Group b Report- Bacillus Cereus 20july

8/3/2019 Group b Report- Bacillus Cereus 20july

1/62

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:

1


2/62

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.

2


3/62

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

3


4/62

4

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


5/62

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

5


6/62

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.

6


7/62

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

7


8/62

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

8
http://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/Gram.html


9/62

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

9
http://www.microbiologybytes.com/video/microscopy.htmlhttp://www.microbiologybytes.com/video/microscopy.htmlhttp://www.microbiologybytes.com/video/microscopy.htmlhttp://www.microbiologybytes.com/video/microscopy.html


10/62

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

10


11/62

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

11


12/62

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.

12


13/62

TAXONOMIC CLASSIFICATION

Kingdom :- Bacteria

Phylum :- Firmicutes

Class :- Bacilli

Order :- Bacillales

Family :- Bacillaceae

Genus :- Bacillus

Species :- cereus

13


14/62

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.

14


15/62

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.

.

15


16/62

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.

16


17/62

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].

17


18/62

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 .

18


19/62

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.

19


20/62

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.

20


21/62

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

21


22/62

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

22


23/62

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

23


24/62

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.

24


25/62

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

25


26/62

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).

26


27/62

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.

27


28/62

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.

28


29/62

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.

29


30/62

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.

30


31/62

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

31


32/62

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:

32


33/62

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.

33


34/62

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.

34


35/62

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.

35


36/62

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).

36


37/62

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.

37


38/62

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).

38


39/62

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.

39


40/62

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.

40


41/62

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

41


42/62

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.

42


43/62

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-

43


44/62

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

44


45/62

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

45


46/62

Fig - Different colonies ofB. subtilis & B.cereus

Grown on Bacillus differential media

Table-2; Data of Biochemical Tests

46


47/62

S.No. Sample

Code

Catalase

Test

Nitrate

Test

Oxidase

Test

Blood Haemolysis

Test

1. R-1 +ve +ve +ve +ve





Out of the 5 collected samples all were identified asB.cereus, through biochemical

tests.

BIOCHEMICAL TEST RESULTS -

1. CATALASE TEST

47


48/62

2. OXIDASE TEST

3. NITRATE REDUCTION TEST

48


49/62

4. STARCH HYDROLYSING TEST

49

Hemolysis on Blood Agar


50/62

OBSERVATIONS OF PCR FOR EMETIC TOXIN PRODUCING B.

CEREUS

Gel Electrophoresis of PCR Amplification

Lane-1: Marker (M)

Lane-2: Sample no.1 (R1)





Table-3; Data Of PCR emetic toxin producingB. cereus

50


51/62

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

51


52/62

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.

52


53/62

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.

53


54/62

REFERENCES

1. Aas, N. Gondrosen, B. and Langeland, G. (1992). Norwegian Food Authorities

Report on Food Associated Diseases in 1990. SNT Report Oslo.

2. Abram D., Vatter, A. E. and Koffler, H. (1966). Attachment and structural

features of flagella of certain bacilli.J. Bacteriol. 95: 2045-2068.

54


55/62

3. Agata N, Mori M, Ohta M, Suwan S, Ohtani I, Isobe M (1994). A novel

dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole

formation in HEp-2 cells.FEMS Microbiol Lett., 121: 3134.

4. Agata N, Ohta M, Mori M, Isobe M (1995). "A novel dodecadepsipeptide,

cereulide, is an emetic toxin ofBacillus cereus".FEMS Microbiol Lett, 129 (1):

1720

5. Agata, N., Ohta, M., Mori, M. and Isobe, M. (1995) A novel

dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS

Microbiol. Lett. 129: 17-20.

6. Allison, C., P. Jones, N. Coleman, and C. Huges. (1992). Ability of Proteus

mirabilis to invade human urothelial cells is coupled to motility and swarming

differentiation.Infect. Immun. 60: 4740-4746.

7. Andersson, M., Mikkola, R., Helin, J., Andersson, M.C. and Salkinoja Salonen,

M. (1998). A novel sensitive bioassay for detection of B. cereus emetic toxin

and related depsipeptide ionophores.Appl. Environ. Microbiol. 64: 1338-1343.

8. Aviram, M., Dornfeld, L. (2001). "Pomegranate juice consumption inhibits

serum angiotensin converting enzyme activity and reduces systolic blood

pressure".Atherosclerosis. 158 (1): 1958

9. Aviram M, Rosenblat M, Gaitini D. (2004). "Pomegranate juice consumption

for 3 years by patients with carotid artery stenosis reduces common carotid

intima-media thickness, blood pressure and LDL oxidation". Clin Nutr., 23 (3):

42333.

10.Beveridge, T. J. (1997). The response of S-layered bacteria to the Gram stain.

FEMS Microbiol Rev.20: 101110.

55


56/62

11.Drobniewski, F.A. (1993).Bacillus cereus and related species. Clin. Microbiol.

Rev. 6: 324-338.

12.Ellar, D. J. and Lundgren, D.G. (1967). Ordered structure in the cell wall of

Bacillus cereus. J Bacteriol. 94: 17781780

13.Finlay, W.J., Logan, N.A. and Sutherland, A.D. (1999). Semiautomated

metabolic staining assay for Bacillus cereus emetic toxin. Appl. Environ.

Microbiol. 65: 1811-1812.

14.Girisch, M., Ries, M., Zenker, M., Carbon, R., Rauch, R. and Hofbeck, M.

(2003). Intestinal perforations in a premature infant caused byBacillus cereus.

Infection 31: 192-193.

15.Granum, P.E. (1994). Bacillus cereus and its toxins. J. Appl. Bacteriol. 23

(Symp. Suppl.), 61S-66S.

16.Granum, P.E. (2001). Bacillus cereus. In: Food Microbiology: Fundamentals

and Frontiers (Doyle, M.P. et al., Eds.), pp. 373-381. ASM Press, Washington,

D.C.

17.Guinebretiere, M.H., Broussolle, V. and Nguyen-The, C. (2002).

Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus

strains. J. Clin. Microbiol. 40: 3053-3056.

18. Becker, H., Schaller, G., Von Wiese, W., Terpian, G. (1994). Int. J. Food

Microbiol. 23: 115.

19.Haggblom, M.M., Apetroaie, C., Andersson, M.A. and Salkinoja- Salonen,

M.S. (2002). Quantitative analysis of cereulide, the emetic toxin ofBacillus

cereus, produced under various conditions.Appl. Environ. Microbiol. 68: 2479-

2483.

56


57/62

20.Hansen, B.M. and Hendriksen, N.B. (2001). Detection of enterotoxic Bacillus

cereus and Bacillus thuringiensis strains by PCR analysis. Appl. Environ.

Microbiol. 67: 185-189.

21.Hocking, A.D. et al. (1997). Foodborne Microorganisms of Public Health

Significance. 5th ed. North Sydney. AIFST NSW Branch Food Microbiology

Group.

22. Kramer, J. M., Gilbert, R. J. (1989).Bacillus cereus and other bacilli species.

In: Food-Borne Pathogens, M. P. Doyle (Ed.), Marcel Dekker, New York pp.

2170.

23.Jimenez, E.A., Rincon, M., Pulido, R., Saura, C.F. (2001). Guava fruit (Psidium

guajava) as a new source of antioxidant dietary fiber. Journal of Agricultural

and Food Chemistry.49(11): 54895493

24.Jo, C., Lee, N.Y., Kang, H.J., Shin, D.H. and Byun, M.W. (2004). Inactivation

of food-borne pathogens in marinated beef rib by ionizing radiation. Food

Microbiol. 21: 5438

25.Kevin, M. Devine, et al. (2008). A matter of life and death: cell wall

homeostasis and the WalKR (YycFG) essential signal transduction pathway.

Mol. Microbiol.

26.Kotiranta, A., Lounatmaa, K., Haapasalo, M. (2000). "Epidemiology and

pathogenesis of Bacillus cereus infections". Microbes Infect 2 (2): 18998.

27.Lawrie, R.A., (1998). Lawries Meat Science (6th ed.). Wood-head Publishing

Limited, Cambridge.

28.Mahfuzul Hoque, M.D., Bari, M.L., Inatsu, Y., Juneja, V.K., and Kawamoto, S.

(2007). Antibacterial activity of guava ( Psidium guajava) and Neem

57


58/62

( Azadirachta indica A. Juss.) extracts against food borne pathogens and

spoilage bacteria.Foodborne Pathogens and Disease. 4(4): 481488

29.Mahler, H., Pasi, A., Kramer, J.M., Schulte, P., Scoging, A.C., Bar, W. and

Krahenbuhl, S. (1997). Fulminant liver failure in association with the emetic

toxin ofBacillus cereus.New Engl. J. Med. 336: 1142-1148.

30.McCarter, L., Hilmen, M. and Silverman, M. (1988). Flagellar dynamometer

controls swarmer cell differentiation of V. parahaemolyticus. Cell. 54: 345-351

31.McKillip, J.L. (2000). "Prevalence and expression of enterotoxins in Bacillus

cereus and otherBacillus spp., a literature review". Antonie Van Leeuwenhoek.

77 (4): 3939.

32.Melendez P.A., Capriles V.A. (2006). Antibacterial properties of tropical plants

from Puerto Rico.Phytomed. 13: 272-276.

33. Menezes, S.M., Cordeiro, L.N., Viana, G.S. (2006). "Punica granatum

(pomegranate) extract is active against dental plaque". Journal of herbal

pharmacotherapy. 6 (2): 7992.

34.Messner, P., Sleytr, U.B. (1992). Crystalline bacterial cell-surface layers. Adv

Microbiol Physiol. 33: 214275.

35.Mikkola, R., Saris, N.E., Grigoriev, P.A., Andersson, M.A. and Salkinoja

Salonen, M.S. (1999). Ionophoretic properties and mitochondrial effects of

cereulide: the emetic toxin ofB. cereus.Eur. J. Biochem. 263: 112-117.

36.Mosupye, F.H. and A. Von Holg, (2000). Microbiological hazard identification

and exposure assessment of street food vending in Johannesburg, South Africa.

Int. J. Food Microbiol. 61: 13745

58


59/62

37.National Committee for Clinical Laboratory Standards (1999). Molecular

Diagnostic Methods for Infectious Diseases. Approved Guideline MM3-A.

NCCLS, Wayne, PA.

38.Noanao, B. and Trust, T. J(1997). The synthesis, secretion and role in virulence

of the paracrystalline surface protein layers of Aeromonas salmonicida and A.

hydrophila.FEMS Microbiol Lett. 154: 17.

39.Nortje, G.L., Vorster, S.M., Greebe, R.P. and Steyn, P.L. (1999). Occurrence of

Bacillus cereus and Yersinia enterocolitica in South African retail meats.Food

Microbiol., 16: 2137

40.Notermans S, Dufrenne, J., Teunis, P., Beumer, R., Te Giffel, M., Peeters

Weem, P. (1997):Food Microbiol. 14: 143151.

41.Paananen, A., Mikkola, R., Sareneva, T., Matikainen, S., Hess, M., Andersson,

M., Julkunen, I., Salkinoja-Salonen, M.S., Timonen, T. (2002). Inhibition of

human natural killer cell activity by cereulide, an emetic toxin from Bacillus

cereus. Clin Exp Immunol. 129: 420428.

42.Paananen, A., Mikkola, R., Sareneva, T., Matikainen, S., Hess, M., Andersson,M., Julkunen, I., Salkinoja-Salonen, M.S. and Timonen, T. (2002). Inhibition of

human natural killer cell activity by cereulide, an emetic toxin from Bacillus

cereus. Clin

43.. Exp. Immunol. 129: 420-428.

44.Pru, B.M., Dietrich, R., Nibler, B., Martlbauer, E. and Scherer, S. (1999). The

hemolytic enterotoxin HBL is broadly distributed among species of theBacillus

cereus group.Appl. Environ. Microbiol. 65: 5436-5442.

45.Satin, M., (2002). Use of irradiation for microbial decontamination of meat:

situation and perspectives. Meat Sci. 62: 27783

59


60/62

46. Schaechter, M., Ingraham, J.L., Neidhardt, F.C. (2006). Microbe. (ASM Press,

Washington, DC,).

47.Schmidt, K. and Tirado, C. (2001) WHO Surveillance Program for Control of

Foodborne Infections and Intoxications in Europe (Seventh report 1993-1998),

p. 110. WHO, Berlin.

48. Seeram, N.P., Aronson, W.J., Zhang, Y., et al. (2007). "Pomegranate

ellagitannin-derived metabolites inhibit prostate cancer growth and localize to

the mouse prostate gland".J. Agric. Food Chem. 55 (19): 77327735.

49.Shinagawa, K., Konuma, H., Sekita, H. and Sugii, S. (1995) Emesis of rhesus

monkeys induced by intragastric administration with the HEp-2 vacuolation

factor (cereulide) produced byBacillus cereus.FEMS Microbiol. Lett. 130: 87-

90.

50.Srivastava, J., Lambert, J. and Vietmeyer, N. (1996). Medicinal plants: An

expanding role in development. World Bank Technical Paper. No. 320.

51. Stachelhaus, T. and Marahiel, M.A. (1995). Modular structure of genesencoding multifunctional peptide synthetases required for non-ribosomal

peptide synthesis.FEMS Microbiol. Lett. 125: 3-14.

52.Stockwell, C. (1988). Natures pharmacy. London, United Kingdom. Century

Hutchinson Ltd.

53.Thomson, W.A.R., (1978). Medicines from the Earth. Maidenhead, United

Kingdom. McGraw-Hill Book Co.

54.Toba, T., Virkola, R., Westerlund, B., Bjorkman, Y., Sillanpa, J., Vartio, T.,

Kalkkinen, N., Korhonen, T. K. (1995). A collagen-binding S-layer protein in

Lactobacillus crispatus.Appl. Environ Microbiol. 61: 24672471.

60


61/62

55.Uniyal, S.K., Singh, K.N., Jamwal P. and Lal, B. (2006). Traditional use of

medicinal plants among the tribal communities of Chhota Bhangal, Western

Himalayan. J. Ethnobiol. Ethnomed., 2: 1-14.

56.Noto,Yuka et al. 2005 "The Relationship Between Salivary Biomarkers and

State-Trait Anxiety Inventory Score Under Mental Arithmetic Stress: A Pilot

Study.". Anesthesia & Analgesia (United States: Lippincott Williams &

Wilkins) 101 (6): 18731876. 22.

57. Ottemann, K. M., and J. F. Miller. 1997. Roles for motility in bacterial-host

interactions. Mol. Microbiol. 24:1109-1117

58.Overhoff B. 1991. Identification of a gene fragment which codes for the

364 amino-terminal amino acid residues of a SecA homologue from Bacillus

subtilis: further evidence for the conservation of the protein export apparatus

in gram-positive and gram-negative bacteria. Mol. Gen. Genet. 228:417-423

59. Pandya et al., 2005. Studies on the activity and stability of immobilized lpha-

amylase in ordered mesoporous silicas. Microporous and Mesoporous

Material, Biotchnol. Appl. Biochem 77: 67-77.

60. Pandey, A., P. Nigam, C. R. Soccol, V. T. Soccol, D. Singh, and R. Mohan.

2000.Advances in microbial amylases. Biotechnol. Appl. Biochem. 31:135-152

61. Perez, A.R., A. Abanes-De Mello, K. Pogliano 2000. "SpoIIB Localizes to

Active Sites of Septal Biogenesis and Spatially Regulates Septal Thinning

during Engulfment in Bacillus subtilis". Journal of Bacteriology. 2000

February; 182(4): 1096 1108.

61
http://www.anesthesia-analgesia.org/cgi/content/full/101/6/1873http://www.anesthesia-analgesia.org/cgi/content/full/101/6/1873http://www.anesthesia-analgesia.org/cgi/content/full/101/6/1873http://www.anesthesia-analgesia.org/cgi/content/full/101/6/1873http://www.anesthesia-analgesia.org/cgi/content/full/101/6/1873http://www.anesthesia-analgesia.org/cgi/content/full/101/6/1873


62/62

62. Priest, F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus.

Bacteriol. Rev. 41:711-753.

63. Ryan KJ, Ray CG 2004. Sherris Medical Microbiology (4th ed. ed.). McGraw

Hill. ISBN 0-8385-8529-9

64. Schaechter, M., J.L. Ingraham, F.C. Neidhardt. 2006 Microbe. (ASM Press,

Washington, DC, 2006).

65.Takeshita M, Hehre E (1975) The capacity of Alpha-amylase to catalyze the

nonhydrolytic degradation of starch and glycogen with formation of novel

glycosylation products, Arch Biochem. Biophys. 169: 627-637

66. Tayeb O. El 2008 et al. Optimization of the industrial production of bacterial

alpha amylase in Egypt. IV. Fermentor production and characterization of the

enzyme of two strains of Bacillus subtilis and Bacillus amyloliquefaciens,

African Journal of Biotechnology Vol. 7 (24), pp. 4521-4536,

67. Todar Kenneth 2008 University of Wisconsin Department of BacteriologyGram-positive, Aerobic or Facultative Endospore-forming Bacteria ,

www.textbookofbacteriology.net
http://en.wikipedia.org/wiki/Special:BookSources/0838585299http://www.textbookofbacteriology.net/http://en.wikipedia.org/wiki/Special:BookSources/0838585299http://www.textbookofbacteriology.net/

Group b Report- Bacillus Cereus 20july

Documents

Transcript of Group b Report- Bacillus Cereus 20july