By Roy Betts, Fellow at Campden BRI, UK
When microbiologists are asked to do microbiological testing, they often begin by asking themselves one simple question: “What should I test for?”
Why this question?
Specific microorganisms can be associated with specific food categories and origins, so understanding which organisms are present can provide an indication of what’s leading to a contamination issue.
With over 35 years’ experience as a food microbiologist at Campden BRI, here I’ll detail how identifying the microorganism present in specific food categories, through testing, can help you understand the potential root cause of contamination. Of course, we know that testing does not and will never assure safety. Safety is managed through correct risk assessment and implementation of HACCP, but testing can provide a useful adjunct to this, helping to verify that these systems are operating well.
Identifying microbiological issues with testing: A microbiologist’s thought process
Let’s return to the question of, “What to test for?”
Although basic, this general question requires a huge amount of knowledge, expertise and wisdom to answer. We must also consider why a series of microbiological tests are being done. Is it for routine monitoring, hygiene determination in production, HACCP verification, or as a search for an ongoing incident with a spoilage organism or pathogen?
This helps us to determine what to test for and where to test. Next, we must consider the item(s) being tested. What type of food or ingredient are we looking at, where did it come from, how was it produced and how is it going to be used? This will play a major part in allowing the microbiologist to determine the risk of certain pathogens (disease-causing microorganisms) being present and the correct tests to set up. When we are considering pathogen tests, it is also critical to understand the legislation in the country of production and the country of sale of any food item. Are any pathogen tests mandatory in the country of production or sale?
Homing in on the organism
When it comes to defining pathogen testing, we must ask more questions:
• What organisms are we likely to find in particular foods and ingredients?
• Which of these will constitute a risk to consumers?
• Which may survive in particular conditions or retain the ability to grow?
• Which have caused problems in the past?
If we start by looking at various groups of foods, we begin to see the information required in the decision-making process. This depends on good microbiological knowledge, an understanding of foods, their properties and their methods of production and a good share of historical understanding of what has caused issues here before.
We should also remember that the food is just an ecological environment to the bacteria contained on and within it. So, changes we make to the food will probably impact its microflora - changing those that survive, those that can grow and potentially changing the risk to the consumer.
Let’s start with raw meats. These will generally have a high water activity (aw) and few controlling factors that will limit microbial growth. Within an intact animal, whole muscle will virtually always be sterile. Raw meats become contaminated during the slaughter and butchering process, potentially from the hide or gut contents. Hides may carry various types of environmental contamination, namely faeces, hence why our major organisms of concern will include enteric pathogens such as Salmonella and pathogenic Escherichia coli (E.coli). If we’re investigating raw poultry, we may discount pathogenic E.coli due to the little evidence of it causing problems in this food. But we may add in Campylobacter as it’s commonly linked to poultry meat in food poisoning cases. Chilling correctly will slow or stop enteric pathogen growth but will not eliminate these organisms.
Of course, most raw meat will be cooked before eating, and this - if performed properly with a validated cook process - will eliminate these organisms. However, we still must consider the risks associated with kitchen cross-contamination from raw meat to ready-to-eat (RTE) products and the small amounts of raw meat that may be deliberately consumed undercooked.
Turning away from raw meats, we face issues even with those that are cured – albeit different issues. Water activity (aw) measures the amount of water available in a food. A higher value for aw means that here is plenty of moisture for microbial growth, whereas a lower value indicates that there may not be sufficient water available to support the growth of bacteria. Curing salts will often reduce the aw of cured meat, but as long as this is above 0.88, bacterial growth is possible. Lower aw will, however, reduce the types of species that can grow.
What about the enteric, faeces-originating pathogens, such as Salmonella or Shiga-toxin producing E.coli (STEC)? We may still have concerns about these, but less concern with their growth. However, we may start to consider organisms more capable of growth at low water activities. Classic ‘culprits’ would include enterotoxin-forming Staphylococci and Listeria. While Staphylococci will be prevented from growing at chilled temperatures, Listeria will continue to grow slowly even in chilled conditions. If the temperature is poorly controlled, some Staphylococci will grow and may form a pathogenic toxin that is very heat resistant and not eliminated by cooking, therefore it will remain active long after the organism has been killed.
Ready-to-Eat (RTE) cooked meats offer a different challenge. If well produced in a hygienic environment, these should have been rendered free from enteric pathogens by the validated cooking process used. However, such items will then be subject to handling in a chilled “high-risk” environment. These areas can often home to Listeria that can thrive under cold, damp conditions and can be particularly difficult to eliminate from even the most hygienically operated production plants.
Often, RTE cooked meats will be packed in low oxygen modified atmosphere or vacuum packs. As in any low oxygen environment, the hazard that must be considered is that posed by anaerobic bacteria, particularly Clostridium botulinum (C. botulinum) and its potent toxin. The growth of this organism must be controlled in such foods. The other Clostridium species to note in cooked meats is C. perfringens. Its spores will survive normal cooking processes but can germinate and grow during cooling, producing high numbers of vegetative cells which, if consumed, will cause food poisoning. Rapid cooling of such meats is critical to prevent growth and control the risks associated with this organism.
Fish and seafood products
Microbiological hazards associated with fish will depend on the product type and how it is produced. Raw fish may be caught at sea or farmed, but after harvest, temperature control will be critical in reducing microbial risk. Even if a fish is going to be fully cooked before consumption, some types (the scombroid fish) can allow production of histamine due to Enterobacteriaceae growth. When consumed, this organic compound will cause histamine poisoning. Worst still, histamine will remain after cooking, meaning the only control is prevention of microbial growth after the catch.
Fish have also been associated with C. botulinum toxin poisoning. It’s presumed that muds at the base of many bodies of waters may have anaerobic conditions which would explain the origin of this potent pathogen on fish. Storing fish products containing this organism under low oxygen conditions would subsequently allow the growth of C. botulinum and production of its toxin. So, how can it be controlled? In short, good temperature control will minimise the ability of C. botulinum to grow. Temperatures below 10°C will prevent proteolytic C. botulinum from growing, whereas non-proteolytic C. botulinum will have to be subjected to temperatures below 3°C to prevent growth. Overall, this organism must be considered a risk in fish (raw or cooked) that is going to be vacuum or modified atmosphere packed and marketed under chilled conditions.
Listeria is another threat that must be considered. Processed fish and fish products may become contaminated by Listeria that originates in the processing area, particularly if the fish is destined to be consumed raw, such as smoked salmon. Finally, another consideration for fish is contamination with Vibrio species; in such cases, testing for this group may be considered.
Shellfish are filter feeders and may contain any pathogen present in the water in which they are found. Enteric bacteria and viruses such as Hepatitis A, E and Human norovirus must be considered. Cooking can eliminate these organisms, but in the case of Hepatitis A, the cook temperature may be recommended to be higher than that we would normally use for other food types.
Raw milk may be contaminated with a variety of human pathogens that will originate from the animal. Salmonella, pathogenic E. coli and Campylobacter have all caused outbreaks linked to raw milk. For products made with raw milk, these key pathogens may still present a threat, but then we also start to think about Listeria which is often associated with chilled dairy products, and again the toxin forming Staphylococci in products with a lower aw.
The correct pasteurisation of milk and prevention of recontamination from raw milk will eliminate enteric pathogens. Our focus, therefore, shifts to the production area and the environmental contaminants that could get into the pasteurized product due to poor hygiene. Fermented dairy products may have both a low pH and sometimes a reduced aw helping to control bacterial growth. Yet, Listeria may still be a real issue, surviving well in chilled production areas and able to grow at lower water activities.
Produce covers a wide array of different foods. Lettuce, tomatoes, beans, potatoes, cabbage, carrots, cucumbers, onions, fruits etc. all fall into this category. Many will be grown in an open, outdoor environment exposed to all the natural animal inhabitants, and some may come in contact with the soil.
Any product that is grown outdoors and may have contact with soil must be considered a risk with respect to enteric pathogens - predominantly Salmonella and pathogenic E. coli. These may arise from the soil, from animals (both ground-dwelling and birds), and irrigation waters. There have been food poisoning outbreaks traced to zoonotic contamination of field crops, either by direct contact or from faecally-contaminated irrigation water that is sprayed onto crops. We should also not discount crop contamination from humans - in the form of those tending or harvesting crops - which may contaminate produce with viral pathogens such as Norovirus and Hepatitis A.
As well as produce that is directly harvested and sold, there is also produce that receives further treatment before sale as an RTE product to the final consumer. The production of RTE salad packs involves harvest, then usually some form of washing to remove soils and will also have a microbial reduction effect. Produce may then be cut/shredded before packing. These activities introduce factory-style activities in a chilled wet environment and start to increase concerns about Listeria contamination.
Sprouted seeds are another type of fresh produce that may be considered. Seeds such as alfalfa or mung bean, are steeped in warm water for periods of many hours to allow germination. Of course, these are ideal conditions for microbial growth and, if the seeds harbour pathogens such as Salmonella or pathogenic E. coli, then high levels of pathogenic growth could occur. Some very large outbreaks have been traced to contaminated sprouted seeds that are consumed raw.
Dry (Low aw) materials
This covers a wide range of materials ranging from dairy powders, chocolate, snack products, cereals and nut butters to herbs and spices. The key thing about organisms in dry environments is that they cannot grow. However, it also means they’ll be extremely resistant to various antimicrobial activities, such as heating, and will survive for very long time periods. The organisms that may be found in such materials will depend on how they have been produced and processed but are likely to be varied. A full risk assessment of their production method will help determine the potential hazards.
If untreated, herbs and spices will contain a microflora similar to that of fresh produce (potentially including enteric pathogens). This may be considered acceptable if they are added to products before a validated cooking process is used, but completely unacceptable if added as a garnish to ready-cooked products. Chocolate and nut butters have been associated with Salmonella outbreaks presumably occurring through cross-contamination during production. Dairy powders likewise have been associated with Salmonella, and - in the special case of powdered infant formula - Cronobacter must also be considered an issue.
Spores are often present in high numbers in dry materials; they can form a resistant state that survives drying and will remain able to germinate even after a long period. Spores are not a particular issue within the dry material unless they have grown to high numbers before the drying process has occurred. In such cases, spore formers such as Bacillus cereus may have produced emetic toxin which:
• is very heat resistant,
• will survive heating processes, and
• can cause food poisoning when consumed.
Likewise, the end-use of dried materials needs to be considered with respect to spore loading. Whilst stable in their dry state, as soon as a dry material is wetted the spores will germinate and grow and may cause problems in foods that use these as ingredients.
Chilled ready meals are a large and varied market. The microbiological hazards depend on the ingredients and methods of manufacture – and these hazards can be defined by a risk assessment for each product. Generally, due to the chilled nature of these product’s production, Listeria must be considered a potential problem. Often Salmonella will also be considered; however, cooking processes and hygienic production that prevents cross-contamination will usually have eliminated this organism, and testing will be used as a HACCP verification activity.
Attempting to define which organisms may present a clear risk in foods is a challenging pastime. It will depend on how the food is grown, harvested, handled, processed, and packaged. It will change as different ingredients are added and if it is presented to consumers as an RTE or a ready to cook product. This article is not exhaustive and is certainly not a guide to what to test for or consider as a risk in all food products. Instead, it simply provides a food microbiologist’s view on how such an issue may be approached, some of the thought processes that can be used (based on a good knowledge of the biology of the organism), and of the type of processes used in producing foods. Each food is different, each factory is different, and each assessment of hazards and risks is different.
If there is ever doubt that the correct hazards have been identified, then producers should contact an experienced food microbiologist that can help them in their ongoing quest to produce the safest foods with the lowest risk.