ISOLATION AND IDENTIFICATION OF MICROBIAL FAUNA FROM

URINE SAMPLES OF URINARY TRACT INFECTED (UTI) CHILDREN

SIDRA TUFAIL
38155129
Prof. Dr. Farkhanda Manzoor

DEPARTMENT OF ZOOLOGY
LAHORE COLLEGE FOR WOMEN UNIVERSITY
2019
 INTRODUCTION
 Urinary tract infection (UTI) is an infection in urinary system or presence of organisms in
the urinary tract. It is an infection involving the kidneys, ureters, bladder, or urethra.

 Clinical symptoms: wanting to urinate more often and urgently, burning pain, urine appear
bright pink or cola colour and a sign of blood in the urine. A feeling that the bladder is still
full after passing urine, cloudy strong smell in urine (Car, 2006).

 The most common pathogen is Escherichia coli.

 The prevalence of UTI is 11.6% among symptomatic patients; and 8.9% in males and 13.8%
in females in Pakistan.

 Girls should wipe from front to back (not back to front) after bowel movements. This helps
prevent bacteria from the anus entering the urethra. Drink plenty of fluids, especially water.
 SCOPE

 Urinary tract infection is most prevalent among Pakistani population because people
never bother the hygienic conditions especially children under the age of 3 to 7 years
old.

 Ignorance of personal hygiene methods used after urinating.

 Urine culture of UTI infected patients not only contains E.coli but also Klebsiella etc.

 These bacteria causes UTI infection which is either lower urinary tract infection or upper
urinary tract infection.

 Several antibiotics are used for the treatment of UTI.

 Material and methods
 Samples collection
Samples were collected from Microbiology paediatric laboratory of Mayo hospital Lahore and
experiment was conducted in and Entomology laboratory of Lahore College for Women
University Lahore.

 Urinalysis of urine samples:

Physical parameters of urine samples pH, color, specific gravity, protein, blood, sugar, pus cells,
RBCs and epithelial cells were analyzed by urinalysis (urine complete test) reagent strips.
 Media preparation
Different types of media were used to characterize isolated microbial fauna from urine samples.
These are:
 Nutrient Agar (NA)
 Cysteine Lactose Electrolyte Deficient (CLED) Agar
 MacConkey agar
 Eosin Methylene blue agar
 Pouring of media
 Media was autoclaved then media plates of nutrient agar, cysteine lactose electrolyte
deficient (CLED), MacConkey agar and eosin methylene blue agar were prepared.

 Using a sterile pipette 0.1ml from each urine sample was transferred onto the surface of
nutrient agar plates separately.

 Using a sterile L rod spreader, the samples were spread evenly on the surface of each
nutrient agar
 Streaking of cultured samples
 After overnight incubation, colonies were picked from the cultural plates and streaked on
the MacConkey agar and eosin methylene blue agar.

 These plates were incubated over night at 37°C for 24 to 48 hours.

 For streaking on CLED agar. 1 µl wire loop was sterilized and dipped of into urine sample.
 The loop was then streaked on the plate of Cysteine Lactose Electrolyte Deficient (CLED)
Agar. (Karza et al., 2017; Sajed et al., 2014).

 Identification of microbial fauna

 Cultural observation
 Color, size, and colony morphology were observed from the incubated plates of agars.

 Microbiological analysis of urine specimen

The bacterial pathogens were identified by streaking the urine specimens on these media
based on their color morphology after the incubation period.

 Statistical analysis

Data was analyzed using the Microsoft excel software.

 RESULTS
 102 children's specimens were suspected patients with UTI according to diagnosis made
by physician.

 In which 62(60.78%) of specimens was confirmed bacterial infection with positive

urine culture while 40(39.21%) of specimen was confirmed bacterial infection with
negative urine culture.

 Microscopically, they were examined and physical properties recorded as a primitive

stage of detecting inflammation.

 Samples were cultured and thirty six different bacteria obtained from urine samples of
UTI infected patients.
Figure: Showing the concentration of proteins and blood present in urine samples of UTI
patients.
%age of proteins and blood
90

80 77

70

60
60

50

40

30

20

10

0
protein blood

Figure: Showing %age population of 77% proteins and 60%

blood is detected in urine samples of UTI patients.
Figure: Showing the concentration of pus cells present in urine samples of UTI patients

%age of pus cells

60

51
50

40
percentage of pus cells

30 27

20

12

10
5 6

0
0 to 4 4 to 8 8 to 12 12 to 16 16 to 20
Range of pus cells

Figure: Showing the %age population of pus cells

detected in urine samples of UTI patients.
Figure: Showing the concentration of epithelial cells in the urine samples of UTI patients.

%age of Epithelial cells

70
65

60

percentage of epithelial cells 50

40

30

21
20
14

10

2
0
0 to1 1 to 2 2 to 3 3 to 4
Range of epithelial cells

Figure: Showing the %age population of epithelial cells

detected in urine samples of UTI patients.
 Culturing of UTI samples on nutrient agar:
The samples were cultured on nutrient agar plates, which considered as differential medium for
the isolation, purification and identification of microbial fauna.

Figure: Showing growth of microbial fauna from urine

samples of UTI infected patients on nutrient agar.
 Culturing of urine samples Eosin methyl blue agar:

The urine samples of UTI patients were cultured on the EMB plates. Showing the growth of
Enterobacter aerogenes, Escherichia coli, Proteous vulgaris and Pseudomonas aeruginosa with
their typical colony morphology.

Figure:

Showing growth of bacteria on EMB media. Growth of

Pseudomonas aeruginosa on sample no 10, growth of
Escherichia coli on sample 11, 12 and growth of Enterobacter
aerogenes on sample 9

NOTE: (Pink, colony without metallic green sheen =

Enterobacter aerogenes)

(Green metallic sheen colonies = Escherichia coli)

 Culturing of UTI samples on MacConkey agar:
The samples were cultured on the MacConkey agar plates. The growth of Enterobacter aerogenes,
Escherichia coli, Proteous vulgaris and Klebsiella pneumonia with their typical colony
morphology

Figure

Showing growth of bacteria on MacConkey medium. Growth of

Escherichia coli on sample 2 and growth of Proteus vulgaris on
sample 3.

NOTE: (Lactose-fermenting 'shiny, rose pink = Escherichia

coli)

(Lactose-non fermenting 'colorless colony = Proteus

vulgaris)
 Culturing of UTI samples on Cysteine Lactose
Electrolyte Deficient (CLED) agar:
The samples were cultured on the Cysteine Lactose Electrolyte Deficient (CLED) agar plates.
Which showed the growth of Escherichia coli, Klebsiella pneumonia and Pseudomonas
aeruginosa with their typical colony morphology.

Figure:

Showing the growth of Escherichia coli on sample 1, growth of

Klebsiella pneumonia on sample 3 and growth of Pseudomonas
aeruginosa on sample 4 from urine samples of UTI infected
patients.
 Percentage prevalence of UTI among children:

 One hundred two of the children's specimens were suspected patients with UTI
according to diagnosis made by physician.

 where in which 62(60.78%) of specimens was confirmed bacterial infection with

positive urine culture while 40(39.21%) of specimen was confirmed bacterial
infection with negative urine culture.
 Percentage prevalence of UTI among children:

Result of urine culture Children with UTI

No. (%)

Confirmed bacterial infection 62 60.78%

with positive bacterial culture

Confirmed bacterial infection 40 39.21%

with negative bacterial
culture
Total 102 100
Demographic data of UTI infected Children:

 Data in the showed that highest %age

prevalence of UTI is found in age group of
1-10.

 Ratio of UTI is higher in girls as

compared to boys.
 Demographic Distribution of UTI infection among children.

Positive urine culture cases

Children with UTI

Demographics
Number of Positive urine % age
samples (n=102) culture
Age group 1-5 years 58 36 62.06%
(years) 5-10 years 26 16 61.53%

10-15 years 18 10 55.55%

Children Girls 64 34 62.74%

gender Boys 38 28 37.25%
Morphological identification of isolated bacteria on the basis
of colony morphology:

Various types of specific media were prepared to cultivate and

characterize isolates, such as; Nutrient agar for all isolates.

 Eosin methylene blue agar and MacConkey agar for gram

negative bacteria,

while Cysteine Lactose Electrolyte Deficient (CLED) agar

used for growth and enumeration of urinary tract
microorganisms.
 Media Bacterial pathogen Colony morphology
Escherichia coli Lactose-fermenting 'shiny, rose pink
Klebsiella pneumonia Pink, colony, large 'glistening and mucoid

Proteus vulgaris Lactose-non fermenting 'colorless colony

MacConkey
Agar
Enterobacter aerogenes Lactose fermenter, light pink, unbonate small and uneven pink
colonies

Enterobacter aerogenes Pink, colony without metallic green sheen

Eosin methylene blue agar Escherichia coli Green metallic sheen colonies
Proteous vulgaris Small color less colony
Pseudomonas aeruginosa Irregular colorless colonies
Mix growth Circle, small colonies, , small mucoid colonies, creamy,
Nutrient agar swarming white to creamy colony

Escherichia coli Opaque yellow colonies with a slightly deeper yellow center
Cysteine Lactose Electrolyte Deficient
Klebsiella pneumonia Yellow to whitish-blue colonies, extremely mucoid
(CLED) agar plates

Pseudomonas aeruginosa Green colonies with typical matted surface and rough
periphery
 Distribution of isolated bacteria among study groups:

 The highest prevalence of specific bacteria was found to be

Escherichia coli recorded among all the paediatric UTI to be
(55.55%).

 The least percentage among the bacteria were Klebsiella

pneumonia, Proteus vulgaris, Pseudomonas aeruginosa and
Enterobacter aerogenes which recorded 16.66%, 5.55%, 11.11%
and 11.11% respectively.
Distribution of isolated bacteria among study groups:
+ve UTI cases

Isolated Bacteria Children with UTI

No %

Escherichia coli 20 55.55%

Klebsiella pneumonia 6 16.66%

Proteus vulgaris 2 5.55%

Pseudomonas 4 11.11%
aeruginosa

Enterobacter 4 11.11%
aerogenes

Total 36 100%
 CONCLUSION

 Out of 102 urine specimens of children with suspected UTI 62(60.78%) of

specimens was confirmed bacterial infection with positive urine culture
while 40(39.21%) of specimen was confirmed bacterial infection with
negative urine culture

The rate of girls having UTI were higher 62.74% in comparison with boys
37.25%.

 The highest %age prevalence of UTI is found in age group of 1-10. Ratio of
UTI is higher in girls as compared to boys.
 Urine samples were cultured and thirty six bacteria were obtained.

 The highest prevalence of specific bacteria was found to be Escherichia coli

recorded among all the paediatric UTI to be (55.55%).

 The least percentage among the bacteria were Klebsiella pneumonia,

Proteus vulgaris, Pseudomonas aeruginosa and Enterobacter aerogenes
which recorded 16.66%, 5.55%, 11.11% and 11.11% respectively.