MMIC - Follow up on Specimen 3.

Specimen 3:
Preliminary Information:

Patient: Maisy Hong (67 years)
Sample: Urine
Symptoms: Fever, chills, bladder distension; on indwelling catheter
Bladder distension: Inability to urinate. May be due to obstruction.
Indwelling catheter: prevent catheter from being pulled out of the bladder

Diagnosis: Urinary Track Infection

From week 1:

Possible causative agents: catheter- associated UTI

Most probable agents: according to incidence seen in UTI patients
1) Escherichia coli – 53-72%
2) Klebsiella spp. (pneumoniae) – 6-12%
3) Proteus spp. (mirabilis) – 4-6%
4) Enterococcus spp.(faecalis) – 2-12%


Additional suspected agents:
5) Pseudomonas spp. (aeruginosa)
6) Enterobacter spp. (aerogenes)
7) Serratia spp. (marcescens)
8) Candida spp. (albicans): may cause UTI, associated with use of catheter

Note: Chlamydia and Mycoplasma were excluded as these are typically sexually transmitted, highly unlikely to be the cause in a 67 year old woman with a catheter

Preliminary tests:
Specimen: Urine (dried on slide)

Stains:
Fungal stain
* Periodic acid-Schiff (PAS) can be used to detect if causative agent is fungi.
Gram stain
*Used to detect if bacteria is
- gram negative
- Escherichia coli (rod)
- Proteus Species (rod)
- Enterobacter Species (bacilli)
- Klebsiella Species (bacilli)
- Pseudomonas Species (bacilli)
*gram positive
- Enterococcus Species (cocci)

After the above test have been done, it would be clearer as to whether it is a fungi or bacteria. If it is a bacteria, it would have been narrowed down to gram type. Further screening as shown below for each individual suspected agent will then be performed if agent falls under the initial testing results.

Individual tests:

Escherichia coli
Morphology (microscopy): Gram negative bacilli, singly.
Pathogenesis: Adherence factor P fimbria binds specific galatose disaccharide found on uroepithelial cells surfaces. Siderophores produce cytotoxic alpha and beta hemolysins for lysis of urinary track cells for iron acquisition.
Key Characteristics: Rapidly ferment lactose, positive indole test, positive B-glucaronidase (using substrate MUG), facultative anaerobic (mixed-acid fermentation)
Tests:
1) EMB (Eosin Methylene Blue). Detect lactose fermenting colonies. Contains eosin and methylene blue.
· Black colonies with green sheen (positive, only E.coli can produce this colour due to acid production)
2) MacConkey Agar. Contains bile salts, crystal violet dye (to inhibit Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose).
· Red colonies (positive)
3) Indole test. Measures the ability of the microorganism to degrade tryptophan into indole, ammonia and pyruvic acid. Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48 hours prior to test. Following incubation, add 5 drops of Kovac's reagent.
· Red or red-violet color (positive)
4) Methyl Red (MR).
· Red (positive)
5) Citrate test. Utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source
· Green (negative)
6) MUG (on nutrient agar). Nutrient Agar with methylumbelliferyl-ß-D glucuronide (MUG) detects Escherichia coli (E.coli).
· Colonies with bright blue fluorescence
7) Blood agar plates (β-hemolytic)
· Hemolysis (Positive)
8) Urease Test
· Orange (Negative).
9) Triple Sugar Iron (TSI). Contains phenol red, high concentrations of lactose and sucrose, and a low concentration of glucose as well as sodium thiosulfate and ferric citrate . Provides aerobic and anaerobic conditions. Detects lactose fermentative bacteria – any bacteria that can ferment any of the three sugars.
· Red agar turns yellowish (positive). Acidic slant & butt (lactose and glucose fermenter)
· Stormy fermentation – production of hydrogen gas
10) Antibiotic Susceptibility :
· Amoxicillin, cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin and the aminoglycosides
11) Serology. Detection of O & H antigens (ELISA)
· Slide agglutination – Positive
· Tube agglutination - Positive
Biochemical summary:
IMViC
++--


Klebsiella spp. (pneumoniae)
Morphology (microscopy): Encapsulated gram negative rod (pH 5, acidic) may be cocci in more alkaline conditions (pH 7)
Pathogenesis: Type 1 fimbriae consisting of building block protein (FimA) extend beyond the capsular matrix and mediate adhesion to host epithelial cells.
Capsular polysaccharide matrix encases cell surface to provide resistance against host defence mechanisms.
Key characteristics: Negative indole test. Positive Voges Proskauer (Vi). Lactose fermenting, facultative anaerobic.
Tests:
1) MacConkey Agar.
· Red colonies (positive)
2)Triple Sugar Iron (TSI).
· Red agar remains red (negative). Alkaline slant & butt. (non-fermenter)
3) Indole test.
· Yellow (negative)
4) Voges-Proskauer (VP) test. Determines if neutral products such as ethyl alcohol, acetoin and butanediol are formed
· Pink-burgundy color (positive), 30mins to develop colour
5) Methyl Red (MR) test:
· Yellow (negative)
6) Citrate test. Utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source
· Prussian blue color (positive)
7) Urease Test.
· Orange (Negative).
8) Antibiotic Susceptibility :
· Third generation cephalosporin
9) Serology. Capsular serotyping
· K antigens
Biochemical summary:
IMViC
--++


Proteus spp. (mirabilis)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Mostly positive indole test, except P. mirabilis. Deaminate phenylalaine. Grow on potassium cyanide medium. Ferment xylose. Motile, swarming effect on agar plates. urease-positive. Facultative anaerobe.
Tests:
1) Oxidase test
· Negative
2) Triple Sugar Iron (TSI).
· Black precipitation (H2S production), red slant (alkaline). Glucose fermenter, non-lactose fermenter.
3) Indole Test.
· Most strains positive (red), mirabilis negative (yellow).
4) Methyl Red Test
· Red (positive)
5) Voges-Proskauer (VP) test.
· Negative.
6) Citrate test.
· Prussian blue color (positive)
7) Urease Test.
· Pink (Positive).
8) MacConkey Agar
· Light/colourless colonies (negative)
9) OILM medium. Test for ornithine decarboxylase utilization.
· Green/blue upper portion (Positive).
10) Blood agar plates
· Swarming observed
11) Antibiotic Susceptibility
· Penicillin derivatives, cephalosporins, quinolones, aminoglucosides
Biochemical summary:
IMViC
-+-+


Enterococcus spp.(faecalis)
Morphology (microscopy): Gram positive cocci. (diplococci)
Key Characteristics: catalase negative, colonies appear grey. Group D streptococci. gamma hemolytic (no hemolysis)
Tests:
1) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Negative
2) Blood plate agar
· No hemolysis
3) Bile-Esculin Hydrolysis Test. Determine the ability of an organism to hydrolyze the glycoside esculin to esculatin and glucose in the presence of bile (10 - 40%).
· Black to dark brown slant (Positive)
4) 6.5% salt broth. Test salt tolerance.
· Positive, growth.
5) PYR (pyroglutamyl aminopeptidase) disc
· Positive
6) Xylose fermentation test. performed with D-xylose tablets.
· Positive
7) MacConkey Agar. Without crystal violet for inhibition of gram positive bacteria.
· Pink (positive)
8) Serology. Western blot (IgG from patient’s sera). ELISA.
· Polysaccharide antigen extracted from bacterial cell walls detected
9) Antibiotic Susceptibility
· Quinupristin, ampicillin and vancomycin


Pseudomonas spp. (aeruginosa)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Aerobic (classified facultative anaerobe), rod-shaped bacterium with unipolar motility (due to single polar flagellum), opportunistic pathogen (seen in immunocompromised individuals). Pearlescent appearance and grape-like odor
Tests:
1) MacConkey Agar
· Colourless colonies - Negative (non glucose fermenter)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Blood plate agar
· swarming
4) Oxidase test
· Positive
5) Urease Test
· Positive
6) Indole Test
· Negative
7) Methyl Red
· Negative
8) Voges Proskauer Test
· Negative
9) Citrate test.
. Prussian blue color (positive)
10) TSI
· red slant (alkaline), red butt (alkaline) . Non fermenter of glucose
11) Antibiotic Susceptibility
· Aminoglycosides, Quinolones, Cephalosporins
12) Serology. Detect A, B , O antigens on cell wall
· Slide agglutination – Positive
· Tube agglutination - Positive
Biochemical summary:
IMViC
---+


Enterobacter spp. (aerogenes)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Facultative anaerobe, oxidase negative, catalase positive. Typically caused by catheter insertions
Tests:
1) MacConkey Agar
· Pink colonies - Positive, may be weak (glucose fermenter)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Oxidase test
· Negative
4) Urease Test
· Variable
5) Indole Test
· Negative
6) Methyl Red
· Negative
7) Voges Proskauer Test
· Positive
8) Citrate test.
· Prussian blue color (positive)
9) TSI
· red slant (alkaline), yellow butt (acidic) . Fermenter of glucose
10) Antibiotic Susceptibility
. Ciprofloxacin, Tazobactem
Biochemical summary:
IMViC
--++


Serratia spp. (marcescens)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Produces lactose-fermenting colonies on differential agar, but is a late lactose fermenter may seem like a negative reaction (considered negative). Forms red-pigmented colonies. Able to perform casein hydrolysis and degrates tryptophan and citrate.
Tests:
1) MacConkey Agar
· Red colonies. Negative (initial)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Oxidase test
· Negative
4) Urease Test
· Positive
6) Indole Test
· Negative
7) Methyl Red
· Negative
8) Voges Proskauer Test
· Positive
9) Citrate test.
· Prussian blue color (positive)
10) TSI
· red slant (alkaline), yellow butt (acidic) . Fermenter of glucose
11) Antibiotic Susceptibility
. Aminoglycoside , Amikacin, Gentamicin, Tobramycin and Quinolones
Biochemical summary:
IMViC
--++



Candida spp. (albicans)
Morphology (microscopy): chlamydospores.
Pathogenesis: Produces extracellular proteinases from SAP genes, phospholipases B enzymes and lipases that are secreted for host infection.

Tests:
1) Southern Blot. Detect SAP genes using SAP1 probe.
· Positive.
2) Serology. Detect cell wall antigens - ELISA (CWP, PPM, CW)
· Positive.
3) Oxoid OBIS albicans Test. Rapid card-based chromogenic test that detects the presence of two enzymes specific to C. albicans and C. dubliniensis: b-galactosaminidase and L-proline aminopeptidase
· Magenta colour (Positive).



References:
textbookofbacteriology.net > e.coli.html
http://www.cpg-biotech.com/ > userguides > tech1 > p35681
http://www.mc.maricopa.edu/ > johnson > labtools > Dbiochem > imvic.html
iai.asm.org > cgi > content > abstract > 73 > 8 > 4626
aem.asm.org > cgi > reprint > 28 > 4 > 534.pdf
http://www.blackwellpublishing.com/ > eccmid15 > abstract.asp?id=36567
http://www.clevelandchiropractic.edu/ > ClassFiles-LA > Microbiology > entero.htm
www2.austin.cc.tx.us > microbugz > html > catalase_test.html

Cheers,
Debra, TG02