MDN 2341: Medical
Microbiology and
Immunology
Dr. Kherie Rowe
MDN 2341 : Medical Microbiology and
Immunology
*
1
Bacteriology
Upper Respiratory Tract Infections (URTIs)
Learning objectives
• Identify the most common bacteria that cause infections in the
upper respiratory tract
• Compare the major characteristics of specific bacterial
diseases of the upper respiratory tract
• Identify the means of diagnosis and culture of the bacteria that
could cause upper respiratory tract infections.
• Describe the possible means of treatment of prevention of
infection.
Upper respiratory tract infections (URTIs)
• Streptococcus pyogenes (strep
throat)
• Corynebacterium diphtheria
• Haemophilus influenzae
• Streptococcus pneumoniae
• Bordetella pertussis
PHARYNGITIS
Streptococcal pharyngitis (Strep throat)
• High fever – >100.4 0F ( 38oC)
• Intense pharyngeal pain (sore throat)
• Erythema (associated with pharyngeal
inflammation)
• Swollen tonsils with exudate (pus)
• Petechiae (pin-point red patches) on the
soft or hard palate
• Cervical lymphadenopathy (swollen
submandibular lymph nodes)
• Swollen uvula
Streptococcal pharyngitis (strep throat)
The mucosal membranes are damaged by the release of
a variety of exoenzymes and exotoxins by this
extracellular pathogen.
Many strains of S. pyogenes can degrade connective
tissues by
using hyaluronidase, collagenase and streptokinase.
Streptokinase activates plasmin, which leads to
degradation of fibrin and, in turn, dissolution of blood
clots, which assists in the spread of the pathogen.
Released toxins include streptolysins that can destroy
red and white blood cells.
Streptococcus pyogenes
Pathogenesis
Streptococcus pyogenes
GAS: Group A streptococcus
5-15% of individuals harbor S. pyogenes
within the respiratory tract
Pathogenesis (inflammation and cell
destruction) in pharyngitis is mediated by
the release of several exoenzymes and
exotoxins:
Hyularonidase
Collagenase
Streptokinase
Streptolysin
DNAse
Protein
Activity
Interest
Hyaluronic acid capsule
Antiphagocytic
Same as human hyaluronic acid in basement
membrane; antibody cross-reactivity
M protein
Fimbral protein binds fibronectin,
blocks complement C3b (inhibiting
opsonization and lysis)
Cross reactive epitope with cardiac myosin
F protein
Adhesin
Binds fibronectin, associated with M protein.
Hyaluronidase
Degrades hyaluronic acid
Invasion
Streptokinase
Dissolves fibrin clots
“Clot buster” drug
Pyrogenic exotoxins
(Erythrogenic toxins)
Spe A
SpeB Cysteine protease
SpeC
Scarlet fever, TSST of GAS, necrotizing fasciitis
Superantigens
erythematous skin reactions, pyrogenicity,
specific and nonspecific T-cell mitogenicity,
immunosuppression
Streptolysin O
Oxygen labile hemolysin
Hemolysis
Streptolysin S
Oxygen stable hemolysin
Responsible for beta hemolysis.
Transmission
• Can be spread through direct contact or via aerosol droplet
spread (coughing and sneezing)
• Children (5-15 years old) are usually at higher risk for
contracting strep throat.
– Overcrowded conditions are a predisposing factor
– Prevalent during winter and spring
Clinical presentation
Differential diagnosis
Lab diagnosis and identification
• Rapid enzyme immunoassay for
Group A antigen (false negatives
occur frequently ~30%)
• Specimens: throat swab or pus.
– Culture (growth on sheep blood
agar)
– Microscopy
Gram positive cocci in chains
Beta-hemolysis on SBA
Bacitracin sensitive
Catalase negative
Lab diagnosis and identification
Lab diagnosis and identification
Treatment and prevention
•
•
•
Antibiotic resistance is limited.
Beta-lactams continue to be effective:
– Oral amoxicillin (children)
– Intramuscular penicillin G
For those with penicillin allergies:
– Cephalosporin
– Macrolides (Erythromycin)
Scarlet Fever
• Caused by strains of streptococci
which carry a lysogenic
bacteriophage that codes for
pyrogenic toxins which are also
erythrogenic.
• 1 or 2 days after pharyngitis,
streptococci release toxins that
trigger fever and a rash that
starts on the chest and spreads
across the body.
Sandpaper-like rash
Strawberry tongue
Complications
• Non-suppurative sequelae
– May occur 1-4 weeks following inadequate treatment of GAS
infection of the respiratory tract and skin.
– This period of latency suggests a hypersensitivity response (TYPE II
and III) to streptococcal products
– Glomerulonephritis may follow throat or skin infections, whilst
rheumatic fever follows only throat infections.
Rheumatic fever
• Rheumatic fever can result from
infection by any of the serotypes of S.
pyogenes.
• Onset of the condition usually occurs 14 weeks (20 days) after pharyngitis
caused by GAS.
• It may result in damage to the heart
valves and muscle.
• Characterized by fever, polyarthritis,
carditis, erythema marginatum,
Sydenham chorea.
• Common in children between 5-15. Rare
in children under 3 and adults.
• Type II hypersensitivity
Rheumatic Fever
• Major and minor criteria
diagnosis for rheumatic fever
• Rising antistreptolysin O (ASO)
titer
• Management:
– Aspirin
– Corticosteroids
– Positive throat culture: antibiotics
• Jones criteria
Rheumatic Fever
Glomerulonephritis
• Develops 3 weeks after infection with
Streptococci (types 2, 4, 12 or 49)mostly associated with skin infections.
• Children are more likely to develop this
complication
• Characterized by fever, edema,
elevated BUN, hematuria (smoky
urine), high blood pressure, low serum
complement levels. Blood casts in the
urine.
• Condition is due to Ag-Ab complex
deposition on the glomerular
basement membrane. (Type III
hypersensitivity)
Glomerulonephritis
• Anti-DNase B titer
OTITIS MEDIA
OTITIS MEDIA
• Otitis media is an inflammatory
condition that affects the middle
ear.
• Acute otitis media is an infection
with rapid onset that usually
presents with ear pain (otalgia).
• In young children, this may result in
the pulling of the ear, increased
crying and poor sleep.
• Fever, anorexia, vomiting and
lethargy may also present.
Red, bulging
tympanic membrane
OTITIS MEDIA
• Most common causes of bacterial infection are Haemophilus
influenzae (non-typeable strains) and Streptococcus
pneumoniae.
• Infection is primarily seen in children between 6 months to 12
years of age.
• More than 90% of H. influenza strains are non-typeable.
Streptococcus pneumoniae
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•
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•
•
Encapsulated
Non-pyogenic (non-pus forming bacterial infections)
Alpha-hemolytic
Normal inhabitant of the upper respiratory tract
Carrier rate is 20-40%
Streptococcus pneumoniae
•
•
•
•
•
•
•
•
•
•
•
•
Gram positive
Catalase negative
Lancet shaped diplococci
Facultative anaerobe
Non-motile
Do not display M protein
Capsulated in animal tissues
Ferment glucose to lactic acid
Bile soluble
Optochin sensitive
Quellung test positive
Capnophilic (grows best in 5% carbon dioxide)
Risk factors for infection
•
•
•
•
•
•
•
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Reduced disease resistance
Viral respiratory infections
Excessive smoking
Alcoholism
Malnutrition
COPD (chronic obstructive pulmonary disease)
Complement deficiencies (C3b)
Splenectomy
Sickle cell anemia
Leading cause of bacterial disease in children and the elderly
Pathogenesis
•
•
•
•
Does not produce toxins
Pathogenicity is related to the presence of the capsule
Enables tissue invasion and evasion of the immune response
Other important virulence factors include:
– IgA protease
– Teichoic acid
– Pneumolysin O: released with bacterial death, damages respiratory
epithelium..
Pathogenesis
Lab diagnosis and identification
• Specimens: throat swab or pus:
– Culture (growth on sheep blood
agar)
– Microscopy
• Quellung reaction
• Latex agglutination
• Colonies are bile soluble
Gram positive diplococci
Alpha-hemolysis on SBA
Optochin sensitive
Catalase negative
Lab diagnosis and identification
• Quellung reaction
– Swelling of the capsule on
exposure to serum containing anticapsule antibodies.(Serotyping)
• Latex particle agglutination
– Antibody is adhered to latex
particles and then used to detect
antigen.
Prevention
• The mortality rate is high in the elderly and infants.
• Elderly, immumocompromised and asplenic individuals can be
immunized with polyvalent (23) polysaccharide vaccine. (PPV)
• It is safe, effective and provides at least 5 years protection.
• In infants younger than 5 years, a polyvalent (13)
polysaccharide vaccine conjugated to a carrier protein
(diphtheria toxoid). (PCV)
Haemophilus influenzae
• Gram-negative coccobacillus (pleomorphic rod)
• Facultative anaerobe
• Most strains are opportunistic pathogens
– they usually live in their host without causing disease, but cause
problems only when other factors (such as a viral infection, reduced
immune function or chronically inflamed tissues, e.g. from allergies)
create an opportunity.
– Transmission is via respiratory droplets; shared toys
Haemophilus influenzae
• Bacterial culture is achieved on chocolate blood agar with
hemin (factor X) and NAD (factor V).
• Incubation is best at 37°C in a CO2 enriched environment.
• Catalase positive
• Oxidase positive
• Quellung reaction
Quellung reaction
Pathogenesis
• Haemophilus can be either encapsulated or unencapsulated.
• The polysaccharide capsule is the most important determinant
of virulence.
• There are six capsular serotypes (a-f). Most infections are
caused by strains belonging to capsular serotype b.
• Unencapsulated strains are part of the normal flora of the URT
and cause local mucosal infections but not invasive infections.
– Sinusitis, otitis media or bronchitis
• IgA protease is also produced.
Lab diagnosis and identification
• Culture on chocolate agar with
factors X and V.
• Culture with Staphylococcus
aureus on blood agar with X
factor (produces satellite
colonies)
• Latex particle agglutination for
capsular antigen detection.
• PCR
Treatment
• Antibiotic sensitivity tests must be done on organisms isolated
from serious infections.
• Most are susceptible to penicillins (high-dose amoxicillin)
• In penicillin-allergic patients, erythromycin or one of its
derivatives (azithromycin) can be used.
• Vancomycin is the drug of choice for penicillin resistant
species.
Prevention
• Vaccine (protein conjugate) against Haemophilus influenzae
type b (Hib).
• Antibodies produced against serotype b promote opsonization,
complement fixation and phagocytosis and killing.
• Vaccination against type b H. influenza is recommended by 2
months old.
• Type b is also associated with disseminated disease,
pneumonia, epiglottitis and meningitis.
Question
1. Microscopic examination of a sputum sample from a 34-yearold male with fever and cough reveals Gram positive lanctshaped cocci in pairs. The bacteria are likely to be :
A.
B.
C.
D.
E.
Catalase positive
Optochin resistant
Bile soluble
Capable of complete hemolysis
Bacitracin sensitive
Gram positive
Lancet shaped
diplococci is
suggestive of S.
pneumoniae.
All streptococci are
catalase negative.
S. pneumoniae is
optochin sensitive,
exhibits alpha
hemolysis and is
bacitracin resistant.
Hence the answer is:
C.
DIPHTHERIA
Diphtheria
Regional lymph nodes are inflamed, sore throat and
dysphagia (difficulty swallowing)
Pseudomebrane
Pseudomembrane is composed of dead cells, fibrin
and bacterial pigment.
Profuse bleeding occurs if the pseudomembrane is
dislodged.
Extension of the pseudomembrane into the trachea
can cause obstruction.
Diphtheria
• In severe cases, the
exotoxin may diffuse into
the neck tissue.
• This produces severe
edema (bull neck).
• Enlarged cervical lymph
nodes ( cervical
lymphadenopathy)
Corynebacterium diphtheriae
• Aerobic, Gram-positive bacillus
• Irregularly shaped rod (club-shaped)
– arranged in V or L shapes
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•
•
•
•
Non-motile
Non-spore forming
Non-capsulate
Causative agent of diphtheria
Grows on Löffler’s medium
Corynebacterium diphtheriae
• Pre-school age children are at highest risk of contacting the
bacterium
• Prevalent in autumn and winter months
• Humans are the chief reservoirs (throat, nasopharynx)
• Transmission is via secretions from an infected person (aerosol
droplets) or by direct contact.
• May enter by the respiratory tract or by the conjunctiva or an
open wound.
Risk Factors
• Low VACCINE coverage among infants and children
• Lack of mass immunization programs amongst children and
adults at high risk
• Poor nutrition
• Community outbreaks
• Crowded or unsanitary living conditions
Clinical manifestations
• Incubation period : 2-5 days
• Prodrome: fever (39°C), malaise, anorexia (loss of appetite),
sore throat, headache.
• Child appears very ill and toxic
• Complications:
– Airway obstruction
– Delirium
– Circulatory collapse (myocarditis)
Pathogenesis
• Upon entry, the bacilli multiply in the throat and secrete a
powerful EXOTOXIN.
• This exotoxin production stimulates both local and systemic
inflammation.
• Local:
– Causes necrosis of epithelial cells and liberates serous and fibrinous
material which forms a grayish-white PSEUDOMEMBRANE.
– The membrane bleeds on being dislodged.
– Surounding tissues are inflamed and edematous
Pathogenesis
• Toxin production only occurs when the bacterium is infected with
bacteriophage carrying the tox gene (toxigenic strains).
• It is an ADP- ribosyltransferase that inhibits host protein synthesis.
• Inactivates EF-2 by ADP ribosylation
• Diphtheria toxin is an A-B toxin.
– Portion A mediates the enzymatic activity that prevents protein synthesis
– Portion B binds to the cell receptor and mediates translocation of Portion
A into the cytosol.
Other examples of lysogenic infection
Pathogenesis
Pathogenesis
Pathogenesis
• Systemic:
– Exotoxin can affect the heart, kidneys and CNS.
– Heart: Conduction disturbance (myocardial fibers are degenerated
and the heart is dilated)
– Kidneys: Renal tubular necrosis
– CNS: Polyneuritis
Diagnosis and identification
• Albert’s staining [specific to
volutin (metachromic) granules
found in Corynebacterium
diphtheriae]
• Club shaped rods present
• Non-motile
• Non-encapsulated
• Tellurite (Loeffler’s medium) agar
(gray-to-black colonies)
• Elek test (toxigenic strains)
Diagnosis and identification
Elek test
• The test colony, along with a
positive and negative control are
streaked onto the Elek agar plate.
• Filter paper strip with the antitoxin
is placed on an agar plate.
• As the antitoxin diffuses away from
the strip, precipitin lines form at the
zone of equivalence (Ag-Ab
precipitation).
• ELISA test is now more commonly
used.
Treatment
• Neutralization of free circulating toxin by the administration of
antitoxin.
• Administration of antibiotics to eradicate bacteria (erythromycin)
• Supportive and symptomatic therapy
• Management of complications.
Treatment
• Antitoxin administration
– Pharyngeal or laryngeal diphtheria of 48 hours duration (20, 00040,000 units)
– Nasopharyngeal lesions: (40,000-60,000 units)
– Extensive disease of 3 or more days with swelling of the neck
(80,000-120,000 units)
– Antitoxin may be repeated if the clinical improvement is slow
Treatment
• Antibiotic administration
– Procaine penicillin (3-6 lac units IM at 12 hour intervals)-endocarditis
– Oral penicillin (Penicillin G) 125-250mg four times a day
– Erythromycin (25-30 mg/kg/day) for 14 days
• Three negative cultures at 24 hour intervals should be
obtained before the patient is declared cured.
Complications
Prevention
• Vaccination: immunization with diphtheria toxoid combined
with tetanus and pertussis toxoid (DTaP) should be given to all
children at 2, 3 and 4 months of age.
• Booster doses are given between the ages of 3 and 5.
• Further booster is given before leaving school and is
considered protected for a further 10 years.