CorynebacteriumCorynebacterium, , ListeriaListeria,,

ErysipelothrixErysipelothrixAerobic Gram-Positive Bacilli Aerobic Gram-Positive Bacilli

(Non-Spore-Forming) (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– Veterinary pathogen

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?