3.  Pregnant migrant farm worker  Fever, headache, chills, diarrhea  Right costovertebral tenderness  Abdominal pain and tenderness  Pregnant migrant farm worker  Fever, headache, chills, diarrhea  Right costovertebral tenderness  Abdominal pain and tenderness  Intrauterine fetal demise – stillborn infant – stillborn infant  Patient started on Ampicillin- subactam and Gentamicin  WBC of 21,300/uL

4. Gram (+) rod shaped bacteria Clear, colorless, beta-hemolytic, raised colonies

5. What is the suspected organism? Listeria Monocytogenes Classification of the organism: - Small gram (+) rods that may appear pleomorphic. - Non-spore former - Umbrella motility at room temperature, motile by polar flagella at 37 C - Umbrella motility at room temperature, motile by polar flagella at 37 0 C Biochemical tests for isolation: - CAMP - 6.5% NaCl - Catalase - TSI: A/A - Esculin Hydrolysis Where does it live? Where does it live? - Soil and water How do you get it? How do you get it? - Eating contaminated food, water, unpasteurized dairy products, and raw meat. - It is possible that the patient acquired the organism while working in the farm. What disease(s) can it cause? - Listeriosis

6. Common Listeriosis symptoms:  Fever  Muscle ache  Gastrointestinal symptoms (nausea or diarrhea)  Headache  Stiffneck  Monocytosis For pregnant women:  Stillbirth  Premature delivery  Infection of the newborn Common Listeriosis symptoms:  Fever  Muscle ache  Gastrointestinal symptoms (nausea or diarrhea)  Headache  Stiffneck  Monocytosis For pregnant women:  Stillbirth  Premature delivery  Infection of the newborn Patient checklist:

7. Virulence Factors: - Growth at low temperature This allows it to grow in food stored in refrigerator. - Motility At 20-25 C, it uses its flagella to swim against the bowels’ peristaltic movement and to pierce the mucosal lining into which At 20-25 0 C, it uses its flagella to swim against the bowels’ peristaltic movement and to pierce the mucosal lining into which they attach. At 37 C, it moves between and into host cells by polymerization of host cell actin at one end of the bacterium, which can propel it throughout the cytoplasm. This is one of the organisms’ special characteristics for spreading. they attach. At 37 0 C, it moves between and into host cells by polymerization of host cell actin at one end of the bacterium, which can propel it throughout the cytoplasm. This is one of the organisms’ special characteristics for spreading. - Monocytosis producing agent This lipid causes monocytosis in the cell; WBC count greater than 950/uL. Patient had a WBC of 21,300/uL – shows possible monocytosis.

8. - Listeriolysin or LLO A toxin that forms pores to help the pathogen escape from endosome to the cytosol. A toxin that forms pores to help the pathogen escape from endosome to the cytosol. - Internalins or InA Possibly mediates invasion; surface proteins that helps to introduce pathogen in epithelial cells. Possibly mediates invasion; surface proteins that helps to introduce pathogen in epithelial cells. - ActA Surface protein that helps the pathogen to propel on adjacent cell by rearrangement of actin. - Phospholipase Also helps the pathogen in escaping endosome to the cytosol.

9. Mechanism of Invasion

10. Who is susceptible to Listeriosis? –Pregnant women are more susceptible in acquiring this disease - 20 times more likely than other healthy adults. –Newborns other than pregnant women themselves suffer the serious side effects of infection from pregnancy. –Immunocompromised and elderly patients can easily get this disease. Who is susceptible to Listeriosis? –Pregnant women are more susceptible in acquiring this disease - 20 times more likely than other healthy adults. –Newborns other than pregnant women themselves suffer the serious side effects of infection from pregnancy. –Immunocompromised and elderly patients can easily get this disease.

11. Antibiotics Ampicillin is the antibiotic of choice against L. monocytogenes and Gentamicin is frequently added for its synergistic effects. This is another hint from the case that the patient has listeriosis because she was started on Ampicillin-subactam and Gentamicin. Prevention  Handwashing before eating  Avoiding deli meat, soft cheeses, and unpasteurized dairy products  Thoroughly cooking raw food from animal sources  Washing raw vegetables before eating  Consuming perishable goods as soon as possible  Education and knowledge of Listeriosis especially for pregnant women

12. Ability of Listeria Monocytogenes Phospolipase C to facilitate escape from the macrophage phagosome is dependent on host Protein Kinase C Mathilde A. Poussin a,1, Michael Leitges b,2, Howard Goldfine a,* a Department of Microbiology, School of Medicine, University of Pennsylvania, 19104-6076 Philadelphia, USA b Signal Transduction Laboratory, Department of Nephrology, Medical School Hannover, Carl-Neuberg-Str.1, 30625 Hannover, Germany Introduction L. monocytogenes is a facultative intracellular pathogenic bacteria that also infects macrophages and phagocytes. After entry in the host cell, the bacteria escapes from the phagosome to the cytoplasm. From previous research by the same group, it was found that pharmacological inhibition of Protein Kinase C resulted in significant reduction of phagosome permeabilization by L. monocytogenes. Why and how? Methods Bone marrow macrophages obtained from PKC knockout and PKC producing mice were infected with L. monocytogenes. A strain of L. monocytogenes lacking LLO was also introduced in both but different

13. sets of bone marrow macrophages. Results The absence of LLO inhibited the organisms’ escape. L. monocytogenes enters and escapes the phagosome with the same efficiency in PKC KO (Fig B) and PKC producing mice (Fig A). This indicates that host PKC is not essential for the escape of the organism. However, the escape of Phospolipase C is not reduced in PKC KO mice. These findings strongly support the hypothesis that Phospolipase C promotes escape from the phagosome of macrophages through redistribution of host PKC.

14. sets of bone marrow macrophages. Conclusion Bacterial PLC promotes escape through the production of diacylglycerol leading to the activation of host PKC.These findings suggest that L. monocytogenes PI-PLC through the formation of diacylglycerol activates a pathway that affects endosomal recycling pathways, which could interfere with phagosome maturation. Bacterial PLC promotes escape through the production of diacylglycerol leading to the activation of host PKC. These findings suggest that L. monocytogenes PI-PLC through the formation of diacylglycerol activates a pathway that affects endosomal recycling pathways, which could interfere with phagosome maturation. This concludes that Phospholipase C promotes escape through mobilization of host PKC. Future goal A better understanding of signaling pathways in these cells.

15. Brouwer MC, van de Beek D, Heckenberg SG, Spanjaard L, & de Gans J. (2006). Community-acquired Listeria monocytogenes meningitis in adults. Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. 43(10), 1233-8. Cone LA, Somero MS, Qureshi FJ, Kerkar S, Byrd RG, Hirschberg JM, & Gauto AR. (2008). Unusual infections due to Listeria monocytogenes in the Southern California Desert. International Journal of Infectious Diseases : IJID : Official Publication of the International Society for Infectious Diseases. 12(6), 578-81. Delgado AR. (2008). Listeriosis in pregnancy. Journal of Midwifery & Women's Health. 53(3), 255-9. Goulet V, Hedberg C, Le Monnier A, & de Valk H. (2008). Increasing incidence of listeriosis in France and other European countries. Emerging Infectious Diseases. 14(5), 734-40.

16. Poussin, MA, Leitges M, Goldfine H. (2009). The ability of Listeria monocytogenes PI- PLC to facilitate escape from the macrophage phagosome is dependent on host PKCb. Journal of Microbial Pathogenesis. 46(1), 1-5. And thank you for Dr. McQueens’ informative lecture slides regarding Listeria Monocytogenes for some of the pictures and key information.