In this article we will discuss about:- 1. Introduction to Clostridium Perfringens 2. The Organism of Clostridium Perfringens and its Characteristics 3. Pathogenesis and Clinical Features 4. Isolation and Identification 5. Association with Foods.
- Introduction to Clostridium Perfringens
- The Organism of Clostridium Perfringens and its Characteristics
- Pathogenesis and Clinical Features of Clostridium Perfringens
- Isolation and Identification of Clostridium Perfringens
- Clostridium Perfringens Association with Foods
1. Introduction to Clostridium Perfringens:
Clostridium perfringens, formerly welchii, has been known as a cause of the serious wound infection, gas gangrene since 1892 when it was first described by the American bacteriologist Welch.
Although accounts appeared shortly after, in 1895 and 1899, linking it with outbreaks of gastroenteritis in St. Bartholomew’s Hospital, London, it was not until the mid-1940s that outbreaks associated with school meals in England (1943) and pre-cooked chicken dishes in the USA (1945) firmly established Clostridium Perfringens as a cause of food poisoning.
The species is classified into five types, designated A-E, based on the production of four major exotoxins, α, β, ε, and t, and eight minor ones (Table 7.5). Clostridium Perfringens type A which is responsible for food poisoning and gas gangrene produces only the α major toxin which has lecithinase (phospholipase C) activity.
Its ability to hydrolyse lecithin and some other phospholipids plays an important role in the pathogenesis of gas gangrene; by attacking cell membranes it produces local tissue disruption in the wound and its absorption into the circulation causes a serious toxaemia. It does not however have any role in the food poisoning syndrome.
Clostridium Perfringens type C which produces α and β toxins causes enteritis necroticans, a more severe, but far more rare, enteric disease in which the β toxin damages the intestinal mucosa causing necrosis. Illness is preventable by active immunization against the β toxin. Outbreaks were reported in Germany in 1946 and 1949, but it is nowadays particularly associated with Papua New Guinea where it is known as pigbel.
Symptoms of abdominal pain and bloody diarrhoea develop several days after a high-protein meal, often pork consumed on festive occasions. Low levels of intestinal proteases are a predisposing factor in victims.
This could arise from a poor diet low in protein, as with the European outbreaks after the Second World War, and may be compounded in Papua New Guinea by protease inhibitors consumed with other foods in the diet such as sweet potatoes.
Clostridium Perfringens type A ranks second to Salmonella as a cause of bacterial food poisoning in the UK, although the incidence is much lower, less than one tenth the number of salmonella cases in the period 1980-90. (Campylobacter infections although more numerous than salmonella are not currently classified as food poisoning.)
There has been no marked increasing trend in recent years; between 1980 and 1990 outbreaks in England and Wales have numbered between 46 and 69 each year with 896 to 1624 cases; corresponding figures for Scotland over the same period were 5 to 11 outbreaks and 75 to 364 cases.
2. The Organism of Clostridium Perfringens and its Characteristics:
Clostridium perfringens is a Gram-positive, rod-shaped anaerobe which forms oval sub-terminal spores. It differs from most other clostridia in that the relatively large rods (1 x 3-9 µm) are encapsulated and non-motile. Though a catalase-negative anaerobe, Clostridium Perfringens will survive and occasionally grow in the presence of oxygen.
Growth occurs over the temperature range 12 to 50 °C although it is very slow below about 20 °C. At its temperature optimum, 43-47 °C, growth is extremely rapid with a generation time of only 7.1 min at 41 °C. Vegetative cells show no marked tolerance to acid (minimum pH 5, optimum 6.0-7.5), have a minimum aw for growth of 0.95-0.97, depending on the humectant, and will not grow in the presence of 6% salt.
The heat resistance of vegetative cells is comparable to that of non-spore-forming bacteria with D values at 60 °C in beef of a few min. D values of spores at 100 °C show a wide inter-strain variation with recorded values from 0.31 min to more than 38 min.
This may, in part, be due to differences in the culture methods used since some workers included lysozyme in their media to improve the recovery of heat- damaged spores.
Distribution of type A Clostridium Perfringens is widespread in the environment. In soil, where it can be found at levels of 103– 104 + g-1, it persists much longer than types B,C,D, and E which are obligate animal parasites and of more limited distribution.
It can be isolated from water, sediments, dust, raw and processed foods and is a common inhabitant of the human gastrointestinal tract. Spore counts of 103 -104 g-1 are common in faeces from healthy individuals and surveys in Japan and the UK have found levels of up to 108– 109 cfu g-1 in healthy elderly people in long-stay care.
3. Pathogenesis and Clinical Features of Clostridium Perfringens:
Clostridium Perfringens food poisoning is generally a self-limiting, non-febrile illness characterized by nausea, abdominal pain, diarrhoea and, less commonly vomiting.
Onset is usually 8 to 24 h after consumption of food containing large numbers of the vegetative organism; the median count of Clostridium Perfringens in foods implicated in UK outbreaks is 7 x 105 g-1 and the required ingested dose has been variously estimated at 106-108cfu.
In otherwise healthy individuals, medical treatment is not usually required and recovery is complete within 1-2 days, although occasional fatalities occur in the very old or debilitated.
Ingested vegetative cells that survive the stomach’s acidity pass to the small intestine where they grow, sporulate and release an enterotoxin. The enterotoxin is synthesized by the sporulating cells, although low levels of production have been observed in vegetative cultures.
The toxin is closely associated with the spore coat, but is not thought to be an important structural component, and is released into the intestinal lumen on lysis of the sporangium.
Toxin production can also occur in vitro. Low levels have been detected in foods, including a sample involved in an outbreak, and a few reported cases with incubation periods less than 2 h suggest that, on occasion, ingestion of pre-formed toxin may cause illness. This is generally held to be rare however.
The enterotoxin is a 35 kDa protein with an isoelectric point of 4.3. It is inactivated by heating in saline at 60 °C for 10 min and is sensitive to some proteolytic enzymes. It acts like cholera toxin by reversing the flow of Na+, CI–, and water across the gut epithelium from absorption to secretion, though it does so by a different mechanism.
Rather than increase the level of intracellular cyclic nucleotides, it acts at the cell membrane. It first binds to specific protein receptors on the epithelial cell and is then inserted into the cell membrane producing morphological changes within a few min.
It changes cell permeability, inhibits synthesis of cell macromolecules, and produces pores in the membrane of the cell which eventually dies as a result of membrane damage.
Diagnosis of Clostridium Perfringens food poisoning is normally based on a number of factors:
1. case history and symptoms;
2. large numbers (> 106 g-1) of Clostridium Perfringens spores in the patient’s faeces;
3. large numbers of vegetative cells of the same serotype in the incriminated food (> 106 g-1);
4. presence of enterotoxin in faeces.
Faecal count data should be treated with some caution since the level of excretion can be very high in aged, healthy, institutionalized patients.
4. Isolation and Identification of Clostridium Perfringens:
In the investigation of outbreaks, enrichment culture is rarely necessary since Clostridium Perfringens will invariably be present in high numbers in implicated foods or clinical samples. Similarly, in routine quality assurance of foods, there is generally little value in being able to detect very low numbers in view of its ubiquity in the environment.
In the examination of foods, the total count (vegetative cells plus spores) is determined but with faecal specimens a spore count, obtained after heating a suspension at 80 °C for 10 min, is also performed.
The most commonly employed selective plating media used to enumerate Clostridium Perfringens employ antibiotic(s) as the selective agent and, most commonly, sulfite reduction to produce black colonies as the differential reaction.
The most popular combinations are tryptose /sulfite/cycloserine (TSC) medium and oleandomycin / polymyxin /sulfadiazine / perfringens (OPSP), incubated anaerobically for 24 h at 37 °C. A better diagnostic reaction is obtained if pour plates are used since colonies on the agar surface of spread plates can appear white.
Suspect colonies can be confirmed by the absence of motility, their ability to reduce nitrate to nitrite, lactose fermentation, and gelatin liquefaction. A traditional confirmatory test, the Nagler reaction, which looks for the production of α toxin and its neutralization by antitoxin on lactose egg-yolk agar is less favoured now because of its lower specificity and the occurrence of lecithinase-negative strains.
Serotyping based on capsular antigens is employed for epidemiological purposes. Most isolates from outbreaks can be serotyped and this can be usefully supplemented by typing with bacteriocins, particularly when serotyping is not possible.
A number of methods are available for the detection of enterotoxin. Traditional biological tests such as the ligated ileal loops and mouse challenge have been superseded by more sensitive, rapid, convenient and humane serological techniques. A commercially available kit employs reverse passive latex agglutination.
The name derives from the fact that in a standard agglutination assay, soluble antibody reacts with a particulate antigen such as bacterial cells. In a reversed passive latex agglutination assay, a soluble antigen reacts with antibody attached to latex particles. These play no part in the reaction and are therefore passive, but they do provide a visual signal when they cross-link as a result of the antigen-antibody reaction.
5. Clostridium Perfringens Association with Foods:
For an outbreak of Clostridium Perfringens food poisoning, the typical scenario includes the following events:
1. a meat dish containing spores of Clostridium Perfringens is cooked;
2. the spores survive the cooking to find themselves in a genial environment from which much of the competitive flora has been removed;
3. after cooking, the product is subjected to temperature/time abuse, such as slow cooling or prolonged storage at room temperature. This allows the spores to germinate and multiply rapidly to produce a large vegetative population;
4. the product is either served cold or reheated insufficiently to kill the vegetative cells. Some of the ingested cells survive through into the small intestine where they sporulate and produce enterotoxin.
From the above outline it is clear that Clostridium Perfringens food poisoning is more likely to occur where food is being prepared some time in advance of consumption and that adequate refrigeration is the key to its control.
Most cases (>70% in the United States and >87% in England and Wales) are associated with meat products such as stews, meat gravies, roast joints and pies. This is partly due to the frequent association of the organism with meats, but major contributory factors are the low redox potential, mode of preparation and consumption which can give Clostridium Perfringens the opportunity to multiply to dangerous levels.
Cured meats are rarely involved in Clostridium Perfringens food poisoning. This is a fine example of the hurdle concept in action; individual preservative factors such as salt content, nitrite level and heat processing are insufficient on their own to assure safety but effectively control growth of Clostridium Perfringens in combination.
Most outbreaks occur in connection with institutional catering such as schools, old people’s homes and hospitals. The association between Clostridium Perfringens and hospital food in particular goes back a long way, to the outbreaks at St. Bartholomew’s Hospital in the 1890s, but here at least there are signs of progress.
In the UK, the 1991 Richmond Report on the safety of food noted that outbreaks in hospitals fell from a peak of more than 20 per year in the 1970s to about half this number in the 1980s: a decline attributed to improvements in facilities and staff training.