RESEARCH METHODOLOGYThe recent research work was carried out in MicrobiologyResearch Laboratory (MRL), in the Department of Microbiology and Biotechnology,Abasyn University Peshawar and in Microbiology and MicrobiologyLaboratory of Combined Military Hospital (CMH) Peshawarfrom june 2016 to november 2017. In this study we were focused on the Multidrug resistant genes of pseudomonas aeruginosa.EquipmentFollowing Equipment and apparatus was used in this researchAutoclave, Centrifuge, Deep-freezer, Digital camera, Electricalbalance, Hot plate with magnetic stirrer, EDTA tube, , Incubator with temp 37C, Laminar air flow hood, light microscope, Centrifuge, Micropipette, Powersupply, Refrigerator, Screw capped test tubes, Shaker Incubator, computer, UVLight, Vortex, Water bath, wire loop,PCR.GlasswareFlasks, petri dishes, beakers, test tubes, glass pipettes,glass rod, EDTA tubes, Test tubes, eppendrof tubes, droppers, syringes,falcontubes.3.2 Collection, transport and Processing ofsamples3.2.1 samples collectionA total 200 Clinicalisolates of P.
aeruginosa culturedfrom urine, blood, bronchial washing, pus swabs, abiotic surface specimens suchas urinary catheters, intravenous catheters and chest tube tips etc. will beincluded. 3.2.3 Identification of isolatesAt least 10 representative growthcolonies from each culture plate were subculture on appropriate media by streakplating technique.
Purified colonies were characterized and identified usingstandard microbiological and biochemical portocols (Cowan1985; Holt et al, 1994).All isolated bacteria were discriminated using Gram’s reaction. Culture mediaVarious kinds of culture media were used for the growth ofdifferent microbes isolated from different samples. The different culture Mediawas used i-e, Nutrient agar, blood agar, CLED agar, MacConkey agar.
MacConkeyagar is a selective and differential media so it was used for the growth ofGram negative bacteria (Uttiya et al., 2016).Identification of isolatedbacteria The pathogenicbacteria will be isolated and form a colony, from colony different bacterialspecies will be isolated according to their morphology, Gram’s reaction andbiochemical test. (Graciela et al., 2015)3.2.4 Gram stainingGramstaining is a technique of staining used to distinguish between bacterialspecies into two large groups Gram-positive and Gram-negative. Gram stainingdifferentiates bacteria by the biochemical and physical properties of theircell walls by identifying peptidoglycan, which is present in the cell wall ofGram-positive bacteria.
Gram-positive bacteria retain the crystal violet dye,and thus are stained violet, although the Gram-negative bacteria do not; afterwash, a counter stain, safranine were added and that stain these Gram-negativebacteria a pink colour. Mutually Gram-positive bacteria and Gram-negativebacteria preference up the counter stain. The counter stain is hidden onGram-positive bacteria for the reason that of the darker crystal violet stain.Requirements Clean glassslides, Inoculating loop, Bunsen burner, Bibulous paper, Microscope, Lens paperand lens cleaner, Immersion oil, Distilled water, 18 to 24 hour cultures oforganismsGram Staining Procedure Smear was preparedand heat fixed then the smear was flooded gently with crystal violet andlet onstand 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 1minute followed by wash with distilled water. The smear was appeared as apurple circle on the slide.
The smear was decolorized using 95% ethyl alcoholusing drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Thesmear was then flooded with safranin to counter-stain and let the stand for 45seconds and again wash with distilled water. The slide was blot dried withbibulous paper.
The smear was viewed with a light-microscope underoil-immersion at 40 and 100.3.3 Biochemical testsFor the confirmation of organisms the following biochemicaltest were conducted.oxidase, catalase, coagulase, etc.
according to theClinical and Laboratory Standards Institute (CLSI) 2015.3.3.
2 Oxidase testBacteria, which have aerobic respiration, often havecytochrome c and a cytochrome c oxidase. The presence of these components canin combination typing. A commercial test, which contains an artificial electronacceptor (N, N, N’, N’-tetramethyl-p-phenylenediamine is often used. Thisartificial electron acceptor change colour depending upon redox state. Thesubstance is also referred to as a redox indicator and it can be oxidized bythe oxidized form of cytochrome c. Cytochrome c oxidase is the last enzyme ofthe electron transport chain, where it normally reduces oxygen to water andpump protons to the outside according to the following net reaction:4 Fe2+-cytochrome cred + 8H+in + O2 ? 4 Fe3+-cytochrome cox+ 2H2O + 4H+outCytochrome c oxidase is a Tran’s membrane protein complex(Complex IV), which is also present in the cytoplasmic (inner) membrane ofmitochondria.MethodA piece of filter paper was soaked with a little drops ofnewly prepared oxidase reagent.
A group of the test microorganism was smearedon filter paper with help of wire loop. When the microorganism isoxidase-producing, the phenylenediamine in the reagent oxidized to deep purplecolor.· Positive testresult at: Dark blue-purple colour change within 10-30 sec.· Negative test result at: No colour change or colour change after morethan 30 sec.
Note that the oxidase reagent is not stable after that theampoule has been opened. It may be used for a couple of hours, but eventuallyit will be oxidized by the oxygen in the air.ApplicationsThe oxidase test is used for identification of gramnegative bacteria.
For instance to identify members of the familyEnterobacteriacae, which are oxidase negative, except members of the genusPlesiomonas (oxidase positive).Members of the family Pseudomonadaceae, and thegenera Aeromonas and Campylobacter are oxidase positive3.3.4 Coagulase testRequirementsTest tube, bacterial culture and plasmaProcedureA single colony from the pure culture was suspendedin 0.5 ml of plasma from horse, rabbit or man. The media was incubate at 37 ºCfor 4 hours. The result was analyzed after incubation period. If the result wasfound negative after 4 hours then kept the incaution continue till 24 hrs andresult was checked at intervals.
3.3.5 Catalase testCatalase test is done for the presence of catalase inbacteria with the use of H2o2. A sample of bacteria was spread onto amicroscope slide. A few drops of h2o2 were added to the bacteria. Presence ofcatalase enzyme was observed by bubble appearance for positive results.· Positive testresult at: Gas formation (O2) in the form av bubbles shows that thebacterium has a catalase.· Negative testresult at: No gas formation.
ApplicationsThe Catalase test is primarily used for gram positivebacteria and can for instance be utilized to distinguish Staphylococcus spp.and Micrococcus spp., which are catalase positive from Streptococcus spp. andEnterococcus spp., respectively, which are catalase negative.1. CoagulasetestSome bacteria yield coagulase, which is an enzyme that changesfibrinogen to fibrin, which means that it can coagulate plasma. The capacity toyield coagulase is supposed to be linked to the virulence of staphylococci.
Thetest is used to differentiate between coagulase positive and coagulase negativestaphylococci.MethodØ Suspendsingle colony from the suspected pure culture in 0.5 ml of plasma from horse,rabbit or man.Ø Incubateat 37ºC.Ø Read thetest afterward 4 h. If the end result is negative (see below), carry on withthe incubation.Ø Completethe final read after 24 h.
· Positivereaction if the plasma coagulates and coagulate isstable. It must not be dissolved upon stirring.· Negativereaction if the plasma does not coagulate or if thecoagulate is dissolved again upon stirring.UseThe coagulasetest is widely used to differentiate among Staphylococcus aureus fromcoagulase negative Staphylococcus spps.
Note on the other handthat some strains of S. aureus can be coagulase negative, butit is unusual. Certain strains of S. hyicus and S.
intermedius can be coagulase positive. S. pseudintermedius iscoagulase positive, but not until after 24 hrs3.4 Antimicrobial ActivityDisc diffusion assayAntibiotic sensitivity was done through Kirby-Bauer discdiffusion technique. Standard protocols mentioned in Clinical and LaboratoryStandards 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 concentrationswas evaluated.
According to the type of isolated bacteria various types ofantibiotics was tested against isolated pathogenic bacteria according to theClinical and Laboratory Standards Institute (CLSI) 2015 protocol.In the disk diffusion test, the bacterial isolate isinoculated uniformly onto the surface of an agar plate and a filter diskimpregnated with a standard amount of an antibiotic is applied to the surfaceof the plate, resulting in a gradient of the antibiotic surrounding the disk.Following incubation, a bacterial lawn appears on the plate and zones ofinhibition of bacterial growth would be present around the antibiotic disk. Thetest is performed under standardized conditions hence the size of theinhibition zone is dependent on the degree of sensitivity of the microorganismto the antibiotic (Mayer. 2007).
3.4.1 Maintenance ofcultureAll of the identified bacterial strains obtained from microbiology Laboratory, AbasynUniversity, Peshawar were sub cultured on the prepared nutrient agar media andincubated for 24 hrs. Nutrient agar slants were prepared and struck with freshculture and then incubated for 24 hrs. The strains were further preserved at 4oCfor further processing.
3.4.2 Inoculumpreparation into broth mediaFreshNutrient broth was prepared into test tubes and autoclaved then a single colonywas taken from 24 hrs old culture with a sterile wire loop and inoculated in aprepared Nutrient broth and kept in an incubator at 37oC to get themaximum growth.3.4.
3Test bacterial strainsEnteric bacterial pathogenic wereidentified during research work in laboratory of Microbiology, AbasynUniversity Peshawar. Identified enteric bacterial pathogen were collected fromlab and processed for antibiotics test. The bacterial species included in thestudy were Pseudomonas eurogenosa.3.4.4 Bacterial lawnpreparation onMueller Hinton agar(MHA)Theturbidity of 24 hour broth culture was maintained by normal saline to a0.5McFarland standard, furthermore, uniform lawn was prepared onMueller Hintonagar (MH), a sterile swab was taken and dipped in broth culture thereafter theswab was gently pressed with the wall of test tube to squeeze extra broth. Theswab was gently rubbed on the MHA plates to spread the colonies evenly in afour quarter.
Antimicrobial Susceptibility TestingAntimicrobialsusceptibility of isolates was done by Kirby-Bauer disc diffusion technique onMueller-Hinton agar, according to Clinical Laboratory Standards Institute(CLSI) 2015 guidelines. Initially inoculum was prepared by suspending a singlewell isolated colony from overnight blood agar or MacConkey agar plates in distilledwater to the final turbidity of a 0.5 McFarland standard. The bacterialsuspension was laid over the agar evenly and antimicrobial disks were dispensedonto the agar plates and incubated for 18 hours at 37? C.Different Antimicrobial agents used againstthe P.earoginosaspecies and theresensitivity and resistance are shown in the table 4.
1. The diameter of zone ofinhibition was measured for all and interpreted as recommended by CLSI 2015guidelines. Storage of Bacterial Strains:For future study bacterialstrans were stored for research purpose , experimental study , survey,learning, or commercial use. Appropriate preservation should be done for itrequires the organisms to remain viable, free of contaminants, and without anymodification in genotype or phenotype.
idyllically, the organism should be easyto recover and restore to its original condition. Bacteria is store accordinglyi.e depending on bacterial compatibility, experimental purpose and cellcapability. Storage depends on temperature range as temp lowers storageincreases as a general rule, the viable storage period of bacteria increases asthe storage temperature decreases. Viabank™ is a well-situated, easy-to-usecryoprotection container for the storageof concern microbe. The organism to be preserved is added to thecryopreservative solution in a vial of 20 (approximately) coloured glass beads. Molecular Identification of Aminoglycosides and FluoroquinolonesGenes: Molecular detection of aminoglycoside-modifying enzymes (AMEs) including: theacetyltransferasesaac(6′)-Ib, thenucleotidyltransferasesant(2″)-Ia andthe plasmid-mediated quinolone resistance (qnrB) genes was performed byPCR using specific primers.Multiplex PCR for detecting aac(6′)-Ib,ant(2″)-Ia and qnrB genes:Steps:A) Extraction of DNA from Bacterial colony.
B) Amplification of DNA in thermal cycler.C) Agarose Gel electrophoresis and Visualization underUV lights by transilluminator.A) Extraction of DNA from Bacterial colony was doneby alkaline lysis methodA single colony of each organism was inoculated fromMacConkey agar into 5ml of Luria-Bertanii broth (LB) and incubated for 20 h at37º C. Cells from 1.5ml of the overnight culture was harvested bycentrifugation at 12,000 rpm for 5 min. 1.5 ml from LB media containing cellswas taken appendrof tube, than 100 µl TNE buffer was mixed.
The mixture wascentrifuged for 1 min at 10000 rpm and supernatant was discarded. Again 100 µlNaOH (50 mM) was added to pellet. After heating at 400C in water bath for 1min, 60 µl of IM TrisHCl (PH 6.
7) was added. Vortex, centrifuge at 10000 rpmfor1 min was done. Then supernatant was used as template (1µl) (Medici et al.2003 with some modification).
B) Amplification of DNA in thermal cyclerPCR analysis for beta lactamase genes of the family aac(6′)-Ib,ant(2″)-Ia, and Quinolone resistant gene of the familyqnrBwere 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 mixturecontaining 4 µl of Dntp mixture (2.
5mM of each) was mixed with 1 µl of templateDNA, 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 wasfulfilled by neuclease free water (Takara Japan).AmplificationThe master mixture and ready made PCR tubes were sitedin the eppendrof thermal cycler. According to the following thermal and cyclingcondition the amplification process was done:C) Gel electrophoresis and Visualization under UVlights by transilluminatorAgarose gel electrophoresisAfter electrophoresis in 1.
0% agarose gel the PCRproducts were evaluated in order to observe the definite products ofamplification 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 Trisborate EDTA Buffer inorder to prepare 1% agarose gel.
The agarosegell which wasmelted was allowed to cool down about 50ºC after that 20 µl ethidiumbromide wasmixed with them after appropriate shaking it was poured into the gel tray andcombs were placed in it. the comb was removed after solidification of the gel.The gel was sited in a Horizontal electrophoresis apparatus which containsethidium bromide and TBE buffer all time through electrophoresis.
Loading and electrophoresis of the sampleLoading buffer approximately 2.0 µl was mixed with 5.0µl of amplified PCR product. The mixture was loaded slowly into the well withthe help of disposable micropipette tips.
A marker of about 100 bp molecularweight was loaded in one well in order to analyze the product size of theamplified PCR .The gel Electrophoresis was carried out for 35 minutes at 100 volts.Gel VisualizationThe amplified gens of the samples which were under studywere visualized by trans-illuminator. And the photograph of the gel was takenby a digital camera and the data was transferred to computer for additionaldocumentation (Lalet al., 2007;Sharma et al., 2010;).