Case Study Listeria monocytogenes 2009

1. Case StudyListeria monocytogenes2009 Case 43 Roubina Tatavosian James Muro Jae Kim

2. Case Summary • 3 1/2 –weeks old male wan born by Cesarean section • At birth he had a left diaphragmatic hernia, which was repaired soon. • He required intubation and respiratory support. • Over 24-h period, the infant developed following symptoms; • Bulging anterior fontanelles (A bulging fontanelles is an outward curving of an infant's soft spot ) • Increasing heart and respiratory rate • Wide fluctuation in blood pressure • Difficulties maintaining adequate tissue perfusion • WBC count increased from 6300 to 13700/μl • Child began to have focal seizures • Examination of cerebrospinal fluid (CSF) is as following; • 92% neutrophils • 2 mg/dl glucose • 350 mg/dl protein • 3900 WBC/ μl

3. Key Information Pointing to Diagnosis • Symptoms of the patientindicates that he had bacterial cause neonatal meningitis. • There are two major kind of bacteria that my cause neonatal meningitis; Listeria monocytogenes and Streptococcus agalactiae in new born. • Patient was born by Cesarean section not a vaginal delivery so this will eliminate Streptococcus agalactiae (exist in vagina canal). • Meningitis occur in the patient after he was born (Late onset kind of meningitis) • Patient required intubation and respiratory support • Results of CSF test (92% neutrophils,2 mg/dl glucose, 350 mg/dl protein, and 3900 WBC/ μl ) • Characteristics of CSF Gram Stain; Gram positive rod • Blood test results • Isolated colonies from CSF and Gram stain of the male infant proof of existence of Listeria monocytogenes

4. Listeria monocytogenes Gram Stain on CSF

5. The Diagnosis for Case # 43 • This patient was diagnosed by bacterial neonatal meningitis, caused by Listeria monocytogenes . • In neonates, neonatal meningitis occur in one of the two forms (Dr McQueen, Lec note); • Early onset: Disease acquired in utero; • The infant is infected transplacentally with the production of septicemia and granulomatous foci in many organs which may cause , stillbirth, premature delivery, or death soon after birth. • The baby is born with cardio and respiratory distress, vomiting, diarrhea, and skin lesions.

6. The Diagnosis for Case # 43 • Late onset: Infection occur at or soon after birth. “In this case study patient was infected with Late onset neonatal meningitis” • Infection usually begins 1-4 weeks after birth and is manifested as meningitis with a high fatality rate.

7. Classification, Gram Stain Results, and Microscopic Appearance of Listeria monocytogenes • Listeria monocytogenes belongs to genus Listeria, Family Listeriaceae. • Differential characteristics; • Gram positive, non-spore forming, motile, facultative anaerobic, β-hemolytic, coccobacillus bacterium (Micro lab book) . • Cultural characteristics; • Colonies are small, round, smooth, and translucent • Cell may be found singly, in short chain, or in palisades.

8. Pathogenesis of L. monocytogenes • L. monocytogenes is an important human pathogen • It’s wide separated in environment (soil, water, vegetation, and animal products) • Virulence factor(Mahon, 2007): • Hemolysin (listeriolysin O • Catalase • Superoxide dismutase • Phospholipase C • Surface protein (P 60)

9. Diseases and Pathogenesis of Disease Caused by L. monocytogenes • Listeria monocytogenes causes listeriosis, septicemia, meningitis, infections in pregnant women, which may result in spontaneous abortion (2nd/3rd trimester) or stillbirth. • In immunosuppressed and older adults, and patient receiving chemotherapy cause invasive listeriosis. • In adults symptoms start as mild flu or GI distress.

10. Diagnosis of Listeria monocytogenes • In direct CSF smear, L. monocytogenes appear Gram-positive • Grow well in SBA and Chocolate agar (1-2 days). • Prefer slightly CO2 condition for isolation • Colonies are surrounded by narrow zone of β-hemolysis

11. Identification of Listeria monocytogenes • Isolation of colonies from cerebrospinal fluid (CSF), blood, or swabs of lesions. • Biochemical Characteristics (Mahon, 2007); • Catalase + • Esculin Hydrolysis + • Motility + • Growth in NaCl + • Hippurate + • CAMP + • Streptococcus agalactiae is very similar to • L. monocytogenes; • Differentiation: • S. agalactiae; Catalase -, Motility - • L. monocytogenes; Catalase + , Motility +

12. Isolation of Listeria monocytogenes • Optimal growth temperature is 30˚C to 35 ˚C • Can grow in the wide range of 0.5 ˚C to 45˚C • Cold enrichment technique may use to isolate organism from clinical specimen. (Inoculation of the specimen into broth and incubation at 4 ˚C for several weeks).

13. Therapy, Prevention and Prognosis of Patient Infected with Pathogen L. monocytogenes • Therapy for L. monocytogenes; • Ampicillin individually or combined with Gentamicin, • Penicillin G individually or combined with Gentamicin (Micro Lab book, 2005)

14. Vaccine? • Vaccine for Listeria monocytogenes is under development.

15. Primary Research Article Contributing to the Understanding of the Disease caused by Listeria monocytogenes • S.A.A. Jassim et al, March 2005, The attachment efficiency of cell-walled and L-forms of Listeria monocytogenes to stainless steel, Agriculture & Environment ,Vol.3 (2) : 9 2 - 9 5. • “Department of Food Science, University of Guelph, Guelph, Ontario, Canada”

16. Primary Research Article Contributing to the Understanding of the Disease caused by Listeria monocytogenes Purpose: • To detect the attachment efficiency of cell walled of L-form of Listeria monocytogenes to stainless steel surfaces. Material and method: • L-form phenotype of Listeria monocytogenes was induced, propagated and recovered in both broth and plate culture by exposure to sub-lethal concentrations of Ampicillin.

17. Primary Research Article Contributing to the Understanding of the Disease caused by Listeria monocytogenes Results of experiment: • L-form cells had the capacity to attach well to stainless steel (0.3% of cells attached) with a higher efficiency than parental cells (0.002%) after 8 h exposure to culture. • After 18 h, the parental cells attached with slightly higher efficiency (0.8%) than L-forms cells (0.625%).

18. Primary Research Article Contributing to the Understanding of the Disease caused by Listeria monocytogenes Figure 1. L-form colonies of L. monocytogenes visible after 72 h on TSA supplemented with 0.5 ng/ml ampicillin Figure 2. Scanning electron micrographs of classical and L-forms of L. monocytogenes. (2a) classical cells with flagella (arrows), Mag: 10,200x; (2b) L-form cell, Mag: 30,000x.

19. Primary Research Article Contributing to the Understanding of the Disease caused by Listeria monocytogenes Conclusion: • The ability of Listeria monocytogenes L-forms to attach to stainless steel may suggest that a classical rigid cell wall structure is not a requirement for cell adhesion in vitro. • This article relates and supports my case about the virulence factorBacterial adhesion of L. monocytogenes.

20. Take Home Message • Meningitisis an inflammation of the meninges, the lining surrounding the brain and spinal cord • Typical symptoms are: • Headache, neck stiffness , back stiffness, focal seizures, nausea, fever, and bulging fontanelles (soft spot on an infant's head). • Bacteria cause disease: • Listeria monocytogenes causes meningitis • Diagnostics: • Cerebrospinal fluid (CSF) test, and blood test. • Therapy is based on: • Patient age, the organism that cause meningitis, and extinct on the disease.

21. Take Home Message • Prognosis: • Is poor in neonates; infected mother should be treated as soon as disease is diagnosed • Prevention : • Food safety (cook all food from animal sources, wash raw vegetables very well, and avoid eating or drinking unpasteurized milk products). • Transmission: • From infected pregnant mother to fetus in utero (early onset) • Infected from the genital tract during delivery (late onset) • Treatment: • Ampicillin, Penicillin

22. References • Connie R. Mahon, Donald C. Lehman, George Manuselis. Diagnostic Microbiology; 3rd ed.;Saunders, an imprint of Elsevier Inc.:2006; Chapter 16. • Michael J. Leboffe, Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratry; 3rd ed. Morton Publishing Company, 2005; page 144,153. • S.A.A. Jassim et al, March 2005, The attachment efficiency of cell-walled and L-forms of Listeria monocytogenes to stainless steel, Agriculture & Environment ,Vol.3 (2) : 9 2 - 9 5. • http://www.aafp.org/afp/990515ap/2761.html • http://www.cfsph.iastate.edu/Factsheets/pdfs/listeriosis.pdf • http://www.son.org.tw/db/Jour/2/199806/2.pdf