The following points highlight five major bacterial agents of foodborne illness. The agents are:- 1. Aeromonas Hydrophila 2. Brucella 3. Plesiomonas Shigelloides 4. Shigella 5. Scombrotoxic Fish Poisoning.
Bacterial Agent # 1. Aeromonas Hydrophila:
Currently, Aeromonas (principally A. hydrophila, but also A. caviae and A. sobria) has the status of a foodborne pathogen of emerging importance.
Like Listeria monocytogenes, Plesiomonas, and Yersinia enterocolitica, it has attracted attention primarily because of its ability to grow at chill temperatures, prompting the concern that any threat it might pose will increase with the increasing use of chilled foods (Table 7.1).
Present uncertainty over its significance however, is reflected in much of the information available which does not, as yet, present a coherent picture. It was first isolated from drinking water by Zimmerman in 1890 and the following year from frog’s blood by Sanarelli. They called their isolates Bacillus punctata and Bacillus hydrophilus respectively and it was not until the 1930s that the genus Aeromonas was first described.
Although the taxonomy is still not settled, more recent studies have led to the recognition of two major groups within the genus: the Salmonicida group which contains the non-motile Aeromonas salmonicida, and several sub-species, and the Hydrophila-Punctata group containing a number of motile species, including A. hydrophila, A. sobria, and A. caviae.
A. salmonicida is not a human pathogen but causes diseases of freshwater fish which can be an important economic problem in fish farming. Members of the Hydrophila group can cause extra-intestinal infections, commonly in the immunosuppressed or as a result of swimming accidents where the skin is punctured.
The first report of gastroenteritis due to Aeromonas came from Jamaica in 1958, but evidence of its ability to cause gastroenteritis in otherwise healthy individuals is patchy.
Epidemiological investigations in several countries have reported higher rates of isolation of aero monads from patients with diarrhoea than from control groups, although this does not necessarily indicate a causal relationship.
In one study, the incidence of A. hydrophila in American travellers to Thailand with diarrhoea was significantly higher than in unaffected individuals. Interestingly, isolation rates in the Thai population were similar for both groups of patients suggesting that Aeromonas may be a cause of ‘travellers’ diarrhoea’ in these regions.
Good supporting evidence from sources other than epidemiological studies has proved difficult to obtain. In a feeding trial involving 50 volunteers with doses as high as 5 x 1010, only two cases of diarrhoea resulted, although a laboratory accident has been reported where approximately 109 cells were ingested by a worker mouth pipetting who later suffered acute diarrhoea.
The Organism and its Characteristics:
Aeromonads are Gram-negative, catalase-positive, oxidase-positive rods which ferment glucose. They are generally motile by a single polar flagellum.
A. hydrophila is neither salt (< 5%) nor acid (min. pH ≈ 6.0) tolerant and grows optimally at around 28 °C. Its most significant feature with regard to any threat it may pose in foods is its ability to grow down to chill temperatures, reportedly as low as —0.1 °C in some strains. Its principal reservoir is the aquatic environment such as freshwater lakes and streams and wastewater systems.
The numbers present will depend on factors such as the nutrient level and temperature but can be as high as 108 cfu ml-1 in a relatively nutrient rich environment such as sewage.
Although it is not resistant to chlorine, it is found in potable water, where it can multiply on the low level of nutrients available in the piped water systems. It has also been isolated from a wide range of fresh foods and is a transient component of the gut flora of humans, and other animals.
Pathogenesis and Clinical Features:
Gastroenteritis associated with Aeromonas occurs most commonly in children under five years old. It is normally mild and self-limiting mosdy characterized by profuse watery diarrhoea, although dysenteric stools may sometimes be a feature. Vomiting is not usually reported.
Aeromonas spp., particularly A. hydrophila and A. sobria, produce a range of potential virulence factors including a number of distinct cytotoxic and cytotonic enterotoxins. Most clinical strains of A. hydrophila and A. sobria produce aerolysin, a heat- labile, β-haemolytic, cytotoxic enterotoxin with a molecular mass of 52 kDa.
Three cytotonic enterotoxins have also been described which act like cholera toxin, stimulating accumulation of high levels of cAMP within epithelial cells. Only one of these shows any marked structural similarity to cholera toxin as measured by cross reactivity with cholera toxin antibodies.
Isolation and Identification:
In some instances enrichment media such as alkaline peptone water are used, but where high numbers are present direct plating is usually sufficient. Species of the Hydrophila group grow on a wide range of enteric media but may often be misidentified as ‘coliforms’ since many strains can ferment lactose.
Most cannot ferment xylose and this is a useful distinguishing feature used in several media. As well as bile salts, ampicillin is used as a selective agent in media such as starch ampicillin agar, blood ampicillin agar and some commercial formulations. Colonies which give the characteristic appearance of Aeromonas on the medium concerned and are oxidase-positive are then confirmed with biochemical tests.
Association with Foods:
Apart from their possible role in gastroenteritis, food and water are also probably the source of the severe extra-intestinal Aeromonas infections associated with immuno-compromised individuals.
Aeromonads of the Hydrophila group have been isolated from a wide range of fresh foods including fish, meat, poultry, raw milk, and salad vegetables as well as water.
The ability of some strains to grow at very low temperatures can lead to the development of high numbers under chill conditions and they can be an important part of the spoilage flora of chilled meats. They are unlikely to survive even mild cooking procedures but may be introduced as post-process contaminants from uncooked produce or contaminated water.
Bacterial Agent # 2. Brucella:
The genus Brucella is named after Sir David Bruce who in 1887 recognized it as the causative organism of undulant fever (brucellosis, Malta fever, Mediterranean fever). Each of the four species that are human pathogens is associated with a particular animal host, B. abortus (cattle), B. melitensis (sheep and goats), B. suis (pigs), and B. canis (dogs) (Table 7.2).
Brucellosis is principally contracted from close association with infected animals and is an occupational disease of farmers, herdsman, veterinarians and slaughterhouse workers. It can also be contracted by consumption of milk or milk products from an infected animal, although the risk is louver.
The illness has been effectively eliminated from the United States, Scandinavia, the UK and other countries by campaigns to eradicate the organism in the national dairy herds through a programme of testing, immunization of young calves and compulsory slaughter of infected cattle.
The Organism and its Characteristics:
Brucella are Gram-negative, catalase-positive, oxidase-positive, short oval rods (0.3 µm x 0.4 µm) which are non-motile and usually occur singly, in pairs, or, rarely, in short chains. It grows optimally around 37 °C and is killed by heating at 63 °C for 7-10 min. When shed in the milk of an infected animal it can survive for many days provided the acidity remains low (<0.5% as lactate).
Pathogenesis and Clinical Features:
Brucellosis is a protracted and debilitating illness characterized by an incubation period of from one to six weeks followed by a chronic, relapsing fever with accompanying lassitude, sweats, headache, constipation, anorexia, pains in the limbs and back, and weight loss.
After the temperature has returned to normal for a few days, another bout of fever may ensue and such episodes recur a number of times over several months. Treatment is commonly with a mixture of tetracycline and streptomycin.
It is a facultative parasite and can live intracellularly or in extracellular body fluids. During the febrile stage, caused by circulating endotoxin, the organism may be isolated from the bloodstream but in the majority of laboratory-confirmed cases diagnosis is based on serological tests rather cultural techniques.
Isolation and Identification:
Brucella are quite fastidious organisms and do not grow in conventional laboratory media. Liver infusions or calf serum are normally added. The organism grows slowly and cultures are normally incubated for three weeks before they are considered negative. In view of this, testing foods for the organism is not practically feasible or useful.
Cattle are tested for the presence of antibodies to the organism in the ‘Ring Test’. Stained antigen is mixed with the test milk, if antibodies to Brucella are present (indicative of infection) then they will cause the antigen to clump and rise with the milk fat on standing to form an intense blue-violet ring at the top of the milk.
Association with Foods:
Although brucellosis has sometimes been associated with the consumption of inadequately cooked meat from an infected animal, raw milk or cream are the principal food vehicles. Brucella is readily killed by normal milk pasteurization conditions so there is no risk from pasteurized milk or products made from it.
Cheeses made from unpasteurized milk can sometimes pose a problem since the organism can survive the cheese-making processes and subsequent storage in the product.
Bacterial Agent # 3. Plesiomonas Shigelloides:
Plesiomonas shigelloides is the only species of the genus whose name is derived from the Greek word for neighbour; an allusion to its similarity to Aeromonas. Its position as a causative agent of foodborne illness also bears some similarity to Aeromonas. It is not normally recovered from human faeces, except in Thailand where a carriage rate of 5.5% has been reported.
The association with diarrhoea is largely based on its isolation from patients suffering from diarrhoea in the absence of any other known pathogens and the strongest of this evidence has come with isolation from several patients in the same outbreak. However volunteer feeding trials have failed to demonstrate a causal link.
The Organism and its Characteristics:
A member of the family Vibrionaceae, P. shigelloides is a short, catalase-positive, oxidase-positive, Gram-negative rod. It is motile by polar, generally lophotrichous flagella in contrast to Aeromonas and Vibrio which are monotrichous.
It grows over a temperature range from 8-10 °C to 40-45 °C with an optimum at around 37 °C. It is not markedly heat resistant and is readily eliminated by pasteurization treatments. Growth is possible down to pH 4.5 and the maximum salt concentration it will tolerate is between 3 and 5% depending on other conditions.
The organism is ubiquitous in surface waters and soil, more commonly in samples from warmer climates. Carriage in cold-blooded animals such as frogs, snakes, turtles, and fish is common and it has been isolated from cattle, sheep, pigs, poultry, cats and dogs. It is not normally part of the human gut flora.
Pathogenesis and Clinical Features:
Cases of P. shigelloses infection are more common in warmer climates and in travellers returning from warmer climates. The usual symptoms are a mild watery diarrhoea free from blood or mucus. Symptoms appear within 48 h and persist for several days.
More severe colitis or a cholera-like syndrome have been noted with individuals who are immunosuppressed or have gastrointestinal tumours. Little is known of the pathogenesis of P. shigelloides infections. Motility appears to be an important factor and evidence has been presented for an enterotoxin causing fluid secretion in rabbits’ ligated ileal loops.
Isolation and Identification:
The relatively recent growth of interest in P. shigelloides is reflected in the use of ‘second-hand’ media in its isolation. Alkaline peptone water and tetrathionate broth have both been used for enrichment culture of P. shigelloides at 35-40 °C and salmonella-shigella and MacConkey agars have been used as selective plating media.
Selective plating media have been developed such as inositol/brilliant green/bile salts, Plesiomonas agar. Isolates can be readily confirmed on the basis of biochemical tests.
Association with Foods:
Fish and shellfish are a natural reservoir of the organism and, with the exception of one incident where chicken was implicated, they are the foods invariably associated with Plesiomonas infections. Examples have included crab, shrimp, cuttle fish and oysters.
Bacterial Agent # 4. Shigella:
The genus Shigella was discovered as the cause of bacillary dysentery by the Japanese microbiologist Kiyoshi Shiga in 1898. It consists of four species Sh. dysenteriae, Sh. flexneri, Sh. boydii, and Sh. sonnei all of which are regarded as human pathogens though they differ in the severity of the illness they cause.
Sh. dysenteriae has been responsible for epidemics of severe bacillary dysentery in tropical countries but is now rarely encountered in Europe and North America where Sh. sonnei is more common. Sh. sonnei causes the mildest illness, while that caused by Sh. boydii and Sh. flexneri is of intermediate severity.
Although Shigella is relatively inactive biochemically when compared with Escherichia species, studies of DNA relatedness have demonstrated that they do in fact belong to the same genus. The separate genera are retained however, because, unlike Escherichia, most strains of Shigella are pathogenic and a re-designation might cause confusion with potentially serious consequences.
Laboratory reports of Sh. sonnei infections in England and Wales rose to 9830 in 1991 compared to 2319 and 2228 in the previous two years. In the United States annual reports over recent years have ranged between 300 000 and 450 000.
Shigellas are spread primarily person-to-person by the faecal-oral route although foodborne outbreaks have been recorded. Some experts consider that the problem of foodborne shigellosis is greatly underestimated.
The Organism and its Characteristics:
Shigellas are members of the family Enterobacteriaceae. They are non-motile, non-spore forming, Gram-negative rods which are catalase-positive (with the exception of Shiga’s bacillus, S. dysenteriae serotype 1), oxidase-negative, and facultative anaerobes.
They produce acid but usually no gas from glucose and, with the exception of some strains of S. sonnei, are unable to ferment lactose; a feature they share with most salmonellas.
Shigellas are generally regarded as rather fragile organisms which do not survive well outside their natural habitat which is the gut of humans and other primates. They have not attracted the attention that other foodborne enteric pathogens have, but such evidence as is available suggests that their survival characteristics are in fact similar to other members of the Enterobacteriaceae.
They are typical mesophiles with a growth temperature range between 10-45 °C and a heat sensitivity comparable to other members of the family. They grow best in the pH range 6-8 and do not survive well below pH 4.5.
A number of studies have reported extended survival times in foods such as flour, pasteurized milk, eggs, and shellfish. The species are distinguished on the basis of biochemical tests and both serotyping and phage typing schemes are available for further subdivision of species.
Pathogenesis and Clinical Features:
Shigellas cause bacillary dysentery in humans and other higher primates. Studies with human volunteers have indicated that the infectious dose is low; of the order of 10-100 organisms. The incubation period can vary between 7 h and 7 days although foodborne outbreaks are commonly characterized by shorter incubation periods of up to 36 h.
Symptoms are of abdominal pain, vomiting and fever accompanying a diarrhoea which can range from a classic dysenteric syndrome of bloody stools containing mucus and pus, in the cases of Sh. dysenteriae, Sh. flexnert and Sh. boydii, to a watery diarrhoea with Sh. sennei.
Illness lasts from 3 days up to 14 days in some cases and a carrier state may develop which can persist for several months. Milder forms of the illness are self-limiting and require no treatment but Sh. dysenteriae infections often require fluid and electrolyte replacement and antibiotic therapy. Shigellosis is an invasive infection where the organism’s invasive property is encoded on a large plasmid.
Isolation and Identification:
Lack of interest in Shigella as a foodborne pathogen has meant that laboratory protocols for its isolation and identification from foods are relatively underdeveloped. A pre-enrichment procedure has been described based on resuscitation on a non-selective agar before overlaying with selective media.
Selective enrichment in both Gram-negative broth and selenite broth has been recommended. Selective plating media used are generally those employed for enumerating the Enterobacteriaceae or Salmonella although neither are entirely satisfactory.
Rapid techniques for identification based on immunoassays which detect the virulence marker antigen, and on the polymerase chain reaction to detect the virulence plasmid by DNA/DNA hybridization have also been applied.
Association with Foods:
Foodborne cases of shigellosis are regarded as uncommon though some consider the problem to be greatly underestimated. The limited range of hosts for the organism certainly suggests that it is relatively insignificant as a foodborne problem when compared with say Salmonella.
In foodborne cases, the source of the organism is normally a human carrier involved in preparation of the food. In areas where sewage disposal is inadequate the organism could be transferred from human faeces by flies.
Uncooked foods which may have received extensive handling such as prawn cocktail or tuna salad have been implicated in a number of outbreaks. In one, which occurred in Cambridge shire, England, in 1992, 107 out of 200 guests at a buffet meal developed diarrhoea and Sh. sonnei was isolated from 81 of 93 faecal samples taken.
The organism was also isolated from two of the catering staff. Investigation revealed a strong association between illness and consumption of two prawn dishes for which both infected caterers had been involved in the preparation.
Bacterial Agent # 5. Scombrotoxic Fish Poisoning:
Scombrotoxic fish poisoning differs from those types of foodborne illness described above in that it is thought to be an example of where bacteria act as indirect agents of food poisoning by converting food components into harmful compounds.
Fish is almost always the food vehicle, particularly the so-called scombroid fish such as tuna, bonito and mackerel, but non-scombroid fish such as sardines, pilchards and herrings have also been implicated. In some cases canned fish has been responsible indicating that the toxic factor(s) is heat stable.
It is a chemical intoxication with a characteristically short incubation period of between 10 min and 2 h. Symptoms include a sharp, peppery taste in the mouth, itching, dizziness, flushing of the face and neck, often followed by a severe headache, feverishness, diarrhoea, nausea and vomiting. A rash may develop on the face and neck and cardiac palpitations may occur.
The symptoms are those of histamine toxicity and can be alleviated with antihistamines. Histamine is produced by bacterial amino acid decarboxylases acting on histidine which occurs in high concentrations in the tissues of dark-fleshed fish. The bacteria themselves increase in numbers as a result of long storage at inappropriate temperatures and freshly caught fish have not been implicated in this type of poisoning.
When making judgments on the risk of scombrotoxic fish poisoning posed by particular products, regulatory authorities usually rely on a measure of the histamine content of the fish. In the United States, the level of histamine deemed hazardous in tuna is 50 mg%. Some guideline values published by the Public Health Laboratory Service in the UK are presented as Table 7.11.
The problem is not as clear cut as it may at first seem, however. It has, for instance, not proved possible to reproduce the symptoms in volunteers fed histamine, and cases have also been reported where the fish contained low levels of histamine.
Although histamine poisoning can occur when clearance of dietary histamine from the body is slowed by monoamine oxidase inhibitors, histamine is generally metabolized efficiently in the human gut and not adsorbed per se.
Among the explanations offered are that other biologically active amines are present in the fish which potentiate the toxicity of histamine or that algal toxins may be involved causing the release of endogenous histamine in the body.