Corynebacterium, Listeria, Erysipelothrix Aerobic Gram-Positive Bacilli (Non-Spore-Forming)

Corynebacterium: Characteristics “club” “small rod” Gram(+), small, club shaped rod, diphtheroid Related to Mycobacterium, Nocardia – diaminopinelic acid, mycolic acid in cell wall Some saprophyte, plants, animals; some disease in animals, humans Majority NF of animals, humans

Corynebacterium: Genera - Human Disease C. diphtheriae Toxigenic strains – diphtheria; respiratory, cutaneous Nontoxigenic strains – pharyngitis, endocarditis Human pathogen C. ulcerans Respiratory, diphtheria Veterinary pathogen C. jeikeium (group JK) Septicemia, endocarditis, wound infection, foreign body (catheter, shunt, prosthesis) infection Human skin NF C. pseudotuberculosis Lymphadentitis, ulcerative lyphangitis, abscess formation

Corynebacterium: Lab Culture 370 C, 24 hours, pH 7.8-8.0, require oxygen CBA – raised, translucent, gray colonies Cystine Tellurite Blood Agar CTBA) Enriched, selective, differential media SRBC, bovine serum, cystine, tellurite Tellurite inhibits RT normal flora Corynebacterium colonies black, brownish due to tellurite reduction Loeffler Coagulated Serum slant Enriched media; serum, egg Enhances formation metachromatic granules characteristic of Corynebacterium, visualized  by methylene blue stain

C. diphtheriae : Virulence Factors - Exotoxin Must be  produced to cause diphtheria Lysogenic bacteriophage carries "tox” gene Trypsin  cleaves toxin - fragment A (toxic  activity), fragment B (tissue binding) Toxin  inhibits protein  synthesis  by  ADP-ribosylating host cell ribosome elongation factor-2 (EF-2) Systemic effects - heart failure, paralysis, adrenal hypofunction leading to Addison’s-like disease C. ulcerans, C. pseudotuberculosis – some strains also make diphtheria-like toxin

C. diphtheriae: Exotoxin

Elek Plate DiphtheriaToxin Test Immunodiffusion test in agar plate Streak MO isolate on plate, place filter strip of antitoxin (antibody) perpendicular to streak If MO makes toxin, at zone of equivalence (antigen + antibody) precipitate forms Newer rapid tests: Enzyme-linked immunosorbent assay (ELISA) PCR DNA amplification to detect tox gene

C. diphtheriae : Virulence Factors Capsule – protein, antiphagocytic. Phospholipase D – dermonecrotic toxin, spreading factor Antibiotic resistance – select for resistance during antibiotic therapy

Diphtheria: Respiratory “leather” “skin” MO in throats of healthy carriers MO infects only humans, limited capacity to invade host Disease starts as local infection of mucous membranes, causing pharyngitis Local toxin results in degeneration epithelial cells Inflammation, edema, pseudomembrane (fibrin clots, leukocytes, dead epithelial cells, MO) in throat

Diphtheria: Respiratory Membrane obstructs airway, may result in suffocation Recovery ~1 week, membrane dislodged, expectorated Dangerous when toxin spreads systemic via blood: heart (heart failure) peripheral nerves (paralysis) adrenal glands (hypofunction)

Diphtheria: Cutaneous More  common tropical, subtropical areas Skin contact with infected person MO colonizes skin surface, enters subcutaneous tissue through break on skin (e.g. insect bite) Papule, chronic non-healing ulcer Exotoxin with systemic signs

C. diphtheriae: Treatment and Prevention Treat by giving specific antitoxin (antibody) binds and neutralizes toxin Penicillin or erythromycin to eliminate MO, stop toxin production DTaP vaccine - immunize with toxoid (inactivate toxin by formalin) to elicit neutralizing antibody Remains epidemic in developing countries due to inadequate vaccination

Other Corynebacterium: Infection and Disease Normal flora of skin, URT May occasionally cause disease, particularly immunocompromised C.  ulcerans ­  toxigenic strains produce disease similar to diphtheria, , but less severe C. jeikeium ­ those with underlying disease; bacteremia, meningitis, peritonitis, wound infection C. pseudotuberculosis ­ those with exposure to animals (cattle, sheep, horses, goats, deer); pneumonia, lymphadenitis

Listeria monocytogenes: Characterisitcs “blood cell” “produce” monocytosis in rabbits G(+) short rods; singly, pairs, chains Isolated from soil, water, vegetation, animals (bird, fish, insect) Disease in wild, domestic animals; uncommon human infection Facultative intracellular pathogen in humans; grows in macrophage, epithelial cells

L. Monocytogenes: Lab Culture Aerobic, microaerophilic Grows well on ordinary lab media CBA – weak beta hemolysis Able to grow slowly in cold (1°C) Motile: Peritrichous flagella, RT (umbrella motility) Polar flagella, 370 C

L. monocytogenes: Lab ID Catalase(+) Oxidase (-) TSI= A/A, H2S(-) Esculin hydrolysis(+) CAMP(+) reaction - enhanced “block” type hemolysis with Staphylococcus aureus Grows in 6.5% NaCl Serogroups based on O, H antigen

L. monocytogenes: Virulence Factors Listeriolysin O (LLO) – hemolysin, pore forming toxin; escapes from phagocytic endosome to cytosol; required for intracellular growth Phospholipase – also involve in escape of MO from endosome to cytosol Listeric polysaccharide – capsule component

L. monocytogenes: Virulence Factors Internalins – surface associated proteins; uptake MO into epithelial cells ActA – surface protein; rearrangement of actin, propel MO through cell into adjacent cell (very invasive) LPS-like substance – high fever in host CNS Tropism - invasive

L. monocytogenes: Listeriosis Found in environment - soil, decaying vegaetable, animal feces Ingest contaminated animal products (milk, cheese, undercooked meat & poultry), unwashed vegetables (especially cabbage) Disease usually mild, flu-like or GI distress Individuals underlying chronic primary disorder Disease - widely disseminated abscesses, granulomas; lesions may be found in liver, spleen, adrenals, respiratory tract, CNS, skin Also meningitis with septicemia, pneumonia High mortality may occur

Listeriosis Uncommon disease - restricted to elderly, pregnant women, immunocompromised Healthy children and adults – AS carriage Pregnant moms AS carriage, septicemia, neonatal disease Pregnancy renders mom more susceptible (immune suppressed) Effect on mom usually minimal, can be devastating for fetus or newborn Immunocompromosed: AS carriage, meningitis, septicemia, other infections

Listeriosis: Neonates Early onset - infected transplacentally: Septicemia, granulomatous foci in many organs May result in abortion, stillbirth, premature delivery, death soon after birth Baby born with cardio and respiratory distress, vomiting, diarrhea, meningitis, hepatosplenomegaly, skin lesions Fatality rate 70-90% in untreated cases Late onset – infected from genital tract during delivery: Usually 1-4 weeks after birth Manifested as meningitis High fatality rate, but less than Early onset infection

L. monocytogenes: Treatment and Prevention Poor prognosis in neonates Infected moms treated as soon as disease is diagnosed to prevent transmission to fetus/neonate Most drugs only bacteriostatic with Listeria - treatment of choice is combination penicillin and gentamycin At risk individuals, avoid eating raw or partially cooked foods (soft cheese, turkey franks, cold cuts, vegetables)

Erysipelothrix rhusiopathiae “red” “skin” “hair”; “red” “disease” G(+) slender, pleomorphic, small bacilli; form filaments Worldwide wild, domestic animals; swine main reservoir Survives well in environment – water, soil, plant material Animal disease widely recognized, human disease uncommon

Erysipelothrix rhusiopathiae: Lab Culture Growth on CBA – alpha or gamma hemolysis, two types of colonies: Smooth – rod, coccobacilli Rough – long, thin filamentous rod Usually 48 hours for growth Microaerophilic, better growth in CO2 or AnO2

Erysipelothrix: Virulence Factors Polysaccharide capsule – protect from phagocytosis Adherence – especially heart valves Neuraminidase – spreading Hyaluronidase – spreading

Erysipelothrix: Clinical Significance Primarily pathogen swine, turkey, fresh water fish Swine - cutaneous, reddish rash; occasional complications of septicemia, endocarditis, arthritis Humans - uncommon pathogen, zoonotic spread; erysipeloid most common form of disease

Erysipeloid Resembles Streptococcus erysipelas skin infection Reddish-blue, edematous lesion at site of inoculation, following trauma (abrasion, wound) to hands Occasionally disseminates -septicemia, endocarditis, arthritis Occupation associated disease: Butchers - handle contaminated meat, poultry,fish animals Farmer, veterinarian – contact with infected animals

Erysipelothrix: Treatment and Prevention Penicillin, tetracycline, erythromycin can be used At risk workers - should cover exposed skin when handling animals or animal products Swine herds - should be vaccinated

Class Assignment Textbook Reading: Chapter 16 Aerobic Gram-Positive Bacilli Corynebacterium Listeria Erysipelothrix Key Terms Learning Assessment Questions

Case Study - Gram(+) Coccobacilli (Listeria) A 35-year-old man was hospitalized because of headache, fever, and confusion. He had received a kidney transplant 7 months before, after which he had been given immunosuppressive drugs to prevent organ rejection.

Case Study - Gram(+) Coccobacilli (Listeria) CSF was collected, which revealed a white-blood cell count of 36 cells/mm3 with 96% polymorphonuclear leukocytes, a glucose concentration of 40 mg/dl, and a protein concentration of 172 mg/dl. A Gram stain preparation of CSF was negative for organisms, but gram-positive coccobacilli grew in cultures of the blood and CSF.

Case Study - Questions 1. What is the most likely cause of this patient’s meningitis? 2. What are the potential sources of this organism? 3. What virulence factors are associated with this organism? 4. How would this disease be treated? Which antibiotics are effective in vitro?