III. RESEARCH METHODOLOGY
The recent research work was carried out in Microbiology
Research Laboratory (MRL), in the Department of Microbiology and Biotechnology,
Abasyn University Peshawar and in Microbiology and Microbiology
Laboratory of Combined Military Hospital (CMH) Peshawar
from june 2016 to november 2017. In this study we were focused on the Multi
drug resistant genes of pseudomonas aeruginosa.
Following Equipment and apparatus was used in this research
Autoclave, Centrifuge, Deep-freezer, Digital camera, Electrical
balance, Hot plate with magnetic stirrer, EDTA tube, , Incubator with temp 37
C, Laminar air flow hood, light microscope, Centrifuge, Micropipette, Power
supply, Refrigerator, Screw capped test tubes, Shaker Incubator, computer, UV
Light, Vortex, Water bath, wire loop,PCR.
Flasks, petri dishes, beakers, test tubes, glass pipettes,
glass rod, EDTA tubes, Test tubes, eppendrof tubes, droppers, syringes,falcon
3.2 Collection, transport and Processing of
3.2.1 samples collection
A total 200 Clinical
isolates of P.aeruginosa cultured
from urine, blood, bronchial washing, pus swabs, abiotic surface specimens such
as urinary catheters, intravenous catheters and chest tube tips etc. will be
3.2.3 Identification of isolates
At least 10 representative growth
colonies from each culture plate were subculture on appropriate media by streak
plating technique. Purified colonies were characterized and identified using
standard microbiological and biochemical portocols (Cowan1985; Holt et al, 1994).
All isolated bacteria were discriminated using Gram’s reaction.
Various kinds of culture media were used for the growth of
different microbes isolated from different samples. The different culture Media
was used i-e, Nutrient agar, blood agar, CLED agar, MacConkey agar. MacConkey
agar is a selective and differential media so it was used for the growth of
Gram negative bacteria (Uttiya et al., 2016).
Identification of isolated
bacteria will be isolated and form a colony, from colony different bacterial
species will be isolated according to their morphology, Gram’s reaction and
biochemical test. (Graciela et al., 2015)
3.2.4 Gram staining
staining is a technique of staining used to distinguish between bacterial
species into two large groups Gram-positive and Gram-negative. Gram staining
differentiates bacteria by the biochemical and physical properties of their
cell walls by identifying peptidoglycan, which is present in the cell wall of
Gram-positive bacteria. Gram-positive bacteria retain the crystal violet dye,
and thus are stained violet, although the Gram-negative bacteria do not; after
wash, a counter stain, safranine were added and that stain these Gram-negative
bacteria a pink colour. Mutually Gram-positive bacteria and Gram-negative
bacteria preference up the counter stain. The counter stain is hidden on
Gram-positive bacteria for the reason that of the darker crystal violet stain.
slides, Inoculating loop, Bunsen burner, Bibulous paper, Microscope, Lens paper
and lens cleaner, Immersion oil, Distilled water, 18 to 24 hour cultures of
Gram Staining Procedure
Smear was prepared
and heat fixed then the smear was flooded gently with crystal violet andlet on
stand for 1 minute followed by a wash with distilled water using a wash bottle.
The smear was flooded gently with Gram’s iodine and allowedon stand for 1
minute followed by wash with distilled water. The smear was appeared as a
purple circle on the slide. The smear was decolorized using 95% ethyl alcohol
using drop by drop for 5 to 10 seconds until the alcohol runs almost clear. The
smear was then flooded with safranin to counter-stain and let the stand for 45
seconds and again wash with distilled water. The slide was blot dried with
bibulous paper. The smear was viewed with a light-microscope under
oil-immersion at 40 and 100.
3.3 Biochemical tests
For the confirmation of organisms the following biochemical
test were conducted.
oxidase, catalase, coagulase, etc. according to the
Clinical and Laboratory Standards Institute (CLSI) 2015.
3.3.2 Oxidase test
Bacteria, which have aerobic respiration, often have
cytochrome c and a cytochrome c oxidase. The presence of these components can
in combination typing. A commercial test, which contains an artificial electron
acceptor (N, N, N’, N’-tetramethyl-p-phenylenediamine is often used. This
artificial electron acceptor change colour depending upon redox state. The
substance is also referred to as a redox indicator and it can be oxidized by
the oxidized form of cytochrome c. Cytochrome c oxidase is the last enzyme of
the electron transport chain, where it normally reduces oxygen to water and
pump protons to the outside according to the following net reaction:
4 Fe2+-cytochrome cred + 8H+in + O2 ? 4 Fe3+-cytochrome cox
+ 2H2O + 4H+out
Cytochrome c oxidase is a Tran’s membrane protein complex
(Complex IV), which is also present in the cytoplasmic (inner) membrane of
A piece of filter paper was soaked with a little drops of
newly prepared oxidase reagent. A group of the test microorganism was smeared
on filter paper with help of wire loop. When the microorganism is
oxidase-producing, the phenylenediamine in the reagent oxidized to deep purple
· Positive test
result at: Dark blue-purple colour change within 10-30 sec.
· Negative test result at: No colour change or colour change after more
than 30 sec.
Note that the oxidase reagent is not stable after that the
ampoule has been opened. It may be used for a couple of hours, but eventually
it will be oxidized by the oxygen in the air.
The oxidase test is used for identification of gram
negative bacteria. For instance to identify members of the family
Enterobacteriacae, which are oxidase negative, except members of the genus
Plesiomonas (oxidase positive).Members of the family Pseudomonadaceae, and the
genera Aeromonas and Campylobacter are oxidase positive
3.3.4 Coagulase test
Test tube, bacterial culture and plasma
A single colony from the pure culture was suspended
in 0.5 ml of plasma from horse, rabbit or man. The media was incubate at 37 ºC
for 4 hours. The result was analyzed after incubation period. If the result was
found negative after 4 hours then kept the incaution continue till 24 hrs and
result was checked at intervals.
3.3.5 Catalase test
Catalase test is done for the presence of catalase in
bacteria with the use of H2o2. A sample of bacteria was spread onto a
microscope slide. A few drops of h2o2 were added to the bacteria. Presence of
catalase enzyme was observed by bubble appearance for positive results.
· Positive test
result at: Gas formation (O2) in the form av bubbles shows that the
bacterium has a catalase.
· Negative test
result at: No gas formation.
The Catalase test is primarily used for gram positive
bacteria and can for instance be utilized to distinguish Staphylococcus spp.
and Micrococcus spp., which are catalase positive from Streptococcus spp. and
Enterococcus spp., respectively, which are catalase negative.
Some bacteria yield coagulase, which is an enzyme that changes
fibrinogen to fibrin, which means that it can coagulate plasma. The capacity to
yield coagulase is supposed to be linked to the virulence of staphylococci. The
test is used to differentiate between coagulase positive and coagulase negative
single colony from the suspected pure culture in 0.5 ml of plasma from horse,
rabbit or man.
Ø Read the
test afterward 4 h. If the end result is negative (see below), carry on with
the final read after 24 h.
reaction if the plasma coagulates and coagulate is
stable. It must not be dissolved upon stirring.
reaction if the plasma does not coagulate or if the
coagulate is dissolved again upon stirring.
test is widely used to differentiate among Staphylococcus aureus from
coagulase negative Staphylococcus spps. Note on the other hand
that some strains of S. aureus can be coagulase negative, but
it is unusual. Certain strains of S. hyicus and S.
intermedius can be coagulase positive. S. pseudintermedius is
coagulase positive, but not until after 24 hrs
3.4 Antimicrobial Activity
Disc diffusion assay
Antibiotic sensitivity was done through Kirby-Bauer disc
diffusion technique. Standard protocols mentioned in Clinical and Laboratory
Standards Institute (CLSI) 2015 will be followed. Different antibiotics
(Amoxicillin, cyclosporine, Vancomycin, Gentamycin, Tazobactum, Cefotaxime,
Ciprofloxacin, Rifampicin, Doxycycline, Chloramphenicol, Polymaxin-B,
Carbapenam, tetracycline, etc.)of various concentrations
was evaluated. According to the type of isolated bacteria various types of
antibiotics was tested against isolated pathogenic bacteria according to the
Clinical and Laboratory Standards Institute (CLSI) 2015 protocol.
In the disk diffusion test, the bacterial isolate is
inoculated uniformly onto the surface of an agar plate and a filter disk
impregnated with a standard amount of an antibiotic is applied to the surface
of the plate, resulting in a gradient of the antibiotic surrounding the disk.
Following incubation, a bacterial lawn appears on the plate and zones of
inhibition of bacterial growth would be present around the antibiotic disk. The
test is performed under standardized conditions hence the size of the
inhibition zone is dependent on the degree of sensitivity of the microorganism
to the antibiotic (Mayer. 2007).
3.4.1 Maintenance of
All of the identified bacterial strains obtained from microbiology Laboratory, Abasyn
University, Peshawar were sub cultured on the prepared nutrient agar media and
incubated for 24 hrs. Nutrient agar slants were prepared and struck with fresh
culture and then incubated for 24 hrs. The strains were further preserved at 4oC
for further processing.
preparation into broth media
Nutrient broth was prepared into test tubes and autoclaved then a single colony
was taken from 24 hrs old culture with a sterile wire loop and inoculated in a
prepared Nutrient broth and kept in an incubator at 37oC to get the
3.4.3Test bacterial strains
Enteric bacterial pathogenic were
identified during research work in laboratory of Microbiology, Abasyn
University Peshawar. Identified enteric bacterial pathogen were collected from
lab and processed for antibiotics test. The bacterial species included in the
study were Pseudomonas eurogenosa.
3.4.4 Bacterial lawn
preparation onMueller Hinton agar(MHA)
turbidity of 24 hour broth culture was maintained by normal saline to a
0.5McFarland standard, furthermore, uniform lawn was prepared onMueller Hinton
agar (MH), a sterile swab was taken and dipped in broth culture thereafter the
swab was gently pressed with the wall of test tube to squeeze extra broth. The
swab was gently rubbed on the MHA plates to spread the colonies evenly in a
Antimicrobial Susceptibility Testing
susceptibility of isolates was done by Kirby-Bauer disc diffusion technique on
Mueller-Hinton agar, according to Clinical Laboratory Standards Institute
(CLSI) 2015 guidelines. Initially inoculum was prepared by suspending a single
well isolated colony from overnight blood agar or MacConkey agar plates in distilled
water to the final turbidity of a 0.5 McFarland standard. The bacterial
suspension was laid over the agar evenly and antimicrobial disks were dispensed
onto the agar plates and incubated for 18 hours at 37? C.
Different Antimicrobial agents used against
the P.earoginosaspecies and there
sensitivity and resistance are shown in the table 4.1. The diameter of zone of
inhibition was measured for all and interpreted as recommended by CLSI 2015
Storage of Bacterial Strains:
For future study bacterial
strans were stored for research purpose , experimental study , survey,
learning, or commercial use. Appropriate preservation should be done for it
requires the organisms to remain viable, free of contaminants, and without any
modification in genotype or phenotype. idyllically, the organism should be easy
to recover and restore to its original condition. Bacteria is store accordingly
i.e depending on bacterial compatibility, experimental purpose and cell
capability. Storage depends on temperature range as temp lowers storage
increases as a general rule, the viable storage period of bacteria increases as
the storage temperature decreases. Viabank™ is a well-situated, easy-to-use
cryoprotection container for the storage
of concern microbe. The organism to be preserved is added to the
cryopreservative solution in a vial of 20 (approximately) coloured glass beads.
Molecular Identification of Aminoglycosides and Fluoroquinolones
Molecular detection of aminoglycoside-modifying enzymes (AMEs) including: the
the plasmid-mediated quinolone resistance (qnrB) genes was performed by
PCR using specific primers.
Multiplex PCR for detecting aac(6′)-Ib,ant(2″)-Ia and qnrB genes:
A) Extraction of DNA from Bacterial colony.
B) Amplification of DNA in thermal cycler.
C) Agarose Gel electrophoresis and Visualization under
UV lights by transilluminator.
A) Extraction of DNA from Bacterial colony was done
by alkaline lysis method
A single colony of each organism was inoculated from
MacConkey agar into 5ml of Luria-Bertanii broth (LB) and incubated for 20 h at
37º C. Cells from 1.5ml of the overnight culture was harvested by
centrifugation at 12,000 rpm for 5 min. 1.5 ml from LB media containing cells
was taken appendrof tube, than 100 µl TNE buffer was mixed. The mixture was
centrifuged for 1 min at 10000 rpm and supernatant was discarded. Again 100 µl
NaOH (50 mM) was added to pellet. After heating at 400C in water bath for 1
min, 60 µl of IM TrisHCl (PH 6.7) was added. Vortex, centrifuge at 10000 rpm
for1 min was done. Then supernatant was used as template (1µl) (Medici et al.
2003 with some modification).
B) Amplification of DNA in thermal cycler
PCR analysis for beta lactamase genes of the family aac(6′)-Ib,ant(2″)-Ia, and Quinolone resistant gene of the familyqnrB
were carried out. Primers obtained from Thermo Fisher Scientific Company, USA.
Primer used for aac(6′)-Ib, ant(2″)-Ia andqnrB were Target gene Primers Sequences Amplicon size.Preparation of reaction mixtureFor amplification by PCR, 50 µl of master mixture
containing 4 µl of Dntp mixture (2.5mM of each) was mixed with 1 µl of template
DNA, 1 µl of each primer stock solution (50pmol/ µl), 0.5 µl of Taq polymerase
(250 U) , 10X PCR buffer 5 µl (Ex Taq), and remaining 38.5 µl volume was
fulfilled by neuclease free water (Takara Japan).Amplification
The master mixture and ready made PCR tubes were sited
in the eppendrof thermal cycler. According to the following thermal and cycling
condition the amplification process was done:C) Gel electrophoresis and Visualization under UV
lights by transilluminatorAgarose gel electrophoresisAfter electrophoresis in 1.0% agarose gel the PCR
products were evaluated in order to observe the definite products of
amplification and compare it with the standard molecular weight marker.Agarose gel PreparationWith the help of
Microwave oven, 2.0 gm of agarose was melted in 200 ml of diluted Tris
borate EDTA Buffer inorder to prepare 1% agarose gel. The agarosegell which was
melted was allowed to cool down about 50ºC after that 20 µl ethidiumbromide was
mixed with them after appropriate shaking it was poured into the gel tray and
combs were placed in it. the comb was removed after solidification of the gel.
The gel was sited in a Horizontal electrophoresis apparatus which contains
ethidium bromide and TBE buffer all time through electrophoresis.Loading and electrophoresis of the sampleLoading buffer approximately 2.0 µl was mixed with 5.0
µl of amplified PCR product. The mixture was loaded slowly into the well with
the help of disposable micropipette tips. A marker of about 100 bp molecular
weight was loaded in one well in order to analyze the product size of the
amplified PCR .The gel Electrophoresis was carried out for 35 minutes at 100 volts.Gel Visualization
The amplified gens of the samples which were under study
were visualized by trans-illuminator. And the photograph of the gel was taken
by a digital camera and the data was transferred to computer for additional
documentation (Lalet al., 2007;
Sharma et al., 2010;).