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Heat and acid sensitivity of motile Aeromonas: a comparison with other food-poisoning bacteria
International Journal of Food Microbiology, 18 (1993) 271-278
271
Elsevier Science Publishers B.V.
F O O D 00586
Heat and acid sensitivity of motile Aeromonas: a comparison with other food-poisoning bacteria Yoshikazu Nishikawa, Jun Ogasawara and Teruo Kimura Department of Epidemiology, Osaka Ci~ Institute of Public" Health and Em'ironmental Sciences. Tojo-cho, Tennoji, Osaka, Japan
The present study was undertaken to compare the heat and acid sensitivity of aeromonads with those of other food-poisoning bacteria. It became obvious that aeromonads were more sensitive to heat than Escherichia coli 0157: H7, Staphylococcus aureus, and Salmonella typhimurium. Aeromonads were killed in peptone water within 2 min at 55°C, while the other bacteria survived heating at 55°C for more than 15 rain. Aeromonas ceils were also less resistant to heat in hamburger steaks. These findings suggest that Aeromonas infection can easily be prevented by heat treatment, although correct handling of food is required to avoid recontamination since aeromonads are very common in various kinds of food. E. coli, S. aureus and S. typhimurium cells survived in buffer at pH 3.2 and in foods seasoned with vinegar. By contrast, Aeromonas cells were found to be highly sensitive to acid. However, the resistance of Aeromonas to acid may be sufficient to allow it to infect the gastrointestinal tract since Vibrio parahaemolyticus, which causes numerous outbreaks of food-poisoning every year in Japan, was susceptible to acid to the same extent as Aeromonas. Key words: Aeromonas; Temperature; pH; Cooking; Food
Introduction In recent years motile Aeromonas strains have been incriminated in diarrhoeal disease (Holmberg and Farmer, 1984; Janda and Duffey, 1988; Cahill, 1990). Since members of the genus Aeromonas are autochthonous inhabitants of aquatic environments, the infection has generally been regarded as water-borne (LeChevalier et al., 1982; Burke et al., 1984). Previous studies have shown, however, that aeromonds are ubiquitous not only in aquatic environments but also in various kinds of food; they seem to form part of the natural flora (Toule and Murphy, 1978; Palumbo et al., 1985; Callister and Agger, 1987; Nishikawa and Kishi, 1988; Kirov et al., 1990). It was also reported that aeromonads can multiply in foods stored in the refrigerator (Toule and Murphy, 1978; Palumbo et al., 1985; Callister and Agger, 1987; Kirov et al., 1990). These findings suggest that some Aeromonas infections might be food-borne.
Correspondence address." Y. Nishikawa, Department of Epidemiology, Osaka City Institute of Public Health and Environmental Sciences, Tojo-cho, Tennoji, Osaka 543, Japan. Fax: + 81-6-772-0676.
272 There are many reports on the survival and growth of Vibrio cholerae (Roberts and Gilbert, 1979; Kolvin and Roberts, 1982), Salmonella (Humphrey et al., 1989, 1990), Staphylococcus aureus (Batish et al., 1989) and Campylobacter (Svedhem et al., 1981; Waterman, 1982) in foods. There are also some reports concerning the thermal resistance of Aeromonas (Palumbo et al., 1987; Condon et al., 1992) and the effects of physico-chemical conditions on the growth of Aeromonas (Grau, 1981; Palumbo et al., 1985). However, we are not aware of any studies that directly compared the heat resistance of Aeromonas with other food-poisoning bacteria. In order to evaluate the resistance to heat of motile Aeromonas cells, their survival at 55°C was compared with that of Escherichia coli, Salmonella typhimurium, S. aureus and Vibrio parahaemolyticus. Furthermore, the fate of aeromonads in egg yolk and in hamburger steaks during cooking was investigated and compared with that of the other bacteria. The effect of pH on the viability of cells and the influence of vinegar in foods was also examined.
Materials and Methods
Test organisms A total of 25 strains of motile Aeromonas were tested: 13 strains (five strains of A. hydrophila and four strains each of A. sobria and A. caL,iae) were obtained from faeces, and 12 strains (four strains of each of A. hydrophila, A. sobria and A. cauiae) were obtained previously from foods and river water as described in earlier reports (Nishikawa and Kishi, 1987, 1988). Unless otherwise stated, a faecal strain (No. 189) of A. hydrophila was used as a reference strain. One strain each of V. parahaemolyticus, E. coli serotype O.157:H7, Salmonella typhimurium and Staphylococcus aureus was also included. All these strains were originally isolated from faecal specimens in our laboratory. Stock cultures of the organisms were stored at room t e m p e r a t u r e in two-fold diluted nutrient agar (Nissui, Tokyo, Japan). To prepare inocula, all strains were seeded into brain-heart infusion broth (Difco, Detroit, MI, USA) and incubated statically for 18 h at 37°C (in the case of V. parahaemolyticus, the medium was supplemented with 2.5% ( w / v ) sodium chloride). These cultures containing organisms in stationary phase were used for the experiments described below. Heat resistance For each test, the bacterial culture was diluted 10-fold in 0.1% peptone water (in the case of U. parahaemolyticus, the peptone water was supplemented with 3.0% ( w / v ) sodium chloride), and distributed in 2-ml aliquots to each of six test tubes (180 mm × 18 rnm i.d.) with aluminium-caps. These test tubes were then placed in a rack in a water bath set at 55°C. The water came half way up each tube i.e., well above the surface of the bacterial suspensions and the bath was gently agitated (60 oscillations/min, 30-ram strokes). Without allowing time for the contents to reach 55°C, test tubes were removed at intervals and immediately placed in chilled water. On completion of each set of experiments the contents of
273 the tubes were removed, diluted as appropriate with 0.1% peptone water and the number of surviving organisms was estimated by performing colony counts on Tryptosoya agar plates (Nissui, Tokyo, Japan) incubated for 24 h at 37°C. To evaluate the heat resistance of A. hydrophila in eggs, yolks were aseptically removed from eggs and used instead of 0.1% peptone water, and the survival of aeromonads was compared with that of S. typhimurium. To investigate the influence of heat on the bacteria in meat, 10 ml of each culture broth of Aeromonas, E. coli, Salmonella and S. aureus were mixed with hamburger ingredients, which consisted of 800 g of ground beef, 300 g of chopped onions, 100 g of whole eggs, 100 g of crumbs, some milk and seasonings. Then the hamburgers, which each weighed 120 g, were broiled on an electric hot plate at 200°C for 8 rain, then the thermostat was set at 150°C. The temperature of central portion of the hamburger was monitored with an electric t h e r m o m e t e r YN100E 105 (Yamatake-Honeywell, Tokyo, Japan). Hamburgers were removed at intervals of 2 rain. Approximately 10 g of the central portion of each were macerated in 90 ml of saline. Then 0.1 ml of a 10-fold dilution of each solution was spread on Bile salts Brilliant green Starch Agar (Nishikawa and Kishi, 1987), Mannitol Lysine Crystal violet Brilliant green Agar (Nissui), MacConkey (Nissui) and Salt Egg Yolk Agar (Nissui) plates, which were used for the detection of Aerornonas, Salmonella, E. coli and S. aureus, respectively. Acid resistance The effects of acidity on survival of the organisms were determined in a series of McIlvaine's phosphate buffers. To obtain the buffers with p H values of 7.0, 5.2, 4.8, 4.4, 4.0, 3.6, 3.2, 2.8, 2.4 or 2.0, solution of 0.1 M citric acid and 0.2 M disodium phosphate were mixed according to the formula, then checked with a pH m e t e r M-7 (Horiba Ltd., Kyoto, Japan). A 1.0-ml aliquot of bacterial culture was transferred to a sterile E p p e n d o r f tube and centrifuged for 1 min in an Eppendorf centrifuge (model 5414). The supernatant was aspirated by suction, and the bacteria were resuspended in 1.0 ml of the buffer. The bacterial suspensions were incubated for 1 h at 37°C, and the number of surviving cells was estimated as described above. To evaluate the influence of vinegar, 1 ml of culture broth was added to Japanese seaweed salad which consisted of 80 g of sliced cucumber and 30 g of seaweed, seasoned with 10 g of vinegar (acidity 4.2% (w/v), 1 g of salt and 4 g of sugar. A 10-g sample of salad was removed at intervals during storage at room temperature and examined by the abovementioned method.
Results
Heat resistance The numbers of viable cells of Aeromonas and of V. parahaernolyticus were rapidly reduced after heating for 2 rain in a water bath set at 55°C while the numbers of E. coli and S. aureus gradually declined after 20-30 min (Fig. 1). All
274 [LogCFU) 9-8--
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Fig. 1. Numbers of viable bacteria recovered from peptone water after the incubation in a water bath al 55°C. Dotted lines show survival of the bacteria suspended in egg yolk. The results are the mean values of five separate experiments or more. Each bar represents the standard deviation.
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275
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Fig. 3. Numbers of viable bacteria recovered after incubation for 1 h at 37°C in McIlvaine's buffer at the indicated pH. The results are the mean values of five separate experiments or more. Each bar represents the standard deviation.
tested strains of Aeromonas were killed in 2 min under these conditions (data not shown). Although viable Salmonella cells suspended in 0.1% peptone water were killed within 15 min, the cells appeared to be more resistant to heat in egg yolk
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Fig. 4. Numbers of viable bacteria in seaweed salad seasoned with vinegar and stored at room temperature. Dotted lines show numbers of viable bacteria in salad prepared without vinegar. The results are the mean values of five separate experiments or more. Each bar represents the standard deviation.
276 (Fig. 1). However, the aeromonads were still highly sensitive to heat and were apparently killed in 4 min (Fig. 1). In hamburgers (Fig. 2), aeromonads showed a marked decrease in numbers of viable cells at 8 rain, when the temperature exceeded 55°C, and none were recovered after 10 rain. By contrast, Salmonella. Staphylococcus and E. co# cells were still recovered in a viable state after 12 rain (Fig. 2).
Acid resistance No Aeromonas (25 strains) or V. parahaemolyticus cells were recovered after incubations below p H 3.6 but E. coli, S. aureus and S. typhimurium were found to be comparatively more resistant to acid (Fig. 3). No aeromonads survived for more than 7 h in Japanese seaweed salad seasoned with vinegar. The other bacteria failed to proliferate in the salad, but they were not killed by the vinegar in the salad (Fig. 4).
Discussion
It is clear that Aeromonas cells are extremely sensitive to heat, as compared with other food-poisoning bacteria. Since Salmonella ceils were found to be more heat-resistant in egg yolk ( H u m p h r e y et al., 1990), aeromonads could also be heat-resistant in foods. However, no marked protective effect of egg yolk was recognized in the case of Aeromonas. Although many sporadic cases have been reported, Aeromonas has not yet been clearly shown to be the cause of an outbreak of gastrointestinal disease among healthy persons (George et al., 1985; Moyer, 1987). A e r o m o n a d s are mainly detected in meat products, which are usually cooked before they are eaten. Compared with other bacteria, aeromonads were also more heat-sensitive in hamburger steaks. This heat-susceptible nature, as recognized in the present experiments, may account for the fact that outbreaks due to aeromonads are few in numbers irrespective of their prevalence in foods. V. parahaemolyticus, which was also highly sensitive to heat, is the most frequent causative agent of food-poisoning in Japan, perhaps because the Japanese frequently consume uncooked seawater fish and shellfish (sushi, sashimi) and V. parahaemolyticus is an inhabitant of sea water. The present study suggested that aeromonads are sensitive not only to heat but also to acid, and that vinegar is effective in inhibiting the growth of aeromonads at the levels used in the salads. However, the resistance of Aeromonas to gastric acid may be sufficient to allow intestinal infection because V. parahaemolyticus was equally acid-sensitive, and it is one of the most important food-poisoning agents in Japan. Susceptibility to citric acid or vinegar, as examined in vitro, may not reflect the sensitivity of aeromonads to gastric acid in the stomach. Water may be advantageous as a vehicle for aeromonads since it dilutes the gastric acid and causes rapid passage of the bacteria through the stomach. Most strains of E. coli are non-pathogenic in the intestine, although some can produce diarrhoea by a number of distinct mechanisms. Just like E. co6, most of
277
the aeromonads in foods might be non-pathogenic in the intestine and only some strains may possess a set of virulence factors. Gray et al. (1990) reported that many isolates of Aeromonas from water and animals had the full range of virulence factors, but the public-health significance of these organisms in foods is unclear at present (Palumbo et al., 1989). Recently, we found that aeromonads which adhered to intestinal cells were present at lower levels among strains isolated from samples of food and river water than among strains isolated from faecal specimens (Nishikawa et al., 1991). Both the heat- and acid-susceptible nature and the rarity of adhesive strains in food may account for the rarity of outbreaks of infection, despite the pervasiveness of aeromonads in food and the environment. In conclusion, it is clear that aeromonads are readily destroyed by heat, as compared with other food-poisoning bacteria. Thus, raw foods that are contaminated with Aeromonas should be safe to eat after normal cooking. The hazard lies in the recontamination of cooked food and subsequent storage since aeromonads are able to grow at low temperature (Toule and Murphy, 1978; Palumbo et al., 1985; Callister and Agger, 1987; Kirov et al., 1990).
Acknowledgement We thank Haruko Nishihara, a student at Osaka University of Pharmaceutical Sciences, for her voluntary assistance.
References Batish, V.K., Nataraj, B. and Grover, S. (1989) Variation in the behaviour of enterotoxigenic Staphylococcus a u r e u s after heat stress in milk. J. Appl. Bacteriol. 66, 27-35. Burke, V., Robinson, J., Gracey, M., Peterson, P. and Partridge, K. (1984) Isolation of Aeromonas hydrophila from a metropolitan water supply: seasonal correlation with clinical isolates, Appl. Environ. Microbiol. 48, 361-366. Cahill, M.M. (1990) Virulence factors in motile Aerornonas species, J. Appl. Bacteriol. 69, 1-16. Callister, S.M. and Agger, W.A. (1987) Enumeration and characterization of Aeromonas hydrophila and Aeromonas cat,iae isolated from grocery store produce. Appl. Environ. Microbiol. 53, 249-253. Condom S., Garcia, M.L., Otero, A. and Sala, F.J. (1992) Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aerornonas hydrophila. J. Appl. Bacteriol. 72, 322-326. George, W.L., Nakata, M.M., Thompson, J. and White, M.L. (1985) Aeromonas-related diarrhea in adults. Arch. Intern. Med. 145, 2207-22l l. Grau, F.H. (1981) Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative gram-negative bacteria on beef. Appl. Environ. Microbiol. 42, 1043-1050. Gray, S.J., Stickler, D.J, and Bryant, T.N. (1990) The incidence of virulence factors in mesophilic Aeromonas species isolated from farm animals and their environment. Epidemiol. Infect. 105, 277-294. Holmberg, S.D. and Farmer, J.J. II. (1984) Aeromonas hydrophila and Plesiomonas shigelloides as causes of intestinal infections. Rev. Infect. Dis. 6, 633-639. Humphrey, T.J., Greenwood, M., Gilbert, R.J., Rowe, B. and Chapman, P.A. (1989) the survival ot salmonellas in shell eggs cooked under simulated domestic conditions. Epidemiol. Infect. 103, 35-45.
278 Humphrey, T.J., Chapman, P.A., Rowe, B. and Gilbert, R.J. (1990) A comparative study of the heat resistance of salmonellas in homogenized whole egg, egg yolk or albumen. Epidemiol. Infect, 104, 237-241. Janda, J.M. and Duffey, P.S. (1988) Mesophilic aeromonads in human disease: current taxonomy, laboratory identification, and infectious disease spectrum. Rev. Infect. Dis. 10. 980-997. Kirov, S.M., Anderson, M.J. and McMeekin, T.A. (1990) A note on Aeromonas spp. from chickens as possible food-borne pathogens. J. Appl. Bacterial. 68, 327-334. Kolvin, J.L. and Roberts, D. (1982) Studies on the growth of fibrio cholerur biotype eltor and biotype classical in foods. J. Hyg. 89, 243-252. LeChevalier, M.W., Evans, T.M., Seidler, R.J., Daily. O.P., Merrell, B.R., Rollins, D.M. and Joseph, SW. (1982) Arrornonas sobriu in chlorinated drinking water supplies. Microb. Ecol. 8, 3255333. Moyer, N.P. (1987) Clinical significance of Arromonas species isolated from patients with diarrhea. J. Clin. Microbial. 25, 2044-2048. Nishikawa, Y. and Kishi, T. (1987) A modification of bile salts brilliant green agar for isolation of motile Aeromonas from foods and environmental specimens. Epidemiol. Infect. 98, 331-336. Nishikawa, Y. and Kishi, T. (1988) Isolation and characterization of motile Arromonas from human, food and environmental specimens. Epidemiol. Infect. 101, 213-223. Nishikawa, Y., Kimura, T. and Kishi, T. (1991) Mannose-resistant adhesion of motile Arromonus to lNT407 cells and the differences among isolates from humans, food and water. Epidemiol. Infect. 107, 171-179. Palumbo, S.A., Maxino, F., Williams, A.C.. Buchanan, R.L. and Thayer, D.W. (1985) Starch-ampicillin agar for the quantitative detection of Arromonas hydrophilu. Appl. Environ. Microbial. 50, 10271030. Palumbo, S.A., Morgan, D.R. and Buchanan, R.L. (1985) Influence of temperature, NaCI, and pH on the growth of Aeromonus hydrophila. J. Food Sci. 50. 1417-1421. Palumbo, S.A., Bencivengo, M.M., Corral, F.D., Williams, A.C. and Buchanan, R.L. (1989) Characterization of the Aeromonas hydrophilu group isolated from retail foods of animal origin. J. Clin. Micribiol. 27, 854-859. Palumbo, S.A., Williams, A.C., Buchanan, R.L. and Phillips, J.G. (1987) Thermal resistance of Aeromonas hydrophila. J. Food Prot. 50, 761-764. Roberts, D. and Gilbert, R.J. (1979) Survival and growth of non-cholera vibrios in various foods. J. Hyg. 82, 123-131. Svedhem, A., Kaijser, B. and Sjogren, E. (1981) The occurrence of Cumpylobacter jejuni in fresh food and survival under different conditions, J. Hyg. 87, 421-425. Toule, G. and Murphy, 0. (1978) A study of bacteria contaminating refrigerated cooked chicken; their spoilage potential and possible origin. J. Hyg. 81, 161-169. Waterman, S.C. (1982) The heat-sensitivity of Campylobuctrr jejuni in milk. J. Hyg. 88, 529-533.
271
Elsevier Science Publishers B.V.
F O O D 00586
Heat and acid sensitivity of motile Aeromonas: a comparison with other food-poisoning bacteria Yoshikazu Nishikawa, Jun Ogasawara and Teruo Kimura Department of Epidemiology, Osaka Ci~ Institute of Public" Health and Em'ironmental Sciences. Tojo-cho, Tennoji, Osaka, Japan
The present study was undertaken to compare the heat and acid sensitivity of aeromonads with those of other food-poisoning bacteria. It became obvious that aeromonads were more sensitive to heat than Escherichia coli 0157: H7, Staphylococcus aureus, and Salmonella typhimurium. Aeromonads were killed in peptone water within 2 min at 55°C, while the other bacteria survived heating at 55°C for more than 15 rain. Aeromonas ceils were also less resistant to heat in hamburger steaks. These findings suggest that Aeromonas infection can easily be prevented by heat treatment, although correct handling of food is required to avoid recontamination since aeromonads are very common in various kinds of food. E. coli, S. aureus and S. typhimurium cells survived in buffer at pH 3.2 and in foods seasoned with vinegar. By contrast, Aeromonas cells were found to be highly sensitive to acid. However, the resistance of Aeromonas to acid may be sufficient to allow it to infect the gastrointestinal tract since Vibrio parahaemolyticus, which causes numerous outbreaks of food-poisoning every year in Japan, was susceptible to acid to the same extent as Aeromonas. Key words: Aeromonas; Temperature; pH; Cooking; Food
Introduction In recent years motile Aeromonas strains have been incriminated in diarrhoeal disease (Holmberg and Farmer, 1984; Janda and Duffey, 1988; Cahill, 1990). Since members of the genus Aeromonas are autochthonous inhabitants of aquatic environments, the infection has generally been regarded as water-borne (LeChevalier et al., 1982; Burke et al., 1984). Previous studies have shown, however, that aeromonds are ubiquitous not only in aquatic environments but also in various kinds of food; they seem to form part of the natural flora (Toule and Murphy, 1978; Palumbo et al., 1985; Callister and Agger, 1987; Nishikawa and Kishi, 1988; Kirov et al., 1990). It was also reported that aeromonads can multiply in foods stored in the refrigerator (Toule and Murphy, 1978; Palumbo et al., 1985; Callister and Agger, 1987; Kirov et al., 1990). These findings suggest that some Aeromonas infections might be food-borne.
Correspondence address." Y. Nishikawa, Department of Epidemiology, Osaka City Institute of Public Health and Environmental Sciences, Tojo-cho, Tennoji, Osaka 543, Japan. Fax: + 81-6-772-0676.
272 There are many reports on the survival and growth of Vibrio cholerae (Roberts and Gilbert, 1979; Kolvin and Roberts, 1982), Salmonella (Humphrey et al., 1989, 1990), Staphylococcus aureus (Batish et al., 1989) and Campylobacter (Svedhem et al., 1981; Waterman, 1982) in foods. There are also some reports concerning the thermal resistance of Aeromonas (Palumbo et al., 1987; Condon et al., 1992) and the effects of physico-chemical conditions on the growth of Aeromonas (Grau, 1981; Palumbo et al., 1985). However, we are not aware of any studies that directly compared the heat resistance of Aeromonas with other food-poisoning bacteria. In order to evaluate the resistance to heat of motile Aeromonas cells, their survival at 55°C was compared with that of Escherichia coli, Salmonella typhimurium, S. aureus and Vibrio parahaemolyticus. Furthermore, the fate of aeromonads in egg yolk and in hamburger steaks during cooking was investigated and compared with that of the other bacteria. The effect of pH on the viability of cells and the influence of vinegar in foods was also examined.
Materials and Methods
Test organisms A total of 25 strains of motile Aeromonas were tested: 13 strains (five strains of A. hydrophila and four strains each of A. sobria and A. caL,iae) were obtained from faeces, and 12 strains (four strains of each of A. hydrophila, A. sobria and A. cauiae) were obtained previously from foods and river water as described in earlier reports (Nishikawa and Kishi, 1987, 1988). Unless otherwise stated, a faecal strain (No. 189) of A. hydrophila was used as a reference strain. One strain each of V. parahaemolyticus, E. coli serotype O.157:H7, Salmonella typhimurium and Staphylococcus aureus was also included. All these strains were originally isolated from faecal specimens in our laboratory. Stock cultures of the organisms were stored at room t e m p e r a t u r e in two-fold diluted nutrient agar (Nissui, Tokyo, Japan). To prepare inocula, all strains were seeded into brain-heart infusion broth (Difco, Detroit, MI, USA) and incubated statically for 18 h at 37°C (in the case of V. parahaemolyticus, the medium was supplemented with 2.5% ( w / v ) sodium chloride). These cultures containing organisms in stationary phase were used for the experiments described below. Heat resistance For each test, the bacterial culture was diluted 10-fold in 0.1% peptone water (in the case of U. parahaemolyticus, the peptone water was supplemented with 3.0% ( w / v ) sodium chloride), and distributed in 2-ml aliquots to each of six test tubes (180 mm × 18 rnm i.d.) with aluminium-caps. These test tubes were then placed in a rack in a water bath set at 55°C. The water came half way up each tube i.e., well above the surface of the bacterial suspensions and the bath was gently agitated (60 oscillations/min, 30-ram strokes). Without allowing time for the contents to reach 55°C, test tubes were removed at intervals and immediately placed in chilled water. On completion of each set of experiments the contents of
273 the tubes were removed, diluted as appropriate with 0.1% peptone water and the number of surviving organisms was estimated by performing colony counts on Tryptosoya agar plates (Nissui, Tokyo, Japan) incubated for 24 h at 37°C. To evaluate the heat resistance of A. hydrophila in eggs, yolks were aseptically removed from eggs and used instead of 0.1% peptone water, and the survival of aeromonads was compared with that of S. typhimurium. To investigate the influence of heat on the bacteria in meat, 10 ml of each culture broth of Aeromonas, E. coli, Salmonella and S. aureus were mixed with hamburger ingredients, which consisted of 800 g of ground beef, 300 g of chopped onions, 100 g of whole eggs, 100 g of crumbs, some milk and seasonings. Then the hamburgers, which each weighed 120 g, were broiled on an electric hot plate at 200°C for 8 rain, then the thermostat was set at 150°C. The temperature of central portion of the hamburger was monitored with an electric t h e r m o m e t e r YN100E 105 (Yamatake-Honeywell, Tokyo, Japan). Hamburgers were removed at intervals of 2 rain. Approximately 10 g of the central portion of each were macerated in 90 ml of saline. Then 0.1 ml of a 10-fold dilution of each solution was spread on Bile salts Brilliant green Starch Agar (Nishikawa and Kishi, 1987), Mannitol Lysine Crystal violet Brilliant green Agar (Nissui), MacConkey (Nissui) and Salt Egg Yolk Agar (Nissui) plates, which were used for the detection of Aerornonas, Salmonella, E. coli and S. aureus, respectively. Acid resistance The effects of acidity on survival of the organisms were determined in a series of McIlvaine's phosphate buffers. To obtain the buffers with p H values of 7.0, 5.2, 4.8, 4.4, 4.0, 3.6, 3.2, 2.8, 2.4 or 2.0, solution of 0.1 M citric acid and 0.2 M disodium phosphate were mixed according to the formula, then checked with a pH m e t e r M-7 (Horiba Ltd., Kyoto, Japan). A 1.0-ml aliquot of bacterial culture was transferred to a sterile E p p e n d o r f tube and centrifuged for 1 min in an Eppendorf centrifuge (model 5414). The supernatant was aspirated by suction, and the bacteria were resuspended in 1.0 ml of the buffer. The bacterial suspensions were incubated for 1 h at 37°C, and the number of surviving cells was estimated as described above. To evaluate the influence of vinegar, 1 ml of culture broth was added to Japanese seaweed salad which consisted of 80 g of sliced cucumber and 30 g of seaweed, seasoned with 10 g of vinegar (acidity 4.2% (w/v), 1 g of salt and 4 g of sugar. A 10-g sample of salad was removed at intervals during storage at room temperature and examined by the abovementioned method.
Results
Heat resistance The numbers of viable cells of Aeromonas and of V. parahaernolyticus were rapidly reduced after heating for 2 rain in a water bath set at 55°C while the numbers of E. coli and S. aureus gradually declined after 20-30 min (Fig. 1). All
274 [LogCFU) 9-8--
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Fig. 1. Numbers of viable bacteria recovered from peptone water after the incubation in a water bath al 55°C. Dotted lines show survival of the bacteria suspended in egg yolk. The results are the mean values of five separate experiments or more. Each bar represents the standard deviation.
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Fig. 2. Fate of bacteria in hamburger steaks broiled on a hot plate. Open circles indicate th( temperature of the central portion of the hamburger. The results are the mean values of five separat~ experiments or more. Each bar represents the standard deviation.
275
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Fig. 3. Numbers of viable bacteria recovered after incubation for 1 h at 37°C in McIlvaine's buffer at the indicated pH. The results are the mean values of five separate experiments or more. Each bar represents the standard deviation.
tested strains of Aeromonas were killed in 2 min under these conditions (data not shown). Although viable Salmonella cells suspended in 0.1% peptone water were killed within 15 min, the cells appeared to be more resistant to heat in egg yolk
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I 6
I 7 / Hours)
Fig. 4. Numbers of viable bacteria in seaweed salad seasoned with vinegar and stored at room temperature. Dotted lines show numbers of viable bacteria in salad prepared without vinegar. The results are the mean values of five separate experiments or more. Each bar represents the standard deviation.
276 (Fig. 1). However, the aeromonads were still highly sensitive to heat and were apparently killed in 4 min (Fig. 1). In hamburgers (Fig. 2), aeromonads showed a marked decrease in numbers of viable cells at 8 rain, when the temperature exceeded 55°C, and none were recovered after 10 rain. By contrast, Salmonella. Staphylococcus and E. co# cells were still recovered in a viable state after 12 rain (Fig. 2).
Acid resistance No Aeromonas (25 strains) or V. parahaemolyticus cells were recovered after incubations below p H 3.6 but E. coli, S. aureus and S. typhimurium were found to be comparatively more resistant to acid (Fig. 3). No aeromonads survived for more than 7 h in Japanese seaweed salad seasoned with vinegar. The other bacteria failed to proliferate in the salad, but they were not killed by the vinegar in the salad (Fig. 4).
Discussion
It is clear that Aeromonas cells are extremely sensitive to heat, as compared with other food-poisoning bacteria. Since Salmonella ceils were found to be more heat-resistant in egg yolk ( H u m p h r e y et al., 1990), aeromonads could also be heat-resistant in foods. However, no marked protective effect of egg yolk was recognized in the case of Aeromonas. Although many sporadic cases have been reported, Aeromonas has not yet been clearly shown to be the cause of an outbreak of gastrointestinal disease among healthy persons (George et al., 1985; Moyer, 1987). A e r o m o n a d s are mainly detected in meat products, which are usually cooked before they are eaten. Compared with other bacteria, aeromonads were also more heat-sensitive in hamburger steaks. This heat-susceptible nature, as recognized in the present experiments, may account for the fact that outbreaks due to aeromonads are few in numbers irrespective of their prevalence in foods. V. parahaemolyticus, which was also highly sensitive to heat, is the most frequent causative agent of food-poisoning in Japan, perhaps because the Japanese frequently consume uncooked seawater fish and shellfish (sushi, sashimi) and V. parahaemolyticus is an inhabitant of sea water. The present study suggested that aeromonads are sensitive not only to heat but also to acid, and that vinegar is effective in inhibiting the growth of aeromonads at the levels used in the salads. However, the resistance of Aeromonas to gastric acid may be sufficient to allow intestinal infection because V. parahaemolyticus was equally acid-sensitive, and it is one of the most important food-poisoning agents in Japan. Susceptibility to citric acid or vinegar, as examined in vitro, may not reflect the sensitivity of aeromonads to gastric acid in the stomach. Water may be advantageous as a vehicle for aeromonads since it dilutes the gastric acid and causes rapid passage of the bacteria through the stomach. Most strains of E. coli are non-pathogenic in the intestine, although some can produce diarrhoea by a number of distinct mechanisms. Just like E. co6, most of
277
the aeromonads in foods might be non-pathogenic in the intestine and only some strains may possess a set of virulence factors. Gray et al. (1990) reported that many isolates of Aeromonas from water and animals had the full range of virulence factors, but the public-health significance of these organisms in foods is unclear at present (Palumbo et al., 1989). Recently, we found that aeromonads which adhered to intestinal cells were present at lower levels among strains isolated from samples of food and river water than among strains isolated from faecal specimens (Nishikawa et al., 1991). Both the heat- and acid-susceptible nature and the rarity of adhesive strains in food may account for the rarity of outbreaks of infection, despite the pervasiveness of aeromonads in food and the environment. In conclusion, it is clear that aeromonads are readily destroyed by heat, as compared with other food-poisoning bacteria. Thus, raw foods that are contaminated with Aeromonas should be safe to eat after normal cooking. The hazard lies in the recontamination of cooked food and subsequent storage since aeromonads are able to grow at low temperature (Toule and Murphy, 1978; Palumbo et al., 1985; Callister and Agger, 1987; Kirov et al., 1990).
Acknowledgement We thank Haruko Nishihara, a student at Osaka University of Pharmaceutical Sciences, for her voluntary assistance.
References Batish, V.K., Nataraj, B. and Grover, S. (1989) Variation in the behaviour of enterotoxigenic Staphylococcus a u r e u s after heat stress in milk. J. Appl. Bacteriol. 66, 27-35. Burke, V., Robinson, J., Gracey, M., Peterson, P. and Partridge, K. (1984) Isolation of Aeromonas hydrophila from a metropolitan water supply: seasonal correlation with clinical isolates, Appl. Environ. Microbiol. 48, 361-366. Cahill, M.M. (1990) Virulence factors in motile Aerornonas species, J. Appl. Bacteriol. 69, 1-16. Callister, S.M. and Agger, W.A. (1987) Enumeration and characterization of Aeromonas hydrophila and Aeromonas cat,iae isolated from grocery store produce. Appl. Environ. Microbiol. 53, 249-253. Condom S., Garcia, M.L., Otero, A. and Sala, F.J. (1992) Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aerornonas hydrophila. J. Appl. Bacteriol. 72, 322-326. George, W.L., Nakata, M.M., Thompson, J. and White, M.L. (1985) Aeromonas-related diarrhea in adults. Arch. Intern. Med. 145, 2207-22l l. Grau, F.H. (1981) Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative gram-negative bacteria on beef. Appl. Environ. Microbiol. 42, 1043-1050. Gray, S.J., Stickler, D.J, and Bryant, T.N. (1990) The incidence of virulence factors in mesophilic Aeromonas species isolated from farm animals and their environment. Epidemiol. Infect. 105, 277-294. Holmberg, S.D. and Farmer, J.J. II. (1984) Aeromonas hydrophila and Plesiomonas shigelloides as causes of intestinal infections. Rev. Infect. Dis. 6, 633-639. Humphrey, T.J., Greenwood, M., Gilbert, R.J., Rowe, B. and Chapman, P.A. (1989) the survival ot salmonellas in shell eggs cooked under simulated domestic conditions. Epidemiol. Infect. 103, 35-45.
278 Humphrey, T.J., Chapman, P.A., Rowe, B. and Gilbert, R.J. (1990) A comparative study of the heat resistance of salmonellas in homogenized whole egg, egg yolk or albumen. Epidemiol. Infect, 104, 237-241. Janda, J.M. and Duffey, P.S. (1988) Mesophilic aeromonads in human disease: current taxonomy, laboratory identification, and infectious disease spectrum. Rev. Infect. Dis. 10. 980-997. Kirov, S.M., Anderson, M.J. and McMeekin, T.A. (1990) A note on Aeromonas spp. from chickens as possible food-borne pathogens. J. Appl. Bacterial. 68, 327-334. Kolvin, J.L. and Roberts, D. (1982) Studies on the growth of fibrio cholerur biotype eltor and biotype classical in foods. J. Hyg. 89, 243-252. LeChevalier, M.W., Evans, T.M., Seidler, R.J., Daily. O.P., Merrell, B.R., Rollins, D.M. and Joseph, SW. (1982) Arrornonas sobriu in chlorinated drinking water supplies. Microb. Ecol. 8, 3255333. Moyer, N.P. (1987) Clinical significance of Arromonas species isolated from patients with diarrhea. J. Clin. Microbial. 25, 2044-2048. Nishikawa, Y. and Kishi, T. (1987) A modification of bile salts brilliant green agar for isolation of motile Aeromonas from foods and environmental specimens. Epidemiol. Infect. 98, 331-336. Nishikawa, Y. and Kishi, T. (1988) Isolation and characterization of motile Arromonas from human, food and environmental specimens. Epidemiol. Infect. 101, 213-223. Nishikawa, Y., Kimura, T. and Kishi, T. (1991) Mannose-resistant adhesion of motile Arromonus to lNT407 cells and the differences among isolates from humans, food and water. Epidemiol. Infect. 107, 171-179. Palumbo, S.A., Maxino, F., Williams, A.C.. Buchanan, R.L. and Thayer, D.W. (1985) Starch-ampicillin agar for the quantitative detection of Arromonas hydrophilu. Appl. Environ. Microbial. 50, 10271030. Palumbo, S.A., Morgan, D.R. and Buchanan, R.L. (1985) Influence of temperature, NaCI, and pH on the growth of Aeromonus hydrophila. J. Food Sci. 50. 1417-1421. Palumbo, S.A., Bencivengo, M.M., Corral, F.D., Williams, A.C. and Buchanan, R.L. (1989) Characterization of the Aeromonas hydrophilu group isolated from retail foods of animal origin. J. Clin. Micribiol. 27, 854-859. Palumbo, S.A., Williams, A.C., Buchanan, R.L. and Phillips, J.G. (1987) Thermal resistance of Aeromonas hydrophila. J. Food Prot. 50, 761-764. Roberts, D. and Gilbert, R.J. (1979) Survival and growth of non-cholera vibrios in various foods. J. Hyg. 82, 123-131. Svedhem, A., Kaijser, B. and Sjogren, E. (1981) The occurrence of Cumpylobacter jejuni in fresh food and survival under different conditions, J. Hyg. 87, 421-425. Toule, G. and Murphy, 0. (1978) A study of bacteria contaminating refrigerated cooked chicken; their spoilage potential and possible origin. J. Hyg. 81, 161-169. Waterman, S.C. (1982) The heat-sensitivity of Campylobuctrr jejuni in milk. J. Hyg. 88, 529-533.