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