CHAPTER 4
MATERIALS AND METHODS
4. 1. CULTURE MEDIA USED
The following culture media were used during the study:
4. 1. A. Blood Agar
Blood agar supports the growth of most common pathogen.
Sterile defibrinated sheep blood: 7 ml
Nutrient agar (melted)
:100ml
Nutrient agar first sterilized and cooled to 40 – 50ºc. than sterile sheep blood was added
and mixed well. Quantity of 15 ml of blood agar poured on each Petridis.
4. 1. B. Mac Conkey Agar
This is a differential medium used for differentiation of lactose fermenting and nonlactose fermenting gram-negative bacteria.
Peptone
:
2.0 gm
Sodium chloride
:
0.5 gm
Bile salt
:
0.5 gm
Lactose
:
1.0 gm
Agar
:
1.5 gm
Distilled water
:
1000 ml
The ingredients were dissolving in distilled water by heating; the pH was adjusted to 7.6.
About 1 ml of 1% neutral red solution was added to every 100 ml of medium and
sterilized by autoclaving at 121ºC for 15 minute.
4. 1. C. Muller-Hinton Agar
Mueller- Hinton agar preparation includes the following steps:
Mueller- Hinton agar was prepared from a commercially available dehydrated base
according to the manufacturer‟s instructions.
Immediately after autoclaving, it was allowed to cool in a 45 to 50 º C water bath.
Freshly prepared and cooled medium was poured into glass or plastic, flat- bottomed
Petri dishes on a level, horizontal surface to give a uniform depth of approximately 4
mm. ( This corresponds to 60 to 70 ml of medium for plates with diameters of 150mm
and 25 to 30 ml for plates with a diameter of 100mm).
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.. Materials & Methods
The agar medium was allowed to cool to room temperature and, unless the plate is used
the same day, otherwise stored in a refrigerator (2 to 8º C). Plates were used within seven
days after preparation unless adequate precautions, such as wrapping in plastic, have
been taken to minimize drying of the agar. A representative sample of each batch of
plates was examined for sterility by incubating at 30 to 35º C for 24 hours or longer.
4. 1. D. Nutrient Agar for slants and plates
Tryptone: 10gm
Yeast Extract: 5gm
Nacl: 10gm
Agar: 15gm
4. 1 .E. Nutrient Broth
Tryptone: 10gm
Yeast Extract: 5gm
Nacl: 10gm
4. 2 MEDIA INCLUDED FOR IDENTIFICATION OF E.coli
4. 2. A. Peptone Water
Peptone
:
1.0 gm
Sodium chloride
:
0.5 gm
Distilled water
:
100 ml
The ingredients were dissolved in distilled water and pH was adjusted to 7.6. It was
distributed into small test tubes in 2 ml quantities and autoclaved. It is used as a base for
sugar fermentation
test
and also
used to test
for
Indole
production by
Enterobacteriaceae.
4. 2. B. Simmons Citrate Agar
Sodium chloride
:
5.0 gm
Magnesium sulphate
:
2.0 gm
Ammonium dihydrogen phosphate
:
1.0 gm
Potassium dihydrogen phosphate
:
1.0 gm
Sodium citrate
:
5.0 gm
Agar
:
20.0 gm
Bromothymol blue (1/500 aqueous solution):
40 gm
Distilled water
1000 ml
:
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.. Materials & Methods
4. 2. C. Christensen’s Urea Agar
Urea Solution
Sodium chloride
:
5.0 gm
Dextrose
:
1.0 gm
Tripticase
:
1.0 gm
Monopotassium phosphate
:
2.0 gm
Urea
:
20.0 gm
Distilled water
:
100 ml
Phenol red 1% solution (In alcohol)
:
1.2 ml
Agar
:
1.5 gm
Distilled water
:
90 ml
4. 2. D. Urea Agar Base
The above ingredients were dissolved in distilled water and pH was adjusted to 6.8. The
phenol red was added and sterilized by filtration. The stock solution was kept in the
cooler. The agar was dissolved in distilled water and sterilized by autoclaving. After
cooling to 45ºC, 10 ml of urea solution was added. They were dispensed in 3-4 ml
quantities in 12X 100 mm test tubes and allowed to solidify to form a small butt and a
long slant. (To test the ability of an organism to hydrolyze urea).
4. 2. E. Triple Sugar Iron Agar.
Sodium chloride
:
0.5 gm
Yeast extract
:
0.5 gm
Peptone
:
2.0 gm
Agar
:
1.5 gm
Distilled Water
:
100 ml
The above ingredients were dissolved by keeping in boiling water bath and the following
ingredients were added.
Lactose
:
1.0 gm
Sucrose
:
1.0 gm
Dextrose
:
0.1 gm
Sodium thiosulphate
:
0.03 gm
Ferrous sulphate
:
0.02 gm
After adjusting the pH to 7.6, phenol red – 0.024 gm (Add 2.4 ml of 1% solution) was
added and distributed into small test tubes in 4 ml quantities and autoclaved. The tubes
were kept in a slanting position immediately after that, so as to get a deep and a short
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.. Materials & Methods
slant. (This is an important screening medium used for the identification of Gramnegative bacilli by detecting their ability to ferment glucose, sucrose and lactose. The
production of hydrogen sulphide can also be detected).
4. 2. F. Adjustment of pH of media
The pH of each batch of Mueller- Hinton agar was checked when the medium
was prepared. The agar medium with a pH between 7.2 and 7.4 at room temperature after
gelling was used. If the pH is too low, certain drugs will appear to lose potency (eg:
Aminoglycoside, quinolones, macrolides), while other agents may appear to have
excessive activity (eg: tetracycline). If the pH is too high, the opposite effects can be
expected. Just before use, excess surface moisture in the plates was removed by placing
in an incubator(35ºC) or a laminar flow hood at room temperature with lids ajar until
excess surface moisture is lost by evaporation( usually 10 to 30 minutes).
The Kirby Bauer method is used for antimicrobial susceptibility testing, which is
recommended by the NCCLS. The accuracy and reproducibility of this test are
dependent on maintaining a standard set of procedures as described here.
4. 2. G. Media sterilization
Media for culture and biochemical identification of microorganisms are sterilized
in autoclave at a temperature of 121ºC for 15 minutes and plating of the sterilized media
was done in a dust free room. The prepared media were used if the results of quality
control tests were satisfactory. Each batch of medium was used for sterility.
4. 3. ANTIBIOTIC DISKS (THE FOLLOWING DISKS WERE USED)
Amikacin (30μg), ceftazidime (30μg), cefotaxime (30μg), imipenem (10 μg),
meropenem
(10
μg),
gentamicin
(10μg),
cotrimoxazole
(1.25/23.75
μg),
chloramphenicol (30 μg), ciprofloxacin (5μg), piperacillin tazobactum(100/10μg) and
cefoxitin (30μg). nalidixic acid (30μg), nitrofurantoin (300 μg) and norfloxacin (30μg),
ceftazidime 30μg, ceftazidime/ clavulanic acid 30/10μg), Ceftazidiome/ tazobactum
(30µg/10µg), imipenem+EDTA(10µg).
4. 3. A. Preparation of McFarland Standard
A barium chloride 0.5 McFarland density standard solution is prepared by adding 0.5 ml
of a 1.175 % (v/v) barium chloride dehydrate (BaCl2.2H2O) to 99.5 ml of 1% sulfuric
acid. The resulting mixture is placed in a tube identical to that used for preparing the
dilution for the antimicrobial susceptibility tests. The same size tube (screw–capped) and
volume of liquid were used and Stored in the dark at room temperature when not in use.
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.. Materials & Methods
4. 4. COLLECTION OF URINE SPECIMEN:
Midstream urine samples were collected. Every patient was given a sterile wide
mouth container and explained the proper method of collection of urine to avoid
contamination. Male patients were instructed to clean their genital area before voiding.
Female patients were instructed to clean the vulva and perineum with soap and water,
dry the area. They were told to provide 10 ml of urine sample. Collected urine samples
were processed without delay. The specimens were processed according to standard
bacteriological methods and identified by standard conventional methods [3,6,23,45]. .
4. 4. A. Macroscopic Examination of Urine
a) Urine was observed with naked eye for altered colour, turbidity, odour.
b) Urine was tested for pH.
c) Presence of protein, sugar and nitrite was seen.
A drop of uncentrifuged urine was placed on the slide and a cover slip was put
and examined under low and high power microscopy. Several fields were searched to
identify and count number of cells. More than 10 pus cells per high power field were
considered as significant. Similarly presence of bacteria, casts, crystals, RBCs was noted.
Another drop of uncentrifuged urine sample was placed on a clean slide and was allowed
to dry. This smear was Gram stained and examined under oil immersion. Presence of at
least one organism per field was considered significant [1,3,5,6,22,23,34,46,47].
4. 4. B. Plating of Urine Sample by Standard Loop Procedure
The sample was inoculated by a standard loop (with an internal diameter of 4
mm) on well dried plates of blood agar and Mac Conkey agar. Plates were incubated
overnight at 37o C. Next day number of colonies and their morphology were noted and
recorded. The colonies of same type were counted on Blood agar. Presence of more than
100 colonies of similar morphology was considered significant. Only those samples that
produced a single type of colony resembling to that of E.coli were selected. Samples
containing multiple types of organisms were not included in this study [6,12,22,23,59].
4.5. ISOLATION, IDENTIFICATION AND CONFIRMATION OF E.
COLI.
4. 5. A. Indole Test: Peptone water was inoculated with test organism and incubated at
37ºC for 24 to 48 hours. 5 ml of KOVAC‟s reagent was added along the side of the test
tube to form a layer on the top. A positive reaction was indicated by the formation of
pink ring at the junction [1,3,5,6,31,59].
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.. Materials & Methods
4. 5. B. Carbohydrate Fermentation: Pure cultures were inoculated from the agar
plates to sugar media and inoculated at 37ºC for 1-2 days. Positive test was shown by
acid and gas production by change in colour of the media (pink with indicator) and gas
inside the Durham‟s tube [1,3,5,6,31,59].
4. 5. C. Citrate Utilization Test
Test organism was inoculated in Simmons citrate agar and incubated at 37ºC for
2 days. Blue medium with a streak of growth was indicated in citrate utilizing bacteria
(positive reaction) [1,3,5,6,31,59].
4. 5. D. Urease Test -Christensen Urea Agar
The test organism was inoculated heavily over the entire slope surface and
incubated at 37. A positive reaction was indicated by a pink colour of the medium. The
alkaline pH produced changes the colour of the medium to pink or red [1,3,5,6,31,59].
4. 5. E. Triple Sugar Iron Agar Test
TSI agar was stabbed in the center of the butt and streaked on the slope with a
needle charged with a single colony of the test organism. The tube incubated at 37 for
24-48 hours. A yellow butt and red slant showed glucose fermentation, yellow butt and
yellow slant showed glucose, lactose and sucrose fermentation. Gas produced was
trapped inside the medium [1,3,5,6,31,59] .
4. 5. F. Methyl Red Test
Test organism was inoculated on glucose phosphate broth and incubated at 37 for
48 hrs. 5 to 6 drops of methyl red reagent was added to the culture. A red colour
indicated positive reaction. negative tests were yellow in colour. Positive reaction
indicated the ability of the organism to produce and maintain an acid Ph [1,3,5,6,31,59].
4. 5. G. Voges-Proskauer Test
The test organism was inoculated in glucose phosphate broth and incubated at 37
for 48 hours. Then VP reagent (1ml of 40 % potassium hydroxide 3 ml of 5% alpha
napthol in absolute ethanol) was added. The tube was shaken vigorously to ensure
maximum aeration. A positive result was indicated by the development of pink colour in
2-5 minutes becoming crimson in 30 minute [1,3,5,6,31,59].
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.. Materials & Methods
4. 6. MAINTENANCE OF BACTERIAL STRAINS
All the strains of E. coli were maintained in nutrient agar slants/deeps. All strains
were also stored on 15% glycerol-supplemented Luria-Bertani medium at -80ºCfor
further molecular test [5,6,59].
4. 7. SPECIMENS INCLUDED IN THE STUDY
A total 3891 urine samples collected over the period of 30 months from January
2009 to June 2011. The study was carried out in the Department of Microbiology of
Pad.Dr.D.Y.Patil Medical College & Hospital Pimpri, which is a 1370 bedded teaching
and tertiary care hospital in Pune-Maharashtra, Western-India.
Samples recovered from inpatient and outpatients of the hospitals were received
from various specialties like Medicine, Surgery, Obstetrics and Gynecology, Pediatrics,
Orthopedics, Dermatology, Nephrology, and Intensive Care Units. Patient‟s history and
provisional diagnosis of the infection were obtained from hospital records.
The study protocols were approved by the Institutional Ethics Committee of the
Dr. D. Y. Patil Medical College, Pune. Out of 3891 bacterial samples obtained, 337 were
uropathogenic E. coli and hence these were the bacterial samples included in the further
work carried out for this study along with the 50 E. coli from stool samples of healthy
volunteers.
4. 8. ANTIMICROBIAL SUSCEPTIBILITY TESTING
Kirby-Bauer disk diffusion method of antimicrobial susceptibility testing is the
most practical method and is still the method of choice for diagnostic microbiology
laboratories. The Kirby- Bauer method recommended by the CLSI guidelines (2007) was
used for antimicrobial susceptibility testing. Antimicrobial disk susceptibility tests were
performed for 337 study isolates. The accuracy and reproducibility of the test are
dependent on maintaining a standard set of procedures as described here.
4. 8. A. Procedure for Performing the Disc Diffusion Test (Inoculam Preparation)
At least three to five well isolated colonies of the same morphological type were
selected from an agar plate. The top of each colony was touched with a loop, and the
growth was transferred into a tube containing 4-5 mL of a suitable broth medium, such
as nutrient broth. The broth culture was incubated at 35º C until it achieved or exceeded
the turbidity of the 0.5 McFarland‟s standard (usually 2 to 6 hours). The turbidity of the
actively growing broth culture was adjusted with sterile saline or broth to obtain turbidity
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.. Materials & Methods
optically comparable to that of the 0.5 McFarland‟s standard by visually comparing the
inoculam tube and the 0.5 McFarland‟s standard against a card with a white background
and contrasting black lines. That resulted in a suspension containing approximately 1to 2
x 108 CFU /mL for E. coli ATCC 25922 [11,12,14,26,60].
4. 8. B. Inoculation of Test Plates
Optimally, within 15 minutes after adjusting the turbidity of the inoculam
suspension, a sterile cotton swab was dipped into adjusted suspension. The swab was
rotated several times and pressed firmly on the inside wall of the tube above the fluid
level. This will remove excess inoculam from the swab. The dried surface of MuellerHinton agar plate was inoculated by streaking the swab over the entire sterile agar
surface. This procedure was repeated by streaking two more times, rotating the plate
approximately 60º each time to ensure an even distribution of inoculam. As a final step,
the rim of the agar was swabbed. The lid was left apart for 3-5 minutes, but no more than
15 minutes, to allow for any excess surface moisture to be absorbed before applying the
antibiotic disks.
4. 8. C. Application of Discs to Inoculated Agar Plates
Antimicrobial discs were dispensed onto the surface of the inoculated agar plate
and were pressed down to ensure complete contact with the agar surface distributed
evenly so that they were no closer than 24 mm from centre to centre. The plates were
inverted and placed in an incubator set to 35º C within 15 minutes after the discs were
applied.
4. 8. D. Detection of Extended Spectrum Β-Lactamases-Screening Test (CLSI, 2007)
Initially screening test for ESBL production was done as part of routine
susceptibility testing. Two antibiotic discs, ceftazidime (30 μg) and cefotaxime (30 μg)
were used for screening for ESBLs. Plates with Mueller- Hinton Agar (MHA) were
prepared and inoculated with the test organism (turbidity corresponding to 0.5
McFarland‟s tube) to form a lawn culture. The above discs were applied on the surface
of the agar. The plates were incubated at 37 º C overnight and sensitive pattern and
resistant pattern were recorded by reading the zone diameter of the test organism. If a
zone diameter of ≤ 22mm for Ceftazidime and ≤27 mm for cefotaxime was recorded the
strain was considered “Suspicious” for ESBL production.
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.. Materials & Methods
4. 8. E. Double Disk Approximation Test (DDAT)
Mueller- Hinton Agar plates with a depth of 4 mm were prepared. With aseptic
precautions 2 to 3 pure colonies of bacterial isolates were selected and inoculated into
2ml of nutrient broth. This was incubated at 37ºC for 2-3 hours to get moderate turbidity
equivalent to 0.5 McFarland standards. A sterile swab was dipped into standardized
inoculam and the soaked swab was rotated against the upper inside wall of the tube to
express excess fluid. The entire surface of the Mueller – Hinton Agar was swabbed to
form a lawn culture and the inoculam was allowed to dry for 15 minutes with lid in
place. With sterile forceps, cefotaxime disk was placed on the agar plate near the centre.
Giving a centre to centre distance of 15 mm, Ceftazidime/clavulonic acid (30µg/10µg),
disc was placed in line with ceftazidime. This result was found to give best result for
detection of ESBL in our laboratory and was based on twice the radius of the inhibition
zone produced by cefotaxime on its own. The plates were inverted and incubated at 37ºC
in ambient air for 16-18 hours. Each plate was examined for enhancement of zone of
inhibition for cefotaxime disk at the side facing augmenting disk. If the strain was an
ESBL producer, then the zone around cefotaxime disk was extended towards augmenting
disk. ATCC Escherichia coli -25922 was used as negative control and ATCC Klebsiella
pneumoniae -700603 was used as positive control [11,12,26,60].
4. 8. F. Detection of Metallobeta-Lactamases by Imipenem-EDTA-Disk Synergy
Test
The IMP-EDTA combined disk test was performed as described by Yong et al.
Test organisms were inoculated on to plates with Mueller- Hinton agar as recommended
by the CLSI. Two 10 μg imipenem disks (Hi Media) were placed on the plate, and
appropriate amounts of 10 μL of EDTA solution were added to one of them to obtain the
desired concentration (750 μg).
The inhibition zones of the imipenem and imipenem-EDTA disks were compared
after 16 to 18 hours of incubation at 35°C.
In the combined disc test, if the increase in inhibition zone with the Imipenem
and EDTA disc was ≥ 7 mm than the Imipenem disc alone, it was considered as MBL
positive [11,12,,14,17,26,60].
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.. Materials & Methods
Table 4.1 CLSI criteria for screening and Confirmatory Tests for ESBLS in E. coli
Method
Initial Screen Test
Phenotype
Confirmatory Test
Medium
Muller-Hinton Agar
Muller-Hinton Agar
Antimicrobial Disk
Cefodoxime 10µg or
Ceftazidime 30 µg
Concentration
Ceftazidime 30µg or
Ceftazidime/clavulanic acid 30/10
Aztreonam 30µg or
µg and Cefotaxime 30 µg
Cefotaxime 30µg or
(confirmatory testing requires use
Ceftriaxone 30 µg
of
(The use of more than one
Ceftazidime,
antimicrobial
agent
for
screening
both
Cefotaxime
alone
and
and
in
combination with Clavulanic acid)
improves the sensitivity of detection)
Inoculum
Incubation
Standard
disk
diffusion
recommendations
Standard
disk
diffusion
recommendations
conditions, length
Results
Cefodoxime zone ≥ 22 mm
A zone ≥ 5 mm increase in a zone
Ceftazidime zone ≥ 22 mm
diameter for either antimicrobial
Aztreonam zone ≥ 27 mm
agent tested in combination with
Cefotaxime zone ≥ 27 mm
clavulanic acid versus its zone
Ceftriaxone zone ≥ 5.25 mm
when tested alone = ESBL (e.g.,
= Suspicious for ESBL production
ceftazidime
zone
=
16;
ceftazidime/clavulanic acid zone =
21
QC
Recommendations
E. coli ATCC 25922 ( see control
E. coli ATCC ® 25922: ≤ 2 mm
limits)Klebsiella pneumoiniae ATCC
increase in zone diameter for
® 700603:
antimicrobial agent tested alone
Cefodoxime zone 9-16 mm
versus its zone-when
Ceftazidime zone 10-18 mm
combination with clavulanic acid
Aztreonam zone 9-17 mm
Klebsiella
Cefotaxime zone 17-25 mm
700603 : ≥ 5 mm increase in
Ceftriaxone zone 16-24 mm
ceftazidime/ clavulanic acid zone
pneumoiniae
tested in
ATCC
diameter, ≥ 3mm increase in
cefotaxime/clavulanic acid zone
diameter
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.. Materials & Methods
4. 9. SEROTYPING
The strains were serotyped by agglutination assays [] by using 96-well microtiter
plates and with the rabbit serum obtained against 173 somatic antigens (O).
Typing of somatic antigens was performed at the National Salmonella and Escherichia
Centre, Central Research Institute, Kasauli, India, using antisera against O antigens - O1
to O173 [55,56,57].
4.10. PHENOTYPIC IDENTIFICATION OF VIRULENCE PROPERTIES
4. 10. A. Aalpha-hemolysin production:
10 ml alkaline meat extract broth was inoculated with 5 x 107 /ml (2 McFarland)
of E. coli and incubated at 37o C. for 2 ½ hour. Culture in alkaline meat extract broth was
centrifuged (6000 rpm for 30 min.) 0.5 ml of supernatant was collected, and equal
amount of 2% sheep RBCs, washed 3 times in normal saline was added. One drop of
streptomycin (100 g) was added and the mixture was then incubated at 37o C for 2 hr
with intermittent agitation and was seen under high power microscope [90,91,92].
4. 10. B. Detection of P-fimbriae and Type-1 fimbriae:
Materials required:
1)
E. coli grown on Nutrient agar
2)
Phosphate buffered saline (PBS) pH 7.4 (appendix II).
3)
Human blood of A Rh +ve blood group.
4)
Colonization Factor Antigen agar (CFA agar) (appendix II).
5)
Ice pack / refrigerator
6)
VDRL slide
7)
Centrifuge machine
8)
Rotator
Human blood A +ve Group:-5 ml of A +ve venous blood was collected using a
disposable syringe from voluntary donors and added to an equal amount of Alsever
solution (app). This was washed 3 times and 3% erythrocyte suspension was made with
phosphate buffer saline pH 7.4 [93,94,95].
Controls used with each test:
Negative control- ATCC E. coli 25922 for MSHA.
Positive control -
A known strain of E. coli repeatedly giving MRHA positive is
taken as control for MRHA.
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.. Materials & Methods
Method: Single colony of E. coli grown on nutrient agar was inoculated into 5 ml
phosphate buffered saline pH 7.4.This was incubated for 5 days to get pellicle on the
surface. The pellicle contained E. coli enriched with fimbriae. From the pellicle, bacteria
were inoculated on to CFA agar and incubated overnight at 37 oC. 40 l of phosphate
buffered saline pH 7.4 was taken and added in a VDRL cavity slide and labelled as „S‟.
40 l of 3% D. mannose was taken simultaneously in another well and labelled „M‟. 40
l of human A +ve blood was added to both these wells and using a nichrome wire,
colonies from the CFA agar were mixed in both wells.
The slide was then placed on VDRL rotator and rotated for four minutes and
haemagglutination reaction with saline and mannose were recorded [93,94,95,97,98].
4. 10. C. Cell Surface Hydrophobicity (CSH) :
Materials used:
1) E. coli grown on nutrient agar
2)Ammonium sulphate in molar concentrations of 2.0, 1.4, 1 (appendix II).
3) Phosphate buffer saline (PBS) pH 6.8(appendix II).
4) VDRL slide
Method used: Improved salt Aggregation Test [98,99] .
E.coli on nutrient agar plate was inoculated into 1 ml of 0.2 M PBS, pH 6.8
(match to Macfarland 6 or7 for 5 x 109 colonies/ml). 40 l of 1M, 1.4 M, 2 M
ammonium sulphate stained with methylene blue was taken in three different wells of
VDRL tile. 40 l of bacterial suspension in PBS pH 6.8 was added in each of these three
wells. The clumps formed in different concentrations of ammonium sulphate were seen
by naked eyes [98,99].
4. 10. D. Serums Bactericidal Assay:
The assay was done according to Siegfried et al with some modifications.
1) Use of Macconkey agar plates for subculture instead of blood agar plates.
2) Use of Nutrient agar plates for inoculation instead of blood agar.
3) Use of water bath with intermittent shaking instead of roller angle shaker.
Materials used:
1)
E. coli on Nutrient agar
2)
MacConkey agar
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.. Materials & Methods
3)
Hanks balanced salt solution (HBSS).
4)
Nutrient agar
5)
Pooled human serum
6)
Incubator at 37 degree centigrade
7)
Water bath at 37 degree centigrade
Preparation of human pooled serum:
Pooled human serum was obtained from the healthy donor attending blood bank.
Control strains for serum bactericidal assay:
E. coli isolate which was consistently serum resistant and another E. coli isolate which
was consistently serum sensitive were used as controls.
Method [100,101,102]:
E. coli from nutrient agar was subculture on MacConkey agar and incubated at 37o C
overnight.Single colony on MA was inoculated into 10 ml HBSS. Incubated for 2 hrs, at
37ºC. 10 l of this bacterial suspension + 9.90 ml HBSS (this gives bacterial count of
1.64 x 104) 25 l of above suspension + 75 l of pooled serum was taken in a sterile test
tube. The mixture was kept in water bath at 37o C with intermittent agitation. It was then
inoculated on three plates of NA at 0 hr, 1 hr and 2 hr of incubation in water bath. The
plates were incubated at 37o C overnight. Viable count was determined. The plate
inoculated at 0 hr was taken as control.
Interpretation:
Susceptibility of bacteria to serum bactericidal activity =
Colony count after treating with serum
----------------------------------------------------
X 100
Colony count from the control well
E. coli was considered sensitive if count dropped to 1% and was considered resistant if
>90% of organisms survived after 2 hrs of incubation. The above mentioned tests were
performed on the E. coli strains isolated from the patients and controls and the results
were recorded.
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.. Materials & Methods
4. 11. MOLECULAR METHODS
4. 11. A. PCR based fingerprinting (REP PCR) [103,104] :
Forward Primer- REP 2 a 2
: 5‟-ACGGCTTATCGGGCCTAC-3‟
Reverse Primer- REP 1 Ra 1 : 5‟- GCGACGGCATCAGGC-3‟PCR cycle:
95˚c
92˚c
40˚c
65˚c
65˚c
7 min
45 sec
1 min
8 min
16 min
{
30 cycle
}
PCR Reaction Mixture:
10 X Buffer+ (NH4)4SO4
2µl
25mM MgCl2
2.1µl
10mM dNTP
2.5µl
100%DMSO
1µl
100pm Primer 1
0.4µl
100pm Primer 2
0.4µl
5 units/µl Taq
0.4 µl
DNA template
5µl
Milli q
6.2µl
Total
20µl
Master Mix was poled into the PCR tubes and was set for PCR reaction.
The amplified products obtained were checked by running on a 1.5% Agarose gel.
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.. Materials & Methods
4.11.B. Study of virulence profile by PCR based detection method [105,106]
Table 4.2 Primer sequences used for virulence gene detection:
Gene
TM(0C)
Oligonucleotide Sequence
Product
size
pap3
5‟ GCAACAGCAACGCCTGGTTGCATCAT 3‟
59
pap4
5‟ AGAGAGAGCCACTCTTATACGGACA 3‟
58
sfa1a
5‟ CTCCGGAGAACTGGGTGCATCTTAC 3‟
61
sfa2a
5‟ CGGAGGAGTAATTACAAACCTGGCA 3‟
58
afa1a
5‟ GCTGGGCAGCAAACTGATAACTCTC 3‟
59
afa2a
5‟ CATCAAGCTGTTTGTTCGTCCGCCG 3‟
61
aer1
5‟ TACCGGATTGTCATATGCAGACCTT 3‟
56
aer2
5‟ AATATCTTCCTCCAGTCCGGAGAAG 3‟
58
hly1
5‟ AACAAGGATAAGCACTGTTCTGGCT 3‟
56
hly2
5‟ ACCATATAAGCGGTCATTCCCGTCA 3‟
58
cnf1F
5‟ AAGATGGAGTTTCCTATGCAGGAG 3‟
56
cnf1R
5‟ CATTCAGAGCCTGCCCTCATTATT 3‟
58
336bp
410bp
750bp
555bp
1177bp
498bp
4.11.C. PCR Master Mix (Simplex Polymerase Chain Reaction mixture to detect
urovirulent genes)
Components
PCR volume (μL)
Final
reaction
10X Dream Taq Buffer
1
1X
dNTPs (10mM) Fermentas
0.2
200μM
Forward primer (10pM)
0.2
0.2pM
Reverse Primer (10pM)
0.2
0.2pM
Template
0.2
200 ng
Taq Polymerase (5 u/μl)
0.1ul
0.5u
Milli Q
7.75
Total reaction volume
10
88
conc.
in
a
.. Materials & Methods
Cycler Conditions
The PCR was performed with an eppendorf vapo-protect thermal cycler in 96 well PCR
plates (AXYGEN) under the following conditions:
The PCR conditions are as described below:
Initial denaturation
5 min
94°C
Denaturation
45 sec
94°C
Annealing
45 sec
63°C
Extension
45 sec
72°C
Final extension
7 min
72°C
30 cycles
4. 11. D. Agarose gel electrophoresis:
The amplified products were resolved in 1% agarose gel (1XTAE buffer) stained with
EtBr and the bands detected were documented using „SIMI‟ gel documentation unit.
Amplified fragments that were reproducible and consistent in performance were chosen
for data analysis.
4. 12. PCR DETECTION OF O25B-ST-131-CLONE OF ISOLATES
Only isolates with serigroups O25 were tested to determine whether they belonged to the
O25b-ST-131 clone by utilizing a PCR-based assay (allele-specific PCR for pabB gene)
according to the methods described by Clermont et al [38,60 ].
For O25b PCR detection the set of 2- primer pairs were employed as follows;
gndbis.f (5-‟ATACCGACGACGCCGATCTG -3‟)
rfbO25.r (5‟-TGCTATTCATTATGCCGCAGC -3‟) were used.
The reaction mixture (total volume 10ul) contained 1ul of 10X PCR buffer, 0.2 µl of 10
mM dNTP mixture, 0.2 ul of 10 p mol/ul primers, 100 ng of genomic DNA and 0.5 unit
of Taq DNA polymerase. PCR amplification was performed in eppendorf thermo cycler
with the following temperature profile: initial denaturation at 95˚C for 5 mins, followed
by 35 cycles each of denaturation at 94˚C for 45 sec, annealing at 59˚C for 45 sec and
extension at 72˚C for 45 sec, and a final extension at 72 ˚C for 10 min.
89
.. Materials & Methods
4.13 STATISTICAL ANALYSIS
Statistical analysis was performed by using BIOSTATISTIC PRIMER software
(by standard normal variate) z-test and Chi-square tests was done for evaluation of
variables correlation between different antimicrobial resistance phenotype. P value of
<0.05 was considered statistically significant.
For statistical analysis of urovirulence factors, t-test and Chi-square tests was
done for evaluation of variables correlation between different virulence factors in
different clinical manifestations of UTI of clinical UPEC phenotype.P value of <0.05
was considered statistically significant.
90