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Production of Bacteriocin from Soil Micro Organisms to Inhibit Different Pathogens By Aditi S. Ambekar MITCON Biopharma, Pune. Under the Guidance of Miss Priya Bhande MITCON Biopharma, Pune. Production of Bacteriocin Page 1 CERTIFICATE This is to certify that Miss. Aditi Satish Ambekar student of Industrial Biotechnology has successfully completed her project work entitled “production of bacteriocin from soil microorganisms to inhibit pathogens” during 11 july 2011 to 16 feb 2012 at MITCON biopharma centre, Pune. Miss Priya Bhande (project guide) Production of Bacteriocin Page 2 ACKNOWLEDGEMENT It gives me immense pleasure to express my deep sincere gratitude to my guide Miss. Priya Bhande for her suggestions, guidance, encouragement and support throughout the period of project. I am grateful to our head of department Mr. Kulkarni sir, for giving me opportunity to join this esteemed institute and extending all laboratory facilities. I am grateful to Miss Angha and Miss Neha for the positive support in my whole project work and the guidance for handling the instruments and in technical work and for all help, understanding me and my problems, supporting me in all possible way throughout my work. I am thankful to our lab assistants Mr. Amit and Mr. Sandeep for the cooperation and providing all necessary things in all possible way. I wish to extend my thanks to my family, especially my parents, my elder brother and my elder sister without whose love and support I could not be here. Last but not least my final thanks to my all lovely room mates my all friends in MITCON, and specially my friend Uma Shinde. They understood mi and tried to help mi in all possible way they could. Thank you very much for being by my side, believing in me and putting up with me in all my good and bad times. Production of Bacteriocin Page 3 Index Introduction ......................................................................................................................... 5 Methods and Principles ....................................................................................................... 8 Observations ..................................................................................................................... 21 Conclusions ....................................................................................................................... 27 Results ............................................................................................................................... 27 Summary ........................................................................................................................... 36 Production of Bacteriocin Page 4 Introduction There are billions to hundreds of billions of soil microorganisms in a mere handful of a typical, garden soil. That single handful might well contain thousands of different species of bacteria (most of whom have yet to be classified), hundreds of different species of fungi and protozoa, dozens of different species of nematodes plus a goodly assortment of various mites and other microarthropods. Almost all of these countless soil organisms are not only beneficial, but essential to the life giving properties of soil. The work of these soil microorganisms is exceedingly complex and extends into nearly every section of this FAQ. The ways the soil bacteria and fungi break down plant and animals residues and wastes are addressed in Section C, "Composting & Use of Compost". The ways the soil bacteria and fungi breakdown and then convert materials into plant nutrients as well as the ways the soil bacteria, fungi, and amoeba hold these nutrients in place and then make them available to the plants are addressed in Section D, "Plant Nutrition". The ways some of the soil microorganisms assist the plants in their physiology are addressed in Section F, "Botany for the Home Gardener". The ways some of the soil bacteria and SOME of the fungi both cause and control plant disease are addressed in Section G, "Plant & Soil Disease; Treatment and Prevention" and the ways some of the nematodes, some of the soil insects, and some of the various micro-arthropods such as mites attack and/or protect plants are addressed in Section H, "Plant & Soil Pests; Prevention and Treatment". This sub-section is limited to the ways the soil microorganisms impact on the physical, chemical, and bio-chemical properties of soil. An extracellular bacteriocidal substance is produced by a serotype c strain of Streptococcus mutans in liquid meduim during the stationary phase of growth. The lethal effect of the substance was demonstrated by the decrease in viable counts of a standardized suspension of group A streptococci in broth. No lysis of affected cells was observed and no changes in appearance of these cells was seen in electron micrographs. The material was effective against certain strains of immmunological groups A, C, D, G, H, L, and O streptococci. It was inactive against strains of S. mutans belonging to the a, b, c, and d serotypes, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The factor was purified 273-fold from the culture fluid by column chromatography. It was sensitive to trysin and Pronase and resistant to catalase. It possessed a molecular weight of more than 20,000 and was not dialyzable. The properties of this substance indicate that it is a Production of Bacteriocin Page 5 bacteriocin. Group A streptococci, which had been treated with antiserum specific for the cell wall group and type antigens, were susceptible to the bacteriocin. Streptococcal strains resistant to the lethal action of the bacteriocin adsorbed the bacteriocin from the solutions, as did the sensitive cells. The bacteriocin was not adsorbed at 0 C. Production of Bacteriocin Page 6 Production of Bacteriocin Page 7 Methods and Principles As the soil was selected as the raw material for the source of micro organisms, the soil with compost was obtained from the Agricultural college, Pune. The tests, materials and methods are mentioned in detail as follows.: :PART A: Isolation of soil organisms Preparation of media: Nutrient agar: composition Peptone- 0.5 % Yeast extract-0.3% Sodium chloride-0.5% Agar powder-2.5% PH-6.8 The soil from the Agricultural College, Pune containing compost was obtained to isolate. The compost soil was taken so as to get maximum organisms. 1 gm soil was added into 9 ml sterile saline. Serial dilutions were made to get well isolated colonies. 1 ml of suspension was spread on media and plates were incubated at 37ºc for 24-48 hours. Production of Bacteriocin Page 8 Next day the plates were observed for the zone of inhibition. The zone of inhibition was observed on the plates from the soil from agricultural college. The zone of inhibition was comparatively small. These colonies were sub cultured on the nutrient plates and slants for pure cultures and for stock cultures respectively. : PART B: Stock cultures of 6 pathogens provided were sub cultured on the nutrient agar. The pathogens were E-coli, Pseudomonas fluorescence, Klebsciella pneumonae, Candida, Salmonella typhi, Shigella decentry. The reaction of the obtained organisms from soil is done to inhibit the growth of these pathogens in the biological way. : PART C: Reaction of organisms on the pathogens. 1. Salmonella typhi: A rod-shaped flagellated, facultative anaerobic, Gram-negative bacterium, and a member of the genus Salmonella. The given salmonella typhi was inoculated in sterile saline. This 1 ml saline was spread on Nutrient agar media and kept for 5 min in laminar air flow. The obtained inhibition forming organisms were streaked on the media, and kept in incubator at 37ºc for 24 hours. Production of Bacteriocin Page 9 2. Shigella dysenteriae: Shigella dysenteriae is a species of the rod-shaped bacterial genus Shigella.[1][page needed] Shigella can cause shigellosis (bacillary dysentery). Shigellae are Gram-negative, non-spore-forming, facultatively anaerobic, non-motile bacteria.[2] This strain also spread alike salmonella, and organism forming inhibition zone was streaked and kept for incubation. 3. Klebsiella pneumoniae: Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines.[ Same procedure as like salmonella and shigella is done and kept for incubation. 4. Pseudomonas Fluroscence: Pseudomonas fluorescens is a common Gram-negative, rod-shaped bacterium.[1] It belongs to the Pseudomonas genus; 16S rRNA analysis has placed P. fluorescens in the P. fluorescens group within the genus,[2] to which it lends its name. The inhibiting organism is streaked on plate on which suspension of pseudomonas is spread,and incubated overnight. Production of Bacteriocin Page 10 5. E-coli: ( Escherichia coli) Escherichia coli (commonly abbreviated E. coli) is a Gram-negative, rodshaped bacterium that is commonly found in the lower intestine of warmblooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls.[2][3] The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2,[4] and by preventing the establishment of pathogenic bacteria within the intestine.[5 The pathogenic strain of E-coli was spread on nutrient agar and the inhibition forming organism was streaked and incubated overnight. 6. Candida: Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans.[3][4] Systemic fungal infections (fungemias) including those by C. albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). C. albicans biofilms may form on the surface of implantable medical devices. In addition, hospital-acquired infections by C. albicans have become a cause of major health concerns. Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans.[3][4] Systemic fungal infections (fungemias) including those by C. albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). C. albicans biofilms may form on the surface of implantable medical devices. In addition, Production of Bacteriocin Page 11 hospital-acquired infections by C. albicans have become a cause of major health concerns. The given strain of Candida was spread on nutrient agar and streaked with the same organism streaked in all above plates. And incubated at 37º c for 24 hours. :PART D : : Identification of the bacteria obtained from soil: For the identification of the bacteria some common tests are done. For the identification of the specific bacteria, physical characters are studied first and then accordingly the chemical tests are done. : Study of physical properties: The study of physical character includes colony characters and gram staining i.e. morphology of the organism. The gram staining of the organism is done as well as the characters of the formed colonies are noted down. : Biochemical tests: -: Principle:According to the physical characters, the biochemical tests are done. When we come to know the gram’s nature, motility and colony structure, we can conclude the biochemical tests referring to the Bergey’s manual. The presence of the particular enzyme in a micro organism can be tested by incorporating a specific substrate in a medium, (if necessary), and then Production of Bacteriocin Page 12 detecting the products formed or even checking the disappearance of the substrate from the medium. These biochemical tests employ various media (having different substrate) which when inoculated with a particular species of bacteria will follow a specific metabolic pathway to hydrolyze the substrate available to them. Some of the routine biochemical tests used for determining metabolic activities of bacteria can be broadly classified as Utilization of carbohydrates and acids Utilization nitrogenous compounds Decomposition of large molecules Miscellaneous tests According to the physical characters, as the bacteria are gram negative, the biochemical tests for the obtained bacteria are mentioned below: Procedures 1. Utilization of carbohydrates and acids : a) Carbohydrate fermentation (sugar utilization) test Principle: Sugars are metabolized through different metabolic pathways (Depending type of species and aerobic and anaerobic environment) to form various acids like pyruvate, lactase, succinate, formate, acetate ect. These acids so formed may further break down to gases (formic hydrogenlyase will split formic acid to H2 and CO2 ) Due to acid formation, the PH of the medium is lowered and phenol red indicator is being faint pink to colorless. Gas formation is demonstrated by the use of Derham’s tube (a small tube inverted in the sugar solution.) which collect gas Requirements: Production of Bacteriocin Page 13 Test cultures, sugar solutions. Composition of sugar solutions Peptone waterPeptone – 1 gm Sodium chloride – 0.5 gm Distilled water – 100 ml Add o.4 ml of 1 % solution of Neutral red indicator Procedure : Sugars used here are Glucose, Sucrose, Maltose, Mannitol, Rhamnose, Lactose. 1 % solution of each sugar was prepared. Distribute the peptone water into the 6 test tubes, 9 ml to each test tube. 1 ml of each sugar solution is added to peptone water containing Derham’s tubes inverted without an air bubble. The tubes are autoclaved at 10 lbs pressure for 10 min. These tubes are inoculated with test culture and kept for incubation at 37º c over night. b) Methyl Red testPrinciple: Only mixed acid fomenters ( e.g. Escherichia coli) produces sufficient quantity of acids during initial phase of incubation (PH below than 4.4) which can be detected by methyl red indicator. This is because the fact the medium glucose phosphate broth is strongly buffered, hence minute quantities of acids if produced, will not permit the PH of Production of Bacteriocin Page 14 the medium to drop down. Moreover, methyl red is a PH indicator having ranges between 6.2 (yellow) to 4.4 (red), so the PH at which Methyl red detects acid is considerably lower than the PH for other indicators used in bacteriological medium. Requirements: Glucose Phosphate Broth(GPB) , Methyl Red Indicator, test tubes. Composition of GPB: Peptone – 10 gm K2HPO4 - 5 gm Glucose – 5 gm d/w – 1 lit. PH – 7.5 Procedure: Inoculate GPB with test culture and incubate at 37ºc for 24-47 hours. After incubation, add about 5 drops of methyl red indicator to the medium c) Indole Production Test: Principle: Indole, a benzyl pyrrole, is one of the metabolic degradation products of the amino acid tryptophan. Organisms that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvate and ammonia. Indole so produced react with the aldehyde group of a weakly acid alcoholic solution of Þ-dimethylaminobenzaldehyde (Kovac’s reagent)in presence of heat to form pink colored rose-indole complex. The reaction can also occur without heat, if the reagent is prepared with HCL. Production of Bacteriocin Page 15 Indole is a substance which reduces surface tension and hence it is concentrated in the surface layer of the medium. Moreover, because indole is soluble in organic compounds. It is recommended that chloroform or xylene be added prior to adding Ehrlich’s reagent. This serves two purposes, firstly it extracts indole from whole of the medium and secondly it forms a separate layer above the medium. As a result, reagent reacts with the indole extracted in the xylene and forms a pink color. Organic solvents like chloroform, ether, and light petroleum can be used instead of xylene. This step is not necessary with kovac’s reagent because the amyl alcohol is used for the diluent is capable of extracting sufficient indole from the aqueous medium to produce a positive reaction. Requirement: 1% Tryptone broth. PH-7.5 Procedure: Inoculate the tryptone broth with a loopful of test culture and incubate 37º c for 24 hours. Add slowly, 1 ml of Kovac’s reagent, on top of the broth and observe for pink ring. d) Vogous Proskauer (VP) testPrinciple: In presence of alkali and air (vigorous shaking) acetoin is oxidized to diacetyl which reacts with guanidine nucleus of arginine present in proteins present in proteins of peptone to produce pink color. At times a pinch of creatine is added to provide an additional source of guanidine nucleus and Production of Bacteriocin Page 16 thus accelerate pink color formation. Test is made sensitive by adding αnaphthol,which serves as catalyst. Requirement: 1.Glucose phosphate broth (similar to the methyl red test) 2.40% KOH solution 3.Test tubes Procedure: Inoculate the loopful of the test culture to the GPB medium, and incubate the tubes at 37°c for 24 hours. After incubation add 0. 5 ml of Borrit’s reagent (40% KOH solution) to the broth. Shake well and slope the tubes to increase the aeration and observe the results. e)Citrate utilization test: Principle: The test determines the ability of the bacteria to use citrate as sole of carbon and energy. This ability depends on the presence of a citrate permease that facilitates transport of citrate into the bacterium. Once inside the cell, citrate is converted to pyruvate and CO2 . Citrate agar slant contain sodium citrate as the sole source of carbon, ammonium phosphate as a sole source of nitrogen, and bromothymol blue as a PH indicator [PH 6 (yellow)-PH 7.6 (blue)]. This test is done on slant since o2 is necessary for citrate utilization. When bacteria oxidise citrate, they remove it from the medium and liberate CO2. This CO2 combines with sodium (Supplied by sodium citrate and water to form sodium carbonate – an alkaline product. Similarly,bacteria that utilize citrate can also extract nitrogen from the ammonium salt, with the production of ammonia, which is convertwd to ammonium hydroxide (NH4OH). These alkaline products raise PH, and turn pH indicator to a blue color and represents a positive citrate test Requirement: Simmon’s citrate agar salnt ,test culture Procedure: Production of Bacteriocin Page 17 Streak test culture on the surface of the slant and incubate at 37°c for 24 hours. Record the color change of the slant after incubation. f)Urea Hydrolysis Test (Urease Test): Principle: A strongly buffered medium in which urea is only nitrogen source is used for the test. Urease is an enzyme possessed by many species of micro organisms that can hydrolyze urea. The ammonia so produced reacts in solution to form ammonium carbonate, resulting in alkalinization and an increase in pH of medium. This is indicated by change in color of the indicator phenol red (pH 6.8-8.4 yellow to purple red) Due to high buffering capacity of the medium, only those organisms possessing vigours urease activity (proteus vulgaris) can given test positive. Requirement: Test culture, Stuart’s urea agar CompositionSolution 1: Peptone- 1gm NaCl- 5 gm Dextrose- 1 gm KH2PO4- 2 gm Phenol red- 6 ml (1:500 solution) Agar-15gm D/w- 900ml Solution 2: Urea- 20 gm d/w- 100 ml Preparation: Add these solution 1 and solution 2 aseptically to make whole volume 1000 ml. Procedure: Prepare the slants of the prepared media and streak the test culture on the media. Production of Bacteriocin Page 18 Incubate the slants at 37°c for 24 hours. Observe the color change in the slant. f)Catalase test Principle: Catalase is an enzyme that splits up hydrogen peroxide into oxygen and water. Chemically catalase is a hemoprotein, similar in structure to hemoglobin. Catalase is present, often in high concentrations in the majority of aerobic organisms but is absent from most obligate anaerobes. Thus when H2O2 is added externally in the medium,catalase activity results in the production of molecular gaseous oxygen. Catalase activity can be tested either by slide test or tube test. Requirement: Test culture, test tubes, hydrogen peroxide. Procedure: Inoculate test sample in 5 ml of H2O2 solution. Observe the effervescences for the oxygen. g)Oxidase test: Principle: Gordon and McLeod (1) introduced oxidase test for identifying Gonococci based upon the ability of certain bacteria to produce indophenol blue from the oxidation of dimethyl-p-phenylenediamine and α-naphthol. Gaby and Hadley (2) introduced amore sensitive method by using N,N-dimethyl-p-phenylenediamine oxalate where all Staphylococci were oxidase negative. In presence of the enzyme cytochrome oxidase (gram-negative bacteria) the N,N-dimethyl-p-phenylenediamine oxalate and α-naphthol react to indophenol blue. Requirement: Production of Bacteriocin Page 19 Test culture, oxidase papers Procedure: Streak the test culture on the given oxidase paper Observe quickly the color change on the paper. Production of Bacteriocin Page 20 Observations : Observations For The Tests Done: : Part A : Observations: The soil spread on the plate of nutrient agar when observed next after incubation, the zone of inhibition was observed around 3-4 colonies. The zone was very small and restricted because of the crowd on the plate. Sr. No. Characters 1. 2. 3. 4. Size Shape Color Opacity 5. 6. 7. 8. 9. 10. Consistancy Margin Elevation Surface Motility Gram’s nature Production of Bacteriocin Observed characters 1-2 mm Circular Faint pink to yellow Transparent, becomes opaque on continuous incubation Translucent Entire Convex Smooth Motile -ve Page 21 : Part B: Observation: The colonies of pathogens when subcultured on the slants of nutrient agar, the well isolated growth was found on the slant. Part C: Observation: The inhibition zone was found on only two plates of pathogens namely Salmonella and Shigella. Other pathogens named Klebsciella, Pseudomonas, E-coli, candida were not inhibited by the direct streaking of the organism. Production of Bacteriocin Page 22 Inhibitionzone found on shigella pathogen Production of Bacteriocin Page 23 Inhibition zone found on salmonella pathogen Part D: : Observation of the biochemical tests done: Before the biochemical tests, the morphological study is done, which include Gram’s staining, motility testing etc. The morphology of the bacteria is when studied,the morphology wasIt was a short, ovoid, Gram –ve bacillus, plump, about 1.5-0.7 µm in size, with rounded ends and convex sides, arranged ,arranged singly, short chains, or in small groups. Production of Bacteriocin Page 24 Gram –ve bacteria having safty pin like appearance : Observations for the biochemical tests on inhibition forming bacteria.: Sr. No. Test name 1. 2. Indole test Methyl red test 3. 4. Vogos proskrous test Citrate test 5. 6. Urease test Oxidase test 7. Catalase test Production of Bacteriocin Observations on organism inhibiting Salmonella typhi No change occured Ring of red color on the surface No color change No change in color of slant Color changed Paper showed no color change No effervesces of O2 Observation on organism inhibiting Shigella dysenteriae Ring of orange color Ring of red colour on the surface No color change No change in color of slant No color change in slant Paper showed voilet color Bubbles are observed Page 25 Observations for the sugar (Utilization of carbohydrate) Sr No. Test name (sugars used) 1. 2. 3. 4. 5. 6. Glucose Sucrose Maltose Mannitol Rhamnose Lactose Production of Bacteriocin Observations on organism inhibiting Salmonella typhi Faint pink color No change Red orange color Orange color No change No change Observation on organism inhibiting Shigella dysenteriae Faint orange Orange color No change Orange color No change No change Page 26 Conclusions According to above observations,we can conclude that the organisms inhibiting the pathogens Salmonella and Shigella are Yersinia pseudotuberculosis and Pasteurella multocida. The biochemical tests done on the organisms obtained, they give tests as observes above,when compared to Bergey’s manual, we have concluded that the bacteria found on plates are pasteurella multocida and yersinia pseudotuberculosis,which are the pathogens. Results According to whole protocols done during the project work,and title of the project, we have worked with the soil organisms which gave the inhibition zone on the pathogens, which when tested we have concluded that the organisms were pathogens i.e. yersinia pseudotuberculosis and pasteurella multocida. : Results for biochemical tests: According to the observation tables, the both organisms fermented glucose, maltose and mannitol but did not fermented lactose, rhamnose and sucrose. All the sugars did not show gas production. P. multocida produced acid in sucrose but not in maltose and vice versa about Y. Pseudotuberculosis. Indole, oxidase, citrate, vogos proskrous, uease and motility tests are found negative in Y. Pseudotuberculosis. And methyl red, catalase tests were found positive. Production of Bacteriocin Page 27 In P. Multocida Indole Oxidase methyl red and catalase are found to be positive whereas citrate, vogos proskrous, urease and motility tests were found negative. Pasteurella multocida Pasteurella multocida is a Gram-negative, non-motile coccobacillus that is penicillin-sensitive and belongs to the Pasteurellaceae family [1]. It can cause avian cholera in birds and a zoonotic infection in humans, which typically is a result of bites or scratches from domestic pets. Many mammals and fowl harbor it as part of their normal respiratory microbiota, displaying asymptomatic colonization. Pasteurella multocida was first found in 1878 in cholera-infected birds. However, it was not isolated until 1880, by Louis Pasteur - the man in whose honor Pasteurella is named.[2]. The bacteria is found in many environments but the associated cholera outbreaks are usually found in central California, the Midwest, and Texas. P. multocida causes disease in wild and domesticated animals as well as humans. The bacterium can be found in fowl, felines, canines, rabbits, cattle and pigs. In birds, P. multocida causes avian cholera; the disease has been shown to follow migration routes, especially of snow geese. The P. multocida serotype-1 is most associated with avian cholera in North America, but the bacterium does not linger in wetlands for extended periods of time. [3]. P. multocida causes atrophic rhinitis in pigs [4]; it also can cause pneumonia or bovine respiratory disease in cattle [5]. In humans, P. multocida is the most common cause of infection from animal injuries (pneumonia in cattle and pigs, atrophic rhinitis in pigs and goats, and wound infections after dog/cat-bites.) The infection usually shows as soft tissue inflammation within 24 hours. A high leukocyte and neutrophil count is typically observed, leading to an inflammatory reaction at the infection site (generally a diffuse localized cellulitis).[6] It can also infect other locales, such as the respiratory tract, and is known to cause regional lymphadenopathy (swelling of the lymph nodes). In more serious cases, a bacteremia can result, causing an osteomyelitis or endocarditis. The bacteria may also cross the blood-brain barrier and cause meningitis.[7] P. multocida mutants are being researched for their ability to cause diseases. “In vitro” experiments show that the bacteria responds to low iron. Vaccination against progressive atrophic rhinitis was developed by using a recombinant derivative of P. multocida toxin. The vaccination was tested on pregnant giltsin (sows without previous litters). The piglets that were born were inoculated, while the piglets born to non-vaccinated mothers developed atrophic rhinitis. [13] Other Production of Bacteriocin Page 28 research is being done on the effects of protein, pH, temperature, NaCl and sucrose on P. multocida development and survival. The research seems to show that the bacteria survive better in waters that are 18 degrees Celsius compared to 2 degrees Celsius. The addition of NaCl by 0.5% also aided the bacterium’s survival, while the sucrose and pH levels had minor effects as well. [14]. Ongoing research has also been done on the response of P. multocida to the host environment. These tests use DNA microarrays and proteomics techniques. P. multocida-directed mutants have been tested for their ability to produce disease. Findings seem to indicate that the bacteria occupy host niches that force them to change their gene expression for energy metabolism, uptake of iron, amino acids and other nutrients. “In vitro” experiments show the responses of the bacteria to low iron and different iron sources, such as transferring and hemoglobin. P. multocida genes that are upregulated in times of infection are usually involved in nutrient uptake and metabolism. This shows that true virulence genes may only be expressed during the early stages of infection. Yersinia pseudotuberculosis: Yersinia pseudotuberculosis is a Gram-negative bacterium that causes Pseudotuberculosis (Yersinia) disease in animals; humans occasionally get infected zoonotically, most often through the food-borne route.[1] It is urease positve. In animals, Y. pseudotuberculosis can cause tuberculosis-like symptoms, including localized tissue necrosis and granulomas in the spleen, liver, and lymph node. In humans, symptoms of Pseudotuberculosis (Yersinia) are similar to those of infection with Yersinia enterocolitica (fever and right-sided abdominal pain), except that the diarrheal component is often absent, which sometimes makes the resulting condition difficult to diagnose. Y. pseudotuberculosis infections can mimic appendicitis, especially in children and younger adults, and, in rare cases, the disease may cause skin complaints (erythema nodosum), joint stiffness and pain (reactive arthritis), or spread of bacteria to the blood (bacteremia). Pseudotuberculosis (Yersinia) usually becomes apparent 5–10 days after exposure and typically lasts 1–3 weeks without treatment. In complex cases or those involving immunocompromisedpatients, antibiotics may be necessary for resolution; ampicillin, aminoglycosides, tetracycline, chloramphenicol, or a cephalosporin may all be effective. The recently described syndrome Izumi-fever has been linked to infection with Y.pseudotuberculosis.[2] Production of Bacteriocin Page 29 The symptoms of fever and abdominal pain mimicking appendicitis (actually from mesenteric lymphadenitis) [3][4][5] associated with Y. pseudotuberculosis infection are not typical of the diarrhea and vomiting from classical food poisoning incidents. Although Y. pseudotuberculosis is usually only able to colonize hosts by peripheral routes and cause serious disease in immunocompromised individuals, if this bacterium gains access to the blood stream, it has an LD50 comparable to Y. pestis at only 10CFU.[6] Thus if these bacteria are given directly to the human then they may cause another infections to the humans,which may be very harmful. But it is well known that the any bacteria do nit or can not kill any other bacteria directly. There must be some proteins or some toxins produced by the microbes which kills the other microbes. This protein or toxin produced by microbe is known as bacteriocin. This bacteriocin when reacted with the pathogens with well diffusion method,the y must give some reaction on it. Production of bacteriocin Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are typically considered to be narrow spectrum antibiotics, though this has been debated.[1] They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. Production of Bacteriocin Page 30 Bacteriocins were first discovered by A. Gratia in 1925[2][3]. He was involved in the process of searching for ways to kill bacteria, which also resulted in the development of antibiotics and the discovery of bacteriophage, all within a span of a few years. He called his first discovery a colicine because it killed E. coli. Bacteriocins are of interest in medicine because they are made by nonpathogenic bacteria that normally colonize the human body. Loss of these harmless bacteria following antibiotic use may allow opportunistic pathogenic bacteria to invade the human body. Bacteriocins have also been suggested as a cancer treatment.[14][15] They have shown distinct promise as a diagnostic agent for some cancers,[16][17][18][19][20] but their status as a form of therapy remains experimental and outside the main thread of cancer research. Partly this is due to questions about their mechanism of action and the presumption that anti-bacterial agents have no obvious connection to killing mammalian tumor cells. Some of these questions have been addressed, at least in part.[21][22] Bacteriocins (which?) were tested as AIDS drugs (around 1990) progressed beyond in-vitro tests on cell lines. [23] but not Members of the genus Bacillus are known to produce a wide arsenal of antimicrobial substances, including peptide and lipopeptide antibiotics, and bacteriocins. Many of the Bacillus bacteriocins belong to the lantibiotics, a category of post-translationally modified peptides widely disseminated among different bacterial clades. Lantibiotics are among the best-characterized antimicrobial peptides at the levels of peptide structure, genetic determinants and biosynthesis mechanisms. Members of the genus Bacillus also produce many other nonmodified bacteriocins, some of which resemble the pediocin-like bacteriocins of the lactic acid bacteria (LAB), while others show completely novel peptide sequences. Bacillus bacteriocins are increasingly becoming more important due to their sometimes broader spectra of inhibition (as compared with most LAB bacteriocins), which may include Gram-negative bacteria, yeasts or fungi, in addition to Gram-positive species, some of which are known to be pathogenic to humans and/or animals. The present review provides a general overview of Bacillus bacteriocins, including primary structure, biochemical and genetic characterization, classification and potential applications in food preservation as natural preservatives and in human and animal health as alternatives to conventional antibiotics. Furthermore, it addresses their environmental applications, such as bioprotection against the pre- and postharvest decay of vegetables, or as plant growth promoters. Production of Bacteriocin Page 31 : Procedure for bacteriocin activity: Requirements: Nutrient broth, test culture, Nutrient agar, borer, centrifuge tubes etc. Procedure: Prepare nutrient broth, inoculate the culture inhibiting Salmonella, and shigella Keep it for incubation at 37º c overnight on incubater shaker, for proper aeration and and fast growth Next day after incubation centrifuge the broth for 7000 rpm for 10 min Now prepare nutrient agar, spread pathogens on the nutrient plates, (pathogens are – salmonella, shigella, pseudomonas, E- coli, candida and Klebsciella.) Keep them for 5 min then make 3 wells with the help of borer,(the borer must be sterile.) Now pour the two supernatents in two wells and keep one as controle. Kellp these plates in fridge for 30 min. Then keep them undistrubing into the incubator at 37ºc for 24 hours Observe the inhibition zone and measure its size and record it. Observation: The zone of inhibition was found on four plates of pathogens except E-coli and Klebsciella. Yersinia pseudotuberculosis inhibited pseudomonas, salmonella, shigella and candida, whereas pasteurella multocida inhibited only shigella. Production of Bacteriocin Page 32 The zone of inhibition found were measured and zones were recorded. Sr. No. Species 1. 2. 3. 4. Pseudomonas Shigella Salmonella Candida Radius of zone of Radius of zone inhibition by Y. of inhibition by pseudotuberculosis p. Multocida 1.2 cm 0.6cm 0.7cm 0.9cm 0.6cm - zone of inhibition on salmonella Production of Bacteriocin Page 33 Zone of inhibition on shigella Zone of inhibition on candida Production of Bacteriocin Page 34 Zone of inhibition on pseudomonas Inhibition zone on four pathogens Production of Bacteriocin Page 35 Summary After bacteria are mechanically removed from solid media, the remaining viable cells can be killed by exposure to chloroform vapors. Until recently, the applicability of this procedure was restricted to glass petri dishes. Here a procedure is described in which plastic petri dishes are used and remain stable in the presence of chloroform vapors. This review focuses on the use and potential of Lactobacillus to prevent infections of the urogenital and intestinal tracts. The presence and dominance of Lactobacillus in the vagina is associated with a reduced risk of bacterial vaginosis and urinary tract infections. The mechanisms appear to involve anti-adhesion factors, by-products such as hydrogen peroxide and bacteriocins lethal to pathogens, and perhaps immune modulation or signaling effects. The instillation of Lactobacillus GR-1 and B-54 or RC-14 strains into the vagina has been shown to reduce the risk of urinary tract infections, and improve the maintenance of a normal flora. Ingestion of these strains into the gut has also been shown to modify the vaginal flora to a more healthy state. In addition, these strains inhibit the growth of intestinal, as well as urogenital pathogens, colonize the gut and protect against infections as shown in mice. Other probiotic strains, such asLactobacillus GG, have been shown to prevent and treat gastroenteritis caused by rotavirus and bacteria. Given that lactobacilli are not the dominant commensals in a gut which comprises around 1010 organisms, much work is still needed to define the mechanisms whereby GR-1, RC-14, GG and other strains contribute to health restoration and maintenance. Such critically important studies will require the medical science community to show a willingness to turn away from pharmaceutical remedies as the only solution to health and disease.. Here, we have used the soil to get desired bacteria and to get baceriocin so as to kill the pathogens we are working with, and observed the activity of bacteriocin by well diffusion method. Well diffusion method: Production of Bacteriocin Page 36 The extracts obtained from the plants or any antibiotic preparations were used for studying their antibacterial activity. A loop full of bacterial strain was inoculated in 30 ml of Nutrient broth in a conical flask and incubated for 72 hrs to get active strain by using agar well diffusion method. Muller Hinton Agar was poured into Petri dishes. After solidification 0.25 ml of test strains were inoculated in the media separately. Care was taken to ensure proper homogenization. The experiment was performed under strict aseptic conditions. After the medium solidified, a well was made in the plates with sterile borer (5mm).The extract compound (50 μl) was introduced into the well and plates were incubated at 37°C for 72 hrs. All samples were tested in triplicates. Microbial growth was determined by measuring the diameter of zone of inhibition14. A control with standard antibiotic was kept for all test strains and the control activity was deducted from the test and results were recorded. Production of Bacteriocin Page 37 References 1.R Ananthnarayn and c k Panikar’s textbook of microbiology sixth edition. 2.Experimental Microbiology by J patel Volume 1 and 2 3.^ Farkas-Himsley H (1980). "Bacteriocins--are they broadspectrum antibiotics?". J. Antimicrob. Chemother. 6 (4): 424– 4. doi:10.1093/jac/6.4.424. PMID 7430010. 5.^ Gratia A (1925). "Sur un remarquable example d'antagonisme 6.^ Gratia JP (October 2000). "André Gratia: a forerunner in microbial and viral genetics". Genetics 156 (2): 471– 6.PMC 1461273. PMID 11014798. 7. ^ Cascales E, Buchanan SK, Duché D, et al. (March 2007). "Colicin Biology". Microbiol. Mol. Biol. Rev. 71 (1): 158–229. doi:10.1128/MMBR.0003606. PMC 1847374. PMID 17347522. 8. ^ Prema P, Bharathy S, Palavesam A, Sivasubramanian M, Immanuel G (2006). "Detection, purification and efficacy of warnerin produced by Staphylococcus warneri". World Journal of Microbiology and Biotechnology 22 (8): 865– 72.doi:10.1007/s11274-005-9116-y. 9. ^ Cotter PD, Hill C, Ross RP (2006). "What's in a name? Class distinction for bacteriocins". Nature Reviews Microbiology 4 (2). doi:10.1038/nrmicro1273-c2. is author reply to comment on article :Cotter PD, Hill C, Ross RP (2005). "Bacteriocins: developing innate immunity for food". Nature Reviews Microbiology 3 (?): 777– 88.doi:10.1038/nrmicro1273. PMID 16205711. 10.^ HENG, C. K. N., WESCOMBE, P. A., BURTON, J. P., JACK, R. W., & TAGG, J. R. (2007). The diversity of bacteriocins in Gram-positive bacteria. In: Bacteriocins: Ecology Production of Bacteriocin Page 38 and Evolution. 1st ed., Riley, M. A. & Chavan, M. A., Eds. Springer, Hildberg, p. 45-83. 11.^ NETZ, D. J. , POHL, R., BECK-SICKINGER, A. G., SELMER, T., PIERIK, A, J. , BASTOS, M. C. F. & SAHL, H. G. (2002).Biochemical characterisation and genetic analysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus aureus. J. Mol. Biol., 319: 745-756. 12.^ NETZ, D. J. A., SAHL, H.-G., MARCOLINO, R., NASCIMENTO, J. S., OLIVEIRA, S. S., SOARES, M. B. & BASTOS, M. C. F. (2001). Molecular characterisation of aureocin A70, a multiple-peptide bacteriocin isolated from Staphylococcus aureus. J. Mol. Biol., 311: 939-949. 13.^ Bastos M.C.F., Coutinho B.G., Coelho M.L.V. Lysostaphin: A Staphylococcal Bacteriolysin with Potential Clinical Applications. Pharmaceuticals. 2010; 3(4):1139-1161. 14.^ de Jong A, van Hijum S A F T, Bijlsma J J E, Kok J, Kuipers O P (2006). "BAGEL: a web-based bacteriocin genome mining tool". Nucleic Acids Research 34 (9): W273– W279. doi:10.1093/nar/gkl237. PMID 1538908. 15. ^ Hammami R, Zouhir A, Ben Hamida J, Fliss I (2007). "BACTIBASE: a new web-accessible database for bacteriocin characterization". BMC Microbiology 7: 89. doi:10.1186/1471-2180-7-89. 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