Infective endocarditis - Laboratory Diagnosis, Culture, Biochemical test, Colony

Laboratory diagnosis of infective endocarditis

The laboratory diagnosis of infective endocarditis begins with collection of sample.

Sample

Blood

Culture

Dilution of blood and culture medium is done at the ratio of 1:10 ratio of blood. However, the 1:5 ratio is used nowadays.

Media

Blood culture media includes:

• Soybean Casein Digest Broth

• Trypticase Soy Broth

• Brain Heart Infusion (BHI) broth

• Supplemented peptone broth

• Thioglycolate broth

• Columbia broth (specialized broth)

• Brucella broth (specialized broth)

Incubation

Incubation of inoculated media is done at 35-37°C. Cultures are examined and sub-cultured daily for 7 days and then twice a week for up to 4 weeks.

Constant agitation of the bottle during the first 24 hours of incubation enhances the growth of most aerobic bacteria.

Interpretation of Blood culture results

Growth is indicated by changes in turbidity, pellicle formation, gas bubble formation, odor, hemolysis, clot formation, etc.

Subculture

A loop full of incubated medium is sub-cultured in MA, BA, and CA for further identification. Incubation of subculture is done at 35°C in blood agar (BA), chocolate agar (CA) in anaerobic conditions, and MacConkey agar (MA) for 24-48 hours in gas.

Colony observation

Presumptive identification is done after viewing the characters of colonies on MA, BA, and CA.

* Due to the presence of crystal violet and bile salts, only gram-negative bacteria grow in MA. Thus it is called differential and selective media.

Gram-positive bacteria

Staphylococcus aureus

BA: opaque, golden yellow, mucoid colonies with β-hemolysis

CA: white, moist colony, darkened edges on media

Coagulase Negative Staphylococcus (CoNs)

BA, CA: minute white colony, no hemolysis

Enterococcus spp.

BA, CA: minute paper white colonies, β-hemolysis in some species

Streptococcus pneumoniae

BA, CA: small, grey, moist colony, alpha-hemolysis (green) observed

Streptococcus agalactiae

BA: translucent white colony with β-hemolysis

CA: translucent white, slightly mucoid colony

Streptococcus pyogenes (group A Streptococci)

BA: tiny paper white colonies with large β-hemolysis

CA: flat, opaque, white colonies

Viridans streptococci

BA: α-hemolytic, small transluscent colonies

CA: tiny white colonies showing α-hemolysis

Gram-negative bacteria

Escherichia coli

MA: pink to dark pink, dry, donut-shaped, dark pink area of precipitated bile salts on the periphery of the colony

BA: smooth, circular, flat colonies, no hemolysis occurs

CA: mucoid, beige, or off-white color with a shiny texture

Pseudomonas aeruginosa

MA: flat, smooth, elliptical-shaped colonies which are 2-3mm in diameter. They are usually colored ranging from blue-green, yellow-green, to red-brown.

BA: dark, translucent, flat, colonies with irregular edges

CA: flat, smooth, dark white colonies with irregular edges

Haemophilus influenzae

MA: No growth

BA: visible growth only by Satellitism test

CA: tiny, flat, smooth white colonies with no hemolysis

Proteus spp.

MA: small, dark, flat colonies with irregular edges, may slightly swarm

BA: translucent colonies with swarming growth (in form of waves), covers the entire surface of an agar plate with no hemolysis

CA: same as in blood agar

Klebsiella pneumoniae

MA: Large, circular, convex, highly mucoid, pink colonies

BA, CA: white, mucoid, convex colonies with no hemolysis

Salmonella spp.

MA: flat, colorless, colonies with irregular edges measuring 2-4mm in diameter

BA, CA: white, flat, smooth, glistering colonies with no hemolysis

Neisseria meningitidis

MA: due to its fastidious nature, does not grow in MA

BA, CA: translucent white, smooth, concave colonies with regular edges, no hemolysis

Confirmatory identification, Biochemical Tests

Gram-positive bacteria

Staphylococcus aureus

Catalase: Positive

Oxidase: Negative

Coagulase: Positive

Deoxyribonuclease (DNase): Positive

*can be confirmed by growth on Mannitol Salt Agar

Coagulase Negative Staphylococcus (CoNs)

Catalase: Positive

Oxidase: Negative

Coagulase: Negative

Enterococcus spp.

Catalase: Negative

Oxidase: Negative

Coagulase: Negative

Growth in Bile Esculin Agar: Positive

Streptococcus pneumoniae

Catalase: Negative

Oxidase: Negative

Coagulase: Negative

Optochin test: Sensitive to Optochin

Streptococcus agalactiae

Catalase: Negative

Coagulase: Negative

Oxidase: Negative

CAMP: Positive

Streptococcus pyogenes

Catalase: Negative

Coagulase: Negative

CAMP: Negative

Oxidase: Negative

PYR (Pyrrolidonyl Aminopeptidase) test: Positive

Streptococci viridans

Catalase: Negative

Coagulase: Negative

CAMP: Negative

PYR (Pyrrolidonyl Aminopeptidase) test: Negative

Optochin test: Resistant

Gram-negative bacteria

*All above organisms reduce nitrate

* Catalase and Oxidase are positive for Pseudomonas aeruginosa

Bacillus spp. (except Bacillus anthracis), Coagulase-negative Staphylococcus (CoNs), Corynebacterium spp., Propionibacterium acnes are probable contaminants. However, isolation of commercial microbial flora from blood cultures of immunocompromised patients can also be the causative agent of infection.

For HACEK/AACEK (gram-negative bacilli)

Both colony morphological characters and biochemical tests are used to identify the causative agent.

• If Aggregatibacter spp. and Actinobacillus actinomyetemcomitans are suspected: small less than 0.5mm colony on BA after 24 hours of incubation.

• If Cardiobacter hominis is suspected:

  Catalase +ve

  Indole +ve

  Oxidase +ve

  gram -ve pleomorphic rod

• If Eikenella corrodens is suspected:

  produces colonies that pit the agar surface

  corroding/pitting colony on BA

  do not grow in MA

  has twitching motility, musty odor,

  oxidase +ve

• If Kingella kingae is suspected

  1-2mm colonies on BA after 48 hours

  short 2-3 µm, gram negative bacilli

  twitching motility