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Microbiology and Biotechnology Letters

Research Article(보문)

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Environmental Microbiology (EM)  |  Microbial Ecology and Diversity

Microbiol. Biotechnol. Lett. 2024; 52(2): 179-188

https://doi.org/10.48022/mbl.2402.02003

Received: February 13, 2024; Revised: April 10, 2024; Accepted: April 15, 2024

Catecholamines (DOPAMINE) Increases the Virulence of Aeromonas hydrophila ATCC AH-1N, the Causative Agent of Motile Aeromonas Septicemia (MAS)

Yan Ramona1,2*, Ida Bagus Gede Darmayasa1, Ni Putu Widiantari3, Ni Nengah Bhawa Dwi Shanti1, Ni Luh Hani1, Pande Gde Sasmita Julyantoro4, Adnorita Fandah Oktariani5, and Kalidas Shetty6

1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Udayana, Bukit Jimbaran Campus, Badung-Bali 80631, Indonesia
2Integrated Laboratory for Biosciences and Biotechnology, Universitas Udayana, Bukit Jimbaran, Badung-Bali 80631, Indonesia
3Universitas Bali International, Denpasar, Bali-Indonesia
4Faculty of Marine Science and Fisheries, Universitas Udayana, Bukit Jimbaran, Badung-Bali 80631
5Department of Biology, Faculty of Mathematics and Sciences, Universitas Negeri Padang, West Sumatra, Indonesia
6Global Institute of Food Security and International Agriculture, North Dakota State University, USA

Correspondence to :
Yan Ramona,       yan_ramona@unud.ac.id

Abstract

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It has been widely documented that stress conditions in aquatic ecosystems could trigger the release of stress hormone (dopamine) in fishes. Such hormone could attract pathogens (such as Aeromonas hydrophila) to initiate its infection in fishes. The major focus of this study was to investigate the effect of the catecholamine derived stress hormone (dopamine) on the motility and hemolytic activity associated with the virulence of A. hydrophila ATCC AH-1N, the causative agent of Motile Aeromonas Septicemia (MAS). The density of bacterial cells used in this study was adjusted at 10p>6p> cells/ml. The results showed that dopamine increased swimming motility of A. hydrophila ATCC AH-1N and was proportional to both dopamine hormone concentration and the incubation period. Dopamine concentration of 100 μM in the medium resulted in the highest increment of swimming ability of A. hydrophila ATCC AH-1N. The dopamine hormone was also found to affect the hemolytic activity of A. hydrophila ATCC AH-1N. The optimum hemolytic activity of the pathogen was found at 50 μM dopamine concentration in the medium, and this hemolytic activity was found to decrease when the concentration of dopamine at greater than 50 μM. It can be concluded from this study that dopamine hormone increased the motility and hemolysis capability, as well as the growth rate of A. hydrophila, and hence increased its virulence.

Keywords: Aeromonas hydrophila, aeromonas septicemia, aquaculture, dopamine, hemolysis

Introduction

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The productivity of aquaculture-related food production system could be influenced by many factors, such as the quality of fish seed culture, production efficiencies linked to labour, production systems, environmental conditions and more importantly pathogen infection [1]. Among these factors, the challenge of pathogens in aquaculture-related production is one of the most important factors causing fish death, leading to decreased production and economic value. An important pathogenic bacterium commonly found to infect in freshwater aquaculture production environments is Aeromonas hydrophila [2]. A. hydrophila is an opportunistic bacterial pathogen that can infect fishes, amphibians, reptiles, and mammals in aquatic environments, and causing a disease known as Motile Aeromonas Septicemia (MAS) in domesticated and wild fishes, in particular [3].

Development of such infections by opportunistic bacterial pathogens in aquaculture systems involves complex interactions among pathogen’s virulence, pathogen’s host, and stress factors of the pathogen’s hosts [2]. The motility and hemolysis activity of the pathogens are among many factors commonly used to measure the pathogen’s virulence [4]. Motility capability is an advantageous factor for the pathogens to rapidly approach their host prior to causing infection to their hosts. Once the pathogens reach their host, they will release proteindegrading enzymes and cause lesions on the body surfaces of their hosts followed by entering the circulation system of the hosts [5]. In the host’s circulatory system, such pathogens and A. hydrophylla, in particular, start their activity to lyse the protective erythrocytes of the hosts [6]. The level of environmental stress has been reported to increase the virulence of A. hydrophylla, as in such conditions the immune system status of its hosts is at the lowest or weakest condition [7, 8]. This will result in difficulty for the fish to fight the invading pathogens.

Under such environmental stress conditions in the aquatic environment, fishes tend to release high levels of stress hormones, such as norepinephrine and dopamine. These hormones will stimulate pathogens through increasing their motility capability, to approach and infect those stressed fishes, such as observations made in Vibrio anguillarum and Vibrio campbellii [9]. A similar phenomenon was also observed by Gao et al. [8] who reported that the existence of norepinephrine increased the motility activity of A. hydrophila in their in vitro experiments. These phenomena indicate that these two stress hormones appeared to attract these bacterial pathogens to approach their hosts and cause severe infection in fish.

In Indonesia, including Bali where this research was undertaken, MAS was first recorded in 1980, and killed 125 tons of catfish in the aquaculture system in West Java [10]. Similar incidence of fish mortality due to MAS was also reported by Nitimulyo et al. [11] who found approximately 173 tons of goldfish death in aquaculture systems in Indonesia. This indicates that strategies to cope with such infections need to be developed. Therefore, comprehensive data obtained from basic research including how stress hormones affect the infection rate and to what extent such stress hormones affect the virulence level of the pathogens need to be generated so that the infection challenges can be strategically addressed in aquaculture systems.

Based on the above rationale, the effect of the stress hormone catecholamine (particularly dopamine) on the in vitro motility, hemolysis activity, and growth rate of A. Hydrophila ATCC AH-1N were elucidated. The information from this research is targeted to be important to generate basic data for developing future strategies to counter A. hydrophila infections in aquaculture systems. Therefore, the main objective of this research was to elucidate how catecholamine (dopamine) increased the virulence factors such as motility, hemolysis activity, and growth rate of A. hydrophila ATCC AH-1N. The novelty of this study is to advance the effect of stress hormone (Catecholamines or Dopamine in particular) on the virulence of Aeromonas hydrophila AH-1N, the causative agent of MAS in aquaculture system. This is a major challenge in fish processing industry in Indonesia and in fisheries exporting countries. By understanding the role of this hormone in the severeness of infection caused by this pathogen, we can advance and develop strategies to overcome such infection and improve food safety in fish processing industries.

Materials and Methods

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Isolate of A. hydrophila ATCC AH-1N and dopamine hormone

Pure culture isolate of A. hydrophila ATCC AH-1N was obtained from stock culture collections of the Microbiology Laboratory, Faculty of Marine Science and Fishery, Udayana University, Bali-Indonesia. Before use in the experiments, some important characteristics of the isolate were confirmed by conducting some important biochemical tests, including Gram staining, hemolysis test, motility test, oxidase, and catalase tests, towards the understanding the characteristics of the isolate [9]. Other materials such as dopamine (stress hormone), SAPI medium, and Luria Bertani Broth (LB Broth) used in our study were products of Sigma Aldrich, Germany and purchased from chemical suppliers in Jakarta, Indonesia.

Preparation of 5 mM Dopamine Stock Solution

Stock solution of dopamine (5 mM) was prepared by diluting 76.59 mg of dopamine (a product of Sigma- Aldrich, Germany, with a molecular weight of 153.18 g/ mol) in distilled water, and the final volume was adjusted to 100 ml [9]. This stock hormone solution was filter sterilized and stored at 4℃ until required in the bioassays. In the assays, sterile stock hormone solution was added into soft Nutrient Agar medium (Oxoid) with 30% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich- Germany), and the working concentrations were adjusted to 25 μM, 50 μM, 75 μM, and100 μM. The soft agar medium with 30% (v/v) FBS only (without the addition of dopamine) served as control.

Preparation of A. hydrophila suspension

This procedure adopted the method of Pande et al. [9]. Stock culture of A. hydrophila ATCC AH-1N was first streak inoculated on Luria Bertani Agar (LBA) medium and incubated at 37℃ overnight until single colonies of this isolate appeared on the plate. Suspension of this isolate was then prepared by inoculating a pure single colony into Luria Bertani Broth (LBB) medium. The inoculated medium was incubated at 37℃ for 24 h, centrifuged at 1500 × g for 5 min, and decanted. The pellet was washed twice with saline solution (0.9% w/v NaCl), centrifuged at 1500 rpm for 5 min, decanted, and re-suspended in saline solution to obtain bacterial turbidity of 0.5 according to McFarland scale (approximately equal to cell density of 108 cells/ml). For the motility and hemolysis tests, the bacterial suspension was diluted 100 times with saline solution to achieve cell density of 106 cells/ml. To estimate the generation time, the A. hydrophila AH-1N suspension was further diluted 10 times to achieve cell density of 105 cells/ml.

Swimming motility test

Swimming motility tests of the A. hydrophila were conducted on soft agar (0.3% b/v) + FBS 30% (v/v) medium with various working concentrations of dopamine (25 μM, 50 μM, 75 μM, and 100 μM) in the medium, following the method specified by Pande et al. [9]. Medium without the addition of dopamine served as control. A volume of 10 μl bacterial suspension previously prepared was subsequently spot inoculated in the center of the plates, incubated at 37℃ for 10 h, and observed for its growth, and the diameter of its growth was measured with a Vernier caliper. Five replicates per treatment were prepared, and the diameter of bacterial growth was measured from 4 different angles to obtain representative data.

Hemolysis activity test of A. hydrophila ATCC AH-1N

Hemolysis activity of the A. Hydrophila ATCC AH-1N was conducted on a blood agar medium in which the working concentrations of dopamine were adjusted to 25 μM, 50 μM, 75 μM, dan 100 μM. Blood agar medium without hormone in it served as control. The bacterial suspension at 10 μl was spot inoculated as in the motility test above, incubated at 37℃ for 48 h, and observed for inhibition zones appeared around the colonies. Each treatment was replicated 5 times with 4 angles of measurement on the inhibition zones, and the results were averaged to obtain representative data.

The effect of dopamine supplementation on the growth rate of A. hydrophila ATCC AH-1N

The generation time (doubling time) of A. hydrophila ATCC AH-1N was investigated in various types of medium (Lauria Bertani medium, Lauria Bertani+30% fetal bovine serum (FBS) medium, SAPI (Standard American Petroleum Institute) medium, and SAPI+30% FBS), all supplemented with 100 μM dopamine hormone. Standard American Petroleum Institute (SAPI) medium is a medium with minimal nutrients in it. This experiment was performed in Erlenmeyer flasks of 250 ml capacity with a total working volume of 50 ml. A volume of 100 μl of each medium was first removed from the flask then replaced with the same volume of bacterial suspension with cell density of 105 cells/ml previously prepared to achieve an initial cell density in each medium of 200 cells/ml. Following inoculation, all flasks were homogenized and subjected to optical density measurement with a spectrophotometer at 600 nm. All inoculated flasks were then incubated in a water bath at 37℃, with periodic sample collection (every 2 h) until the stationary phase of the isolate growth was reached. The generation time (Td) of the A. hydrophila ATCC AH-1N was estimated from its growth curve (on semi log determination) generated from each medium composition, during its exponential growth phase.

Based on the A. hydrophila ATCC AH-1N generation time (Td), the specific growth rates of the isolate were then estimated using the formula of μ=Ln2Td as specified in Ciawi et al. [12]. Five replications per treatment were conducted and the results were averaged to obtain representative data.

Data analysis

Quantitative data obtained in this experiment were analyzed using analysis of variance (ANOVA) with help of SPSS software for Windows version 23. If a significant difference was indicated at p < 0.05, the data was further analyzed using a multiple range analysis of Duncan at p < 0.05.

Results

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Tests for confirmation of A. hydrophila ATCC AH-1N

The bacterial isolate used in this study was confirmed as A. hydrophila ATCC AH-1N following specific tests for its characteristics, and the results are shown in Table 1. Colony (on McConkey agar medium) and cell morphologies of the isolate are shown in Figs. 1A and 1B, respectively, and this confirmed that the isolate used in our experiments was A. hydrophila ATCC AH-1N.

Table 1 . Some important characteristics of A. hydrophila ATCC AH-1N observed in the confirmation tests.

Characteristics testedObservation
Morphology of colony:
Colony morphologyround
Edgesmooth
Elevationconvex
Diameter of colony (mm)2.0
Colour of the colony on Blood Agar (BA) mediumgrey
Colour of the colony on MacConkey Agar mediumpink
Cellular morphology:
Shaperod
Gram stainGram-negative (-)
Biochemical tests:
Oxidase+
Catalase+
Citrate Simon+
TSIA test+
Motility test+
Glucose fermentation+
Maltose fermentation+
Manitol test+


Figure 1.Colony morphology of A. hydrophila ATCC AH-1N on McConkey agar medium (A) and cell morphology of the isolate following Gram staining and visualized under a microscope with 1000x magnification (B).

Motility test of A. Hydrophila ATCC AH-1N on soft agar medium with various hormone concentrations

The swimming ability of A. hydrophila ATCC AH-1N indicated by the colony's diameter on a soft agar medium supplemented with various concentrations of dopamine is shown in Table 2 and Fig. 2. Dopamine concentration in the medium appeared to significantly affect or proportionally related to the motility of the isolate.

Table 2 . The effect of dopamine concentration in the soft medium on the diameter growth of A. hydrophila ATCC AH- 1N, was measured after 7 and 10 h of incubation at 37℃, in the swimming capability test (motility test).

The concentration of dopamine (μM)Diameter of growth (mm)*
After 7 h of incubationAfter 10 h of incubation
024.15 ± 1.21a32.07 ± 0.83a
2525.63 ± 1.01a,b34.83 ± 1.06b
5026.29 ± 0.85b35.80 ± 0.47b
7526.86 ± 0.78b38.51 ± 1.12c
10036.56 ± 0.88c51.91 ± 1.07d

*Values in Table 2 ± standard deviation are averages of 5 replicate experiments with 4 different angles of measurement. Values in the same column followed by the same letter(s) are not statistically significant at p < 0.05 according to the multiple range test of Duncan, following Analysis of Variance (ANOVA).

Figure 2.Diameter of A. hydrophila ATCC AH-1N growth on soft agar medium supplemented with various concentrations of dopamine, 10 h of incubation at 37℃. Growth diameters of the isolate on control (1); 25 μM (2); 50 μM (3); 75 μM (4); and 100 μM (5).

When compared to the control (medium without hormone addition), the addition of dopamine into the culture medium significantly increased (p < 0.05) the diameter of growing colonies following incubation for 7 or 10 h, indicating that it’s swimming ability increased (Table 2). Application of dopamine at a concentration of 100 μM produced colonies with growth diameters of 36.56 ± 088 mm and 51.91 ± 1.07 mm following incubation for 7 and 10 d, respectively. These values are statistically significant when compared to all other treatments.

Hemolysis capability of A. hydrophila ATCC AH-1N due to the effect of dopamine

Hemolysis activity of the A. hydrophila ATCC AH-1N tested on Blood agar medium indicated that this isolate had the capability to induce lysis of red blood cells contained in the medium, which was indicated by the formation of clear zones around the bacterial colonies (Fig. 3). The capability of this isolate to induce lysis of red blood cells appeared to be affected by the addition of dopamine, and its concentration in the medium appeared to significantly affect the activity of the bacterial isolate to induce lysis of the red blood cells (Table 3).

Table 3 . Diameter of hemolytic zones of A. hydrophila ATCC AH-1N due to supplementation of blood agar medium with various concentrations of dopamine, measured at 24 and 48 h of incubations.

Dopamine concentrations (μM)Diameter of the haemolytic zone (mm)*
After 24 h incubationAfter 48 h incubation
0 (control)6.96 ± 0.19a9.38 ± 0.16a
258.29 ± 0.26b12.84 ± 0.64c,d
509.44 ± 0.16c13.20 ± 0.39d
757.64 ± 0.21b12.24 ± 0.26b,c
1007.26 ± 0.17a11.72 ± 0.40b

*Values in Table 3 ± standard deviation are averages of 5 replicate experiments with 4 different angles of measurements. Values in the same column followed by the same letter(s) are not statistically significant at p < 0.05 according to multiple range test of Duncan, following Analysis of Variance (ANOVA).

Figure 3.Diameter zones of hemolysis by A. hydrophila ATCC AH-1N on blood agar medium supplemented with various dopamine concentrations after 48 h incubation at 37℃. Control (1); 25 μM (2); 50 μM (3); 75 μM (4); and 100 μM (5).

When compared to control (medium without dopamine supplementation), all treatments produced significantly higher (p < 0.05) hemolytic activity of A. hydrophila ATCC AH-1N, following both 24 h and 48 h incubation (Table 3). The optimum dopamine concentration to induce hemolytic activity of A. hydrophila ATCC AH-1N was found to be around 50 μM. The concentration of dopamine of higher than 50 μM (i.e. 100 μM) appeared to reduce its hemolytic activity (Fig. 3, Table 3), although it is still significantly higher (p < 0.05) than the control (Table 3).

The effect of dopamine on the generation time of A. hydrophila ATCC AH-1N

Supplementation of the working concentration of 100 μM dopamine into all media examined was found to decrease doubling time or increase the specific growth rate of the A. hydrophila ATCC AH-1N isolate by 15.56% to 27.63% (Table 4). All values of doubling times as indicated in Table 5 are significantly affected (p < 0.05, when compared to control) by the supplementation of 100 μM dopamine in the medium.

Table 4 . Effect of dopamine supplementation on the doubling time of A. hydrophila ATCC AH-1N in various media.

Dopamine concentrationsGeneration times of A. hydrophila ATCC AH-1N in various media (hours)*
LBLB+30% FBSSAPI mediumSAPI+30% BFS
0 μM (control)2.75 ± 0.20a1.80 ± 0.24a3.90 ± 0.47a3.46 ± 0.50a
100 μM1.99 ± 0.14b1.52 ± 0.18b1.52 ± 0.18b2.65 ± 0.26b

*Values in Table 4 ± standard deviation are averages of 5 replicate experiments. Values in the same column followed by different letter(s) are significantly different at p < 0.05 according to multiple range test of Duncan, following Analysis of Variance (ANOVA). LB = Lauria Bertani; FBS = Fetal bovine serum; SAPI = Standard American Petroleum Institute.

Table 5 . The effect of dopamine supplementation on the specific growth rate (μ) of the A. hydrophila ATCC AH-1N grown in various types of media.

Dopamine concentrationsSpecific growth rate (μ) of A. hydrophila ATCC AH-1N in various media (per h)*
LBLB+30% FBSSAPI mediumSAPI+30% BFS
0 μM (control)0.25 ± 0.01a0.39 ± 0.05a0.17 ± 0.02a0.20 ± 0.02a
100 μM0.34 ± 0.02b0.46 ± 0.06b0.23 ± 0.04b0.26 ± 0.03b

**Values in Table 5 ± standard deviation are averages of 5 replicate experiments. Values in the same column followed by different letter(s) are significantly different at p < 0.05 according to multiple range test of Duncan, following Analysis of Variance (ANOVA). LB = Luria Bertani; FBS = Fetal bovine serum; SAPI = Standard American Petroleum Institute.

Types of the medium used also affected the growth rate of the isolate. The addition of BFS at a working concentration of 30% increased the specific growth rate (reduced the doubling time) of the isolate both in the LB medium and SAPI medium, and these values are statistically significant (p < 0.05) with their corresponding control (Table 4). The generation time of the A. hydrophila ATCC AH-1N presented in Table 4 was estimated from its growth curve (on semi-log estimation) within its exponential growth phase, and these are shown in Fig. 4.

Figure 4.Estimation of the generation time (doubling time/Td) of the A. hydrophila in various types of growth media. (A) Growth curve of A. hydrophila ATCC AH-1N in LB medium with 100 μM dopamine, but without addition of FBS; (B) Growth curve of A. hydrophila ATCC AH-1N in LB medium with 100 μM dopamine and 30% FBS; (C) Growth curve of A. hydrophila ATCC AH-1N in SAPI medium with 100 μM dopamine; (D) Growth curve of A. hydrophila ATCC AH-1N in SAPI medium with 100 μM dopamine and 30% FBS.

Based on the data in Table 4, the values of the specific growth rates (μ) of the A. hydrophila ATCC AH-1N grown in various types of media were calculated using the formula stated in the “Materials and Methods” section above, and the results are shown in Table 5. It is clear in Table 5 that the values of the specific growth rate (μ) are conversely correlated with the generation time. Both generation time and specific growth rate of A. hydrophila ATCC AH-1N are significantly affected by environmental factors, including substrates where the isolate is grown or cultivated.

Discussion

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Aeromonas hydrophila is an important pathogen in aquatic environments [13] with capability to infect various types of freshwater fishes, such as Nile tilapia [14] and cat fish [15] in aquaculture systems. Some important characteristics of this pathogenic isolate, as shown in Table 1, are in line with those specified in the Bergey’s Manual of Determinative Bacteriology [16] and those described by Pauzi et al. [17]. These important characteristics indicated that the isolate used in our study was confirmed to be A. hydrophila ATCC AH-1N. More recent studies conducted by Arwin et al. [18] also reported similar characteristics of A. hydrophila. This pathogen is known to have a wide range of tolerance to salinity changes, and therefore it can survive in almost all aquatic ecosystems [19].

The pathogenic level or virulence of this species is affected by many factors, in which the level of stress hormones in the body of its hosts has been reported as an important signal to infect its host [20, 21]. Dopamine, for example, has been reported by Pande et al. [9] to play an important role in increasing the virulence of several aquatic bacterial pathogens belonging to genus of Vibrio (such as Vibrio campbellii and Vibrio anguillarum). This is in line with the results of our study where dopamine supplemented in the medium increased the swimming capability of this pathogen (Table 2 and Fig. 2). A high level of dopamine in the host appeared to be an indicator for the pathogen to initiate its infection of its hosts. In other words, such hormone seems to function as an attractant (a signal) for the pathogen to initially attack its hosts. Under stress conditions, the level of such hormone tends to increase, and this is closely correlated with the immune level of the organisms or hosts of pathogens [8]. Under such stress conditions, the level of immunity of an organism tends to be at the lowest [7, 20, 22], and this will be used by the pathogens as signal to initiate their infection. The pathogen will increase their swimming ability (motility) to rapidly approach their host so that they can utilize the body components of their host as sources of energy [23]. Stress conditions also induce organisms (vertebrate or invertebrate) to release stress hormones. In addition to dopamine, they also release stress hormone belong to catecholamine class, such as epinephrine and norepinephrine (NE) [8].

The response (increase in its virulence) of A. hydrophylla to the existence of the stress hormone catecholamine is controlled by two genes, qseB and qseC, and the involvement of these genes were proven by Qin et al. [24]. Deletion of these two genes from the bacterial chromosome by these researchers was found to weaken the NEpromoted growth, biofilm formation, and hemolytic activity of this pathogen.

In the last decade, the role of the catecholamine stress hormone such as dopamine has been well documented to affect the motility, growth, as well biofilm formation of pathogens. Verbrugghe et al. [7] and Dong et al. [25] for examples reported that the growth rate, motility, and biofilm formation ability of Escherichia coli and Salmonella spp. increased due to elevated levels of the host’s stress hormone. This report is supported by our findings that supplementation of various media at 100 μM working concentration of dopamine significantly reduced its generation time or increased its specific growth rate (Table 4 and 5, Fig. 4). This indicated that the stress hormone dopamine applied at this level significantly increased the growth rate of the isolate. The mechanisms by which this stress hormone stimulates A. hydrophila ATCC AH-1N growth was not conclusive in our research. However, some previous studies, such as Dong et al. [25] and Perraud et al. [26] reported that the bacterial growth stimulation in response to catecholamines (norepinephrine, epinephrine, dopamine) could be due to an increase in the bacterial ability to chelate/ uptake ions of iron (Fe3+) in their surroundings. It was further stated by these two studies that the aromatic rings of the stress hormone structure could play as auxiliary siderophores (iron chelating agents) for the bacteria (Gram-negative in particular, including A. hydrophila). This could result in an increase in their ability to chelate iron (Fe3+), in iron-limiting environments. In more recent study by Gao et al. [8] also reported that catecholamine stress hormone norepinephrine (NE), in particular, had significant effect on the pathogenic capability of A. hydrophila. In their study, they also elucidated those changes in the expression of 13 out of 16 genes encoding virulence capability in this pathogen (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur genes) played significant roles in the enhancement of growth and virulence of the A. hydrophila.

In addition to improved motility and growth rate, the virulence factor of aquatic bacterial pathogens (e.g. A. hydrophila) is measured from their hemolytic capability (their capability to induce lysis of red blood cells). In our current study, addition of dopamine hormone at 50 μM into the medium was found to increase the hemolytic capability of A. Hydrophila ATCC AH-1N by 26.27% and 28.93%, following incubation times of 24 h and 48 h, respectively (Fig. 3 and Table 3). Our results are not in line with those reported by Gao et al. [8] who found that stress hormone norepinephrine did not affect hemolytic capability of A. hydrophylla, but it improved its protease activity. The reasons behind these different results are not really clear. Addition of dopamine into the medium at concentrations of higher than 50 μM (75 was 100 μM) was found to reduce hemolytic activity of A. hydrophylla ATCC AH-1N in our study (Table 3 and Fig. 3). This trend could be due to saturated concentration of the hormone in the medium has been reached. Similar result (effect of hormone concentration on the hemolytic activity) was also reported by Torabi Delshad et al. [27] who found that supplementation of 100 μM dopamine did not have any effect on the hemolytic activity of Yersinia ruckeri, although generally it increased the virulence of this pathogen.

Conclusion

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Dopamine hormone increased the motility and in vitro hemolytic activities of A. hydrophila ATCC AH-1N, and these indicated an increment of its virulence. The highest motility and hemolytic activities of this isolate occurred at the dopamine concentrations of 100 μM and 50 μM, respectively.

Acknowledgment

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The authors acknowledge the head of Microbiology, Faculty of Fishery and Marine Science and head of Microbiology of Faculty of Medicine, Udayana University for the provision of A. hydrophila ATCC AH-1N isolate and laboratory facilities, respectively during research. The financial support by the Udayana University research center and North Dakota State University for this research should also be acknowledged.

Author Contributions

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Conceptualization, Y.R., K.S., P.G.S.J., and I.B.G.D; methodology, K.S., P.G.S.J., and Y.R.; Software; Validation, K.S., P.G.S.J., I.B.G.D. and Y.R.; Formal analysis, K.S. and Y.R.; Investigation, A.F.O., N.P.W., N.N.B.D.S., and I.B.G.D.; Data curation, I.B.G.D., N.L.H. and AFO; Writing-original draft preparation, Y.R., and P.G.S.J.; Writing review and editing, Y.R. and K.S.; Visualization, N.P.W. A.F.O., and N.N.B.D.S.; Supervision, K.S., P.G.S.J., and Y.R.; Project administration, N.L.H., N.N.B.D.S, and A.F.O.; Funding acquisition, K.S. and Y.R. All authors have read and agreed to the published version of the manuscript.

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