BACILLACEAE

DR SO EBEDE
Consultant clinical
microbiologist/Lecturer
UNTH/UNN
 Learning objective
• Meaning of bacillacae- shape & chinese letter

• Members
• Medically important disease
• Treatment
• Prevention
• control
 Outline
• Introduction
• Classification
• Characteristics
• Epidemiology
• Risk/ predisposing factors
• Diseases
• Laboratory diagnosis
• Treatment, prevention and control
 BACILLACEAE
• BACILLUS species
• CLOSTRIDIUM species
• CORYNEBACTERIUM species
• LISTERIA species
 Family bacillaceae
• Gram positive bacillus
• Resembles Latin stick / Chinese writing
• Endospore forming- very characteristic
• Habitat- water, soil, animal intestine
• Ubiquitous in nature
 1.) BACILLUS
• Gram positive
• Aerobic- strict or facultative
• Endospore forming
• Primary habitat-soil
• Non-motile rods- B. anthracis / Motile- B. cereus …peritrichous flagella
• Catalase positive
-Practical applications ;
• Bioterrorism/Bio-warfare – B. anthracis
• Validation of sterilization process – spores of B. stearothermophilus, B. subtilis
• Source /production of antibiotics- bacitracin, polymyxin, gramicidin.
• Biological control in folic acid assays –B. coagulans, aflatoxin –B. meganterium, hexachlorophane-
B. subtilis
• Excellent model for study of genetics and development of biotechnological processes
• Wide use as insecticide due to its selective action on insects and environmentally friendly – B.
thurigiensis

Two spp of medical importance

• B. anthracis
• B. cereus
Bacillus subtilis
 Bacillus species:
=Pathogenic
• B. anthracis

=Saprophytic
• B. cereus
• B. subtilis
• B. megaterium
• B. circulans

=Others
• B. stearothermophilus,
• B. coagulans
Two spp of medical importance
•Bacillus Anthracis
•Bacillus cereus
 1.1)Bacillus anthracis
•B. anthracis is the causative agent of anthrax
•Zoonosis/ zoonotic disease.
•Enzootic and epizootic diseases.
•Anthrax is a highly infectious animal disease
that can be transmitted to humans by direct
contact with infected animals (cattle, goat,
sheep) or their products, especially hides and
wool.
 Epidemiology

• Between 20,000 and 100,000 cases are estimated to

occur worldwide annually.
• People at risk to anthrax include:
• People who work in close association with animals
• Herdsmen
• Factory workers [e.g. wool sorters in wool factory]
• Veterinarians
• Abattoir workers
 Morphology
• Bacillus anthracis are blunt/ square ended rods/
bacilli, measures1.0 x3.5μm
• Form endospores- oval and centrally located
• Non-motile
• Capsulated –anti-phagocytic
• Occur singly, in pairs or frequently in long chains
Arranged as:
• single or paired bacilli in clinical specimen
• long serpentine chains and clumps in culture.
• Although spores are readily observed in 2-3 day cultures, they are not seen in
clinical specimens.
• polypeptide capsule -very prominent
consisting of glutamic acid is observed in
clinical specimens but is not produced in vitro
unless special growth conditions are used.
In addition to the capsule, B. anthracis has a
• polysaccharide cell wall antigen and a
• toxin
 Anthrax toxin
Anthrax toxin consists of 3 antigenically distinct
materials, namely:
• Protective antigen (PA)
• Lethal factor (LF)
•Edema factor (EF)
Note : None of the components is active alone,
the combination of protective antigen and either
of the other two antigens have toxic properties.
 Clinical pictures/significance
• Cutaneous anthrax- human infection is usually by
entry of spores through a cut/abrasion on skin
resulting in anthrax.
• Pulmonary anthrax known as Woolsorter’s disease
result from inhaling spores.
• Gastrointestinal anthrax results when the spores
reach the gastrointestinal tract following ingestion of
contaminated animal meal
• B. anthracis bacteraemia can develop from any form
of anthrax.
Cutaneous anthrax
Inhalational anthrax
 Pathogenesis
• For a successful infection, B. anthracis must
invade the host’s innate immune system by
killing macrophages.
• Macrophages have many anthrax toxin
receptors on their plasma membranes to
which the PA portion of the exotoxin
systematically attaches.
• Attachment continues until 7PA-ATK
complexes gather in a doughnut-shaped ring.
• The ring acts like a syringe boring through the plasma
membrane of the macrophage.
• The ring then binds EF and LH after which the entire
complex is engulfed by the macrophages’ plasma
membrane and shuttled to an endosome inside the
cell.
• Once there, the PA molecules form a special pore
that pierces the endosomes’ membranes and lets EF
and LF into the cytoplasm.
• EF has adenylate cyclase activity similar to diphtheria
pertussis toxin increasing intracellular cAMP.
• Toxin activity results in fluid release or formation of
• edema.
• Additionally, LF interferes with a transcription factor
which regulates numerous cytokines and other
immunity genes promoting macrophage survival.
• As thousands of macrophages die, they release their
lysosomal contents leading to fever, internal bleeding,
septic shock and rapid death.
 Clinical Manifestation
• cutaneous form-In humans, accounts for more than 95% of
naturally occuring anthrax
• The incubation period of cutaneous anthrax is 1-15 days.
• Infection is initiated with introduction of spores through a break
in the skin.
• After ingestion by the macrophages at the site of entry, the spores
germinate and give rise to the vegetative cells which multiply
extracellularly and form a capsule and produce the exotoxin.
• Skin infections initially resemble insect bites, then develop into a
papular vesicle and finally into an ulcer with a necrotic center
called eschar. The eschar dries and falls off in 1-2 weeks with little
scaring.
 Treatment of cutaneous anthrax
• Without antibiotic treatment, mortality can be
as high as 20%.
• Therapy is with Ciprofloxacin, Penicillin or
• Doxycycline. Treatment should continue for 7-
10 days with the naturally-acquired disease
or for 60 days in the case of bioterrorism.
• With antibiotic treatment, mortality for
cutaneous anthrax is very rare.
• In inhalational anthrax or woolsorter’s disease,
the spores (1-2μ diameter) are inhaled and
lodged in the alveolar spaces where they are
engulfed by alveolar macrophages.
• The spores survive phagocytosis and germinate
within the endosome.
• The bacteria then spread to the regional lymph
nodes and eventually, the blood stream.
• Pulmonary anthrax results in massive
pulmonary edema, haemorrhage and
respiratory arrest.
• Once the bacteria enter the blood stream, they
begin producing exotoxin.
• The medial lethal inhalation dose for humans
has been estimated to about 8,ooo spores.
• The classic clinical description of inhalational
anthrax is that of a two-phase illness.
• In the initial phase, which follows an incubation period of 1-6
days, the disease appears as a nonspecific illness characterized
by mild fever, malaise, non-productive cough, and some chest
pain.
• The second phase begins abruptly and involves a higher fever,
acute dyspnoea and cyanosis.
• This stage progresses rapidly with septic shock, associated
hypothermia and death occurring within 24-36 hours from
respiratory failure.
• Treatment (with the same antibiotics as for cutaneous anthrax)
is successful only if began before a critical
• concentration of toxin has accumulated.
• Treatment (with the same antibiotics as for
cutaneous anthrax) is successful only if began
before a critical concentration of toxin has
accumulated.
• One reason this form of anthra [pulmonary/inhalational] is so
difficult to treat is that the symptoms appear only after B.
anthracis has already multiplied and started to produce large
amounts of the tripartite toxin.
• Thus, although antibiotics may kill the bacteria or suppress its
growth, the exotoxin can still eventually kill the patient.
• About 16 of the 18 cases of inhalation anthrax reported in the
U.S. between 1900 and 1978 were fatal. About 5 of the 22
cases in 2001 were fatal.
• The symptoms of the gastrointestinal anthrax
appear 2-5 days after the ingestion of undercooked
meat containing spores. They include: nausea,
vomiting, fever and abdominal pain.
• The manifestations progress rapidly to severe bloody
diarrhoea. The primary lesions are ulcerative
enabling B. anthracis to become blood-borne.
• Mortality is greater than 50%.
 Laboratory Diagnosis

Specimens include:
•Materials from the eschar
•Sputum
•Faeces
Media:
•The organism grows well on regular media such as blood agar
(BA), nutrient agar.
•The selective medium is PLET medium. Polymyxin, Lysozyme,
EDTA & Thallous acetate [PLET] are usually added to the regular
medium (e.g. BA) to make it selective for Bacillus.
• Specialized Stain: Mac Fadyean’s stain [comprising
Mercury chloride and polychrome methylene blue]
• The smear is fixed with mercury chloride [a fixative]
and stained with polychrome methylene blue.
• On examination, the Bacilli appear blue in colour,
surrounded by purplish material.
• PCR is the basis for confirming the identity of the
organism.
• Positive Macfadyean’s stain = blue bacilli
surrounded by purplish material
 Prevention and Control
• Vaccination of animals, primarily cattle is an
important control measure.
• However, people with a high occupational risk
such as those who handle infected animals or
their products should be immunized with the
cell-free vaccine obtainable from the CDC
(Centre for Disease Control).
 1.2 ) Bacillus cereus
• Bacillus species other than B. anthracis are
primarily opportunistic pathogens that have
relatively low capacity for virulence
• Of these, B. cereus is clearly the most
important pathogen.
Bacillus cereus cycle
 Epidemiology
• B. cereus, B. subtilis and other Bacillus species
are ubiquitous organisms present in virtually
all environment.
• Isolation of the bacteria from clinical
specimens in the absence of characteristic
disease usually represents insignificant
contamination.
 Bacillus cereus infections
• Of these, B. cereus is clearly the most
important pathogen with
• Gastroenteritis / food poisoning,
• ocular lesions,
• intravenous catheter-related and artificial
devices related sepsis.
Bacillus cereus ocular infections include:
•Severe keratitis
•Endophthalmitis
•Panophthalmitis
=They usually follow traumatic, penetrating
injuries but infections in intravenous drug
abusers have also been documented.
• In patients with traumatic injuries, the
organism can originate from contaminated
soil or on the object penetrating the eye.
• Bacillus Panophthalmitis is a rapidly-
progressing disease that almost always ends
in the complete loss of light perception within
48 hours of injury.
 1.2) Other infections with B. cereus, B. subtilis and
other
Bacillus species include:
• Intravenous catheter and central nervous
system-shunt infection
• Endocarditis, especially in drug abusers
• Pneumonitis
• Bacteraemia
• Meningitis in severely immuno-suppressed
patients
Treatment, Prevention and Control
• Usually, symptomatic treatment is adequate because of the
short and complicated course of Bacillus cereus
Gastroenteritis.
• Treatment of other Bacillus infections is complicated by the
fact that they have rapid progressive course and a high
incidence of multidrug resistance.
• Generally, Vancomycin, Ciprofloxacin, and Gentamycin have
been used.
• Food poisoning can be prevented by the proper refrigeration
of food before serving, when practicable and after cooking.
 2. Clostridium species
Introduction
•They are large, obligate anaerobic , gram-positive ,
blunt ended rods
•Most species motile
•Form endospores
•Spores resistant to chemical disinfectant, UV, boiling
for sometime but not to autoclaving temperature.
•Synthesize the most potent exotoxin known
 Major pathogenic Clostridia and their
associated Diseases.
SPECIES DISEASES
•Cl. tetani Tetanus / lock jaw
•Cl. Botulinum Botulism
•Cl. barati Botulism
•Cl. Butyricum Botulism
•Cl. Difficile Pseudomembranous colitis + antibiotics

•Cl. Perfringes Gas gangrene, soft tissue infections, food poisoning,

necrotic enteritis, septicemia
•Cl. septicum Gas gangrene
•Cl. histolyticum Gas gangrene
•Cl. novvi Gas gangrene
•Cl. sordelli Gas gangrene
•Cl. tertium Opportunistic infection
 Morphological & Identification
• Clostridia form spores which are of wider
diameter than the vegetative cells, i.e. they
bulge.
• The location of the spore varies among the
species occuring centrally, terminally or
subterminally.
• Many species are motile with peritrichous
flagella.
 Habitat
• Found in the soil, aquatic settings, sewage and
intestinal tract flora of animals and humans.
= Epidemiology
• A number of Clostridial species produces
destructive and invasive infections when
introduced into tissues from surgery and
trauma
• Thrives in conditions of low oxygen tension
 Clostridium tetani
• C. tetani is the causative agent of tetanus
• one of the most important killer diseases in
developing countries.
=Types :
a) Neonatal tetanus which results from unhygienic
care of umbilical stump as wells
b) Adult tetanus are
• widely prevalent and are diseases of paramount
public health importance.
 Morphology
• The typical bacteria is long, thin and straight
measuring about 2-5μ in width and 3-8μ in length.
• The bacilli have rounded ends.
• Bacilli strongly Gram positive in young cultures
• but may be gram-negative in older cultures
• Terminally located spores, of greater diameter than the
vegetative cell giving the typical drumstick appearance
• Organism un-capsulated
• Motile by peritrichous flagella.
 Cultural characteristics
• Obligate anaerobe
• optimum temperature for growth of 37°C.
• Like all Clostridia, it has complex nutritional
requirements.
-Blood agar- Haemolysis is evident on blood agar medium and
- Robertson cooked meat agar (RCM) are commonly
used.
• Colonies of irregular, 2-5μ in diameter, glistening
translucent and greenish-yellow
 Sensitivity to Physical & Chemical
Agents
• The spores of C. tetani show variable resistance to
environmental agents.
• Boiling water within 5-15 minutes kills some, but some strains
have to be boiled for up to 3 hours before inactivation.
• Some spores survive dry heat, 5% phenol, 0.1% mercury
chloride.
• spores however are sensitive to1% iodine solution and H2O2.
• The vegetative cells are sensitive to penicillin and
• various other antibiotics including Clindamycin and
Metronidazole.
 Virulence factors

Two products liberated by C. tetani are:

•The classical neurotoxin (tetanus spasmin)
•A haemolysin (tetanolysin) which is heat labile and O2 labile.
•All the symptoms in tetanus are attributable to the neurotoxin.
•Tetanus toxin is one of the most poisonous substances known.
It is heat-labile, being destroyed at 65°C within 5 minutes.
• Treatment of the toxin with formaldehyde results in a toxoid
which is non-toxic but immunogenic
• Tetanus spasmin act by blocking the release of
neurotransmitters (e.g. GABA, glycine) for inhibitory
synapses, thus causing excitatory synaptic activity to
be unregulated.
• This leads to spastic paralysis. It does not affect
Acetylcholine transmission.
• The toxin binding is irreversible so recovery depends
on the formation of new axonal terminals.
 Pathogenesis
• Following wound contamination due to wide distribution of C. tetani
spores [commonly found in soil and animal manures]. The condition
of the wound must be such that an
• Anaerobic environment exist with some dead tissue present [low
tissue oxidation-reduction potential].
• These conditions allow the spores to germinate and the vegetative
bacteria to proliferate and produce the toxins.
• Such conditions are often seen in puncture wounds, other kinds of
wound and conditions resulting in tissue damage may also offer a
suitable environment for the growth of C. tetani.
• An important example of this is the unhygienic care of the umbilical
stump in developing countries which gives rise to the high incidence
of tetanus neonatarum (neonatal tetnus).
 Clinical features

• Early symptoms are muscle stiffness with muscles of the jaw

often being the first to develop spasms. This condition gives
the disease its common name, ‘lock jaw’.
• As the disease progresses, spasms develop in other muscles.
• The spasms may be brief but can occur frequently and can
cause great pain and exhaustion
• Respiratory conditions are many and death rate is high
especially in young children and the elderly.
• In non-fatal cases, recovery takes several weeks but is often
complete.
 Types and manifestations of tetanus
= Generalized tetanus
•[most common]
•Involvement of bulbar and paraspinal muscles
•[trismus/lock-jaw, risus sardonicus, difficulty in
•swallowing, opisthotonus, irritability];
•Involvement of the autonomic nervous system
•[sweating, hyperthermia];
•Cardiac arrhythmia;
•Fluctuations in blood pressure.

=Cephalic tetanus
Primary infection in head particularly ear;
•Isolated or combine involvement of cranial nerves,
•particularly 7th cranial nerve;
•Very poor prognosis.
= Localized tetanus Involvement of muscles in area of
primary injury;
•Infection may precede generalized disease;
•Favorable prognosis.

= Neonatal tetanus Generalized disease in neonates;

•Infection typically originates from umbilical stump;
•Very poor prognosis in infants whose mothers are
non-immune.
 Epidemiology

• C. tetani is ubiquitous being found in fertile soil and

colonizing the gastrointestinal tract of many animals
including humans.
• Tetanus is responsible for many deaths in people
living in underdeveloped areas where vaccination is
unavailable or practices are lax.
• It is estimated that more than 1 million cases occur
worldwide with a mortality ranging from 20-50%.
• At least half the deaths occur in neonates.
 Laboratory diagnosis
=Specimens:
•wound exudates, swabs, tissue removed from wounds.

•=Direct Gram staining: direct smears made from exudates or

swabs and gram stained may show drumstick bacilli. This is not
confirmatory as the bacilli may be found in wound contaminated
with soil but the patient may not have tetanus.

•Direct Immunofluorescent Test: can also be employed for the

demonstration of the tetanus bacilli.
•Conjugated immunoglobulins are commercially available.
 Animal pathogenicity

•Two mice can be used for the test.

•The supernatant of the cooked meat broth culture is
injected intramuscularly into the hind limbs of the
mice, one of which had received a prior (1 hour earlier)
intra-peritoneal injection of 500-1500 units of tetanus
antitoxin.
• Signs of tetanus develop in the test animal and the
protected animal remains normal.
 Treatment, prevention and control

• Tetanus can be prevented by good management of wound.

• Debridement of the primary wound (which may appear
innocuous)
• Use of metronidazole (prophylactic/active use)
• Passive immunization with human tetanus immunoglobulin
and vaccination with tetanus toxoid.
• Wound care and metronidazole therapy eliminate vegetative
bacteria that produce toxin and the antibody bind free
tetanus spasmin molecules.
• Active immunization is achieved by the use of tetanus toxoid.
This toxoid is often adsorbed to the suitable adjuvant. It can
be used alone but often with Diphtheria and Pertussis
antigens as a triple vaccine or even with inactivated polio
vaccine as a quadruple vaccine.
• Toxin-bound nerve endings are protected from antibodies,
thus the toxic effect must be controlled symptomatically until
the normal regulation of synaptic transmission is restored.
• Vaccination with a series of 3 doses of tetanus toxoid and
booster doses every ten years is highly effective in preventing
tetanus.
 Clostridium botulinium
• Cl. botulinum is the causative agent of botulism.
Others include:
• Cl. barati
• Cl. Butyricum
• Botulinism caused by a most potent known neurotoxin
• Causes flaccd paralysis unlike spastic paralysis of tetanus
• Contact with the organism not required as it is pure intoxication
• Heterogenous group of fastidious spore-forming,
• Anaerobic bacilli.
• The organisms are subdivided into four groups I-IV on the
• basis of toxin produced and their proteolytic activity.
• Most human diseases are caused by types I and II strains.
Clostridium botulinium
 Epidemiology
• Worldwide in soil, aquatic enviroments, and
spores contaminate vegetables, meat or fish.
• Under appropriate anaerobic enviroment with
neutral or alkaline Ph , organism germinate
and toxin produced during vegetative phase.
• Produced toxin elaborated in foods, outbreaks
frequently occur in families orother eating
groups.
 Botulinium toxin
• Designated A-G, but human disease is almost always due to types A, B,or E
• Cl. botulinum toxin is a progenitor protein (A-B toxin) consisting of the
neurotoxin subunit (light/A chain) and one or more non-toxic subunits
(heavy/B chain).
• The non-toxic subunits protect the neurotoxin from being inactivated by
stomach acids.
• Botulinum toxin is one of the most poisonous substances known to man.
1mg of the toxin is estimated to kill 30 million mice and lethal dose for
man is 0.1-1.0μg.
• Affects peripheral cholinergic synapses by blocking neuromuscular
junctions and inhibiting release o neurotransmitter, acetylcholine
preventing contraction leading to flaccid paralysis
 Clinical significance

• Three forms of botulism has been identified.

• a) Classical (food-borne)
• b) Infant botulism
• c) Wound botulism
 a.) Classical/food-borne botulism
• Fewer cases of food-borne botulism are seen
annually and most are associated with the
consumption of home canned foods (types A
& B toxins) and occasionally with preserved
fish (type E toxin).
• The food may not show any sign of spoilage
but even a small taste can cause full blown
clinical disease.
 b.) Infant botulism
• Infant botulism is more common and has been associated
with the consumption of food (particularly honey)
contaminated with botulinium spores.
• A cause of floppy baby syndrome
• Occurs when Clostridium botulinium colonizes the large gut of
babys 3-24 wks of age, toxin produced slowly absorbed
• Constipation, feeding problems, lethargy, poor muscle tone
are common early signs
• May cause sudden infant death
 c.) Wound botulinism
• Wound botulism is rare but not unknown
• Occurs rarely when wound become
contaminated with the organism and toxin is
absorbed from that site
• Although the symptoms of the disease are
identical to those of food-borne disease, the
incubation period is generally longer (4 days
or more) and the gastrointestinal symptoms
less prominent.
 Cultural characteristics
• Culture is done on
- egg yolk agar, and
- 2 bottles of cooked meat broth.
• One of the bottles is heated to 80°C for 10 minutes to
eliminate contaminated vegetative cells.
• The cultures are incubated anaerobically at 30°C for 3-5 days
during which periodic screening is carried out for the
presence of toxin in the cooked meat broth.
• Fluorescent antibody test is used to identify the organism
growing in culture.
 Laboratory diagnosis
=Specimens include stool, blood, suspected
food, gastric fluid and wound exudates.
= Direct demonstration of the organism from
the clinical material can be done using
fluorescent antibody procedure.
 Treatment, prevention and control

• Patients with botulism require adequate ventilatory support.

• Elimination of the organism from the gastrointestinal tract
through the judicious use of gastric lavarge and
metronidazole or penicillin therapy
• Use of trivalent botulinum antitoxin [versus toxins A,B &E] to
bind toxins circulating in the blood stream.
• Protective levels of antibody do not develop after disease so
patients are susceptible to secondary infections.
• Disease is prevented by destroying the spores in food when
practicable.
• Disease is prevented by destroying the spores in
food when practicable.
• Preventing spore germination [by maintaining the
food in an acid pH or storage at 4°C or colder] or
destroying the preformed toxin [by heating the
food for 20 minutes at 80°C or above].
• Infant botulism may be prevented by not
including honey in the diet of every young
children.
 Clostridium difficile
• Cl. difficile is responsible for antibiotics-associated
pseudomembrane colitis as well as many cases of antibiotic-
associated diarrhoea
• After introduction to a site, enviroment, dust, beddings,
toilets- become persistently contaminated with spores and
new residents are easily colonized.
Clostridium difficile pseudomembranous colitis
 Morphology
• Cl. difficile is typically a large, rectangular, Gram
positive bacillus measuring 6-8μ in length and 0.5μ in
width.
• The spores are terminal, elongated and slightly wider
than the bacillus.
• Colonies are 2-3mm in diameter, irregular, flat or
slightly raised, semi-translucent and white with a
glossy but rough surface
 Virulence factors and pathogenesis

= virulence factors:
C. difficile produces two toxins:
•an enterotoxin [toxin A]
•a cytotoxin [toxin B]

= Pathogenesis :
•The enterotoxin is chemotactic for neutrophils with infiltration
of polymorphonuclear leucocytes into the ileum resulting in the
release of cytokines,hypersecretion of fluids and development of
haemorrhagic necrosis.
• C. difficile is part of the normal intestinal flora in a minority of
healthy people and hospitalized patients.

• The disease develops in people taking antibiotics because the

agent alter the normal enteric flora permitting the
overgrowth of these relatively resistant organisms or making
the patient more susceptible to the exogenous acquisition of
C. difficile.

• The disease develops when the organism proliferates in the

colon and produce their toxin there.
 Laboratory diagnosis
= specimen :
•Fresh stool specimen is very essential since the vegetative organisms are rapidly killed
on exposure to O2.
•Swabs
•supernatant of the feces can be investigated for the presence of
cytotoxin.
•Culture
• selective medium, CCFA— cycloserine, cefoxitine,fructose agar
facilitates the isolation of this organism.
The sample may be pre-treated with alcohol to reduce the number of non-sporing bacteria.
•The medium is incubated anaerobically and examined after 24 hours.
• cooked meat broth medium can be used as enrichment to increase the yield. After
incubation, subculture is made unto CCFA.
 Treatment, prevention and control

• Discontinuation of implicated antibiotics, e.g.Clindamycin,

Ampicillin is generally sufficient to alleviate mild disease.
• However, specific therapy with metronidazole or Vancomycin
is necessary for the management of serious disease.
• Relapses may occur in as many as 20-30% of patients after
completion of therapy because spores of Cl. difficile are
resistant to antibiotic treatment.
• Retreatment with the same antibiotic is frequently successful
• spores of Cl. difficile are difficult to destroy, contaminate the
environment, a major source of nosocomial outbreaks of Cl.
difficile.
 Feacal transplant

- Feacal transplant
• high recovery rate ~ 92%
• low recurrence rate ~ 6%
• safe
• cost effective
• simple
• fast
• potentially life-saving
- Suitable donors:
• In close contact with the patient, but he or she does not live in the same
household
• Healthy
• Young
• Voluntary
 Indications for feacal transplant
• First serious relapse after a successful
treatment of severe pseudomembranous
colitis
• Third recurrence after a successful treatment
of pseudomembranous colitis
• Treatment-resistant chronic
pseudomembranous colitis, which causes
protein losing enteropathy
 Clostridium perfringes
• Clostridium perfringes causes a spectrum of diseases
from a self-limitpng gastroenteritis to an
overwhelming destruction of tissue gas gangrene
(Clostridial myonecrosis) associated with a very high
mortality even in patients who receive early medical
intervention and some strains also cause a common
form of food poisoning.
• When introduced in tissues, c. perfringes can cause
anaerobic cellulitis and gas gangrene (myonecrosis)
Clostridium perfringes gas gangrene
 Morphology
• Clostridium Perfringes is a large, rod shaped, non-
motile, Gram-positive, encapsulated bacillus with
spores rarely observed in vivo or after in vitro
cultivation.
• ubiquitous in nature, with its vegetative form as part
of the normal flora of the vagina and GIT .
• It grows rapidly in tissue and culture; It is haemolytic
and metabolically active
 Virulence factors
• Clostridium perfringes secrete a variety of exotoxins,
enterotoxins and hydrolytic enzymes that facilitates
its disease processes.
• The production of four major lethal toxins (alpha-,
beta-, epsilon, & iota- toxins) is used to sub-divide
isolates into 5 types (A-E).
• Type A Clostridium perfringes causes most of the
human infections.
 pathogenesis
• Clostridial spores reach tissues either by contamination of
traumatized areas or from the individual’s intestinal tract.
• The spores germinate a low tissue oxidation-reduction potential
and vegetative cells multiply.
• Tissue glycogen is fermented and the large amounts of gas
produced leads to distension of tissue and to interference with
blood supply and of course ischemia.
• Alongside these effects is the secretion of the wide array of
toxins which actions favor the spread of infection..
• Tissue necrosis extends providing an opportunity for increased
bacterial growth, hemolytic anemia leading to severe toxemia
and death.
 Pathogenesis cont.
• Gas gangrene is often caused by a mixture of
organisms―toxigenic Clostridia, proteolytic
Clostridia, various cocci and Gram-negative
organisms.
• Cl. perfringes occurs in the genital tracts of 5%
of women and can cause uterinemyonecrosis
following instrumental abortions.
 Clinical syndrome
a) Gas gangrene (myonecrosis):
•This is a life-threatening disease which onset is
characterized by intense pain that develops one
week after the introduction of Clostridial spores
into tissue by trauma or surgery.
•This is followed rapidly by extensive muscle
necrosis, shock, renal failure and death
frequently within 2 days of initial onset
• Macroscopic examination of muscle reveals
devitalized, necrotic tissue with gas found in tissues.
• Microscopically, abundant rectangular gram-positive
bacilli in the absence of inflammatory cells (because
of the activity of other toxins, e.g. lecithinase) are
seen.
• The Clostridial toxin characteristically causes
extensive hemolysis and bleeding.
 b.) CELLULITIS, FASCITIS & OTHER SOFT TISSUE
INFECTIONS
• Cl. perfringes can initiate cellulitis or a rapidly
progressive destructive process in which the organism
spreads through fascial planes causing suppuration and
formation of gas.
•Even though there is no muscle involvement, fasciitis
also has a dismal outcome.
•Surgical intervention is generally unsuccessful because
of the rapidity with which the organisms spread.
c.) FOOD POISONING:
• Clostridial food poisoning is characterized by a short
incubation period of 8-24 hours, a clinical presentation that
includes abdominal cramps, watery diarrhoea but no fever,
nausea or vomiting, and a clinical course of less than 24 hours.
•Disease usually results from ingestion of meat products
contaminated with about 108-109 organisms of type A
Clostridium perfringes.
•The enterotoxin produced after spore germination acts as a superantigen stimulating
the release of cytokines from lymphocytes.
• The refrigeration of food after preparation prevent enterotoxin production.
• Alternatively, reheating the food can destroy the toxin
• d.) NECROTIZING ENTERITIS (Enteritis
necroticans):
• This is a rare acute necrotizing process in the
jejunum,characterized by abdominal pain,
bloody diarrhoea, shock, peritonitis. The
mortality in patients with this disease
approaches 50%.
e) SEPTICAEMIA:
•The isolation of C. perfringes or other Clostridia spp.
From blood cultures can be alarming. However, in
many instances, the isolates are clinically insignificant
and may represent a transient bacteremia or
contamination of culture with Clostridia colonizing the
skin. The significance of the isolate must be viewed in
the light of other clinical findings.
 Laboratory diagnosis
The laboratory performs only a confirmatory role in the
diagnosis of Clostridial diseases, because therapy must
be initiated immediately in affected patients.
• Specimens: material from wound, extract, swabs, pus,
tissue.
• Gram-stained smears will show large rectangular
gram-positive rods.
•There is notable absence of leucocytes.
 C. perfringes grows rapidly
= Media include
• Robertson
•Cooked meat medium,
• Glucose broth,
• Thioglycolate broth or
•Blood agar incubated anaerobically.
- Rapidly spreading growth with marked hemolytic
activity on blood agar is suggestive of Clostridium
 Biochemical Reactions
• On inoculation into a tube of litmus milk, and incubation overnight, a
‘stormy clot’ reaction is observed.
• Nagler reaction: this demonstrates the activity of α-toxin.
The suspected organism is inoculated unto a plate of egg yolk or
serum agar medium(provides lecithin), half of which is smeared with
antitoxin.
• Inoculate and incubate.
• On the half of the plate without antitoxin, a halo/opalescence is
seen caused by the lecithinase action of the α-toxin.
• The role of C. perfringes in food poisoning is documented by
recovering more than 105 organisms per gram of food or 106
bacteria per gram of faeces collected within one day of onset of
disease
Treatment, prevention and control
• Systemic Clostridium perfringes infection such as fasciitis,
gangrene must be treated aggressively with surgical
debridement and high dose penicillin therapy.
• The use of anti-serum against α-toxin and hyperbaric O2 has
been ascribed with some benefits
• Less serious localized Clostridium diseases can be successfully
treated with resistance only rarely reported for species other
than C. perfringes.
• Antibiotic treatment for Clostridial food poisoning is usually
unnecessary.
• Prevention and control of C. perfringes infection is difficult
because of the ubiquitous distribution of the organism.
 3.) Corynebacterium species
• The bacterium causing diphtheria was
described for the first time by Kleb (1883)
• Loeffler in 1884 demonstrated its aetiological
significance
• Therefore and also known as Kleb-Loeffler’s
bacillus or KLB.
Five(5) medically important Corynebacterium species
causing human diseases and include

• Corynebacterium diphtheriae - diphtheria

• Corynebacterium haemolyticum - pharyngitis
• Corynebacterium xerosis- endocarditis
• C. pseudotuberculosis- TB like illness
• Corynebacterium ulcerans - pharyngitis
 Corynebacterium diphtheriae
• Corynebacterium diphtheriae is the causative agent of diphtheria.
• It is a pleomorphic organism.
• The bacterium measures 3-6 μm × 0.6-0.8 μm, is slender and sometimes has
swollen ends giving it “club-shaped” appearance.
• Gram positive, non- motile, non-sporing bacilli, non acid fast
• Generally resemble the Chinese writing as adjacent bacteria lie at various angles to
each other giving V or L appearances which collectively resemble arrangement of
Chinese letters. This unusual arrangement is because of incomplete separation of
daughter cells at the moment of division
• It contains 2-3 granules at the swollen ends which give reddish purple colour when
stained with Loeffler’s alkaline methylene blue.
• The granules are also known as metachromatic granules, Babes Ernst’s granules
and volutin granules. These granules store energy sources for the bacterium
• The Rest of the bacterium is unevenly stained with this dye.
 Epidemiolology
• A disease primarily of non-immune children under the age of 15 years
• Being detected more and more in adults due to waning of the clinical disease which served
as a source of subclinical infection
• Infection usually spreads by contact with a patient or carrier or rarely contact with soiled
articles with discharges from the infected patient
• Incubation period 2-5 days

• Humans—both carriers and cases are the reservoir

• Effective antibiotic therapy immediately reduces infectivity
• Passive immunity received transplacentally protects newborns upto 6 months of age
• Disease or subclinical infection, usually, but not always induces life long immunity
• Immunization with toxoids leads to prolonged but not lifelong immunity
• Most important source of infection are asymptomatic carriers
 Culture media
• Corynebacterium require serum for their growth.
• Several media which have been enriched with serum have been used.
• The selectivity of the media can be increased by the addition of sodium
tellurite.
a) Growth on Loeffler’s Inspissated Serum Medium
• Loeffler’s inspissated serum medium is a solid enriched medium without
agar.
• It has been used extensively to grow C. diphtheriae. It gives luxurious
growth of bacterium in a short period of 6-8 hours.
• The colonies are small, circular, creamy and glistening.
• However, differences in colonial morphology cannot be recognised on it.
• This medium is of great use to obtain large growth in short periods. The
morphology of the bacterium is best developed in this medium.
B) Growth in Hiss's serum water
• The organism grows easily in this liquid medium.
• A pellicle forms on the surface of the otherwise uniformly turbid growth.

C) Growth on Blood Tellurite Agar Medium

• Mcleod’s heated blood tellurite agar medium is the medium of choice
(selective medium) to study the colony characters.
• Potassium tellurite present in the medium in the concentration of 0.04%
provides it selectivity
 Susceptibility to physical and
chemical agents
• C. diphtheriae is extremely sensitive to heat
and suspensions are killed at 60°C in less than
10 minutes.
• It however, resists dry environment for
months together.
• Most strains are highly susceptible to various
disinfectants and chemotherapeutic agents.
 Diptheria toxin
• The pathogenicity of C.diphtheriae is attributed to release of a potent exotoxin by
the bacteria.
• The production of toxin is dependent upon the presence of tox gene in the
chromosome of the bacterium (Lysogenic conversion).
• In vitro production of this toxin depends largely on the concentration of iron.
• Toxin production is optimal at 0.14 μg of iron per ml. of medium but is virtually
suppressed at 0.5 μg/ml. The factors that control toxin production in vitro are not
well understood.
Properties of Diphtheria Toxin
• Diphtheria toxin is extremely potent and lethal dose for a guinea pig weighing 250
grams is 0.0001 mg.
• With the help of trypsin it can be fragmented into two dissimilar fragments called
A and B
 Mechanism of Action
• Neither fragment A nor B is toxic on its own, even in
very high concentrations.
• Fragment A gains entry into the cell and catalyses
ADP ribosylation of diphthamide, a novel amino acid
on elongation factor 2 (EF2).
• This leads to inhibition of protein synthesis and
deathof cell which clinically manifests as the necrotic
lesion of diphtheria.
• On its own, fragment A cannot enter into cell
cytoplasm. It needs fragment B for the same.
Animal Toxigenicity
• Guinea pigs and rabbits are susceptible to the action of diphtheria toxin
and death results within 96 hours of inoculation of toxigenic strain or
toxin itself
Formation of Toxoid
• The diphtheria toxin can be converted into toxoid wherein it retains its
immunogenicity but loses the virulence by prolonged storage, incubating
it at a temperature of 37°C for 4-6 weeks or by subjecting it to the action
of formaldehyde or acidic pH.
• This toxoid is extensively used to induce active immunity in children
against diphtheria
 Terms used in Relation with Toxins and Antitoxins

• Minimum lethal dose (MLD): of the diphtheria toxin is defined as the least amount of the
toxin required to kill a guinea pig weighing 250 grams within 96 hours after subcutaneous
inoculation.
• One unit of antitoxin: was defined as the smallest amount of anti-toxin required to neutralise
100 MLD of toxin.
• LO (Limes null) dose of the diphtheria toxin is the largest amount of toxin that when mixed
with one unit of anti-toxin and injected subcutaneously into a 250 gram guinea pig will on
average produce no or minimal local oedema.
• L+ (Limes tod): dose of diphtheria toxin is the smallest amount of toxin that when mixed with
one unit of anti-toxin and injected subcutaneously into a 250 gram guinea pig will on an
average kill the animal within
96 hours.
• Minimum reacting dose (MRD): is the least amount of toxin that when injected intradermally
in a guinea pig, causes an erythematous flush 5 mm in diametre visible after 36 hours.
• Lf unit: The flocculating or Lf unit of diphtheria toxin is the amount of toxin which flocculates
most rapidlywith one unit of anti-toxin. It is the only method used
for titration of toxoids.
 Pathogenesis
• Man (clinical case and asymptomatic carrier) is the only source and reservoir of C.
diphtheriae.
• In classical diphtheria the site of infection is nasopharynx. The
• bacilli multiply here and produce exotoxin.
• The toxin causes local necrosis.
• The combination of cell necrosis and exudative inflammatory response leads to accumulation
of red blood cells, necrosed cells, bacteria and fibrin and these all mesh together to form the
characteristic pseudomembrane which is white to grey in colour. This membrane first appears
on tonsils, and may spread to larynx and trachea
• Toxaemia and systemic manifestations of diphtheria result due to absorption of toxin from
the site of membrane
• . Toxin can result in death of cells because of damage to the protein synthesis.
• Clinical manifestations and death are usually due to neural and cardiac involvement
• Primary cutaneous involvement is also not infrequently seen in diphtheria.
• Other sites where diphtheritic membrane can be formed include lips, conjunctivae, ears,
vagina and rarely uterine cavity
 Clinical manifestations
The clinical features include:
• High fever due to toxaemia
• Pain in the throat because of the stretching
• membrane.
• Very severe cases of diphtheria are often termed as
malignant or hypertoxic in which there is striking cervical
adenopathy (bull neck), extreme toxaemia and a poor
response to antitoxic treatment.
• Death in diphtheria occurs due to circulatory failure
 Diagnosis
• Clinical diagnosis:
• The laboratory diagnosis is based upon:
a. Demonstration of organism
b. Isolation of organism and
c. Confirmation of toxigenicity of isolate.
• Clinical Sample:
• Collection and Transportation
• Two throat swabs are collected from the patients pseudomembrane in the throat
and preferably peel off a part of it
- one for making smears for Gram and Albert staining and
- the other for culture purposes.
Collection ofthroat swabs requires good illumination and hence tongue is depressed
using a spatula while taking the sample.
Swabs are immediately transported to the laboratory
Demonstration of Organism
• Two smears are prepared using one of the throat swabs on two clean, grease free glass slides.
• One smear is stained with Gram’s stain and the other with Albert’s stain.
• These slides are examined immediately under the microscope and characteristic features of C.diphtheriae
observed.
• These are:
a. Thin, slender, gram-positive bacilli with clubbing at ends
b. Metachromatic granules (seen better with Albert’sstain)
c. Bacilli arranged at acute angles giving the appearance of Chinese letters or cuneiform writing
The Gram’s stained smear is examined first. The Albert’s stained smear is examined only if Gram’s staining
shows gram-positive bacilli. Since some of the gram negative bacilli such as E. coli and P. aeruginosa also
contain metachromatic granules, examining only
• Albert’s smear for such bacteria can give false positive diagnosis of diphtheria.
Isolation of Organism
• The second throat swab should be used to culture on different media. The advantages and disadvantages
of various media that should be employed are given
 Host susceptibility testing
Schick Test
• It is no longer in use and is being described for academic interest only.
Principle
• Schick test operates on the principle that when diphtheria toxin is injected intradermally into a susceptible person, it causes a local reaction, while in
an immune individual, no reaction ensues as the toxin is neutralised by the antitoxin in circulation.
• This test was introduced by Schick in 1913 and is performed to assess the immunity against diphtheria in children above2 months of age.
• The test comprises of injecting intradermally 0.2 ml of diphtheria toxin which contains 1/50 MLD of toxin in the left forearm. Similar dose of heat
inactivated toxin is injected in the right forearm.
• Readings are taken after 24-48 hours and then after 5-7 days of inoculation. Any of the following types of reactions may be observed:
• In negative reaction there Is no reaction of any kind in either forearm. This indicates that person is immune to diphtheria and the antitoxin
concentration in the serum of the individual is 0.01 unit or more/ml.
• In positive reaction an erythematous reaction appears in the test arm within 24-36 hours (1-3 cm diameter) and persists for 7 days whereas there is
no reaction on the control arm. This status is indicative of susceptibility of the individual to diphtheria.
• The pseudoreaction develops in both the arms in less than 24 hours, is not sharply circumscribed and usually fades away within four days. This is also
indicative of immunity to diphtheria.
• In combined reaction both the arms show reaction during first 24 hours, after which in test arm, reaction continues to develop whereas in control
arm it fades.
• By fourth day, a clear distinction can be seen in two arms.
• This status is indicative of susceptibility to diphtheria.
• Similarly a localised swelling may occur after the subcutaneous injection of diphtheria toxoid in some individuals.
• This test is known as Moloney test and such individuals should not be given injections of diphtheria toxoid since:
a. They may have violent local or systemic reaction to injection
b. Most are already immune
c. The test itself would have stimulated more antitoxin production.
 Treatment
• The mainstay of treatment are diphtheria anti-toxin
and antibiotic therapy at the first clinical suspicion
without waiting for the laboratory confirmation.
• The dosage of ADS varies between 20000 to 100000
units.
• Alongwith this a course of penicillin therapy is given.
• For treatment of carriers, erythromycin is more
effective
 Immunization
• Active immunization using diphtheria toxoid is the mainstay.
• Passive immunization alongwith antibiotic therapy is given in the clinical cases.
• Diphtheria toxoid is usually given in children as a triple vaccine “DPT”.
• 10-25 Lf units of diphtheria toxoid (indicated as Ddose) is used and for older children and
adults smaller dose of 1-2 Lf units (indicated as d dose) is used.
• Three (3) doses are given with an interval of 4 weeks between the doses beginning at 6
weeks of age followed by boosters a year later and at school entry. ADS, after skin testing, is
given in clinical cases.
• Ideally all individuals recovering from clinical disease should
• receive active immunization.
 OTHER CORYNEBACTERIA

• Apart from C. diphtheriae, four other species

of this genus are known to produce diseases
in human beings.
• Corynebacterium haemolyticum - pharyngitis
• Corynebacterium xerosis- endocarditis
• C. pseudotuberculosis- TB like illness
• Corynebacterium ulcerans - pharyngitis
 4.) Listeria monocytogenes
• Short, Gram positive, non-sporing bacillus
•Motile ( tumbling motility) with peritrichous flagella @ 22-
300Cand non-motile @ 370C
•Selective incubation @40C on Mueller Hinton agar, blood agar
and MacChonkey agar.
•Growth at @40C
•B haemolysis on blood agar
•Catalase positive
•Acid production from glucose, trehalose, salicin but not from
lactose
•Acetoin production +
 Common infections of Listeria
monocytogens
• Neonatal sepsis
• Neonatal meningitis
• Spontaneous abortion or stillbirth
• Sepsis in immunocompromised patients
• Meningitis in immunocompromised patients
• Puerperal sepsis.
• Listeriosis

• Of all the above mentioned, meningitis is the most common presentation and pregnant
women, newborns, or organ transplant recipients are most likely to be involved.
 Listeriosis
• Listeriosis is a serious but preventable and treatable food-borne poisoning due to contamination by
Listeria monocytogene.
• Food borne listeriosis one of the most serious and severe foodborne diseases but unlike mostcommon
foodborne disease causing bacteria, that due to Listeria monocytogens multiply at low refrigeration
temperature.
• It is relatively rare disease with 0,1-10 cases per million (WHO)
• Pregnant women, elderly, immunocompromised individuals are at risk
• High risk food includeready to eat food, deli meat, smoked fish, meat abacha cure and fermented meals,
saucages, soft cheese, fishery products and vegeatables
• Vegeatables may be contaminated by the offending agent
• Listeria monocytogens widely distributed in nature soil, water, animals feaces
• Foods mostly associated with listeriosis include long shelf live under refrigeration, foods consumed
without further treatment eg frankfurters, smoked salmon, unpasterized milk and ice cream
• Two (2) main types : Invasive and non- invasive (febrile listerial gastroenteritis) listeriosis
• Non- invasive (febrile listerial gastroenteritis) listeriosis – mild form, affecting otherwise heaithy people
with diarrhoea, fever ,headache and myalgia. Outbreak follows ingestion of high dose of the organism
• Invasive listeriosis is more severe and affect certain high risk group as outlined ealier, characterized with
severe symptoms and high mortality rate 0f 20-30%. Symptoms include fever, myaigia,septicaemia,
meningitis. Incubation period 1-2 weeks but can reach 90 days
 Treatment
• All strains are sensitive to ampicillin and this
drug, either alone or in combination with
aminoglycosides,remains the treatment of
choice.
• This organism is resistant to cephalosporins.
 Conclusion/ Summary
Family: Bacillaceae
a.) Bacillus species
•Bacillus anthracis – anthrax
•Bacillus cereus – food poisoning
b.) Clostridium species
•Clostridium tetani – tetanus
•Clostridium botulinium – botulism
•Clostridium perfringes – gas gangrene/ myonecrosis, soft tissue infections, food poisoning, necrotic enteritis & septicemia
•Clostridium difficile- pseudomembranous colitis
c.) Corynebacterium species
•C diphtheriae - diphtheria
•C. haemolyticum - pharyngitis
•C xerosis- endocarditis
•C. Pseudotuberculosis- TB like illness
•C. ulcerans - pharyngitis
d.) Listeria species
Listeria monocytogens, the causative agent of:
•Neonatal sepsis
•Neonatal meningitis
•Spontaneous abortion or stillbirth
•Sepsis in immunocompromised patients
• Meningitis in immunocompromised patients
• Puerperal sepsis.
•Listeriosis
 Thank you for listening

• Questions ??? / Ajuju ???