Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Letters in Applied Microbiology 1997, 25, 269–273

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapia nilotica A.H. Khalil and E.H. Mansour Department of Food Science and Technology, Faculty of Agriculture, Menofiya University, Shibin El-Kom, Egypt 1319/96: received 22 October 1996 and accepted 26 March 1997 A .H . K H AL IL A ND E. H . M AN S OU R. 1997. Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic and proteolytic activity were studied with respect to temperature and time of incubation as well as the lethal toxicity on tilapia, Tilapia nilotica. The highest production of the haemolysin product was achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocyte was found to be more susceptible than sheep erythrocyte for determining the haemolytic activity. The haemolytic activity against tilapia erythrocyte was completely inactivated after heating the ECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized when the bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme was performed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonas hydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD50 2·1 × 104 cell/fish), as well as heat stable unknown virulent factors that were responsible for 20% mortality. The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min.

INTRODUCTION

Aeromonas hydrophila is widely distributed in nature and has been found as part of the normal microbial flora in fish and other aquatic animals (Kaper et al. 1981; Len 1987). Aeromonas hydrophila produces a variety of biologically active extracellular products (ECP), including enzymes and haemolysin compound (Wretlind et al. 1973; Shotts et al. 1985; Pansare et al. 1986; Gudmundsdo´ttir 1996). Allan and Stevenson (1981), Thune et al. (1986) and Santos et al. (1988) have reported that the toxic fraction of extracellular products is associated with the haemolytic activity, while Kanai and Wakabayashi (1984) and Sakai (1985) have shown that the proteases are the main virulence factors implicated in fish toxicity. The bacterium has been recognized as an active spoiler of fish under refrigeration (Sakai 1985; Pansare et al. 1986). The spoilage potential of Aer. hydrophila is assumed to be correlated to its ability to produce extracellular products that have a haemolytic and proteolytic activity. Since the conditions for the maximum observed haemoCorrespondence to: Dr A. H. Khalil, Department of Food Science and Technology, Faculty of Agriculture, Menofiya University, Shibin El-Kom, Egypt. © 1997 The Society for Applied Bacteriology

lysin and protease activity were not identified, the present study was conducted to determine the potential haemolytic and proteolytic activity of the crude extracellular products secreted by Aer. hydrophila with respect to incubation time and temperature, as well as to determine the heat stability. The lethal toxicity in Tilapia nilotica was also evaluated using live and heat-killed Aer. hydrophila and different preparations of ECP.

MATERIALS AND METHODS Bacterial strain and growth conditions

Aeromonas hydrophila N 122, isolated from mullet (Mugil cephalus), was kindly provided by Dr Soliman (Faculty of Veterinary Medicine, University of Alexandria, Egypt). The bacterium was usually subcultured on nutrient agar at 25°C for 48 h. Culture from slants was used to inoculate 250 ml of nutrient broth in 500 ml Erlenmeyer flasks. The flasks were incubated by shake culture at three different temperatures (25, 30 and 35°C) for 36 h. Samples were removed at specified time intervals (0, 6, 12, 18, 24, 30 and 36 h) and examined for bacterial growth by determining the optical density at

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Haemolytic activity of ECP was measured using a modification of the Kwapinski (1965) technique. Erythrocytes were washed with phosphate-buffered saline (PBS) at pH 7·2 and separated by centrifugation at 4000 g for 5 min. A standard 2% erythrocyte suspension was prepared so that 0·5 ml of completely lysed cells in a total assay volume of 5·0 ml would give an absorbance of 0·4 at 540 nm. The assay system contained 0·5 ml of standard erythrocyte suspension and 4·5 ml of appropriately diluted extracts containing the crude toxin. The mixtures were incubated for 1 h at ambient temperature. Reaction mixtures were centrifuged at 4000 g for 5 min and the absorbance of the supernatant fluid at 540 nm was measured. One unit of haemolytic activity was defined as the amount of toxin that gave an absorbance of 0·4 at 540 nm. Appropriate dilutions of the samples were prepared for measuring the haemolytic activity units. Proteolytic activity

Proteoltyic activity of ECP was measured as described by Sakai (1985). Casein was dissolved at 1% (w/v) in 0·1 mol l−1 glycine-sodium hydroxide buffer (pH 9·6) to be used as a substrate solution. One millilitre of ECP was added to 1 ml substrate solution and incubated at 30°C for 10 min. The reaction was stopped by adding 0·1 ml of 1 mol l−1 trichloroacetic acid and the precipitate was removed by centrifuging at 6000 g for 10 min. The absorbance of the supernatant fluid was measured at 280 nm. As a control, trichloroacetic acid was added at zero time. One unit of proteolytic activity was expressed as an increase of 0·01 in the absorbance value at 280 nm with casein as a substrate. Heat stability

The stability of haemolysin and proteases was measured by subjecting the samples to heat treatment ranging from 40 to 90°C for 10 and 15 min. After the heat treatment, the residual haemolytic and proteolytic activity was measured as described above.

RESULTS AND D ISCUSSION Haemolytic activity of ECP

The effect of incubation time and temperature on bacterial growth and haemolytic activity of ECP on tilapia erythrocytes is shown in Fig. 1. The optimum temperature for bacterial growth in nutrient broth medium was 30°C. At this temperature, the bacterial growth reached its maximum after 24 h of incubation time. The highest haemolytic activity was observed in the supernatant fluid of culture grown at 35°C for 30 h. For cultures grown at 30 and 35°C, the extracellular haemolysin products were secreted into the medium during the early exponential growth phase, progressively increased throughout the remainder of the exponential phase and degraded during the stationary phase. Cultures grown at 25°C showed the lowest bacterial growth and haemolytic activity. The haemolytic activity appeared in the supernatant fluid in almost mid-log phase, increased exponentially throughout 2

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which were serially diluted 10-fold with saline solution (0·85% NaCl). Control fish were injected with 0·1 ml of PBS. Fish were maintained for 5 d at room temperature (approx. 25°C) and mortalities were recorded. LD50 was calculated by the method of Reed and Muench (1938). Lethal toxicity to tilapia of various preparations from Aer. hydrophila culture grown at 30°C and 35°C was estimated. Culture supernatant fluid was sterilized using a 0·45 mn membrane filter. A portion of the membrane filtrate from both cultures was heated at 60, 80 and 100°C for 10 min. The precipitated cells from Aer. hydrophila culture were resuspended in PBS at a concentration of 1 mg ml−1. A portion of the resuspended cells was heated at 100°C for 10 min. PBS was used as a control. The lethal toxicity of each preparation was measured by injecting 0·1 ml intraperitoneally into tilapia.

Bacterial growth (optical density)

610 nm. Bacterial cells were removed from culture broth samples by centrifuging at 9750 g for 10 min at room temperature. The supernatant fluids were used as a source of crude extracellular products (ECP) for the determination of the haemolytic and proteolytic activity.

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Fig. 1 Effect of incubation time and temperature on Aeromonas

Tilapia nilotica was used for determining the LD50. Groups consisting of 10 fish (30–35 g) each were injected intraperitoneally with 0·1 ml of viable bacterial cell suspensions,

hydrophila growth and haemolytic activity of ECP against tilapia erythrocytes. , growth at 35°C; , 30°C; r, 25°C; ž, haemolytic activity at 35°C; R, 30°C; Ž, 25°C

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the log phase and was followed by a slow decline during the stationary phase. These results indicated that Aer. hydrophila N 122 grown in nutrient broth medium at 35°C for 30 h had a high haemolytic activity against tilapia erythrocytes. Similar results were reported by Sakai (1985), who found that the supernatant fluid from Aer. salmonicida haemolysed the erythrocytes of sockeye salmon. Data illustrated in Fig. 2 indicate that tilapia erythrocytes had a higher sensitivity to culture supernatant fluids than sheep erythrocytes. These results are consistent with the findings of Toranzo et al. (1983), who showed different susceptibility between rainbow trout blood and sheep blood when exposed to culture filtrate from Vibrio anguillarum. Heat stability of haemolytic ECP

Heat stability of haemolytic ECP from Aer. hydrophila at various temperatures from 35 to 100°C for 10 and 15 min is shown in Fig. 3. The results indicated that the haemolytic activity against tilapia erythrocytes gradually decreased with

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Proteolytic activity of ECP

The proteolytic activity of ECP was evaluated from cultures grown at different temperatures and incubation times (Fig. 4). The results indicated that the highest bacterial growth and proteolytic activity of ECP were obtained when the bacterium was grown at 30°C. The proteolytic enzyme was excreted into the medium during the late exponential growth phase and continued to increase sharply until early in the stationary phase. The ECP from cultures grown at 35°C did not show any proteolytic activity, while cultures grown at 25°C showed moderate growth and proteolytic activity. It is interesting to note that the highest haemolytic activity of ECP was obtained from cultures grown at 35°C (Fig. 1), which did not show any proteolytic activity. From these results, it can be concluded that the potential toxicity of the ECP might vary due to culturing at different incubation temperatures (Venugopal et al. 1983). Heat stability of proteolytic ECP

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Fig. 2 Haemolytic activity of ECP from cultures grown at 35°C

against tilapia and sheep erythrocytes. , tilapia; Ž, sheep. One unit of haemolytic activity was defined as the amount of toxin that gave an absorbance of 0·4 at 540 nm

Figure 5 shows the thermal inactivation profile for the proteolytic activity of ECP from cultures grown at 30°C for 36 h. The proteolytic activity of ECP was relatively stable when heated for 10 min at 60°C (80% activity) or for 15 min at 50°C (75% activity). Increasing the temperature to more than 60°C caused a sharp decrease in the proteolytic activity. Complete inactivation of the proteolytic enzymes was observed after heating the ECP at 70°C for 15 min or 80°C for 10 min. Similar thermal inactivation of ECP was reported by Pansare et al. (1986) and Santos et al. (1988), who stated

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Fig. 3 Heat stability of extracellular haemolysin treated at different temperatures for 10 and 15 min. , 10 min; Ž, 15 min. 100% haemolytic activity  the highest haemolytic units of ECP from cultures grown at 35°C

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increasing temperature. It was completely inactivated at 55 and 60°C for 15 and 10 min, respectively. Thermal inactivation of ECP from Aeromonas spp. was also observed by Santos et al. (1988).

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Fig. 4 Effect of incubation time and temperature on Aeromonas hydrophila growth and proteolytic activity of ECP. , growth at 35°C; , 30°C; r, 25°C; ž, proteolytic activity at 35°C; R, 30°C; Ž, 25°C

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Proteolytic activity (%)

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Fig. 5 Heat stability of extracellular protease treated at different

temperatures for 10 and 15 min. , 10 min; Ž, 15 min. 100% proteolytic activity  the highest proteolytic units of ECP from cultures grown at 30°C

that protease activity was gradually and thoroughly lost after heating the ECP of Aeromonas spp. at 80°C for 15 min. The LD50 at 5 d for the viable cells of Aer. hydrophila in tilapia was 2·1 × 104 cells. The strain used in the present study was included in the high virulent category (LD50 104– 105) by Mittal et al. (1980). The toxicity of Aer. hydrophila to tilapia was greater than that reported by Thune et al. (1986), who found the LD50 in channel catfish ranged from 2·8 × 104 to 1 × 109 cells. Lethal toxicity of bacteria and ECP

Table 1 records the lethal toxicity of different preparations from culture supernatant fluid from live and killed cells of Aer. hydrophila. The sterilized supernatant fluid from cultures grown at 35°C and 30°C killed all tilapia injected (100% mortality) at different rates, while live cells caused 90% mortality. On the other hand, heat-killed cells and phosphate buffer used as a negative control did not cause any mortality. Heat-treated ECP at 60°C and 80°C for 10 min showed less Table 1 Lethal toxicity of Aeromonas hydrophila to tilapia

— ––––––––––––––––––––––––––––––––––––––––––––––––––––– Mortality Death time Treatment (%) (h) — ––––––––––––––––––––––––––––––––––––––––––––––––––––– ECP (35°C) Unheated 100 12 ECP (35°C) Heated 60°C 20 72 Heated 80°C 20 72 Heated 100°C 0 0 ECP (30°C) Unheated 100 24 ECP (30°C) Heated 60°C 60 72 Heated 80°C 20 72 Heated 100°C 0 0 Live cells 90 29 Heat-killed cells 0 0 PBS 0 0 — –––––––––––––––––––––––––––––––––––––––––––––––––––––

mortality (60% and 20%, respectively) than that of untreated ECP. The active principle of the ECP was completely lost after boiling for 10 min. Fish mortality occurred from 12 to 72 h after intraperitoneal injection. Also, the unheated ECPs from cultures grown at either 30°C or 35°C were more effective in killing tilapia than the respective live cells (higher mortality and shorter time to death). Similar mortality results were obtained by Inamura et al. (1984) for Vibrio anguillarum against goldfish. Liu et al. (1996) reported that Vibrio harveyi exhibited strong protease, phospholipase and haemolytic activities and caused mortality in tiger prawn. It is interesting to note that the ECP obtained from cultures grown at 35°C, which had a haemolytic active principle only, was more potent in tilapia than that of the ECP obtained from cultures grown at 30°C, which had haemolytic and proteolytic activity. These results could suggest that the lethality of ECP of Aer. hydrophila is associated with haemolytic activity rather than proteolytic activity. The ECP from cultures grown at 35°C, which had haemolytic activity, showed 100% mortality, whereas ECP heated at 60°C for 10 min, which had no haemolytic activity (Fig. 3), showed 20% mortality. Increasing the temperature up to 80°C for 10 min showed 20% mortality, which might be due to some other heat-stable virulent factors in ECP. Complete inactivation of the haemolysin toxin and unknown virulent factors was achieved by heating the ECP to 100°C. The ECP from cultures grown at 30°C, which had proteolytic and haemolytic activity, showed 100% mortality, whereas ECP heated at 60°C for 10 min, which had no haemolytic and 81% proteolytic activity (Figs 3 and 5), showed 60% mortality. Increasing the temperature up to 80°C for 10 min (no haemolytic or proteolytic activity) showed 20% mortality, which might be due to some other virulent factors in ECP. Complete inactivation of the toxins was achieved by heating the ECP to 100°C. It can be concluded that Aer. hydrophila produces haemolytic and proteolytic exotoxin lethal to tilapia, as well as unknown heat-stable virulent factors that are responsible for 20% mortality. The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. These decreases in lethality were mainly due to the inactivation of haemolytic and proteolytic activities. The virulence of ECP was closely associated with the production of the haemolysin compounds.

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