Download Production of bacteriocine from soil micro organisms to inhibit

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.
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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)
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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.
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Index
Introduction ......................................................................................................................... 5
Methods and Principles ....................................................................................................... 8
Observations ..................................................................................................................... 21
Conclusions ....................................................................................................................... 27
Results ............................................................................................................................... 27
Summary ........................................................................................................................... 36
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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
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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.
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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.
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 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.
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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.
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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,
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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
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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:
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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
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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.
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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
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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:
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 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.
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 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:
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Test culture, oxidase papers
Procedure:
 Streak the test culture on the given oxidase paper
 Observe quickly the color change on the paper.
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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
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Observed characters
1-2 mm
Circular
Faint pink to yellow
Transparent, becomes
opaque on continuous
incubation
Translucent
Entire
Convex
Smooth
Motile
-ve
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: 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.
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Inhibitionzone found on shigella pathogen
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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.
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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
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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.
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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
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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
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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
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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
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: 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
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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
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Zone of inhibition on shigella
Zone of inhibition on candida
Production of Bacteriocin
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Zone of inhibition on pseudomonas
Inhibition zone on four pathogens
Production of Bacteriocin
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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
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References
1.R Ananthnarayn and c k Panikar’s textbook of microbiology
sixth edition.
2.Experimental Microbiology by J patel Volume 1 and 2
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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
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Production of Bacteriocin
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11.^ NETZ, D. J. , POHL, R., BECK-SICKINGER, A. G.,
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