Gram positive rods of medical importance

• Non Endospore formers

2
Corynebacterium
 General characteristics
• large diverse group of bacteria of animals and • Stain unevenly with Gram
human pathogens and saprophytes • Cells contain metachromatic granules
• there are more than 80 species in the genus (visualize with methylene blue stain)
• most of the species are found as normal biota • Metachromatic granules (often near the
on skin and mucous membranes of upper poles) give the rod a beaded irregularly-
respiratory tract, GIT, Urogenital tract of shaped appearance. "Club shaped".
humans and animals. • Arranged in palisades or in V- or L-
• >50 are clinically significant shaped formations
• Lipid-rich cell wall contains meso-
• Do not form spores
diaminopimelic acid, arabino-galactan
• Non motile polymers, and short-chain mycolic acids
• Aerobic or facultative anaerobes • Lysogenic bacteriophage encodes
potent exotoxin in virulent strains
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Corynebacterium spp
Pathogenic Species
• found in the throat and nasopharynx of
carriers and in patients with diphtheria
• Toxin producing strains - gravis,
intermedius and mitis
• Corynebacterium diphtheria - major
pathogen of medical importance
Non pathogenic species
• morphologically resemble diphtheriae
• common commensals of the nose,
throat, nasopharynx, skin, urinary tract,
and conjunctiva
• do not produce Exotoxin
• may cause disease in
immunosuppressed individuals
• do not contain metachromatic granules 5
• Corynebacterium diphtheriae

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 Epidemiology

• Causes Diphtheria (child killer), a serious disease world wide

• Common in crowded poor urban children
• Common in countries where the population has not been immunized
• High mortality; 5-10 % even in properly treated patients.
• Untreated patients are infectious for 2-3 weeks.
• Unless immunized, children and adults may be infected repeatedly
• No seasonal incidence
• The development of effective vaccination protocols and widespread
immunization beginning in early childhood has made the disease rare in
developed countries
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 Transmission
Humans are the only natural host of C. diphtheriae.
Both toxigenic and non toxigenic organisms reside in the upper respiratory tract.

organism is primarily spread by

• respiratory droplets, usually by convalescent or asymptomatic carriers.
• introduction of organism into the subcutaneous through a puncture wound or cut in
the skin
• direct contact with an infected individual or a contaminated fomite (less frequently
spread by )
• droplet infection or hand-to-mouth contact

do not spread beyond their initial site of infection

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 Virulence Factors
The organism must first establish and maintain itself in the throat.

Toxins (a heat-labile polypeptide )

– Produced by strains of C. diphtheriae infected with a lysogenic β-phage, which
carries the tox gene for diphtheria toxin.
– Composed of two fragments, A and B linked together by a disulfide bridge.
– The binding (B) fragments mediates binding of the toxin to glycoprotein
receptors on cell membranes
– The active (A) fragment possesses enzymatic activity that cleaves nicotinamide
from nicotinamide adenine dinucleotide (NAD)
– The toxin is exceedingly potent and is lethal for humans
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 Pathogenesis
The bacilli grow rapidly on mucous membranes or in skin abrasions, and those that are
toxigenic produce toxin. The toxins are absorbed into the mucous membranes

• toxin binds to a receptor (heparin-

binding epidermal growth factor) on
the surface of cells
• triggers the entry of the toxin into
the cell through receptor-mediated
endocytosis
• on reaching the cytoplasm
trypsinization cleaves the toxin into
the two fragments
• Fragment A disrupts protein
synthesis
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 Pathogenesis
The host response to C. diphtheriae consists of the
following
• Local toxigenic effects:
• Locally the Toxin induces tissue necrosis and
exudate formation
• Also trigger an inflammatory reaction
• results in a pseudomembrane composed of
fibrinous exudate, necrotic respiratory epithelial
cells, leukocytes, and organisms
• formed commonly over the tonsils, pharynx, or
larynx
• may spread down the bronchial tree causing
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respiratory tract obstruction and dyspnea
 Pathogenesis
• The regional lymph nodes in the neck
may enlarge
• marked oedema of the entire neck,
with distortion of the airway
• referred to as “bull neck” clinically
Wound or skin diphtheria
• a membrane may form on an infected
wound that fails to heal
• The small amount of toxin that is absorbed
during skin infection promotes
development of antitoxin antibodies

• The toxin also is absorbed and can produce various systemic effects involving the
kidneys, heart, and nervous system 12
 Diseases
Body Sites

13
 Disease
Respiratory (Nasopharyngeal diphtheria)
• Pharyngeal, Laryngeal Systemic
– sore throat and low-grade fever • Cardiac conduction defects and myocarditis
usually develop may lead to congestive heart failure and
– Prostration and dyspnea soon follow permanent heart damage
because of the obstruction caused by • Neuritis of cranial nerves and paralysis of
the membrane. muscle groups, such as those that control
movement of the palate or the eye, are seen
– obstruction may even cause
late in the disease
suffocation
• There may be difficulties with vision, speech,
swallowing, or movement of the arms or legs
Cutaneous diphtheria • Death often is a result of cardiac failure.
a chronic (nonhealing) ulcer with a gray
membrane
tissue degeneration and death (rare).
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 Diagnosis

• Clinical
– Presumptive
– Diphtheria is considered in patients with pharyngitis, low-grade fever,
and cervical adenopathy (swelling of the neck)
– Erythema of the pharynx progressing to adherent gray
pseudomembranes

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 Diagnosis
Toxingenicity Tests
• Animal inoculation

• In Vivo – rabbit skin test-necrosis

– Animal inoculation – guinea pig challenge test- lethal
– 0.8 ml emulsified bacteria injected
into 2 guinea pigs(GP)
• In Vitro GP A-has dipht. antitoxin (injected 2
– Elek test hours before)
GP B-Doesn't have antitoxin

Result: Guinea pig B dies

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 Lab diagnosis
Specimen

• Swab from the nose, throat, or other

suspected lesions
• Obtained swab before antimicrobial
drugs are administered.
• Swabs should be collected from
beneath any visible membrane.

The swab should then be placed in

semisolid transport media.

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 Diagnosis
Gram stain
• Gram -positive rods Special stains for metachromatic granules
• small, slender pleomorphic (club-shaped)
that appear in short chains

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 Diagnosis
Culture
• Routine media
– Blood agar
• Selective /Differential media
– Cysteine-tellurite blood agar
– Tinsdale's agar contains potassium tellurite (an inhibitor of
other respiratory flora) organism produces distinctive black
colonies with halos
• Loeffler’s
– Suspected isolates must be tested for virulence using an
immunologic precipitin reaction to demonstrate toxin
production
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Laboratory diagnosis

The bacteria grow into convex and semi-

opaque colonies.

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 Diagnosis
All C. diphtheriae isolates should be subjected to testing for toxigenicity.

Elek (an immunoprecipitation)

method

• organisms (controls and unknowns)

are streaked on medium
• A filter paper strip containing
antitoxin is placed on a cultured agar
plate.
• After 48 hours of incubation, the
antitoxin diffusing from the paper
strip has precipitated the toxin
diffusing from toxigenic cultures
• results in precipitin bands between
the strip and the bacterial growth. 21
 Diagnosis
Serology Molecular
• Detection of toxin. • Detection of C. diphtheriae tox
gene

– Rapid enzyme-linked – Polymerase chain reaction

immunosorbent assays (PCR).
– Immunochromatographic strip – The PCR assay directly on
assays clinical specimens

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 Treatment
Requires
• prompt neutralization of toxin
– A single dose of serum antitoxin inactivates any circulating toxin, although it
does not affect toxin already bound to a cell-surface receptor.
• eradication of the organism
– C. diphtheriae is sensitive to several antibiotics, such as erythromycin or
penicillin
– Antibiotic treatment slows the spread of infection and, by killing the organism,
prevents further toxin production.
– Antibiotic treatment usually renders patients noninfectious within 24 hours.

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 Prevention

• The control of an epidemic outbreak of diphtheria involves rigorous

immunization and a search for healthy carriers among patient
contacts
– immunization with toxoid, usually administered in the triple
vaccine, together with tetanus toxoid and pertussis antigens DPT
– the initial series of injections should be started in infancy
– booster injections of diphtheria toxoid (with tetanus toxoid)
should be given at approximately ten-year intervals throughout
life
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Listeria

25
 General characteristics
• contains 10 species.
• Listeria monocytogenes is an important human pathogen,
• Listeria ivanovii is primarily an animal pathogen.
• widespread in the environment and has been recovered from soil; water; vegetation;
and animal products raw milk, cheese, poultry, and processed meats).
• cause illness in wild and domestic animals, including sheep, cattle, swine, horses,
dogs, cats, rodents, birds, and fish

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• Listeria monocytogenes

27
 General characteristics
• capable of growing and surviving over a
wide range of environmental conditions
• Intracellular parasites
• It can survive at refrigerator
• Short Gram-positive rods temperatures (4°C)
• Non–spore-forming • conditions of low pH and high salt
• Catalase positive conditions
• Has a tumbling end-over-end motility • able to overcome food preservation
at 22–28°C but not at 37°C and safety barriers

making it an important foodborne

pathogen

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 Epidemiology

• Listeria infections, may occur as sporadic cases or in small epidemics

• Infections are usually foodborne
• Studies have shown that
– Listeria infections are most common in pregnant women, fetuses or newborns,
and in immunocompromised individuals, such as the elderly or patients
receiving corticosteroids
• One to fifteen percent of healthy humans are asymptomatic intestinal carriers
of the organism

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 Transmission
• At risk
– Pregnant women
– Foetus/Neonates
– Elderly patients
– Immunocompromised patients

Listeria infections occur primarily in two clinical settings:

(1) in the fetus or in a newborn as a result of transmission across the placenta or

during delivery
(2) in pregnant women and immunosuppressed adults, especially renal transplant
patients through the gastrointestinal tract after ingestion of contaminated
foods such as cheese, fruit, or vegetables.
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 Virulent factors
• Growth at low temperature
• Motility
• Catalase, superoxide dismutase, phospholipase C
• Surface protein (p60)
– Protein p60 induces phagocytosis through increased adhesion and penetration
into mammalian cells
• Hemolysin (listeriolysin O)
– pore forming toxin, damages the phagosome membrane, effectively preventing
killing of the organism by the macrophage. Required for intracellular growth
• Internalins protein A and B

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 Pathogenesis
The adhesin proteins (Ami, Fbp A, and flagellin
proteins) facilitate bacterial binding to the host cells L monocytogenes can move from cell
to cell without being exposed to
Cell wall surface proteins- internalins A and B antibodies, complement, or
interact with E-cadherin, a receptor on epithelial polymorphonuclear cells
cells, promoting phagocytosis into the epithelial
cells.

After phagocytosis, the bacterium is enclosed in a

phagolysosome, where the low pH activates the
bacterium to produce listeriolysin O.

Listeriolysin O along with two phospholipases,

lyses the membrane of the phagolysosome and
allows the listeriae to escape into the cytoplasm of
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the epithelial cell
 Diagnosis
CAMP test Esculin hydrolysis

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 Diseases
In
• Immunocompromised individuals
(those with defects in cellular • Newborn
immunity)
• acut meningitis
– severe invasive disease ie • Fetus
Septicemia and meningitis
spontaneous abortion, stillbirth,
• Healthy individuals
granulomatosis infantiseptica, a
– febrile gastrointestinal illness devastating illness characterized
(diarrhoea, nausea, vomiting)
by microabscesses and granulomas
• Pregnant women usually in the third in the liver and spleen
trimester
– milder flulike•illness.

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 Diagnosis
• Specimen
– Blood
– Cerebrospinal fluid

• Microscopy
– Gram-positive, short rods
– may occur as slender diplobacilli
or in short chains
– or arranged in V- or L-shaped
formations similar to
Corynebacteria.
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 Laboratory diagnosis
Gram stain: The appearance of Gram-positive rods
resembling diphtheroids

Culture: Formation of small, gray colonies with a

narrow zone of β-hemolysis on a blood agar
plate suggest the presence of Listeria.

The isolation of Listeria is confirmed by the presence of distinctive tumbling motility by

light microscopy in liquid medium, which is most active after growth at 25°C which
differentiate them from the non motile Corynebacteria.
Identification of the organism as L. monocytogenes is made by sugar fermentation tests.
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