Incidence and toxigenicity of Aeromonas hydrophila in seafood

International Journal of ELSEVIER

Food

Microbiology

31 (1996) 121-131

Incidence and toxigenicity of Aeromonas hydrophila in seafood Guo-Jane

Tsai*, Tzy-Huah

Chen

Department of Marine Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, Taiwan 20224, ROC Received

21 February

1995; revised

31 July 1995; accepted

30 October

1995

Abstract

Three selective media, Oxoid Aeromonus agar (OA), blood ampicillin agar (BA) and starch ampicillin agar (SA) were used to evaluate the presence of Aeromonas hydrophila in 66 samples of oyster, shrimp, fish and surimi products. Oyster had the highest incidence, with 50% positive, whilst no A. hydrophilu was found in the surimi. Of the three selective media, BA displayed the highest recovery rate of A. hydrophilu from seafood. Forty-eight isolates from this survey were tested for their capability to produce hemolysin and cytotoxin. Hemolysin was produced by 79.2% of the isolates and cytotoxin was produced by 91.7% of the isolates in brain heart infusion broth. One of the toxin-producing isolates from oyster, strain 8-169, was further tested for growth and toxin production in oyster, shrimp and fish at various temperatures. This particular isolate grew best and had highest toxin production in oyster. Hemolysin and cytotoxin were produced earlier at 28°C than at 37°C and titers of hemolysin were also higher at 28°C. At 5°C it was able to grow and produce hemolysin in oyster. Keywords:

Aeromonas

* Corresponding

0168-1605/96/$15.00

hydrophila;

Seafood; Cytotoxin;

0 1996 Elsevier

Science B.V. All rights

Hemolysin

author.

PII SO168-1605(96)00972-S

reserved

122

G.-J. Tsai, T.-H. Chen 1 Int. J. Food Microbiology 31 (1996) 121-131

1. Introduction Aeromonus hydrophila is a well-known pathogen for aquatic animals (De Figueiredo and Plumb, 1977; Austin and Allen-Austin, 198.5; Len, 1987). Recently it has been reported as a cause of gastroenteritis in humans (Goodwin et al., 1983; Holmberg and Farmer, 1984; Agger et al., 1985; Ljungh and Wadstrom, 1985) and food is suspected as the vector in the dissemination of this pathogen (Abeyta et al., 1986). It was announced as a ‘new’ foodborne pathogen in 1984 by the FDA (Buchanan, 1984). A. hydrophiia is widely distributed in various foods (Hood et al., 1983; Okrend et al., 1987; Alur et al., 1989; Knochel and Jeppesen, 1990; Hudson and de Lacy, 1991) and various water supplies (Clark et al., 1982; Burke et al., 1984; Slade et al., 1986; Krovacek et al., 1989; Krovacek et al., 1992). It can grow well at 4-5X (Palumbo et al., 1985b; Palumbo, 1987; Palumbo, 1988), in vacuum-packaged food (Hudson et al., 1994) or in 36% CO,-content atmosphere (Berrang et al., 1989), thus certain preservation methods for food, such as refrigeration, vacuum package or controlled atmosphere storage, cannot inhibit its growth effectively. The mechanism for this organism in food to elicit gastroenteritis is not well documented. In food, such preformed toxins as cytotoxin or hemolysin are considered a potential cause of its pathogenicity (Burke et al., 1981; Todd et al., 1989; Majeed and Mac Rae, 1991; Kirov and Brodribb, 1993). It has been the objective of this study to survey the distribution of A. hydrophilu in seafood in Taiwan by using three selective media, and to test the ability of A. hydrophila isolates to produce cytotoxin and hemolysin, and to investigate the effect of temperature on growth and toxin production by A. hydrophila in seafood.

2. Materials and methods 2.1. Seafood samples Oysters (Cvassotreas gigas), shrimps (grass shrimp and sword prawn), fish fillets (Alaska pollock, sword fish and tuna), and surimi products (fish balls) were purchased from local retail markets in regular consumer packages. All samples were stored in a portable refrigerator and analyzed within 24 h of purchase. 2.2. Media Starch-ampicillin agar (SA) containing 10 pg/ml of ampicillin was prepared based on the method of Palumbo et al. (1985a). Oxoid Aeromonas ampicillin agar (OA) (Oxoid CM833 with 5 pgg/ml of ampicillin) and blood ampicillin agar (BA) (Oxoid CM 271 with 5% defibrinated sheep blood and 10 pg/ml of ampicillin) were prepared as per the manufacturer’s instructions (Oxoid, Basingstoke, UK).

G.-J. Tsai, T.-H. Chen 1 Int. J. Food Microbiology 31 (1996) 121-131

123

2.3. Bacterial analysis Twenty-five grams of sample were homogenized with 225 ml of 0.1% peptone water using a Stomacher (Seward, Lab-Blender 400, USA) for 1 min. Serial dilutions were made with 0.1% peptone water and plated on the various media. The SA plates were incubated at 28°C and the OA and BA plates at 35°C. After 24 h of incubation, the SA plates were flooded with 5 ml Lug01 iodine solution (Palumbo et al., 1985a). The yellow to honey colored colonies surrounded by a clear zone were scored as presumptive Aeromonus spp. The colonies on BA plates with a clear surrounding zone, and the dark green colonies on OA plates, were also enumerated as presumptive Aeromonas spp. Five presumptive Aeromonas spp. colonies were picked randomly from each plate and transferred to nutrient agar (NA) (Difco, Detroit, MI) slants for further tests. After overnight incubation at 28°C the Gram’s stain and oxidase reaction were determined. All Gram negative and oxidase positive isolates were further analyzed by the following biochemical tests: O/F test, esculin hydrolysis, L-arabinose utilization and salicin fermentation. Isolates with positive reactions for the above biochemical tests were further confirmed by API 20E kit (bioMCrieux, Marcy-l’Etoile, France) according to the manufacturer’s in$ructions. Isolates confirmed as A. hydrophih were investigated further for toxin production.

2.4. Culture conditions for toxin production Isolated strains were inoculated into 2 ml tryptic soy broth (TSB, Difco) and incubated at 28°C for 18-24 h; thereafter, 0.1 ml culture was transferred to 10 ml brain heart infusion broth (BHIB, Difco) in a 50 ml flask and incubated in a shaker (100 rpm) for 20 h at 37°C. The cultures were then centrifuged (12 000 x g) at 5°C for 45 min. The supernatants were filtered through a 0.22 pm membrane (Gelman, Ann Arbor, MI, USA). The sterile filtrates were stored at 5°C for hemolysin and cytotoxin testing.

2.5. Hemolysin assay The protocol used for hemolysin detection was as described by Kozaki et al. (1987) with some modifications. Fifty microlitres volumes of two-fold dilutions of cell-free filtrate in 0.01 M Tris-HCl buffered saline (pH 7.2) were added to equal volumes of a 2% suspension of sheep erythrocytes in microtiter plates (Corning, New York, USA) and incubated at 37°C for 1 h. Then the cells were spun down (400 x g, 15 min) and the supernatant in each well was transferred to another microtiter plate, and read at 570 nm by a Plate Reader (Dynatech, Model 5000, Wan Chai, Hong Kong). The absorbance from the lytic reaction of 0.05% saponin on sheep erythrocytes was calculated as 100% hemolysis. The titer of hemolysin was defined as the reciprocal of the highest dilution of culture filtrate that caused 50% hemolysis.

124

2.6. Cytotoxin

G.-J. Tsai, T.-H.

Chen / Int. J. Food Microbiology

31 (1996) 121-131

assay

Tests for cytotoxin were performed in 96-well microtiter plates with 100 ~1 of culture filtrate added to equal volumes of HeLa cell suspension ( lo5 cells/ml) maintained in eRDF medium (Kyokuto, No. 6090, Tokyo, Japan) with 4.77 g/l HEPES added. After the plates were incubated at 37°C under 5% CO, for 24 h, the cell viability was tested using MTT (3-[4,5-dimethylthazol-2-y1]2,5_diphenyltetrazolum bromide) assay, as described by Mosmann (1983). The titer of cytotoxin was defined as the reciprocal of the highest dilution of culture filtrate that caused the death of all cells in each well. 2.7. Effect of temperature on growth and toxin production in oyster, shrimp and fish A. hydrophila isolate No. 8-169 was grown in BHIB at 28°C for 18 h. After centrifugation (12 000 x g, 45 min) and washing twice with sterile phosphate buffer diluent, the cell pellet was suspended in sterile phosphate buffer at a concentration of lo3 cfu/ml. Oyster, shrimp and fish were cut into small pieces (about 2 g per piece) and 20 samples of 25 g each were weighed into flasks. After sterilization at 121°C for 20 min and cooling down to room temperature, each flask was inoculated with 1 ml cell suspension (lo3 cfu/ml) and incubated at 5°C 28°C or 37°C without agitation. At various intervals thereafter, two duplicate flasks were sampled and 25 ml phosphate buffer diluent added to each flask. These samples were homogenized using a Stomacher for 1 min and a portion of sample homogenate was used for enumeration of A. hydrophilu on NA plates. The rest was centrifuged (12000 x g, 45 min) and the supernatant was filtered through a 0.22 pm membrane. The filtrate was analyzed for hemolysin and cytotoxin as described above. In another trial, growth and toxin formation was examined in fish homogenates. Fish meat (25 g) was added to 225 ml deionized water and homogenized for 2 min using a blender (Osterizer, USA). This fish homogenate was divided into 25 g samples and placed into flasks. Each flask was then sterilized, inoculated with cells, incubated and sampled as described above. Samples were enumerated for A. hydrophifu on NA plates and examined for its titers of hemolysin and cytotoxin.

3. Results Of the 66 seafood samples examined, 17 (25.8%) were found to be contaminated with A. hydrophila (Table 1). The contamination rates of A. hydrophila in oyster, fish and shrimp were 50, 22.2 and 14.3%, respectively. None of the surimi products tested were found to be contaminated with A. hydrophilu (Table 1). In comparing the efficiency of the three selective media for the isolation of A. hydrophila, it was found that BA was the most efficient. The detection rates of A. hydrophila in 66 seafood samples for BA, SA and OA were 22.7 (15/66), 7.6 (5/66) and 1.5 (l/66)%,

G.-J. Tsai, T.-H. Chen / Int. J. Food Microbiology 31 (1996) 121-131 Table Percent

125

1 of Aeromonus hydrophilu in samples

of distribution

Samples

Number

Oyster Fish Shrimp Surimi Total

22 18 14 12 66

sampled

Number

positive

11 4 2 0 17

of oyster,

Percentage

positive

50.0 22.2 14.3 0.0 25.8

shrimp,

fish and surimi

Range of A. hydrophila count (log cfu/g) 3.2-5.8 1.9-3.9 2.1-2.1

respectively (results not shown). Forty-eight isolates of A. hydrophila from this survey were tested for cytotoxin and hemolysin production (Table 2). Of 30 isolates from oyster, 22 (73.3%) strains released hemolysin into the culture medium (BHIB) and 28 strains (93.3%) could produce cytotoxin. Fourteen (87.5%) out of 16 isolates from fish fillet could produce extracellular hemolysin and cytotoxin. Both of two isolates from shrimp could also produce both toxins. Of the 48 isolates examined, 38 (79.2%) produced extracellular hemolysin and 44 (91.7%) produced cytotoxin (Table 2). Isolate No. 8-169 was chosen for the following experiment because it had the highest titers for both hemolysin and cytotoxin, 111 and 256, respectively. As seen from Fig. 1 similar growth rates were obtained for A. hydrophila isolate No. 8-169 cultured in sterile oyster at 28°C and 37°C; the highest titers of cytotoxin were identical at both temperatures except that the peak for cytotoxin appeared earlier at 28°C. However, more hemolysin was produced at 28°C than at 37°C (Fig. IA and Fig. 1B). This strain grew and produced some hemolysin in oyster at 5°C (Fig. 1C). In sterile shrimp (Fig. 2), the cell counts of this isolate at 28°C and 37°C were similar during the first 40 h, but the number at 28°C continued to increase thereafter. The titers of cytotoxin at 28°C were also higher than at 37°C (Fig. 2A and Fig. 2B). Although no detectable toxin was produced, this strain also grew in shrimp at 5°C (Fig. 2C). However, it could not grow in heat-sterilized fish cubes at any of the temperatures tested (Fig. 3). It grew well and produced both cytotoxin and hemolysin in fish homogenate at 28°C but not at 37°C or 5°C (Fig. 4). Table 2 Percent of hemolysin types of foods Source

of isolate

Oyster Fish Shrimp Total “Number

and cytotoxin

Number

30 16 2 48 in parenthesis

indicates

producing

examined

strains

Hemolysin

of Aeromonas hydrophila isolated

positive

Cytotoxin

from different

positive

WI

Titer range

(“Al)

Titer range

73.3 (22) 87.5 (14) 100 (2) 79.2 (38)

2-111 8-91 4-5 2-111

93.3 (28) 87.5 (14) 100 (2) 91.7 (44)

8-256 64-256 16-32 8-256

the number

of positive

isolates.

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G.-J. Tsai, T.-H.

Chen / Int. J. Food Microbiology

31 (1996) 121-131

140 120 100 80 60

8

40 20

.-

0

1-0 0

20

20

40

40

60

60

80

80

100

100

120

140

120

160

0 140

180

Time (h) Fig. 1. Changes in cell numbers and titers of hemolysin and cytotoxin in oyster spiked with Aeromonas isolate (No. 8-169) and incubated at 37°C (A), 28°C (B) and 5°C (C).

hydrophila

4. Discussion Recently, various media have been suggested for the isolation and enumeration of A. hydrophilu from clinical specimens or foods, such as BA (Robinson et al., 1984) SA (Palumbo et al., 1985a) and OA (Pin et al., 1994). Ampicillin was added as a selective agent in these media. The results from the present survey showed that the detection rates of A. hydrophila using SA and OA plates were lower than when BA plates were used. This might result from the fact that SA and OA could not differentiate A. hydrophilu from A. caviae or A. sobria. When we randomly selected the presumptive colonies from these plates, there were high probabilities that these colonies would, in fact, be A. caviae or A. sobria; especially probable were colonies of A. sobriu on OA plates. The concentration of ampicillin suggested by the manufacturer for OA was half (5 ppm) of that in BA or SA (10 ppm). This lower antibiotic concentration favored the growth of A. sobriu on the OA plates (Pin et al., 1994). On the other hand, both A. hydrophila and A. caviae are known to grow well on SA plates. The recovery rates of pure cultures of A. hydrophila and A.

G.-J. Tsai, T.-H. Chen 1 Int. J. Food Microbiology 31 (1996) 121-131

127

caviue from food by SA plates were 95.8% and 94.6%, respectively (Pin et al., 1994). Callister and Agger (1987) found 48 and 26% of isolates from SA plates were A. hydrophila and A. caviae, respectively. They also found that 90% of A. hydrophilcr isolates were hemolysin positive while only 6% of A. cauiue isolates produced hemolysin. Therefore, since both ampicillin (10 ppm) and hemolysin production are used effectively for the selection and differentiation of A. hydrophilu, this could account for the higher detection rates on the BA plates. Several reports have indicated a high incidence of Aeromonas spp. in poultry, beef, fish and shellfish (Abeyta et al., 1986; Okrend et al., 1987; Fricker and Tompsett, 1989; Hudson and de Lacy, 1991; Hanninen, 1993). However, there are few systematic studies on the incidence and toxigenicity of A. hydrophila in seafood from retail stores. The contamination rate of A. hydrophilu in oyster and fish were 50 and 22.2%, respectively (Table 1). Similar contamination rates of 58% in shellfish in New Zealand (Hudson and de Lacy, 1991) and 19.1% in fish in the Reading area in the UK (Fricker and Tompsett, 1989) have been reported. The high incidence of (A)

::L-

0

SO

1

20

40

60

80

100

I20

140

1

10 9 8 7 6 5 4 3 2 1

20 40 30 50 10

% Ii

0 0

1-o 0

20

20

40

40

60

60

80

80

100

loo

120

120

140

160

180

Time @)

Fig. 2. Changes in cell numbers and titers of hemolysin and cytotoxin in shrimp spiked with Aeromonas hydrophilu isolate (No. 8-169) and incubated at 37°C (A), 28°C (B) and 5°C (C).

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G.-J. Tsai, T.-H. Chen / Int. J. Food Microbiology 31 (1996) 121L131

(4 7

SO

6

40

5

30

4 20

3

10

2 I a,,, 0

20

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80

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4 3

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0 IO0 I20 140 160 180

Fig. 3. Changes in cell numbers, and titers of hemolysin and cytotoxin in fish spiked with Aeromonas hydrophila isolate (No. 8-169) and incubated at 37°C (A), 28°C (B) and 5°C (C).

A. hydrophila in oyster indicates the high potential

of infection from eating raw oyster. Moreover, as very high percentages of the isolates from oyster can produce hemolysin (73.3%) and cytotoxin (93.3%), the risks associated with eating raw oyster are further increased. Although the exact mechanisms for A. hydrophila to produce hemolysin and/or cytotoxin are unclear, it seems that the source of A. hydrophila isolate is critical for the selection of toxin-producing strains. Fricker and Tompsett (1989) found the percentage (61.5%) of cytotoxin-producing isolates of Aeromonas species from fish was much higher than those from red meat or poultry. Mateos et al. (1993) found 100% of the isolates from infected fish produced both hemolysin and cytotoxin whilst only 55.5% of clinical isolates produced these toxins. The results from our survey also showed that very high percentages of the isolates from seafood are able to produce hemolysin (79.2%) and cytotoxin (91.7%). Therefore, seafood seems to be a good source for toxin-producing isolates of A. hydrophila.

Food composition and temperature both significantly affected the toxin productivity of A. hydrophila isolate No. 8-169. Oyster was a better medium than shrimp

G.-J. Tsai, T.-H. Chen / Inl. J. Food Microbiology 31 (1996) 121-131

129

for both toxin production and growth. More hemolysin in oyster (Fig. 1) or cytotoxin in shrimp (Fig. 2) was produced at 28°C than at 37°C. Mateos et al. (1993) also found higher hemolysin titers were obtained at 28°C than at 37°C for the environmental isolates of A. hydrophila. Although refrigeration (5OC) retarded the growth of A. hydrophila, some detectable hemolysin was produced at this temperature. Therefore, prolonged cold storage of oyster should not be encouraged. The reason that this strain could not grow in heat-sterilized fish cubes (Fig. 3) might be related to the availability of nutrients, as the surface texture of the fish became tough after the sterilization at 121°C. In contrast, at least at 28°C it was able to grow well in fish homogenate (Fig. 4B) because the nutrients had been extracted with deionized water during homogenization and were therefore available. That this isolate could not grow in fish homogenates at 37°C or 5°C (Fig. 4A and Fig. 4C) may also be linked with the availability of nutrients at marginal growth conditions. Mateos et al. (1993) found the environmental isolates of A. hydrophila produced caseinase and elastase at 28°C but not at 37°C. Before a conclusive explanation can be drawn, more research is needed, in particular into the physical (4 7

50

6

40

5

30

4

20

3

10

2 1u 0

20

40

60

80

100

120

,400

70 60 50 40 30

B %

20 10 v

0

0 20

40

60

Fig. 4. Changes in cell numbers, and titers of hemolysin and cytotoxin in fish homogenates inoculated with Aeromonas hydrophila isolate (No. 8-169) and incubated at 37°C (A), 28°C (B) and 5°C (C).

130

G.-J. Tsai, T.-H. Chen 1 Int. J. Food Microbiology 31 (1996) 121L131

and biochemical changes of fish meat after heating, the growth factors found in fish meat, and changes in the enzymatic systems of this A. hydrophilu isolate at different temperatures.

Acknowledgements We would like to thank Dr. Z.L. Kong for his valuable assistance in cell culture techniques. This study was funded in part by the Ministry of Health (DOH 83-TD-053).

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Hudson, J.A., Mott, S.J. and Penney, N. (1994) Growth of Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica on vacuum and saturated carbon dioxide controlled atmosphere-packaged sliced roast beef. J. Food Prot. 57, 2044208. Kirov, S.M. and Brodribb, F. (1993) Exotoxin production by Aeromonas spp. in foods. Lett. Appl. Microbial. 17, 2088211. Knochel, S. and Jeppesen, C. (1990) Distribution and characteristics of Aeromonas in food and drinking water in Denmark. Int. J. Food Microbial. 10, 317-322. Kozaki, S., Kato, K., Asao, T., Kamata, Y. and Sakaquchi, G. (1987) Activities of Aeromonas hydrophila hemolysins and their interaction with erythrocyte membranes. Infect. Immun. 55, 15941599. Krovacek, K., Peterz, M., Faris, A. and Mansson, I. (1989) Enterotoxigenicity and drug sensitivity of Aeromonas hydrophila isolated from well water in Sweden: a case study. Int. J. Food Microbial. 8, 149-154. Krovacek, K., Faris, A., Baloda, S.B., Peterz, M., Lindberg, T. and Mansson, I. (1992) Prevalence and characterization of Aeromonas spp. isolated from foods in Uppsala, Sweden. Food Microbial. 9, 29936. Len, P.P. (1987) Mesophilic spoilage of marine fish: bay trout (Arripis trutta), bream (Acanthopagrus butcheri) and mullet (Aldrichetta forsteri). Food Technol. Aust. 39, 2777282. Ljungh, A. and Wadstrom, T. (1985) Aeromonas and Plesiomonas as possible cause of diarrhea. Eur. J. Clin. Study Treatment Inf. 13, 1699175. Majeed, K.N. and Mac Rae, I.C. (1991) Experimental evidence for toxin production by Aeromonas hydrophila in a meat extract at low temperature. Int. J. Food Microbial. 12, 181- 188. Mateos, D., Anguita, J., Naharro, G. and Paniagua, C. (1993) Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila. J. Appl. Bacterial. 74, 1I 1~118. Mosmann, T. (1983) Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J. Immunol. Methods 65, 55563. Okrend, A.J.G., Rose, B.E. and Bennett, B. (1987) Incidence and toxigenicity of Aeromonas species in retail poultry, beef and pork. J. Food Prot. 50, 5099513. Palumbo, S.A. (1987) Can refrigeration keep our foods safe? Dairy and Food Sanitation 7, 56-60. Palumbo, S.A. (1988) The growth of Aeromonas hydrophila k144 in ground pork at 5°C. Int. J. Food Microbial. 7, 41-48. Palumbo, S.A., Maxino, F., Williams, A.C., Buchanan, R.L. and Thayer, D.W. (1985a) Starch-ampicillin agar for the quantitative detection of Aeromonas hydrophilu. Appl. Environ. Microbial. 50, 1027m- 1030. Palumbo. S.A., Morgan, D.R. and Buchanan, R.L. (1985b) Influence of temperature, NaCl and pH on the growth of Aeromonas hydrophih. J. Food Sci. 50, 1417 1421. Pin, C., Marin, M.L.. Garcia, M.L., Tormo, J. and Casas, C. (1994) Comparison of different media for the isolation and enumeration of Aeromonas spp. in foods. Lett. Appt. Microbial. 18, 190- 192. Robinson, J., Burke, V., Worthy, P.J., Beaman, J. and Wagener, L. (1984) Media for isolation of Aeromonas spp. from faeces. J. Med. Microbial. 18, 405-411. Slade, P.J., Falah, M.A. and Al-Ghady, A.M.R. (1986) Isolation of Aeromonas hydrophila from bottled waters and domestic water supplies in Saudi Arabia. J. Food Prot. 49, 471-476. Todd, L.S.. Hardy, J.C., and Stringer, M.F. (1989) Toxin production by strains of Aeromonas llydrophila grown in laboratory media and prown puree. Int. J. Food Microbial. 9, 1455156.