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In Silico Drug Design of Biofilm Inhibitors of Staphylococcus epidermidis

by Al-mulla, Aymen Faraoun, MS


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B- Culture Media:
Culture media Company (Origin)
Blood agar base Himedia (India)
Brain Heart Infusion Agar Oxoid (England)
Muller-Hinton broth Himedia (India)
Mannitol salt agar Himedia (India)
Nutrient agar Oxoid (England)
Nutrient broth Oxoid (England)
Trypticase soy broth Himedia (India)
C- Chemicals:
Material Company (Origin)
Acetaminophen SDI (Iraq)
Acetic acid (glacial) Riedel-Dehaeny (Germany)
Acetylsalicylic acid Riedel-Dehaeny (Germany)
Congo Red dye Alfa Aesar (America)
Diacetyl (Switzerland)
Ferric ammonium citrate Shreenath chemicals (India)
Glucose Oxoid (England)
Glycerol BDH (England)
Gram stain kit Syrbio (Syria)
Ibuprofen SDI (Iraq)
Sucrose Oxoid (England)
Thymol Riedel-Dehaeny (Germany)

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3.1.1 Preparation of Media
The following media were prepared as recommended by the
manufacturing company. The pH was adjusted to (7.0-7.3) and sterilized
by autoclaving at 121 C for 15 min. under 1.5 Ib pressure.
I. Media used for isolation and identification of bacteria:
A- Nutrient broth:
This medium was used for the activation of bacteria.
B- Nutrient agar:
This medium was used for the isolation and preservation of bacteria for
routine work.
C- Muller-Hinton agar:
Medium used for the detection of novobiocin sensitive staphylococcal
strains.
D- Blood agar:
This medium was prepared by solubilizing 40 g of blood agar base in
1litre of distilled water then autoclaved and cooled to 50 C; human blood
was added and mixed, poured in plates. This medium was used for
bacterial activation.
E- Mannitol salt agar:
A medium used to differentiate between mannitol fermintor and nonfermintor
staphylococci.


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II. Media used for detection of biofilm production:
A- Trypticase soy broth (TSB):
A medium used to activate the growth of bacteria and enhance the slime
production.
B- Congo Red Agar (CRA):
A medium composed of brain heart infusion agar 37 g/l, sucrose 50 g/l,
and Congo red dye 0.8 g/l. Congo red dye was prepared as a concentrated
aqueous solution and autoclaved (121°C for 15 minutes) separately from
the other medium constituents, and was then added when the agar had
cooled to 55°C (Freeman et al., 1989). This medium was used to detect and
identify slime producing Staphylococcus epidermidis.
III. Reagents and Solutions:
A- Phosphate Buffer Saline (PBS)
Buffer solution was used in washing tissue culture plates before staining.
B- Catalase reagent:
Hydrogen peroxide (H2O2) 3% was prepared for the detection of catalase
production (Atlas et al., 1995).
C- Coagulase Plasma reagent:
This test is used to differentiate Staphylococcus aureus (positive) from
Coagulase negative staphylococci.
3.2 Methods
3.2.1 Bacteriological study
I. Isolation of bacteria:
Eighty seven isolates of staphylococci were collected from wound
swaps, ear swaps and from urine samples of patients from different


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hospitals of Baghdad city (Baghdad hospital ,AL-Yarmook hospital, AL-
Karama hospital and Ibn Al-Baladi hospital), during 18-10-2012 to 28-2-
2013.
These isolates were subjected to different routine tests: microscopic and
biochemical tests. They were grown on nutrient agar and blood agar after
24 hours of incubation at 37 C. The colonies of S. epidermidis appeared
white pin point colonies on nutrient agar and milky pin point colonies on
blood agar.
II. Identification of bacteria:
1. Morphological identification:
Morphological features of bacterial colonies and cells (after gram stain)
were studied. These include: shape, color and arrangement.
2. Biochemical tests:
Biochemical tests were done to differentiate S.epidermidis from other
Staphylococci.
A- Coagulase test:
One drop of bacterial broth was added to (0.5 ml) rabbit plasma in test
tube and incubate at 37 C overnight. Coagulation of plasma indicates a
positive test; coagulase negative isolates are unable to form a clot (Rossney
et al., 1990).

B- Catalase test:
One drop of H2O2 (3%) was added to a touch of bacterial culture and was
transferred by a sterile wooden stick on a clean slide. Formation of gaseous
bubbles indicates a positive result (production of catalase) (Benson, 2002).


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C- Novobiocin susceptibility test:
Novobiocin antibiotic discs were used to detect bacterial susceptibility.
A novobiocin disc was placed at the centre of the Mueller-Hinton plate
seeded with bacteria. The plate was then incubated for 24 hours at 37 C.
Inhibition zone diameter was measured. Any measurement equal to or less
than 16 mm indicates resistance to Novobiocin. A zone of 17 mm or larger
indicates susceptibility to novobiocin (Zephania, 2012).
D- Mannitol fermentation test:
The bacterial inoculum was streaked on a mannitol salt agar plate and
incubated overnight at 37 C. When bacterial fermentation occurs, the plate
agar turns yellow. If not ,the bacteria is considered as a negative mannitol
fermentor and the media color remains pink (Atlas et al., 1995).
III. Preservation of cultures:
1- Short period preservation:
The nutrient agar slants and plates (for daily usage) were inoculated with
the bacteria by the streaking method. The slants and plates were incubated
at 37 C for 18 hours. Then, they were sealed with parafilm and stored at 4
C in refrigerator.

2- Preservation for long period:
A- Preservation with glycerol (20%):
Bacteria were inoculated in nutrient broth and incubated for 24 hours at
37 C; then glycerol was added at 20% concentration and stored at 20 C
(Branco et al. , 2010).


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B- Preservation by agar stabbing:
Screw capped vials of agar medium were prepared by dissolving 1.5 %
of nutrient agar in distilled water, sterilized by autoclave, cooled at room
temperature and inoculated with previously activated bacterial culture by
the stabbing method. The vials were wrapped with parafilm and aluminum
foil and preserved in a dark place at refrigerator temperature (Al-Mulla,
2010).
IV. Biofilm detection methods:
All methods were done under microaerobic conditions to enhance
biofilm production.
A- Tube adherence method:
A loopful of test organisms was inoculated in 10 mL of trypticase soy
broth with 1% glucose in test tubes. The tubes were incubated at 37 C for
24 h. After incubation, tubes were decanted and washed with phosphate
buffer saline (pH 7.3) and dried. Tubes were then stained with crystal violet
(0.1%). Excess stain was washed with distilled water. Tubes were dried in
an inverted position. Biofilm formation was considered positive when a
visible film lined the wall and the bottom of the tube. The amount of
biofilm formed was scored as 1-weak/none, 2-moderate and 3-high/strong
(Christensen et al., 1982).

B- Tissue culture plate method:
Overnight culture diluted to 1:100 and 200 μL was introduced in each
well as triplicate. After 24 h. of incubation at 37 C, the contents of each
well were removed by gentle tapping. The wells were washed with
phosphate buffer saline (pH 7.2) four times. This removed free floating
bacteria, then plates were kept to dry. Biofilm formed by bacteria adherent


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to the wells were stained by crystal violet (0.1%). Excess stain was
removed by using distilled water and plates were kept for drying. Optical
density (OD) was measured by using micro ELISA autoreader at
wavelength 570 nm. The experiment was performed in triplicates. The
result was considered weak/non biofilm producer when the (OD) was less
than 0.12, while the result was considered moderate when (OD) values
were between 0.12-0.24. The result was considered strong when (OD) was
more than 0.24 (Christensen et al., 1985).
C- Congo red agar method:
Congo red agar plates were inoculated and incubated for 24 to 48 hours
at 37 C. Positive result was indicated by black colonies with dry crystalline
consistency (Freeman et al., 1989).
V. Bacterial growth curve estimation:
Two biofilm producing isolates were used to estimate the growth curve
of S. epidermidis and were compared with the growth curve of a nonproducer
isolate. The growth curve experiments for each of the three
isolates were duplicated. An overnight 10 ml TSB bacterial culture was
used as initial inoculum for the experiment. From that culture, 0.5 ml was
added to 300 ml TSB (in 500 ml flask) and the OD of that culture was read
representing zero time reading. Simultaneously, the viable count of
bacteria at that time was done by making serial decimal dilutions and using
the drop method (Slack and Wheldon, 1978) to grow bacteria on a nutrient
agar plate. This was done every hour and the plates were incubated
overnight at 37 C. The OD values and the bacterial count [colony forming
unit (CFU)/ ml] with time (every hour) were plotted for a growth curve.


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VI. Measurement of antibiofilm activity:
This measurement was done by a modification to the tissue culture plate
method (Christensen et al., 1985). Five ml TSB broth tubes were
inoculated with 100μL previously incubated bacterial culture plus
different concentrations of the compounds to be tested. Then 200μL of this
mixture was put in tissue culture polystyrene wells, triplicates for each
concentration with negative controls of medium, compound alone and
bacterial culture alone. The plates were incubated overnight at 37 C and
wells were aspirated and washed with phosphate buffer saline four times
and stained with 0.1% crystal violet. After drying, the plates were read with
a micro ELISA autoreader at (570 nm).
3.2.2 Computer aided drug design programs and databases:
Each program or database was used for a different purpose.
A- NCBI Database: http://www.ncbi.nlm.nih.gov/ for the retrieval of
protein sequence and information (Matthiesen, 2010).
B- Uniprot Database: http://www.uniprot.org/ for protein information
(Apweiler et al., 2004).
C- Mega5.1 software: For alignment purposes (Tamura et al., 2011).
D- RaptorX: http://raptorx.uchicago.edu/ for protein modelling
purposes (Källberg et al., 2012).
E- Qmean server: http://swissmodel.expasy.org/qmean/cgi/index.cgi
for protien model quality estimation (Benkert et al., 2009).
F- Swissdock: http://www.swissdock.ch/ for molecular docking purposes
(Hetal et al., 2013).
G- Discovery Studio software v.2.5: For multidrug design and
bioinformatics purposes (Tsai et al., 2009).


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H- ZINC Database: http://zinc.docking.org/ for cheminformatic
purposes (Irwin and Shoichet, 2005).
I- ZINCPharmer: http://zincpharmer.csb.pitt.edu/ for pharmacophore
screening purposes (Koes and Camacho, 2012).
J- T.E.S.T software v.4.1: For toxicity and mutagenicity estimation
(Sushko et al., 2010).
K- Cello: http://cello.life.nctu.edu.tw/ for DNA and protein localization
(Yu et al., 2006).
L-BTXpred: http://www.imtech.res.in/raghava/btxpred/index.html
for the prediction of bacterial toxins (Saha and Raghava, 2007).
M- VaxiJen: http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html
for the Prediction of
Protective Antigens and Subunit Vaccines (Doytchinova and Flower,
2007).
3.3 Statistics
Most of statistics and functions built in the programs have been used.
Other statistical tests were used upon requirements.


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Chapter four

Results and Discussion

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