Practical of

Urinary Tract Infection

Department of
Microbiology
Medical Faculty
University Of
Indonesia

The common features of UTI are

an :
1. Urgent desire to urinate (urgency)
2. Frequent urination (frequency)
3. Painful urination (dysuria)
In children, UTI may present as bedwetting

Laboratory confirmation of the diagnosis is

by microscopy and culture of a urine specimen,
Which should be collected without contamination
by urethral, perineal or enteric flora.

Important aspects of
Microbiologic Examination of
UTI:

Specimen collection and handling

Specimen processing and culture
Interpretation of microbiology

laboratory result

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

Laboratory examination of urine

URINE :
Prone to contamination
Quantitative test to differentiate causative
agent
Samples:
- Mid stream urine
( Clean catch urine, Mid-stream urine)
- Supra pubic puncture
especially for anaerobic culture
- Catheterized urine

Collection :

Miller JM, A guide to specimen management in clinical

microbiology. ASM Press, washington DC, 1996, 2-20, 23-29, 57

1. Mid stream urine ( Clean catch urine )

First morning specimen (prefered)
Take the specimen after the first portion of urine
has been voided
Pediatric patients: screening by using a
strapped-on bag device after careful cleansing

Collection of a midstream specimen of urine

(MSU)
Prepare specimen container,
water, soap, tissues
Female patient :
- spread labia, using plain soap
or antiseptic
- wipe front to back
- dry with tissues
1.
2.
3.
4.
5.
6.
7.

Male patient :
- retract prepuce, using plain
soap or antiseptic
- cleans glans
- dry with tissues

Began passing urine

Stop flow in midstream
Pass several mL into open container without touching rim
Stop flow before it ends
Recap container
Pass remaining urine into lavatory
Send specimen to laboratory immediate (refrigerate if
prolonged transport time)

URINE
SUPRA PUBIC
PUNTURE

The urinary catheter

Specimens of bladder urine for laboratory investigations can be collected
From catheterized patients as shown. The second port (above) is for
putting fluids into the bladder.
Urine from the drainage bag should not be tested because it may have
been standing for several hours.

Identification label /
request form:
Patient data (name, age, sex, address/ ward)
Clinician/requester data (name, address, phone number)
Specimen type
Urine : midstream, SPP, catheterized
date and time of collection
antibiotics therapy
working diagnosis
requested test
Other relevant data : e.g. post-surgery, immunodeficiency

TRANSPORT

Urine container
criteria :
Sterile, dry,
wide-necked,
leak proof
Send to lab as soon as possible
Urine must be tested within 30 minutes.
If >30 min. keep at 4-8oC for maximal 24 h.

Diagnosis
A. Microscopic methods
fresh clinical specimens is a quickly way of determining the
cause of some infectious diseases.
significant pyuria :
> 10 8 cells/mL urine (indicate of UTI)
UTI can occur in the absence of pyuria
epithelial cells in specimens from female patient usually
indicate vaginal contamination
white cells cast maybe the result of infection in upper UT

Microscopic of urine
a. Urine microscopy can
be used to determine
the presence of white
blood cells (WBs) and
red blood cells
(RBs), epithelial cells,
and bacteria.
b. A volume of urine is
plated out onto a
selective medium such
as Mc-Conkey agar

Direct Urine Microscopic Examination:

Sobel JD and Kaye D. Urinary tract infection. In: Bennetts
Principles and practice of infectious diseases. Mabdell GL,
Bennett JE, and Dolin R. 6th ed, Elsevier Churcil Livingstone
Philadelphia, 2005, vol 1, 875-905

Unstained
(x400)

Stained (x1000)

Uncentrifuged
sample

106

105

Centrifuged
sample

105

104

Mikroskopik :
-Untuk melihat jumlah lekosit dan mikroorganisme
-- menilai apakah urin tersebut terkontaminasi

B. Culture methods
depending upon the source and origin of
clinical
specimen, a decision is made as to which
culture
protocol to follow.

Day 1 :

Laboratory examination of
urine

a urine sample is streaked on surface of

Blood Agar plate and medium Mc Conkey
agar with a dispossable loop (0.01ml)
Over night incubation
Quantitative (Colony
counts)
Colony count
Isolation of colonies, Biochemical tests,
Drug susceptibility test
Over night incubation

RESULT

Day 2 :

Sum of colonies = 125

Per ml = 100 x 125 = 12.500

Refer to table
Interpretation of
Colony count

Day 2 :

Sum of colonies = 1.715

Per ml = 100 x 1.715 = 171.500

Refer to table
Interpretation of
Colony count

Interpretation of colony count

urine specimen
and patient b

Likely to be significantb

Not Likely to be
significantb

Midstream, female
with cystitis

> 102 CFU of potensial

pathogen/mL, Leucocyte
Esterase (LE) is positive

Quantity of potential
pathogen quantity of
contaminating flora

Midstream, female
with pyelonephritis

> 105 CFU of potensial

pathogen/mL, Leucocyte
Esterase (LE) is positive

Quantity of potential
pathogen quantity of
contaminating flora

Midstream,
asymtoptomatic
bacteria

> 105 CFU of potensial

pathogen/mL, Leucocyte
Esterase (LE) is negative

> 105 CFU of potensial

Confirm by repeating urine
pathogen/mL, Quantity of when clinically indicated
potential pathogen
quantity of contaminating
flora

Midstream, male
with UTI

> 103 CFU of potensial

pathogen/mL, Leucocyte
Esterase (LE) is positive

> 103 CFU of potensial

Gram stain demonstrates
pathogen/mL, Quantity of potential pathogen in
potential pathogen
neutrophils and/or casts
quantity of contaminating
flora

Staraight catheter,
all patients

> 102 CFU of potensial

pathogen/mL, Leucocyte
Esterase (LE) is positive
for symptomatic patients

> 102 CFU of potensial

pathogen/mL, Leucocyte
Esterase (LE) is negative

Indwelling kcatheter, > 103 CFU of potensial

, all patient
pathogen/mL, (multiple
pathogensw may be
present)

Adsitional data
suggesting that isolate
is significant

Gram stain demonstrates

potential pathogen in
neutrophils and/or casts

Gram stain demonstrates

potential pathogen in
neutrophils and/or casts

Bacteriuria detected in a
No reason to culture unless
sympthomatic patient; uri patient is asymptomatic

Biochemical test

Commercially identification

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

API-20E
Test

The API-20E test is used

to ID Gram-negative
bacilli from the family
Enterobacteriacea

The one we are using is a

system of 20 individual,
miniturized tests used to
determine the metabolic
capabilities of the
organism

The tests allow us to

come up with a numerical

7-digit profile, based on
which tests are positive
and which are negative.

Antibiotic susceptibility tests

1. Disc Method (Diffusion technique)
Disc method are used by most laboratories to test
routinely for antimicrobial sensitivity
A disc of blotting paper is impregnated with a known
volume and appropriate concentration of an
antimicrobial, an this is placed on a plate of
sensitivity testing agar inoculated wit the test
organism.

The antimicrobial diffuses from the disc into the

medium. After overnight incubation, the culture is
examined for areas of no growth around the disc.
Standar of dilution: Mc Farland 0,5

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.

Disc Diffusion

Disc Diffusion: Interpretation

2. Tube method (Dilusion technique)

Dilution technique measure
the minimal inhibitory concentration (MIC)
The minimal bactericidal concentration
(MBC) which the lowest concentration of
antimicrobial required to kill bacteria

Broth Dilution

MBC determination

Breakpoint Susceptibility
Testing

Sample of NCCLS Breakpoint

Chart

NA, not applicable.

Note that for some antibiotics, breakpoints vary depending on the
pathogen

AML

C
S

Diameter zona = 20 mm

Alternative Susceptibility Test

E-test

E-test: Interpretation

Today's Work..

Specimen management
Microbiology examination of urine specimen
Microorganisms causing urinary tract infection
Gram stain of urine specimen

Direct Urine Microscopic Examination:

Unstained
(x400)

Stained (x1000)

Uncentrifuged
sample

106

105

Centrifuged
sample

105

104

Mikroskopik :
- melihat adanya lekosit , bakteri atau kontaminan

Mac-Conkey 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.
Mac-Conkey 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.

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

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.

Proteus sp

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.