Corynebacterium

Genus
Gram positive non-sporing forming
rods

Introduction
Corynebacterium diphtheriae infects
the nasopharynx or skin.
Toxigenic strains secrete a potent
exotoxin which may cause diphtheria.
The symptoms of diphtheria include
pharyngitis, fever, swelling of the neck or
area surrounding the skin lesion.

Introduction
Diphtheritic lesions are covered by a
pseudomembrane.
The toxin is distributed to distant
organs by the circulatory system and
may cause paralysis and congestive
heart failure.

C. diphteriae group
C. diphteriae
C. ulcerans
C. pseudotuberculosis

Defintion
History: records back to 4th century BC,
recognized as specific disease in 1821.
Gram-positive, rod-like organism.
Often club-shaped, sometimes in
pallisades, "Chinese letters" .
No spores, no flagella.
Sometimes encapsulated.

Definition
Corynebacterium diphtheriae is a
nonmotile, noncapsulated, club-shaped,
Gram-positive bacillus.
Toxigenic strains are lysogenic for one of
a family of corynebacteriophages that
carry the structural gene for diphtheria
toxin, tox.

Special staining to
reveal volutine granules

Corynebacterium
diphteriae Gram
staining

Corynebacterium
diphteriae Gram
staining

Definition
Corynebacterium diphtheriae is classified
into biotypes (mitis, intermedius, and
gravis) according to colony morphology,
as well as into lysotypes based upon
corynebacteriophage sensitivity.

Cultivation
Growths in special culture media
(Tinsdale) with potassium tellurite
Black colonies
Loeffler culture medium (with serum
bovine) white colonies (bacteria growth
as typical rods with largest extremities and
typical Chinese letters arrangement).

Classification
Biochemical properties and cultivation
growth classify the species in three types
gravis* (13 subtypes** );
intermedius (4 subtypes);
mitis (40 subtypes)
* no relation with diseases severity
** according with their antigenic structure

Pathogenesis

The pathogenesis of diphtheria is based

upon two primary determinants:
the ability of a given strain of C.
diphtheriae to colonize in the
nasopharyngeal cavity and/or on the
skin,
local invasion
its ability to produce diphtheria toxin.

Pathogenesis
Since those determinants involved in
colonization of the host are encoded by
the bacteria, and the toxin is encoded by
the corynebacteriophage, the molecular
basis of virulence in C. diphtheriae
results from the combined effects of
determinants carried on two genomes.

Pathogenesis
Nontoxigenic strains of C. diphtheriae are
rarely associated with clinical disease;
however, they may become highly
virulent following lysogenic conversion
to toxigenicity.

Pathogenic factors
Diphtheria Toxin (DT) / protein
- secreted as 58.4 kDa protein
- cleaved to yield A/B chains
- A has catalytic activity
- B has receptor binding/translocation
activities

Diphtheria toxin
Fragment A catalyzes the NAD
dependent ADP-ribosylation of
elongation factor 2, thereby inhibiting
protein synthesis in eukaryotic cells .
Fragment B binds to the cell surface
receptor and facilitates the delivery of
fragment A to the cytosol.

Diphtheria Toxin (DT)

Cells death necrosis
(pseudomembrane in oro-pharynx)

Clinical Manifestations
There are two types of clinical
diphtheria:
- nasopharyngeal
- cutaneous.
Symptoms of pharyngeal diphtheria
vary from mild pharyngitis to hypoxia
due to airway obstruction by the
pseudomembrane.

Pseudomembranes

Clinical Manifestations
The skin lesions in cutaneous diphtheria
are usually covered by a gray-brown
pseudomembrane.

Clinical Manifestations
The involvement of cervical lymph nodes
may cause profound swelling of the
neck (bull neck diphtheria), and the
patient may have a fever.

Local lymphadenopathy
(bull neck diphtheria)

Clinic
Inflammation results in pseudomembrane
formation (fluid portion of exudate - mainly
fibrin - and cells coagulate on surface).
Pseudomembrane may cause blockage
Toxin diffuses throughout body via blood:
- cardiac, neurologic complications
- heart/respiratory damage, paralysis
Death by suffocation
Survivors usually have neurologic, cardiac
complications.

Pathonecity
Bacteria remain localized:
- no blood culture positive
- typical disease is toxemia only

Complications
Life-threatening systemic complications,
principally
- loss of motor function (e.g., difficulty in
swallowing) and
- congestive heart failure, may develop
as a result of the action of diphtheria toxin
on peripheral motor neurons and the
myocardium.

Colonization
Little is known of the colonization factors of C.
diphtheriae.
However, it is apparent that factors other than
the production of diphtheria toxin contribute to
virulence.
Epidemiologic studies have demonstrated that
a given lysotype may persist in the population
for extended periods.
It may later be supplanted by another lysotype.

Colonization
The emergence and subsequent
predominance of a new lysotype in the
population are presumably due to its ability to
colonize and effectively compete in their
segment of the nasopharyngeal ecologic niche.
Corynebacterium diphtheriae may produce a
neuraminidase that cleaves sialic acid from the
cell surface into its pyruvate and N-acetylneuraminic acid components.

Diagnosis
The clinical diagnosis of diphtheria
requires bacteriologic laboratory
confirmation of toxigenic C. diphtheriae in
throat or lesion cultures.
For primary isolation, a variety of media
may be used: Tinsdale tellurite agar
and Loeffler agar for subcultivation of
the strain isolated in primoculture.

Samples prelevations
(swabs)
Sterile cotton-tipped applicators are used to
swab the pharyngeal tonsils or their beds.
Calcium alginate swabs may be inserted
through both nares to collect nasopharyngeal
samples for culture.
Since diphtheritic lesions are often covered
with a pseudomembrane, the surface of the
lesion may have to be carefully exposed before
swabbing with the applicator.

Lesion may have to be carefully

exposed before swabbing with the
applicator

Diphteria lesions

Diagnosis
Exsudate prelevation (3 swabs from pharynx
and one from noso-pharynx)
Smear examination (typical bacteria
morphology) from 1st swab
Cultivation:
- Tinsdale and blood agar (2nd swab)
- Enrichment culture media (ECT - eggs,
cisteine, tellurite) 3rd swab (as the swab from
naso-pharynx also).

Smear examination

Tinsdale agar

Loeffler
culture
media

Diagnosis
Identification biochemical
identification tests:
- differentiate C. diphteriae from others
corynebacteria and
- differentiate serovars of C. diphteriae:
mitis, gravis and intermedius
Toxin presence (in vitro Elek test; in
vivo, animal model or Vero cells test).

Identification
Following initial isolation, C. diphtheriae
may be identified as mitis, intermedius,
or gravis biotype on the basis of
- carbohydrate fermentation patterns and
- hemolysis on sheep blood agar plates.

Toxigenicity
The toxigenicity of C. diphtheriae strains
is determined by a variety of in vitro and
in vivo tests.
The most common in vitro assay for
toxigenicity is the Elek immunodiffusion
test.

Elek test
This test is based on the double
diffusion of diphtheria toxin and
antitoxin in an agar medium.
A sterile, antitoxin-saturated filter paper
strip is embedded in the culture medium,
and C. diphtheriae isolates are streakinoculated at a 90 angle to the filter
paper.

Elek test
The production of diphtheria toxin can be
detected within 18 to 48 hours by the
formation of a toxin-antitoxin precipitin
band in the agar.

Elek test

Elek test

In vivo test
Alternatively, many eukaryotic cell lines (e.g.,
African green monkey kidney, Chinese hamster
ovary) are sensitive to diphtheria toxin,
enabling in vitro tissue culture tests to be used
for detection of toxin.
Several sensitive in vivo tests for diphtheria
toxin have also been described (e.g., guinea
pig challenge test, rabbit skin test).

Treatment
Antitoxin serum
Antibiotics: penicillin, erythromycin,
tetracycline, gentamycine, cefalotin,
ciprofloxacin.
Erythromycin to cure the healthy
carriers (no-toxin producer strains
conversion by lysogenic conversion see
genetic).

Prevention
Immunization of animals with altered toxin, producing
antitoxin, was first done in 1890, 1st used in humans in
1891.
Toxin-antitoxin (neutralized in vitro) introduced by
Theobald Smith in 1909, used little.

Toxoid introduced in 1923, now widely used

Passive protection from immune mother
Active immunization (vaccination) at early age
universally advocated.

Prevention
Carrier rate
- without immunization ~ 5%
- with immunization ~ 0%

Corynebacterium
ulcerans
Upper respiratory tract pathogen of humans
Mastitis, abscesses in cattle

Can produce diphtheria toxin (if lysogenized) with

similar effect but different antigenically
Treatment: antitoxin serum and penicillin or
erythromycin.

Corynebacterium
pseudotuberculosis
Caseous lymphadenitis in sheep, goats
Ulcerative lymphangitis in horses

Infection of superficial site (wound)

extends to lymph node (human).
Treatment: penicillin, erythromycin,
tetracycline and surgical drainage.

C. minutissimum :
Disease: eriytrasma, localized infection
of skin
Special examination under UV light (Wood
lamp) special lesions aspect of coral.
Treat: erythromycin

C . jeikeium :
The most frequent isolated from Europe,
and North America.
Commensal in healthy persons (skin and
mucosa)
and patients colonization (isolated from
blood, CSF, etc)
Contamination of different i.v. devices

Causes heart abscesses and pyelone-phritis in

immuncompromissed hosts.
Treatament: sensitive to glycopeptides
(vancomycin, teichoplanin).
Resistent to: penicillin, cephalosporins and
aminoglycosides.
Endocarditis: penicillin and genta- or vancomycin
and gentamycin, with sinergistic effect.
CNS: genta-, rifampicin, cotrimoxazole.

Listeria

Listeria
Small, Gram-positive, non-sporing rods.
End-over-end tumbling motility when
grown at 20-25o C, not at 37o C.
Facultative anaerobe, beta-hemolytic.

Listeria - smear from

pathological product

Listeria - smear from

pathological product

Listeria - EM (flagella)

Habitat
Habitat
- primary habitat soil, decaying vegetation
(saprophyte)
- animal feed
- water
- fecal material of domestic animals, birds
and fish
- sewage

Classification
Genus divided into 6 speciies
- based on biochemical characteristics,
relatedness of DNA
- pathogens in group 1 and 2

Species isolated from

human

L. monocytogenes
L. ivanovii
L. seeligeri

L. monocytogenes
A particular property of L. monocytogenes
is the ability to multiply at low
temperatures (limite: 0 40 0 C).
Bacteria therefore can accumulate in
contaminated food stored in the
refrigerator.

Pathogenesis
Listeria monocytogenes is presumably
ingested with raw, contaminated food .

Pathogenesis
An invasion factor secreted by the pathogenic
bacteria enables them to penetrate host cells of
the epithelial lining.
Since this microorganism is widely distributed,
this event may occur rather often.
Normally, the immune system eliminates the
infection before it spreads.
Most adults who have no history of listeriosis
have T lymphocytes primed specifically by
Listeria antigens.

Pathogenesis
If the immune system is compromised,
however, systemic disease may develop.
Listeria monocytogenes multiplies not only
extracellularly but also intracellularly within
macrophages after phagocytosis and even
within parenchymal cells which are entered by
induced phagocytosis.
It therefore belongs to the large group of
facultatively intracellular pathogens.

Listeria - smear from

pathological product

Pathogenesis
Survival within the phagosomes and
eventual escape into the cytoplasm are
mediated by a toxin, which also acts as
a hemolysin.

Clinical aspects of
infection
Listeria monocytogenes causes
- intra-uterine infection,
- meningitis (even, encephalitis; very rare for
bacterial infections)
- septicaemia.
The incubation period: 1 to 90 days, with an
average for intra-uterine infection of around
30 days.

Infection in pregancy
and the neonate
Listerios in pregancy is classified by fetal
gestation at onset.
Neonatal infection is devided into:
- early (< 2 days old)
- intermediate (3-5 days) and
- late (> 5 days)-onset diseases.

Maternal infection
Rare before 20 weeks.
Simptoms: asimptomatic, but often have
very mild symptoms (chills, fever, back pain,
sore throat, headache, sometimes with conjunctivitis,
diarrhoea).

Symptomatic women may have

positive blood cultures.

Maternal infection
Consequences: with the onset of fever,
fetal movements are reduced, and
premature labour occur withn about one
week.
Culture of amniotic fluid, placenta or high
vaginal swab after delivery invariably
yelds a heavy growth of L. monocytogenes.

Maternal infection
While the outcome of infection for the
mother is usually bening, the outcome for
infant is more variable.

Neonate infection
Abortion, stillbirth and early-onset
neonatal diseases are common.
Early neonatal listeriosis is
predominantly a septicaemic illness,
contracted in utero.
In contrast, late neonatal infection is
predominantly meningitic.

Neonate infection
Early-onset diseases represents a
spectrum of mild to severe infection.
Those neonate who die of infection usually
do so within a few days of birth and have
pneumonia, hepatosplenomegaly,
petechiae, abscesses in the lever o r
brain, peritonitis and enterocolitis.

Neonate infection
Late-onset disease is he third
commonest form of meningitis in
neonate.
Meningitis is often complicated by
encephalitis, which is exceptional
among bacterial infections.

Clinical Manifestations
In adults and juveniles the main
syndromes are:
- CNS infection;
- septicaemia;
- endocarditis.

Host Defenses
Because it multiplies intracellularly, L
monocytogenes is largely protected
against humoral immune factors such
as antibodies, and the effective host
response is cell- mediated, involving
both lymphokines (especially interferon)
produced by CD4+ (T-helper) cells and
direct lysis of infected cells by CD8+
(cytotoxic) T lymphocytes.

Diagnosis
Listeria monocytogenes is implicated
when monocytosis is observed in the
peripheral blood as well as the cerebrospinal fluid.
Early diagnosis may be obtained by
finding pleocytosis with Gram-positive
rods in a Gram stain of smears of the
cerebrospinal fluid.

Smear

Microscopy

Cocobaccilari form from young culture

Filamentous forms from old culture.
Mobility: 18 25 0 C (flagella appearance).

Cultivation (complex medium)

Blood agar plate

Chromogenic culture media

Diagnosis
Final proof is obtained by culture.
Serologic tests are highly unreliable.

Diagnosis
Antigenic identification: thirteen
serotypes (serovars) are recongnized.
Most cases of human infections are
caused by serovars 4b, 1/2a and 1/2b.
Large food-borne outbreaks have
predominantly been caused by serovar
4b strains.

Treatment
Most of the common antibiotics, except
cephalosporins, are active against L.
monocytogenes in vitro.
In practice, ampicillin or penicillin
combined with an aminoglycoside has
given the best results.

Treatment
However, because infection occurs mainly in
infirm patients and because intracellular
bacteria are hardly accessible to most drugs,
the cure rate is low.
Furthermore, Listeria cells, although inhibited,
are not killed by ampicillin.
High doses for prolonged periods are
indicated.

Epidemiology
Sources: soil, sewage, water and dacying
plant material, where it can survive for more
than 2 years.
Numerous types of raw, processed, cooked
and ready-to eat foods contain L. monocytogenes.
The unusual tolerance of the bacterium to
sodium cloride and the ability to multiply at
refrigeration temperatures makes L.
monocytogenes of particular concern as a
post-processing contaminant of foods.

Transmission
The consumption of contaminated
foods is the principal route of
transmission.
The second, mother to child infection.
The direct contact (animal material or
infected animal) is rare.
No vaccine available.
Prevention: unspecific.