Dr. Rhodri Evans, Department of Industrial Microbiology, University College, Dublin, Belfield, Dublin 4.
Clostridium botulinum can hardly be regarded as a new food-borne pathogen. Kerner first documented botulism between 1815 and 1828 where he recorded 234 cases. The organism however was not isolated until 1895 by Van Ermigen who named the organism Bacillus botulinus, derived from the word botulus, which is Latin for sausage, this being the main food that was poisoned at the time. It is an anaerobic spore former that produces a characteristic neurotoxin. It is the organism that is responsible for the illness botulism. The species can be subdivided in to seven types depending on the serological nature of the toxin produced. The types are designated A to G. Of these, A, B, E and F produce a toxin that is harmful to man. Indeed, botulinum toxin is one of the most potent toxins know to man, the lethal dose of type A toxin for humans being circa 1ng/Kg body weight, this value being slightly higher for the other toxin types.
The seven types can be further subdivided in to four groups depending on their physiological and biochemical characteristics. Those organisms that produce toxins that affect man constitute groups I and II. Group I consists of the proteolytic strains of types B and F and also type A. All strains in this group are proteolytic. Group II strains are all non-proteolytic and it consists of those strains of types B and F that are non proteolytic and also Type E.
The first difference between strains of groups one and two is that those of group one are proteolytic whilst those of group two are non-proteolytic. They also have different thermal characteristics in that those of strains of group one are all mesophilic with a minimum growth temperature (GT) of 100C, a maximum GT of 450C -500C and an optimum GT of 400C. Those of Group II on the other hand are all psychrotolerant. This means that they are able to grow at lower temperatures and have a minimum GT of 3.30C, a maximum GT of 40-450C and an optimum GT of 30-350C. The two groups have different lower limits for growth in terms of pH. The proteolytic strains will grow down to pH = 4.6-4.8, whilst those of group two will only grow down to pH = 5.0. They also have different minimum water activities inhibitory to growth. Group 1 strains will grow in conditions where the Aw is as low as 0.94, but those of group two will only grow as low as Aw = 0.97. These values equate to 10% and 5% (w/v) aqueous NaCI respectively.
The spores produced by the members of the two groups are also different. Those of Group I strains are rather heat resistant but those of Group II are sensitive to heating and are rapidly killed. Type A spores have a D110 of 2.8 min whilst Type F spores have a D77 of 0.77-1.95 min. This comparison can be made more clearly by looking at the differences between non-proteolytic and proteolytic strains of the organism that produce the same toxin type. Proteolytic type B strains require heating for 483 minutes at 82.20C or for 1.36 min at 1100C to achieve the log reduction, but non-proteolytic strains of the same type are killed much more rapidly. For example, they only survive for as little as 1.49 minutes at 82.20C.
The production of an extremely potent neurotoxin by the organism is what makes it of concern to the food industry. The toxin works by blocking the release of the neurotransmitter acetyl choline from the presynaptic vesicles. The illness is characterised by a general weakening of the muscles followed by flaccid paralysis. Death is usually as a result of suffocation through failure of the thoracic muscles.
The level of toxin required is so low that it is conceivable that during the shelf life of the product, growth from a single spore could lead to the production of sufficient cell numbers that enough toxin could be produced to kill someone. The fact that it is an anaerobic spore former is also important. Food has been packaged in a number of ways for many years in order to help prolong the shelf life of the product. For many years, the major method of packaging was canning. Clostridium botulinum was recognised as a potential source of contamination many years ago and since then much information has been gleaned from experiments to determine what time/temperature combinations of heat processes were required to destroy the spores of C. botulinum. It is usually the spore of the organism that causes contamination of foods since even mild heat treatments are usually sufficient to kill vegetative cells.
The spores of proteolytic strains are quite heat-resistant and it is these that were taken in consideration when spore destruction studies were performed. Eventually a value known as the botulinum cook was determined and this involved heating the cans to an internal temperature of 1210C for 3 minutes. This is also know as the 12-D kill, i.e. enough to kill 1012 spores of a botulinum population.
If cans are properly processed then there is no risk of contamination but incidents have occurred where there is either a failure of he heating process or where post-processing contamination has occurred.
Non-proteolytic strains i.e. Group ll, possess the ability to grow at low temperatures (known as psychrotolerance). Because the spores of these strains are relatively sensitive to heating, they are not a problem in most foods. However, if a product is minimally processed, as is the case more and more these days, and relies mainly on refrigeration to control growth then strict control of chill must be maintained. If the products are not given a large heat treatment and rely on a high NaCI content, then particularly the proteolytic strains are a problem. They are able to withstand 10% (w/v) NaCI and this equates to a water activity of 0.94. This value may be lower if different solutes are used to lower the Aw. Also, in salted products, there is a period when the salt must diffuse to the centre of the product. This time may be sufficient to allow growth and toxin production by the organism.
A factor which is often forgotten is just how common in the environment spores of C. botulinum actually are. The distribution is ubiquitous. As a result all food types are potential sites of contamination. Although it is ubiquitous it is not to say its distribution is uniform. Indeed there is definite regionalisation of the different types. Type A spores are found predominantly in soils of Western USA, Brazil, Argentina and China. The distribution of type B spores seems more uniform but those found in the soils of eastern USA are proteolytic whilst those found in the UK and Europe are non-proteolytic. Type E spores are found in aquatic sediments of colder regions such as Alaska, Canada, Scandinavia, the countries of the former Soviet Union and also Japan.
Why there is such diversity isn't obvious but for instance type A is favoured by neutral to alkaline soils with low organic content, hence its virtual absence from eastern USA and Europe where the soil is heavily farmed. Type E is found in cold regions because of its psychrotolerance.
In the USA there continues to be a problem with food poisoning by C. botulinum. In the period 1971-1989, there were a total of 272 outbreaks, of which 252 were typed. 61% of these were type A, 21% were type B and 17% were type E. 1 % were caused by type F and so, although this type is able to poison humans, it is rarely implicated in an outbreak. The data can be subdivided in to eastern and western USA. In the east, the incidents of A and B were roughly equal, whilst in the west the number of type A was eight times that caused by type B. This is consistent with the predominance of type A in the soils of western USA. The highest number of cases occurred in fruit and vegetables with the majority of these being in vegetables. There were equivalent numbers in meat and fish, with most of the outbreaks associated with fish occurring in Alaska. 92% of the outbreaks were from foods which had been canned or processed in the home.
In countries where type E predominates in environmental surveys, it is also the type responsible for food poisoning outbreaks. For example, in Denmark, all eleven food poisoning incidents over a five year period were attributed to type E However, in all cases the types of food involved all were meat. Normally, type E poisonings are associated with fish products because of predominance of these spores in aquatic sediments. This is what is seen when one considers the data for Japan where a large amount of fish and fish products are consumed. Of the 97 outbreaks that were typed, 96% were type E and 99% were attributable to fish or fish products. However, 98% of the incidents involved food of domestic origin. It appears that there is a definite link between the type of botulinum responsible for poisoning and the predominant type in the area.
There have been very few reported outbreaks in the UK. There have only been four since 1955, and in two of these, food that had been imported was determined to be responsible. In two cases in particular, we can highlight where the processing of the food broke down. One incident in 1978 was caused by post-processing contamination of a can of Canadian salmon. There was a hole in the seal of the can and when it was cooled in water drawn from the Great Lakes, a spore or spores entered the can which became scaled with food debris, an anaerobic environment developed and growth and toxin production ensued.
The most recent outbreak occurred in 1989 which involved hazelnut yoghurt and it was by far the largest. The hazelnut puree supplied to the dairy was manufactured with a reduced sugar content which affected the Aw. The spores were able to germinate and growth and toxin production occurred before the puree was added to the yoghurt. There have never been any reported incidents of botulism in Ireland.
Traditional food processing procedures have been well enough elucidated to eliminate the risk of botulinal poisoning unless there is a breakdown in the processing procedure. However, methods of production are constantly changing and so one must assume that new processes introduce the potential for botulinal poisoning. The main area of concern that has been identified as a potential new risk are those foods that are collectively know as the REPFEDs, refrigerated processed foods of extended durability. This is a name that covers a wide range of products including sous-vide processed and cook-chill foods. A subsection of these products is also those which are packaged under a modified atmosphere (MAP products).
The basic characteristics of all REPFEDs are:
- that they are pre-cooked, packaged products intended to receive little or no additional treatment prior to consumption
- they have an extended refrigerated shelf life
- they are not usually protected by conventional preservation systems such as low water activity or low pH
- they are marketed under vacuum/modified atmosphere packaging and/or in sealed containers.
So why do these products present a special hazard in terms of risk from botulinal poisoning. The most important factor that must be considered is the temperature of storage. These foods are primarily designed to be preserved by refrigeration. However, the reported minimum growth temperature for non-proteolytic strains of this organism is 3.30C, but growth at this temperature is relatively slow. The temperature at which these products are stored is seldom as low as this. Also these products are classed as luxury items in that they are relatively expensive. The consumer dictates what form the product takes. Consumer preference dictates that the product should be delivered in as natural a condition as possible. Also there are those actions of the consumer over which the producer has no control. This can be called the consumer syndrome. One factor of this syndrome is the abuse of temperature of storage by the consumer once they get the product home. So here we have the scenario of the major preservation method being abused.
These products also receive mild heat-treatments. The reasons behind this are two fold. Firstly, because of the demand for the product to be as natural as possible, the heat treatment applied is minimal so that the flavour/colour/nutritional value are retained. Also, proteolytic strains of botulinum are not considered to be a risk if the
Temperature is below 100C and so the 12-D kill is not employed. Another factor is the heat treatment given to the product by the consumer before consumption. Botulinal neurotoxin is quite heat labile and so if it had been formed in the food, even a mild warming up would destroy it.
In conclusion, outbreaks of botulism are very rare, mainly due to the good processing practices employed by the food industry. However, with the advent of new foods which are minimally processed and rely on refrigeration as the main preservation factor, there possibly exists the potential for an increased risk of botulinal poisoning.
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