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	positive	positive	negative
Coagulase test	positive	negative	nil

Possible Organisms	Escherichia coli	Enterobacter sp	Pseudomonas aeruginosa	Klebsiella pneumoniae	Proteus mirabilis
Gram Stain: negative	bacilli	bacilli	bacilli	bacilli	bacilli
Culture on Mac Conkey	pink lactose fermenting colonies	very weak lactose fermenters	non-lactose fermenting colonies producing blue-green pigments	pink lactose fermenting colonies	non-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 production	brown dark-centered colonies indicating lactose fermentation and acid production	colorless colonies indicating no lactose fermentation and acid production
oxidase	negative	negative	positive	negative	negative
*Triple sugar iron (TSI)	acidic slant/acidic deep	alkaline slant/acidic deep	No change	alkaline slant/acidic deep	alkaline 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 pneumoniae	Proteus 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 Test	Serology test	Culture
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)
Shigella	Gram 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.Coli	Gram 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	nil	Selective “CAMP” agar at 42ºC in microaerophilic environment (grow at 5% oxygen + 10% carbon dioxide)
Clostridium difficile	Gram Stain: Gram positive bacilli	nil	nil	Blood 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 pneumoniae	Gram negative (pink) bacillus, a large capsule can be observed	1. 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 aeruginosa	Gram-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 marcescens	Gram-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 mirabilis	Gram-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 - positive	Enterococcus faecalis	Staphylococcus saprophyticus
Morphology	Cocci( in pairs)	Bacilli
Catalase test	-	+
coagulase	Nil	-
Laboratory diagnosis /culture	Blood agar: non-hemolysis	Mac Conkey’s agar: Spherical, irregular grape-like cluster in culture

Other microbes	Trichomonas vaginalis	Candida albicans	Mycoplasma hominis
Morphology	acridine orange : pear-shaped, motile, flagellated protozoan	single-celled, diploid fungus	round, 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