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PRAKTIKUM

MIKROBIOLOGI
INFEKSI SALURAN
URINARIA (UTI)

Aspek Penting Pemeriksaan


Mikrobiologi Pada UTI :
- Pengumpulan spesimen &
penanganannya
- Perlakuan spesimen and kultur
- Interpretasi hasil Lab Mikrobiologi.

Microbiology of Community-Acquired
Urinary Tract Infection
Dysuria-Pyuria Sydrome in Females
Children

Adults

More frequent

. Escherichia coli

. Escherichia coli
. Staphylococus
saprophyticus (young,
sexually active patient)

Less Frequent

. Other Enterobacteriaceae
. Enterococci
. Streptococcus agalactiae

. Other Enterobacteriaceae
. Enterococci

Other Community-Acquired Infection


Children

Adults

More frequent

. Escherichia coli

. Escherichia coli

Less Frequent

. Other Enterobacteriaceae
. Enterococci

. Other Enterobacteriaceae
. Enterococci

Microbiology of Nosocomially Acquired


Urinary Tract Infection in Children or Adult
Catheter-Associated Short-Term (< 30 d) Catheterization
More frequent

. Escherichia coli

Less Frequent

. Other Enterobacteriaceae
. Pseudomonas Spp.
. Staphylococcus epidermidis

Catheter-Associated Long-Term (> 30 d) Catheterization


More frequent

. Providencia stuartii
. Pseudomonas Spp.
. Escherichia coli

. Other Enterobacteriaceae
Less Frequent

. Staphylococcus epidermidis

GENERAL INFORMATION
Urine dpt terkontaminasi oleh Mo yang
terdpt di uretra/ Periuretral Proliferasi
Misleading
Pasien Simptomatik : 1 spesimen utk
diagnosis, & 1 spesimen diambil setelah
terapi 48 72 jam.
Pasien asimptomatik (2-3 spesimen).
Renal Tuberculosis spesimen diambil 3
hari berturut2 (pagi hari).

Pengumpulan spesimen
Sampel urine diambil pd pagi hari, utk
memperoleh bakteri tahan asam, fungi & Mo
patogen lain.

Midstream urine Mo yg tdp di bladder

Continue
Pediatric: diambil dr kantung urine kultur
segera mungkin.
Urine dikumpulkan menggunakan tempat yg
steril, & tahan bocor.

Continue.
Hindari Kontaminasi
Pasien Simptomatik / Asimptomatik:
Interpretasi kultur kuantitatif , dan
perhitungan spesimen urine

SPECIMEN TRANSPORT
Segera (1-2 jam) tanam pd medium
transport
Jika transportasi terlambat, maka spesimen:
disimpan di lemari es: tidak lebih 24 jam
Inokulasi: medium primer sbl medium
transport
ditambahkan larutan pengawet
Perhitungan koloni dinyatakan Invalid jika
spesimen tdk diproses selama 30 menit

PROSES SPESIMEN
100,000 CFU/ml Infeksi UTI.
Pasien Simptomatik, perhitungan
dibawah < 100.000 CFU/ ml sering
di validasikan dgn leukosit pd spesimen
Kultur Anaerob sulit diperiksa, Jika
diduga (suspected) suprapubic
bladder aspiration

Continue
Foley catheter tidak bisa di kultur
Sampel Urin kantung kateter dgn
menggunakan syringe & jarum utk
Aspirasi, melalui sampling port stl di
desinfektan

Sampling
Port

Urine collection can be


performed without needle
Avoidance of needle stick
injury

TRANSPORT MEDIA

Urine specimen is
best collected
Soon after a
patient wakes
when Organisms
have had the
opportunity
to multiply over
several hours.
Urine container criteria :
Sterile, dry, wide-necked,
leak proof

Laboratory examination of urine

Quantitative (Colony counts)

+1
+2
+3
+4

a urine sample is streaked on surface of


Blood Agar plate ( for control) and medium
Endo agar / Mc Conkey agar with a special
loop calibrated to deliver a known volume.
Over night incubation
Isolation of colonies,Biochemical tests,
Drug susceptibility test,
Over night incubation

RESULT
Urinary Tract infection Module05 ..

Endo C agar cat. No. 4044


Selective culture medium for detection and isolation
Of fecal Escherichia coli and coliform bacteria in
Various material according to ENDO
Sodium sulfide and fuchsin inhibit the growth of Gram
positive bacteria.
E. coli and coliform bacteria metabolize lactose with the
production of aldehyde and acid. The aldehyde liberates
fuchsin from the fuchsin-sulfide compound, the fuchsin
then colors the colonies red.
E. coli will giving the permanent greenish metallic
sheen (Fuchsin sheen)

Agar Endo

MacConkey Agar
A differential medium for detection, isolation and
enumeration of coliform and intestinal pathogen in
water, dairy product and biological specimens.
This agar is particularly recommended for the
cultivation of pathogens which may be present in
a variety of specimens such as urine, faeces and
wound swabs.
MacConkey agar should be used in parallel with other
selective indicator media such as Desoxycholate
Citrate Agar, Bismuth sulphite agar, Briliant
Green Agar and Brilliant Green bile broth, and non
selective medium such as Blood Agar.

MCA (Mac Conkey Agar)


Escherichia coli (koloni merah muda)
Salmonella sp (koloni putih)

INTERPRETATION OF COLONY COUNTS


Score

Number of
colonies

Number of bacterial
per ml

< +1

50

5.000

+1

100

10.000

>+1

150

15.000

+2

200

20.000

>+2

250

25.000

+3

300

30.000

>+3

500

50.000

+4

1000

100.000

> +4

> 1000

> 100.000

Urinary Tract infection Module05 ..

Diagnostic
significance

Significant
bacteriuria

Gram Stain
Escherichia coli

Microscopic :
Gram negative rod

C.L.E.D Medium
(Cystine-Lactose-Electrolyte Deficient)
This medium recommended for urinary bacteriology,
supporting the growth of all urinary pathogens. The present
of important contaminants such as diphtheroids, lactobacilli
and micrococci is also clearly elicited giving indicate and
indication of the degree of contamination.
This medium prevent the swarming phenomenon by
having the low electrolyte content.
Discrete colonies of motile and non motile bacteria
may thus be urinary bacteriology where motile
species, notably Proteus spp. may be present a
problem in identifying the species responsible to UTI.

Proteus
This species causes urinary infection, especially following
catheterization or cystoscopy.
Mostly Proteus strains produce a characteristic swarming growth
over the surface of blood agar and several other culture media.
Proteus culture have a distinctive smell.
In laboratory C.L.E.D medium (electrolyte deficient) provide for
plate culture of urinary organisms for prevents the swarming
phenomenon
Proteus spp. posses urease which produces ammonia in
the urine. This raises the the pH and causes precipitation
of phosphate crystals, leading to stone formation.
Urinary Tract System Module05 ..

Ureasa test
Test the ability of microorganisms to
metabolize urea and produce ammonia
using urease (indicator: methyl red)
Result :
Positive pink
Negative yellow

NOVOBIOCIN TEST
This test using for differentiated between Staphylococcus
epidermidis and Staphylococcus saprophyticus from
Urine specimens.
A. Staphylococcus epidermidis (sensitive-Novobiocin),
occasionally cause of urinary infection in hospital,
sometimes associated with catheterization.
B. Staphylococcus saprophyticus (resistant-Novobiocin),
it causes cystitis in young women patients in general
practice beside Escherichia coli

Urinary Tract System Module05 ..

Gram Stain
Staphylococcus sp.
Microscopic :
Gram positive cocci
of uniform size
That occur
characteristically in groups
but also singly and in pairs

Pseudomonas
Pseudomonas aeruginosa:
This species causes urinary infection, usually following
catheterization or associated with chronic urinary
infections.
This species is usually resistant to many antibiotics
and is particularly associated with recurrent urinary
tract infection in hospital patients.

Urinary Tract System Module05 ..

SABOURAUD DEXTROSE AGAR


Sabouraud dextrosa
agar is used to culture
Candida albicans and
other fungi .
pH medium should be
within the rage 5.4 5.8
at room temperature,

candida albicans
on sabouraud agar

Gram Stain
Candida sp.

Microscopic :
Gram positive

BIOCHEMICAL TEST

Carbohydrate fermentation

Urinary Tract infection Module05 ..

Commercially identification

The API 20Etm (produced by Biomerieux) system for bacteria such as coliform
(ONPG : orthonitrophenyl- -D-galactopyramoside; VP : Voges-Proskauer)

Three API 20E strips :


a. Immediately after inoculation
b. After 24 hours incubation
c. That in ( b) after addition of reagents to certain wells.
The organisms here is Escherichia coli. Here the first carbohydrate well (glucose)
is also used for the nitrate reduction test

Antibiotic susceptibility tests


Disc susceptibility test
A.

Using wire loop or Dacron-tipped


swab, touch the tops of 4 to 5
bacterial colonies of the same
morphology on an agar plate
culture.

E.

Swab the surface of the plate


three times, rotating the plate
60 0 each time.

B.

Transfer the bacteria to a tube


containing 4 to 5 mL of Brain Heart
Infusion broth or NaCl.

F.

Allow 3 to 5 minutes. Using a


forceps or multidisk dispenser
plate antibiotic impregnated
disk on surface of the agar.

C.

Match the density of the bacterial


suspension to the 0,5 Mc. Farland
density standard.

G.

Tamp the disks down firmly


onto the surface of the agar.
Incubate at
35 0 C for 24
hours.

D.

Insert a cotton-tipped swab into the


suspension, the swab firmly
against the wall of tube to express
excess fluid and streak the surface
of dried Mueller-Hinton agar plate.

H.

After overnight incubation, use


a ruler or caliper to measure the
diameter of incubation zones.

Urinary Tract System Module05 ..

Gentamycin (CN)
: 12 - 15
Chloramphenicol (C) : 12 - 18
Penicillin (P)
: 28 - 29
R (Resistant) ; I (Intermediate) ; S
(Sensitive)

Staphylococcus aureus
CN

CN

MIC result : Escherichia coli = 2 g

E-test strip

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