Stress induced virulence of Staphylococcus aureus and Listeria monocytogenes

Abstract

Control of the dissemination of pathogens in the food production chain is an on-going process with new challenges emerging as pathogenic bacteria evolve and express enhanced virulence characteristics. Staphylococcus aureus and Listeria monocytogenes are two notorious pathogens responsible for staphylococcal food poisoning (SFP) and listeriosis: a foodborne intoxication and an infectious disease respectively. In this work, S. aureus was investigated regarding the regulatory mechanism of the prophage-encoded enterotoxin A (SEA), and the impact of the food environment on the SEA produced. Moreover, L. monocytogenes’ capacity to survive and grow was evaluated under conditions of osmotic and acid stress. The expression of stress and virulence-related genes under these conditions was evaluated, and related to acquired resistance towards further stress.

In the S. aureus studies, a strain-dependent SEA production was demonstrated, and linked to the different sea gene alleles (sea1, sea2). The investigated strains were grouped as high and low SEA-producers, carrying the sea1 and sea2 allele respectively. Furthermore, it was shown how the life cycle of the sea-carrying phages, and specifically prophage induction, increases the levels of SEA produced in some of the high SEA-producers by increasing both sea gene expression and the number of copies of the gene in the induced cells. A phage-activated transcript, alongside the transcript from the endogenous P1 sea promoter, characterized these strains, which were further categorized as inducible high SEA-producers. Prophage induction was found to be linked to the SOS response, through the activation of the RecA protein. A study of S. aureus on pork sausages led to the observation that SEA production and growth rates should not be considered as coupled events, and that food components may lead to prophage induction and increased SEA formation in the food product. A study of the impact of NaCl and sorbic acid on prophage induction and SEA production showed that increased NaCl activates the phage and increases the potential risk for SFP, however, the presence of sorbic acid in pH conditions critical for S. aureus growth lowers this risk.

L. monocytogenes was found to be more susceptible to sequential stress applications, where acid and osmotic shifts combined with low temperature delayed the initiation of growth. Strain dependence characterized the survival and growth potential of the pathogen under adverse conditions, and adaptive responses were triggered only under mild stress habituation that allowed growth. However, stress adaptation could potentially increase virulence, even in cases where the phenotypic responses indicate a low risk of pathogen survival.

In summary, the genetic complexity of bacterial phenotypic responses when exposed to stress in the food processing environment was demonstrated in this work. The obtained knowledge could be incorporated into predictive modeling for more accurate hazard evaluation and effective control of pathogenic bacteria in food.

Abstract (Swedish)

Popular Abstract in English

The food we consume is hardly, if ever, sterile. From fresh produce to processed products, foods harbor one or more types of microorganisms. The presence of some ‘beneficial’ microorganisms is desirable in food production, because they contribute to the characteristics of the final product, as, for example, in the production of wine, beer or cheese. In other cases, microorganisms can be ‘spoilage’ or ‘pathogenic’, and have negative effects on food quality and safety because they cause spoilage and foodborne diseases respectively.

Foodborne diseases constitute global health and economic issues and every sector relevant to food production and public health care has at some time focused on developing ways to control the dissemination of pathogenic bacteria in food. The measures employed include preservation through the application of physical (heating, freezing, drying) and chemical (low pH, preservatives) treatments (stresses) during food production in order to eliminate any pathogens present. Although these treatments have been proven effective at delivering safe and high quality products to consumers, bacteria have found ways to adapt and survive their application.

The evolution of foodborne pathogens in response to preservation treatments has brought new challenges for food safety. In recent decades, new pathogenic strains have emerged, exhibiting enhanced virulence and resistance to preservation treatments. Therefore, for effective control of their dissemination in food, it is necessary to understand the stress response mechanisms bacteria employ, and the events which lead to the development of enhanced virulence and resistance. In this way, hazard analysis before and during food production will be improved and the risk of disease more efficiently controlled and reduced.

The work presented in this thesis concentrates on understanding the virulence relevant to foodborne diseases of the pathogenic bacteria Staphylococcus aureus and Listeria monocytogenes. The primary interest was the impact of adverse food-related environmental conditions on gene expression. The first part of the thesis discusses S. aureus and the disease it causes through the formation of enterotoxins, staphylococcal food poisoning (SFP), with a particular focus on the production of enterotoxin A (SEA). In the second part, the adaptation and survival of L. monocytogenes in acidic and osmotic environments is investigated, and related to the expression of key stress and virulence genes.

Staphylococcal food poisoning is a common foodborne disease. It is caused by consumption of food containing pre-formed enterotoxins, without live bacterial cells necessarily being present. Therefore the challenge in controlling SFP is to prevent and control the production of enterotoxins in food. Common preservation methods that are effective in the elimination of bacterial cells, have no impact on the staphylococcal enterotoxins, as they are stable against thermal treatment, acidity and proteolysis. Enterotoxin A has been reported as the cause of 80% of SFP outbreaks worldwide. A key characteristic of SEA is that it is encoded by a gene (sea) carried on Siphoviridae temperate bacteriophages. These phages are able to enhance the virulence of the host bacterial cell by altering its life cycle and expressing virulence factors such the sea gene when conditions in the environment diverge from optimal. The work presented in this thesis explored the relationship between the phage life cycle, expression of the sea gene, and production of SEA. The impact of food composition and preservatives such as salt and weak acids was assessed on the phage and the SEA produced, to highlight food processing treatments that could potentially increase the risk of SFP through the formation of high levels of SEA. In summary, it was found that the life cycle of the sea-carrying phages has a critical role in the expression of the gene and the levels of SEA produced by the S. aureus strains, and it is further affected by the conditions of the cell environment and food preservation treatments.

The second part of the thesis studies another major foodborne pathogen, L. monocytogenes, which is responsible for the infectious disease, listeriosis. The pathogen is robust and can survive in harsh environments and at refrigeration temperatures, and thus constitutes a high risk for food safety. It can also adapt to adverse environments and develop resistance, which increases its persistence in food and in the processing environment. The focus of this work was the response of the pathogen to osmotic and acid stress, and how the intensity and sequence of these stresses can affect its capability for survival and growth in a new environment. The genetic events behind the phenotypic responses of the pathogen were also investigated, in order to potentially establish a correlation between them that could be used to improve control of L. monocytogenes in food.

The knowledge obtained during this thesis work could be used to improve existing predictive models in the assessment of hazards during food production, and contribute to the design of effective food safety strategies.

• author: Zeaki, Nikoleta
• supervisor: Jenny Schelin
• opponent: Dr. Le Loir, Yves, Science and Technology of milk & eggs (STLO), INRA-Agrocampus Ouest, Rennes, France
• organization: Applied Microbiology
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Engineering and Technology
• categories: Higher Education
• pages: 203 pages
• defense location: Kemicentrum, Lecture Hall C, Getingevägen 60, Lund University Faculty of Engineering, LTH.
• defense date: 2015-05-29 10:15:00
• ISBN: 978-91-7422-398-9
• language: English
• LU publication?: yes
• id: b8ed2826-2f12-4a06-8c35-953d66f30161 (old id 5364870)
• date added to LUP: 2016-04-04 10:36:05
• date last changed: 2018-11-21 20:59:42