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Various bacteria in blood culture bottles showing their visible growth

Various bacteria like Staphylococcus aureus, Streptococcus pyogenes, Baclillus species, Pseudomonas aeruginosa and Escherichia coli in blood culture bottle showing their visible growth as shown above picture.

Visible signs of growth caused by organisms commonly encountered in blood culture:

Macroscopic observation                           Associated microorganisms

1. Haemolysis-  Streptococci,Staphylococci, Listeria species, Clostridium species , Bacillus species
2. Turbidity – Aerobic gram-negative bacilli, anaerobes
3. Pellicle formation – Bacillus species, Pseudomonas species, Yeast cells
4. Clotting – Staphylococcus aureus
5. Visible colonies ( puffballs) -Staphylococci, Streptococci

Laboratory Diagnosis  of Various bacteria

Staphylococcus aureus

Samples/ specimens collection

• It depends on the site of infection and the nature of the lesion. e.g.
• Pus (Suppurative lesion)
• CSF ( meningitis)
• Blood (septicemia)
• Sputum( respiratory infection)
• Nasal swab (detection of carriers)
• Feces and remains of food  (food poisoning)

Gram stain: Gram-positive cocci in the cluster

Culture

Media -for routine Nutrient agar and blood agar

for Selective

• 7-10% salt agar
• Mannitol salt agar

• Tellurite glycine agar
• Phenolphthalein phosphate agar
• Polymyxin B agar (75 μg/ml)

Colony characteristics

on nutrient agar

• Smooth, circular, often yellow-pigmented colonies and non-diffusible.
• 1-2 mm in diameter
• Butyrous inconsistency

On blood agar

Beta hemolytic

Biochemical tests

• Catalase test: Positive
• Oxidation and fermentation (OF) test: Fermentative
• Coagulase test: Positive
• DNAse test: Positive
• From these features, the organism is identified as Staphylococcus aureus.
• Coagulase test
• Coagulase brings about the clotting of plasma which is similar to thrombin -catalytic conversion of fibrinogen into fibrin.

Types

1. Free coagulase: This is an extracellular enzyme of bacteria secreted into the medium. It is a thrombin-like substance that can change fibrinogen to fibrin. A tube coagulase test is performed for its detection.
2. Bound coagulase: This is closely bound to the cell wall. On its surface, it has receptors for fibrinogen so that fibrin forms links between the bacteria. This causes the clumping of Staphylococci. Hence, bound coagulase is also known as the clumping factor. Slide coagulase test is done for its detection.

Laboratory Diagnosis OF Streptococcus pyogenes

Specimen: Throat swab, pus swab, or exudates

 Microscopy: Gram-staining of pus with the presence of Gram-positive cocci in chains

Culture: Collect swab from the affected area and either plate immediately or send to the laboratory in Pike’s medium. Inoculate specimen on blood agar and incubated at 37˚C anaerobically or under 5-10% CO2, as hemolysis develops better (pinpoint colonies with beta hemolysis).

 Isolation of organisms: 

• Rapid diagnostic test kits are available for the detection of streptococcal group A antigen from throat swabs.
• Bacitracin (0.04U) sensitivity test: Bacitracin is a polypeptide antibiotic derived from Bacillus subtilis that functions to block cell wall formation by interfering with the dephosphorylation of the lipid compound that carries peptidoglycans to the growing microbial cell wall. It uses to differentiate Streptococcus pyogenes from other ß hemolytic streptococci, Streptococcus agalactiae . S. pyogenes is sensitive whereas S. agalcatiae is resistant.

Serology

1. Anti-streptolysin O (ASO)  test: ASO titers higher than 200 are indicative of prior streptococcal infection. High levels are usually found in acute rheumatic fever but in glomerulonephritis, times are often low.
2. Antideoxyribonuclease B (anti-DNAase B): Commonly used titers higher than 300 are taken.
3. Streptozyme test: A passive slide hem-agglutination test using erythrocytes sensitized with a crude preparation of streptococci and It is a convenient, sensitive and specific screening test.

Laboratory  Diagnosis of Pseudomonas

Diagnosis of P. aeroginosa infection depends upon isolation and laboratory identification of the bacterium. It grows well on most laboratory media and commonly is isolated on blood agar or eosin-methylthionine blue agar. It is identified on the basis of its Gram morphology, inability to ferment lactose, a positive oxidase reaction, its fruity odor, and its ability to grow at 42°C. Fluorescence under ultraviolet light is helpful in the early identification of P. aeruginosa colonies. Fluorescence is also used to suggest the presence of P. aeruginosa in wounds.

Culture characteristics of Pseudomonas aeruginosa 

They are obligate aerobe but grow anaerobically if nitrate is available. Growth occurs at a wide range of temperatures 6 to 42°C the optimum being 37 °C. Growth on ordinary media producing large opaque irregular colonies with distinctive musty mawkish or earthy smell. Iridescent patches with metallic sheen are seen in cultures on nutrient agar. In broth forms dense turbidity with a surface pellicle. Many organic compounds are used as carbon and nitrogen sources, but only a few carbohydrates by oxidative metabolism. Glucose was used oxidatively and lactose negative on MacConkey’s agar.

Pigment production of Pseudomonas

Some strains produce diffusible pigments like Pyocyanin (blue), pyoverdin or fluorescein ( greenish-yellow), pyorubin (reddish-brown), and pyomelanin ( brown to black).

 Biochemical reactions of Pseudomonas

They are oxidative and non- fermentative. Glucose is utilized oxidatively. Indole, Methyl Red (MR)  and  Voges-Proskauer (VP)  and  hydrogen sulphide (H₂S) tests are negative. Catalase, oxidase, and arginine tests are positive.

Toxins and enzymes

Toxic extracellular products in culture filtrates, exotoxin A and S. Exotoxin A acts as NADase resembling Diphtheria toxin. Proteases, elastase hemolysins, and enterotoxin,  Slime layer and Biofilms

 Typing and its importance

An important cause of Hospital Infections and important for epidemiological purposes. Serotyping, bacteriocins typing, pyocyanin, restriction endonuclease typing with pulse-field gel electrophoresis

 Resistance

They are Killed at 55°C in 1 hour.  High resistance to chemical agents. Resistance to quaternary ammonium compounds like chloroxylenol, resistant to hexachlorophene. They also grow in antiseptic bottles like Dettol as cetrimide as a selective medium. and sensitive to acids silver salts, beta glutaraldehyde.

Lab Diagnosis of E . coli

Specimens: Blood, urine, pus, CSF, etc

Gram stain: Gram-negative rods having a size of 1-3 x 0.4-0.7 µm. There is no evidence of spores but and some strains may have capsules.

Hanging drop preparation: Motile by peritrichous flagella and occasionally strains are non-motile.

Culture Characteristics

• Aerobe and facultative anaerobe
• Growth occurs between 10-40°C (37°C) •
• Nutrient agar: colonies are large, thick, greyish white, moist, smooth, opaque, or partially translucent discs.
• Blood agar: Many strains especially pathogenic ones are hemolytic on blood agar.
• MacConkey Agar: colonies are bright pink due to lactose fermentation.
• Broth ( Peptone water or tryptone soy broth): Generally turbidity with heavy deposit.
• EMB Agar: Blue and violent colonies with a metallic sheen

Biochemical Reactions of E. coli 

•  Catalase test: positive
• Oxidase test: negative
• Reduces nitrates to nitrites
• Indole test: Positive
• Methyl-Red (MR) test: Positive
• Voges-Proskauer (VP) test: Negative
• Citrate utilization test: Negative
• IMViC: + + – – ( i.e. Indole and MR test positive while VP and citrate utilization test negative)
• Gelatin liquefaction: Negative
• ONPG test: Positive
• The acid produced from carbohydrates (Some atypical strains of E. coli do not ferment lactose or have a delayed reaction.)
• H₂S production: Negative
• Urease test: Negative
• Sugar fermentation: glucose, lactose, mannitol, maltose, and many other sugars fermented with acid and gas production.
• Sucrose is generally not fermented.

References for Identification of Various Bacteria

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.