Case Study Listeria monocytogenes 2009 Roubina Tatavosian James Muro Jae Kim

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

Key Information Pointing to Diagnosis Symptoms of the patient indicates 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

Listeria monocytogenes Gram Stain on CSF

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.

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.

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.

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)

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.

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

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 +

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).

Therapy for L. monocytogenes; 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)

Vaccine? Vaccine for Listeria monocytogenes is under development.

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”

Purpose: Material and method: 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.

Results of experiment: 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%).

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.

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 factor Bacterial adhesion of L. monocytogenes.

Take Home Message Meningitis is 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.

Take Home Message Prognosis: Prevention : Transmission: Treatment: 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

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