group b report- bacillus cereus 20july
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DECLARATION
I hereby declare that the work which has been presented in the dissertation entitled
Isolation & characterization of B. cereus isolated from soil samples,
identification of emetic toxin producing gene in B. cereus at molecular level
and to check the antimicrobial activity of medicinal plants against B
.cereus.
Submitted for the partial fulfilment of the B.E. Biotech is an authentic record my
work carried out under the supervision of -----------------------.
The matter embodied in this dissertation submitted by me has not been submitted
for a degree of my any other academic in any other university or examination
body in India & abroad.
Place: Agra SHWETA DASS
Date:
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ACKNOWLEDGEMENT
Commencing with the name of almighty, the most beneficent, most merciful who
do we worship and thine aid we seek.
I am highly grateful and feel my proud privilege to take this opportunity to express
my deepest and heartful sense of gratitude to, Miss. Deepti Tiwari, Director, ITS
& RC, Agra for his keen interest, affectionate behavior, continued forbearance,
valuable guidance, constructive criticisms and suggestions without his stimulating
guidance tremendous encouragement it would have not been possible to carry out
the present work.
I am also grateful toMrs. Rashmi Sharma (H.O.D.), Mrs. Anuradha Chauhan,
Miss. Shilpi Gupta, Mr. Arvindra Kumar Jadaun, for their valuable
suggestions different aspects of the present research work.
I take this opportunity to express my hearty grateful to Dr. Sanjeev Kumar
Sharma, Director, I.E.T. Khandari Campus, Agra for providing requisite
facilities for the study.
It seems quite formal to thank my fatherShri Raghuvar Dayal and mother Smt.
Renu Devi, what is mine is there and what I will be in the near future is certainly
will because of them.
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I heartily feel deep regards to my dear madam Miss. Garima Sharma, who gave
me inspiration, affection and always prays for my better future.
I am also immensely thankful to my elder brother Hardeep Singh & Harendra
Kumar Singh, and sister Pinki kumari for their affection, bondless co-operation and
inspiration.
I am fortunate to have friends like real gem; I am very much grateful to Pooja &
Anjali for their valuable help during the ups and downs of the life.
Many of my colleagues helped me both morally and academically at various stages
during the period of my study. For this I wish to record my gratitude and heartiest
thanks toKalpana, Gaurav, Manisha, And other colleagues but the number is
too great to name them all the number is too great to name them all.
At last but not least, I express my deep sense of gratitude to my friends Ved,
Anu, Archarna, Amita, for their affection & encouragement during the course of
my study.
(Shweta Dass)
B.E. biotechnology
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TABLE OF CONTENTS
S. NO. TITLE PAGE NO.
1 ABBRIVATION 5
2 AIM OF STUDY 6
3 INTRODUCTION 7-10
4 REVIEW OF LITERATURE 11-40
5 METHOD & MATERIALS 40-63
6 RESULTS 63-71
7 DISCUSSION & CONCLUSION 71-74
8 REFFERENCE 74-82
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ABBRIVATION
B . cereus Bacillus Cereus
gm gram
Mg milli gram
l micro litre
ml milli litre
rpm revolution per minute
d/w distilled water
UV LIGHT ultra violet light
C degree centrigrate
EDTA Ethylene diamine tetra
acetic acid
TAE BUFFER tris acetic acid EDTA
bufferTE BUFFER tris EDTA buffer
DNA Dioxy ribonucleic acid
Tm melting temperature
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AIM OF STUDY
On considering the role ofB-cereus in several diseases we selected the study
Isolation & characterization of B. cereus isolated from soil samples ,identification of the emetic toxin producing gene in B. cereus at molecular level
and to check the antimicrobial activity of medicinal plants against B .cereus
with following objectives.
Isolation ofB.cereus from different soil sample.
Characterization of isolatedB.cereus at Biochemical level.
Identification of Emetic toxin producing strains ofB.cereus at Molecular level.
To check out the antibacterial activity of several plants against
isolatedB.cereus.
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INTRODUCTION
Bacillus cereus is a normal inhabitant of the soil, but it can be regularly isolated
from foods such as milk, milk products , grains and spices. B. cereus causes two
types of food-borne intoxications (as opposed to infections). One type is
characterized by nausea and vomiting and abdominal cramps and has an incubation
period of 1 to 6 hours. It resembles Staphylococcus aureus food poisoning in its
symptoms and incubation period. This is the "short-incubation" or emetic form of
the disease. The second type is manifested primarily by abdominal cramps and
diarrhea with an incubation period of 8 to 16 hours. Diarrhea may be a small
volume or profuse and watery. This type is referred to as the "long-incubation" or
diarrheal form of the disease and it resembles food poisoning caused by
Clostridium perfringens. In either type, the illness usually lasts less than 24 hours
after onset.
The short-incubation form is caused by a preformed, heat-stable emetic toxin,
ETE. The mechanism and site of action of this toxin are unknown, although the
small molecule forms ion channels and holes in membranes. The long-incubation
form of illness is mediated by the heat-labile diarrheagenic enterotoxin Nhe
and/or hemolytic enterotoxin HBL, which cause intestinal fluid secretion,
probably by several mechanisms, including pore formation and activation ofadenylate cyclase enzymes.
Bacillus cereus is a Gram-positive, spore-forming microorganism capable of
causing foodborne disease at present three enterotoxins, able to cause the diarrheal
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syndrome, have been described: hemolysin BL (HBL), nonhemolytic enterotoxin
(NHE) and cytotoxin K. HBL and NHE are three-component proteins, whereas
cytotoxin K is a single protein toxin. Symptoms caused by the latter toxin are more
severe and may even involve necrosis. In general, the onset of symptoms is within
6 to 24 h after consumption of the incriminated food.
In microbiology, the term bacillus means any rod-shaped microbe (and coccus
means a spherical microbe). However, Bacillus (written with a capital letter and
italicized) refers to a specific genus of bacteria. The family Bacillaceae are all
Gram-positive, rod-shaped bacteria which form endospores, with two main
divisions:
the anaerobic spore-forming bacteria of the genus Clostridium
the aerobic or facultatively anaerobic spore-forming bacteria of the genus
Bacillus
Characteristically, Bacillus cultures are Gram-positive when young, but may
become Gram-negative as they age.Bacillus species are aerobic, sporulating, rod-
shaped bacteria which are ubiquitous in nature. Gram-stained cells, 1 m wide, 5-
10 m long, arranged singly or in short chains. The organism produces heat
resistant spores and these may germinate if cooling is too slow [1]
Bacillus endospores are resistant to hostile physical and chemical conditions, but in
addition various Bacillus species have a wide range of physiologic adaptations
which enable them to survive or thrive in harsh environments, ranging from desert
sands and hot springs to Arctic soils and from fresh waters to marine sediments.
Because the spores of many Bacillus species are resistant to heat, radiation,
disinfectants, and desiccation, they are difficult to eliminate from medical and
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pharmaceutical materials and are a frequent cause of contamination. Bacillus
species are well known in the food industry as spoilage organisms. At the start of
this video, spores can be seen as the bright, refractile objects seen underphase
contrast microscopy. The second part of the video show green spores differentiated
from pink vegetative cells by a spore staining procedure:
Fig 1: Bacillus Cereus
Only a few genera of bacteria such as Bacillus and Clostridium are capable of
forming endospores. These are dormant form of the bacterium that allows it to
survive sub-optimal environmental conditions. Spores have a tough outer coveringmade of keratin and are highly resistant to heat and chemicals. The keratin also
resists staining, so specialized procedures are necessary to stain endospores.
Diarrheal poisoning is caused by heat-labile enterotoxins produced during
vegetative growth ofB. cereus in the small intestine whereas the emetic type of
food poisoning is caused by the small, heat- and acid-stable cyclic
dodecadepsipeptide cereulide [2][3]. While enterotoxins are comparatively well
characterized at the molecular and the expression level [4], far less is known about
the emesis causing toxin. The chemical structure and characteristics of cereulide
have been studied in some detail but the molecular basis for its synthesis remains
unknown. Cereulide causes cellular damaging effects in animal models [5] is toxic
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to mitochondria by acting as a potassium ionophore [6] and it was involved in
fulminant liver failure in a human case [7]. Recently, it has been reported that
cereulide inhibits human natural killer cells and might therefore have an
immunomodulating effect [8].
In general, the incidence ofB. cereus food poisoning is underestimated since B.
cereus is not a reportable disease and reporting procedures vary between countries.
There is a tendency for many more B. cereus food poisoning cases to be reported
in northern countries. In NorwayB. cereus was the most common microbe isolated
from food-borne illnesses in 1990 [9] and it was responsible for 14% of the
outbreaks in Finland in which the causative agent was identified [10].B. cereus is
a major problem in convenience food and mass catering. Due to heat and acid
resistance of its spores it is not eliminated by pasteurization or sanitation
procedures. Investigation of food-borne outbreaks in the German Federal Armed
Forces showed that B. cereus was by far the most frequently isolated pathogen in
the retained food samples. It was responsible for 42% of the outbreaks reported
between 1985 and 2000.
Since B. cereus is a ubiquitous spore former that cannot be totally avoided, it is
necessary to develop rapid methods to discriminate hazardous strains from non-
toxic strains. The utility of polymerase chain reaction (PCR) based methods is
evident by the 1999 guidelines issued by NCCLS [11] encouraging the use of
molecular methods in clinical laboratories performing bacterial identification
assays. Such an assay would also be advantageous for quality control in the food
industry and could improve food safety substantially. While for enterotoxic B.
cereus strains molecular diagnostic PCRass ays have been described [12] [13] [14]
and commercial immunological assays are available, for emetic strains such tools
are still missing. The presented PCR system may fill that gap by providing a
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molecular assay to rapidly detect emetic toxin producingB. cereus strains.
REVIEW OF LITERATURE
Bacillus cereus is one of around 60 representatives of the widely varied Bacillus
genus. Along with the very similar species B. mycoides, B. thuringiensis and B.
anthracis, it comprises the so called Bacillus cereus group. The differences
between these four species are very small. B. cereus is found frequently as a
saprophyte in soil, water, vegetation and air, from where it is easily transferred to
food, either from the original raw material or during the food processing. It iscommon in dried foodstuffs, spices, cereals, meat, eggs, milk and milk products,
cooked and inappropriately kept food. [15][16][17]
Bacillus cereus is a causative agent of gastrointestinal and non-gastrointestinal
diseases.Bacillus cereus causes two distinct food poisoning syndromes:
Rapid-onset emetic syndrome characterized by nausea and vomiting.
Nausea and vomiting begins one to five hours after contaminated food is
eaten. Boiled rice that is held for prolonged periods at ambient temperature
and then quick-fried before serving is a frequent cause, although dairy
products or other foods may also be responsible.
Slow-onset diarrhoeal syndrome. Diarrhoea and abdominal pain occurs 8
to 16 hours after consumption of contaminated food. This is associated with
a variety of foods, including meat and vegetable dishes, sauces, pastas,
desserts, and dairy products.
Besides its food poisoning potential, B. cereus has been shown to be responsible
for wound and eye infections, as well as systemic infections [18]. Recently, it has
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been reported that systemic complications ofB. cereus infections in premature
neonates might be at least partly related to enterotoxins [19]. However, in general
the role of the diverse toxins and virulence factors of B. cereus in systemic
infections is poorly studied. The development of molecular tools will be necessary
to allow a rapid characterization of virulence mechanisms of clinical B. cereus
isolates.
SCIENTIFIC CLASSIFICATION
Bergeys Manual contains six sections that describe all Gram positive bacteria
except the actinomycetes. Most of these bacteria are distributed among the first
sections on the basis of their general shape (weather they rods or bacilli, cocci or
irregular) and their ability to form endoscope.
In Bergey's Manual of Systematic Bacteriology (1st ed. 1986), the G+C content of
known species ofBacillus ranges from 32 to 69%. This observation, as well as
DNA hybridization tests, revealed the genetic heterogeneity of the genus.
In Bergey's Manual of Systematic Bacteriology (2nd ed. 2004), phylogenetic
classification schemes landed the two most prominent types of endospore-forming
bacteria, clostridia and bacilli, in two different Classes of Firmicutes, Clostridia
and Bacilli. Clostridia includes the OrderClostridiales and Family Clostridiaceae
with 11 genera including, Clostridium. Bacilli include the OrderBacillales and the
Family Bacillaceae. In this family there are 37 new genera on the level with
Bacillus.
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TAXONOMIC CLASSIFICATION
Kingdom :- Bacteria
Phylum :- Firmicutes
Class :- Bacilli
Order :- Bacillales
Family :- Bacillaceae
Genus :- Bacillus
Species :- cereus
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HISTORY
In 1887, Bacillus cereus isolated from air in a cowshed by Frankland and
Frankland. In 1906, B. cereus was first associated with food poisoning in Europe.
Outbreaks of food poisoning caused by aerobic, sporeforming bacilli termed
anthracoid or pseudoanthrax were reported.
In 1950, Steinar Hauge in Norway provided the first complete account ofB.
cereuspoisoning, and proved that this microorganism is a human pathogen.
From 19471949, Hauge investigated four outbreaks of food poisoning with
a total of 600 persons affected. The food vehicle in all four outbreaks was
vanilla sauce prepared from corn starch, rich in B. cereus spores. Hauge
found that the corn starch used in this case had ~104 spores ofB. cereusper
gram. The dessert was prepared and stored at room temperature until it was
served and eaten the next day. All individuals who ate the dessert had
clinical symptoms of food poisoning. To provide evidence that B. cereus
was the cause of food poisoning, Hauge demonstrated Kochs postulates by
consuming a culture of the isolatedB. cereus strain. He grewB. cereus to a
level of 4106 cells per ml, and drank 200 ml of bacterial suspension. After
13 hrs, the symptoms of food poisoning started.
Since 1950, many outbreaks from a variety of foods including meat and
vegetable soups, cooked meat and poultry, fish, milk and ice cream were
described in Europe. In 1954, experiments with volunteers in USA failed to confirm Hauges
observations.
In 1969, the first well-characterized B. cereus outbreak in the USA was
documented.
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Since 1971, a number ofB. cereuspoisonings of a different type, called the
vomiting type, were reported. This type of poisoning was characterized by
an acute attack of nausea and vomiting 15 hrs after consumption of the
incriminated meal. Sometimes, the incubation time was as short as 1530
min or as long as 612 hrs. Almost all the vomiting type outbreaks were
associated with consumption of cooked rice. This type of poisoning
resembled staphylococcal food poisoning.
.
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SOURCES OFBACILLUS CEREUS
1. Wide distribution in soil, dust and air
B. cereus is widely distributed in nature and can be found in soil, dust, air,
water and decaying matter. Its ability to form spores allows survival through
all stages of food-processing, other than retorting.
2. Carried by humans and animals
Human: Humans are not a significant source of food contamination by B.
cereus. This organism already exists on many foods and can therefore be
transiently carried in the intestine of healthy humans (0-43%).
Animal: Animals can carry B. cereus on parts of their body. May
occasionally cause mastitis in cows.
3. In many food products
Raw foods of plant origin are the major source ofB. cereus. The widespread
distribution of the organism, the ability of spores to survive dried storage and
the thermal resistance of spores, means that most ready-to-eat foods will
contain B. cereus and will require control measures to prevent growth,
especially after cooking has eliminated competing flora. Strains producing
emetic toxin grow well in rice dishes and other starchy foods, whereas strains
producing diarrhoeal toxin grow in a wide variety of foods from vegetables
and salads to meat and casseroles. Numerous dried herbs and spices and
dehydrated foods have been shown to containB. cereus.
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4. Dairy products
Rice and cooked oriental foods
Its not just rice, this is just the most well known example of foods that
can become contaminated. Other cooked cereals such as cous and bulghur
wheat can also be affected, as can pasta, potatoes, pastries, any foods with
sauces, such as casseroles and pies. Even salads have been found to harbor
Bacillus cereus spores and actively growing bacteria.
Spices and spice mixes
Dried products (flour, dry milk, pudding, soup mix)
5. Meats
Microorganisms control in meat products is the major concern in the
preparation of high quality foods [20]. The hygienic state of animals prior,
during and after slaughter can be critical to the finished product quality [21].
During slaughtering process the meat is exposed to many sources ofBacillus
cereus contamination [22]. The incidence ofBacillus cereus is higher in cooked
and processed (ground beef) meat than in raw meat samples [23] [24].
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STRUCTURE OF BACILLUS CEREUS
Like most Gram-positive bacteria the surface of theBacilluscereus is complex and
is associated with their properties of adherence, resistance and tactical responses.The vegetative cell surface is a laminated structure that consists of a capsule, a
proteinaceous surface layer (S-layer), several layers of peptidoglycan sheeting, and
the proteins on the outer surface of the plasma membrane.
Fig2: Surface of aBacillus cereus Transmission E.M. C=Capsule; S=S-layer;
P=Peptidoglycan.
Surface layer (S-layer) :-
A regularly ordered protein or glycoprotein layer (S-layer) has been detected as
the outermost component of several gram-negative and gram-positive
organisms [25] [26]. The functions of the S-layer in bacteria are not completely
understood. It has been suggested that the S-layer mediates the adhesion to
avian intestinal epithelial cells in Lactobacillus acidophilus and to collagen in
Lactobacillus crispatus [27] Increased virulence and resistance to phagocytosis
[28] have been associated with the presence of the S-layer in animal pathogens.
Ellar and Lundgren [29]described the presence of an S-layer on the surface of
B. cereus .
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Capsule :-
Capsule synthesis in Gram positive bacteria falls into two catagories;
production of polyglutamic acid and polysaccharide capsule. While mostlaboratory strain of B.subtilis do not produce significant capsule material, the
genome sequence indicates that they possess the genes required for production
of each type of capsule. [30].
Cell Wall :
The variability of cell wall structure that is common in many Gram-positivebacteria does not occur in the genusBacillus. The vegetative cell wall of almost
all Bacillus species is made up of a peptidoglycan containing meso-
diaminopimelic acid (DAP). This is the same type of cell wall polymer that is
nearly universal in Gram-negative bacteria, i.e., containing DAP as the diamino
acid in position 3 of the tetrapeptide. In some cases, DAP is directly cross-
linked to D-alanine, same as in the Enterobacteriaceae; in other cases, two
tetrapeptide side chains of peptidoglycan are spanned by an interpeptide bridge
between DAP and D-alanine, which is characteristic of most Gram-positive
bacteria.
In addition to peptidoglycan in the cell wall, all Bacillus species contain large
amounts of teichoic acids which are bonded to muramic acid residues. The
types of glycerol teichoic acids vary greatly between Bacillus species and
within species. As in many other Gram-positive bacteria, lipoteichoic acids are
found associated with the cell membranes ofBacillus species.
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The cell wall forms the barrier between the environment and the bacterial cell.
It is also responsible for maintaining the shape of the cell and withstanding the
cell's high internal turgor pressure [31].
Fig3: Mechanism of cell wall
The cell wall synthetic enzymes (eg. penicillin binding proteins and
autolysins) are produced intracellularly but their sites of action are extracellular,
i.e. within the cell wall. Therefore cell wall synthesis requires signaling
between the cell wall and the cytoplasmic compartments to coordinate the
production of precursors/enzymes with their utilization. [32].
Flagella :-
The flagellum is essential for active movement of individual cells in a liquid
environment (swimming) and for chemotaxis and plays an important role in
interaction with surfaces asa sensor of medium viscosity [33] .
When bacterial flagella are examined by electron microscopy [34] they are
found to be composed of three morphologically distinguishable sections: a long
flagellar filament, a hook like terminal structure, and a basal region which is
attached to the cell membrane.
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Swarming can be considered a strategy for rapid spread over solid surfaces in
the environment and for active colonization of mucosal surfaces in infected
hosts[35].
Fig5: Electron microscopic Structure of Flagella
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Fig4: Different type of B.cereus
Endospore :-
Endospores were first described by Cohn in Bacillus subtilis and later by
Koch in the pathogen, Bacillus anthracis. Cohn demonstrated the heat
resistance of endospores in B. subtilis, and Koch described the developmental
cycle of spore formation in B. anthracis. Endospores are so named because
they are formed intacellularly, although they are eventually released from this
mother cell or sporangium as free spores. Endospores have proven to be the
most durable type of cell found in Nature, and in their cryptobiotic state of
dormancy they can remain viable for extremely long periods of time, perhaps
millions of years.
fig 6- spores ofbacillus
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Pathogenesis of Bacillus cereus
B. cereus is responsible for a minority of food borne illnesses (25%), causing
severe nausea, vomiting and diarrhea [36]. Generally speaking,Bacillus foodborneillnesses occur due to survival of the bacterial endospores when food is improperly
cooked. This problem is compounded when food is then improperly refrigerated,
allowing the endospores to germinate [37]. Bacterial growth results in production
of enterotoxins, one of which is highly resistant to heat and to pH between 2 and
11, ingestion leads to two types of illness, diarrheal and emetic (vomiting)
syndrome.
The diarrheal type is associated with a wide-range of foods, has an 816.5 hour
incubation time and is associated with diarrhea and gastrointestinal pain. Also
known as the long-incubation form ofB. cereus food poisoning, it might be
difficult to differentiate from poisoning caused by Clostridium perfringens.
The emetic form is commonly caused by rice that is not cooked for a time and
temperature sufficient to kill any spores present, then improperly refrigerated.
It can produce a toxin which is not inactivated by later reheating. This form
leads to nausea and vomiting 15 hours after consumption. It can be difficult to
distinguish from other short-term bacterial food borne pathogens, e.g.,
Staphylococcus aureus).
If rice is cooked at, or over 100 degrees Celsius for 20 minutes or more bacillus
cereus cannot survive, therefore eliminating possible food-poisoning. It was
previously thought that the timing of the toxin production might be responsible for
the two different types, but in fact the emetic syndrome is caused by a toxin called
cereulide that is found only in emetic strains and is not part of the "standard
toolbox" ofB. cereus. Cereulide, a dodecadepsipeptide produced by non-ribosomal
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peptide synthesis (NRPS), which is somewhat unusual in itself. Cereulide is
believed to activate 5-HT receptors leading to increased afferent vagal stimulation
[38].
Toxins Production
Bacillus cereus produces one emetic toxin (ETE) or Cereulide and three different
enterotoxins: HBL, Nhe, and EntK.
Two of the three enterotoxins are involved in food poisoning. They both consist of
three different protein subunits that act together. One of these enterotoxins (HBL)
is also a hemolysin; the second enterotoxin (Nhe) is not a hemolysin. The third
enterotoxin (EntK) is a single component protein that has not been shown to be
involved in food poisoning. All three enterotoxins are cytotoxic and cell membrane
active toxins that will make holes or channels in membranes.
Cereulide is a small, heat and acid stable cyclic dodecadepsipeptide which is
chemically closely related to the potassium ionophore valinomycin [39]. It is toxicto mitochondria by acting as a potassium ionophore and has been reported to
inhibit human natural killer cells [40]. According to its chemical structure it has
been shown that this toxin is produced by a nonribosomal peptide synthetase
(NRPS), but its exact genetic organization and biochemical synthesis is unknown.
The non-hemolytic enterotoxin (Nhe) is one of the three-component enterotoxins
responsible for diarrhea in Bacillus cereus food poisoning. Nhe is composed of
NheA, NheB and NheC. The three genes encoding the Nhe components constitute
an operon. The nhe genes have been cloned separately, and expressed in either
Bacillus subtilis orEscherichia coli. Separate expression showed that all three
components are required for biological activity.
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The hemolytic enterotoxin, HBL, is encoded by the hblCDA operon. The three
protein components, L1, L2 and B, constitute a hemolysin. B is for binding; L1 and
L2 are lytic components. This toxin also has dermonecrotic and vascular
permeability activities, and it causes fluid accumulation in rabbit ileal loops.
APPLICATIONS OF B. CEREUS
Symbiosis
B. cereus competes with other microorganisms such as Salmonella and
Campylobacter in the gut, so its presence reduces the numbers of those
microorganisms. In food animals such as chickens [41], rabbits, and pigs, some
harmless strains ofB. cereus are used as a probiotic feed additive to reduce
Salmonella in the intestines and cecum. This improves the animals' growth as well
as food safety for humans who eat their meat.
Antibiotic Production
Bacillus antibiotics share a full range of antimicrobial activity: bacitracin, pumulin,
laterosporin, gramicidin and tyrocidin are effective against Gram-positive bacteria;
colistin and polymyxin are anti-Gram-negative; difficidin is broad spectrum; and
mycobacillin and zwittermicin are anti-fungal.
As in the case of the actinomycetes, antibiotic production in the bacilli is
accompanied by cessation of vegetative growth and spore formation. This has led
to the idea that the ecological role of antibiotics may not rest with competition
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between species, but with the regulation of sporulation and/or the maintenance of
dormancy.
Antibiotics produced by the aerobic sporeformers are often, but not always,
polypeptides. Known antibiotic producers are Bacillus cereus (e.g. cerexin,
zwittermicin), Bacillus circulans (e.g. circulin), Brevibacillus laterosporus (e.g.
laterosporin),Bacillus licheniformis (e.g. bacitracin),Paenibacillus polymyxa (e.g.
polymyxin, colistin), Bacillus pumilus (e.g. pumulin) and Bacillus subtilis (e.g.
polymyxin, difficidin, subtilin, mycobacillin).
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MATERIAL & METHODS
REQUIREMENT
Conical flask
15 Vile
Pipette
Water bath
Centrifuge
Electronics analytical balance
Autoclave
Agarose gel electrophoresis
assembly
Casting tray
Comb
Balancer
Deep freezer
PCR( Thermal cycle)
Beakers
Aluminium foil
Oven
Incubator loop
Cotton
Matching box
WASHING
Firstly we discard the Petri dish. In which Petri dishes are wrap with Paper and
Aluminum foil. And tapping with tap on to the wrapped Petri dish.
Then placed it in to the Autoclave.
Set the Autoclave at 121C for 15 min. The temperature was 15 psi.
Now we use the detergent for washing the Petri dishes.
To dry the Petri dish we use the Hot air oven at 80C for 30 min. Before drying
we wrap the Petri dish by Paper.
Store the wrapped Petri dishes for further use.
We use the detergent for washing the Tip.
To dry the Tip we use the Hot air oven at 37C for 30 min. Before drying, place
all the tips in to the tip box. Then we wrap the tip box by Paper.
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Store the wrapped Tip box for further use.
STERILIZATION
GLASSWARE:
To take the glassware like Petri dishes, conical flasks, Jars, Test tubes, etc.
Wrap the glassware by Paper and Aluminum foil. And tapping by tap on to the
wrapped glassware.
Take some water in to the Autoclave and place the wrap glassware.
Set the Autoclave at 121C for 15 min. And Pressure was 15 Psi.
Store the wrapped glassware for further use.
PLASTIC WARE:
To take the plastic ware like tips of pipette, Eppendrofs or vial, etc.
All tips are place in to the tip box and vile are in to the vile box.
Wrap the boxes by Paper and Aluminum foil. And tapping by tap on to the
wrapped box.
Take some water in to the Autoclave and place the wrap glassware.
Set the Autoclave at 121C for 15 min. And Pressure was 15 Psi.
Store the wrapped boxes for further use.
Sterilize the platinum loop by the Flame (direct heat).Whenever the loop was
red hot.
CHEMICAL STERILIZATION:
Before doing practical we wash our hand by Alcohol.
Wipe the surface area of performing experiment by the Alcohol.
Some time we wiped the glassware like Petri dish, Slide, etc. with alcohol also.
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SAMPLE COLLECTION:-
5 samples were collected from different region of Agra.
S.No. Area of Collection Type of Sample Type of Sample
1. Shastripuram , Agra milk R 1
2. Khandari , Agra milk R 2
3. Shahganj ,Agra milk R 3
4. Kargil, Agra milk R 4
5. Sikandra ,Agra milk R 5
SAMPLE PREPARATION:
Taken 10 ml. Of milk in test tubes. Mix the samples properly and heat it at 80C
in hot air oven for one hour. This step allows the killing of all vegetative cells
present in the sample, only spores will remain.
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CULTURING:
Bacillus cereus was isolated from the above sample by streaking the sample on
Nutrient agar Medium which is Basal media forall microorganisms.
PREPARATION OF NUTRIENT AGAR MEDIA:
Ingredients gm/literPeptic digest of animal tissue :- 5.00
Beef extract :- 1.50
Yeast extract :- 1.50
Sodium chloride :- 5.00
Agar :- 15.00
Final pH (at 25C) :- 7.4 0.2
PROCEDURE-
All the ingredients were suspended in desired amount in the flask containing
distilled water, stirred well to dissolve. Heat to boiling to dissolve the medium
completely. The pH was adjusted to 7.4 0.2 by adding 10N Sodium hydroxide.
This medium was dispensed into culture flasks, autoclaved at 121oC at 15 lb
pressure for 15 min and then allowed to cool at room temperature and poured in
petridish. After solidification the medium was streaked with samples collected.
The colonies which appeared abundant, forming opaque, creamy on agar (pH 7.0)
were further grown on Bacillus differential media. This media is used to
differentiate Bacillus subtilis and Bacillus cereus based on their capability to
ferment Mannitol.
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PREPARATION OF BACILLUS DIFFERENTIAL MEDIA
Ingredients gm/liter
Yeast autolysate :- 0.20
Mannitol :- 5.00
Phosphate :- 1.00
Potassium :- 0.20
Magnesium :- 0.20
Bromo cresol purple :- 0.0075
Agar :- 15.40
Final pH (at 25C) :- 72
PROCEDURE-
All the ingredients were taken in the flask, stirred well to dissolve.
The pH was adjusted to 7.40.2 by adding NaCl or HCl.
This medium was dispensed into culture flasks, autoclaved at 121oC at 15 lb
pressure for 15 min.
Then allowed to cool at room temperature and poured in petridish.
After solidification the medium was streaked with samples collected
The colonies which appeared white on Bacillus differentiation agar were collected
and preserve as pure culture in nutrient broth. These pure cultures were further
assayed by biochemical test and Gram staining.
IDENTIFICATION
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1. GRAMS STAINING:
Reagents-
Grams stain :- Crystal Violet
Moderant :- Grams Iodine
Decolorizing agent :- 70% Alcohol
Counter stain :- Safranin
Procedure:-
The smear was prepared on sterilized glass slide.
The smear was fixed by passing over the flame.
The smear was flooded with crystal violet and incubated for 2 min.
The smear was washed with tap water.
The smear was flooded with grams iodine for 2 min.
The smear was washed with tap water.
The smear was decolorized with 70% alcohol for 30 sec.
The smear was washed with tap water. The smear was counter stained with safranin for 2 min.
The smear was washed with tap water, air dried and observed under
oil immersion microscope.
2. ENDOSPORE STAINING:
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Reagent
Grams stain Crystal Violet
Counter stain Safranin
Procedure-
Place a strip of blotting paper over the slide.
Place the covered slide over a screened water bath and then saturate
blotting paper with primary stain malachite green.
Allow the slide to sit over the steaming water bath for 5 minutes,
reapplying stain if it begins to dry out.
Remove blotting paper and rinse slide with water until water runs
clear.
Flood slide with the counterstain safranin for 20 seconds and then
rinse.
View specimen under oil immersion lens with light microscope.
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BIOCHEMICAL TEST
1.CATALASE TEST:
Catalase test is used to detect the presence of the enzyme Catalase. Catalase
enzyme is found in most bacteria. It catalyses the breakdown of hydrogen peroxide
(H2O2) with the release of free Oxygen. Catalase is found in most aerobic and
facultative anaerobic bacteria.
Reagent -
3% H2O2.
Procedure-
1. The sterile glass slide was taken.
2. 1 drop of 3% H2O2 was placed on slide and the single colony was
mixed with sterile loop.3. The slide was observed for immediately and vigorous bubbling.
4. A positive result was the rapid evolution of O2 as evidenced by
bubbling.
5. A negative result was no bubbles or only a few scattered bubbles.
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2.OXIDASE TEST
The oxidase test identifies organisms that produce the enzyme cytochrome
oxidase. Cytochrome oxidase participates in the electron transport chain bytransferring electrons from a donor molecule to oxygen. The oxidase reagent
contains a compound that changes color when it becomes oxidized. If the test
organism produces cytochrome oxidase, the colorless reagent used in the test will
detect the presence of the enzyme oxidase and, reacting with oxygen, turn violet to
purple.
Reagent
N, N, N`N`-Tetra methyl-p-phenylenediamine dihydrochloride.
Procedure
1. Take 2-3 drops of (C6H4 [N (CH3)2]2.2HCl) oxidant on separate slides.
2. Using aseptic technique, inoculate culture of assigned bacteria on slides andmixed it.
3. Observe for the presence or absence of a color change from pink to maroon
and finally to purple (lower portion of the plate). If the change occurs in 10-30
seconds after adding the reagent, the bacterium is considered positive for
oxidase enzyme activity. If no color change takes place, or the change is a
slightly darker pink, the bacterium is considered negative for oxidase activity.
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3. NITRATE TEST :
During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to
ammonia. No denitrification products were observed. B. subtilis wild-type cellsand a nitrate reductase mutant grew anaerobically with nitrite as an electron
acceptor.
NO3 ----> NO2 ----> NH3 or N2
Reagents -
Nitrate broth.
Sulfanilic.
Alpha-naphthylamine.
Powdered zinc.
PROCEDURE -
1.Inoculate separate tubes of nitrate broth with each of assigned bacteria.
2. Incubate the tubes at 37C for 24-48 hours.
3. After incubation, add five drops of sulfanilic acid and then five drops of alpha-
naphthylamine to each tube.
4. Observe whether or not a red coloration develops in the cultures. The
development of a red color indicates the reduction of nitrates to nitrites. If no color
develops, either the bacterium cannot reduce nitrates to nitrites OR any nitrites
produced were rapidly further reduced to ammonia or other end products (that
would not impart the red color).
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5. To determine if nitrites were produced, but then some or all were reduced past
the nitrite stage, add a minute quantity of powdered zinc to any tubes that are
colorless after the sulfanilic acid and alpha-naphthylamine were added.
6. If a red color then appears afterthe addition of the zinc, this is interpreted as NO
reduction of nitrates (can't tell if the other result, further reduction of all nitrites,
has occurred). The zinc actually reduces the nitrates to nitrites, which then produce
the red color in the presence of the sulfanilic acid and alpha-naphthylamine.
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4.HEMOLYSIS ON BLOOD AGAR
Hemolysis on blood agar is used for the preliminary or confirmatory
identification of many types of clinically important bacteria. While it is factored
into the differential diagnosis of a specific infectious agent, hemolysis type is not
specific enough to be a final diagnosis criterion.
The three hemolysis conditions continue to be described by terms that are
somewhat confusing.
Alpha-hemolysis is a greenish discoloration of the blood agar surrounding a
bacterial colony; it is a characteristic ofStreptococcus pneumoniae.
Beta-hemolysis indicates a zone of clearing in the blood agar in the area
surrounding a bacterial colony. It is a characteristic of Streptococcus
pyogenes, Bacillus cereus as well as some strains ofStaphylococcus aureus.
Gamma-hemolysis is actually a lack of hemolysis in the area surrounding a
bacterial colony growing on blood agar. In fact, culture of bacteria on blood
agar for the purpose of hemolysis classification is performed at 37o
C in thepresence of 5% CO2. This results in an overall brownish discoloration of the
blood agar, from its original blood-red hue. An uninoculated blood agar
plate (BAP) is shown on the left, above. Gamma-hemolysis would therefore
describe bacterial growth that results in neither a greenish tinge to the
discoloration (alpha-hemolysis) nor a clear zone that the observer "could
read a newspaper through" (beta-hemolysis).
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ISOLATION OF DNA
Reagents and Solutions:-
T.E Buffer (pH 8.0)
o 0.1M Tris HCl
o 0.01M EDTA
5M NaCl (29.3g of NaCl was dissolved in 1000ml of distilled water,
autoclaved and stored at room temperature).
CTAB/NaCl (4.1g NaCl and 10g CTAB was dissolved in 1000 ml distilled
water at 650C and stored at temperature).
Chloroform/Isoamyl alcohol (mix 24 volume of chloroform with 1 volume of
isoamyl alcohol (24:1). It should be prepared fresh).
10% SDS (10g SDS was dissolved in 100 ml distilled water by heating at
650C in water bath for 20 min. do not autoclaved, stored at room temperature).
Lysozyme (20mg lysozyme was dissolved in 1ml deionized distilled water.
The solution is stored in small aliquots at 200C)
Proteinase-k (10mg of proteinase was dissolved in 1ml deionized distilled
water and the solution is stored at 200C).
70% Ethanol.
Isopropanol.
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PROCEDURE:-
1 or 2 loops full of microbial growth was scraped from culture media
and suspended into 400 l of T.E .buffer in a vial. The vial was freezed and thaw by 200C for 15 minutes and heated it
immediately up to 80 1000C for 5 min. and again snap cooled at by keeping
the vial in ice for 15 min.
40 l lysozyme was added in the vial, mixed well and incubate for 2 hours at
370C in shaking water bath.
56 l of 10% SDS and 5 l of proteinase k was added in the vial, mixed well
and incubated at 65oC in shaking water bath for 30 minutes.
80 l of 5M NaCl and 64 l of CTAB/NaCl solution were added in the vial
and incubate at 650C in water bath for 30 minutes.
Equal volume of freshly prepared Chloroform/Isoamyl alcohol solution (24:1)
was added in vial, mixed well and centrifuge at 10,000 rpm for 15 minutes.
Three layers become visible. The upper aqueous layer contains DNA, which
is taken into another fresh micro centrifuge tube.
0.6 volume of Isopropanol was added in vial in the supernatant and incubated
at 200C for 30 minutes.
The tube was centrifuged at 8000xg (10,000rpm) for 5 min.
The supernatant was discarded without losing pellet.
150 l of 70% chilled ethanol was added in tube and centrifuge the tube
at 8000xg (10,000rpm) for 5 min.
The supernatant was discarded and air dried the pellet.
The white pellet observed after centrifuged the tube.
30 l d/w. was added in the tube and stored at 200C till use.
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AGAROSE GEL ELECTROPHORESIS
Chemicals and Reagents:-
Tris Acetate EDTA Buffer(TAE Buffer) 50X :-
Tris base: 242g
Glacial Acetic Acid: 37.1ml
EDTA: 37.2g
The final volume was made up to 1000 ml with deionised distilled water. pH was
maintained up to 8.0, autoclaved at 1210C and stored at room temperature.
Ethidium bromide dye :-
Ethidium bromide 10 mg
Distilled water 1ml
Agarose Gel (2%):-
Agarose 0.8 g
50X TAE 0.8 ml
Ethidium bromide dye 3 l
Distilled water 39.2 ml
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DNA loading dye:-
Bromo Phenol Blue 0.25%
Xylene cynol 0.25%
Glycerol 30%
The dye was prepared in d.w. and it should be stored at 4oc.
PROCEDURE:-
2% Agarose was dissolved in TAE Buffer.
The solution was boiled in a water bath mixing occasionally by swirling with
hands.
Agarose gel was boiled gently till it dissolved.
The solution was cooled up to 55oC and Ethidium bromide (0.5/ml) was added
into the solution and the solution was dispensed in casting tray with appropriate well
forming comb and was allowed to solidify.
250 ml TAE Buffer was poured in electrophoretic unit.
Prepared gel was placed in such a way that the wells are towards cathode. The
sample were loaded in wells and run the gel at 32V for 2 hours.
The gel was observed on U.V. Transilluminator.
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PCR (POLYMERASE CHAIN REACTION)
Reagent & chemicals
Distilled water :- 276.5 l
10x PCR buffer :- 35.0 l
dNTPs 200 M :- 7.0 l
primer(forward ) :- 7.0 l
primer(Reversed) :- 7.0l
Taq DNA polymerase :- 3.5 l
DNA sample :- 2.0 l
Sequence of Primer-
EM1F: 5-GACAAGAGAAATTTCTACGAGCAAGTACAAT-3
EM1R: 5-GCAGCCTTCCAATTACTCCTTCTGCCACAGT-3
PCR cycle-
Initial denaturation at 940C for 5min. following 45 cycles with denaturation at 940C
for 1 min, annealing at 550C for 1 min, extension at 720C for 1 min the final
extension at 720C for 10 min.
.
PROCEDURE-
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1. The master mix was prepared by mixing all the components given above. This
was done on ice. then 48l of master mix were added in each 6 PCR tubes.
2. DNA template 2 l was added in PCR tubes and the tubes were placed in
thermocycler and the program was set and started with the appropriate
temperatures, time and number of cycles.
3. The PCR product was stored at -200C till use.
RESULTS
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5 sample were collected from different regions and cultured on nutrient agar media
and then on Bacillus differential agar media which were tested through various
biochemical test for the identification ofBacillus cereus.
S.
No.
Area of
Collection
Sample
Code
Type of
Sample
Colony
Colour
Grams
Stain
Endospore
Stain
1. Shastripuram ,
Agra
R1 milk White+
Yellow
+ve +ve
2. Khandari ,
Agra
R2 milk White+
Yellow
+ve +ve
3. Shahganj,Agra
R3 milk White +ve +ve
4. Kargil, Agra R4 milk White +ve +ve
5. Sikandra
,Agra
R5 milk White +ve +ve
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Fig - Different colonies ofB. subtilis & B.cereus
Grown on Bacillus differential media
Table-2; Data of Biochemical Tests
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S.No. Sample
Code
Catalase
Test
Nitrate
Test
Oxidase
Test
Blood Haemolysis
Test
1. R-1 +ve +ve +ve +ve
2. R-2 +ve +ve +ve +ve
3. R-3 +ve +ve +ve +ve
4. R-4 +ve +ve +ve +ve
5. R-5 +ve +ve +ve +ve
Out of the 5 collected samples all were identified asB.cereus, through biochemical
tests.
BIOCHEMICAL TEST RESULTS -
1. CATALASE TEST
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2. OXIDASE TEST
3. NITRATE REDUCTION TEST
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4. STARCH HYDROLYSING TEST
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Hemolysis on Blood Agar
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OBSERVATIONS OF PCR FOR EMETIC TOXIN PRODUCING B.
CEREUS
Gel Electrophoresis of PCR Amplification
Lane-1: Marker (M)
Lane-2: Sample no.1 (R1)
Lane-3: Sample no.2 (R2)
Lane-4: Sample no.3 (R3)
Lane-5: Sample no.4 (R4)
Lane-6: Sample no.5 (R5)
Table-3; Data Of PCR emetic toxin producingB. cereus
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Results
S.No. Sample Code PCR result
1. R1 Amplified
2. R2 Amplified
3. R3 Amplified
4. R4 Not Amplified
5. R5 Amplified
Out of 5 samples only 4 samples (R1, R2, R3, R5,) identified asB. cereus amplified
through PCR which confirms the presence ofB. cereus at molecular level.
DISCUSSION & CONCLUSION
Tables 1, 2, and 3 show that, using cultural characteristics, and biochemical
characteristics, ofB. cereus. It is ubiquitous, saprophytic, soil bacterium and its
ability to produce a wide variety of enzymes. This latter feature of the
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microorganism has been commercially exploited for over a decade. B. cereus has
been used for industrial production of proteases, amylases, antibiotics, and
specialty chemicals[63].
One of the degradative enzymes synthesized early in stationary phase in B.
cereus alpha-amylase, an exo-enzyme responsible for the degradation of starch to
simpler sugars which can be assimilated by the cell
We have identify a gene of an extra cellular -amylase from the mesophilic
strain ofB. cereus. The extra cellular -amylase enzyme is not very closely related
to any other amylases of family 13 of glycosyl hydrolases.
On the other hand it canbe aligned to the other enzymes, and it has the conserved regions I-IV found in
other amylases.
The use ofB. cereus in an industrial setting should not pose an unreasonable
risk to human health or the environment. First, human health and environmental
hazards ofB. cereus are low. Second, the number of microorganisms released from
the fermentation facility is low. In addition, B. cereus is ubiquitous in the
environment, and the releases expected from the fermentation facilities will not
significantly increase populations of this bacterium in the environment.
The B. cereus genome contains several genes that are predicted to code for
proteins that belong to the cupin super family. Cupins are proteins that are related
to plant seed storage proteins that fold into small beta-barrels. Several of the B.
cereus cupins share identity with the secreted oxalate-degrading enzymes of fungi
and plants. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding
genes.
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In addition, theavailability of the complete genome sequence [64]and about
3,000 "y"-mutants constructed within the B. cereus Functional Analysis program
[65] make B. cereus an ideal model organism for research on gram-positive
bacteria.
Plants are important source of potentially useful structures for the
development of new chemotherapeutic agents. The first step towards this goal is
the in vitro antibacterial activity assay [66]Many reports are available on the
antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and anti-
inflammatory properties of plants [67] Some of these observations have helped in
identifying the active principle responsible for such activities and in the developing
drugs for the therapeutic use in human beings. However, not many reports are
available on the exploitation of antibacterial property of plants for developing
commercial formulations for applications in crop protection. In the present study,
the methanol leaf, root/bark extracts ofAcacia nilotica, Tinospora cordifolia,
Withania somnifera andZiziphus mauritian showed the activity against B. cereus.
The results of present investigation clearly indicate that the antibacterial and
antifungal activity vary with the species of the plants and plant material used.
Thus, the study ascertains the value of plants used in ayurveda, which could be of
considerable interest to the development of new drugs.
In conclusion, the use of B. cereus in fermentation facilities for the
production of enzymes or specially chemicals has low risk. Although notcompletely innocuous, the industrial use ofB. cereus presents low risk of adverse
effects to human health or the environment.
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