Microbiol. Immunol., 39(9), 655-661, 1995

Isolation, and Virulence Profiles, of Aeromonas hydrophila Implicated in an Outbreak

of Food Poisoning in Sweden

Karel Krovacelc*'1, Stefano Dumontet2, Erik Eriksson3, and Suraj B. Baloda1

1 Section of Bacteriology & Epizootology, Department of Veterinary Microbiology, Swedish University of Agricultural Sciences,Biomedical Center, Box 583, S-751 23 Uppsala, Sweden, 'University of Potenza, Potenza, Italy, and Svelab Laboratory, Box

6123, S-700 06, Orebro, Sweden

Received February 27, 1995. Accepted June 6, 1995

Abstract: A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A

group of 27 people consumed a typical Swedish food "landgfing" which is a type of smOrgfisbord containing

shrimps with mayonnaise, liver pate, ham, sausage, and legume salad which was purchased from a food

store. Twenty-two of the 27 persons became ill within 20-34 hr of consumption of the food and reported the

symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One per-

son also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who con-

sumed the left-over food the next day, 2 became ill with similar symptoms. The bacteriological examina-

tion of left-over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise,

smoked sausage, liver pate and boiled ham. The total number of A. hydrophila in these foods were log 106

to log •„107 organisms per gram of food sample. A. hydrophila was however, not isolated from legume/may-

onnaise salad samples. All the food samples tested showed low numbers of other expected food contami-

nating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus, fungi and

yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food

samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to pro-

duce P-hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human

intestinal (Henle 407) cells in culture.

Key words: Aeromonas hydrophila, Foods, Food poisoning, Sweden, Virulence factors, Invasion

During the last years, Aeromonas species have received increased recognition as human intestinal

pathogens both in immunocompromised patients as well as in healthy individuals (15). Investigators have also indicated that some members of the motile Aeromonas spp. are emerging food- and water-borne pathogens of increasing importance (18, 19, 21, 22). Aeromonas spp. are widely distributed in the environment and in a wide range of fresh foodstuffs. These microorganisms have been isolated from different foods such as meats, sea foods, dairy products, fish, vegetables as well as from surface water, drinking water and chlorinated drinking water (2, 21, 22, 26).

It seems that foods of animal origin are probably contaminated by environmental sources. Aeromonas spp. are often found in the fecal flora of animals, although as

a minor component. However, in literature, there are only a few reports on food-borne outbreaks caused by Aeromonas spp. It could be explained by the fact that a large part of the unidentified cases of food-borne out-breaks are probably caused by organisms which are rou-tinely not tested for identifying the etiology of food

poisoning. According to Buchanan and Palumbo (6), about 40% of the reported food-borne outbreaks which occur in the U.S.A. each year are of unknown etiology. It is thus possible that Aeromonas spp., which are rarely reported as food-borne pathogens, might be one of these unidentified agents responsible for food-borne outbreaks.

In this report, we describe the isolation and virulence

profiles of A. hydrophila implicated in an outbreak of food poisoning in Sweden.

* Address correspondence to Dr. Karel Krovacek, Section of

Bacteriology and Epizootology, Department of Veterinary Micro-

biology, Swedish University of Agricultural Sciences, Biomedical

Center, Box 583, S-751 23, Uppsala, Sweden.

Abbreviations: BHI, brain heart infusion; CHO-K1 cells, Chi-

nese hamster ovary cells; SMT, suckling mouse test.

655

656 K. KROVACEK ET AL

Materials and Methods

Food poisoning incident. On 28 December 1993, a

group of 27 persons attending a funeral ceremony, were served with individual portions of a typical Swedish food namely "landgang" which is a type of smorgasbord containing shrimps, mayonnaise, liver pate, ham, sausage and legume salad which was purchased from a food store. The food was served at 13.00 p.m.. During the night,

22 of the 27 persons who consumed this food became ill and experienced a variety of symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever, and vomiting. One person reportedly also fainted. On 29 December, the remaining 5 healthy persons ate the left-over portion of the food, and as a result 2 of these got ill with similar symptoms. The symptoms continued for a couple of days. The samples of food were collected for microbiological analysis on 30 December and were subjected to analysis within 6 hr of collection.

Microbiological analysis of food samples. Five spec-imens from the left-over food namely, shrimps with mayonnaise, liver pate, boiled ham, smoked sausage and legume/mayonnaise salad, were subjected to micro-biological analysis. Samples were carefully investigat-ed following the routine laboratory standards for inves-tigation of food samples, for the presence of total num-ber of aerobes, coliforms at 37 C and 44 C, fecal strep-tococci, staphylococci, Clostridium perfringens, Bacillus cereus, Salmonella, Aeromonas, fungi and yeasts. These samples were handled and analyzed by following the standard norms of microbiological analysis of foods. In brief, the samples were transported to the laboratory in an ice box maintaining a temperature of 0 C to + 4 C and were analyzed within 6 hr of sampling. Ten grams of the respective food samples were homogenized for 30 sec in 90 ml of peptone water (Oxoid L34). The homogenates

were diluted tenfold in peptone water and plated on dif-

ferent growth culture media for determining the micro-

biological flora listed below (Table 1).

For Salmonella analysis, 25 g of samples were homog-

enized in 225 ml of peptone water (Oxoid CM509) and

incubated at 37 C for 24 hr. A 0.1-ml aliquot of this pre-

enriched culture was transferred to 10 ml of Rappaport-

Vassiliadis broth (RVS broth, Merck 334 V 375100)

and incubated for 24 hr at 42 C. The enriched cultures

were then inoculated on Brilliant green agar plates

(Oxoid CM329).

Isolation and identification of Aeromonas hydrophila.

The bacteriological growth from the respective food

samples, upon preliminary observations, showed pure

culture growth of a single type of bacteria on blood

agar plates. A close examination of colony morphology

of blood agar plates showed pure cultures ofƒÀ-hemolyt-

ic organisms. Further examination showed that the

organisms were Gram-negative rods, motile, oxidase-pos-

itive, glucose-fermenting and were resistant to Vibrio-

static agent-O129. Furthermore, different isolates from

respective food samples were biochemically character-

ized using API 20NE and all were confirmed as A.

hydrophila (16).

Culture conditions for detection of potential viru-

lence factors. The A. hydrophila isolates were streaked

on blood agar (5% horse erythrocytes in Brain heart

infusion (BHI) agar from Difco Lab., Detroit, Mich.,

U.S.A.) and incubated at 37 C for 20 hr. The bacteria

were further inoculated into BHI broth and incubated at

37 C for 20 hr under agitation (60 rpm). Cultures were

harvested by centrifugation (16,000•~g) at 4 C for 30 min

and the supernatants were membrane-filtered (Milli-

pore, 0.22 rim). The sterile cell-free culture filtrates

were tested for the presence of hemolysins, cytotoxins,

cytotonic toxins and enterotoxins.

Detection of exotoxins. The enterotoxic activity was

detected by the suckling mouse assay (7) while the

Table 1. Culture media used for analysis of food samples for the presence of different microorganisms

OUTBREAK OF A. HYDROPHILA FOOD POISONING IN SWEDEN 657

cytotoxic and cytotonic toxins were detected using Chi-nese hamster ovary (CHO-K1) cells (Flow Labs., Irvine, Scotland) as described earlier (20). Hemolysin produc- tion was assayed using horse erythrocytes as described previously (26).

In vitro adhesion to and invasion of human intestinal (Henle 407) cells. The A. hydrophila isolates were cul-tured at 37 C in BHI for 24 hr under static conditions, and the capacity of bacteria to bind to and invade the human intestinal cells was investigated using methods described earlier (20).

Results

Food Poisoning Incident As a result of food poisoning, 24 members of a group

of 27 persons became ill within 20-34 hr of consumption of contaminated food. They experienced a number of symptoms (and with a varying degree of severity) name-ly, acute diarrhea, abdominal pain, headache, fever, vomiting and fainting. All age groups were represented in this outbreak including children (aged one year and six years); teenagers (15 and 17 years), and adults (between 30-73 years). Some of the patients reportedly sought medical advice at night. Unfortunately, fecal samples for

microbiological examination were not taken.

Bacteriological Analysis of Food Samples

A. hydrophila was isolated in high numbers from the

following food samples: shrimps with mayonnaise, liver

pate, boiled ham and smoked sausage. The total number

of A. hydrophila in these foods was •„107 organisms

per gram of food sample. A. hydrophila was, however,

not isolated from legume salad samples (Table 2).

All the food samples tested showed low numbers of

other expected food contaminating organisms such as

coliforms at 37 C and 44 C, fecal streptococci, Staphy-

lococcus aureus, fungi and yeast. Furthermore, Bacillus

cereus, Clostridium perfringens and Salmonella spp.

were not detected from the food samples (Table 2).

Virulence Properties of Aeromonas hydrophila Isolated

from Food

The A. hydrophila isolates produced 13-hemolysis on

horse blood agar. Cytotoxic and cytotonic toxins were

measured by the rounding and elongation of CHO-K1

cells in tissue culture, respectively. Each of the three iso-

lates produced cytotoxin titers of 1:4 while cytotonic

toxin titers ranged from 1:64 to 1:128 (Table 3). Fur-

thermore, sterile cell-free culture supernatants of two

Table 2. Microbiological examination of the food samples

Table 3. Virulence properties of Aeromonas hydrophila implicated in food poisoning incident in Sweden

658 K. K.ROVACEK ET AL

a

b

Fig. 1. Light microscopy photograph showing the invasion of Henle 407 cells by Aeromonas hydrophila (L2301 V) isolated from foods. Figure la shows the initiations of invasion of Henle 407 cells by the Aeromonas, while the intracellular presence of Aeromonas cells is seen in Fig. lb.

OUTBREAK OF A. HYDROPHILA FOOD POISONING IN SWEDEN 659

of the isolates (L2301 I and L2301 V) gave positive results in the suckling mouse test (SMT), indicating the presence of enterotoxin. The two isolates produced gut weight to remaining body weight ratios of 0.070 and 0.082 which, according to Burke et al (7) are + and++, respectively. The third isolate, however, wasneg-

ative in the SMT assay as it gave a ratio of 0.065 (Table 3). The cells of the three A. hydrophila isolates also pos-

sessed the capacity to bind to human intestinal (Henle 407) cells to varying degrees i.e., between 2-10 bacteri-al cells/intestinal cell (Table 3). Of the three isolates, one isolate of A. hydorphila (L2301 V) also possessed the capacity to invade Henle 407 cells (Fig. 1). It is also interesting to note that this isolate showed the highest cytotonic toxin titer in the CHO-K1 cell assay and max-imum ratio of the gut weight over remaining body weight in the SMT for the assay of enterotoxin (Table 3).

Discussion

Outbreaks of food-borne infections caused by Aero-monas are infrequently reported. A brief summary of the published reports of Aeromonas-associated food-borne disease has been given by Kirov (18). The reports show that suspect foods were mainly seafoods (predominant-ly oysters) that had either been pre-frozen, and presum-ably inadequately cooked, or were foods that were con-sumed with minimal cooking. In our study too, similar types of food (cold stored) were involved in the Aeromonas-associated food poisoning and it included both, seafoods (e.g., shrimps) as well as liver pate, boiled ham and smoked sausage.

A number of investigators (1-3, 5, 6, 8, 11, 13, 14, 19, 21, 22, 27) have indicated that the aeromonads form a group of pathogens which are emerging as food-borne organisms of increasing importance. Our studies in the past have shown the presence and isolation of aeromon-ads from foods and drinking water in Sweden (21, 22, 26). It is thus not surprising that the direct involve-ment of A. hydrophila in a food poisoning incident in Sweden has been finally reported by this study. A high number of A. hydrophila in pure culture has been found in the food samples involved in this incident of food poi-soning. It is well known that Aeromonas spp. can survive and multiply at low temperatures (storage at 4 C or below) (23). Furthermore, these organisms may pos-sess the capacity to produce a variety of virulence factors, e.g., exotoxins, at these temperatures (23). In the present study, the A. hydrophila has been isolated from cold food, which is normally stored at refrigeration tempera-ture prior to consumption. Since the food poisoning resulted in a variety of symptoms in the infected indi-

viduals such as severe diarrhea together with abdominal pain and vomiting, it is possible that A. hydrophila could not only grow in number, but also produce exotoxins and other virulence factors at these storage temperatures.

A report on the involvement of seafood-based food cocktails in the Aeromonas-associated food poisoning has been described by Altwegg et al (3). They report an inci-dent of food poisoning associated with the consumption of Aeromonas-contaminated shrimp cocktail. These investigators presented strong evidence linking a strain from contaminated food with the clinical isolate from a patient with diarrhea and thus demonstrating that food can be responsible for the transmission of Aeromonas gastrointestinal infections. Our study corroborates the findings of Altwegg et al (3) and the reports by Kirov (18), indicating the potential of involvement of Aeromonas in a variety of sea foods, including shrimps. It is thus interesting to speculate from our studies that the route of A. hydrophila transmission could have, in fact, been through contaminated shrimps. Since the prepara- tion of smorgasbord involves handling of different foods at the same time, this process could have led to the cross contamination of other food types involved in this incident. Many of Aeromonas found in foods produce exotox-ins. Approximately, 20% of New Zealand A. hydrophi-la food strains were reported to be exotoxin-positive (18). In the United States, more than 90% of A. hydrophila strains were cytotoxin producers and in Den-- mark, 37% of A. hydrophila showed hemolysin produc- tion (8, 19, 31). We have also reported that majority of Aeromonas strains isolated from different foods in Swe-den, were capable of producing exotoxins. Some of these isolates were also able to cause fluid accumulation in rabbit ileal loop assay (21).

In the present study, the three isolates have been shown to produce exotoxins such as hemolysin, cytotox-in, cytotonic toxin and enterotoxin. In the SMT for the detection of enterotoxins produced by the three A. hydrophila isolates, two isolates produced a gut weight/remaining body weight ratio of 0.070 and 0.082, respectively, which according to Burke et al (7) are + and++ reactions. On the other hand, the third isolate gave

a negative ratio of 0.065 in the SMT. Furthermore, two of the isolates (L2301 I and L2301 V) also showed a cor-relation between the production of hemolysin, cytotox-in, cytotonic toxin, and enterotoxin in the SMT. Such a correlation has also been reported by Cumberbatch et al (10) and Johnson and Lior (17).

For invasive enteropathogens, the ability to adhere to gastrointestinal mucosa is an essential requirement for tis-sue invasion. Aeromonas spp. have been shown to adhere to human erythrocytes and buccal epithelial cells

660 K. KROVACEK ET AL

(4) and to rabbit intestinal brush border cells (29). We

have also reported that A. hydrophila strains isolated

from the marine environment and from human diarrhea!

cases have the capacity to adhere to human intestinal

cells in vitro and that the majority of the isolates were

classified as low adherers with •ƒ 10 bacteria binding per

intestinal cell (25). On the other hand, Carello et al (9)

reported that human diarrhea! Aeromonas isolates bound

to larynx carcinoma cells in a higher number than the

environmental isolates, none of which bound at more

than 20 bacteria per laryngeal cell. In the present study,

the three isolates of A. hydrophila bound to Henle 407

cells with a range of 2-10 bacteria per cell.

The ability to invade epithelial cells by Aeromonas can

be associated with their ability to cause dysentery-like

diarrhea (32). However, there are only a few studies on

this aspect of Aeromonas pathogenicity (12, 28, 32).

Gray et al (12) reported that 14-36% of Aeromonas

strains isolated from animals and the environment were

able to invade HEp-2 tissue culture cells. We have also

reported earlier that A. hydrophila and A. sobria strains

isolated from fish and hare had the capacity to invade

HEp-2 tissue culture cells (24). On the other hand,

Morgan et al (30) investigated 5 strains of A. hydrophi-

la for the ability to invade tissues by the Sereny methods

and none of these 5 strains produced keratoconjunctivi-

tis in guinea pigs, though three of the strains produced

hemorrhagic, purulent fluid accumulation in ligated ileal

loop. In our study, we have demonstrated that one of the

investigated isolates (L2301 V) showed invasion of

human intestinal cells in vitro.

Aeromonas hydrophila may cause two types of diar-

rhea namely, mild and self-limiting diarrhea, and a

dysentery-like illness with bloody diarrhea, vomiting

and abdominal pain. It is interesting to note from our

study that the patients who suffered from food poisoning

caused by A. hydrophila, experienced the latter type of

disease symptoms, which were probably accentuated

by the production of cytotoxic and cytotonic toxin reac-

tions in CHO-K1 cells, enterotoxic activity in SMT, and

binding to and/or invasion of human intestinal 407 cells

by these isolates (Table 2).

A common source of Aeromonas spp. in outbreaks of

gastroenteritis is water supplies. A source other than

water can be different kinds of foods of animal origin, sea

foods and vegetables. Although the numbers of food-

borne outbreaks caused by Aeromonas spp. are quite

limited so far, the presence of Aeromonas spp. in the food

chain should not be ignored. Some of the food isolates

may pose a risk for human health, particularly for elder-

ly and immunocompromised persons. Additionally, to

understand the complete picture of food-borne Aero-

monas-associated disease, it is of great importance to

know whether a specific Aeromonas isolate from food

possesses virulence factors responsible for the symp-toms of Aeromonas-associated disease as well as for

colonization and growth in the host.

The authors would like to thank the Swedish Council for Forestry and Agricultural Research, Stockholm, for financial support. We would also like to thank the members of the family

BjOrkman at Orebro, Sweden, for their feedback regarding this incident.

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