Determining the Effects of Propionate on Listeria Monocytogenes Susceptibility to Lysozyme Degradation

Jeanne Paula E. Sering1,2,3 University of Dayton

300 College Park, Dayton, OH 45469

1. Department of Biology

2. Berry Summer Thesis Institute

3. University Honors Program

Thesis Mentor: Yvonne Sun, Ph.D.

Department of Biology

Abstract

Listeria monocytogenes is a harmful pathogen transmitted through contaminated food. Listeriosis, the infection associated with L. monocytogenes, is rare but potentially fatal, with a twenty to thirty percent mortality rate. For that reason, the lack of safe strategies to prevent infections can be detrimental. Current infection preventative strategies rely on stringent food surveillance and recalls, but we want to determine alternative tactics to further protect the public from L. monocytogenes. More specifically, we want to identify environmental factors that can compromise the ability of L. monocytogenes to cause infections before the pathogen reaches the intestines. For example, propionate is generally recognized as safe by the FDA and is used as an additive in various food products. Our lab has previously demonstrated that propionate exposure in L. monocytogenes can lead to changes in growth and pathogenesis. To determine how propionate exposure affects L. monocytogenes survival and fitness in the gastrointestinal tract, my thesis project therefore studies the effects of propionate on L. monocytogenes resistance to the lysozyme found in our saliva. If propionate enhances L. monocytogenes lysozyme resistance, the use of propionate in food products might contribute to L. monocytogenes survival during transmission between food and our gastrointestinal tract. However, if propionate decreases L. monocytogenes resistance to lysozyme, it could be beneficial to use propionate as an efficient infection preventative strategy. To better understand the functions of propionate in L. monocytogenes lysozyme resistance, I performed a literature review in the following areas: the importance of oral health, antimicrobial mechanisms in the oral cavity, lysozyme, and Listeria monocytogenes

The Importance of Oral Health

Oral health is often placed on a different plane of existence by the general public in comparison to physical health. When people commit to a lifestyle change, they often start by eating healthier and exercising while overlooking oral hygiene. The importance of oral health is therefore taken less seriously. The reality remains that oral health is an essential component of an individual's overall wellbeing. For example, according to the CDC, oral diseases may lead to issues regarding impairments of speaking, eating, and learning (Oral Health Fast Facts, 2022). Quality of life can be significantly impacted by cavities, severe gum disease, and severe tooth loss (Oral Health Fast Facts, 2022). Moreover, multiple health conditions are connected to oral health in some manner that people are unaware about.

For example, improper care of one’s oral health may lead to health problems elsewhere in the body. Propionibacterium acnes is a bacterium that is a part of the normal microbiota of the skin, oral cavity, gastrointestinal tract, and genitourinary tract (Achermann et al., 2014). Moreover, the bacterium operates as an opportunistic pathogen that causes invasive infections; these include implant-associated infections which turn heads to dental implants in the oral cavity (Achermann et al., 2014). Implants are a common option that dental patients resort to in order to increase the longevity of the image of their teeth. Dental infections associated with these implants have the possibility to develop into a more serious concern affecting an individual’s overall health. An individual’s overall health is also impacted by the state of one’s oral microbiome, and having an infection located in the oral cavity indicates an unbalanced microbiome. Despite the oral cavity containing one of the most diverse and unique microbiomes, it is also found to be relatively understudied in the field of microbiology today.

A popular topic of study, being the microbiome of the gut, is explored heavily in comparison to the microbiome of the oral cavity (Willis & Gabaldón, 2020). Food that is consumed by individuals enters the oral cavity prior to entering the gut. For the second destination of food to be perceived as more important than the gatekeeper of the human body seems almost retrogressive. Studies have found associations between multiple diseases with increases or decreases of the abundances of organisms in the oral cavity (Willis & Gabaldón, 2020). Along with these findings, particular microbiomes within habitats of the oral cavity have a direct implication with disease and infection. For example, individuals with worse dental health had tongue microbiomes enriched in pneumonia-associated bacteria (Willis & Gabaldón, 2020). Such research reinforces the idea that oral health plays a detrimental part in one’s overall health.

While health problems can originate in the oral cavity, there are instances in which health problems lead to poor oral health. For example, diabetes is a health condition where one has irregular blood glucose levels and results in the individual having less resistance to infection. Individuals diagnosed with this condition are more susceptible to dental issues and gum disease (Diabetes and Oral Health - Better Health Channel, n.d.). This finding further implies that oral health and medical health are constantly intertwined with one another. While these two disciplines constantly intersect, dental health and general health are still disconnected physically.

More often than not, hospitals and dental offices are their own separate entity. At one point in time, dentistry wasn’t a profession but a trade, and the field was reliant on medicine to build a strong foundation (Simon, 2016). The tie between oral health and medical health was severed with the development of health insurance (Simon, 2016). In fact, dental insurance and medical insurance serve two entirely different purposes; medical insurance is intended to cover unpredictable, highcost expenses, and dental insurance is intended to cover predictable, low-cost preventative care (Simon, 2016). This method implicates low-income populations by placing them at a higher risk for being unable to afford needed, high-cost dental care. The historical separation of dentistry and medicine remains prevalent today, and now, people are becoming more aware of the strong correlation between oral and medical health. More and more people are advocating for the need to change and reunite the two fields to make healthcare more accessible to everyone.

Antimicrobial Mechanisms in the Oral Cavity - Oral Microbiome

Numerous antimicrobial mechanisms in our mouth are constantly at work. Over 700 bacterial species reside in the oral cavity and play a pivotal role in regard to human health and the immune system (Fan et al., 2018). Major oral bacteria phylum containing 96 percent of the oral taxa in the human oral microbiome are Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria (Dewhirst et al., 2010). Within the various habitats in the oral cavity, the composition of such species is subject to change. For instance, studies have shown that alcohol consumption can affect the overall composition of the oral microbiome (Fan et al., 2018). If a commonly consumed beverage by adults can alter the oral microbiota, there is cause for concern about what other substances can affect the oral microbiome.

The microbiome is often responsible for fighting off disease and infections. If thrown off balance, the oral microbiome may encounter disease that can potentially escalate to a systemic disease, not only affecting the mouth. To counter this, saliva and gingival crevicular fluid provide nutrients to maintain a balanced microbiota. For example, hypothiocyanite production is catalyzed by lactoperoxidase reinforcing antimicrobial activities such as inhibiting bacterial glycolysis (Kilian et al., 2016). To add on, proteins form a protective coating that protects the tooth surfaces from acid attacks (Kilian et al., 2016). At the same time, our microbiome can be perturbed by diseases in other parts of the body. For example, obesity, metabolic syndrome, multiple sclerosis, among other chronic health conditions are associated with perturbations of the gut microbiome (Durack & Lynch, 2019). A more diverse microbiome is an indicator of a healthy one, and the previously listed diseases are directly associated with a less diverse microbiome. To further explain, a less diverse microbiome indicates the lack of defenses against infection and disease. Since multiple reactions are occurring in the oral microbiome, the use of more defense mechanisms is essential in the protection of a healthy microbiome.

As a result of the metabolic activities of the oral microbiome, acid, which was previously discussed, is potentially yielded. Acid, the product of the chemical reaction catalyzed by the introduction of sugar to the microbiome in our mouth, is what breaks down our tooth enamel eventually resulting in decay (Understanding the Chemistry of Your Oral Health, n.d.). Our oral microbiome can cause both oral and systemic diseases when not properly protected from pathogens (Deo & Deshmukh, 2019). Therefore, maintaining a healthy microbiome, in which the “good” bacteria outnumber the “bad” bacteria, is essential for living a healthy life without disease.

In relation to oral health and the mechanisms prevalent in our mouth, there are billions of chemical interactions that occur in our mouth. With that being said, there are both advantageous and disadvantageous chemical occurrences that impact our oral health as a whole. An important course of action to note is that there are numerous strategies to maintain excellent oral health, such as eating a healthy diet with limited sugar and balanced acidity.

Antimicrobial Mechanisms in the Oral Cavity - Lysozyme

A specific antimicrobial mechanism found in the saliva is lysozyme, which is an enzyme that catalyzes the degradation of bacterial cell walls. This enzyme is produced and secreted through bodily discharge such as tears, saliva, human milk, and mucus; lysozyme can also be found in egg white, macrophages, and polynuclear neutrophils (Joel et al., 2016). The process in which lysozyme breaks down bacterial cell walls involves the cleavage of peptidoglycan by catalyzing the hydrolysis of β-(1,4) linkages between the NAM and NAG saccharides (Primo et al., 2018). Lysozyme works alongside other molecules in the mouth to maintain the equilibrium of the oral microbiota.

In the oral cavity, lysozyme acts as a defense mechanism against pathogens from spreading infection. Previous studies have shown that lysozyme has the ability to constrain the growth of invasive bacteria, such as L. monocytogenes, in culture broth as well as milk and cheese food products (Ramos & Malcata, 2017). Such findings prove lysozyme a notable factor to consider in antimicrobial studies.

The Foodborne Pathogen Listeria monocytogenes

L. monocytogenes is a gram-positive bacteria that is responsible for causing the infection called listeriosis. One can contract the disease by consuming contaminated food or by handling contaminated food and transferring the bacteria from their hands to their mouth. Foods that are particularly susceptible to contamination include produce, dairy products, and pre-packaged foods. Listeriosis is a rare but potentially fatal infection with a mortality rate of twenty to thirty percent (Medicine, 2021). Several groups of people are more at risk of contracting the disease; these groups include the elderly, the pregnant, and the immunocompromised (Commissioner, 2020). We want to find ways to further protect these individuals from anything we consume. While there are infection preventative strategies in place, we want to discover ways to weaken L. monocytogenes ability to infect before the bacteria reaches the gastrointestinal tract.

L. monocytogenes is unique as a bacterium since it has developed ways to persevere even in extreme conditions. To explain further, L. monocytogenes has a tolerance for both acidic and salty conditions, high and low temperatures, and low moisture content (Medicine, 2021). Such adaptations enable the bacteria to survive in prepackaged food products and processing plants. Essentially, the bacteria is adopting different mechanisms to persist in different environmental conditions using its own structure and resources. Results of a previous study found that L. monocytogenes is able to utilize three enzymes (PgdA, PbpX, and OatA) and two regulators of gene expression (DegU and Rli31) in order to resist lysozyme (Burke et al., 2014). Furthermore, my project focuses on lowering this lysozyme resistance of L. monocytogenes

Final Perspectives

Studying how to compromise L. monocytogenes lysozyme resistance benefits the food industry in terms of making food safer for consumption. The use of food additives has mainly been for extending shelf life and increasing flavors. There might be an opportunity to explore food additives that can help render foodborne pathogens more susceptible to antimicrobial strategies already in place. More research is needed to examine how environmental factors, alternative food additives, and food storage tactics influence pathogen fitness and pathogenesis. I personally would like to further investigate the connection between oral hygiene and L. monocytogenes lysozyme resistance. While the connection between oral and general health has been professionally separated, I am aspired to look at one’s health in a holistic manner. After all, oral hygiene is eminently important to our health.

This research experience has contributed to my personal growth in many ways that I am still continuously discovering. I learned how to design survival experiments tailored to my project and analyze scientific literature that allowed me to broaden my perspective on microbiology studies. To add on, I was introduced to the research process and the tediousness of repeating experiments. Something that Dr. Sun has taught me this summer that finding no significant differences within your results is still a finding. I’ve learned to be proud of what I have accomplished in the time I had and to look at problems I faced from different angles. All the while, I fostered friendships within the lab and with my peers doing research in other disciplines. I’m extremely grateful for the opportunity to begin my research project with insurmountable support from the university.

Acknowledgements

Firstly, I would like to thank my mentor, Dr. Yvonne Sun, for her intellect and guidance in my project. I would also like to thank the University Honors Program, the Berry Family, the Berry Family Foundation, and the Berry Summer Thesis 2022 cohort. Finally, I would like to thank my family and friends for their unending love and support.

References

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