آنتاگونیسم (antagonism)

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seminar subject: Inhibitory effects of microorganisms on each other The professor : Doctor. Majid Alipur Skandani Provider: zahra nabavi 

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introduction The interaction between microorganisms If the food , bacteria, mold and yeast were present, competition between | crucial The theme is dominant over the other and which are causing food spoilage . 4 types behave differently towards each other microorganisms are : 

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a | introduction >» Symbiotic > Metabiotic > Synergestic > antagonestic 

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| introduction In other cases bacteria may contribute to the growth and form of living symbiotic together in coexistence would be Metabiotic Alternatively , a condition known as metabiotic , it provides the means for a micro-organism , growing conditions for other microorganisms provides both growth initially , but eventually one will prevail over the other. 

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Synergestic Bacteria may have synergistic effects with respect to antagonestic Bacteria may be hostile to each other or have the effect of growth. inhibit the growth of one another . Such as the effects of lactic acid bacteria fermentation agent , due to the metabolites produced , inhibit the growth of Gram-negative bacteria are saprophytes. 

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introduction Microbial antagonism is a common phenomenon for most microorganisms and microbial specify by unlocking Peru (AMP) occurs . The reason for this : aggression is, competition 7” Place Y Foodstuffs Y production and secretion of extracellular D polymerase Y The antimicrobial activity of the host 

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| introduction Identify inhibitor microrganism through : Y The morphology and biochemical tests based on microbial appearance ¥ Gram stain ۷ System bacteriological Done ¥ PCR 

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1) Agar spot assay 5) microtitre plate well 9) Doul culture method 2) Cross streak method 6) Agar well diffusion 10) agar slab method 3) Agar disc diffusion 7) agar plug diffusion 11) Spot on lown assay 4) oxford cup method 8) point inoculation 

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Agar disk diffusion Agar disk-diffusion testing developedin 1940 is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing. 

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Agar disk-diffusion method 1 lAgar plates are inoculated with a Standardized inoculum of the test microorganism. 2 [Then, filter paper discs(about6mmindiameter), containing the test compound at a desired concentration are placed 3 The Petridishes are incubated under suitable conditions Press down disc 11 

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Generally,antimicrobial agent diffuses into the agar and inhibits germination and growth of the test microorganism at the end the Diameters of inhibition growth zones are measured v 02 

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Antifungal activity by disc diffusion method Y Differences in the quality of the paper used in making the disks may affect the diffusion characteristics of the antibiotic. 

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Agar well diffusion ۷ Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts . ¥ is similar to that used in the disk-diffusion method but Well method and disks are very similar , but the effect is more wells 

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1.the agar plate surface is inoculated by spreading a volume of indicator microrganism 2.Then,a hole with a diameter of 6to8mm is punched aseptically with a sterile cork borer or a tip 3.a_volume(20-100 pL) of the inhibitor microrganism solution at desired concentration is introduced into the well 4. Then,agar plates are incubated under suitable conditions depending upon the test microorganism 5.The antimicrobial agent diffusesin the agar medium and inhibits the growth of the microbial strain tested. 

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Inhibition of Escherichia coli by treated supernatant of Bacillus pumilus in a agar well diffusion assay. A-well containing supernatant treated with 1 mg/ml trypsin. B-well containing supernatant treated with NaOH. C-well containing supernatant treated with 0.5 mg/ml catalase. D-well containing untreated culture supernatant (control) 16 

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ببس streak method ac oak & Cross ’ Cross streak method is 2 used to rapidly screen EB 85 8 for microorganisms antagonism 

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2. After = an incubation period depending upon the microbial strain — A | Cross streak qd method / 2 [a 

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A) Plate showing ACTK2 inhibiting the growth of tested bacteria B) ACTK2 inhibited the growth of F, proliferatum by perpendicular streak method Screening of Antibacterial Activity ‏ع‎ ‎cout by Cross Streak Method Against Five Bacterial Pathogenic Strains 

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Three pL of inhibitor microrganism overnight culture was spotted on the surface of agar plates and incubated overnight at 37 °C Next day 200 pL of indicator _. microrganism overnight culture was added into 7 mL of soft agar (0.7%) The mixture was overlaid on the MRS agar plate containing the spots of inhibitor microrganism The zone free of bacterial growth observed around the spots was measured in millimeters 

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Y This method is also used for yeast 3 ۱ Yeast spot assay Spot of salmonella 

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22 Spot-on-lawn Assay St Spot-on-lawn assay to demonstrate inhibition of LB42 by pediocin CP2 

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OWN: plate 120 ml of overnight culture on a agar plate Take 3 ml of liquid soft agar; let cool down until you can touch it Add 120 ml of overnight culture and mix Put on plate and make sure that the soft agar is distributed equally Let dry under the fumehood 

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24 “0ك 

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Result of the agar spot lawn diffusion assay: (A) A drop of C. perfringens type A strain (Cp31) supernatant was stabbed on a lawn of an indicator type A strain (Cp88) and partially through the agar beneath, no inhibition was observed. When type E supernatant was used, CpE218 and CpE132 (B and C respectively) a clear inhil zone was observed 25 

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Comparison v on the Spot agar method , indicator microrganism overlead on spot of inhibiting microorganisms but in agar lown method inhibitor microrganism spot on lown derive from indicator microrganism and soft agar. Yin Agar spot assay both Orleay and spot done but the in spot on lown method just the spot. 

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Agar plug diffusion method —Sss— ‏مسح‎ > making an agar culture of the strain of inhibitor microrganism > During their growth,microbialcells secrete molecules which diffuse in the agar medium. > After incubationan agar- plot or cylinder is cut aseptically with a sterile cork borer and deposited on the agar surface of An other plate previously inoculated by the test microorganism. > The substances diffuse from the plug to the agar medium. > Then,the antimicrobial activity of the microbial secreted moleculesis Detected by the appearance of the inhibition zone around the agar 

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Press dram vial into agar 

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Y In this way a deterrent to inhibitor microorganisms inoculated into the wells , The wells to fill Az saft agar or melting agar. 

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vVxtora cup » 00 il culture supernatant ( (CFS) from the | “Indicator microrganism inhibition microrganism * Plates were incubated (8000 g, 20 min, 4 C) for 48 h at 37 C and cultures (250 ml, 108 added into the Oxford the diameter of the CEU/mL) were plated on | ‏جرخ‎ inhibition zone around each well Suitable culture ‏سا‎ 1 ( measured. 30 

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< ‏نی‎ Microtitre 8۱۵۲۶ ۷۷6۱۱۵۵۵۷ 7 4 ¥ The assays were performed in sterile multiwell microdilution plates Y Conidial suspensions (10 Il) containing 104 conidia per ml were added to 190 ul of the CFS of each inhibitor microrganism Y Conidia of each strain were used as control 

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“> Fungal growth was determined by measuring the optical densit (OD580nm) at 30 C after 48 h in a spectrophotometer 

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Y The antifungal activity of the inhibitor microrganism was expressed as the fungal growth inhibition (%) measured as described above. Y Inall trials, the percentages of inhibition [Inhib% = 100 (OD inhibition 100/ OD Control)] was determined. 

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Dual culture Dual culture in vitro bacterial-fungal assays. a Control R 

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Exploitation of Fungal and Endophytic Bacteria Y Screening of the antagonistic microorganism was carried out by inoculating each isolates in dual culture with inhibitor microrganism in Petri dishes Y The microbial isolate was streaked on the medium, 20 mm from the dish edge 

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Y An 8-mm-diameter mycelial disk of indicator microrganism was placed 30 mm from the opposite edge of the dish Y The radial growth of the mycelium was measured after incubation at room temperature (30° + 2 °C) for 7d ¥ the percent inhibition of radial growth (PIRG) was recorded 

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Y PIRG was defined using the equation indicated below. /\ ey / \ (percent inhibition of radial gr @IRG = R1-R2 x 100 81 R1= average of colony radius of pathogen in control plate R2= average of radial colony of fungus in dual culture plate 

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The reason for using PDA FOR fungi The solid culture medium used for the detection and enumeration of yeast and mold. Y high concentration of sugar and acid pH Y Production Air pigments and fibers are spread by peptone Potato , especially in species of Fusarium, Aspergillus And Penicillium 

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POINT INOCULATION | METHOD Each of the bacterial isolates was point inoculated at sides 3cm from the center of the plate and after that culture of indicator microrganism was point inoculated in the center of the plate. | Control plate was only point inoculated with indicator microrganism 1 The plates incubated at 28+2°C for 4-5 days | Antagonistic activity was investigated for four to seven days after incubation at room temperature (282°C). 

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‘Some points ۹ 1 

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Inhibition diameters are divided into 4 categories : atte > 3 mm diameter b) ++ > 10-13 mm diameter ‏(ع‎ + > 8-0 mm diameter d) - < 8 mm diameter 

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Bacterial growth in agar plate and measurement of lone ameter 

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44 3 gE 

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Statical analysis » For statistical analysis software SPSS » to draw ChartS software EXCEL » The impact of strains Bacterial different on each others Statistical analysis ANOVA > Means comparison Least The statistical difference(LSD) 

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starch agar MC-Congo red DNase test agar lipase medium skim milk agar xylanase agar METABOLITS To screen the bacteria for production of amylase To test the bacteria for cellulase production» agar ‏سس‎ ‎‘To check the bacteria for Dnase production To test for lipase production Production of protease To screen the bacteria for xylanase 5 5 

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Important fungal metabolites that are commercially produced include: » antibiotics > organic acids > enzymes 

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Strains were also tested for antibacterial activity against human pathogenic bacteria such as: > Bacillus subtilis > Escherichia coli > Vibrio cholera > Pseudomonas aeruginosa > Staphylococcous aureus > Salmonella typhi » Candida albicans 

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PDA & PSA .: is the most widely used medium for growing fungi and bacteria. LURIA BROTH :: It to be one of the most common media used for maintaining and cultivating laboratory recombinant strains of Escherichia coli Xylanases : fungi, bacteria, yeast 

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Some cultures Blood agar medium was used because it allowed good growth of microrganisms specially Corynebacterium And staphilococos and because the zones of inhibition were easily seen. The blood also provided a source of catalase. 

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Miiller-Hinton agar : isa MICROBIAL GROTH MEDIUM that is commonly used for ANTIBIOTIC SUSCEPTIBILITY testing. Itis used for almost all organisms such as: Neisseria Moraxella Streptococoos Jactic acid bactria Entrococoos sp 

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This means that almost all organisms plated on here will grow. Additionally, it contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics. Second, it is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a truer zone of inhibition. 

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Specific mediums for lactic acid bacteria: > MRS agar > MRS broth > BHI > MHA Specific medium for Jactococoos sp: M17 agar 

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Bacterioides bile esculin agar (BBE) Laked Kanamycin-vancomycin blood agar (LKV) Anaerobic phenylethyl alcohol agar (PEA) Cycloserine cefoxitin fructose agar (CCFA) Thioglycollate broth 

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One of the most important microorganisms control Aspects of food preservation . Remove organisms Of food spoilage and pathogenic goal for many Research. Microbial pathogens in foods 6 to 33 million / year . Since the control of bacteria in Foods is important 

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refrences 1.Alomar, J., Lebert, A., Montel, M.C., 2008a. Effect of temperature and pH on growth of Staphylococcus aureus in co-culture with Lactococcus garvieae. Curr. Microbiol. 56, 408e412. Alomar, J., Loubiere, ۶, Delbes, C., Nouaille, S., Montel, M.C., 2008b. Effect of Lactococcus garvieae, Lactococcus lactis and 2.Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk. Food Microbiol. 25, 502e508.Ananou, S., Garriga, M., Hugas, M., Maqueda, M., Martinez-Bueno, M., Galvez, A., Valdivia, E., 2005. Control of Listeria monocytogenes in model sausages by enterocin AS-48. Int. |. Food Microbiol. 103, 179e190. 3.Aoki, S.K., Pamma, R., Hernday, A.D., Bickham, J.E., Braaten, B.A., Low, D.A., 2005.Contact- Dependent inhibition of growth in Escherichia coli. Science 309,1245¢1248. Bavananthasivam, J., Dassanayake, R.P., Kugadas, A., Shanthalingam, S., Call, D.R., Knowles, D.P., Srikumaran, S., 2012. Proximity-dependent inhibition of growth of Mannheimia haemolytica by Pasteurella multocida. Appl. Environ. Microbiol. 78, 6683e6688. 4.Benkerroum, N., Daoudi, A., Hamraoui, T., Ghalfi, H., Thiry, C., Duroy, M., Evrart, P,Roblain, D., Thonart, P,, 2005. Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective 5.adjuncts to control Listeria monocytogenes in dry-fermented sausages. J. Appl. Microbiol. 98, 56e63 Charlier, C., Cretenet, M., Even, S., Le Loir, ‏,ل‎ 2009. Interactions between Staphylococcus aureus and lactic acid bacteria: an old story with new perspectives. Int. J. 5.Food Microbiol. 131, 30€39. Delbes-Paus, C., Dorchies, G., Chaabna, Z., Callon, C., Montel, M.C., 2010. Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community. Food Microbiol. 27, 924e932. 

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refrences 6.Diner, E,J., Beck, C.M., Webb, J.S., Low, D.A., Hayes, C.S., 2012. Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI). 7.Genes. Dev. 26, 515e525. Dubey, G.P,, Ben-Yehuda, S., 2011. Intercellular nanotubes mediate bacterial communication. Cell 144, 590e600. Dubois,M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F.,1956. Colorimetricmethod for determination of sugars and related substances. Anal. Chem. 28, 350e356. 8.El-Ziney, M.G., van den Tempel, T., Debevere, J., Jakobsen, M., 1999. Application of reuterin produced by Lactobacillus reuteri 12002 for meat decontamination and preservation. J. Food Prot. 62, 257e261. 9.Fall, P.A., Leroi, F., Chevalier, F., Guerin, C., Pilet, ۷۸-۴, 20108, Protective effect of a non- bacteriocinogenic Lactococcus piscium CNCM I-4031 strain against Listeria 10.monocytogenes in sterilized tropical cooked peeled shrimp. J. Aquat. Food Prod. Technol. 19, 84e92. Fall, P.A., Leroi, F., Cardinal, M., Chevalier, F., Pilet, M.F., 2010b. Inhibition of Brochothrix thermosphacta and sensory improvement of tropical peeled cooked 11.shrimp by Lactococcus piscium CNCM 1-4031. Lett. Appl. Microbiol. 50, 357e361. Fall, P.A., Pilet, M.F., Leduc, F., Cardinal, M., Duflos, G., Guerin, C., Joffraud, JJ., Leroi, F., 2012. Sensory and physicochemical evolution of tropical cooked peeled shrimp 12.inoculated by Brochothrix thermosphacta and Lactococcus piscium CNCM I-4031 during storage at 8 C. Int. J. Food Microbiol. 152, 82e90. Gandhi, M., Chikindas, M.L., 2007. Listeria: a foodborne pathogen that knows how to survive. Int. J. Food Microbiol. 113, 1e15. Ghanbari, M., Jami, M., Domig, KJ., Kneifel, W., 2013. Seafood biopreservation by lactic acid bacteria e a review. Food Sci. Technol. 54, 315e324. Guiral, S., Mitchell, TJ., Martin, B., Claverys, J.-P., 5 ‘Competence-programmed 5 

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Tanks for your attention