SYSTEM IC BACTERIOLOGY

STAPHYLOCOCCUS
Dr. Abdelwahid Ali
 They are spherical, gram +ve cocci arranged
in irregular grapelike clusters.
 All staphylococci produce the enzyme
CATALASE which degrades hydrogen
peroxide (H2O2) into O2 and H2O (catalase
positive).
 Habitat; they inhibit body surfaces and
mucous membranes and disseminate in air
and dust.
 SPECIES;
 1- Staphylococcus aureus: it is the main
pathogen, responsible for pyogenic infections.
 It is identified by a positive coagulase test
(coagulase is an enzyme that clots citrated
blood).
 It is present as a commensal bacteria in the
nose, skin (axilla and perineum), throat, gut,
and the mouth (saliva).
 2- Staphylococcus epidermidis: a common
member of skin flora and less common gut
and upper respiratory tract flora, it is
coagulase negative, sensitive to novobiocin.
It causes hospital acquired infections e.g.;
 It enter the blood-stream causing metastatic
infection especially at site of implants.
 It infects intravenous catheters and
prosthetic
 implants e.g. prosthetic heart valves
(endocarditis) , vascular grafts, and prosthetic
joints (arthritis or osteomyelitis).
 Major cause of neonatal sepsis.
 Peritonitis in renal failure patient undergo
peritoneal dialysis through indwelling
catheter
 Most common cause of cerebrospinal fluid
shunt infections.
 3- Staphylococcus saprophyticus: Similar to
Staph. epidermidis (coagulase negative), but
resistant to novobiocin. It causes community
acquired infections e.g. urinary tract
infections particularly in sexually active
young women after Escherichia coli.
 Staphylococcus aureus is the most
pathogenic, and is distinguished from the
others by;
 1- produces coagulase.
 2- ferments mannitol.
 3- hemolysis blood(lysis RBCs).
 While the others species do not.
 Morphology and staining:- gram +ve cocci
arranged grapelike clusters.
 Culture:- grows well on ordinary media
aerobically, and less well anaerobically.
optimal temperature 37C°.
 Colonial appearance:- Staph. aureus has
typically golden colonies. Pigmentation
varies from orange to white. Staph.
epidermidis has white colonies.
 Selective media:- Staphylococci tolerate Nacl
in concentrations of 5-10%.
 Salt containing media are useful in isolating
staphylococci from samples containing other
bacteria.
 Surface antigens:- Staph. aureus has several cell
components and antigens;
 1- protein A; it is the major protein in the cell
wall. It is an important virulence factor, since it
binds to the Fc portion of IgG preventing the
binding of complement. It is also antiphagocytic
 2- Teichoic acids; they are polymers of ribitol
phosphate. Antibodies to these acids develop
in certain staphylococcal infections e.g.
endocarditis.
 3- Surface receptors for specific
staphylococcal bacteriophages permit the
phage typing of strains of Staph. aureus for
epidemiologic purposes.
 Toxin production:- Staph. aureus forms a
large number of extracellular toxins and
enzymes which may play a role in
pathogenicity. These are listed below;
 1-Haemolysin toxins; lyse RBCs of various
animal species.
 2-Coagulase; clots plasma.
 3-Fibrinolysin; digest fibrin.
 4-Leucocidin; kills leucocytes.
 5-Hyaluronidase; breaks down hyluronic acid
which forms the material that binds cells of
connective tissue. This enzyme facilitate the
spread of staph. aureus in tissues.
 6-DNAase; hydrolyses DNA.
 7-Lipase; lyse lipids (lipolytic).
 8-Protein A; antiphagocytic.
 9-Epidermolytic toxins A and B; they cause
epidermal splitting and exfoliation.
 10- Enterotoxins; cause vomiting and diarrhea.
 11- Toxic shock syndrome toxin; causes shock,
rash and desquamation.
 Not all strains of staphylococci produce the
whole range of toxins listed above. Staph.
epidermidis produces few toxins.
 Pathogenesis:- staphylococci cause disease
both by producing toxins and by multiplying
and invading tissues.
 The organism is characteristic of causing
suppurative and necrotic lesions.
 The typical lesion of Staph. aureus infection is
an abscess, but the organism may
disseminate via the blood stream causing
septicemia as well, with metastatic
abscesses.
 It also causes superficial infections such as
conjunctivitis and wound infections.
 It also causes deep infections like septicemia,
endocarditis, pyemia, osteomyelitis and
pneumonia.
 It also causes toxic mediated diseases such as
food poisoning, skin exfoliation and toxic shock
syndrome.
 Staph. epidermidis is of lower pathogenicity but
it is an important pathogen of implanted metal
or plastic devices and prosthetic heart valves.
 Staph. saprophyticus is a cause of urinary tract
infection.
 Laboratory diagnosis:-
 Specimens; according to type of infection i.e. nasal
swab, throat swab, pus from a wound, blood or
tracheal discharge.
 Direct examination:- make smears from the
specimens on glass slides. Stain with gram’s stain
and examine under the microscope to
visualize gram-positive cocci arranged in
grapelike clusters.
 Culture; specimens are cultured on blood
agar
medium. Growth of Staph. aureus usually
yield white or golden-yellow colonies that are
usually beta-haemolytic. Pathogenic Staph.
aureus is coagulase positive, ferments mannitol
and hemolysis RBCs.
 The other two coagulase negative
staphylococci are distinguished by their
reaction to the antibiotic novobiocin, Staph.
epidermidis is sensitive, whereas Staph.
saprophyticus is resistant.
 There are no useful serologic or skin tests
employed in the diagnosis. Staph. aureus can
also be identified by detection of protein A.
 Identification can also be confirmed by tests
for the production of the enzyme coagulase
or DNAase by the organism.
 Typing:- strains of Staph. aureus can be
distinguished by typing, using the method of
bacteriophage typing.
 Treatment:- Saph. aureus is resistant to
penicillin due to production of the enzyme ß-
lactamase which breaks down the B-lactam
 Ring of penicillin. Production of B-lactamase
is plasmid-coded and transferred via
bacteriophage.
 In hospital, Staph. aureus readily appears in
multiple resistant form, i.e. it is resistant to
other antibiotics.
 Antibiotic sensitivity testing is useful for the
clinical strains as it helps in choosing an
antibiotic for treatment. Staph. aureus was
 Shown to be sensitive to antibiotics such as
vancomycin, cephalosporin and flucloxacillin.
 Abscess treatment requires drainage of pus.
 Staph. epidermidis is often resistant to penicillin.
Vancomycin is particularly useful in the treatment of
resistant strains.
 Prevention and control:- there is no effective
immunization with toxoids or bacterial vaccines against
staphylococcus diseases.
 Cleanliness, frequent hand washing and
aseptic management of lesions and wounds
help to control spread of Staph. aureus.
Treatment of carriers is also helpful in
reducing the spread of the organisms within
the community.
STREPTOCOCCUS
 Gram positive spherical or oval cocci, arranged
in pairs or chains.
 General characteristics:-
 Culture;- grow well on blood agar, enrichment
of media with blood, serum or glucose may be
necessary.
 Selective media containing an aminoglycoside
antibiotic or crystal violet inhibit other
bacteria in a mixed culture but permit growth
Chains of cocci

Streptococcus pyogenes
 of streptococci.
Growth is aerobic but sometimes grow better
anaerobically.
Growth is enhanced by 10% Co2.
The major fermentative product is lactic acid
which accumulates in cultures and rapidly stops
growth.
Colonies are usually small.
 Biochemical reactions:-
 Streptococci give a negative catalase reaction.
 Hemolysis;- according to hemolytic activity,
following culturing on blood agar medium,
streptococci are identified as follows;
 1- Beta(β) hemolytic streptococci; form a clear
zone of hemolysis around their colonies, since
complete lysis of RBCs occurs. Hemolysis is
 produced by hemolysin toxins synthesized by
streptococci.
2- Alpha(α) hemolytic streptococci; form a
green zone around their colonies as a result of
incomplete lysis of RBCs in the agar.
3- Some streptococci are not hemolytic and
designated as gamma(ϒ) streptococci.
 A- Beta-hemolytic Streptococci; these are
arranged into groups A to U (known as Lancefield
groups) on the basis of antigenic differences in
the carbohydrate(c) antigen in the bacterial cell
wall.
 In the laboratory, the group is determined by
precipitin test with specific antisera or by direct
immunofluoresce.
 The main medically important Lancefield groups
 with the species responsible for human
disease are as follows;
Group A: Streptococcus pyogenes.
Group B: Streptococcus agalactiae.
Group C: Enterococci (Streptococcus faecalis),
recently named Enterococcus faecalis.
Groups C,E,F,G,H AND groups K to U
streptococci infrequently (rarely) cause human
disease.
 B- Non-Beta-hemolytic Streptococci:-
 Some of them produce no hemolysis and
others produce alpha-hemolysis.
 The principal alpha-hemolytic streptococci
are Streptococcus pneumonae and viridians
group of streptococci.
STREPTOCOCCUS PYOGENES

 (Lancefield group A streptococci)

 It is the most pathogenic species of the
genus, produces a large number of powerful
enzymes and toxins.
 Present as commensal bacteria on the skin
and in the oropharynx of healthy adults, and
more commonly children.
 General characteristics:-
 Culture; grow on blood agar as small, dry
colonies surrounded by B-hemolysis.
 Some strains produce a hyaluronic acid
capsule during the logarithmic phase of
growth and develop mucoid colonies on
blood agar.
 Toxins and enzymes:- strept. Pyogenes
produces the following enzymes and toxins;
 1- streptokinase; a protease which lysis fibrin
in clots and thrombi.
 2- hyaluronidase; attacks hyaluronic acid,
the cement material binding cells of
connective tissue, causing increased
permeability and spread of the organisms
through tissues.
 3-DNAases (Deoxyribonucleases); disrupt DNA
 4- NADase (nicotinamide adenine
dinucleotidase); it kills leucocytes.
 5- erythrogenic toxin; responsible for the
erythematous rash in scarlet fever. It is
produced only by certain strains of streptococcus
pyogenes lysogenized by a bacteriophage
carrying the gene for the toxin.
 6- hemolysin toxins;these lyse RBCs. 2 of them
are produced by group A streptococci;-
 1- Streptolysin O; it is oxygen labile, causes
beta-hemolysis only when colonies grow under
the surface of blood agar. It is antigenic, and
anti-streptolysin (O) antibody (ASO) develops
after group A streptococcal infection. The titre
of ASO antibody is useful in the diagnosis.
 2-Streptolysin S; it oxygen stable, it is
 not antigenic and not employed in diagnosis.
It is responsible for beta-hemolysis when
colonies grows on surface of blood agar.
 All these enzymes and toxins probably
contribute to the invasiveness and
pathogenicity of Streptococcus pyogenes.
 Serotypes:-
 Streptococcus pyogenes (Lancefield group A)
can be divided into types, depending on
 three surface protein antigens related to the
bacterial cell wall, named protein M, R and T.
protein M antigen is the most important and
it is associated with virulence as it inhibits
phagocytosis.
 Antibodies to M protein provides type-
specific immunity. 65 distinct M serotypes of
Streptococcus pyogenes have been identified
(from 1 to 65).
 Pathogenicity:-
 Strept. Pyogenes is the common bacterial
cause of sore throat (known as pharyngitis),
and its complication such as tonsillitis, ottitis
media, sinusitis, mastoiditis and scarlet fever.
 The organisms can enter wounds and
abraded skin causing lymphangitis and
erysipelas (an acute lymphangitis of the skin).
 The organisms also can enter the uterus after
 delivery to produce endometritis and sepsis
(known as puerperal fever).
 Post-streptococcal non-suppurative
complications may occurs, following Strept.
Pyogenes infection. These complications occur
in organs that were not originally infected by
the streptococci, they include:-
 1- Acute glomerulonephritis; occurs 2-3 weeks
after skin infection by certain serotypes of
 Strept. Pyogenes e.g. M protein type 49.
 The disease is initiated by antigen-antibody
complexes on the glomerular basement
membrane.
 2- Rheumatic fever:- it occurs 1-4 weeks
after infection with any serotype of
Streptococcus pyogenes (usually pharyngitis).
 It affects the joints (polyarthritis), and the
heart (carditis).
 Rheumatic fever is due to an immunologic
reaction resulting from cross-reactions
between streptococcal antigens and antigens
of joint and heart tissues. It is an
autoimmune disease.
 Both complication can be prevented by early
and prompt treatment of Strept. Pyogenes
infection.
 Laboratory Diagnosis:-
 Direct examination of smears of pharyngeal
swabs are not diagnostic as viridans
streptococci are members of the normal flora
of the mouth and can not be distinguished
from the pathogenic Strep. pyogenes.
 However stained smears from skin lesions and
wounds that reveal streptococci are diagnostic
Culture of swabs from the infected pharynx or
 lesion on blood agar plates show small,
translucent beta-haemolytic colonies within 18-
48hours. If growth is inhibited by the
antibacterial agent Bacitracin, they are likely to
be group A streptococci (i.e. they are sensitive
to Bacitracin).
Serology:-
Anti-streptolysin O (ASO)titres are high soon
after group A streptococcal infection.
 Antibody titres to DNAase are high in patients
with group A streptococcal skin infections.
 Treatment:-
 All group A streptococci are susceptible to
penicillin G, but neither rheumatic fever nor
acute glomerulonephritis patients benefit from
penicillin treatment after onset of the disease.
 All strains of Strept. Pyogenes are sensitive to
 Penicillin, but there is often resistance to
tetracycline. Resistance to erythromycin is
increasing.
 Prevention:-
 There are no vaccines available against those
streptococcal infections.
STREPTOCOCCUS AGALACTIAE
(lancefield group B streptococci)
 They are normal inhabitants of the female
genital tract, this may be secondary to
anorectal carriage. Human strain are mainly
type 1.
 Culture:-
 Grows on ordinary and bile-containing media
e.g. MacConkey agar. Colonies are usually
beta-hemolytic, but may be alpha or non-
hemolytic.
 Pathogenicity:-
 Causes neonatal meningitis and septicemia.
The new-borne is usually infected when
passing through the birth canal. The
organism is also associated with septic
abortion and puerperal or gynecological
sepsis.
 Diagnostic features:-
 They are Bacitracin-resistant.
 Treatment:-
 Sensitive to pencillin and erythromycin.
 ENTEROCOCCI
 (Lancefield group D Streptococci)
 These organisms were classified as faecal
streptococci. They occur as part of the normal
flora in the gut and faeces. They have now
been placed in a separate genus called
 Enterococcus faecalis and Enterococcus
faecium.
 General characteristics:-
 Oval cocci, usually in pairs and do not readily
form chains.
 Culture:-
 Grow on ordinary and bile-containing media,
heat-resistant and able to grow at 45c°; also
able to grow in the presence of 6.5 Nacl
 (hypertonic medium).
 Colonies:-
 Large and whitish, hemolysis is variable. Form
pink lactose-fermenting colonies on
MacConkey agar.
 Identification:-
 By colonial and cultural characteristics,
antibiotic sensitivity pattern and
demonstration of Lancefield group D antigen.
 Pathogenicity:-
 Causes urinary and biliary tracts infections,
abdominal wound infection and endocarditis.
 Treatment:-
 Enterococci strains are resistant to penicillin,
cephalosporins, vancomycin and
aminoglycosides. Although some strains may
be sensitive to ampicillin, antibiotic
sensitivity
 testing should be performed using several
antibiotics as it helps in choosing an antibiotic
or an antibacterial agent for treatment.
 VIRIDANS GROUP OF
STREPTOCOCCI
 They are ill-defined group of streptococci which
show alpha-hemolysis on blood agar, but
hemolysis is variable and some strains are non-
hemolytic.
 Viridans streptococci are part of the normal
flora of human upper respiratory tract namely
pharynx, throat and around the teeth.
 They are of low pathogenicity, but may
become opportunistic pathogens.
 If they reach the blood stream, after dental
extraction or tonsillectomy or even mouth
trauma, they may cause subacute bacterial
 endocaditis, particularly in patients with
abnormal or damaged heart valves. This type
of infection is of high fatality rate unless
effectively treated with antimicrobial agents.
 Some of the species in the group may cause
dental caries e.g. Streptococcus mutans
synthesizes polysaccharides (dextrans) that
are found in dental plaque and lead to dental
caries.
 Identification:-
 Species in the Viridans Group of Streptococci
do not possess a characterizing Lancefield
group antigen.
 They are usually not identified in the
laboratory and simply reported as
Streptococus viridans.
 Species in the group can be identified by a
range of biochemical tests.
 The principal species in the viridians group
include:-
 1- Streptococcus mutans.
 2- Streptococcus mitis.
 3- Streptococcus salivaris.
 Viridians streptococci are distinguished from
Streptococcus pneumoniae, which is also
alpha-hemolytic and occurs as commensal
bacteria in the same site i.e. the upper
 respiratory tract, by the following tests:-
 1- viridans streptococci are not encapsulated,
while Streptococcus pneumoniae encapsulated.
 2- viridans streptococci are not are not bile-
soluble (bile does not lyse it), while
Streptococcus pneumoniae lysed by bile (it is
bile soluble).
 3- viridans streptococci are not inhibited by
optochin (ethyl hydrocuprein)
 i.e. resistant to optochin, while Streptococcus
pneumoniae inhibited by optochin i.e. sensitive to
optochin.
 4- pneumococci ferment inulin, while viridans not.
 Treatment:-
 viridans streptococci are sensitive to penicillin
and erythromycin.
 Endocarditis caused by these organisms is curable
by prolonged penicillin treatment.
 Patients with abnormal or damaged heart
valves should be given penicillin, as a
precaution measure, before tooth extraction
or dental surgery.
STREPTOCOCCUS PNEUMONIAE

 Is the most pathogen among the

streptococci. It is also known as
pneumococcus.
 It is a normal commensal of the upper
respiratory tract.
 5-50% of the healthy population are carriers
of it, (i.e. they harbor virulent organisms in
their oropharynx).
 General characteristics:-
 Pneumococci are spherical cocci, arranged in
pairs and sometimes form short chains.
 Normally it is capsulated with carbohydrate
antigenic capsule (i.e. the virulence factor),
which inhibits phagocytosis and favours
invasiveness.
 Culture:- in;
 1-Blood agar.
 2-broth enriched with serum or glucose.
 Colonies:-
 Alpha-hemolytic colonies with sunken center
due to spontaneous autolysis of older
organisms.
 Antigenic structure:
 Capsule; contains the polysaccharide
carbohydrate antigen which is type-specific.
 84 capsular types are recognized.
 The standard method for identification is
capsular swelling(known as quelling
reaction).
 It is observed microscopically when a type of
pneumococci are mixed with their specific
antisera.
 C-substance; a cell-associated antigen
common to all pneumococci and consists of
choline teichoic acid.
 Protein M antigen; resembles M antigens of
Streptococcus pyogenes but it is not
associated with virulence.
 Pathogenicity:-
 Pneumococci cause considerable morbidity
and mortality.
 They cause pneumonia, bronchitis,
septicemia , meningitis, infection of the
upper respiratory
 tract such as otitis media, sinusitis, and
conjunctivitis.
 The organism produce no toxins known to
play a role in pathogenesis.
 They produce IgA protease which enhances
their colonization of the mucosa of the upper
respiratory tract.
 The capsule is the virulence factor for
Streptococcus pneumoniae.
 Specific antibody against its capsule
opsonizes the organism, facilitate
phagocytosis and promotes resistance.
 These antibodies develop in humans as a
result either of infection (asymptomatic or
clinical), or of administration of vaccine.
 A proportion of the healthy population
harbor virulent organisms in the oropharynx,
but
 resistance is high in healthy young people.
 The disease results most often when
predisposing factors are present such as
alcohol intoxication, cerebral impairment that
can depress the cough reflex, abnormality of
the respiratory e.g. viral infections or
respiratory tract injury due to irritants.
 Pneumococcal types:-
 Streptococcus pneumoniae exists in about 84
 serotypes (from 1-84), some of them are very
invasive and cause serious infections such as;
 Pneumonia and septicemia (serotypes 1-3).
 Meningitis (serotypes 7 and 12 ).
 Serotypes 6 and 18 are important in children.
 Lab. diagnosis:-
 Specimens; sputum, blood, cerebrospinal
fluid
 Direct examination; sputum smears are
 prepared and stained with Gram’s stain.
 Identification is performed by the quelling
reaction (capsular swelling) using multitypes
antiserum.
 Culture:-
 On blood agar, pneumococci form small
alpha- hemolytic colonies which are bile-
soluble.
 Their growth is inhibited by optochin.
 Culture of blood specimens are positive in
about 25% of pneumococcal infections.
 Culture of cerebrospinal fluid is usually
positive in cases of meningitis.
 Treatment:-
 Most pneumococci are sensitive to penicillin,
but some strains are resistant to it by
unknown mechanism as the organisms do
not
 produce the enzyme β-lactamase.
 Strains resistant to erythromycin and
tetracycline are increasing.
 Prevention:-
 In spite of the efficacy of antimicrobial drug
treatment, the mortality rate is high in elderly
(i.e. over 65 yrs. old), immunocompromized,
or deblitated persons.
 They should be immunized with the
polyvalent (23-types) polysaccharide vaccine
of Streptococcus pneumoniae.
Gram-negative Cocci
genus: Neisseria
 The genus Neisseria contains several
bacterial species, only 2 of them are
important human pathogens;
 1- Neisseria meningitidis; causes i-meningitis
and ii-meningococcemia.
 They are also called meningococcus.
 2- Neisseria gonorrhoea; also known as
gonococcus. It causes;
 i- the sexually transmitted disease gonorrhea
 ii- neonatal conjunctivitis.
 iii- pelvic inflammatory disease.
 General characteristics:-
 The Neisseriae are non-motile Gram-ve cocci.
 Most of them often growing in pairs.
 They are small, resembling paired kidney
beans.
 Meningococci isolated from blood or cerebro-
 spinal fluid have a polysaccharide capsule.
 Neisseriae grow best aerobically in an
atmosphere containing 5-10% Co2, so cultures
are incubated in a candle jar.
 All Neisseriae (pathogenic and commensal)
are oxidase positive.
 Meningococci and gonococci are unable to
grow at room temperature (22c°) and their
growth is inhibited by; i-toxic trace metals and
 ii- fatty acids.
 Therefore they cultured on chocolate agar
[which contain blood heated at 80c°, that
inactivates the inhibitors- (the fatty acids
found in blood agar media)].
 The pathogenic Neisseriae are fragile, they
are autolytic and cultures may die out in a
few days when conditions are unfavorable
such as refrigerator temperatures.
 Strains are best preserved by freezing at -
20c° or by lyophilization.
NEISSERIA MENINGITIDIS

 Types:-
 Meningococci divided into at least 13
serologic groups based on variations in the
antigenicity of their capsular
polysaccharides.
 These serogroups are designated by letters;
A, B,C, X, Y, Z, 29E, W135, H, I, K, L.
 Bacterial strains in serogroups B and C causes
endemic cases of meningitis, while strains in
 group A caused most epidemics of the
disease
 Strains in other serogroups rarely cause the
disease although they are isolated
sometimes from the nasopharynx.
 Virulence factors:-
 1- polysaccharide capsule, is antiphagocytic.
 2- the endotoxin, causes fever and shock.
 3- IgA protease, which cleaves secretory IgA
 helping the bacteria to attach to the mucosa of
the upper respiratory tract.
 4- pili, which facilitate bacterial adherence to
the mucosa.
 Pathogenesis:-
 Human is the only natural host for
meningococci.
 They are transmitted by airborne droplets.
 They colonize the membranes of the naso-
pharynx and become part of the flora of the
upper respiratory tract.
 Carriers are usually asymptomatic, but they
serve as a source of infection for others.
 From the nasopharynx the organisms can
enter the blood-stream and spread to specific
sites, such as the meninges or joints, or be
disseminated throughout the body causing
meningococcemia.
 The important manifestation diseases are ;
 1- meningitis.
 2- meningococcemia (septicemia).
 Meningococcal meningitis is characterized by
fever, headache, stiff neck and an increased
level of polymorphonuclear leucocytes in
cerebrospinal fluid.
 The meningococcemia is accompanied by
high fever, hemorrhagic rash (purpura) and
 shock with spread of the organisms to many
organs.
Involvement of the adrenal glands leads to
hemorrhage and adrenal insufficiency (the life-
threatening Waterhouse-Friderichsen Syndrome).
Laboratory Diagnosis:-
Specimens; nasopharyngeal swab, cerebrospinal
fluid (CSF) obtained by lumbar puncture &blood.
 Direct Examination:-
 CSF is centrifuged, smears are made from the
deposit and stained with Gram’s stain.
 Presence of Gram-ve cocci arranged in pairs
(diplococci), intracellularly with polymorphs
or extracellularly in the exudate, makes a
presumptive diagnosis.
 Meningococci cause purulent infection in the
meninges. The CSF is usually turbid and
contains increased number of blood leucocytes
mostly polymorphs.
 There is an increase in the concentration of
protein and a decrease in the concentration of
glucose in CSF when compared to normal CSF.
 Meningococci may appear in blood smears in
cases of severe meningococcemia.
 Culture:-
 The organisms grow best on chocolate agar
 incubated at 37c° in a 5% CO2 atmosphere.
 Secretions taken by swabs from nasopharynx
are cultured on Thayer-Martin medium (which is
chocolate agar containing antibiotics to inhibit
the growth of the normal bacterial flora). After
growth, the colonies are examined, smears
prepared from them show gram-ve diplococci.
 Identification:- miningococci can be identified
 by biochemical test such as;-
 i-fermentation of maltose.
 ii- oxidase positive test.
 Or by serological test;
 direct immunofluorescence using specific
antiserum.
 Isolation of meningococci from
nasopharyngeal specimens indicates that the
 person is a carrier rather than having mening-
ococcal disease.
 Serodiagnosis:-
 Tests for serum antibodies are not useful for
clinical diagnosis. However, a procedure that
can assist in the rapid diagnosis of
meningococcal meningitis is the
agglutination test, which detects capsular
polysaccharide of the organisms in CSF.
 IMMUNITY;- resistance to the disease
correlates with the presence of antibody to
the capsular polysaccharide.
 Most carriers develop protective antibody
titres within 2 weeks of colonization of the
nasopharynx.
 Immunity is group specific, and so it is
possibles to have protective antibody to one
group of organisms yet be susceptible to
 infection by organisms of the other groups.
 Complement is an important feature of the
host defenses, because persons with
complement deficiencies, particularly in the
terminal component C6 to C9, have an
increased incidence of meningococcal
bacteraemia.
 Epidemiology:- epidemics of the disease are
mostly caused by strains of group A meningo-
cocci.
 Outbreaks of the disease are associated with
the presence of large number of carriers and
a large non-immune population in the
community.
 The carriage rate in normal populations is
about 10-25%, this may rise to 50% or more
in household contacts of patients and during
epidemics in closed communities e.g. military
 camps, schools, etc.
 Crowding and other stress factors such as
dust , lack of ventilation, host weather may
contribute to the disease.
 Treatment:-
 Penicillin is the drug of choice, cefotaxime or
chloramphenicol are alternatives.
 Strains resistant to penicillin have rarely
emerged, but sulphonamide resistance is
 common.
 Chemoprophylaxis for close contacts of a
patient is necessary, rifampicin is the drug of
choice, ceftriaxone or ciprofloxacin are a
alternatives.
 Prevention:-
 Chemoprophylaxis and immunization are
both used to prevent meningococcal disease.
 Neisseria meningitidis vaccines are prepared
 from the capsular polysaccharides of the
organisms.
 Effective vaccines are available from group A
and C, but not for group B as its
polysaccharide is poorly immunogenic in
humans.
 Vaccination is proved to be effective in
preventing epidemics of meningitis and in
reducing the carrier rate.
 Combined vaccines that include
polysaccharides A, C, Y, and W-135 strains are
also available.
Neisseria gonorrhoeae

 It causes disease only in humans.

 It is usually transmitted sexually as it is fragile,
and sensitive to dryness and cool conditions.
 The disease gonorrhea is usually symptomatic
in men, but often asymptomatic in women.
 Virulence factors:-
 1- Pili ;- mediate attachment of the organisms
to mucosal cell surfaces and they are
antiphagocytic. Nonpiliated strains are
 Avirulent.
 2- Protein 2;- one of the outer membrane
proteins. It facilitates attachment of the
organisms to mucosal cell surfaces.
 3- Endotoxin;- lipooligosaccharide (LOS) of
the outer membrane.
 4- IgA protease;- an enzyme produced by the
organism. It hydrolyses secretory IgA which
blocks attachment of the organisms to the
 Mucosal surface.
 Antigenic Structure:-
 Neisseria gonorrhoeae was devided into
nearly 100 serotypes according to variations
in pilin sequences (protein of the pili). These
variations are caused by chromosomal genes
rearrangement.
 Neisseria gonorrhoea was also divided into
about13 serotypes according to antigenic
 variations of protein 2 of the outer membrane
of the bacterial cell wall.
 Pathogenesis:-
 Gonococci cause both;-
 1- localized infections in the genital tract e.g.
gonorrhoea.
 2- disseminated infections with seeding of
various organs such as septic arthritis.
 Examples for local infections:-
 1- gonorrhoea in men; is characterized by
urethritis accompanied by dysuria and a
purulent discharge. Complication are;
 i-epididymo-orchitis.
 ii- prostatitis.
 2- gonorrhoea in women; is infection located
primarily in the endocervix causing purulent
vaginal discharge. Complication are;
 i- ascending infection of the uterine tubes
(salpingitis) which can result in STERILITY.
 ii- pelvic inflammatory disease.
 3- anorecal infections occur in homosexual
men; they are asymptomatic, but abloody or
purulent discharge can occur (proctitis).
 4- pharyngitis due to oral sex practice; which
is asymptomatic in most patients.
 5- ophthalmia neonatorum; is a purulent
 conjunctivitis in a new-born infants occurs as
a result of infection, acquired from the
infected mother, during passage of the new-
born through the birth canal.
 Laboratory Diagnosis:-
 Speciemens; uretheral and endocervical
purulent discharges taken by swabs, urethro-
prostatic discharge obtained after prostatic
massage from males. Swabs from anorectal
 sites from homosexuals, and pharyngeal swabs
from those who practice oral sex.
 Direct examination; smears are made from
the discharge and stained with Gram’s stain.
 The finding of gram –ve diplococci within
polymorphs in a sample of urethral discharge
from a male is sufficient for diagnosis.
 In females, gram-stained smears are often
falsely negative, as the organisms are present
 in small numbers and difficult to identify as
they are mixed with the normal flora.
 Therefor, specimens from women must be
cultured. Culture is also done to diagnose
suspected pharyngitis or anorectal infections.
 Culture;
 Specimens are cultured on gonococcal
selective medium such as Thayer-Martin
medium, which is chocolate agar containing
 antibiotics to suppress the normal flora.
Cultures are incubated at 37c° in a 5% CO2
atmosphere for 48hours.
 After growth, colonies are examined and
smears are made and stained with Gram’s
stain. The finding of an oxidase-positive
reaction in a colony composing of gram-ve
diplococci is sufficient to diagnose Neisseria.
 Identification; specific identification of
 gonococcus can be made by its fermentation of
glucose but not maltose or by direct fluorescent-
antibody staining using specific antiserum to
gonococcs.
 Agglutination test with monoclonal antibody
is also used for identification of gonococcus.
 Blood culture;
 It is performed when hematogenous spread of
gonococci is suspected eg.arthritis,meningitis.
 Treatment:- N. gonorrhea is sensitive to
ceftriaxone, ciprofloxacin, spectinomycin,
erythromycin and other antibiotics.
 Many strains of gonococci are highly resistant
to penicillin due to a plasmid-coded β-
lactamase.
 Gonococcal conjunctivitis in newborns is
treated by erythromycin eye ointment or by
silver nitrate eye drops.
 Immunity:- the main host defenses against
gonococci are;
 1- antibodies (IgA and IgG),
 2- complement, and
 3- neutrophil polymorphs.
 4- antibody-mediated opsonization.
 5- killing the organisms within phagocytes.
 Repeated gonococcal infections are common
 primarily as a result of the many bacterial
strains present due to antigenic variations of
pilli and the outer membrane proteins.
 If a patient developed resistance against a
certain bacterial strain, he may be infected by
another strain.
 Prevention:- no vaccine is available.
Prevention of gonorrhea involves;-
 1- using condoms during sexual intercourses.
 2- prompt treatment of symptomatic
patients and their contacts.
 3- detection of asymptomatic carriers and
treating them with antibiotics.
 4- chemoprophylaxis can be applied as a
preventing measure.
COMMENSAL NEISSERIAE

 Regularly present in the mucous membranes

of the mouth, nose, pharynx and less
frequently in the genital tract.
 Rarely cause diseases.
 They include species like Neisseria pharyngis.
MORAXELLA

 Gram-ve cocci or short bacilli arranged in

pairs, strictly aerobic, non-motile and
oxidase- positive.
 Species:-
 1- Moraxella lacunata :- causes purulent
conjunctivitis.
 2- Moraxella catarrhalis :- classified recently
as Branhamella catarrhalis. Similar to
commensal neisseriae and grows well on
ordinary media. Can be identifird by
biochemical tests.
Causes lower respiratory tract infection.
Majority of strains are beta-lactamase
producers and resistant to penicillin and
ampicillin, but sensitive to tetracycline and
erythromycin.
GRAM-POSITIVE RODS

 There are 4 medically important genera of

gram-positive rods:-
 Bacillus, Clostridium, Corynebacterium, and
Listeria.
 The table below shows the differences in
characters of these genera;
Genus Anaerobic growth Spore formation Exotoxins
imortant
In pathogenesis
Bacillus -ve +ve +ve
Clostridium +ve +ve +ve
Corynebacterium -ve -ve +ve
Listeria -ve -ve
Genus: Bacillus

 Organisms in the genus Bacillus are spore-

forming, gram+ve rods, aerobic and form
chains.
 They are soil saprophytes, but 2 species are
of medical importance;
 1- Bacillus anthracis: causes the disease
anthrax which is common in animals and can
be transmitted to humans (also called
malignant pustule in humans).
 2- Bacillus cereus: causes food poisoning.

 BACILLUS ANTHRACIS
 Morphology:-
 Large, non-motile, rectangular bacilli, usually
arranged in pairs.
 Spores are oval, central, and they are not
formed in tissue, but develop after the
 organism shed from animal body or if it is
grown on artificial media.
 The bacilli are capsulated in the animal body
and on laboratory culture under certain
conditions.
 The capsule consists of the protein
polypeptide of D-glutamic acid.
 Other members of the genus are motile.
 Staining:-
 Gram-positive, spores can be stained by
modified Ziehl-Neelsen method.
 McFadyean’s reaction is used to demonstrate
Bacillus anthracis on blood films.
 A heated-fixed blood film is stained with
polychrome methylene blue. Observe under
the microscope blue bacilli surrounded by
purplish-pink material due to disintegrated
 capsules and indicating a positive reaction
diagnostic of Bacillus anthracis.
 Culture:-
 Aerobe and facultative anaerobe, grows readily
n ordinary media over a wide temperature
range.
 Optimum temerature is 35c°.Best temperature
for sporulation ranges from 25-30c°. Colonies
are large, dense, grey-white and irregular.
 When cultured on blood agar only slight
hemolysis developes around the colonies. This is a
differential feature because other Bacillus species
are markedly hemlytic. In both cultures they
develop a thick pellicle.
 Antigenic structure:-
 1- Anthrax toxin: it is a complex group of toxins,
consisting of three components;
 I) protective antigen. ii) lethal factor.
 3) oedema factor.
 Oedema factor causes oedema and it is
dependent on the protective antigen for its
binding and entry into the cells.
 Lethal factor in the presence of protective
antigen is rapidly fatal for mice.
 2- Capsular polypeptide.
 Viability of bacterial cells:-
 Bacillus anthracis vegetative cells are readily
destroyed by heat, but spores demonstrate a
high level of heat resistance.
 Spores can remain viable for many years in
contaminated soil.
 Pathogenesis:-
 It causes anthrax, which is characteristically
septicemic, with splenic enlargement.
 Humans are infected by spores on animal
products such as wool and hides or by contact
with sick animals. The portals of entry are:-
 1- The skin; it causes a painless ulcer with a
black necrotic eschar (also called malignant
pustule). Local oedema is evident.
 Untreated cases progress to bacteraemia and
death. Known as cutaneous anthrax.
 2- Respiratory tract; by inhalation of spores.
 It causes pulmonary anthrax which is life-
threatening pneumonia.
 3- Oral route; by ingestion of spores or
infected animal products e.g. meat. The
lesions will appear in the gastrointestinal
tract (intestinal anthrax).
 Laboratory diagnosis:-
 Specimens; exudates from oedematous
malignant pustulles, sputum in cases of
 pulmonary anthrax, faeces in gastrointestinal
infection and blood in cases of septicemia.
 Direct examination; smears are prepared from
specimens and stained with Gram’s stain.
 Smears show large gram +ve rods in chains and
surrounded by capsule.
 Spores are usually not seen in smears of
specimen.
 Culture; colonies develop on blood agar
aerobically, colonies are not hemolytic.
 White mouse usually die within 18-36 hours
after injection with small amount of viable
colonies.
 Identification:-
 Bacillus anthracis is identified from other
bacillus species by being;-
 i- non-motile.
 ii- form non-hemolytic colonies on blood agar.
 iii- it is sensitive to penicillin.
 Treatment:-
 Penicillin is the drug of choice.
 Tetracycline is an alternative for patients who
are allergic to penicillin.
 It is also sensitive to ciprofloxacin,
erythromycin and chloramphenicol.
 In cases of pulmonary anthrax, the disease is
usually recognized after the onset of
bacteremia so mortality rate is extremely high.
 Treatment with both antibiotics and anthrax
antitoxin is advised.
 Prevention:-
 1- Erradication of the disease from animals by
vaccination, deep burial of animals dying of
anthrax to prevent the spread of spores.
 2- Sterilization of suspected animal products
such as wool and hides, condemnation of
infected meat.
 3- Persons at high risk should be immunized
with cell-free vaccine prepared from the
purified protective antigen which is one of
the components of anthrax toxin, it is proved
to be immunogenic in humans.
BACILLUS CEREUS

 Bacillus cereus causes food poisoning when its

spores contaminate grains such as rice.
 The spores germinate when rice is kept warm
during cooking.
 Portal of entry is the gastrointestinal tract. The
organism produces enterotoxins (exotoxins)
causing vomiting, nausea and diarrhea.
 Laboratory diagnosis is usually not done and
symptomatic treatment is given.
CORYNEBACTERIUM

 The genus Corynebacterium contains a

number of species, some of them are
pathogenic while others are commensals in
humans (called diphtheroids) and may be
implicated in opportunistic infections.
 Corynebacterium diphtheria is the most
important species. It causes the disease
Diphtheria in humans.it may be carried in the
throat and nose of healthy people.
Corynebacterium diphtheriae

 General characteristics:-
 Morphology and staining;-
 Pleomorphic G+ve rods.
 They are non-sporing.
 Non-capsulated and non-motile.
 They divide by snapping fission so that
adjacent bacterial cells lie at different angles
to each other forming V, L and W shapes, the
 so called Chinese-letters, arrangement of the
bacterial cells.
 Some strains stain irregularly due to the
intracellular deposition of polymerized
phosphate forming the metachromatic
granules (volutin granules) which are nutrient
storage granules. These granules can show up
with special stains e.g. Neisser’s methylene
blue stains them deep blue.
 Culture:-
 Aerobe and facultative anaerobe, optimum
temperature for growth is 37c°.
 Does not grow well on ordinary agar.
 Media containing blood or serum are required
 Selective media are necessary for isolation
from clinical specimens.
 Selective media:- 2 selective media can be
used for isolation of Corynebacterium diphtheriae:-
 1- Loefflers serum medium:-
 The organisms grow rapidly in it, faster than
other upper respiratory tract bacteria present in
the clinical specimens.
 The morphology of the organisms develops well
and shows a typical appearance.
 2- Blood tellurite agar:- after 48 hrs incubation
on
it, Corynebacterium produce grey-black
coloneis due to their ability to reduce
pottassium telluite to tellurium.
 Colonies:-
 The pathogenic strains of Corynebacterium
diphtheriae are those which produce the
exotoxin.
 This exotoxin is called diphtheria toxin and it
is responsible for virulence.
 Non-toxigenic strains i.e. strains which are
unable to produce the exotoxin are avirulent
and are called Diphtheroids.
 The toxigenic strains are divided into 3
biotypes according to shape, size and colour
of their colonies which are related to the
severity of the clinical disease they cause.
 1- gravis; causes severe disease.
 2- intermedius; causes intermediately severe
disease.
 3- mitis; causes a mild disease.
 Other corynebacterium species also grow on
tellurite media forming colonies that can be
confused with those of Corynebacterium
diphtheriae.
 Identification:-
 By biochemical tests and by demonstration
of
toxin production.
 Biochemical tests such as acid production
from a range of carbohydrates are used to
differentiate Corynebacterium diphtheriae
from other Corynebacterium species.
 Gravis strains but not intermedius or mitis
strains ferment strach and glycogen.
 Typing:- phage typing and bacteriocin typing
haveall been used to subdivide strains of
 Corynebacterium diphtheriae for
epidemiological studies.
 Diphtheria toxin:-
 It is iron-free, heat labile protein exotoxin.
 It is produced only by bacterial strains
carrying a bacteriophage.
 The toxin blocks (inhibits) protein synthesis in
human cells.
 It acts locally on the mucous membranes of the
respiratory tract to produce a grey, adherent
pseudomembrane (called diphtheritic
pseudomembrane).
 After absorption into the blood stream, the toxin
acts systemically on the cells of the myocardium,
the nervous system and adrenal glands.
 The toxin can be rendered non-toxic but still
antigenic by treatment with formaldehyde,
 the toxoid vaccine so formed is used in
prophylactic immunization.
 For demonstration of diphtheria toxin Guinea
pig inoculation (protection test) or gel
precipitation using specific diphtheria
antitoxin (Elek test) can be used.
 Pathogenicity:-
 Corynebacterium dephtheriae transmitted by
airborne droplets i.e. inhalation.
 The organisms then reside in the upper
respiratory tract.
 Although exotoxin production is essential for
pathogenesis, invasiveness is also necessary
because the organisms must first establish
and maintain themselves in the throat and
then starts to produce the exotoxin.
 Local inflammation occurs in the throat, with
a fibrinous exudate that forms the adherent
pseudomembrane.
 Inflammatory reactions may be complicated
by extension of the membrane in the larynx
and trachea, causing airway obstruction and
death.
 Serious systemic manifestations, mainly
carditoxic (heart failure) and neurotoxic
(cranial nerve paralysis), may occur following
absorption of the exotoxin into the blood
circulation. The prominent clinical findings are
 1- extension of the pseudomembrane into
the larynx and trachea, causing airway
obstruction.
 2- myocarditis accompanied by arrhythmias
and circulatory collapse.
 3- recurrent laryngeal nerve palsy.
 In tropical countries the organism may cause
skin lesions.
 Laboratory diagnosis:-
 Specimens; throat swab.
 Direct examination:-
 Smears from throat swabs are stained with
Gram’s stain and with Neisser’s methylene
blue. The finding of pleomorphic gram +ve
rods with metachromatic granules, as
revealed by the methylene blue, may be
 Suggestive for Corynebacterium dephtheriae.
Deffinitive diagnosis is performed by culture of
the specimen and isolating the organisms and
then identifying them by biochemical tests and
demonstration of their toxin.
 Culture:-
 On Loeffler tellurite medium.
 Observe grey-black coloies on tellurite medium
composed of bacteria with distinct microscopic
morphology, using Neisser’s stain, and
arranged in Chinese letter manner.
 Toxic production:-
 Production of diphtheria toxin can be
demonstrated by Elek plate in vitro and by
guinea pig inoculation in vivo.
 Schick’s test:-
 This test is used to assess the immune status
of a person against diphtheria.
 It is performed by intradermal injection of 0.1
ml of purified standardized diphtheria toxin.
 If the patient has no antitoxin (antibody), the
toxin will cause inflammation (erythematous
reaction) at the site of the injection 4-7 days
later. If no reaction occurs, antitoxin (antibody)
is present and the patient is immune.
 The test was used for epidemiological purposes
to assess the degree of immunity against
diphtheria in a population.
 Treatment:-
 As the toxin binds rapidly and irreversibly to
cells, and once bound, it can not be
neutralized by antitoxin, diphtheria antitoxin
should be given immediately to suspected
cases of diphtheria. The function of the
antitoxin is therefore is to neutralize the
unbounded toxin in the circulation.
 Treatment with penicillin or erythromycin is
recommended, but is not a substitute for
antitoxin. Antibiotics inhibit the growth of
the organism which excrete the toxin, so
reduce toxin production, and decrease the
incidence of carriers but do not neutralize the
effect of the antitoxin.
 Tracheotomy may be necessary to relieve
laryngeal obstruction in some patients.
 Epidemiology:-
 Source of infection is respiratory secretions
from the throat of infected persons or
symptomless carriers. Also nasal secretions
may be source of infection.
 Spread of the disease is facilitated by close
contact.
 People at risk are those in poor health and
those living in bad housing conditions.
 Prevention:-
 Using diphtheria toxoid vaccine which is
given in combination with tetanus toxoid
vaccine and killed pertussis vaccine, in the so
called triple vaccine (DPT). Immunization
consists of 3 doses (injections) given at 3, 4
and 5 months of age, with a boaster doses at
1 and 6 yrs of age. Vaccination help in
minimizing incidence of the disease.
LISTERIA

 Six species recognized in the genus Listeria,

but almost all human infection are caused by
the species Listeria monocytogenes.

 Listeria monocytogenes
 It is small gram+ve rods arranged in Chinese
letters configuration similar to
Corynebacterium. The organisms are non-
motile at 37c°, but show active motility at 25c°
 In young cultures. This distinguishes it from
Corynebacteria, which are non-motile. The
organism is distributed widely in animals,
plants and soil. From these reservoirs, it is
transmited to humans by contact with
infected animals or their faeces, by
unpasteurized milk and cheese and by
contaminated vegetables. It causes a food-
borne disease.
 General characteristics:-
 Culture;- aerobic and facultative anaerobic,
optimal growth temperature is 37c°. It can
survive and grow at 6c° which facilitates its
isolation from contaminated specimens.
 Colonies;- on blood agar, colonies are non-
pigmented and surrounded by a narrow zone
of complete beta (β) hemolysis, which
resembles the hemolysis of some streptococci.
 Typing;-
 On the basis of somatic (O) and flagellar (H)antigenic
structure, 13 serotypes of Listeria monocytogenes are
recognized. Almost all infections are caused by 3 serotypes,
type 46 being the commonest.
 Toxins;-
 The organism produces Listeriolysin O which is a hemolysin
toxin similar to streptolysin O.
 Pathogenesis;-
 Listeria infections occur primarily in 2 clinical settings;-
 1- in the fetus or newborn as a result of transmission
across the placenta or during delivery.
 2- in immunosuppresed adults, particularly renal
transplant patients.
 The pathogenesis of Listeria is dependent upon the
organism’s ability to invade mononuclear phagocytic
cells and to induce granuloma
formation.
 Pregnancy associated Listeriosis affects the fetus
as a result of transmission of the organisms
across the placenta. It results in abortion or
premature delivery and acute meningitis in the
newborn.
 Transplacental infection also produces
disseminated abscesses or granulomas in
multiple organs of the fetus. Neonatal meningitis
 And bacteremia may result from perinatal
bacteraemia in the mother or from infection aquired
during vaginal delivery.
 Adults with impaired cellular immunity are
particularly susceptible to this intracellular pathogen;
about 70% of patients with listeria infection have
underlying immunisupression. Infection commonly
involves the central nervous system, manifested by
meningitis and rarely encephalitis with multiple focal
abscesses in the brain stem.
 Laboratory Diagnosis;-
 Specimens;- blood and CSF from adults,
meconium and CSF from newborn, swab from
genital tract, placenta and amniotic fluid of
mother.
 Culture;- on blood agar, observe small colonies
surrounded by a narrow zone of beta (β)
hemolysis. Smears from colonies stained with
Gram’s stain show gram+ve rods resembling
 Diphtheroids. Can be differentiated by their active
motility when grown in both culture at 25c° from
non-motile Corynebacteria.
 Identification;-
 By biochemical tests such as sugar fermentation
tests to distinguish it from other Listeria species
 Treatment;- it sensitive in vitro to a number of
antibiotics. Resistance strains are rare.
 Ampicillin is usually used in treatment, often in
combination with gentamicin.
 Prevention:-
 No available vaccine.
 Prevention by;
 1- limiting the exposure of pregnant women
and immunosuppressed patients to sources
of infection such as infected animals and their
products and contaminated vegetables.
 2-pasteurization of milk.
CLOSTRIDIUM

 Clostridia are anaerobic, spore-forming, gram

+ve bacilli.
 Most species are soil saprophytes, but a few
are pathogens.
 Their natural habitat are;-
 1-human and animal intestine.
 2-soil.
 3-water.
 4-decaying material.
 There are 4 medically important clostridium
species;-
 1- Clostridium tetani; causes tetanus.
 2- Clostridium botulinum; causes botulism
(food poisoning).
 3- Clostridium perfringens; causes gas
gangrene, some strain cause food poisoning.
 4- Clostridium difficile; causes antibiotic-
associated pseudomembranous colitis.
 Other Clostridium species of minor
importance include;-
 Clostridium novyi; causes gas gangrene.
 Clostridium septicum;causes gas gangrene
and neutropenic enterocolitis.
 GENERAL CHARACTERISTICS:-
 Morphology and staining;- large rods,
sometimes pleomorphic, filamentous forms
are common. Gram +ve but older are –ve.
 Spores;-
 All species form spores, which may be
bulging i.e. wider than the bacterial body.
 The spores are round or oval in shape and
situated terminally, subterminally or centrally
in the bacterial cell.
 Clostridium perfringens forms spores with
difficulty (sporulate when cultured on alkaline
medium).
 Motility;- all species are motile with
peritrichous flagella except Clostridium
perfringens is non-motile.
 Capsule;-Clostridium perfringens has a capsule
, most other species are non-capsulated.
 Culture;-
 1- Blood agar anaerobically.
 2- Robertson’s meat medium to which
reducing agents are added.
 Addition of an aminoglycoside makes an excellent
selective medium for clostridia.
 Anaerobic requirements is variable e.g.
 C. tetani is strict anaerobic while C.perfringens can
grow in the presence of limited amounts of
oxygen.
 Colonies of various species may be round and
opaque, irregular and translucent or glossy.
 Some species like C.tetani has tendency to swarm
 Biochemical activity;- many species of
Clostridia are saccharolytic i.e. ferment sugars.
This produces reddening of meat particles in
Robertson’s meat medium, with a rancid smell
 Many species are proteolytic i.e. produce
enzymes that digest proteins. This causes
blackening and digestion of meat particles in
Robertson’s meat medium, with a foul smell.
 Most species of Clostridia liquefy gelatin.
 Toxin;- the medically important species produce
several exotoxins.
 Some of them are neurotoxins, others are
enterotoxins.
 Clostridium’s pathogenicity is toxin-mediated.
 The exotoxins of C.tetani and C. botulinum are
amongst the most toxic substances known i.e.
small amounts are enough to cause disease.
 They also produce many toxic enzymes such
as lecithinase, collagenase, hyaluronidase
that lyse proteins and destroy body tissues.
 Antibiotic sensitivity:- sensitive to penicillin,
clindamycin, tetracycline and erythromycin.
 They resistant to aminoglycosides such as
gentamycin, neomycin and kanamycin.
CLOSTRIDIUM TETANI

 Morphology:-
 A long bacillus with round terminal spores giving a
characteristic drumstick appearance.
 Widely spread in nature (soil), and occurs as a
commensal in the digestive tract of many animal
especially horses and shed in faeces, rarely found
in human faeces.
 In the soil, the bacilli sporulated and survive for
long periods.
 Wounds become contaminated with spores, which
under anaerobic condition germinate to produce
vegetative bacilli which form toxins.
 Toxins;- C. tetani produces potent protein exotoxin.
It is mainly released by bacteria and has 2
components which are polypeptide in nature;-
 1- tetanospasmin; it is a neurotoxin. It is the true
tetanus toxin.
 2- tetanolysin; lyses erythrocytes. Its role in
pathogenicity is not know.
 Pathogenicity:-
 The portal of entry is a wound site. Germination
of spores is favoured by necrotic tissue and poor
blood supply in the wound, causing anaerobic
environment.
 Tetanospasmin is produced by vegetative cells
at the wound site.
 C. tetani does not spread beyond the wound but
the toxin is absorbed at the motor nerve endings
 And then travels via the nerves to the anterior
horn cells in the spinal cord, where it binds to
ganglioside receptors and block the release of
inhibitory mediators (glycine and γ- aminobutyric
acid) that control motor nerve impulses at spinal
synapses. This will lead to severe muscle spasms.
 Mortality rate is high due to exhaustion and
respiratory failure (asphyxiation) despite
intensive therapy.
 Clinical features:-
 Severe and painful muscle spasms, the masseter
muscles are often affected by rigid contraction,
causing “lockjaw” which prevents the mouth from
opening. Characteristic facial grimace is produced
by spasm of the facial muscles. The body becomes
arched, as the spasm progress, with only the
patient’s head and heels touching the bed.
 The incubation period is about 5-15 days. Wound of
the face, neck and upper extremities are more
dangerous than those of the legs and feet, they are
associated with more severe disease and a shorter
incubation period. The elderly are particularly at risk.
 Antigens:-
 C. tetani strains have the same somatic (O)
antigen but differ in their flagellar (H) antigen.
 Different strains produce one antigenic type of the
toxin tetanospasmin but they distinguished
by their (H) antigens.
 Laboratory Diagnosis:-
 The diagnosis is often clinical, attempts to
confirm it bacteriologically frequently fail. C.
tetani is isolated from the wound in 30%.
 Specimens:- swab or exudate from wound.
 Direct Examination:-
 Smears are prepared from wound exudate,
 Then stained with Gram’s stain. Examine
microscopically for characteristic gram +ve
bacilli with round terminal spores (drum sticks)
 Culture:- on blood agar or aminoglycoside
blood agar which contain reducing agents.
Observe typical translucent spreading colonies
 Identification:-
 By biochemical tests, and by demonstration of
the exotoxin by inhibition of hemolysis on
 blood agar by specific antibodies against the
exotoxin (antitoxin). Production of the exotoxin is
confirmed by demonstration of mouse
pathogenicity and its prevention by antitoxin in
the mouse protection test.
 Treatment:-
 Supportive; artificial ventilation, with muscle
relaxants to control spasms, excision and cleaning
of wound.
 Antitoxin:-large doses administered intravenously
to neutralize toxin. It has no effect on toxin already
bound to ganglioside receptors in the nerve cells.
 Antibiotic:- penicillin or tetracycline, to prevent
bacterial multiplication and further toxin
production.
 Prevention:- tetanus is prevented by active
immunization in childhood with tetanus toxoid
vaccine (formaldehyde treated toxin) which is
incorporated in the Triple vaccine (DPT).
 When trauma occurs, the wound should be
cleaned and debrided.
 If the wound is contaminated, tetanus
immune globulin (antitoxin), as well as
tetanus toxoid booster dose, should be given
and penicillin administered to prevent further
bacterial multiplication.
 Tetanus immune globulin (tetanus antitoxin)
is made in humans to avoid serum sickness
reactions that occur when antitoxin made in
horses is used.

Clostridium botulinum
Transmission:- cl. Botulinium causes botulism
which is a rare but severe food intoxication in soil
and water and contaminate vegetables and meat.
When these foods are canned without adequate
sterilization, spores survive and germinate in the
anaerobic environment of the container.
 Toxin is produced (botulinum toxin) within the
canned food and ingested preformed. The
highest-risk foods include alkaline vegetables
such as green beans and smoked fish.
 Spore germination and toxin formation are
inhibited by a low pH (acidic).
 The toxin is heat-labile and is inactivated by
boiling at 100c° for several minutes. Thus, butulisim
can be prevented by sufficient cooking.
 Toxin:-
 Botulinum toxin is a protein exotoxin, more
potent than that of Cl. tetani. There are seven
toxin types designated as A,B,C,D,E,F and G
according to variations in their polypeptide
sequence.
 They are neurotoxins which are serologically
distinct but pharmacologically similar toxins.
 Human botulism is usually due to toxin types A,
Band E.
 Pathogenesis:-
 Botulinum toxin after its ingestion, is absorbed
from the gut and carried via the blood to
peripheral nerve synapses where it blocks release
of acetylcholine at neuromuscular junctions.
Symptoms are neurological rather than intestinal
including weakness and paralysis, diplopia,
dysphagia, vomiting, constipation, and sometimes
difficulty in speaking. The disease is often fatal,
death due to respiratory failure.
 Botulism can also occurs when the spores
contaminate a wound (wound botulism) or when
they are ingested (infant botulism).
 Laboratory Diagnosis:-
 The organism is usually not culture. Diagnosis is
done by demonstration of botulinum toxin in
suspected food, patient’s serum and faeces
by inoculation of mice. Observe paralysis and death of
mice when specimens are positive.
 Identification of the toxin is performed by mouse
protection test. Test mice are protected by antitoxins to
toxin types A, B, E.
 Protection by the corresponding antitoxin identifies the
type of toxin present.
 Treatment:- antibiotics are of no value.
 Treatment is supportive, with artificial ventilation and
the administration of antitoxin to neutralize absorbed
toxin. Trivalent antitoxin to type A,B&E
 Control:- proper sterilization of all canned
foods, a temp. that will kill the heat-resistant
spores of Cl. Botulinum (120c°for 20minutes)
is essential. Canned food must be adequately
cooked to inactivate the toxin. Swollen cans
must be discarded.
CLOSTRIDIUM PERFRINGENS
(or welchii)
 Cl. Perfringens causes 2 distinct diseases, gas
gangrene (also known as myonecrosis) and food
poisoning.
 General characteristics:-
 Gram +ve bacilli, non-motile and capsulated.
 Spores are oval and centrally located. Spores develop
when the organisms are cultured on alkaline medium.
 Culture:- most strains grow well on blood agar
anaerobically, producing hemolytic colonies, but
some strains are non-hemolytic.
 Optimum temp. for growth is 37c°.
 Biochemical Activity:-
 Mainly saccharolytic. In tube cultures of litmus
milk, a characteristic stormy clot is formed due
to the production of acid and large amounts of
gas (known as stormy fermentation).
 Typing:- Cl. Perfringens can be divided into 5
types A,B,C,D and E on the bases of types of
toxins it produces. Type A is the major human
 Pathogen; the other types are important
pathogens of domestic animals.
 Toxins:- strains of Cl. Perfringens produce,
during active multiplication, exotoxins with
necrotizing (cytolytic), hemolytic or lethal
properties. In addition; enzymes such as
collagenase, proteinase, deoxyribonuclease
and hyaluronidase which are elaborated by
the growing organisms, cause accumulation
 of toxic degradation products and gases in the
affected tissues. These toxins include;-
 1- Alpha toxin; an enzyme causes cell lysis due to
lecithinase action on the lecithin in mammalian
cell membranes. It is lethal to cells causing
necrosis and hemolysis.
 2- Theta toxin; it is a protein causing lysis of RBCs.
Responsible of intravascular hemolysis associated
with Cl. Perfringens bacteraemia.
 3- Enterotoxin; it is produced by certain strains
 of Cl. perfringens type A. It is a protein produced
only during sporulation and not during vegetative
growth i. e. spore-coat protein. It causes acute
food poisoning.
 Pathogenesis:-
 Cl. perfringens spores are located in the soil and
the vegetative cells are members of the normal
flora of the colon and vagina.
 1- Gas gangrene; occurs when spores of the
organism gained access into wound.
infection with Cl. perfringens is favored by
extensive wounding in which necrotic tissue,
foreign bodies, and impairment of blood supply to
the wound occur. All these factors develop
anaerobic media which favors the multiplication
of Cl. perfringens and its production of toxins,
particularly alpha toxin which causes tissue
damage and spread of infection in the wound. This
may be followed by toxemia, intravascular
haemolysis and shock.
 Gas gangrene lesions are characterized by edema,
blackening of tissue, and a foul-smelling wound
exudate. Palpable crepitation may be detected under
the skin due to gas production by clostridia degradative
enzymes.
 Cl. Perfringens causes the majority of cases of gas
gangrene (65%). The remainder of cases are caused by
other species of clostridia such as Cl. novyi and Cl.
septicum.
 Food Poisoning; Cl. perfringens is normally present in
numbers of 10³- 10 per gram of
 Direct gram film; smears of tissue and exudate
show large gram-positive rods. Spores are not
usually seen.
 In clinical material, Cl. Perfringens is usually
capsulated but dose not form spores.
 Culture; perfomed anaerobically on blood agar
and on aminoglycoside blood agar, or aerobically
in Robertson’s meat medium which contains
reducing agents. Colonies are round, opaque and
faeces of a normal person. Food poisoning occurs when
the person ingests a food containing 10 organisms per
gram of food (infectious dose).
 Cl. Perfringens produces an enterotoxin during its
sporulation in the gastrointestinal tract.
 The disease is characterized by diarrhoea with cramps
and vomiting.
 Laboratory Diagnosis:-
 Specimens; pus and exudate from wounds, debris of
infected tissues or muscles. Some of the infected food
in case of food poisoning.
surrounded by a zone of beta-haemolsis on blood
agar.
 Identification:- Cl. Perfringens in culture is
identified by its characters; non-motile,
capsulated, formation of zones of beta-
haemolysis around colonies on blood agar and by
its stormy-fermentation of lactose in milk.
 Identification is confirmed by testing ability of the
organisms to produce alpha toxin (lecithinase).
 Laboratory Diagnosis of food-poisoning:-
 No assay for the toxin is made. Large numbers
of Cl. Perfringens is usually isolated from the
infected food.
 Treatment:-
 Cleaning and debridment of affected tissue.
 Amputation of affected tissue should be
performed when it is necessary. Penicillin is the
antibiotic of choice, usually given in large doses.
 Antitoxin could be used although it is of doubtful
value. Hyperbaric oxygen, to reduce anaerobiasis
in tissues, has been reported to be effective.
 Prevention:- wounds should be cleaned and
debrided. Penicillin may be given for prophylaxis.
 Treatment of food poisoning:- symptomatic
treatment is given, no antibiotics are
administered as symptoms resolve in 24hours.
Adequate cooking of food to kill the
organisms(prevention).
  CLOSTRIDIUM DIFFICILE
 Cl. Difficile is part of the normal flora of the
gastrointestinal tract in about 3-5% of health adults. It
is regularly present in the in faeces of 10-50% of health
infants. It is found with its toxins in the faeces of
patients suffering from antibiotic-associated colitis; in
its severe form, this becomes pseudomembrane colitis.
 Toxins:- Cl. Difficile produces 2 toxins within the colon,
exotoxin A and exotoxin B (both are
glucosyltransferases), the main effect of exotoxin
 B is the damage to the gut mucosa (cytotoxin).
 Pathogenesis:-
 Administration of antibiotic, e.g. clindamycin
and ampicillin, suppress drug-sensitive normal
flora in the gut. This allows Cl. Difficile to
multiply and proliferate in the colon producing
its toxins. It causes profuse diarrhea which
associated with pseudomembranes which are
yellow-white plaques or material adhering to
the colonic mucosa.
 Laboratory Diagnosis:-
 Isolation;- Cl. Difficile can be isolated from faeces
using a selective medium. Incubation is anaerobic.
Colonies are rough, irregular and fluoresce under
ultraviolet light.
 Demonstration of toxins;-
 Exotoxin B can be detected directly in stool
samples or in broth culture of Cl. Difficile.
 An ELISA that detects both exotoxins A and B is
available. All enteropathogenic isolates of Cl.
Difficile are toxigenic.
 Treatment;- the causative antibiotic should
be withdrawn and fluids lost should be
replaced (rehydration). Cl. Difficile is sensitive
to vancomycin and to metronidazole (flagyl),
they should be administered orally to kill the
organisms in the colon.
 Prevention;- no specific preventive measures.
HAEMOPHILUS

Habitat:-
Species in the genus Haemophilus are found mainly
in the respiratory tract. Some of the species are part
of the normal flora, while others cause respiratory
diseases, some species are also associated with
diseases at other mucosal surfaces e.g. conjunctiva
and genital tract.
General characteristics:-
Morphology;- small, gram-negative rods
(coccobacilli) non-sporing and non-motile.
 Culture;- require enriched media, such as blood or
chocolate agar (heated blood at 80c°)
 Optimum growth temp. is 37c°. Most species
grow poorly in the absence of oxygen, but growth
is enhanced in an atmosphere with added CO2.
Enriched media are necessary because
Haemophilus species need one or both of two
growth factors, which are present in blood;-
 1- heat-stable (X) factor; haemin or some other
 Iron containing porphyrin.
 2- heat-labile (V) factor; which is diphospho-
pyridine nuleotide.
 Requirement for growth factors can help to
differentiate between species, some require one
growth factor while others require the 2 growth
factors for growth and multiplication.
 Pathogenicity:-the diseases caused by
Haemophilus species are listed below:-
 1- Haemophilus influenzae; causes chronic
bronchitis, sinusitis, ottitis media, meningitis,
epiglottitis and septicaemia.
2- Haemophilus aegypticus:causes conjunctivitis.
3- Haemophilus ducreyi; causes the sexually
transmitted disease (painful chancroid).
4- Haemophilus parainfluenzae, H. haemolyticus and
H. parahaemolyticus; these are commensal species
in the upper respiratory tract and rarely cause
disease.
It is the main pathogenic species among the genus Haemophilus.

HAEMOPHILUS INFLUENZAE
 It’s habitat is the upper respiratory tract; most
strains found in the normal flora are non-
capsulated.
General characteristics; small, Gram-negative
rods (coccobacilli), a minority of strains are
capsulated.
Culture; on blood or chocolate agar.
Colonies; small, translucent and non-haemolytic
 capsulated strains form larger colonies.
Selective medium; the addition of bacitracin to
chocolate agar inhibits the growth of many
Gram +ve bacteria found in the upper
respiratory tract e.g. viridans streptococci, and
the use of this medium facilitates the isolation
of Haemophilus influenzae from sputum
specimens.
 Identification; by testing on nutrient agar for
the growth requirements, using discs
impregnated with (X) and (V) factors
Serotyping; capsulated strains are serotyped
on the basis of variations in capsular
polysaccharide antigens. Six serotypes are
recognized, designated as a, b, c, d, e and f. type
(b) is the main pathogen.
 Pathogenicity: capsulated strains(mainly type
b) cause various invasive infections, mainly in
children from 2 months to 3 years old, in the
upper respiratory tract. The organism can enter
the bloodstream causing septicemia and
meningitis. In adults the organism also cause
pneumonia.
Virulence factors include; endotoxin, production
of IgA protease and the antiphagocytic capsule
 Pathogenicity: capsulated strains(mainly type
b) cause various invasive infections, mainly in
children from 2 months to 3 years old, in the
upper respiratory tract. The organism can enter
the bloodstream causing septicemia and
meningitis. In adults the organism also cause
pneumonia.
Virulence factors include; endotoxin, production
of IgA protease and the antiphagocytic capsule
 the 2 growth factors (X) and (V).
Definitive identification can be made with either
biochemical tests or the capsular swelling
(quellung) reaction using specific antibodies.
Fluorescent-antibody staining of the organisms
or agglutination tests which detects the
capsular polysaccharide can also be used in
identifying encapsulated strains of Haemphilus
influenzae.
 Treatment; the treatment of choice for
Haemophilus influenzae meningitis is
ceftriaxone. Untreated H. influenzae meningitis
has a fatality rate of about 90% . The incidence
of neurologic sequelae is high when treatment
is delayed. H. influenzae upper respiratory tract
infections such as otitis media and sinusitis, are
treated with ampicillin or trimethoprim-
 sulfamethoxazole or erythromycin.
Prevention; meningitis in close contacts of the
patient can be prevented by rifampicin.
Rifampicin is used because it is secreted in saliva
more than ampicillin. Rifampicin decreases the
respiratory carriage of the organism, thereby
reducing transmission. A vaccine contains the
capsular polysaccharide of H. influenzae type (b)
 is used for the control of the disease. It is
usually given between the ages of 2 and 15
months.
BORDETELLA

 Important members of the genus are;

Bordetella pertussis; causes whooping cough
(pertussis) .
Bordetella parapertussis; causes milder form of
whooping cough.
BORDETELLA PERTUSSIS

Habitat; the human respiratory tract, usually

associated with acute disease.
General characteristics;
Morphology; short, sometimes oval, Gram-ve
bacilli. Freshly isolated strains may be
capsulated, it’s polysaccharide capsule is
essential for virulence, strains that lost the
capsule do not cause disease.
 Culture; special enriched medium is required
for primary isolation. The widely used medium is
charcoal blood agar. This replace the traditional
Bordet-Gengou medium which contains 30%
blood. The media are made selective by the
addition of penicillin or cephalexin.
Colonial morphology; colonies are like mercury
drops, they appear after 3 or more days of
 incubation in a moist aerobic atmosphere at
35C°.
Identification; confirmed serologically by slide
agglutination test using a polyvalent antiserum.
Serotypes; 3 main serotypes, recognized by the
presence of specific surface antigens of
Bordetella pertussis.
Pathogenicity; Wooping cough is an acute
 tracheobronchitis which occurs primarily in
infants and young children. The organisms are
transmitted by air-borne droplets (inhalation).
The organisms attach to the ciliated epithelium
of the upper respiratory tract but do not invade
the underlying tissue. Decreased cilia activity
and epithelial cell death occur.
The disease begins with mild upper respiratory
tract symptoms followed by the typical
paroxysmal cough stage, which lasts from 1 to 4
weeks. The paroxysmal pattern is characterized
by a series of severe coughs, accompanied by
production of copious amounts of mucus, that
end with an inspiratory “whoop” as air rushes
past the narrowed glottis. Despite the severity
of symptoms, the organism is restricted to the
 respiratory tract and blood culture are negative.
Central nervous system anoxia and exhaustion can
occur. Most acute deaths are in infants in the first 6
months of age.
Laboratory diagnosis; isolation of the organism
from infected clinical cases is not easy and
diagnosis is usually based on symptoms.
Specimens; nasopharyngeal secretion collected by
swab, and the cough plate which is held in front of
mouth during a paroxysm of coughing.
Culture; inoculate charcoal blood agar or
Bordet-Gengu medium, incubate from 3-5 days
at 35-36C° observe moist “mercury-drop”
colonies.
Identification; by slide agglutination with
specific antiserum or by direct fluorescent-
antibody staining, using specific antibodies.
Serology; detection of rising antibody titer
against Bordetella pertussis in paired serum
specimens may be diagnostic in children over
one year old.by ELISA method.
Treatment; erythromycin reduces the number
of organisms in the throat and decreases the
risk of secondary complications such as
pneumonia. But antibiotics are only of value if
given within the first 10 days of infection.
 oxygen therapy and suction of mucus is
important, particularly in infants.
Prevention; Wooping cough can be prevented
by active immunization with killed Bordetella
pertussis organisms (killed vaccine). Given
combined with diphtheria and tetanus toxoid in
what is known as the triple vaccine. Given in 3
doses beginning at the 1st of the third month
Erythromycin is also given as prophylaxis for
close contacts of patients.
Mycobacteria

 Mycobacteria are aerobic, acid-fast bacilli.

They are neither Gram –ve nor Gram+ve i.e.
they are stained poorly by the dyes used in
Gram’s stain. The term acid-fast refers to the
organism ability to retain the carbol fuchsin
stain despite subsequent treatment with an
ethanol-hydrochloric acid mixture. The lipid
content, approximately 60%, of their cell wall
make mycobacteria acid-fast.
 The main medically important
Mycobacterium species are listed in the table
below, together with some of their
properties:-
Species Habitat and Disease Cultural characters
on Lowenstein-
source Jensen medium
Mycobacterium Infected humans Tuberculosis Rough, dry, yellow
tuberculosis colonies. Slow
grower.
Mycobacterium Infected cattle Tuberculosis White, smooth
bovis colonies, (inhibited
by glycerol). Slow
grower
Mycobacteria Infected humans Leprosy No growth
leprae
General characteristics

Mycobacterium tuberculosis
 Morphology:
It is slender, non-sporing bacilli, beaded i.e.
showing vacuoles darkly stained and others
faintly stained.
• Staining:
Ziehl-Neelsen stain, the bacilli are stained with
heated concentrated carbol fuchsin and then
decolorized with acid-alcohol.
The bacilli retain a bright red colour.
In sputum specimens the organisms can be
detected under fluorescent microscopy with an
uramine stain, the organism appear yellowish to
orange in colour.
Mycobacterium tuberculosis can not be
classified as Gram’s +ve or –ve, the organisms
are designated as acid-fast because of its lipid-
 rich cell wall.
* Culture:
Obligate aerobe, it can grow in simple synthetic
medium containing glycerol, ammonium salts and
a mixture of amino-acids added to improve the
rate of growth.
It is isolated routinely in the laboratory on
Lowenstein-Jensen medium which contains egg-
yolk, serum albumin, glycerol and malachite
green added to inhibit unwanted normal flora
present in the sputum specimen.
The organisms grow slowly, colonies may
appear after 2-3 weeks incubation at 37cͦ.
Cultures should be kept for 6-8 weeks before
being discarded as negative specimens.
The organisms have a doubling time of 18 hrs,
in contrast to most bacteria, which can
double in number in 1 hr or less.
Biologic properties:
Mycobacterium tuberculosis is relatively
resistant to some chemical factors such as
acids and alkalies (Na OH) and to some
disinfectants like 5% phenol (due to its cell
wall structure). It is also resistant to
dehydration and so survives in dried sputum.
This property may be important in its
transmission by aerosol.
In contrast, Mycobacterium tuberculosis is
sensitive to some physical factors such as
sun-rays and ultra-violet radiation.
 Cell-wall structure:
lipid constitute about 60% of cell wall
structure, it is composed of long-chain fatty
acids called mycolic acids, which contribute
to the organism’s acid-fastness. The cell wall
also contain true waxes and glycolipids.
These lipids are linked to polysaccharides and
to proteins by complex routes.
The polysaccharides play role in antibody
formation by the host, while the proteins
when linked to some waxes (wax D), they are
able to elicit delayed type hypersensitivity
which is the reaction of the tuberculin test.
The cell-wall also contains phosphotides
which play role in the formation of caseation
necrosis by the organisms.
 Virulent strains of Mycobacterium
tuberculosis possess a cord factor, which
allows the organisms to grow parallel to each
other in a characteristic (serpentine) cordlike
pattern. Avirulent strains grow irregularly.
 Pathogenicity:
Mycobacterium tuberculosis does not
produce an exotoxin and it does not contain
endotoxin.
 The disease occurs as a result of settlement
and spread of the organisms in the body and
the host response against it. The formation of
lesions, their spread or healing is dependant
on the following factors:-
- The number of the organisms entering the
body, and their site of entry; whether the
respiratory tract(Mycobacterium tuberculosis)
or the digestive tract (Mycobacterium bovis)
 - The virulence of the bacterial strains entering
the body.
 The resistance or the susceptibility of the
host.
* There are two types of lesions;
1- Exudative lesions; consist of an acute
inflammatory response and occur chiefly in
the lungs at the initial site of infection.
2- Granulomatous lesions; consist of a central
area of giant cells (macrophages) containing
the tubercle bacilli and surrounded by a zone
of epithelioid cells and lymphocytes. A
tubercle is formed when the granuloma
become surrounded by fibrous tissue
(fibrosis) and has undergone central
caseation necrosis.
 From the lung the organisms disseminate via
the bloodstream to many internal organs
causing miliary tuberculosis.
 Transmission:-
Mycobacterium tuberculosis is transmitted
from person to person by respiratory aerosol
(inhalation), and its initial site of infection is
the lung.
 Cattle infected with Mycobacterium bovis
constitute the reservoir for human disease.
The organisms are shed milk of infected
cows. If such milk is consumed
unpasteurized, it will cause gastrointestinal
tuberculosis in humans.
 The risk of infection and disease is highest
among socioeconomically disadvantaged
people, who have poor housing and nutrition.
 Clinical features:-
tuberculosis is a slowly progressive, chronic,
granulomatous infection which most often
affects the lungs, other organs and tissues
may also be involved.
Clinically, tuberculosis is seen in two forms:-
1- Primary; generally more invasive, with
marked lymph nodes involvement.
2- Post-primary; in which the development of
delayed-type hypersensitivity modifies the
infection, with limitation of spread and
considerable fibrotic reaction.
- Primary tuberculosis usually involves the lung.
- The commonest form is a local lesion with
marked enlargement of the regional lymph
nodes (Ghon complex).
 Primary infection may progress to cause:-
a- Tuberculous bronchopneumonia: an acute
diffuse extension of the infection throughout
the lung.
b- Miliary tuberculosis: dissemination of the
tuberculous foci (tubercle) widely throughout
the body, as a result of haematogenous
spread of infection (bloodstream).
c- Tuberculous meningitis: blood-borne spread
of infection to penetrate the blood-brain
barrier and involve the meninges.
d- Bone and joint tuberculosis: affects different
sites, a common form is spinal tuberculosis
(Pott’s disease), in which there may be
collapse of the vertebrae.
e- Genitourinary tuberculosis: may includes
renal tuberculosis which presents with
 frequency and painless haematuria,
endometrial tuberculosis in females and
tuberculous epididymitis in males.
• Spread of the tubercle bacilli within the body
usually occurs by two methods:-
1- A tubercle lesion can erode into a bronchus,
empty its caseous contents, and thereby
spread the organisms to other parts of the
 lungs, to the gastrointestinal tract if
swallowed, and to other persons if
expectorated by coughing.
2- It can disseminate via the bloodstream to
many internal organs.
• Immunity:-
The immunity exhibited by the host to
Mycobacterium tuberculosis is cellular type.
 Humeral immune mechanisms (antibodies)
do not play a role in resistance.
 The tubercle bacilli are readily phagocytosed,
but can then multiply within mononuclear
phagocytes and resist digestion. This
intracellular survival of the organisms is
associated with the development of delayed
hypersensitivity (cell-mediated immunity),
 and of activated macrophages with increased
ability to kill the ingested bacilli.
• After treatment and recovery from primary
infection, the host exhibits delayed
hypersensitivity which indicate that
resistance to the organism is acquired by the
host.
• The delayed hypersensitivity reactions in the
host can be demonstrated by tuberculin test.
 Tuberculin test:- it is the intradermal
inoculation of purified protein derivative (a
purified filtrate from cultures of Mycobacterium
tuberculosis). It is administered to the person to
be tested by intradermal injection using a
syringe (Mantoux test) , results:-
1- Positive test (indicating delayed
hypersensitivity), produces induration, followed
by papule formation at the site of the injection
after 48-72 hrs.
2- Negative test: no reaction.
The tuberculin test is positive only in
individuals
who are infected before by Mycobacterium
tuberculosis, or those who have primary
lesions of tuberculosis which healed after
treatment.
Individuals who do not come in contact with
 Mycobacterium tuberculosis before show
negative tuberculin test i.e. they do not react
to the intradermal injection of the purified
protein derivative. These individuals are non-
immune and need to be vaccinated.
• Laboratory Diagnosis:-
Neither the tuberculin test nor other serological
tests can give a definitive diagnosis of active
infection with Mycobacterium tuberculosis.
 Only isolation of the organisms, and other
direct clinical tests which include x-ray, can
give a definitive diagnosis.
 Specimens: Collected as follows from
different sites of tuberculosis;
- Respiratory; sputum, laryngeal swab,
bronchial washings or gastric lavage.
- Meningitis; cerebrospinal fluid (CSF).
- Bone and joint; samples removed at
operation or by aspiration.
- Renal; early morning urine.
When respiratory or renal tuberculosis is
suspected 3 repeated specimens are usually
necessary.
 Direct microscopy:-smears are repeated from
specimens.
Detection of typical tubercle bacilli in smear
stained with Ziehl-Neelsen (examined by
ordinary light microscope).
• Treatment:-
Multiple-drug therapy is used in treatment to
prevent the emergence of drug-resistant
strains during the long duration of treatment.
This prolonged therapy is necessary because
of;
1- Intracellular location of the organisms.
2- Caseous material in the lesion which blocks
penetration by the drug.
Both Mycobacterium tuberculosis and
Mycobacterium bovis are sensitive to a wide
range of drugs.
 Because resistant strains (variants) arise
readily, therapy should always comprise a
combination of 3, or even 4 drugs. The first-
line drugs for treating the disease include;
Isoniazid, Rifampicin, Pyrazinamide and
Ethambutol.
• Prevention:-
Bacille Calmette-Guerin, or BCG vaccine. This
vaccine confers partial immunity to infection
and prevents the invasive disease
characteristic of primary infection such as
milliary tuberculosis and tuberculous
meningitis.
The vaccine is administered intradermally,
usually given to neonates on the first day
after birth.
It also given to non-immune individuals who
showed negative tuberculin test.
Mycobacterium lepry

 It causes leprosy.
 Habitat; found only in cases of human
infection.
 General characteristic:-
 Similar to Mycobacterium tuberculosis in
size, acid fast. Non-motile, non-sporing, and
non-encaspulated.
 In smears prepared from lesions and stained
 with Ziehl-Neelsen stain, the organisms are
seen singly or in small groups within
mononuclear cells (phagocytes).
 Mycobacterium leprae has not been grown in
the lab., either on artificial media, tissue or
cell culture. It can be grown in the mouse
footpad.
 Humans are the natural hosts.
 - the optimal temperature for growth is lower
than body in the skin and superficial nerves.
temperature, so it grow preferentially
• Pathogenesis:-
-leprosy is a chronic granulomatous disease.
- Infection is acquired by prolonged contact
with patients with lepromatous leprosy, who
discharge Mycobacterium leprae in large
 numbers in nasal secretions and from skin
lesions.
- After entry into the body, the organisms
replicate intracellularly within skin histiocytes,
endothelial cells and nerve cells.
There are 2 distinct forms of leprosy:
1- Tuberculoid leprosy: the disease is not
progressive, the lesions are localized to the
skin and peripheral nerve, with skin anasthesia
(which results in disfiguring). There are only
scanty Mycobacterium leprae in the lesions,
which tend to be benign, and the disease is
often self-healing.
2- Lepromatous leprosy:- it is the progressive
form of the disease. Numerous bacilli are
present in the lesions, sensory skin nerves are
affected causing anaesthesia. The lesions are
nodular in form (called leproma) and spread on
the skin and mucous membranes, particulary
nasal mucosa, and other organ like liver, lung
and spleen. These nodular lesions are also
seen on the face, giving the appearance of
lionlike face. This form of leprosy is a
systemic disease, with Mycobacterium leprae
spreading via the bloodstream.
 Lepromin skin test:-
It determines the clinical type of the disease
produced. It is called Mitsuda reaction. It is
similar to the tuberculin test i.e. delayed
hypersensitivity reaction.
The test:-
Heat-killed Mycobacterium leprae are
inoculated intradermally. Induration appearing
at the site of injection up to 28 days after
inoculation indicates a positive reaction.
The test is positive in cases of tuberculoid
leprosy indicating that a high degree of cell-
mediated immunity is operating against the
organisms.
The test is negative in lepromatous leprosy,
when no cellular immune reaction is
operating
against the organisms (immunological anergy).
The test is also positive in individuals
vaccinated with BCG vaccine (tuberculosis
vaccine).
Laboratory Diagnosis:-
Specimens; skin biopsy, scrapings from lesions
in the nasal mucosa, secretions from
ulcerated nodular lesions.
Diagnosis; by direct demonstration of acid-fast
bacilli in smears or sections from lesions
stained with Ziehl-Neelsen stain.
Propagation in the lab.;
Mycobacterium leprae does not grow on
artificial media in the lab. It can be cultivated
in vivo by inoculation into the foot-pads of
mice.
Culture and serology are not used for
diagnosis.
 Treatment:-
Combination therapy to avoid drug resistance.
Triple therapy required for lepromatous
leprosy using; Dapsone, Rifampicin, and
Clofazimine for 2 yrs.
For tuberculoid leprosy Rifampicin and
Dapsone are given for 8 months.
 Epidemiology:-
spread of the disease in the community is slow.
Although pts with lepromatous leprosy shed
numerous organisms from the nose, the
incidence of the disease in contacts is low.
The route of infection is probably mainly
inhalation. The incubation period is long,
often several years and the onset is gradual.
 The disease occurs worldwide, with most cases
in the tropical areas of Asia and Africa.
• prevention:-
Isolation of all lepromatous patients, coupled
with chemoprophylaxis with Dapsone for
exposed children. BCG vaccine may offer a
degree of protection, but at present its use is
not recommended.
Spirochetes

 They are a group of helical (coiled) organisms

which share many properties with Gram –ve
bacteria.
 There are 3 genera which contain pathogenic
species to humans;
1- Treponema.
2- Borrelia.
3- Leptospira.
 Habitat: Most are free-living and non-
pathogenic, but a few species cause
important human disease.
 General characteristic:-
1- Spirochetes are thin-walled, flexible, spiral
rods.
2- Treponemas and Leptospira are so thin that
they stained poorly or not by usual staining.
3- They can not be seen with light microscope,
but can be rendered visible by dark-ground
microscopy, or by staining with heavy metals
e.g. silver impregnation or by
immunofluorescence.
4- The larger spirochetes e.g. Borrelia species
are G-ve, can accept Giemsa’s and other
blood stains and seen in standard light
microscope.
5- Spirochetes are motile by rotation about
their long axis and by flexion.
Treponema

 The most important pathogenic species of

this genus is Treponema pallidum, the cause
of sexual transmitted disease, syphilis.
 Other species are found as commensal, in the
mouth, genital secretions and intestine.
 Treponema pallidum:-
 Habitat; the lesions of primary and secondary
syphilis.
 General Characteristics:-
 Morphology; long, slender, filamentous,
spiral rods with spaced coils.
 Viability; Treponema pallidum are fragile
bacteria that die rapidly outside the body. It is
very sensitive to drying and heat.
 Culture; it can be cultivated in;
1- vitro
2- ordinary bacteriologic media.
3- tissue culture or cell culture.
Although humans are the only natural host,
the organisms can be propagated by
inoculation of testes and scrotal sac of
rabbits.
• Antigenic structure;
1- lipid antigen ; composing of phospholipid
and called Wassermann antigen. It is not
specific
 antigen, beside Treponema pallidum it present
also in many mammalian tissues e.g. tissue of
cattle heart, it is known as cardiolipin.
2- protein antigens; composing of peptides.
They are specific antigens, present only in the
outer coat of pathogenic Treponemes.
• Pathogenicity:-
Treponema pallidum is transmitted from an
infected lesion (e.g. genital organs, mouth or
rectum) by ;
1- Intimate contact.
2- Sexual intercourse.
- The organism does not produce toxins or
enzymes that play role in pathogenicity.
- At the site of entry the organisms multiply
and a hard chancre forms within 2-10 weeks
after
 infection, with multiplication of the organisms in
the adjacent lymph nodes (Primary syphilis).
The hard chancre (non-tender ulcer) heals
spontaneously within 3-8 weeks, but painless
enlargement of local lymph nodes persists.
- In secondary syphilis the organisms pass via the
lymph nodes into the bloodstream causing
systemic lesions in various organs and
 maculopapular rash on the skin and mucous
membranes. These lesions are rich in
spirochetes and are highly infectious.
- At this stage there is generalized
lymphadenopathy in half the patients.
- Mucosal ulcers in the mouth are shown in
about one third of patients.
-About one-third of those early syphilis cases
progress to cure without treatment.
 In another third the infection remains latent
i.e. without apparent lesions and no clinical
symptoms.
 In the last third of cases, if the disease is not
treated, it will progress to the late or tertiary
stage.
- Tertiary syphilis is characterized by
cardiovascular and central nervous system
involvements, but the organisms are rarely
found in the lesions.
- An infected pregnant woman can transmit
Treponema pallidum across the placenta to
her fetus after the third month of pregnancy.
This is known as congenital syphilis.
 - Transmission in congenital syphilis usually
takes place during primary or secondary
syphilis in the mother.
- Early and late manifestations of syphilis can
develope in the newborn.
• Laboratory Diagnosis:-
• Specimens;
1- fluid or scrapings from chancre or ulcerated
secondary lesions.
2- serum (for serological tests).
*Direct demonstration;
Treponema pallidum is demonstrated in fluids
or scrapings of early lesion by preparation of
wet smears and examination by dark-ground
microscopy. The organisms are identified by
 their morphology and motility pattern.
 Smears are also prepared from the lesions
and the organisms can be demonstrated by
direct immunofluorescence.
*Serology;
- The effective laboratory diagnostic methods
are serological. Two methods are used,
depending on nature of antigen used;
1- Nonspecific serologic tests; -
 In these tests, nontreponema antigens are
used.
 Extracts of normal mammalian tissues (e.g.
cardiolipin from beef heart) react with
antibodies in serum samples from patients
with syphilis.
 These antibodies are detected by
Flocculation
 tests according to VDRL (Venereal Disease
Research Laboratory) methods.
- These non-specific tests are, inexpensive,
easy to perform, and rapid, but give false-
positive reactions in some infections such as
leprosy, and in various autoimmune
diseases (SLE) . Therefore positive results
have to be confirmed by specific tests.
- Results of non-specific tests usually become
negative after treatment, therefore used to
determine the response of the patient to the
treatment.
2- Specific serologic tests;-
- they use Treponemal antigens.
- they are more specific and sensitive than the
non-specific tests.
- they are more expensive.
- they are difficult to perform and sometimes
not available.
- specific treponemal antibodies usually arise in
the patient serum within 2-3 weeks after
infection, and so the tests are positive in
most patients with primary syphilis.
- they remain positive after effective treatment
and can not be used to determine the response
of patient to treatment.
- examples of these specific tests include:-
1- Treponema pallidum haemagglutination
assay (TPHA); in which Treponema pallidum
cells are linked to RBCs.
2- Fluorescent Treponemal antibody (FTA); the
antigen here is a suspension of T. pallidum.
3- Treponema pallidum immobilization test
(TPIT); the antigen used is live Treponema
pallidum propagated in the testes of rabbits.
*Treatment:-
Treponema pallidum and other treponemes are
sensitive to penicillin G.
It is usually administered in large doses and
continued for 10-21 days.
- late or latent syphilis is treated by large doses
of penicillin for 21 days.
- if the patient is allergic to penicillin,
tetracycline or erythromycin can be used for
prolonged periods also.
* Prevention:-
- no vaccine against syphilis.
- prevention depends on early diagnosis and
3- Treponema pallidum immobilization test
(TPIT); the antigen used is live Treponema
pallidum propagated in the testes of rabbits.
*Treatment:-
Treponema pallidum and other treponemes are
sensitive to penicillin G.
It is usually administered in large doses and
continued for 10-21 days.
adequate treatment.
- use of condoms in sexual intercourses and
administration of antibiotic after suspected
exposure.
- prohibition of illegal sexual intercourses and
homosexuality and encouragement of
marriage.
Leptospira

 General characteristics:-
 Morphology; spiral organisms with
numerous closely set coils, slender, and
motile.
- they are not stained with dyes, but are seen
by dark-ground microscopy.
*Culture:-- obligate aerobes, grow in enriched
fluid semi-solid media containing blood,
serum or vitamin B12.
- optimum temperature is around 36cͦ. It is slow
grower.
* Viability:- pathogenic strains may survive for
days outside human or animal body in moist
surroundings, which are not acidic.
* Identification: by serological means.
* Species: the genus Leptospira contains a
number of species, some are commensals,
while others are pathogenic.
- the most important human pathogen is
Leptospira interrogans which contains 2
serogroups:-
1- leptospira icterohaemorrhagiae; its reservoir
are rodents particularly rats.
2- leptospire conicola; its reservoir are dogs
and pigs.
 Pathogenicity:- humans are infected by
contact with infected animals or by drinking
water contaminated with urine of infected
animals, which contains large numbers of the
organism.
- human infections occur by oral route or via
the skin and mucous membranes.
- leptospirosis is characterized by fever and
dysfunction of the liver causing jaundice and
hemorrhage.
- the central nervous system may be involved
leading to aseptic meningitis.
* Laboratory Diagnosis:-
* Specimens; blood, cerebrospinal fluid, urine.
Direct Demonstration; leptospira can be seen
in wet smears prepared from the specimens
and
visualized by dark-ground microscopy.
* Culture:- in the first phase of the disease the
organism can be isolated in a blood or cerebrospinal
fluid culture.
- In the second phase of the disease the organisms are
present in large numbers in the patient’s urine and can
be isolated by culturing urine in an appropriate
leptospira medium.
* Serology: serological tests are valuable in
diagnosing human leptospirosis.
- high titers of antibodies are usually
detectable in patient’s serum during the
second week after infection.
- agglutination and complement fixation tests
are used for diagnosis.
 Treatment:-
- it is sensitive to penicillin and tetracycline.
- treatment of choice is penicillin G.
* Prevention:- human leptospirosis is a
zoonotic disease.
- prevention by avoiding contact with infected
animals and contaminated environment.
- avoiding drinking or swimming in water
contaminated by urine of infected animals.