Patient 3 - Maisy Wong

Posted by: Chew Shi Ling & Agnes Chua

Patient Particulars
Maisy Wong,
Female, 66 years old
*Note: It is also known that the patient is an in-patient of the hospital based on the ward and bed number of the patient provided.

Clinical Diagnosis
Complaints: Fever, chills, bladder distension (stretching); on indwelling catheter
Diagnosis: Urinary Tract Infection (UTI)
Antibiotic Treatment (if any): Nil
*Note: A catheter is a hollow tube that is used to drain urine from the bladder. An indwelling catheter stays in place for long periods of time (Long time catheter). There are two kinds of indwelling catheters: urethral and supra pubic. A urethral catheter is inserted into the bladder through the urethra. A supra pubic catheter is inserted into the bladder through a hole in the abdomen, a few inches below the tummy button[8].

Specimen: Urine

Collection of specimen: As an indwelling catheter is in place, the urine should be obtained by sterile aspiration of the catheter with needle and syringe but not from the collection bag. To resolve diagnostic problems, urine can be aspirated aseptically directly from the full bladder by means of suprapubic puncture of the abdominal wall[2].

Suspected Organisms:
(i) Streptococci species (Entercocci)
(ii) Enterobacteriaceae (E. coli, Proteus-Providencia-Morganella, Klebsiella-Enterobacter-Serratia)
(iii)Gram Neg Bacilli (Pseudomonas aeruginosa, Acinetobacter species)

Key Characteristics of respective organisms [2,6]:
(A)Enterococci
· Gram positive cocci arranged in pairs
· Facultative anaerobes2
· Do not contain catalase enzyme, thus, catalase negative
· Non-hemolytic aka gamma hemolytic
· Bile-esculin positive
· Able to grow in 6.5% NaCl

B) Enterobacteriaceae species
· Gram negative rods
· Facultative anaerobes
· Catalase positive
· Oxidase negative

(Bi)E. coli
· Member of normal intestinal flora
· Rapidly ferment lactose
· Beta-hemolytic
· Produce positive indole test
· Positive for b-glucoronidase using the substrate (MUG)
· Ferments mannitol

(Bii)Proteus-Providencia-Morganella
· Does not ferment lactose
· Motile
· Grow on potassium cyanide medium
· Ferment xylose
· “Swarming” on solid media
· Urease positive for Proteus species and Morganella morganii
· Urease negative for Providencia species

(Biii)Klebsiella-Enterobacter-Serratia
Klebsiella species
· Exhibit mucoid growth
· Lacks motility
· Lysine Carbohydrate positive
· Citrate positive
· Have large polysaccharide
· Voges-Proskauer positive
· Rapidly ferment lactose

Enterobacter species
· Motile
· Citrate positive
· Ornithine decarboxylase positive
· Produce gas from glucose
· Voges-Proskauer positive
· Rapidly ferment lactose

Serratia
· Produces DNase, lipase and gelatinase
· Voges-Proskauer positive
· Slow fermenter of lactose

(C) Pseudomonas species
(Ci)P. aeruginosa
· Gram negative motile rods as single/pairs/occasionally short chains
·Grows well at 37-42 degree Celsius (Ability to grow at 42 degree celsius helps in differentiation)
· Oxidase positive
· Able to produce water soluble pigments such as pyocyanin(bluish & nonfluorescent), pyoverdin(greenish & fluorescent) & pyorubin (red-brown).
· Does not ferment lactose

(Cii)Acinebacter species
· Short
· Plump coccobacilli
· Oxidase negative

Preliminary Tests involved:
(A) Culture Medias
· MacConkey agar
MacConkey agar is a culture medium designed to grow Gram-negative bacteria and stain them for lactose fermentation. It contains bile salts, crystal violet dye (to inhibit Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose), lactose and peptone. By utilizing the lactose available in the medium, Lac+ bacteria such as Escherichia coli will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red colonies. Lac- bacteria that cannot utilize lactose will use peptone instead. This forms ammonia, which raises the pH of the agar, and leads to the formation of white colonies[6]. Thus, usage of this agar allows a rapid, presumptive identification of gram negative enteric bacteria[2].

· Blood Agar Plate
Blood Agar Plate contains mammalian blood (usually sheep), typically at a concentration of 5–10%. BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity[6]. 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. γ-hemolysis is the term referring to a lack of hemolytic activity[6]. Most aerobic and facultative anaerobes will grow in blood agar[2].

*Note: On receipt of the urine sample(s), there is a need to utilize the sample and culture them on culture media as soon as possible. The reason being that many types of microorganisms multiply rapidly in urine at room or body temperature and the multiplication of the organisms (contaminants), could interfere with accurate interpretation of the results.
Other then the pathogenic microorganism, Contaminating microorganisms such as the normal flora in the urethra might grow in the cultures as well even when a full voided urine is used. Nevertheless, these microorganisms could be identify as they are usually present at values lower than 10x2 to 10x4 per mL[2].

(B) Gram Staining and Microscopy
Performed using colonies that are cultured above (Gram positive stained blue/violet, Gram Negative is stained red/pink)[2].

(C) Biochemical Tests
When Gram Negative Bacilli is observed, the suspected microbe could be further tested with the oxidase test. Usage of the oxidase test helps to differentiate between the Enterobacteriacease species (Oxidase negative) and Pseudomonas species (Oxidase positive). Further identification of the specific microbe will involve:
1. Oxidase negative: Performance of Triple sugar iron sugar, Indole, Methyl Red, Citrate, Urea and Voges-Proskauer tests. Results of respective microbe is presented in Table 1
2. Oxidase positive: Performance of API 20 NE, Pseudomonas Agar F and Pseudomonas Agar P

Note: Pseudomonas Agar P favours the formation of pyocyanin and/or pyorubin and reduces that of fluorescein, whereas Pseudomonas Agar F stimulates the production of fluorescein and reduces that of pyocyanin and/or pyorubin. Simultaneous use of both culture media allows rapid, preliminary identification of most Pseudomonas species, as some strains can only synthesize pyocyanin, some form only fluorescein and others produce both pigments[1].

When Gram Positive Cocci is observed, the suspected microbe could be further tested with the catalase test. A negative catalase test is an indication for the possibility of the presence of the organisms from the Streptococcus species. Further identification of the specific microbe may involve:

Bile Esculin Agar
The bile esculin agar is a selective and differential agar. Bile salts are the selective ingredient while the esculin is the differential component. Enterococci are able to grow in the presence of 4% bile and hydrolyze the esculin to products that react with ferric citrate in the medium to produce insoluble iron salts resulting in the blackening of the medium[6].

Potassium cyanide medium

It allows the growth of Proteus-Providencia-Morganella

XLD Agar

Xylose lysine deoxycholate agar (XLD agar) is a selective growth medium used in the isolation of isolation of enteric pathogens species from clinical samples and from food. Composition of the medium includes Yeast extract, L-Lysine, Xylose, Lactose, Sucrose, Sodium deoxycholate, Sodium chloride, Sodium thiosulfate, Ferric ammonium citrate, Phenol red & Agar It has a pH of approximately 7.4, leaving it with a bright pink or red appearance due to the indicator phenol red. Acid fermentation lowers the pH and the phenol red indicator registers this by changing to yellow. The ability of the microorganisms to metabolise thiosulfate will lead to the production of hydrogen sulfide causing the formation of colonies with black centers.

Table 1: Results of respective tests[2,6]

(D) Serological tests

Capsular swelling tests

Klebsiellae form large capsules consisting of polusaccharides (K Antigen) covering the somatic (O / H) antigens and can be identified by capsular swelling tests[2].

Latex agglutination assays

Latex agglutination is a way to rapidly visualize an antigen-antibody reaction through the easlily observed clumping that occurs between polystyrene latex beads coated with a
specific antibody and the target antigen for the antibody. Latex agglutination assays are available for the rapid presumptive identification of several bacterial pathogens of humans and are easily performed in a physician's office. The speed of the test allows the physician to initiate therapy immediately; changes in therapy might need to be made later based on a more thorough series of diagnostic tests on the patient's isolate and on findings from antibiotic susceptibility testing for the isolate[3].

In this test, the group-specific antibodies are coated onto polystyrene latex beads. When the latex beads are incubated with an extract containing the released corresponding group-specific carbohydrate antigen, a strong antigen-antibody reaction occurs, that crosslinks the beads in a clearly observable agglutination reaction[3].

(E) Disk Diffusion Susceptibility Testing
The disk diffusion test measures the ability of drugs to inhibit the growth of the bacteria by determining the minimum diameter of inhibition zone for each respective drug. The results correlate reasonably well with therapeutic response in those disease processes where body defenses can frequently eliminate infectious microorganisms[2].

Common Drugs that the organisms are susceptible to [2]:
Enterococci:Vancomycin is the drug of choice. Ampicillin and nitrofurantonin is used to treat patients in umcomplicated UTI.

E. Coli: Ampicillin, cephalosporin, aminoglycosides, sulfonamides, trimethoprim-sulfamethoxazole can be used

Proteus-Providencia-Morganella: Penicillin derivatives, aminoglycosides, cephalosporins and quinolones

Klebsiella-Enterobacter-Serratia: Thrid generation cephalosporin

P. aeruginosa: Usually involve combination therapy as bacteria is highly multidrug resistant. The various combination of drugs used includes, Penicillin derivatives (carbencillin, piperacillin or ticarcillin) + aminoglycosides, thrid generation cephalosporin such as ceftazidime, quinolones such as ciproflaxacin, monbactams such as aztreonam, carbepenam such as imipenam.

Acinetobacter species: Selected Aminoglycosides and Cephalosporins

REFERENCES
1. EMD Chemicals Inc. Pseudomonas Agar F Base. On site: http://www.emdchemicals.com> Search. Retrieved on 6th December 2006.

2. Geo FB, Janet SB & Stephen AM. (2004). Jawetz, Melnick, & Adelberg’s Medical Microbiology. 23rd edition. McGraw-Hill.

3. Jacob RJ & Thomson JM. (2000). Culture Media and Biochemical Tests. On site: http://www.mc.uky.edu Retrieved on 5th December 2006.

4. UMDNJ-School of Osteopathic Medicine. (2004). On-site: http://www3.umdnj.edu> case2gramnegatives. Retrieved on 7th December 2006.

5. UMR Microbiology. (2004). On-site: http://web.umr.edu/ > S_marcescens. Retrieved on 7th December 2006.

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

7. IID Laboratory Manual. (2002). On-site: http://medteach.mccs.uky.edu/COM/iid98/manual > Lab 06. Retrieved on 7th December 2006.

8. In contact. (2006).
http://www.incontact.org/ > search > indwelling catheter. Retrieved on 4th December 2006.