BACILLUS

Gram Positive Bacilli

Spore Former Non-Spore Former

GRAM POSITIVE BACILLI

Bacillus spp.
Clostridium spp. - normal shape
Pleomorphic:
Mycobacterium spp.
Corynebacterium spp.

Filamentous:
Nocardia spp.
Erysipelothrix spp.
Actinomyces spp.

Coccobacilli:
Listeria spp.

Normal shape:
Lactobacilli
INTRODUCTION

 Sporogenous ORSpore forming rod shaped

bacteria: 2groups
1. Aerobic – Bacillus – Non Bulging spores.
2. Anaerobic – Clostridia- Bulgingspores.

 Important Bacillusspecies:
 Bacillus anthracis
 Bacillus cereus
 Bacillus stearothermophilus
INTRODUCTION
 Ubiquitous, present in Soil,Air, Dust, & Water.
 Frequently isolatedas “ LAB CONTAMINANTS”.
 B. anthracis, the causative agent of an important Zoonotic
disease called “ANTHRX”.
 B. cereuscan cause “FOOD POISOINING”.
 All members are generally “MOTILE” except B. anthracis,
which is“NON-MOTILE”.
 Temp. range for growth 25-750C.
 Salt conc. 2% -25%.
 Also gained importance recently because of its ability to
be used as “BiologicalWeapon”.
HISTORICAL
INTERE
ST
 First organism observed undermicroscope,
Pollender, 1849.

 First communicable disease shown to be

transmitted by inoculation of infected blood –
Davaine, 1850.

 First bacillus to be isolated in pure culture and

shown to possess spores – Koch, 1876.

 First bacteria to be used for preparation of

attenuated vaccine byPasteur, 1881.
BACILLUS ANTHRACIS
 Causative agent of “ANTHRX”.
 A disease primarily of animals, & man gets
infected secondarily (Zoonosis).
MORPHOLOGY
 Gram positive, Non-Acid fast,Non-Motile.

 Large (3-10 µm X 1-1.6 µm), rectangular

 Capsule is made up of polypeptide, polymer of d-glutamic
acid.

 This capsule is Plasmid coded (pX02).

 It inhibits complement mediatedphagocytosis.

 Capsules not formed under ordinary conditions only if media
containing bicarbonate or are incubated at 10 to 25 %CO2.
 If media contains serum, albumin, charcoal or starch –Capsule
formation may occur in absence of CO2.
MORPHOLOGY
 The bacilli are arranged end to end in long
chains.

 The ends are often concave & somewhat

swollen so that a chain of bacilli presenta
“bamboo-stick” appearance.

 Spore are oval & central in position & are of

the same width as the bacillary body so that
theydo not cause bulging of vegetative cell.

 Spores are formed in culture and in soil but

never in hostbody.
SPORES
 Spores are stain by special methods – Sudan black – B –Fat
globules maybe made out within bacilli. “Hot malachite
green (Ashby’s method) OR 0.25% Sulphuric acidas spores
are Acidfast”.
 When blood films are stained with polychrome methylene blue
for a few seconds and observed – an amorphous purplish
material is noticed around the bacilli.
 This represents the capsular material and is characteristic of
anthrax bacillus.
 This is called as“Mc Fadyean’s reaction” & used
as presumptive diagnosis ofAnthrax in animals.
CULTURE
 B. anthracis is an aerobe & facultative anaerobe, with a
temp. range for growth being 12-450C (optimum 35-
370C).

 NUTRIENT AGARMEDIA
 Colonies are irregular, round, 2-3 mm in diameter, dull,
raised, opaque & grayish white with frosted glass
appearance.

 Under low power microscope, the edge of the colony is

found to be composed of long, interlacing chains of
bacilli, resembling locks of matted hairs, the so called
“Medusa head”appearance.
CULTURE
 BLOOD AGARMEDIA
 Colonies are Non -haemolytic,
 Though occasional strains produce a narrow zone of
haemolysis.

 SELECTIVE MEDIUM
 A selective medium (PLET) consisting of Heart infusion
agar with Polymyxin, Lysozyme, Ethylene diamine
tetracetic acid (EDTA) & Thallous acetate has been devised
for isolation of B. anthracis from mixtures containing other
spore-bearing bacilli.
PATHOGENICITY
o Naturally anthrax is disease of cattle and sheep, less or more
other animals are also susceptible (Zoonosis).
Subcutaneous Inoculation of G.P. with B. anthracis
culture filtrate.
Animal dies within 24-72 hrs.

Autopsy – Site of inoculation – shows local gelatinous

hemorrhagic edema.

Spleen – Extensive subcutaneous congestion, enlarged,dark

red, friable - easily broken into pieces or become powder.

Blood – Dark red & coagulates less firmly than normally.

Bacilli are found in large number in local lesions, heart, blood,
spleen (more than 108 bacilli / ml)
PATHOGENESIS

 Bacilli remain attached to interior capillaries,

number is more – so obstruction occur in
blood flow.
VIRULENCE FACTORS
oTwo major virulence factor–
o Capsular polypeptide
o Anthrax toxin
o Each produced by separateplasmid.

o Capsular polypeptide –
oComposed of poly peptide of a high molecular weight
consisting of D-glutamicacid.
o Inhibits phagocytosis.
oLoss of plasmid (px02) controls production of capsule,
leads toloss of virulence.
oAttenuated anthrax spore vaccine is prepared by this
method (Sternestrain).
ANTHRAX TOXIN

 The toxin is complex of 3fractions:

 1. Edema Factor (OF or Factor I)
 2. ProtectiveAntigen Factor (PAor Factor II)
 3. Lethal Factor (LF or Factor III)

 These are nontoxic if act individually.

 But the complex causes local edema and
generalised shock.
ANTHRAX TOXIN
 These three factors have been characterised and
cloned.

 Protective Antigen Factor or PA- It binds the target

cell surface receptors and in turns provides
attachment sites for Edema factor or OF or Lethal
Factor or LF, facilitating their entry inside the target
cell.

 The antibody to PAis protective because it blocks the

attachment of PAto target cell surface receptor and
inhibit the further action of OF or LF.
ANTHRAX TOXIN

 Edema Factor or OF - It is anAdenyl cyclase which is

activated only inside the target cells, leading to
intracellular accumulation of cyclicAMP.

 This is believed to be responsible for the edema and

other biological effects of the toxin.

 Lethal Factor - Entry of LF into the cell causes cell

death but the mechanism of action is not known.
ANTHRAX TOXIN

 Loss of the plasmid (px01) which encodes the

toxin renders the strain avirulent.

 This is believed to have been the basis for the

original anthrax vaccine developed by Pasteur.

 The avirulent Sterne vaccine strain is devoid of

the plasmid coding for the capsular
polysaccharide.
 PATHOGENESIS – ANIMALS
 Anthrax is primarily a disease of animals like cattle
& sheep, less often of horses & swine.

 Anthrax in animals presented as a fetal septicemia;

however, localized cutaneous lesions may be
produced rarely.

 Infected animals discharge large number of bacilli

from the mouth, nose & rectum.

 The bacilli sporulate in soil & remains as the source

of infection.
HUMAN ANTHRAX

 Humans are occasionally secondarilyinfected

from diseasedanimals.
 There are three clinical types of disease based
on route ofinfection.
 CUTANEOUS
 PULMONARY
 INTESTINALANTHRAX.
 ALLTYPESLEADTO SEPTICAEMIA.
 HUMAN ANTHRX –
CUTANEOUS
 95 % of human cases are of cutaneoustyp e.
 Route of entry: Skin byinoculation.
 Involves face, neck, hands, arms & back.
 Papul Vesicles containing colorless or blood stained
e fluid Malignant Pustule.
 ‘Malignant pustule’ – Satellite lesions filled with serum
or yellow fluid arranged around a central necrotic
lesion which is covered by a black Eschar.
 Also known as ‘Hide Porter’sDisease’.
 Resolves spontaneously, 10-20% of untreatedmay
develop fatal septicemia or meningitis.
HUMAN ANTHRAX – PULMONARY

 Pulmonary anthrax occurs due to inhalation

of the dust or filaments of wool from infected
animals, particularly in wool factories .
 This is also called “Wool – sorter’s Disease”

 A life- threatening hemorrhagic

pneumonia caused by Inhalation of spores.
 HUMAN ANTHRAX –
GASTROINTESTINA
L
 Intestinal anthrax is a rare in man and is found in
those who consume improperly cooked food/
infected meat for e.g.- African Tribes living in
Jungle.

Human anthrax canbe

 Industrial – in meat packing or wool factories
 Nonindustrial – frequent association with
animal handlers likebutchers, veterinarians,
farmers
DIFFERENCE BETWEEN CUTANEOUS AND
PULMONARY ANTHRAX
Features CutaneousAnthrax PulmonaryAnthrax
Other Name Hide porter’s disease (As Wool sorter’s disease (As it is
commonly occurs in dock seen in workers of wool factories
workers carrying loads of hides acquire infection by inhalation of
and skin on their bare backs) dust frominfected wool.

Transmission Cutaneous exposure to spores Inhalation of spores

(enter through abradedski)

Characterized Malignant pustule – the lesion Haemorrhagic pneumonia

by begins as a papule that evolves Bacillus spread by lymphatics or
into a painless vesicle followed by blood, leading to –
the development of a coal-black,
necrotic eschar surrounded by Bacteremia
non-pitting indurated edema.
 The name anthrax, which Haemorrhagic meningitis
means coal, comes fromthe
black colour of the eschar.
 However, it is a non-malignant
condition
DIFFERENCE BETWEEN CUTANEOUS AND
PULMONARY ANTHRAX
Features CutaneousAnthrax PulmonaryAnthrax

Occupation Dock worker, butcher, Workers of the wool factory

al exposure abattoir and farmer

Occurrence Most common(95%) Rare

Prognosis Self- limiting,rarely becomes Fatal

fatal if untreated

Bioterrorism Rarely causes bioterrorism Most common form to cause

bioterrorism
LABORATORY DIAGNOSIS

 A. SPECIMENS–
 Swabs
 Fluids or Pus frompustules
 Sputum &
 Blood from pulmonary & septicaemic anthrax
are generally collected.
 MICROSCOPY
 Gram stained smear from the specimen shows often chain of
largeGram Positive Bacilli.
 Capsule appears as a clear halo around the bacterium by
India-Ink preparation/ staining.
 Capsulesare produced in the presence of bicarbonates
or10-25% CO2
 Sporesare oval and centrally located, non bulging
 Sporesare stained by special stains –Sudan black B.
 MICROSCOP FEATURES
Staining blood films with polychromemethylene
blue:
- Pink or purple amorphous material around blue
bacillus
(M’ Fadyean’s reaction):represents
capsular material – used forthe
presumptive diagnosis ofanthrax in
animals.
CULTURE
 Specimen is inoculated on Nutrient Agar medium &
incubated at 370C for overnight.

 Irregular, round, raised, dull, opaque, grayish white

colonies with a frosted glass appearance.

 Low power – edge of the colony is composed of long,

interlacing chains of bacilli, resembling locks of matted
hair– “Medusa Head Appearance”.

 Gelatin stab culture – “inverted fir tree” appearance,

with slow liquefaction starting from top.
 INVERTED FIR
TREE

Medusa HeadAppearance
-wavy colonies withsmall
projections
 “String of Pearls reaction” – solid medium
containing 0.05-0.5 units of Penicillin/ml, in 3-6
hrs. the cells become large, spherical andoccur
in chains on agar surface, resembling a string of
pearls.

 Selective medium – PLET medium – contains

Polymyxin, Lysozyme, EDTA& Thallous acetate :
to isolate it from mixtures containing other spore
bearing bacilli.
 ANIMAL INOCULATION
 White mouse or Guinea – pigs are inoculated/ injected with
exudate or culture.
 By rubbing contaminated tissue over shaven skin of a
guinea pig.
 Animal dies in24----------------------------------------------------
-------- 48hrs.
 Serology
1. Ascoli’s thermoprecipitation test – to demonstrate
anthraxAg in tissue extracts

2. EIA(using purified anthrax toxinAg)

3. PCRto detect anthrax contamination of animal &

agricultural products
 TREATMENT
Bacillus anthracis treatment :
- Ciprofloxacin or Doxycycline, plus Clindamycin,
and/or Rifampin for60 days.

Antibiotics for post exposure prophylaxis :

Ciprofloxacin for 60days.
Doxycycline for 60 days orAmoxicillin for 60days (given
if strain is penicillin sensitive).

Raxibacumab: It is a monoclonal antibody that

neutralizes anthrax toxin (protective antigen). It is
intended for the prophylaxis and treatment of
inhalation anthrax.
PROPHYLAXIS

 General methods of prevention

1. Improvement offactory hygiene
2. Proper sterilization of animal products
3. Animal carcasses to be buried deep in
quicklime or cremated
PROPHYLAXIS
 Active immunisation of
1. Domestic animals with live attenuated spore
vaccines

2. Persons withoccupational risk (butchers, farmers,

veterinarians) with a cell- free vaccine containing
purified protective antigenas immunogen.

3. 3 doses IM at an interval of 6 weeks with annual

booster injections.
* Anthrax infection in humans give life long
permanent immunity & secondary infections are
very rare.
 ANTHRAX VACCINES
 Original anthrax vaccine – developed by Pasteur
– live attenuated bacilli vaccine – strain rendered
avirulent by the loss of plasmids which encodes
anthrax toxin.

 Live attenuated anthrax spore vaccines-

1. Sterne vaccine – contains spores of a non -
capsulated avirulent mutant strain - loss of
plasmid which controls capsuleproduction.

2. Mazucchi vaccine – contains spores ofstable

attenuated Carbazoostrain.
 Biological warfare
 Anthrax was a long feared as a potential tool in
biological warfare.
 Large epidemics (occasionally)
1. In 1979 – former Soviet Union: due to accidental
release of spores from a military facility engaged
in biological research.

2. In 1980s – Zimbabwe: affected 10,000 persons.

3. In 2001 – USA several died due to mails with

spores of B. anthracis having enhanced virulence.

* Hence the need to develop better human vaccine.

 ANTHRACOID BACILLI
 Aerobic spore bearing bacilli resembling B.
anthracis are called “ANTHRACOID” or
“PSEUDOANTHRAX BACILLI.”

 Some of them are frequent laboratory

contaminants & have to be differentiated from B.
anthracis.

 The main differentiating features between

anthracoid bacilli & B. anthracis are shown in
table.
 DIFFERENTIATING FEATURESBETWEENB. ANTHRCIS&ANTHRACOIDBACILLI
FEATURES B. anthracis Anthracoid bacilli
Motility Non- motile Generally motile
Capsule Capsulated Non –capsulated
Chain formation Long chains Short chains
Colony on NutrientAgar Medusa HeadColony No suchmorphology
Growth in Broth No turbidity Uniform turbidity
Gelatin Stabculture Inverted Fir treeappearance Rapid gelatin liquefaction
& show gelatinliquefaction
Haemolysis on BA Absent Usually well marked
Growth in PenicillinAgar (10 No growth Grow usually
units/ml)
Growth at 450C No growth Grow usually
Susceptibility to Gamma Susceptible Not susceptible
phage
Pathogenicity test inanimals Pathogenic Non-pathogenic
Ascoli’s precipitin test Positive Negative
Fluorescent Antibody test Positive Negative
with anthraxantiserum
BACILLUS CEREUS
 Cause of FOODPOISOINING.
 Ubiquitous in nature.
 Vegetables, milk, cereals, spices, meat & poultry.
 Some spores survive cooking & germinate into vegetative bacilli
which produce ENTEROTOXIN that causes food poisoining.

 Ocular disease :
 It causes severe keratitis and panophthalmitis following trauma to
the eye that may lead to loss of vision.

 Other conditions: Rarely cause systemic infections including –

endocarditis, meningitis, osteomyelitis and pneumonia.
 The presence of medical device or intravenous drug use predispose
to theseinfections.
TYPES OF FOOD POISOINING
 1. SHORT INCUBATION PERIOD TYPE (1-5HRS).

 Characterized by acute Nausea & vomiting, 1-5 hrs

after the meal.

 Diarrhoea is not common.

 It is usually associated with consumption of cooked

rice, usually fried rice from Chinese restaurants.
2. LONG INCUBATION PERIOD TYPE (8-
16)
 Characterised by
 Acute abdominal pain & diarrhoea, 8-16hrs.
after consumption of contaminated food.

 Vomiting is rare symptom in this type.

BACILLUS CEREUS Gastroenteritis
CLINICAL PRESENTATION

DIARRHOEAL EMETIC FORM

FORM

Incubation period > 6

Incubation period < 6
hours Diarrhoea
hours Severe
Lasts 20-36 hours
vomiting Lasts 8-
10 hours
 TYPES O F
GASTROENTERITIS
Bacillus cereus DiarrhealType EmeticType

Incubation period 8-16 hours 1-5hours

Toxin Secreted in intestine(Cl. Performed toxin (formed in

perfringens enterotoxin) diet, similar to Staph. aureus
enterotoxin)

Heat Heat labile Heat stable

Food items contaminated Meat, vegetables, dried Rice (Chinese fried rice)
beans, cereals

Clinical feature Diarrhoea, fever, abdominal Vomiting, abdominal

cramps cramps

Serotypes 2,6,8,9,10,12 1,3,5

 DIAGNOSIS
 Suspected food, faeces & vomitus are cultured on ordinary
media ora special such as –
 MYPA- Mannitol, Egg yolk, Phenol red, Polymyxin agar.
 PEMBA– Polymyxin B, Egg yolk, mannitol, bromothymol blue
agar.
 Spore bearing Gram Positive Bacilli may be seen on smear from
colonies.
 B. cereus is a motile bacilli, non-capsulated, not susceptible to
gamma phage & does not react with fluorescent antibody
conjugate.
TREATMENT
 Disease is mild and self limiting, requiring no specific
treatment.

 Rehydration
 Antibiotics – insystemic infections

 Bacillus cereus susceptible to Clindamycin,

erythromycin vancomycin, aminoglycosidesand
tetracycline.

 It is resistant to penicillin (by producing β-lactamase)

and trimethoprim.
BACILLUS THURINGINESIS

 It is closely related with B. cereus and may

occasionally produce food poisoning.

 It is also used as Larvicidal agent for mosquito

control.
BACILLUS USED AS STERILIZATION
CONTROL

 B. stearothermophilus & B. subtilis both are

used as biological controls for autoclave and
plasma sterilization.

 B. pumilus is used as biological control for

ethylene oxide.
INDUSTRIAL USE OF BACILLUS SUBTILIS
 B. subtilis is used in industries for various purposes –
As cleaning agent (Detergent).

 In paper and textile industries – produces amylase that

breaks down starch.

 For pollution treatment- by breaking downpollutants

(Bioremediation).

 In pesticide industries – for protecting crops against

fungi.

 In food industries – helps in fermentation.

SHORT NOTES

 Malignant pustule.
 B. cereus foodpoisoning.
 Wool sorter’s disease.
 Hide porter’s disease.
 Mc Fadyean’sReaction.
 CLOSTRIDIUM
Characteristics
Gram-positive . obligate
anaerobes capable of
producing endospores which
protect them in harmful
environment . Individual cells
are rod shaped.
The spores are usually wider
than the rods, and are located
terminally or sub terminally.
Most clostridia are motile by
peritrichous flagella.while
others have a capsule like
Clostridium.perfringens
College Of Dentistry - Mosul University 2
CLOSTRIDIUM CONSISTS OF AROUND 100 SPECIES
THAT 
INCLUDE COMMON FREE-LIVING BACTERIA AS WELL
AS IMPORTANT PATHOGENS THERE ARE FIVE MAIN
SPECIES
responsible for disease in
humans.
C. perfringens: gas gangrene; food poisoning
C. tetani: tetanus
C. botulinum: botulism
C. difficile: pseudomembranous colitis
C.Sordellii : can cause a fatal infection in exceptionally rare
cases after medical abortions
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THE SHAPE AN POSITION OF SPORES VARIES IN
DIFFERENT SPECIES AND IS USEFUL THE
IDENTIFICATION OF CLOSTRIDIA
*Central in Cl.bifermentans
*Sub terminal in Cl.perfringens
*Oval or terminal in Cl.tertium
*Spherical and terminal giving
drum stick appearance in Cl.tetani

College Of Dentistry - Mosul University 4

 C. perfringens is a
relatively large Gram-
positive short fat
bacilli with blunt ends.
It is capsulate and
non-motile. Anaerobic.
It grows quickly on
laboratory media on
blood agar ( B –
Haemolytic )

College Of Dentistry - Mosul University 5

 LIQUID MEDIUM FOR
CULTIVATION
COOKED MEAT
BROTH

 Thiglyclolate broth

 CMB contain
unsaturated fatty
acids which take up
oxygen
College Of Dentistry - Mosul University 6
CLOSTRIDIUM. PERFRINGENS
CULTURE & IDENTIFICATION

College Of Dentistry - Mosul University 7

IT DISTINGUISH BETWEEN DIFFERENT

SPECIES OF BACTERIA.
The lactose (milk
sugar), litmus (pH indicator),
and casein(milk protein)
contained within the medium can
all be metabolized by different
types of bacteria.
Milk is the first substrate used to 
maintain bacteria, this test allows
for accurate depiction of bacterial
types. The addition of litmus,
other than explaining the pH type,
acts as an oxidation-reduction
indicator. The test itself tells
whether the bacterium can
ferment lactose, reduce litmus,
form clots, form gas, College Of Dentistry - Mosul University
 THIS TEST IS DONE TO DETECT THE
LECITHINASE ACTIVITY
 The M.O is streaked on the medium containing egg
yolk (contains lecithin)
 The plate is incubated anaerobically at 37 C for 24 h
 Colonies of Cl. perfringens are surrounded by zones
of turbidity due to lecithinase activity and the effect is
specifically inhibited if Cl. perfringens antiserum
containing antitoxin is present on the medium
 `
College Of Dentistry - Mosul University 9
 C. PERFRINGENS NAGLER REACTION

NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes

phospholipids in egg-yolk agar around streak on right.
Antibody against α-toxin inhibits activity around left streak.
College Of Dentistry - Mosul University 10
 Positive Nagler Reaction
Procedure of Nagler Reaction

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 Anaerobic Jar

College Of Dentistry - Mosul University 12

College Of Dentistry - Mosul University 13
 The organisms
associated with gas
gangrene attack soft
tissues by producing
toxins and aggressins,
and some strains of
the bacteria produce
enterotoxins and
cause food poisoning

College Of Dentistry - Mosul University 14

 If there are pieces of necrotic tissue in
the wound, small pieces should be
transferred aseptically into a sterile
screw-capped bottle and examined
immediately by microscopy and culture.
Specimens of exudate should be taken
from the deeper areas of the wound
where the infection seems to be most
pronounced.
College Of Dentistry - Mosul University 15
 VIRULENCE
FACTORS
 toxins –
 alpha toxin – causes RBC rupture,
edema and tissue destruction
 Enterotoxin
 collagenase
 Hyaluronidase
 DNase

College Of Dentistry - Mosul University 16

 CLOSTRIDIUM TETANI
Anaerobic bacteria of the genus species Clostridium it is gram
positive, slender bacillus and it has spherical terminal spores
giving drum stick appearance
It is non capsulated & motile with peritrichus flagella

It produces a potent biological toxin, tetanospasmin, and is the

causative agent of tetanus a disease characterized by painful
muscular spasms that can lead to respiratory failure and, in up
to 40% of cases, death.

College Of Dentistry - Mosul University

 AN INFECTIOUS DISEASE CAUSED BY
CONTAMINATION OF WOUNDS FROM
THE BACTERIA CLOSTRIDIUM TETANI,
OR THE SPORES THEY PRODUCE THAT LIVE
IN THE SOIL, AND ANIMAL FECES

 Infection follows when spores

 become activated and develop
 into gram-positive bacteria that multiply
 and produce a very powerful toxin
(tetanospasmin) that affects the
muscles.

College Of Dentistry - Mosul University

 Tetanus spores are found throughout the
environment, usually in soil, dust, and animal waste.

 Tetanus is acquired through contact with the

environment; it is not transmitted from person to
person.

College Of Dentistry - Mosul University

 THE USUAL LOCATIONS FOR THE BACTERIA TO
ENTER THE BODY:

 Puncture wounds (such as those caused by rusty

nails, splinters, or insect bites.)

 Burns, any break in the skin, and IV drug access sites are
also potential entryways for the bacteria.

College Of Dentistry - Mosul University

1. IT INHIBITS THE RELEASE OF ACETYLCHOLINE THUS
IT 
INTERFERES WITH NEUROMUSCULAR TRANSMISSION.


2. Inhibition of postsynaptic spinal neurons by 

blocking the release of an inhibiting mediator

College Of Dentistry - Mosul University

 Gram +ve stains grow on blood agar media
aerobically
 Inoculation of culture in to 2 mice one is protected
with anti-toxin and the other is unprotected (dies with
typical tetanic spasms )
College Of Dentistry - Mosul University
"C. diff", is a species 
of Gram-positive
bacteria of the genus
Clostridium that
causes diarrhea and
other intestinal
disease when
competing bacteria
are wiped out by
antibiotics.
 Most common cause of nosocomial diarrhea. 

Rate and severity of C. difficile-associated diarrhea 

(CDAD) increasing.

Clostridium difficile is a bacterium that can cause 

symptoms ranging from diarrhea to life-threatening
inflammation of the colon. Illness from C. difficile
most commonly affects older adults in hospitals or
in long term care facilities and typically occurs after
use of antibiotic medication
 characteristic 
features:
Clostridia are 
anaerobic, spore-
forming rods (bacilli).
C. difficile bacteria can be found throughout the 
environment — in soil, air, water, and human
and animal feces. A small number of healthy
people naturally carry the bacteria in their large
intestine. But C. difficile is most common in
hospitals and other health care facilities, where
a much higher percentage of people carry the
bacteria.
 CHAIN OF INFECTION
Infectious Agent
>65 years C.difficile
History of antibiotic use Bowel and
Recent received Contaminated
healthcare environment
Underlying conditions
Reservoir
Abdominal surgery Susceptible Host
Weakened immunity

Contact
transmission
Portal of entry Means of from
Transmission contaminated
Faecal/Oral hands,
equipment or the
environment
Disruption of normal 
colonic flora
Colonisation with C. 
difficile
Production of toxin A 
+/- B
Mucosal injury and 
inflammation
TOXIGENIC STRAINS

PRODUCE 2 MAJOR
toxins:
toxin A 
(enterotoxin)
toxin B (cytotoxin) 
Neutralised by C. 
sordellii antitoxin
Watery diarrhea three or more times a day for two 
or more days
Mild abdominal cramping and tenderness 
Watery diarrhea 10 to 15 times a day 
Abdominal cramping and pain, which may be 
severe
Fever 
Blood or pus in the stool 
Nausea 
Dehydration 
Loss of appetite 
Weight loss 
Pseudomembraneous colitis 

Perforation of the colon 

Sepsis 

Death 
 THE SPECIMEN
Fresh is best (test within 2 hours)
Liquid or loose, not solid
If unable to test within 2 hours, refrigerate
at 4 C for up to 3days
Freeze at -70 C (not -20 C) if testing will
be delayed
Specimen quality will influence test results
 Endoscopy 
(pseudomembranous
colitis)
Culture 

Cell culture cytotoxin 

test
EIA toxin test 

PCR toxin gene 

detection
NOTE : ALCOHOL DOES NOT KILL C. DIFFICILE
SPORES, USE OF SOAP AND WATER IS MORE
EFFICACIOUS
GRAM POSITIVE
OBLIGATE ANAEROBIC BACILLUS
Spores 
Resistant to heat, light, drying and radiation 
Specific conditions for germination 
Anaerobic conditions 
Warmth (10-50oC) 
Mild alkalinity 
 INGESTION

Organism 
Spores 
Neurotoxin 

Wound contamination 

Inhalation 

Person-to-person not documented 

THREE FORMS FOODBORNE WOUND

• Infant
• All forms fatal and a medical emergency
Incubation period: 12-36 hours
 The symptoms of botulism are similar to 
those of Guillain-Barré syndrome, stroke,
and myasthenia gravis.
As a result, botulism is probably 
substantially under-diagnosed.
Serum electrolytes, renal and liver function 
tests, complete blood tests, urinalysis, and
electrocardiograms will all be normal unless
secondary complications occur.
 The incubation period varies according to 
the mode of transmission, rate of absorption
of the toxin, and the total amount and type
of toxin.
Foodborne botulism usually takes 24-36 
hours to manifest itself.
Wound botulism often takes 3 or more days 
to appear.
Inhalation botulism has occurred very rarely, 
but incubation times may range from several
hours to perhaps days, again depending
upon the type and amount of toxin inhaled.
All four types of botulism result in symmetric 
descending flaccid paralysis of motor and
autonomic nerves always beginning with the
cranial nerves. These symptoms are
preceded by constipation in cases of infant
botulism.
Symptoms include: 

Double or blurred vision 

Drooping eyelids 

Dry mouth 

Difficulty Swallowing 

Muscle weakness 
 If left untreated symptoms may expand to 
include paralysis of respiratory muscles as
well as the arms and legs.
Asphyxiation due to respiratory paralysis is 
the most common cause of death in
botulism cases.
 Proper food preparation is one of the most 
effective ways to limit the risk of exposure to
botulism toxin.
Boiling food or water for ten minutes can 
eliminate some strains of Clostridium botulinum
as well as neutralize the toxin as well.
However, this will not assure 100% elimination.
Limiting growth of Clostridium botulinum and 
the production of botulism toxin is an
alternative to their outright destruction.
 NOW MANUFACTURED UNDER THE NAME
―BOTOX‖ 
Experimentally used for treating migraine 
headaches, chronic low back pain, stroke,
cerebral palsy, and dystonias (neurologic
diseases involving abnormal muscle posture
and tension)
Frequent injections allows an individual to 
develop antibodies
Studies carried out to determine feasibility of 
other strains of BoNT
BoNT B manufactured for treatment of 
cervical dystonia in 2000 as ―Myobloc‖
BOTOX INJECTION PATIENT 13 WEEKS AFTER
INJECTION

Sadick, N. and A.R. Herman (2003). “Comparison of Botulinum Toxins A and B in the
Aesthetic Treatment of Facial Rhytides.” Dermatologic Surgery 29:340-347.
CORYNEBACTERIUM DIPTHERIAE
 INTRODUCTIO
N
• Causative agent of Diptheria in humans.
• Small, pleomorphic (club-shaped),
gram-positive bacilli that appear in short
chains (“V” or “Y” configurations) or in clumps
resembling “Chinese letters”.
• Cells contain metachromatic granules
(visualize with methylene blue stain)
• Lipid-rich cell wall contains
meso-diaminopimelic acid, arabino-galactan
polymers, and short-chain mycolic acids.
• Morphology :
• Thin, slender, gram positive, non sporing, non-
motile bacilli with average size 3-6umx0.6-0.8um.

• club shaped due to presence of metachromatic

granules at one or both ends, also called volutin or
babes-Ernst granules.

• Appear in short chains (“V” or “L” configurations) or

in clumps resembling “Chinese letters” or cuneiform
arrangements.
• Special stains like albert [ malachite green &
toludine blue], neisser or polychrome
methylene blue are used for staining.
• The bacilli look green and metachromatic
granules look bluish black by using albert
stain.
• There are 3 biotypes- gravis, intermedius and
mitis.
• Cultural characteristics:
• Media enriched with blood serum or egg.
• Hiss serum water:
• Liquid media containing serum- growth seen as
turbidity and pellicle formation.
• Loefflers serum slope:
• Rapid growth 6-8 hours-small circular, white or
creamy and glistening.
• Tellurite blood agar:
• Potassium tellurite 0.04%- act as selective media
• Black colonies due to reduction of tellurite to
tellurium.
• Colonies appears after 48 hrs.
TELLURITE BLOOD AGAR
• Tinsdale agar
• contains sheep’s blood, bovine serum, cystine
and potassium tellurite is selective medium.
• Brown halo surrounding the colony is
differentiating feature.
• Biochemical reactions:
• Ferment glucose and maltose with acid but no gas.
• Don’t ferment lactose, mannitol or sucrose
• Don’t hydrolyse urea or form phosphatase.
• Gelatin is not liquified
TOXINS:
EXOTOXIN: DIPTHERIA
TOXIN
• Pathogenesis:
• Most common in children of 2-10 years.
• Incubation period-3-4 days
• Mode of transmission- droplet spread
• The diphtheria may be following clinical types:
1. Faucial
2. Laryngeal
3. Nasal
4. Conjunctival
5. Otitic
6. Vulvovaginal
7. Cutaneous mainly around mouth and nose.

Faucial diphtheria is the commonest type.

The toxin has both local as well as systemic effects.

• Local effects:
• Bacilli remain confined to the site of entry
usually upper respiratory tract
• Multiply and produce toxin.
• Toxin causes local necrotic changes along with
superficial inflammatory reactions
• The necrosed epithelium together with
fibrinous exudates, leucocytes, erythrocytes
and bacteria constitute pseudomemebrane.
• Which makes problem in swallowing.
• Systemic effects:

• Toxin diffuses to blood streams and causes toxemia

• The toxin has got affinity for cardiac muscle,
adrenals and nerve endings.
• It acts systematically on the cells of these tissues
• The bacilli themselves do not play any part in the
systemic effects because they neither penetrate in
to the tissue nor pass into blood stream producing
bacteremia.
• Complications:
• Local
• The pseudomembrane may extend to the larynx
which may lead to laryngeal obstruction, asphyxia
and death.
• Systemic
• Diphtheritic myocarditis which may terminate in
heart failure and death.
• Polyneuropathy and post diphtheritic paralysis of
palatine and ciliary muscles
• Degenerative changes in adrenal, kidney and liver
may occur.
• Laboratory diagnosis:

• Isolation of organism

• Demonstration of its toxicity by virulence tests.

• Collection of specimens:
• two Swabs from lesions e.g. throat, nose,
larynx, ear, conjunctiva, or skin.

• Direct microscopy:
• Smears are stained with both gram and albert stain.

• Diphtheria bacilli show beaded slender green rods

in typical chinese letter pattern.
• Culture:
• The swabs are inoculated on the following culture
media:
• Loefflers serum slope:
• Growth appears within6-8 hours.
• Subculture is done on tellurite blood agar and plate is
incubated at 37 c for 48 hours.
• Tellurite blood agar:
• These plates have to be incubated at 37c for at least 48
hours before declaring these as negative, as growth
may sometimes be delayed.
• Blood agar:
• Useful for differentiating streptococcal or
staphylococcal pharyngitis which may simulate
diphtheria.
• Colony morphology and staining:
• Loefflers serum slope: small circular, white or
creamy and glistening.
• Tellurite blood agar: Black or grey coloured
colonies.
• Smears are stained with both gram and albert stain.

• Diphtheria bacilli show beaded slender green rods

with bluish black metachromatic granules, in typical
chinese letter pattern.

• Gram stain is done to identify vincents spirochaetes

and fusiform bacilli.
• Biochemical tests:
• Hiss serum water is used for testing fermentation of
carbohydrates.

• Ferment glucose and maltose with acid but no gas.

• Don’t ferment lactose, mannitol or sucrose

• Don’t hydrolyse urea or form phosphatase.

• Gelatin is not liquified.

• Virulence tests:
• These tests demonstrate the production of
exotoxin by bacteria isolated on culture.
• Virulence testing may be done by:
- In vivo: Guinea pigs and rabbits- by subcutaneous
or intracutaneous.
- In vitro: Eleks gel precipitation test and tissue
culture tests
• Prophylaxis:
• Active immunisation
- Started at 6 weeks of age by toxoid in combination
with pertusis and tetanus vaccine [DPT, Triple
vaccine].
- 3 doses are given by intramuscular route at an
interval of 4-6 weeks.
- Booster dose at 18 months and 5 years.
• Passive immunisation- 500-1000 units of
antitoxin[Anti diphtheric serum,ADS] is
administered subcutaneously.
• Combined immunisation

• Schick test:
• Done to demonstrate circulating diptheria antitoxin.
 TREATMENT:
• Erythromycin (orally or by injection) for 14
days (40 mg/kg per day with a maximum of 2
g/d), or
• Procaine penicillin G given intramuscularly for
14 days (300,000 U/d for patients weighing
<10 kg and 600,000 U/d for those weighing
>10 kg).
• Patients with allergies to penicillin G or
erythromycin can use rifampin or clindamycin.