corynebacterium listeria erysipelothrix_bacillus
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Laboratory diagnosis of infections caused by Gram positive bacilli
Corynebacterium, Listeria, Erysipelothrix, Bacillus
Gram positive and Gram negative bacilli
Genera:• Corynebacterium• Listeria• Erysipelothrix• Bacillus
Family EnterobacteriaceaeGenera:A. Highly pathogenic:
• Yersinia• Salmonella• Shigella
A. Facultatively pathogenic:• E.coli• Klebsiella• Proteus• Enterobacter• Serratia• Citrobacter
Genus Corynebacterium Species Corynebacterium diphtheriae
- high pathogenicity- Clinical significance: diphtheria = disease produced by
the diphtheric toxine
- → symptoms at the entry gate: sore throat, adherent membrane (pseudomembrane) on tonsils, pharynx,
nasal cavity - → general toxic symptoms: fever (hematogenic difusion
of diphtheric toxin)
Left: diphtheric pseudomembrane on tonsilsRight: diphtheric skin lesion
Genus CorynebacteriumOther pathogenic species (zoonosis)
Producers of diphteric toxin:
• Corynebacterium ulcerans – infection of cattle – mastitis; infection transmissible to humans via infected milk
• Corynebacterium pseudotuberculosis – comensal of animals (horses, sheep) – transmissible to humans via direct contact or contaminated milk
Species Corynebacterium diphtheriaeLaboratory diagnosis
• Colection of specimens: throat / nasal / wound swab• Microscopy:
– Gram positive bacilli, aspect of ”Chinese letters” / capital letters– low value (C.diphtheriae – similar to other comensal
corynebacteria in the throat – “diphteroid bacilli”)
• Cultivation:– Blood agar – Selective media with tellurite (Tinsdale, Gundel-Tietz)– Highly selective Loffler medium
Corynebacterium diphtheriae: Gram+, encurved rods, ”swolen” ends
Corynebacterium diphtheriae – Gram+, encurved rods, “swollen” ends
Corynebacterium diphtheriae- Cultivation -
• Blood agar: nonhemolytic, white-grey colonies, striated margins (”daisy flower”)
Corynebacterium diphtheriae- Cultivation - continued
• Tinsdale medium (tellurite): small, black colonies + brown surrounding area (reaction between H2S produced by bacteria and potasium tellurite in medium)
Corynebacterium diphteriae on Tinsdale agar: black colonies with brown halo
Left: Corynebacterium diphtheriae on blood agarRight: Corynebacterium diphtheriae on tellurite
Corynebacterium diphteriae- Cultivation - continued
• Loffler medium – high selectivity: white, creamy colonies on slant
Biochemical tests: Urease test (medium: urea + phenol red indicator)
• Principle: urease producing germs decompose urea in the culture medium: PINK
• Corynebacterium diphteriae – NEGATIVE TEST – tube on the right
• diphteroid bacilli (normal flora) – POSITIVE TEST – tube in the middle
Toxigenic vs Nontoxigenic strains- Toxigenesis tests -
A. In vivo: experimental disease in Guinnea pigs
B. In vitro: Elek´s test: • Elek agar inoculated with
streaks of bacterial culture;• strip of sterile filter paper
impregnated with antidiphteric antitoxin placed perpendicular to bacterial streaks;
• incubate 24 hours; • POSITIVE test = lines of Ag-
Ab precipitate = strain is toxigenic (important differentiation from diphteroids – normal flora of the pharynx)
Elek test - continued
Elek agar: agar + bovine serum
1 and 4 – Elek (+) = double radial immunodiffusion – precipitation lines in each angle formed by bacterial streak and strip impregnated with diphteric antitoxin = toxin producing strains
2 and 3 = Elek (-) – nontoxigenic strains
Corynebacterium diphteriaeAntimicrobial sensitivity
• Sensitive to penicillin, vancomycin, erythromycin – given asap, based on clinical suspicion – before lab confirmation!
• + treatment must include Diphteria ANTITOXIN !!• Lack of/ delayed treatment may cause death (effects of toxin –
inhibition of protein synthesis; organ necrosis – e.g. heart, liver, kidneys; neurologic lesions)
IMPORTANT: Diphteria – vaccine-preventable disease!!• Vaccination protocols e.g.
– trivalent vaccines: (Diphteria, Tetanus, Pertusis);
– pentavalent vaccines (Hemophilus influenzae type B, Pertusis, Tetanus,
Diphteria, Hepatitis B)
Gram positive and Gram negative bacilli
Genera:• Corynebacterium
• Listeria• Erysipelothrix• Bacillus
Family EnterobacteriaceaeGenera:A. Highly pathogenic:
• Yersinia• Salmonella• Shigella
A. Facultatively pathogenic:• E.coli• Klebsiella• Proteus• Enterobacter• Serratia• Citrobacter
Genus Listeria
Common characters: • Gram positive, short bacilli
(rods) /cocobacilli, aerobic, non-spore forming, length: 0.5-1 µm/diameter: 0.5 µm, rounded ends
• Arrangement in palisades / V-shape / chains
• Mobility due to peritrichous flagella (flagella around the rod) – up to 5
Genus Listeria- Clinical significance -
• Natural habitat: soil, water, sewage, plants, foods (raw, undercooked)
• Species involved in human pathology: Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii
• Transmission via contaminated food (milk, diary products, poultry meat) – food poisoning
• Transplacentar transmission → meningitis, sepsis in newborns• Immunosuppressed patients (HIV, blood malignancies): meningitis,
encephalitis, sepsis• Occupational infection in veterinarians, slaughterhouse workers
(from infected animals/animal tissues): pulmonar infections
Genus Listeria- Laboratory diagnosis -
• Collection of specimens: CSF, blood, amniotic fluid, respiratory secretions, food samples, etc.
• Microscopic examination: difficult to differentiate from:– corynebacteria,
– Hemophilus influenzae (excessive decoloration of smear),
– streptococci (short rods/coccobacilli with rounded ends sometimes resemble ovoid cocci)
Listeria – Gram stained smear
Genus Listeria- Laboratory diagnosis – continued
Cultivation:• Normally sterile collection sites (CSF, blood):
– Inoculation in liquid media (enriched nutrient broth), 35°C, 5-7 days (daily examination for bacterial growth)
– Reinoculation from turbid broth tubes on blood agar, incubation for 2 more days
• Nonsterile collection sites (faeces, respiratory secretions, foods):– Initial inoculation on enrichment media– Reinoculation on selective media (e.g. with antibiotic content)
Genus Listeria- Laboratory diagnosis – continued
Colonial characters: • Blood agar: small (up to 1 mm), round, smooth,
translucent colonies, discrete hemolysis (sometimes under the colony, visible only after picking up the colony with loop)
• Selective media (e.g. Oxford agar): black colonies, black halo (formation of iron compounds)
Listeria colonies on blood agar
Listeria monocytogenes on Oxford agar
• Black colonies, black halo
Listeria: main bacteriological diagnosis elements
Listeria- Biochemical tests -
• CAMP test – POSITIVE for L.monocytogenes
• (enhanced hemolysis in the area where Listeria streak meets S.aureus streak)
• CAMP test is negative for nonpathogenic Listeria strains
Gram positive and Gram negative bacilli
Genera:• Corynebacterium• Listeria
• Erysipelothrix• Bacillus
Family EnterobacteriaceaeGenera:A. Highly pathogenic:
• Yersinia• Salmonella• Shigella
A. Facultatively pathogenic:• E.coli• Klebsiella• Proteus• Enterobacter• Serratia• Citrobacter
Genus Erysipelothrix
• Common characters: Gram positive bacilli, non spore forming, tendency to form long filaments
• Clinical significance: zoonosis; human diseases occur as work-related infections (veterinarians, butchers, fishermen): inflammatory skin lesions (erysipeloid)
Gram positive and Gram negative bacilli
Genera:• Corynebacterium• Listeria
• Erysipelothrix
• Bacillus
Family EnterobacteriaceaeGenera:A. Highly pathogenic:
• Yersinia• Salmonella• Shigella
A. Facultatively pathogenic:• E.coli• Klebsiella• Proteus• Enterobacter• Serratia• Citrobacter
Genus Bacillus Species Bacillus anthracis
• Large, Gram positive rods (10 µM),
• straight cut ends, • aerobic, • disposed in chains• Spore forming
Bacterial survival outside host
Spores: reproductive structures adapted
for longtime survival in unfavourable
conditions
(etymology: ancient Greek spora = seed)
Bacterial spores - outer layer of keratin resistant to chemicals, staining and heat → bacterium able to stay dormant for years, protected from temperature differences, absence of air, water and nutrients
Spore forming bacteria: • Clostridium spp (e.g. Clostridium difficile, Clostridium tetani); • Bacillus spp (B. anthracis).
Bacillus anthracis – Gram staining
• Gram positive, long bacilli, squared ends, endo-spores visible in the middle of bacterial cells – spores are resistant to staining
(magnification 1500X)
Bacillus anthracis (continued)
• High pathogenicity• Disease = zoonosis (infection of animals AND humans)• Clinical forms:
– Cutaneous anthrax – spores enter the body via skin lesions– Pulmonary anthrax – inhalatory infection– Digestive anthrax – ingestion of infected undercooked meat – + biological weapon (inhalatory infection) – agent of bioterrorism
Left: anthrax skin lesionRight: ”anthrax attack” letter (1 week after 9/11)
Bacillus anthracis: cultivation
• Nonfastidious germ; grows well on blood agar
• Colonial characters: large (2-5 mm) white colonies, non-hemolytic, irregular margins, sometimes comma shaped (lower image), ground-glass aspect
(above details visible under magnifying glass)
Bacillus anthracis on blood agar
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