Assessment Of Modus Operandi For Phenotypic And
EUROPEAN ACADEMIC RESEARCHVol. IV, Issue 8/ November 2016Impact Factor: 3.4546 (UIF)DRJI Value: 5.9 (B )ISSN 2286-4822www.euacademic.orgAssessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesMUHAMMAD IRFAN AFRIDIDepartment of BiotechnologyMohi ud Din Islamic University, AJ&K, PakistanDepartment of Biotechnology and Genetic EngineeringKohat University of Science and Technology, KP, PakistanKAMRAN IQBAL SHINWARI1Department of Biotechnology and Genetic EngineeringKohat University of Science and Technology, KP, PakistanCollege of Life Sciences, Nanjing Agricultural UniversityNanjing, Jiangsu, ChinaMUHAMMAD YOUNASMUHAMMAD ZEESHAN QURESHIDepartment of BiotechnologyMohi ud Din Islamic University, AJ&K, PakistanABID ULLAH SHAHHAZIQ HUSSAINNASIR AZAMDepartment of Biotechnology and Genetic EngineeringKohat University of Science and Technology, KP, PakistanMARIA ZAIBIRSA ZUBAIRZARA AZAMDepartment of BiotechnologyMohi ud Din Islamic University (MIU), AJ&K, PakistanAbstract:Salmonella sp. is a fastidious gram negative rod shapebacteria. These enteric bacteria are known to be pathogenic and cause1Corresponding author: kamishin@yahoo.com6318
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella Speciesinfections to human and animals. Currently, Salmonella causinginfections are mainly identified at molecular level. Many differentstrategies with recent improvements have been utilized to recognizedifferent species of Salmonella such as colony morphology, biochemicaltests, substrate metabolism, serotyping, phase typing, ELISA andgenomic DNA probing. As in different parts of the world, variousSalmonella causing diseases are rapidly elevating in both humans andanimals; therefore, the need to diagnose or identify the specificSalmonella pathogen is a necessary exploitation for the specific cure orprevention approaches. Hence this succinct review as an enlighteningassessment has been undertaken to be acquainted with the mostreliable methods for the swift detection of contagious Salmonellaspecies all over the world.Key words: Salmonella enteric, salmonellosis, phage typing, plasmidprofiling, ribotyping, multiplex PCRINTRODUCTIONSalmonella are enteric gram negative; rod shaped flagellated,non-spore forming; aerobic and facultative anaerobic organisms(Figure 1). These are classified under the familyEnterobacteriaceae. These are widely spread in nature and canreside in the gastrointestinal tracts of animals and humans(some are animal-host specific) and cause disease state thatranges from self-limited diarrhea to bacteremia with entericfever or invasion of vascular structures, bone or other localizedsites (Hook, 1990). Many bacteria species are reported to bepathogenic (Patchanee et al., 2010) or may be beneficial(Shinwari et al., 2016) but Salmonella is one of the mostnotable pathogens involved in human food-borne illness. Mostof human salmonellosis cases are caused by the consumption ofcontaminated egg, poultry, pork, beef and milk products(Geimba et al., 2004).EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166319
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesSalmonella causes serious health problems in developingcountries and its infective dose can be as low as 15 to 20 cellswhich also depend upon age and health (immunity) of the host.Salmonella infection of humans is a worldwide health problemthrough water consumption and spreads largely as epidemic(FDA, 2003).Salmonella infection is a continuous threat to publichealth and various challenges have been faced to manageSalmonella. The first confront is to stop large spread of foodcontaminations; the second confront is the traceability ofSalmonella in various food supply chains and due to lack of foodmarkers, its origin can’t be traced easily. The third defy is to bein command of the antimicrobial resistance of variousSalmonella strains which have been found to resist drugs inmany countries all over the world. Another imperativechallenge is to augment the Salmonella detection by usingadvanced molecular methods and instantaneous sharing ofinformation about different strains and also establishing greatsurveillance systems for tracking Salmonella in differentvicinities of the world (Khan et al., 2007).Fig 1. Microscopic image of SalmonellaNomenclature of SalmonellaSalmonella classification has altered many times and it is stillunsound. The genus Salmonella was formerly differentiatedinto two species: Salmonella enterica and Salmonella bongori.EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166320
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesHowever, a new species, Salmonella subterranea was identifiedand validated (Shelobolina et al., 2004). Among them, thespecies Salmonella enterica (S. enterica) is further divided intothe six subspecies S. enterica subsp. enterica (I), S. entericasubsp. salamae (II), S. enterica subsp. arizonae (IIIa), S.enterica subsp. diarizonae (IIIb), S. enterica subsp. houtenae(IV) and S. enterica subsp. indica (VI). Fermentation of selectedsubstances such as sorbitol, dulcitol, lactose, malonate,galacturonate and d-tartrate as well as assemblage of enzymessuch as β-glutamyl-transferase or β-glucuronidase, gelatinaseand also lysis by phage O1 allows differentiations between thedifferent species and subspecies (Le Minor, 1984). At this time,Salmonella genus is composed of over 2500 serotypesdifferentiated according to three different types of surfaceantigens, 99% of these serotypes belong to S. enterica andnearly 60% of them are in S. enterica subsp. enterica. Theaverage DNA sequence similarity between Salmonellaserotypes is 96-99% (Edwards et al., 2002).Salmonella Detection StrategiesDetection strategy for Salmonella involves conventionallaboratory techniques, biochemical tests, serological diagnosis,ELISA and DNA based molecular diagnosis. These methods areappraised in detail subsequently.Laboratory TechniquesConventional lab isolation of Salmonella is done by usingcultural methods. These cultural procedures usually have fourdistinct phases i.e. (i) pre-enrichment in a non-selectivemedium (peptone buffer media), for multiplication of the targetorganism and others present in the sample; (ii) selectiveenrichment, to allow the survival or growth of the Salmonella,while inhibiting other accompanying organisms in the selectivebroth; (iii) isolation, using selective agar media (e.g. Mckonkey’sEUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166321
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesAgar) that restrict growth of bacteria other than Salmonella,(iv) confirmation, where isolates are subjected to a variety ofbiochemical and serological tests to confirm that these areSalmonella and to determine their serovar. In broad-spectrum,each individual step needs at least 16 h up to a maximum of 48h. Thus, the whole procedure takes 4–7 days to complete and istherefore laborious and also requires expert staff (Waltman andMallinson, 1995).Biochemical TestsMany biochemical tests can be used to detect Salmonellabecause these microbes are able to ferment many substances inmedia like mannitol, lactose, glucose, lysine, sorbitol, arginine,arabinose etc, their biochemical reactions are specific and give acharacteristic color change for easy detection of differentSalmonella serovars. In one of biochemical test for S. typhi, it isunable to ferment trehalose and so this inability is used mainlyfor its detection. Nowadays, special biochemical kits areavailable for rapid detection of many Salmonella spp. in asingle step. Biochemical kits like API 20E is available whichcan detect reactions with 20 different substances in singleincubation (Fig. 2). After incubation, the color reactions areread and analyzed with the kit manufacturer’s software and the% probabilities of Salmonella are then confirmed (Salem et al.,2007).Fig 2. Typical Salmonella reaction of API 20E test kitEUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166322
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesSerotypingSerotyping is the first step for routine diagnostics of Salmonellastrains and performed with commercially available omni, polyand monovalent antisera. Up to date, more than 2500 serotypesof Salmonella has been identified and classified in theKaufmann-White scheme (Theral and Frost, 1990). This schemedifferentiates between O (somatic) antigens of the cell surface,H1 and H2 (flagellar) antigens and the virulence capsular (Vi)antigens which may be present in some serotypes like such asS. typhi and S. paratyphi. Each Salmonella serogroup has agroup specific O-antigen and within each O-group, differentserovars are distinguished by the combination of O & Hantigens that are present and by presence of these antigenseach serotype has been given a specific antigenic (naming)formula. Serotyping is easy to perform and standardizedantisera are commercially available but it only allows thegrouping of Salmonella strains to a specific serotype and nomore differentiation between strains of the same serotype isobtained (Threfall and Frost, 1990). Serological analysis holdsthe first step in an epidemiological investigation of Salmonellaand is good enough for epidemiological investigations butsmaller labs do not have access to the pools of anitsera requiredfor this analysis and may need to rely on other techniques toanalyze isolates (Theral and Frost, 1990).Phage typingPhage typing is the technique in which lysis of differentSalmonella serotypes is done with bacteriophages. Individualisolates of many serotypes vary in their susceptibility to lysisand yield a specific lytic pattern. Therefore, a Salmonella strainis subjected to a specified set of typing phages and the lyticpattern obtained commonly allows the assignment to a specificphage type. Phage typing is mostly performed for serotypessuch as S. typhi, S. typhimurium, S. enteritidis or S. paratyphi.EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166323
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesPhage typing has led to the identification of over 200 S.typhimurium phage types (Threlfall & Frost, 1990). Phagetyping has proved to be an important tool for straincharacterization, surveillance, source attribution and outbreakinvestigations (Hald et al., 2007). Phage typing is not a commonlab practice; it is only performed at research centers withtrained individuals.ELISAImmunological tests like ELISA have been advancedspecifically for Salmonella detection. Nowadays it involvesautomation and miniaturization technology along withaccessibility of commercially equipped high eminence reagents.The application of ELISA has led to the detection of Salmonellainfection in humans, poultry, pigs and cattle. ELISA based onlipopolysaccharide (LPS) and virulence (Vi) antigens have beenused to detect S. typhimurium and S. typhi infection in rodentsand humans respectively (Nicholas and Cullen, 1991). Bothindirect and sandwhich ELISA methods are used to checkagglutation reaction of IgG, IgM and IgA antibodies with abovementioned antigens. The ELISA is an easy and rapid approachbut it suffer from limited sensitivity and a considerable chanceto produce both false-positive and false-negative results(Nicholas and Cullen, 1991). Widal test a form an old ELISAmethod still is used widely for diagnosis of S. typhi andconfirmation of typhoid fever. The Widal kit includes binding ofdead cells onto a microtiter plate and is dyed red, when blood ofpatient is added to microtiter plate, an immunologic responseoccur due to binding of antibodies which result in color changeand confirmation of typhoid fever (King, 2000).DNA Detection MethodsPhenotypic typing methods requiring large time, personnel andreagents have led to the development of typing methods basedEUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166324
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella Specieson genotypic information. Numerous genotyping methods havebeen applied to the typing of Salmonella. Two of the mainadvantages of these methods are that they do not depend on theexpression of phenotypic properties and that all strains ofSalmonella can be typed by the same method with only smallchanges for use with a particular serovar (Olsen, 2000). FewDNA diagnostic methods have been used largely but no singlemethod can be recommended for all typing purposes forSalmonella. The choice of method depends on the specific caseand sometimes more than one method is applied to improve thequality of the typing. Some important molecular methods areexplained further.Plasmid ProfilingPlasmid profile analysis was one of the first DNA-based typingschemes. It is particularly important, since most of theplasmids have virulence and antimicrobial resistance propertiesin Salmonella. Plasmid content of the host within the sameserotype shows the differentiation according to the profile i.e.the number and molecular sizes of plasmids obtained. Thedifferent plasmid profiles within a serotype indicate the lateraltransfer by gaining or losing the plasmid(s). The plasmidsfound in Salmonella differ in size 2–200 kb with differentfunctions (Rychlik et al., 2006). The detection method is basedon the isolation of plasmids followed by agarose gelelectrophoresis and to view the plasmid pattern, agarose gelmust be stained with ethidium bromide solution and thenvisualized under UV light. Plasmid profiling is most useful inan outbreak which is limited temporally and geographically(Mendoza & Landeras, 1999). Plasmid analysis has somelimitations i.e. plasmids can rapidly be acquired or lost byconjugation or horizontal plasmid transfer to other serotypes inSalmonella.EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166325
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesPFGE (Pulsed Field Gel Electrophoresis)PFGE has been considered as the “gold standard” among othermolecular typing methods. By cutting the bacterial DNA withrare-cutting restriction endonucleases and running with specialelectrophoresis separation technique which use pulsed currentsthat change polarity at defined intervals, it separates the largefragments of DNA up to 12000 kb and yields strain specificpatterns. The most commonly used restriction enzymes inSalmonella are XbaI, SpeI and NotI. Comparisons of patternsfrom multiple enzymes can determine new subtypes andincrease the distinguishing power of this technique (Liebisch &Schwarz, 1996). PFGE of 60 S. enteritidis isolates revealed 28different XbaI restriction profiles and 26 with SpeI but whenthe patterns generated from both enzymes were combined, 32different pulsed-field types could be identified (Ridley et al.,1998). PFGE has also been used to track outbreak strainswithin and across boundaries of other countries. Despite thatPFGE is method of first choice but this method is relativelyslow, often takes three days to complete, and requires thepresence of expensive specialized equipment, high qualitychemicals and a good experience in the preparation of the DNAcontaining agarose gels. Moreover, single genetic events, suchas point mutations, integration, deletion or recombinationevents can result in differences in the fragment patterns(Herschleb et al., 2007).RibotypingRibotyping is the fingerprinting of rRNA coding sequenceswhich describes the hybridization of restriction-digested DNAfragments with probes specific for rDNA. Multiple copies of therRNA operon are present within the Salmonella chromosome(Mendoza & Landeras, 1999). Although rRNA genes themselvesare homologous among many serotypes but differences occur inthe intervening sequences length and nucleotide composition.EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166326
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella SpeciesRibotyping is important for making differentiations of serotypes(like S. enteritidis) in case of recurrent epidemics and outbreaksat particular areas (Ridley et al., 1998). Ribotyping methodbegins with separating endonucleases digested chromosomalDNA on agarose gels. DNA is then transferred to a nylonmembrane and fragments are hybridized to a probe thatrecognizes 16S and 23S rRNA. Analysis of multiple restrictionendonucleases can improve the discriminatory powers ofribotyping (Mendoza & Landeras, 1999). Automated ribotypinghas also been developed and the banding pattern can becompared to data stored in computer databases. Ribotyping isbetter than PFGE because it takes less time and drawback isthat its reagents are very costly and also the riboprinterequipment is very expensive (Fontana, 2004).IS (Insertion Sequence) TypingIS200 is a mobile (transposable) element found in a variety ofeubacterial genera such as Salmonella, Escherichia andShigella. IS200 elements are very small (707-711 bp) andcontain a single gene present in chromosome with multiplecopies. IS200 transposes rarely and is very stable therefore; thisstability makes IS200 a suitable molecular marker forepidemiological and ecological studies especially when thenumber of IS200 copies is high. IS200 typing has been used toevaluate the molecular relationships between Salmonellaisolates e.g. in S. enterica, IS200 fingerprinting is extensivelyused for strain identification. The method involveshybridization of digested chromosomal DNA with an IS200probe. IS200 typing is sometimes considered as superior toPFGE and ribotyping (Jeoffreys et al., 2001).PCR Methods & Multiplex PCRCommonly used PCR based detection methods involve RAPDtechnology whichuses short synthetic 10 bases longEUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166327
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella Speciesprimers which can amplify Salmonella genomic DNA by PCRand then the banding pattern of amplified product is observedon gels. AFLP is also used for Salmonella diagnosis whichinvolves use of restriction enzymes to form fragments thenligation of adapters to genomic restriction fragments followedby PCR amplification with adapter specific primers. Theoptimal number of scorable bands (50–100) can easily be set byselection of the suitable AFLP primers and restriction enzymes(like EcoR1 & MseI). These characteristics make AFLP apowerful fingerprinting technique which can be used inidentification, epidemiology and taxonomy (Folkerstma et al.,1996).Multiplex polymerase chain reaction (mPCR) is avariant of PCR in which two or more loci are simultaneouslyamplified in the same reaction. During the last dercade, a lot ofresearch has shown the practicality of identifying Salmonellaserovars using mPCR (Kim et al., 2006). In addition, thetechnique has been shown to be a powerful and cost-effectivetool for Salmonella detection. This method is based on detectionof genes present in specific Salmonella serotypes and thesegenes are selected from analysis of whole-genome sequencing.Usually set of 3-5 primers are designed for virulence genes ofinfectious agents like S. typhi, paratyphi, enteritidis andprimers for surface antigen genes are also designed to detectintra serovar variation. The specific virulence genes gryA, gryBand cry1 of S. typhi, paratyphi and enteritidis respectively arecommonly used in primer designs and their presence in mPCRresult help in easy detection of Salmonella from otherpathogenic microbes. In order to carry out the mPCR, it has tobe optimized i.e. primers should not dimerize with self andother primers and the melting temperature should not varyotherwise unwanted amplification might occur which will affectthe mPCR results. Due to the rapidness, cost effectiveness, highEUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166328
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella Speciesspecificity and sensitivity, it has become the most used methodnowadays (Kim et al., 2006).CONCLUSIONSalmonella illustrate large phenotypic diversity and severalphenotypic typing techniques like serotyping and phage typinghave been largely developed and used for many years but theyhave some drawbacks. An ideal typing method should fulfill ddiscriminatory power, ease of interpretation, easy to use andlow cost. Any method used currently for typing of Salmonellastrains is an ideal method alone in terms of these criteria, butall methods exhibit benefits and also limitations. So far PCRbased diagnosis are the best available methods and the mostpreferred for detection of Salmonella sp.REFERENCES1. Edwards, R. A., G. J. Olsen and S. R. Maloy. 2002.Comparative genomics of closely related salmonella.Trends in Microbiology, 10:94-99.2. FDA, U.S. Food and Drug Administration. 2003. Foodborne pathogen microrganisms and natural toxinshandbook – Salmonella spp.3. Geimba, M,. E. Tondo, F. Oliveira, C. Canal. 2004.Serological characterization and prevalence of spvRgenes in Salmonella isolated from foods involved inoutbreaks in Brazil. Journal of Food Protection, 67:12291233.4. Folkertsma, R. T., J. N. A. M. Rouppe van der Voort, K.E. de Groot et al. 1996.Gene pool similarities ofEUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166329
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella Speciespotato nematode populations assessed by AFLPanalysis. Molecular Plant–Microbe Interactions, 9:47–54.5. Fontana, J., A. Stout, M. Tyndall, B. Bolstorff, S.Rossiter and R. Timperi. 2002.The use of pulsedfield gel electrophoresis and automated ribotyping tomonitor the increased prevalence of a multidrugresistant SalmonellaserotypenewportinMassachusetts associated with cows. InternationalConference on Emerging Infectious Diseases, Atlanta.6. Hald, T., D. M. Lo Fo Wong and F. M. Aarestrup. 2007.The Attribution of HumanInfectionwithAntimicrobial Resistant Salmonella Bacteria inDenmark to Sources of Anima Origin. FoodbornePathogenic Disease, 4:313- 326.7. Herschleb, J., G. Ananiev, and D. C. Schwartz. 2007.Pulsed-field gel electrophoresis. Nature Protocols, 2:677684.8. Hook, E. W. 1990. Salmonella species (including typhoidfever). In G. L.Mandell,Douglas, R. G. & J. E.Bennett (ed.), Principles and Practices of InfectiousDiseases. Churchill Livingstone, New York, 1700-1715.9. Jeoffreys, N. J., G. S. James, R. Chiew and G. L. Gilbert.2001. Practical evaluation of molecular subtyping andphage typing in outbreaks of infectionduetoSalmonella enterica serotype typhimurium. Pathology,33:66-72.10. Khan, A. A., C. D. Melvin, and E. B. Dagdag. 2007.Identification and molecular characterization ofSalmonella spp. from unpasteurized orange juices andidentification of new serotype Salmonella strain S.enterica serovar Tempe. Food Microbiology, 24: 539-543.11. King, A. L. 2000. Widal agglutination test - 100 yearslater: still plagued by controversy. Postgraduate MedicalJournal. 76:80-84.EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166330
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella Species12. Kim, S., J. G. Frye, J. Hu, P. J. Fedorka-Cray, R.Gautom and D. S. Boyle. 2007. Multiplex PCR basedmethod for identification of common clinical serotypes ofSalmonella enterica subsp. enterica. Journal of ClinicalMicrobiology, 44:3608–3615.13. Le Minor. L. 1984. Facultative anaerobic gram-negativerods. In: Holt J. G. and Krieg N. R. (eds.), Bergey'sManual of Systematic Bacteriology. Williams andWilkins, Baltimore, 9th edition, 1:427-458.14. Liebisch, B. and S. Schwarz. 1996. Molecular typing ofSalmonella enterica subsp enterica serovar enteritidisisolates. Journal of Medical Microbiology, 44:52-59.15. Mendoza, M. C. and E. Landeras. 1999. Molecularepidemiological methods for differentiationofSalmonella enterica serovar Enteritidis strains inhumans and animals: epidemiology, pathogenesis, andcontrol. Saeed, A. M., Gast, R.E., Potter, M.E. and Wall,P.G., Ed., Iowa University Press, Ames, IA, 125-140.16. Nicholas, R. A. J. and G. A. Cullen. 1991. Developmentand application of an ELISA for detecting antibodies toSalmonella enteritidis in chicken flocks. VeterinaryRecord, 128:74-76.17. Olsen, J. E. 2000. Molecular Typing of Salmonella.Veterinary International Journal, 34:345-367.18. Patchanee, P., B. Molla, White, D. E. Line and W. A.Gebreyes. 2010. Tracking Salmonella contamination invarious watersheds and phenotypic and genotypicdiversity. Foodborne Pathogenic Diseaeses, 7:1113-1120.19. Ridley, A. M., E. J. Threlfall. and B. Rowe. 1998.Genotypic characterization of Salmonella enteritidisphage types by plasmid analysis, ribotyping, and robioliolgy, 36:2314-21.EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166331
Muhammad Irfan Afridi, Kamran Iqbal Shinwari, Muhammad Younas, MuhammadZeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, IrsaZubair, Zara Azam- Assessment of Modus Operandi for Phenotypic andGenotypic Recognition of Salmonella Species20. Rychlik, I., D. Gregorova. and H. Hradecka. 2006.Distribution and function of plasmids in Salmonellaenterica. Veterinary Microbiology, 112:1-10.21. Shinwari, K. I., U. S. Abid, M. I. Afridi, M. Zeeshan,H. Haziq, H. Javed, A.Owais and M. Jamil. 2015.Application of plant growth promotingrhizobacteriain bioremediation of heavy metal polluted soil. AsianJournalof Multidisciplinary Studies, 3: 179-185.22. Shelobolina, E. S., S. A. Sullivan, K. R. O'Neill, K. ion, and U(VI)-reducing potential of afacultatively anaerobic, acidresistant bacterium fromlow-pH, nitrate and U(VI)-contaminated subsurfacesediment and description of Salmonella subterranea sp.nov. Applied and Environmental Microbiology, 70:29592965.23. Threlfall, E. J. and J. A. Frost. 1990. The identification,typing andfingerprinting of Salmonella: laboratoryaspects and epidemiological applications. Journal ofApplied Bacteriololgy, 68, 5-16.24. Waltman, W. D. and E. T. Mallinson. 1995. Isolation ofSalmonella from poultry tissue and environmentalsamples: a nationwide survey. Avian Diseases, 39, 45-54.EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 20166332
Zeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, Irsa Zubair, Zara Azam-Assessment of Modus Operandi for Phenotypic and Genotypic Recognition of Salmonella Species EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 2016 6319 infec
(Fraud Report) Forensic Audit Phases Pemaparan Ekstern Modus Operandi Pemaparan Intern Analysis Technical of Evidence Find Read & Interpret . Pelaporan (temuan final) Nasib Padmomihardjo - Presentation 2007Presentation 2008 Pelatihan Fraud Auditing 2 27 Juni 2014
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