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Patient’s Name: Khong Fay Fay
Age: 26 Years
Sex: Female

Clinical Diagnosis
Complaints: Fever, chills, dysuria (pain during urination)
Diagnosis: Urinary Tract Infection
Antibiotic treatment: Nil

Urinary Tract Infection (UTI)
UTI is an infection that can happen anywhere along the urinary tract. It is usually caused by a bacterium from the anus entering the urethra and then the bladder. UTI is more common in women because their urethra is shorter and closer to the anus. (1) It may also be caused by dehydration, obstruction, disturbance of smooth urinary flow or presence of a foreign body, e.g. stone or urinary catheter. Trauma during sexual intercourse may also cause and infection in women. (2)

Usually, an UTI infected patient will experience dysuria, hematuria (bloody urine), fever, cystitis ( pain in the midline suprapubic region and/or frequent urination) and urethritis (discomfort or pain at the urethral or a burning sensation) .

The more common and suspected microorganisms that cause UTI are Escherichia coli (E.coli), Klebsiella pneumoniae, Proteus mirabilis, Psedonmonas aeruginosa. Staphylococcus saprophyticus and Enterococcus spp.

Gram-negative E.coli, Klebsiella and Proteus

E.coli, Klebsiella and Proteus are the most common cause of UTI. These bacteria fall in the family of the Enterobacteriaceae. The Enterobacteriaceae are a large, heterogeneous group of gram-negative rods. They are the most common group of gram-negative rods cultured in the clinical laboratory and the most common bacteria that cause disease. They are facultative anaerobes and are either motile with peritrichous flagella or non motile; they grow on peptone or meat media; grow well on MacConkey’s agar; ferment rather than oxidize glucose, often with gas production; are catalase-positive, oxidase-negative, and reduce nitrate to nitrite. (3)

Gram-negative Pseudomonas aeruginosa

Pseudomonas are commonly found in soil and water. P. aeruginosa sometimes colonizes humans and is the major human pathogen of the group. It is widely distributed in nature and is most found in moist environments in hospitals. P. aeruginosa tend to cause disease in humans with abnormal host defenses. They are gram-negative, motile and aerobic rods that are oxidase-positive and do not ferment lactose. (4)

Gram-positive Staphylococcus saprophyticus
10-20% of sexually active, young women are infected by Staphylococcus saprophyticus causing UTI. Staph. saprophyticus are gram-positive, cocci in irregular clusters and are facultative anaerobes. They are coagulase-negative and catalse-postive. (5, 6)

Gram-positive Enterococcus spp.
Entercococcus spp. are classified under Group D of the heterogeneous group of the Streptococci family. Streptococci are gram-positive spherical bacteria that form pairs or chains during growth. Group D Entercocci are usually non-hemolytics and occasionally alpha-hemolytic and are PYR positive. (7)

Diagnostics Laboratory Tests
The diagnosis of UTI can be confirmed by a urine culture. The urine culture will be sub cultured, isolated to identify the bacteria by running a series of appropriate biochemical tests. An antibiotic susceptibility testing will also be done to see which antibiotic is most suitable to be used to treat the patient. In diagnosis of UTI, if the urine culture yielding a greater than 100,000 colony-forming units (CFU)/mL and pure growth would indicate a significant bacteria growth. Or a urine culture that yield >100,000 CFU/mL with 2 bacteria growth may also indicate significant growth. However, if more than 3 organism is present, it indicates mixed bacteria growth and may be due to bad sample collection or contamination.

The urine culture is sub-cultured into TSA with 5% sheep blood agar (blood agar) and Cysteine Lactose Electrolyte (CLED agar).
The agar plates are left to incubate in the incubator at 37o C for 18-24hrs. The media is streak in a systematic manner as shown below.

http://uths.revealed.net/science/stein/homework/lab_ident_bacteria_intro.htm

After incubation, a gram stain would be done by isolating a colony obtained from the blood agar. A gram stain is done and examined microscopically to determine if the microorganism is a gram-negative or gram-positive bacterium. On microscopic examination, a purple-blue indicates gram-positive organisms while red-pink indicates gram-negative organisms.

GRAM NEGATIVE BACILLI

http://uths.revealed.net/science/stein/homework/lab_ident_bacteria_intro.htm


GRAM POSITIVE COCI

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Gram-negative rods may indicate E.coli, K. pneumoniae, P. mirabilis or P. aeruginosa infection.
Gram-positive cocci in clusters may indicate Staphylococcus saprophyticus while gram-positive cocci in pairs or chains may indicate Enterococcus spp..

If the gram-stain showed a gram-positive cocci, the following tests can be done.

Catalase
With an inoculating loop, pick a small colony and swab it onto a glass slide. Then, add 2-3 drops of hydrogen peroxide to the colony. A catalase positive test will show bubbles form immediately.
The catalase test differentiates the Staphylococci, which are positive, from the Streptococci, which are negative. When a catalase test is positive, it will produce bubbles. This is because Staphylococci produce catalase that will convert hydrogen peroxide to water and oxygen, producing bubbles.

(+) (-)

http://faculty.mc3.edu/jearl/ML/ml-10.htm

Coagulase
With an inoculating loop, pick a small colony and swab it onto a glass slide. Then, add 2-3 drops of coagulase to the colony. Mix the coagulase with the colony well and observe for agglutination.
When a catalase is positive, proceed to do a coagulase test to differentiate S.aureus from other Staphylococci. S.aureus produce coagulase, an enzyme-like protein that clots citrated plasma. Coagulase will bind to prothrombin and initiate fibrin polymerization. Coagulase may deposit fibrin on the surface of Staphylococci. When this happen, S. aureus will produce a clumping factor on its surface for the organism’s adherence to fibrinogen and fibrin. Agglutination indicates coagulase positive, indicating that the organism is most likely a S. aureus. Coagulase negative suggest it might be other species of staphylococci. In the case of UTI, it might be a S. saprophyticus.

(-) (+)

http://faculty.mc3.edu/jearl/ML/ml-10.htm

When a catalase test is negative, observe the blood agar and check for hemolysis. Blood agar is an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. β-hemolytic activity will show complete lysis of red blood cells surrounding colony, while α-hemolysis will only partially lyse hemoglobin and will appear green. non-hemolysis or γ-hemolysis is the term referring to a lack of hemolytic activity.

When a catalase test is negative, observe the blood agar and check for hemolysis. Blood agar is an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. β-hemolytic activity will show complete lysis of red blood cells surrounding colony, while α-hemolysis will only partially lyse hemoglobin and will appear green. non-hemolysis or γ-hemolysis is the term referring to a lack of hemolytic activity.

β-hemolytic

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α-hemolysis

jade.ccccd.edu/mweis/.../media_SD_list_page.htm

γ-hemolysis

www.sph.unc.edu/courses/eric/dd_cs/betahemo.htm

For α-hemolytic results, an Optochin test can be done. Optochin is an antibiotic susceptibility testing. This test is to differentiate between α-hemolytic Streptococci which is resistant to Optochin and S. pneumoniae which is sensitive to Optochin.
If the organism is sensitive to Optochin, it will produce a clear zone surrounding the colony.

For γ -hemolytic results, a Pyrulidonyl Peptidase (PYRase activity) test can be done. PYR is a rapid colourmetric test for use in the differentiation of Enterococci form Lancefield Group D Streptococci. PYR disc is impregnated with PYR, a substrate that is hydrolyzed by pyrase to form β-naphthylamine. They hydrolase forms a red compound upon addition of a colour developer. Pyrase activity is present in Entercocci. If pink or orange-pink colour is formed, it indicates a positive PYR test, showing presence of the organism Enterecocci. If no colour is form, it shows a negative PYR test, showing presence of non-hemolytic Streptococci.

If the gram-stain showed gram-negative bacilli, the following tests can be done.

Oxidase
Oxidase test is done to determine the presence of cytochrome oxidase activity in bacteria. Cytochrome oxidase is an enzyme found in certain bacteria that is able to transfer electrons to oxygen. The enzyme oxidizes reduced cytochrome c to make this transfer of energy. Presence of cytochrome oxidase can be detected through the use of oxidase disk which acts as an electron donator to cytochrome oxidase. If the bacteria oxidize the disk, the disk will turn purple, indicating a positive test. No colour change indicates a negative test. (8)

http://medic.med.uth.tmc.edu/path/oxidase.htm

If oxidase is positive, do a sensitivity testing on Mueller Hinton agar. P. aeruginosa will produce green pigment colonies.

If oxidase test is negative, observe the colonies form on CLED agar. CLED agar is used to differentiate between lactose fermenters and non-lactose fermenters bacteria. It also prevents the swarming of Proteus species. Lactose fermeters produce yellow colonies on CLED agar while non-lactose fermenters appear blue.


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For lactose fermenter bacteria, proceed to do an Indole test. Indole is one of the degradation products of amino acid metabolism. It is useful in identifying E. coli. The test is based on the formation of a red complex when indole reacts with the aldehyde group of an active chemical in kovac reagent. A positive test will produces pink/purple colour at interface and the organism is E. coli. A negative test will remain colourless and further testing can be done to confirm if it is Enterococci or Klebsiella.

References:

1. Wikepidia (2006). On-site: http://en.wikipedia.org > search >. Retrieved on 4th December 2006.

2. Medical Microbiology and Infection at a Glance Pg 94
3. Geo FB, Janet SB & Stephen AM. (2004). Jawetz, Melnick, & Adelberg’s Medical Microbiology. 23rd edition. McGraw-Hill.Pg 248
4. Geo FB, Janet SB & Stephen AM. (2004). Jawetz, Melnick, & Adelberg’s Medical Microbiology. 23rd edition. McGraw-Hill. Pg 262
5. On-site: year2>mmid>bms5300>bugs>stasapro">http://medinfo.ufl.edu>year2>mmid>bms5300>bugs>stasapro
6. Geo FB, Janet SB & Stephen AM. (2004). Jawetz, Melnick, & Adelberg’s Medical Microbiology. 23rd edition. McGraw-Hill. Pg 223
7. Geo FB, Janet SB & Stephen AM. (2004). Jawetz, Melnick, & Adelberg’s Medical Microbiology. 23rd edition. McGraw-Hill.Pg 231, 235
8. On-site: intranet-web>Courses>DMI_8351>Oxidase">http://dentistry.ouhsc.edu>intranet-web>Courses>DMI_8351>Oxidase