CLINICAL BACTERIOLOGY
Dr .Mostafa M. Eraqi
Ph.D. Microbiology and Immunology
A 44-year-old male presented to the emergency room with
complaints of chest pain and was found to have suffered a
myocardial infarction. His past medical history was significant
for
hypertension,
noninsulin-dependent
diabetes,
hypercholesterolemia, and a history of heavy smoking (two
packs per day for 15 years). A cardiac catheterization on
hospital day 3 showed three vessel coronary artery disease,
and he underwent triple coronary artery bypass graft surgery
on hospital day 5. On hospital day 7, he developed septic shock
with acute renal and respiratory failure requiring intubation.
At that time, he had a fever of 39.3C, his arterial blood gas
revealed a pO2 of 89 mmHg on 100% O2, and he had
a white blood cell count of 27000/mm3. Two blood cultures
were obtained. A chest radiograph showed a left lower lobe
infiltrate with pleural effusion. A chest tube was placed to
drain the effusion. On hospital day 11, pus was noted to be
seeping from his sternal wound. His wound was debrided and a
rib biopsy was performed. Blood, drainage from his chest tube,
tracheal aspirates, pus from his sternal wound, and a rib
biopsy were cultured. The bacterium isolated from the clinical
specimens was a gram-positive, catalase-positive, coagulasepositive coccus that was resistant to methicillin.
QUESTION
What is the causative agent, how does it enter the body and
how does it spread
a)
within the body and
b)
b) from person to person?
Causative agent
The patient has an infection caused by methicillin
(oxacillin)-resistant Staphylococcus aureus (MRSA).
Bacteria in the genus Staphylococcus are gram positive
cocci that grow in grape-like clusters and produce the
enzyme catalase. The genus Staphylococcus comprises
some 35 species, many of which are members of the
endogenous microbiota of the skin and mucous
membranes of the gastrointestinal and genitourinary
tracts of humans. Interestingly, a number of these
species have defined habitats on the human body.
Three species account for most human disease, namely
S. aureus, S. epidermidis, and S. saprophyticus. Of
these, S. aureus is the most pathogenic. S. aureus has a
typical gram-positive cell wall that features a

thick peptidoglycan layer extensively cross-linked with
pentaglycine bridges. The extensive cross-linking makes the cell
very resistant to drying so that staphylococci can survive on
fomites (inanimate objects) for long periods of time. The wall also
contains covalently bound teichoic acid and lipoteichoic acid,
which is anchored in the cytoplasmic membrane. There are a
number of molecules that are exposed on the cell surface. These
are either anchored in the cytoplasmic membrane and traverse
the cell wall to the outside or they are anchored in the wall and
extend from it. Among the important cell surface-exposed
molecules of S. aureus are various microbial surface components
that recognize adhesive matrix molecules (MSCRAMMs). These
components recognize and bind various extracellular matrix
proteins such as fibronectin, collagen, and fibrinogen (clumping
factor), and the Fc region of mammalian IgG (protein A). In
addition to wall-associated clumping factor that binds solid-phase
fibrinogen, S. aureus
secretes coagulase, which binds soluble fibrinogen. Coagulase
binds prothrombin in a 1:1 molar ratio to form a complex termed
staphylothrombin, which converts fibrinogen to fibrin. Because S.
aureus is the only staphylococcal species that possesses coagulase
and protein A their detection serves as a method to identify the
bacterium (see Section 4). MSCRAMMs facilitate invasion of
keratinocytes and endothelial cells by S. aureus. Once taken up by
cells, the bacteria survive in vacuoles and free in the cytoplasm
where some persist for several days. Bacterial invasion results in
cytotoxicity and apoptotic cell death.
Almost all S. aureus clinical isolates produce capsular
polysaccharides, which have been divided into 11 serotypes.
Serotype 1 and 2 staphylococci are heavily capsulated and produce
mucoid colonies. The remaining serotypes produce ‘microcapsules’
and form nonmucoid colonies. Interestingly, most clinical isolates
are microcapsule-producing serotypes 5 and 8.
Almost all strains of S. aureus secrete a group of enzymes and
cytotoxins that includes four hemolysins (alpha, beta, gamma, and
delta), nucleases, proteases, lipases, hyaluronidase, and
collagenase. Alpha-toxin is a poreforming toxin that acts on many
types of human cells. It is dermonecrotic and neurotoxic. Betatoxin is a sphingomyelinase C whose role in pathogenesis remains
unclear. Delta-toxin is able to damage the membrane of many
human cells, possibly by inserting into the cytoplasmic membrane
and disordering lipid chain dynamics, although there are
suggestions that it may form pores. Two types of bicomponent
toxin are made by S. aureus, gamma-toxin and Panton-Valentine
leukocidin (PVL). While gammatoxin is produced by almost all
strains of S. aureus, PVL is produced by only a few percent of
strains. The toxins affect neutrophils and macrophages. PVL has
pore-forming activity and has been associated with necrotic
lesions involving the skin and with severe necrotizing pneumonia
in community-acquired S. aureus infections. Also, S. aureus
produces a staphylokinase, which is a potent activator of
plasminogen, the precursor of the fibrinolytic protease plasmin.
S. aureus produces several families of exotoxins that have
superantigen activity. These include heat-stable enterotoxins (A–
E, G–I) that cause food poisoning, exfoliative (epidermolytic)
toxins A (chromosome encoded) and B (plasmid encoded) (ETA
and ETB) that are the cause of staphylococcal scalded skin
syndrome (SSSS) occuring predominantly in children, and toxic
shock syndrome toxin 1 (TSST-1) that causes staphylococcal toxic
shock.
ENTRY AND SPREAD WITHIN THE BODY
S. aureus requires a breach in the skin or mucosa
such as produced by a catheter, a surgical incision,
a burn, a traumatic wound, ulceration or viral skin
lesions to facilitate its entry into the tissues.
Furthermore,
staphylococcal
infections
are
frequently associated with reduced host resistance
brought
about
by
cystic
fibrosis,
immunosuppression, diabetes mellitus, viral
infections, and drug addiction. S. aureus has
several enzymes that aid in invasion of the barrier
epithelia and serve as spreading factors. Among
these are lipases, hyaluronidase, and fibrinolysin.
PERSON TO PERSON SPREAD
Because they colonize the surface of the skin
staphylococci are readily shed and easily
transferred horizontally by direct contact or by
contact with fomites such as bed linen, medical
instruments, and so forth.
Epidemiology:
Transient colonization of the umbilical stump and
skin, particularly in the perineal region, is common
in neonates. About 15% of children and adults are
persistent carriers of S. aureus in the anterior
nares. There is a high rate of carriage in hospital
personnel, IV drug users, diabetics, and dialysis
patients, and MRSA and MSSA (methicillinsensitive S. aureus) colonization is common in the
population.
WHAT IS THE HOST RESPONSE TO THE INFECTION
AND WHAT IS THE DISEASE PATHOGENESIS?
PATHOGENESIS (INVASIVE AND TOXOGENIC)
S. aureus is a versatile pathogen that causes a
wide range of diseases. Disease may be mediated
by invasion, bacterial multiplication leading to
formation of abscesses, and destruction of a variety
of tissues, as well as by a range of exotoxins, some
of which are superantigens. Furthermore, S.
aureus and other staphylococcal species are broadly
antibiotic-resistant
(see
below).
Factors
contributing to the virulence of Staphylococcus
aureus are presented in Table 1.
SKIN INFECTIONS

superficial such
as impetigo


infections of
hair follicles
(folliculitis)
deeper infections like carbuncles

boils
(furuncles)
S. aureus is a frequent cause of bacteremia and
about half of these instances are nosocomial,
associated with surgical wound infection and
foreign bodies such as indwelling catheters, or
sutures. From the blood S. aureus may seed many
organs including the heart, lungs, bones, and
joints. S. aureus endocarditis has a high mortality
and may throw off septic emboli that may infect
other organs. Pneumonia may result if bacteria
reach the lungs via the bloodstream or as a result
of aspiration. Acute and chronic osteomyelitis and
septic arthritis may result from hematogenous
spread or from a skin infection.
TOXIGENIC DISEASE
Enterotoxins (A–E, G–I) produced by S. aureus are the most
common cause of food poisoning, with enterotoxin A causing
most cases. Food poisoning results from the production of
toxin in the foodstuff and its subsequent ingestion. The most
frequently contaminated foods are salted meats, potato salad,
custards, and ice cream. Generally, the food is contaminated
by S. aureus on the hands of food preparers. Holding the food
at room temperature allows the staphylococci to proliferate
and produce toxin. The enterotoxins are heat-stable and
tasteless so that, although heating of the food will kill the
staphylococci, the enterotoxins are unaffected. The
intoxication has a rapid onset, usually within 4 hours, and
the symptoms are nausea, vomiting, diarrhea, and abdominal
pain. Symptoms usually resolve within 24 hours. The
mechanism(s) of action of the enterotoxins are not understood
but may include mast cell activation and release of
inflammatory mediators and neuropeptide substance P in the
gastrointestinal tract and elsewhere.
EXFLOLIATIN TOXIN

Depended on immune state, produced by many
strains of staph. Aurus belonging to phage group
II. It cleaves stratum granulosum layer of
epidermis.
Ritter’s disease
Lyell’s disease
Scrlatina like
rash
Age
Infant and young
childreen
Older childreen and
adult
Older childreen
and adult
Pathology
Toxic epidermal
necrolysis with large
areas of denuded
skin+generalized
bullae (Scalded baby
syndrom)
Localized bulla red,
moist, healing without
scaring , if no 2ry
bad.infection(Scalded
skin syndrom)
Resemle strept.
Scarlet fever except
no infection to
tongue and palate
TOXIC SHOCK SYNDROM(T.S.S)
Caused by enterotoxin F produced by staph.aurus
which is propaply indogenous flora of skin.
 Method of infection:
 Contamination
of
improper
excessively,
prolonged useof expandable(intravaginal tampon
mad of synthetic materials)
 Contamination of wound
 Sing and symptoms:
Haedach, nusea, vomiting, shock, death

DIAGNOSIS
1. Sample from:
pus
folliculitis, frunculosis, corbancle
sputum
pneumonia
blood
bacteremia, endocarditis
facese
food poisoning
2 Examination:
Direct film
Culture: aerobic, ordinary media easly
blood agar B-haemolysis
Selective media
Broth containing 7.5% NaCl
Golden yellow staph. Aureus
White
staph. Epidermidis
White-greyish white
staph. saprophyticus
BIOCHEMICAL REACTION
A 7-year-old boy was well until yesterday when he
developed dysphagia, painful anterior lymph nodes, and a
fever to 40∞C. The patient vomited once and complained
of a headache. Physical examination showed an acutely ill
patient with a temperature of 39oC and a pulse of 104
beats per minute. Examination of his head, eyes, ear,
nose, and throat revealed bilateral tonsillar hypertrophy
with grayish-white exudates and punctate hemorrhages
Bilateral
tender
submandibular
lymph
nodes
were
palpated. The remainder of the physical examination was
within normal limits. A throat swab was obtained.
Laboratory findings were: hemoglobin, normal; hematocrit,
normal; WBC count, 19 000 mm3; differential, 80% PMN,
4% bands, 15% lymphocytes.
1. WHAT IS THE CAUSATIVE AGENT, HOW DOES IT ENTER THE BODY AND HOW
DOES IT SPREAD (A). WITHIN THE BODY AND (B). FROM PERSON TO PERSON?
● S. pyogenes, also known as the group A streptococcus
(GAS), is the most common cause of bacterial pharyngitis.
● The majority of cases (about 70%) of pharyngitis are
caused by viruses.
● GAS is a gram-positive, catalase-negative coccus that
grows in chains.
● GAS grows readily on blood agar incubated at 37C in the
presence of 5% CO2 for 24–48 hours, forming small, gray
opalescent colonies surrounded by a large zone of complete
hemolysis (b-hemolysis).
● GAS has a typical gram-positive-type cell wall that
features a thick peptidoglycan layer with covalently bound
teichoic acid.
● M protein is the most important virulence factor of GAS
and there are about 100 types.
● GAS has many determinants of pathogenesis
that include lipoteichoic acid (pro-inflammatory),
adhesins,
superantigens
(pyrogenic
exotoxins),
cytotoxins (streptolysin O and S), and a capsule.
● GAS is spread person to person via respiratory
droplets, the hands, and fomites.
● GAS adheres to and invades epithelial cells from
where it can invade subcutaneous tissues and seed
the lymphatics and bloodstream.
2. WHAT IS THE HOST RESPONSE TO THE INFECTION AND ITS
PATHOGENESIS?
● The barrier epithelia in concert with innate
immune factors at these surfaces are effective
barriers to the penetration of GAS.
● Host phagocytes are a second line of defense
against GAS invasion.
● GAS is opsonized by activation of the alternate and
lectin innate complement pathways and the classical
pathway in the presence of anti-M protein antibodies in
the plasma and tissue fluid.
● The virulence of GAS results from its ability to adhere
to and invade host cells and to avoid opsonization and
phagocytosis by means of capsule, M protein, and C5a
peptidase.
● The hemolysins, streptolysin S and O, are cytotoxins that
can lyse erythrocytes, leukocytes, and platelets and likely
other host cells.
● The pyrogenic exotoxins (erythrogenic toxins) are
superantigens
that
result
in
the
release
of
proinflammatory cytokines that mediate shock and organ
failure characteristic of streptococcal toxic shock syndrome
and give rise to the rash associated with scarlet fever.
3. WHAT IS THE TYPICAL CLINICAL PRESENTATION
AND WHAT COMPLICATIONS CAN OCCUR?
● Pharyngitis occurs 24–48 hours post-exposure with sudden onset
of sore throat, malaise, fever, headache, and erythematous pharynx
and
tonsils
with
creamy,
yellow
exudates
and
cervical
lymphadenopathy.
● Scarlet fever is caused by GAS strains
lysogenized by a temperate bacteriophage
that specifies production of pyrogenic exotoxin resulting in an
erythematous rash, circumoral pallor, and
strawberry tongue.
● Pyoderma (impetigo) is a highly contagious, superficial
infection of exposed skin, typically the face, arms, and
legs, characterized by vesicles that progress to pustules
that rupture and are replaced by honey-colored crusts.
● Erysipelas is an acute infection of the skin, most
frequently the legs, accompanied by lymphadenopathy,
fever, chills, and leukocytosis in which the infected skin is
painful, inflamed, raised, and clearly demarcated.
Cellulitis is an infection similar in nature to erysipelas
except that it involves not only the skin but the
connective tissues.
● Necrotizing fasciitis is a deep infection of the connective
tissue that spreads along fascial planes and destroys
muscle and fat.
● Streptococcal toxic shock syndrome (STSS) often follows
necrotizing fasciitis; the disease is characterized by shock
and organ failure.
● Acute rheumatic fever (ARF) is an autoimmune
inflammatory disease of the connective tissue, particularly
the heart, joints, blood vessels, subcutaneous tissue, and
central nervous system that occurs about 3 weeks after
GAS pharyngitis in susceptible subjects.
● Acute glomerulonephritis (AGN) is an autoimmune
disease that can follow either pharyngeal or skin infection
causing
inflammation
of
the
glomeruli,
hypertension, hematuria, and proteinuria.
edema,
4.HOW
IS
THE
DISEASE
DIAGNOSED
AND
WHAT
IS
THE
DIFFERENTIAL DIAGNOSIS?
● Microscopy: Examination of infected tissues or body
fluids stained by Gram’s method can provide a rapid
preliminary diagnosis.
● Culture: Swabs from the posterior pharynx, undisturbed
crusted pustules, tissue, pus, blood, and so forth, are
plated onto blood agar to grow the characteristic colonies
surrounded by a large zone of b-hemolysis.
● Determinative tests: Susceptible to bacitracin and PYRpositive.
●
Antigen
detection:
Rapid
tests
based
on
immunological detection of the Lancefield group A
wall carbohydrate antigen are available to directly
detect GAS on throat swabs; a negative rapid test
must be confirmed by culture.
● Serology: Anti-streptolysin O titer (the ASO titer)
is a useful marker for confirming acute rheumatic
fever or acute glomerulonephritis resulting from
GAS pharyngitis; anti-DNase B for AGN following
skin infection.
5. HOW IS THE DISEASE MANAGED AND PREVENTED?
● Penicillin is the antibiotic of choice in patients who are not
hypersensitive.
● Erythromycin or a cephalosporin can be used in patients allergic to
penicillin.
● Antibiotic treatment of GAS pharyngitis prevents the development of
rheumatic fever.
● Serious soft tissue infections require drainage and surgical
debridement as the first line of therapy.
● Prophylactic antibiotic therapy is required for several
years in individuals who have had rheumatic fever.
● Any person with pre-existing damage to their heart
valves should receive prophylactic antibiotics before any
procedure likely to result in transient bacteremia, such as
dental treatment.
PATHOGENICITY
1. Rheumatic Fever
A.
Rheumatic toxins (streoptolysin O)
B.
Autoimmunity
C.
Cross reactivity
D.
Genetic and anatomic aberration
2. Acute glumerulonephritis
3. Migratory arthiritis
Scarlet fever
 Puerperal sepsis
 Acute follicular tonsilitis
 Erysipelas

BACTERIAL DIAGNOSIS
I.
II.
A.
History: Clinical examination
Laboratory Diagnosis:
Direct:
source pus, sputum, urine,
blood, urine, stool,……….
suitable time
preparation( signs of growth)
1. Direct film stain
Gram
Ziehl-Neelsen stain
2. Culture:
a. Cultural character: O2, Co2, O.T , pH
b. Media
Ordinary
Enriched
selective
Differentiated
c. Morphological characters of colonies:
haemolysis, shape,……….

Systemic examination of isolated colonies
pigment,
1.Microscopy stained by : special stain (spor, capsule)
other technigues(motility)
2. Biochemical reaction and antibiotic senstivity test
3. Animal inoculation: Susceptable animal
procedure, route of injection
effect and P.M picture
Ex. Pneumococci (mice)
Diphtheria (G.P and rabite)
Cl. Teteni (G.p , mice, rabbite)
T.B. (G.P, rabbite
4. slide aglutination test by using specific
antisera
Other methodsof typing (Staph., strept.)
B. Indirect:
Serology test: pationt serum detected for Ad
Interadermal tests for T.B, Brucella, Leprosy

Staining methods:
1.
Making of a film
2.
Staining of film by:
a. Simple stain: (Methylen blue or Diluted carbol
fuchsin) .
b. Differential stain
c. Special stain
All cocci are gram positive except Neisseria group
All Bacilli are gram negative except
•
Corynebacterium group
•
Anthrax and Anthracoids
•
Clostridium group
•
Lactobacillus group
ZIEHL-NEELSEN STAIN
Concentrated carbol fuchsin
 Sulphoric acid 20%
 Alcohol 95%
 Methylene blue
