Microorganisms can be classified in four main groups of varying complexity. We are most familiar with Bacteria and moulds since we can either see them (e.g. mouldy fruit), or we can see the effects of their activities (e.g. spoiled meat). Viruses and parasites are less evident but, as with bacteria, we are aware of their effects when we suffer from an infection.
Many bacteria cause foodborne diseases. This list gives you an idea of the variety of organisms that can be transmitted through food. Later, we will discuss the differences between them, where they come from, and how they get into foods.
The main spoilage organisms are bacteria, yeasts and moulds. They may cause food to deteriorate, producing undesirable changes in flavour, odour or taste. Sometimes, these changes may be seen as desirable. In some cheeses, moulds are essential to the production process. However, we would not consider bread or fruit with the same mould growing on the surface to be fit to eat .
Many microorganisms are useful to us. Fermented products exist all over the world. Here are some examples.
Lactic acid bacteria are very common in the natural world and are probably some of the first organisms to grow as plant matter decays. Many natural traditional fermentations contain these organisms. These organisms produce not only lactic acid but other organic acids as by-products of carbohydrate breakdown. They are found in soil, on plants and are natural inhabitants of the human gut. The organic acids reduce the pH of the surroundings (we shall look at pH later). This inhibits the growth of many microorganisms and may help lactic acid bacteria compete in their environment. Organic acids also inhibit many pathogens. Many traditional fermentations used by ancient peoples to preserve food are still used today.
Microorganisms are extremely small . This slide tries to put this statement into perspective by showing you the astounding number of harmless lactic acid bacteria in a cup of yoghurt. (When explaining this concept, try to find an example which is meaningful to your listeners). Source: J.Crowther
Here we see a diagram of a bacterial cell. It has a rigid cell wall with a membrane made of phospholipid and protein. The cell contains the cytoplasm , a semi-liquid matrix of water, enzymes and proteins necessary for the metabolic activities of the organism. There are organelles in the cytoplasm such as ribosomes , where the polypeptides that make up the proteins and enzymes are synthesised from their constituent amino acids. There is a nuclear body of a double-stranded DNA which stores genetic information ready for transcription. Some bacteria may sporulate. This means that they change their cell walls to become more resistant towards heat, drying, etc. to have better chances of survival. Bacterial spores should not be confused with mould spores. Mould spores are used for their reproduction, and are not necessarily very resistant.
Bacteria divide asexually by binary fission. There are four or five points at which the cell gradually splits after first elongating. Bacteria can exchange genetic material in a process called conjugation, where simple circular strands of DNA called plasmids are passed from one to the other. Under optimum conditions, bacteria can duplicate every twenty minutes.
This graph shows the increase of bacteria with time, and its relation to spoilage and toxin production. At first, bacteria adapt to their surroundings and do not divide; this is the lag phase of growth . The next period is called the logarithmic growth phase , because the numbers increase exponentially (we have used a logarithmic scale on the Y axis, so this phase appears as a straight line). The time needed for the number of organisms to double is the generation time . After a while, the production of toxic by-products such as acids, and the depletion of growth substrates such as carbohydrate, essential amino acids, or oxygen, limit further growth. The curve flattens; this is the stationary phase. Toxins are produced towards the end of the logarithmic phase and during the stationary phase of the growth curve. Since toxin formation may occur before microbial growth produces visible changes, a seemingly acceptable food can cause an intoxication.
Here, we summarise what we have just seen in the growth curve. The three phases of growth are: the adaptive lag phase ; the logarithmic growth phase of rapid exponential growth; and the stationary phase where growth slows due to depletion of nutrients and build-up of toxic products, and where cell division is in balance with cell death. Sometimes, if we leave a culture long enough, a fourth period of decline occurs where cells die and the population decreases. Toxin can remain in food even if we can find no detectable cells of the organism.
These are the major factors affecting growth of bacteria. We shall examine them without looking at actual mechanisms. Those of you who wish to have further information can refer to your reading list. Among these factors, microbial interactions are generally less important than the others.
Temperature affects microbial growth. We have already seen the growth curve. Most bacteria found in foods grow best at 28 - 45°C. Some can grow rapidly at 20 - 25°C. Foods should never be kept in warm surroundings for more than one or two hours. In the cold, germs breed slowly. A few can multiply under refrigeration conditions (3-10°). In the freezer, most live but do not breed. Boiling and pasteurising kills germs in a few minutes but it does not kill heat-resistant spores or destroy heat-resistant toxins. That is why cooked food should be eaten immediately. Here we can see a picture of the critical temperatures. This is a good diagram for training people.
Every organism has a minimum, optimum and maximum temperature for growth. Below the optimum, the growth rate (generation time) decreases. The sub-optimum temperatures do not usually kill the organism. At high temperatures, damage to proteins and cell constituents occurs: both simple, reversible structural changes and irreversible denaturation. This is why the curve descends rapidly at temperatures above the optimum.
Here we can see the effect of temperature on the growth of Salmonella typhimurium. At 25°C, it reaches the stationary phase in one day; at 10°C, the stationary phase has not been reached in five days. At temperatures lower than 10°C, growth is negligible.
Here we can see the growth ranges of several foodborne pathogens.
Toxigenic moulds are also affected in the same way.
At the same temperature, different bacteria behave differently. In this graph, we see the logarithmic growth curves of different genera of bacteria. This behaviour is partly the basis of competition among organisms. The selection imposed by factors such as temperature, pH etc, causes the organisms most suited to the environment to predominate.
The effect of temperature is seen in this way.
Adding salt affects toxin production as well as bacterial growth, but the effect also depends on temperature and water activity. Here we can see that at 10°C, adding 2% salt increased time to toxin production from 10 to 16 days. However, if the temperature is above 10°C, the effect of additional salt is minimal. At such temperatures, much higher salt levels are required to produce the same effect as at 10°C.
The effect of pH is different for different organisms. These limits can be affected by the nature of the acid.
The water activity of a food is related to the water vapour pressure and indicates the amount of water available for microbial growth. In general, reducing water activity also reduces microbial growth. Water activity can be decreased by adding solutes such as salt and sugar, which bind water and make it unavailable to bacteria. This can also be achieved by drying or freezing. When a solution becomes more concentrated, the vapour pressure falls and a W falls from a maximum of 1.0 for pure water. Source: J.Crowther, Unilever Research
Here we see the water activity of different concentrations of salt and glucose at 25°C. Weight for weight, salt is more effective than glucose in achieving a given water activity value. However, comparisons should be made on a mole for mole basis, because water activity is a function of the number of molecules or ions in the water. Salt, besides being of a lower molecular weight, also dissociates in two ions in water.
The range of water activities in foods is quite large. This table shows that the water activity is a selective parameter that determines which flora will grow.
Some products of the metabolic activity of moulds (i.e. mycotoxins) may also cause foodborne intoxications. The three genera most often associated with toxin production are listed here. Acute intoxication by mycotoxins seems to be rare, but the result of chronic exposure is of considerable concern. Moulds are found in the environment, often on fruits, nuts and grains where they grow under the right conditions of temperature and humidity. In general, moulds are more likely to cause food spoilage than to produce toxins. We shall have a chance to look at microscopic moulds during our first practical session.
Viruses, unlike bacteria, cannot be cultivated outside a living host. They are much smaller and can be seen only with an electron microscope. Here is a list of viruses associated with foodborne disease. There are no animal or plantborne viruses causing foodborne illness. All foodborne viruses originate from the human gut, and use it as their target. Any food vehicle contaminated with human faeces could be contaminated with these viruses. Shellfish harvested in polluted water are common causes of foodborne viral infections all over the world.
Viruses consist of a protein capsule which may be surrounded by a lipid/protein envelope. Inside there is DNA or RNA, a polymerase and sometimes other specific enzymes associated with the replication of the virus. Viruses cannot replicate outside a host cell . They attach to the host cell where the viral DNA or RNA, and other enzymes enter. Signals are sent that cause the host cell’s replicative apparatus to be used for the replication of the virus.
Very few virus particles are required to infect a host. Sometimes as few as 10 - 100 particles per ml are sufficient. Techniques such as electron microscopy or immunoassays can detect as few as ten thousand particles per gram of sample. Genetic techniques can detect even smaller numbers of virus particles. Source: Dr. K.Bellamy. 1989
Parasites can include bacteria and viruses, but the term is most frequently applied to protozoal and helminthic organisms. These parasites sometimes have complicated life cycles in which the human host is only one stage. To make the proper interventions to prevent transmissions to humans, it is important to understand these life cycles. Some of them are transmitted by eating red meat or fish, others by eating vegetables or drinking water. Parasites are particularly very important &quot;food safety hazards&quot; in tropical countries, although also in colder climates parasites are a major concern to public heath professionals.
In this lecture, we have had an overview of microorganisms. We have seen that FBD is caused by bacteria, moulds, viruses and protozoa. They not only cause infections but some also produce toxins. The behaviour of bacteria depends on the food in which they are found. If we understand contamination, and factors affecting survival and growth of the different bacteria, we may be able to control them in foods.
Bacteria can be harmful or useful and may even be used to preserve some foods. The lactic acid bacteria have been cited as an example but there are many others that may produce substances that are inhibitory to harmful bacteria. Viruses and parasites, unlike bacteria, yeasts and moulds, do not usually grow in food. We cannot control them in the same way but we can manage them using other interventions.
In the preceding lecture, we found out what microorganisms are. Now, we shall look at their ecology. Today we hear a lot about ecology, usually in reference to the environment and pollution. The word is widely used, and misused. Ecology is a science, as the “-ology” indicates. It examines the relationships between living organisms and their environment. This lecture will introduce you to the subject, paying particular attention to the details relevant to foodborne disease.
Microbes are everywhere. Their rapid division allows them to multiply to large numbers, and sometimes produce toxins. Their small size and mass allows them to be transported easily. Certain organisms may be associated with niches where the environment favours their multiplication. For example, organisms able to withstand high temperatures are found in hot springs, a habitat where most microorganisms could not establish themselves. Some may also grow at low temperatures. Some are particularly ‘agile’. Gut organisms are found predominantly in the gut and are spread through the faeces. They can survive, and even multiply, outside the gut.
One problem is that many of the organisms that interest us are not restricted to a single habitat. Here we can see that this causes a cycle of contamination, survival, multiplication and spread in the environment. This diagram shows Salmonella but it could also represent many other organisms. Although the organism may not be restricted in its ability to grow, there are some places where it is particularly likely to be found. We refer to these as reservoirs. The guts of humans and animals are common reservoirs. However, any warm place with sufficient nutrients and water can become a reservoir.
Some organisms such as Campylobacter may survive, but do not grow easily outside their niche. Thus, the number of potential reservoirs is restricted. This has implications for intervention measures. Campylobacter requires special conditions: a low oxygen level, a temperature > 30° and some special nutrients. It is probably a natural resident of the gut of birds. Poultry is a major reservoir for pathogenic campylobacters. Although it needs special conditions to grow, it can survive under less favourable conditions. It also seems to have a low infectious dose. We shall see later that this may contribute to the increasing incidence of infection with this organism.
Many raw materials, each with an associated normal flora, are used as foods or for making foods. Pathogens may enter the flora, either due to the way they are produced or by being transferred from the animal of origin. In certain cases, pathogens may almost be considered part of the normal flora, e.g. Campylobacter in chickens.
Meat and meat products are often associated with pathogenic microorganisms. This is because the gut is the major reservoir for many pathogens. Husbandry and rearing practices contribute to contamination. For example, in industrialised countries, by use of animal feeds made from rendered animal by-products such as bone meal and fish meal. The use of vegetable-based feeds made from soy and other oilseed press-cakes has also contributed to the problem.
Here we can see how meat is contaminated. Cross-contamination between animals on the farm is due to husbandry practices. However, at slaughter, the gut contents can contaminate the surfaces in the abattoir and be spread by the utensils, causing cross-contamination between carcasses. Further cross-contamination can occur at the butcher’s shop. Nowadays, in most developed countries, slaughterhouse hygiene and practices are carefully controlled. Diseased carcasses are usually spotted and discarded at examination. Even so, some contamination will always occur.
Milk is susceptible to contamination, due to the proximity of the udder to the ground and to the back end of the cow. Many pathogens have been associated with milk and milk products. Some of these are pathogenic for cattle, such as Brucella and Mycobacterium ; others are harmless microorganisms living in the soil or in the host.
Intensive poultry-rearing and processing practices have led to reduced prices and increased consumption. However, the risk of contamination of poultry with pathogens has increased. Many pathogens are associated with poultry. Some, such as Campylobacter, may be natural gut inhabitants. Others, such as Salmonella, may be picked up from the environment or from contaminated feed.
By far, the most important organism associated with eggs is Salmonella. Salmonella can contaminate eggs by penetrating the egg shell, especially if it is cracked, and through cross-contamination during preparation of dishes containing eggs. In addition, Salmonella enteritidis (and occasionally other serotypes) can infect the ovaries of hens and contaminate the interior of the egg before the shell is formed. Once inside the egg, Salmonella can grow quickly if the egg is not refrigerated.
Fish and shellfish also have associated hazards. One of the world’s largest foodborne disease outbreaks occurred in Shanghai, China in 1989, where some 300,000 cases of hepatitis A caused by the consumption of contaminated clams were reported. Raw shellfish and raw fish marinated in lime juice (cerviche) have recently been associated with cholera in Latin-America. C. botulinum Type - E is found in the gut of fish and is also common in some marine sediments. It has caused problems in Middle-Eastern dishes prepared by fermentation of uneviscerated fish, and in dry smoked fish in Egypt. V.parahaemolyticus comes from the sea, but the reasons for its association with shellfish are unclear.
Pathogens may be associated with vegetable raw materials. All of these except moulds, are due to cross-contamination from either contaminated water (e.g. waste water, surface water, household water), animal manure or human beings. Fruits and nuts are a source of foreign bodies (shells, pips, etc.).
B. cereus is found in soil and dust and thus commonly occurs in grains. Handling of grains in bulk attracts animals and birds. Their defecation may contaminate the grains with the enteric pathogens. Cereals are naturally associated with moulds and thus there is a potential for mycotoxin production. This will be discussed in module 6. Another risk associated with cereals is the mixing of toxic weeds with wheat. .
Spices are associated with microbial hazards for the same reason as cereals or vegetables. They can become heavily contaminated with Salmonella if they are extensively handled or dried in unprotected areas.
We can see that although some food pathogens come from plants and soil, animals are the main source. In the case of developing countries, waste water / irrigation water plays an important role.
We end this first module with a basic knowledge of what microorganisms are, where they come from, and how they get into raw foods. We shall look later at how they get into prepared foods.
Kuliah 2 kmk
Dept. Kesehatam Lingkungan FKM UI Microorganisms classified, growth, and ecology
<ul><li>pathogenic organisms </li></ul><ul><li>spoilage organisms </li></ul><ul><li>useful organisms </li></ul>Microorganisms classified by their significance
Phases of bacterial growth <ul><li>Lag phase </li></ul><ul><ul><li>(short) period of adjustment to environment </li></ul></ul><ul><li>Logarithmic Growth phase </li></ul><ul><ul><li>growth begins and accelerates to a phase of rapid, constant exponential growth </li></ul></ul><ul><li>Stationary phase </li></ul><ul><ul><li>depletion of nutrients and accumulation of toxic metabolic products growth is slowed to a point where cell division and cell death are in balance </li></ul></ul><ul><li>Death phase </li></ul><ul><ul><li>population decreases due to death of cells </li></ul></ul>
<ul><li>Temperature </li></ul><ul><li>Time </li></ul><ul><li>pH </li></ul><ul><li>Water activity (a w ) </li></ul><ul><li>Oxygen tension </li></ul><ul><li>Preservatives </li></ul><ul><li>Microbial interactions </li></ul>Factors affecting growth of bacteria in food
Temperature 0° 10° 36.5° 60° 72° 100° Boiling point Pasteurising temperature Freezer Fridge Body temperature SAFETY SAFETY SAFETY SAFETY DANGER
How temperature affects growth rate of a bacterial population C (Minimum) B (Optimum) A (Maximum) Temperature Hot Cold
Growth of S. typhimurium at different temperatures Time (Days) 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 25° 20° 15° 10°
Temperature range for growth of pathogens Temperature°C Min. Opt. Max. Salmonella 5 35 - 37 47 Campylobacter 30 42 47 E. coli 10 37 48 S. aureus 6.5 37 - 40 48 C. botulinum (proteolytic) 10 50 C. botulinum (non-proteolytic) 3.3 25 - 37 B. cereus 4 30 - 35 48 - 50 1 43 2
Temperature range for growth of toxigenic moulds Temperature °C Min. Opt. Max. Penicillium verrucosum 0 20 31 Aspergillus ochraceus 8 28 37 Aspergillus flavus 10 32 42 Fusarium moniliforme 3 25 37
Growth of different bacteria at 25°C Time (Days) 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 S. typhimurium L. monocytogenes Ps. fluorescens B. cereus C. bot-Proteolytic G+ve Spoilers Log CFU
Temperature affects bacteria <ul><li>Lag phase </li></ul><ul><li>Growth rate </li></ul><ul><li>Final cell numbers </li></ul><ul><li>Enzymatic and chemical composition </li></ul><ul><li>of cells </li></ul><ul><li>Nutritional requirements </li></ul><ul><li>Limits for other factors influencing growth </li></ul>through the change in
Effect of salt concentration on time to botulinum toxin production Salt Concentration (%) 0 2 4 6 8 10 12 14 16 0 0.5 1 1.5 2 10°C 14°C 18°C 24°C
<ul><li>a w is the ratio of the water vapour pressure of the food (p) to that of pure water (p o ) at the same temperature. </li></ul>Definition of water activity (a w ) a W = p / p o
NaCl and glucose concentrations and corresponding a w values at 25°C <ul><li>1.00 0.00 0.00 </li></ul><ul><li>0.99 1.74 8.90 </li></ul><ul><li>0.98 3.43 15.74 </li></ul><ul><li>0.96 6.57 28.51 </li></ul><ul><li> 0.94 9.38 37.83 </li></ul><ul><li>0.92 11.90 43.72 </li></ul><ul><li>0.90 14.18 48.54 </li></ul><ul><li>0.88 16.28 53.05 </li></ul><ul><li>0.86 18.18 58.45 </li></ul>a W % w/w % w/w NaCl Glucose
Range of a W in foods and their microbial flora > 0.98 Fresh meats Fresh fish Fresh fruits Fresh vegetables Canned vegetables in brine Canned fruit in light syrup (<3.5% salt, 26% sugar) (C. perfringens, Salmonella) (Pseudomonas) 0.93 - 0.98 Fermented sausages Processed cheese Bread Evaporated milk Tomato paste (10% salt, 50% sugar) (B. cereus, C. botulinum, Salmonella) lactobacilli, bacilli and micrococci a w range foods microbial flora
Range of a W in foods and their microbial flora 0.6 - 0.85 Xerophilic fungi Halophiles Osmophilic yeasts Dried fruit Flour Cereals Salted fish Nuts a w range foods microbial flora 0.85 - 0.93 S. aureus Mycotoxin producing moulds Spoilage yeasts and moulds Dry fermented sausages Raw ham (17% salt, saturated sucrose) < 0.6 No growth but may remain viable Confectionery Honey Noodles Dried egg, milk
Key messages <ul><li>Temperature, pH, water activity and oxygen tension are the principal factors affecting microbial growth </li></ul><ul><li>There are optimum ranges for these parameters </li></ul><ul><li>These optima are interdependent </li></ul><ul><li>They can be selected to inhibit the growth of certain organisms within limits related to the palatability of food </li></ul><ul><li>Certain foods are suited for the growth of certain flora </li></ul>
Some toxigenic moulds causing foodborne disease <ul><ul><ul><li>Aspergillus spp. </li></ul></ul></ul><ul><ul><ul><li>Fusarium spp. </li></ul></ul></ul><ul><ul><ul><li>Penicillium spp. </li></ul></ul></ul><ul><li>( Main sources - fruits, nuts and grains ) </li></ul>
Major viruses causing foodborne disease <ul><li>Hepatitis A and E viruses </li></ul><ul><li>Small Round Structured Viruses (e.g. Norwalk agent) </li></ul><ul><li>Rotavirus </li></ul><ul><li>Polio virus </li></ul>
Virus structure Protein Capsule Nucleic acid (DNA or RNA) Poliovirus Hepatitis-A virus Rotavirus Norwalk-like or SRSV
Virus levels required for infection and detection <ul><li>infection </li></ul><ul><ul><ul><ul><ul><li>10 -100 particles / ml </li></ul></ul></ul></ul></ul><ul><li>detection </li></ul><ul><ul><ul><ul><ul><li>10 5 - 10 6 particles / g </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>(by electron microscopy) </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>10 4 - 10 5 particles / g </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>(by radioimmunoassay) </li></ul></ul></ul></ul></ul>
Nature of bacteria, moulds, viruses and parasites - Key messages (1) <ul><li>foodborne diseases are caused by bacteria, moulds, viruses, and parasites </li></ul><ul><li>certain microorganisms are of greater significance than others for humans </li></ul><ul><li>bacteria and moulds multiply on foods and may produce toxins </li></ul><ul><li>understanding the factors controlling growth of microorganisms allows us to control them in food </li></ul>
<ul><li>bacteria may be harmful or useful </li></ul><ul><li>bacteria, yeasts and moulds can be used to preserve foods </li></ul><ul><li>lactic acid bacteria secrete lactic and other organic acids </li></ul><ul><li>organic acids inhibit pathogens in food and in the gut </li></ul><ul><li>viruses and parasites do not grow in food </li></ul>Nature of bacteria, moulds, viruses and parasites - Key messages (2)
Raw materials will have a normal flora <ul><li>meat </li></ul><ul><li>poultry </li></ul><ul><li>fish and shellfish </li></ul><ul><li>cereals </li></ul><ul><li>milk </li></ul><ul><li>egg products </li></ul><ul><li>vegetables, fruits and nuts </li></ul><ul><li>spices </li></ul><ul><li>oils and fats </li></ul><ul><li>water </li></ul>
Ecology of foodborne pathogens Key Messages <ul><li>Some originate from animals </li></ul><ul><ul><li>Salmonella </li></ul></ul><ul><ul><li>Campylobacter </li></ul></ul><ul><ul><li>E. coli </li></ul></ul><ul><li>Some come from plants and soil </li></ul><ul><ul><li>Moulds </li></ul></ul><ul><ul><li>B. cereus </li></ul></ul><ul><ul><li>C. botulinum </li></ul></ul>
Ecology of foodborne pathogens Key Messages <ul><li>Some originate in the sea </li></ul><ul><ul><li>V. parahaemolyticus </li></ul></ul><ul><ul><li>C. botulinum Type E </li></ul></ul><ul><ul><li>V. cholerae </li></ul></ul><ul><li>Some originate from man </li></ul><ul><ul><li>Viruses </li></ul></ul><ul><ul><li>S. typhi </li></ul></ul><ul><ul><li>S. aureus </li></ul></ul><ul><ul><li>Shigella </li></ul></ul>