Stressing bacteria for safer food
By subjecting pathogenic bacteria to massive stress, Tone Mari Rode has gained new knowledge that may make it more difficult for bacteria to survive in food in the future.
On 27 March 2009, Tone Mari Rode had her doctoral disputation, under the title "Stress responses in food related Staphylococcus aureus" at Nofima Mat. Rode’s doctorate has been under the aegis of the Norwegian University of Life Sciences (UMB) but the work was carried out at Nofima Mat. She will continue at Nofima Mat in a post.doc position and will now conduct research into E. coli bacteria.
Most people suffer from food poisoning at some time or other during their life. The probability of being infected by Staphylococcus aureus is great, as this is one of the most common food poisoning bacteria. To be able to combat S. aureus, and bacteria in general, it is important to learn how bacteria respond to different environmental factors.
Many people ill – few studies
In her doctorate work, Tone Mari Rode has been studying how Staphylococcus aureus (also called yellow staphylococcus) reacts to acid stress. When you "irritate" bacteria by subjecting them to acid or other types of stress, the bacteria’s ability to grow and survive is inhibited. When subjected to stress, bacteria employ various defence mechanisms. Studies of which genes are involved during acid stress, so called gene response studies, have been an important part of the work.
"Previous studies of this bacterium have mainly been clinical. Such studies are important, because yellow staphylococcus also creates many problems for hospitals and similar institutions. Even though many people are infected via food, few have previously studied S. aureus in a food context," explains Tone Mari Rode.
Stress turns genes on and off
She used organic acids, such as lactic acid and acetic acid, to stress the bacteria. Organic acids in food lower its pH value. Cured sausage, pickled cucumber and mayonnaise are some examples of foods with a low pH. This type of acid can enter the bacteria and acidify it from within. Other acids, such as hydrochloric acid, do not have this property. "We thought that organic acids would cause more stress – and indeed they did. When the bacteria are stressed, they turn genes on and off in an attempt to raise the pH," explains Rode.
"What makes bacteria stressed?"
"Practically anything! They are easily stressed. As well as low pH, temperature changes, preservatives, the amount of oxygen, the presence of sugar or salt and a number of other factors affect the stress level of bacteria."
Exactly which genes are turned on and off in bacteria under stress tells us something about how that bacterium reacts to defend itself against such attacks. This kind of analysis is very complicated.
"A future goal will be to combine various factors that stress bacteria in the most effective way. With the aid of gene response studies, we may be able to get a step closer to this, so that we can make it more difficult for bacteria to live in foods and on surfaces," hopes the new doctoral candidate.
Bacteria that grow on surfaces can produce a slime and create a coating we call biofilm. Biofilm can be produced on almost any surface and can be extremely difficult to remove. Rode’s research has shown that both bacteria that cause hospital infections and those that cause food poisoning create biofilm. "Biofilm is common in food production; it can form in the pipes of a dairy, for example. But it is also a big problem among patients who have been given a prosthesis. Biofilm can easily develop on a new prosthesis and may result, for example, in a patient having to have a new hip replacement," explains Rode. Preventing the creation of biofilm is therefore of great significance. Tone Mari Rode has also studied what affects a bacterium’s ability to cling to a surface. Her results show that several environmental factors may prevent the formation of biofilm by S. aureus. "It is important to focus on biofilm, both in the food industry and the health sector. Biofilm may develop anywhere, even at low temperatures," stresses Rode.
Most people experience food poisoning several times throughout life. The probability of getting infected by Staphylococcus aureus is large, as this is one of the major food poisoning bacteria. To be able to combat S. aureus, and bacteria in general, it is important to learn how bacteria respond to different environmental factors, and how these factors affect the bacteria at a genetic level.
This thesis focuses on stress responses of S. aureus. The work has included the exploration of biofilm formation among both food-related and clinical strains, an investigation using DNA microarray for gene expression analysis studying organic acid stress responses at pH 4.5, and a comparison of two different technologies for gene expression analysis, MassARRAY and microarray.
The work showed that several environmental factors can induce biofilm production in S. aureus. Different strains responded differently with respect to biofilm formation, but there were no systematic differences observed between food-related and clinical strains. Generally, temperatures suboptimal for growth increased biofilm formation. Sodium chloride, glucose and ethanol increased the strains’ ability to produce biofilm, however the magnitude of the effect was highly strain dependent. A transcriptional analysis of selected genes involved in biofilm production supports the existence of both ica-dependent and ica-independent mechanisms for biofilm formation in S. aureus. Organic acid (lactic and acetic acid) stress induced a marked change in the growth pattern compared with inorganic acid (HCl) stress. Both the transcriptional and metabolic responses supported that lactic acid stressed bacteria increased the production of non-acidic end products from pyruvate. The results indicate that the diacetyl pathway has an important function in S. aureus at low pH. Genes encoding iron and purine regulation, as well as genes involved in transport and synthesis of cell wall associated proteins were differentially expressed due to lactic acid stress compared with HCl stress. In the work with MassARRAY, it was shown that this method has potential for studying bacterial gene expression. A comparison of gene expression during acid stress using MassARRAY and microarray revealed a strong linear relationship between the results. A comparison with quantitative real-time PCR generally confirmed these results.