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By: Cristóbal Domínguez-Borbor, Aminael Sánchez-Rodríguez, Stanislaus Sonnenholzner, and Jenny Rodríguez *
The purpose of this study was to identify specifically essential oils (EOs) that can interfere with the QS of known pathogenic vibrios in P. vannamei farming.
Despite its significant growth, the shrimp farming industry has constantly been hit by viral and bacterial pathogens.
Recently, the emergence of new strains of highly virulent vibrios, capable of causing severe mortalities has been reported within the shrimp farming industry.
Most strains of pathogenic vibrios have shown resistance to common antibiotics, making them difficult to control in aquaculture production systems.
To a large extent, the emergence of resistant strains is due to the misuse of antibiotics, commonly adopted by producers to treat vibriosis.
A promising low-risk and environmentally friendly strategy is antivirulence therapy.
“Antivirulence therapy is based on the interruption of bacterial communication, known as quorum sensing (QS).”
It minimizes the risk of microbial resistance as it inhibits virulence without affecting bacterial growth.
Bacteria communicate through QS using small chemical molecules called autoinducers and acquire collective behaviors to regulate the expression of several virulence factors.
QS is involved in factors such as bioluminescence production, biofilm development, exopolysaccharide production, swarming motility, plasmid transfer, secondary production of metabolites, and interactions with the host and other microbes.
It is well known that bioluminescence, biofilm formation, swarming motility, and toxin production in Vibrio species are mediated by the QS system to distinguish between high or low population density and coordinate the genetic expression of the entire community.
Vibrios can launch a coordinated attack that facilitates overcoming the host’s defense barriers thanks to QS-mediated mechanisms.
“Natural products, specifically essential oils (EOs) at sublethal doses, can alter the QS system and thus the virulence of pathogenic bacteria. As a result, EOs have recently been proposed as an efficient and safe alternative for antibiotics replacement. The purpose of this study was to identify EOs that can interfere with the QS of known pathogenic vibrios in P. vannamei farming”.
QS has been linked to the virulence of pathogenic vibrios important to aquaculture.
Preventing vibrios communication or altering their QS mediated responses are appealing strategies to reduce or even abolish their virulence.
Bacterial strains, growth conditions, and essential oils evaluation
Four vibrios were used in this study, V. harveyi (strain E22), V. campbellii (stain LM2013), V. parahaemolyticus (strain ATCC 27969) and V. vulnificus (strain S2).
All strains were grown aerobically in Luria Bertani agar. Five essential oils (EOs) were evaluated, essential oil of Organum vulgare (EOOv), Melaleuca alternifolia (EOMa), Cymbopogon citratus (EOCc), Cinnamomum verum (EOCv), and Thymus vulgaris (EOTv).
For the anti-QS assays, the EOs were emulsified. It was previously determined that the dosage of the substance used as an emulsifier does not influence the parameters evaluated.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of Eos were determined to establish sublethal doses, affecting only the QS indicators of bioluminescence, biofilm development and swarming motility, without affecting the viability of vibrios.
A positive control (containing inoculum but no EO) and negative control (containing EO but no inoculum) were included on each microplate, in addition to six replicates for each concentration of EOs and controls.
Essential oil effect on the growth of Vibrio strains (A) V. harveyi, (B) V. campbellii, (C) V. vulnificus and (D) V. parahaemolyticus. All assayed concentrations were sublethal to the four Vibrio species.
Effect of EOs on the bioluminescence and biofilm formation
Given that bioluminescence in Vibrio species is one of the phenotypes which is controlled by quorum sensing (Manefield et al., 2000), we examined the possibility that EO may affect bioluminescence in V. harveyi and V. campbellii wild type strains.
“The effect of EOs on biofilms of the four Vibrio strains, V. harveyi, V. campbellii, V. parahaemolyticus and V. vulnificus, was assessed. The bacterial biofilm biomass was stained with violet crystal (CV) and quantified spectrophotometrically. Bacterial suspensions for each Vibrio strain were prepared. A control was performed containing bacterial suspension without EO with six replicates for each concentration. The planktonic cells were removed, and the generated biofilms were carefully washed twice.”
The plates were then rinsed to remove excess dye and dried at room temperature.
The data were transformed to (%) biofilm formation considering 100% biomass of untreated bacterial biofilms (negative control).
Effect of EOs on swarming motility
The effect of the EOs on the swarming motility of the four vibrios was also evaluated.
The LBb supplemented was autoclaved. After the medium LB was cooled to 45 ± 3 °C, the EOs were added separately to each concentration to be evaluated.
The medium LB was dispensed in Petri dishes, and the plates were dried for 15 min and immediately following vibrios inoculum was inoculated in the center of the plates.
The plates were incubated at 26 °C for 72 h, and the migration of the swarming motility was measured in mm.
The swarming motility migration of the treated vibrios with EO was compared with the swarming motility migration of the untreated vibrios.
In addition, the effect of EOs on swarming motility in the presence of the antibiotic was also evaluated.
In vitro and in vivo toxicity of EOs
Initially, the toxicity of the EOs was determined in vitro by MTT reduction assay.
First, hemolymph was extracted from healthy shrimp. Then, the primary culture of hemocytes was carried out with Hanks salts.
“The primary cultures were incubated, and there were six replicates for each concentration evaluated. A control of hemocytes without EO was included. The results were transformed into percentages of cell viability.”
To determine the safety of EOs in vivo, P. vannamei larvae were used in three larval stages, zoea 1 (Z-1), mysis 1 (M-1), and post larva (PL-3).
Shrimp larvae were provided by a commercial hatchery. Each trial was carried out independently.
The water used in the tests was filtered and sterilized in an autoclave.
The EOs were applied every eight hours in relation to the total volume of water of each experimental unit.
As a control, larvae were included under the same conditions but without exposure to EOs.
The larvae were monitored for 96 h. The data were transformed to survival percentages, considering 100% as the survival of larvae that did not receive EOs.
Additionally, the data obtained were determined the dose causing 50% mortality (LD50) for each larval stage of shrimp.
In vivo antivirulence effect of EOs and Effect of EOs application in P. vannamei grow-out ponds
The antivirulence effect of the EOs was verified by a challenge test using healthy P. vannamei larvae of the stage.
The bacterial inoculum was prepared, and as control, a culture of V. campbellii without EO was performed.
The culture flasks were incubated. Subsequently, the cultures were centrifuged, the supernatants were discarded, and the pellet cells were resuspended and immediately inoculated to each treatment assigned.
This bioassay considered 100% of the virulence to the inoculum of V. campbellii cultivated without EO.
“The potential effect of two EOs in shrimp production grow-out systems was evaluated. Each day, the EOs were incorporated into the commercial pelleted feed and were immediately supplied to the assigned ponds.”
Daily feeding was set initially to approximately 3% of the average body weight of the shrimp and adjusted weekly based on observed feed consumption and growth.
Final shrimp survival (%), average weight (g), production yields (kg/ha) and feed conversion ratio (FCR) were evaluated at harvest time.
Statistical analysis
All experiments were done in six replicates, except in the bioassay in grow-out ponds that four replicas were used.
The results were expressed as an average (±standard deviation) of the replicates.
Statistical analyses were performed to determine significant differences using one-way ANOVA after verifying the normality and variance homogeneity assumptions.
When significant differences were detected, a Dunnett’s analysis was applied.
EOs sublethal doses determination and bioluminescence inhibition
EOs exhibited different MIC and MBC values against the four Vibrio strains evaluated.
MIC and MBC values were lowest for EOOv and EOMa, showing that their inhibitory and bactericidal activities were stronger compared to the other essential oils evaluated.
MIC and MBC values were used as references for subsequent tests, and only sublethal concentrations below the MIC were assayed in each case.
Results shown indicate that none of the EOs affected the growth of the four pathogenic vibrios at the highest concentration assayed.
Only EOOv and EOMa significantly reduced the bioluminescence of V. harveyi and of V. campbellii.
The percentage of bioluminescence inhibition in each bacterial strain showed a marked concentration dependency for the case of EOOv and EOMa. EOOv was the most efficient oil to inhibit bioluminescence.
EOs effect on biofilm formation and swarming motility
EOCc, EOCv, and EOTv did not inhibit biofilm formation in the four pathogenic vibrios evaluated, so they were not further considered for the swarming test and in vivo trials.
EOOv and EOMa significantly reduced the biofilms of the four vibrios in a concentration-dependent manner.
EOOv was the most efficient oil in reducing the biofilms of the four pathogenic vibrios by more than 50% in each case.
“The antibiotic oxytetracycline inhibited vibrios biofilm formation less efficiently than the EOs, especially in V. parahaemolyticus.”
The swarming motility of the four pathogenic vibrios was significantly affected by the EOs in a concentration-dependent manner.
For the oxytetracycline swarming motility test, we selected the V. vulnificus strain due to its swarming ability to cover the entire Petri dish in 96 h. In this trial, we found that EOs also affect antibiotic resistance.
Since EOs decreased the swarming motility of V. vulnificus, oxytetracycline inhibition halos in the presence of EOs were kept until the end of the experiment 96 h.
In the control group, inhibition halos were reduced as the hours passed.
In vitro and in vivo toxicity of EOs and beneficial effects of EOs in P. vannamei grow-out ponds
EOs in vivo toxicity tests revealed that the earlier the larval stage, the greater their susceptibility to the EOs.
In the zoea stage, the two EOs significantly affected the survival in most of the doses evaluated.
Regarding the mysis stage, EOs only showed a negative effect on survival at the highest doses evaluated.
Regarding the PLs, EOOv decreased survival only at a concentration of 10.0 μgmL−1, and EOMa did not affect the survival of the PLs at any of the concentrations evaluated.
Significant differences were recorded between the cumulative mortality rates of P. vannamei PLs, challenged with V. campbellii grown in the presence and absence of EOs.
“Cumulative survival and yield improved significantly (P < 0.05) in ponds treated with EOOv at both doses evaluated, compared to the control group.”
Regarding EOMa, only at the highest dose (5.0 mg kg−1) were survival and production performances significantly higher compared to the control group.
Results Analysis
EOs have a well-reported ability to inhibit QS in human and animal pathogenic bacteria.
“The results we obtained showed that EOOv and EOMa can inhibit QS-mediated processes in four pathogenic vibrios related to shrimp farming.”
Observations from the in vitro assays allowed us to determine actives doses for in vivo tests, in which EOOv and EOMa significantly increased survival of shrimp challenged with the V. campbellii pathogen.
EOOv and EOMa also showed encouraging results when used feed supplements in shrimp ponds.
EOOv and EOMa were able to inhibit the bioluminescence of V. harveyi and V. campbellii. Bioluminescence production is positively regulated by the QS and is involved in the establishment of the pathogen in the host.
Luminescent vibrios are widely used as models in the search for anti-QS products because this phenotype is only expressed when bacteria reach the quorum.
We assessed the effect of EOs on QS-mediated processes, such as biofilm formation and swarming motility.
“The two EOs (EOOv and EOMa) that negatively affected the bioluminescence production were also shown to affect biofilm inhibition in the four studied pathogenic vibrios.”
Vibrios form biofilms on the surfaces of a cement slab, plastic, and steel coupons elements widely used in shrimp farming systems.
Adhesion and proliferation within the biofilm are established mechanisms of pathogenesis and infection of some Vibrio species in shrimp.
When biofilm formation capacity is reduced, antibiotic resistance and pathogenesis potential are also reduced in the population of free-living vibrios.
Once a mature biofilm is established, it is very difficult to eliminate since the bacteria embedded in the biofilm exhibit a 1000-fold increased resistance to conventional antimicrobial agents, in this way limiting the possibilities of treatment.
“EOOv and EOMa showed a clear effect on preventing biofilm formation in the four vibrios evaluated in the present study. To our knowledge, it is the first report where EOOv is evaluated as an agent that prevents biofilm formation in pathogenic vibrios of P. vannamei.”
In addition to biofilms, another aspect that must be considered in tissue colonization is swarming motility, through which pathogenic vibrios can move collectively.
Vibrios are highly motile bacteria due to the rotation of the flagella that facilitate movement.
It has been proven that the swarming motility of several pathogenic vibrios of aquaculture interest is also positively regulated by QS, such is the case of V. harveyi, V. campbellii, V. lginolyticus.
The swarming motility of the vibrios allows them to develop a colonial bacterial population both inside and outside the host, form biofilms, and become resistant to antibiotics.
Interfering with vibrios swarming motility is essential to affect their virulence.
EOOv, and EOMa significantly reduced the swarming motility of the four pathogenic vibrios for the control group.
Swarming motility generates resistance to antibiotics since it facilitates close contact of the bacteria with antibiotics, resulting in greater acquired resistance.
In consequence, swarming motility is more significant in the presence of antibiotics.
“We observed EOs ability to inhibit swarming motility even in the presence of the antibiotic oxytetracycline, a result that indicates an additional application of EOs, potentiating antibiotics effectiveness.”
In the present study, the EOOv was the most efficient to inhibit QS-mediated processes in the four vibrios evaluated.
Most likely, the anti-QS activity observed is related to the considerable proportions of carvacrol (45.6%) and thymol (5.2%) present in this EOOv.
EOs have been widely used in food preparation and as a food preservative for human consumption for several decades.
Both EOOv and EOMa were tested in shrimp grow-out ponds, and greater survival and production yields were obtained.
In both in vivo trials, better results were obtained with the EOOv.
This result matches results obtained in vitro, in which the EOOv was more effective in arresting QS indicators.
EOs are aromatic and limpid substances that can be obtained from different parts of the plants, and their effectiveness is given by the proportions of the bioactive molecules being able to vary in a broad and diverse spectrum of action within the same plant genus.
“In addition, EOs from the same plant species can vary in their chemical composition, depending on the environmental and climatic conditions in which they grow, their maturity, and the extraction method.”
In this sense, it is important to mention that a disadvantage of the use of EOs to control vibriosis in shrimp farming is that they do not have a standard composition.
It is advisable to obtain EOs from guaranteed providers and evaluate each batch’s quality by means of controlled in vitro tests.
