Sunday, December 9, 2007

Medical Microbiology- PBL 1 ( second blog)

Case study 1
List of microbes: Staphylococcus aureus, Staphylococcus saprophyticus, Enterococcus faecalis. Escherichia coli, Enterobacter, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Chlamydia trachomatis.

A gram stain will be done first for the suspected bacteria that can cause UTI, before proceeding to any laboratory investigation.

Gram positive bacteria: Staphylococcus aureus, Staphylococcus saprophyticus, Enterococcus faecalis.Gram negative bacteria: Escherichia coli, Enterobacter, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Chlamydia trachomatis.

Biochemical and Culture Testing
Possible Organisms Staphylococcus aureus Staphylococcus saprophyticus Enterococcus faecalis
Gram Stain :positive cocci(clusters) cocci(clusters) cocci(chains)
Culture on Sheep blood agar haemolytic, yellow colonies non-haemolytic white colonies gamma non-haemolytic white colonies( but can show weak alpha haemolysis)
Catalase test positivepositivenegative
Coagulase test positivenegativenil



Possible Organisms Escherichia coli Enterobacter sp Pseudomonas aeruginosaKlebsiella pneumoniaeProteus mirabilis
Gram Stain: negative bacilli bacilli bacillibacillibacilli
Culture on Mac Conkey pink lactose fermenting colonies very weak lactose fermentersnon-lactose fermenting colonies producing blue-green pigmentspink lactose fermenting coloniesnon-lactose fermenting colonies
Culture on eosin methylene blue (EMB) metallic green sheen with dark colonies brown-centered with pale blue colonies colorless colonies indicating no lactose fermentation and acid productionbrown dark-centered colonies indicating lactose fermentation and acid productioncolorless colonies indicating no lactose fermentation and acid production
oxidase negative negative positivenegativenegative
*Triple sugar iron (TSI) acidic slant/acidic deep alkaline slant/acidic deep No changealkaline slant/acidic deepalkaline slant with black precipitate



acidic slant/acidic deep: ferment lactose and glucose
alkaline slant/acidic deep : ferment glucose only
No change: no carbohydrate fermentation
Black precipitate: H2S production

If oxidase test is negative, proceed to IMViC biochemical test

IMViC Escherichia coli Enterobacter sp Klebsiella pneumoniaeProteus mirabilis
Indole + ---
Methyl red+ --+
Voges proskauer- ++-
Citrate test -+ ++
Urease ---+



Antibiotic Susceptibility test
5 antibiotics: Gentamycin, Ceftadizime,Cefuroxime, Ampicillin and Ciprofloxacin. Varying zone diameter size can be observed for both the gram positive and negative bacteria.

If Chlamydia trachomatis is highly suspected, as it is a common STD that can cause UTI, some portion of the urine sample can be send for DNA based analysis method such as polymerase chain reaction(PCR).

Case study 2
Besides Salmonella, there are also several other possible microorganisms that could lead to enterocolitis or cause the diarrhea in the patient. Here are the other possibilities:
1. Enterotoxigenic Escherichia coli
2. Campylobacter jejuni
3. Clostridium difficileShigella: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei



Type of microbe Microscopy test Biochemical TestSerology testCulture
Salmonella Gram Stain: Gram negative bacilli TSI: alkaline slant/acid butt with H2S production
Indole: Negative

Methyl-red: Positive

Voges-Proskauer: Negative

Citrate: Positive

Slide agglutination test: serotyping using O, H and Vi antigens

Tube agglutination test: detect agglutinating Ab to O & H Ag in patient’s serum

MacConkey agar: Observe plate for non-lactose fermenting (clear) colonies

Hektoen agar: Observe plate for clear or green colonies and colonies with black centers (H2S production)

Salmonella-Shigella agar: Observe plate for clear colonies and colonies with black centers (H2S production)

XLD Agar: Observe plate for red colonies and colonies with black centers (H2S production)
ShigellaGram Stain: Gram negative bacilli TSI: Alkaline slant/acid butt but no H2S production
Indole: Negative

Methyl-red: Positive

Voges-Proskauer: Negative

Citrate: Negative
Slide agglutination test
MacConkey agar: Observe plate for non-lactose fermenting (clear) colonies

Salmonella-Shigella agar: Observe plate for clear colonies and colonies WITHOUT black centers (no H2S production)

Hektoen agar: Observe plate for clear or green colonies and colonies WITHOUT black centers (no H2S production)
Enterotoxigenic E.ColiGram Stain: Gram negative bacilli TSI: Alkaline slant/acid butt with gas but not H2S production
Indole: Positive

Methyl-red: Positive

Voges-Proskauer: Negative

Citrate: Negative
Serotyping using O & H Ag
MacConkey agar: Observe plate for red/pink colonies (lactose-fermenting colonies)

EMB agar: Observe plate for greenish metallic sheen
Campylobacter jejuni Gram Stain: Gram negative bacilli that appear either comma or S-
shaped
TSI: Alkaline slant/deep
Oxidase: Positive
nilSelective “CAMP” agar at 42ºC in microaerophilic environment (grow at 5% oxygen + 10% carbon dioxide)
Clostridium difficile Gram Stain: Gram positive bacillinilnilBlood agar at human body temperatures


Antibiotic susceptibility testing:
1. Enterotoxigenic Escherichia coli:
• Ampicillin
2. Campylobacter jejuni
• Erythromycin
3. Shigella:
• Ampicillin
4. SalmonellaAmpicillin


Case study 3


Laboratory investigations:
Urine culture is to test to identify the exact type of bacteria causing infection.
Culture on:
1.Blood Agar Plate (BAP)
2.Eosin Methylene Blue (EMB) agar
3.MacConkey Agar (MAC)
4.Ordinary nutrient agar
5.Triple Sugar Iron (TSI) agar
All are grown under anaerobic conditions except for Pseudomonas spp. such as P. aeruginosa as it is a strict aerobe.

Microscopy
1.Gram stain
2.Fungal stain

Morphology are studied in terms of the microorganisms’ shape, arrangement, response to strain and specific structures.

Biochemical tests are done to indicate the presence or absence of enzyme(s), a group of enzymes or a whole metabolic pathway. This helps to identify microorganisms.


Gram staining Cultures (Under anaerobic conditions)Biochemical tests Antibiotic Susceptibility test
Escherichia coli Gram-negative (pink) bacillus
1. Blood agar: Gamma hemolysis

2. Eosin Methylene Blue agar: Colonies with metallic green sheen

3. MacConkey agar: Pink colonies

1. Indole test: Positive

2. Methyl Red (MR) test: Positive

3. Voges-Proskauer (VP) test: Negative

4. Simmon’s citrate test: Negative

5. Oxidase test: Negative

6. Urease: Negative

7. TSI acid slant/acid butt with gas, no H2S

-Susceptibility depends on the type of strains

- Beta-lactamase resistant strains are not sensitive to penicillin and cephalosporin

- Non-resistant strains are sensitive to ampicillin and trimethoprim-sulfamethoxazole
Enterococcus faecalis Gram-positive (purple) cocci
1. Blood agar: Non hemolytic

2. MacConkey agar: Pink colonies with mucoid appearance

3. Bile Esculin Agar: Ferric citrate indicator will turn black

1.Indole test: Negative

2. Voges-Prokauer test: Positive

-Resistant to aminoglycoside, penicillin and vancomycin when given individually

-A synergistic combination of aminoglycoside and cell wall-active antibiotics such as ampicillin and vancomycin
Klebsiella pneumoniaeGram negative (pink) bacillus, a large capsule can be observed1. MacConkey agar: Pink colonies with mucoid appearance

1. Indole test: Negative

2. Methyl Red test: Negative

3. Voges-Prokauer test: Positive

4. Urease test: Positive

- Isolates from nosocomial infections are frequently resistant to multiple antibiotics

- Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)
Pseudomonas aeruginosaGram-negative (pink) bacillus
1. Blood agar: Beta-hemolysis

2. MacConkey agar: Colourless colonies

3. Ordinary nutrient agar: Blue-green colonies

1. Indole test: Negative

2. Methyl Red test: Negative

3.Voges -Prokauer test: Negative

4. Catalase test: Positive

5. Oxidase test: Positive

6. TSI agar: Negative (Growth with typical metallic sheen)

7. Pyocyanin test: Positive

8. Urease test: Positive/Negative

9. Fluprescein test: Positive

- Highly multidrug resistant

- Combination therapy: Penicillin derivatives, Ceftazidime, Ciprofloxacin, Aztreonam, Imipenam
Serratia marcescensGram-negative (pink) bacillus

1. MacConkey agar: Pink colonies

2. Ordinary nutrient agar: Red colonies

1. Indole test: Negative

2. Methyl Red test: Negative

3. Voges-Prokauer test: Positive

4. Urease test: Negative

- Antibiotic resistance vary greatly

- Isolates from nosocomial infections are frequently resistant to multiple antibiotics

- Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)
Proteus mirabilisGram-negative (pink) bacillus
1. Blood agar with phenylethyl alcohol: Colonies do not have swarming effect

2. MacConkey agar: Colourless colonies

1. Indole test: Negative

2. Methyl Red test: Positive

3.Voges-Prokauer test: Negative

4. Catalase test: Positive

5. Urease test: Positive

6. TSI agar: Black butt
- Sensitive to ampicillin, aminoglycosides and trimethoprim sulfamethoxazole


Case Study 4
1.Chlamydia pneumoniae
• Obligate intracellular bacterium
• Does not gram stain
• Affects adults and children

2. Haemophilus influenza
• Pleomorphic gram-negative bacillus
• Affects children and adults (especially with COPD-Chronic Obstructive Pulmonary Diseases)

3. Moraxella catarrhalis
• Oxidase positive
• Gram-negative diplococcus
• Affects children and adults with COPD

4. Pseudomonas aeruginosa
• Glucose-nonfermenting
• Gram-negative bacillus
• Affects adults and children, diabetic adults, nosocomial, CF (Cystic Fibrosis) patients

5. Streptococcus pneumoniae
• Gram-positive lancet-shaped cocci
• Appear in pairs or short chains
• Affects adults (mainly elderly)

6. Mycoplasma pneumoniae
• Smallest free-living organism
• Lacks a bacterial cell wall
• Does not gram stain

7. Staphylococcus aureus
• Gram-positive cocci in clusters
• Coagulase-positive
• Catalase-positive
• Produces Beta-lactamase

8. Paragonimus westermani
• Fluke (Trematode)
• Affects children and adults in endemic areas

9. Adenovirus
• Enveloped dsDNA (double-stranded DNA)
• Affects children and adults

10. Parainfluenza virus Type I, II, III
• Enveloped ssRNA (single-stranded RNA)
• Affects infants and young children

11. Bordetella pertussis
• Coccobacillary, encapsulated gram-negative rod
• Negative blood culture

Lab investigations

Wet mounts
• Observe for microbe structure – bacillus, cocci, lancet-shaped, size, etc.

Gram stain
• Positive gram stain – microbe will stain purple/ blue
• Negative gram stain – microbe will stain red/ pink

Acid-fast bacterium stain
• Stains mycobacterium that do not gram-stain due to their high lipid content

Direct fluorescent-antibody stain
• Histologic stain to detect spirochetes

Peripheral blood films
• Observe microbial activity in blood
• Most respiratory tract infections would have negative blood smears

Enzyme immunoassay
• Identifies organisms with known antiserum
• Specific antibody linked to its homologous antigen

Latex agglutination assay
• Latex beads coated with specific antibody
• Agglutination will occur in the presence of the homologous bacteria

Blood cultures
• Positive blood culture – microbial growth (gold, yellow colonies, etc.)
• Negative blood culture – no microbial growth

Bacteriologic sputum cultured on enriched agar
• Bordet-Gengou agar

Antibiotic susceptibility tests

Penicillin – a general antibiotic for penicillin sensitive isolates
Ceftriazone
Erythromycin
Tetracycline – eg. Doxycycline
Praziquantel


Case study 5
Possible microorganisms

Microorganism Test Result
Staphylococcus aureus
● Gram-staining

● Culturing on mannitol salt/blood

agar

● Coagulase test

● Catalase test

● TSI

● Gram-positive, cluster-forming cocci

● Yellow or gold

colonies, drop in pH (yellow area) / ß-hemolytic

● Positive



Positive

● Acid slant/acid butt
Enterococci

● Gram-staining

● Catalase test


● Gram-positive cocci, occuring singly, in pairs, or in short

chains
● Negatives
Coagulase-negative staphylococci
● Gram-staining

● Culturing on blood agar



Coagulase test

● Catalase test


● Gram-positive, cluster-forming coccus

● Yellow or

gold colonies

● Negative

● Positive
Escherichia coli
● Gram-staining

● Culture on EMB/ MacConkey's agar



TSI agar

● Urease test

● Indole test

● Citrate

test

● Gram-negative rod

● EMB:Lactose-fermenting, blue-black

colonies with metallic green sheen

● MacConKey:Lactose-fermenting, red

colonies

● Acid slant/acid butt with gas but no H2S

● Negative●

Positive

● Negative
Pseudomonas aeruginosa
● Gram-staining● Culture on EMB/ MacConkey's agar

● TSI●

Oxidase test

● Indole test

● Citrate

● Gram-negative rod

● EMB/MacConkey:Non-lactose fermenting

colonies

● Alkaline slant/alkaline butt

● Positive



Negative

● Positive
Enterobacter species
● Gram-staining

● Culture on EMB/ MacConkey's agar



Urease test

● Vogues-Proskauer test

● Citrate test

● Gram-negative rod

● EMB: Lactose-fermenting, brown dark

-centered, mucoid colonies

● MacConkey:Lactose-fermenting, pink mucoid

colonies

● Negative

● Positive

● Positive
Proteus mirabilis
● Gram-staining

● Culture on EMB/ MacConkey's agar



TSI

● IMVIC

● Urease test

● Gram-negative cocci

● EMB/MacConkey:Non-lactose fermenting

colonies

● Alkaline slant/acid butt with H2S

● Indole:

Negative

● Methyl-red: Positive

● Vogues-Proskauer: Negative



● Catalase: Positive

● Positive
Klebsiella pneumoniae
● Gram-staining

● Culture on EMB/ MacConkey's agar



TSI

● Indole test

● Urease test

● Citrate test

● Gram-negative rod

● EMB: Lactose-fermenting, brown dark

-centered, mucoid colonies

● MacConkey:Lactose-fermenting, pink mucoid

colonies

● Acid slant/acid butt with some gas production, no H2S



Negative

● Positive

● Positive

Antibiotic:
Methicillin
Vancomycin
Penicillin
Oxacillin


Case study 6
Antibiotic Susceptibility test 5 antibiotics:
vancomycin, ciprofloxacin , Erythromycin, bactrim and cefamandole


Gram-negative Test Result
Gardnerella vaginalis
● Morphology

● Oxidase

● TSI

● IMViC

● Laboratory diagnosis /culture

●Bacilli

● negative

● Acidic slant/acidic deep

● Catalase (-)

● Chocolate agar and HBT agar: Small, circular, convex, gray colonies

●Colistin-oxolinic acid blood agar: Beta-hemolysis
Escherichia coli
● Morphology

● Oxidase

● TSI

● IMViC

● Laboratory diagnosis /culture

●Bacilli

● negative

● Acidic slant/acidic deep

● I(+),M(+), Vi(-),C(-), U(-)

● EMB: green sheen,fermenting colonies

●MacConkey agar: fermenting colonies
Neisseria gonorrhoeae
● Morphology

● Oxidase

● TSI

● Laboratory diagnosis /culture

●Bacilli

● positive

● Acidic slant/acidic deep

●Giemsa-stained

●PCR and ELISA

●Immunofluorescence
Chlamydia trachomatis
● Morphology

● Oxidase

● Laboratory diagnosis /culture

●cocci

● positive

●Giemsa-stained

●PCR and
ELISA

●Immunofluorescence
Pseudomonas aeruginosa
● Morphology

● Oxidase

● TSI


IMViC

● Laboratory diagnosis /culture

●Bacilli

● positive

● Alkaline slant/ alkaline butt

●Catalase (+)

●EMB and MacConkey agar: non-fermenting colonies







Gram - positiveEnterococcus faecalis Staphylococcus saprophyticus
Morphology Cocci( in pairs) Bacilli
Catalase test- +
coagulase Nil-
Laboratory diagnosis /culture Blood agar: non-hemolysisMac Conkey’s agar: Spherical, irregular grape-like cluster in culture




Other microbes Trichomonas vaginalis Candida albicansMycoplasma hominis
Morphology acridine orange : pear-shaped, motile, flagellated protozoansingle-celled, diploid fungusround, pear shaped and even filamentous
Laboratory diagnosis /culture
Trichomonas Direct Enzyme Immunoassay and Fluorescent Direct Immunoassaysaline

wet preparation : motile trichomonads and increased PMNs (ratio of PMNs to vaginal epithelial cells)

Blood agar plates: large, round, white or cream colonies
Mycoplasma GU Culture System: ‘fried egg’ and granular appearance colonies


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