may be attributed to low consumption of antimicrobials in aquaculture.
All tested EOs exhibited antimicrobial activity against tested fish pathogens, with Cinnamommum camphora the most active against the majority of isolates. The strong antimicrobial activity of Cinnamomum capphora was identified previously for both Gram-positive and Gram-negative bacteria, with the MIC for E. faecalis being 1.6 mg/mL [26]. Cinnamomum zeylanicum was the most potent against Pseudomonas spp., both with the disc diffusion and MIC methods in fish isolates from Latvia [19]. The low activity of C. camphora against A. salmonicida subsp. salmonicida strains with MIC above 3200 µg/mL found in the study Hayaygheib et al. [27], was in contrast with our results. In our view, differences in the antimicrobial activity of EOs are attributed to the chemical compositions of EOs, with Cinammomum capmhora var. linaloolifera proving the most active against Aeromonas spp. and Entecococcus in the present study.
Malaleuca alternifolia expressed high activity against A. hydrophila and could be a natural alternative for prevention and control of the pathogen [13]. M. alternifolia exhibited antimicrobial activity against A. hydrophila isolates with the MIC method [28]. The EO of M. alternifolia is a well-known agent for local application, with antibacterial properties shown clinically [29]. The antimicrobial activity against A. salmonicida subsp. salmonicida was low in the Hayaybheib et al. study [27], which concluded that limonene and linalool showed a weak antimicrobial activity.
EO of Vetiveria zizanioides mostly expressed a weak antimicrobial activity. Orchard et al. [30], found that the EO of Vetiveria zizanioides was highly effective against P. aeruginosa. However, EOs from the roots of V. zizanioides and V. nigritana showed a low activity against Gram-negative bacilli but strong against Gram-positive cocci, in agreement with our results [31].
Amyris balsamifera showed antimicrobial activity against Gram-negative Aeromonas spp. and Yersinia spp. Antimicrobial activity of sandalwoods was reported previously, with high activity against Klebsiella pneumonia identified in the study of Jirovetz et al. [32]. High antimicrobial activity of sandalwood oil against Gram-positive S. aureus was observed, while different results were observed against E. coli and Ps. aeruginosa [32].
Gaultheria procumbens and Litsea cubeba showed antimicrobial activity against Yersinia enterocolitica. G. procumbens EO was more effective against Gram-negative bacteria than against Gram-positive bacteria; while high antimicrobial activity of L. cubeba against Y. enterocolitica was reported by Ebani et al. [33]-(15.7 ± 0.6 mm) in poultry isolates [34]. Tripolum pannonicum and Origanum vulgare exhibited antimicrobial activity against Y. ruckeri [35]. EO of G. procumbens and L. cubeba were active against Sh. baltica, which is recognized as an H 2 S producer in ice-stored fish from the Danish Baltic sea [36]. Shewanella may serve as an opportunistic pathogen of human and aquatic animals [37].
Despite the weak antimicrobial activity of Citrus aurata identified in the present study, the EOs of citrus were active against human and fish pathogenic strains, able to develop antibiofilm properties previously [38,39]. However, resistance or weak inhibition against Escherichia spp. and Klebsiella spp. of citrus EO in the studies of Moreira et al. [40] and Mancuso et al. [38] were in line with our results.

Chemical Characterization of Essential Oil Samples by Gas Chromatography/Mass Spectrometry (GC/MS) and Gas Chromatography (GC-FID)
GC/MS analyses of selected essential oil samples were performed using an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to quadrupole mass spectrometer 5975B (Agilent Technologies, Santa Clara, CA, USA). A HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm) was used. The temperature program was: 60 • C to 150 • C (increasing rate 3 • C/min) and 150 • C to 280 • C (increasing rate 5 • C/min). The total run time was 60 min. Helium 5.0 was used as the carrier gas with a flow rate of 1 mL/min. The injection volume was 1 µL (essential oil samples were diluted in pentane), while the split/splitless injector temperature was set at 280 • C. With a split ratio at 40.8:1, investigated samples were injected in the split mode. Electron-impact mass spectrometric data (EI-MS; 70 eV) were acquired in scan mode over the m/z range 35-550. MS ion source and MS quadrupole temperatures were 230 • C and 150 • C, respectively. Acquisition of data started after a solvent delay time of 3 min. GC-FID analyses were performed on Agilent 6890N gas chromatograph coupled to FID detector. Column (HP-5MS) and chromatographic conditions were same as for GC-MS. FID detector temperature was set at 300 • C. The individual volatile constituents of injected essential oil samples were identified based of their retention indices [41] and comparison with reference spectra (Wiley and NIST databases). The retention indices were experimentally determined using the standard method [42], which included retention times of n-alkanes (C6-C34), injected under the same chromatographic conditions. The percentages of the identified compounds (amounts higher than 0.1%) were derived from their GC peak areas.

Microbial Isolates
Fish isolates were originated from wild and aquacultured roach (Rutilus rutilus) and bream (Abramis brama) from skin, gills, and gut samples. Wild fish were caught in freshwaters (lake and river) in Latvia, while aquacultured fish were bought from producers after their placement on the market. Microbial isolates were confirmed with Maldi-TOF MS Biotyper (Bruker Daltonics, Bremen, Germany). Isolates of fish health and public health significance were selected for the present study with the following genera were represented: Aerococcus spp., Aeromonas spp., Escherichia spp., Enterococcus spp., Pseudomonas spp., Shewanella spp., Yersinia spp., Vagococcus spp.

Detection of Antimicrobial Activity with Disc Diffusion Method
A 0.1 mL of suspension of tested culture (10 5 cfu/mL) was used for inoculation of Mueller Hinton Agar (MHA, Oxoid, Basingstoke, UK). Blank discs were impregnated with 15 µL of EO and placed onto inoculated agar. The agars were incubated at 4 • C for 2 h then at 30 • C and 37 • C for 24 h. The zone of inhibition was measured and the zone < 10 mm was accepted as not inhibitory. All analyses were done in triplicate.

Detection of Minimum Inhibitory Concentration
Tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) in Mueller Hinton Broth (MHB, Oxoid, Basingstoke, UK). After overnight incubation at 30 • C and 37 • C, a bacterial suspension of 10 6 cfu/mL was used for inoculation. A 96well micro-titer plate was used with 50 µL added to each well, excluding the 10th well where 100 µL was added for sterility control. The EO solution in dimethyl sulphoxide (DMSO, Penta, Prague, Czech Republic) was used for inoculation, with 5% of DMSO added to the 10th well. Suspensions were done by transferring of 50 µL to each next well. MIC values were determined by measuring of turbidity. The MIC values was expected to be the lowest concentration of the EO inhibiting bacterial growth. The test was performed in triplicate, and cefoxitin (30 µL) was used as positive control.

Data Analysis
Mean, standard deviations for the antimicrobial activities of EOs were calculated. One-way analysis of variance (ANOVA) was applied to detect differences for antimicrobial effects of different EOs on microorganisms.

Conclusions
Identified antimicrobial resistance in Pseudomonas spp. isolates shows the emergence of the spread of antimicrobial resistance in aquaculture. Despite the antimicrobial resistance in Aeromonas isolates being lower than reported previously, there is evidence of the presence of resistant isolates in wild isolates of freshwater fish. All EOs tested in the present study exhibited antimicrobial activity against wild isolates of freshwater fish, with EO of Cinnamomum camphora Nees & Eberm, and Cinnamomum caphora var. Linalolifera proving the most active against Aerococcus spp., Aeromonas spp., and Enterococcus spp. EOs of Gaultheria procumbens and Litsea cubeba Pers. exhibited the strongest antimicrobial activity against Shewanella spp., Yersinia spp., and Vagococcus spp. Efficiency of EO against foodborne pathogens may be a promising strategy to prevent and/or treat infectious diseases in fish in a sustainable way by reducing the consumption of antimicrobials in aquaculture. Potential applications of EOs and antimicrobial activity of specific compounds in EOs against fish pathogens need to be further studied.