INDEX:-

INTRODUCTION

CORYNEBACTERIUM

CORYNEBACTERIUM DIPHTHERIAE

Diseases

Morphology

Cultural
Characteris
tics

Biochemical Reactions

Toxin

Resistance

Antigenic Structure

Pathogenesis

Clinical Diseases

Laboratory Diagnosis

Epidemiology

Prophylaxis

Treatment

OTHER MEDICALLY IMPORTANT

CORYNEBACTERIA

DIPHTHEROIDS

OTHER CORYNEFORM GENERA

REFERENCE
INTRODUCTION:-

 The coryneform group consists of Corynebacterium and related genera that

are aerobic.
 non-sporing.
 irregularly shaped.
 non-spore-forming.
 gram-positive rods.

CORYNEBACTERIUM:-

 The genus Corynebacterium comprises 66 species; 38 of them associated

with human disease.
 Corynebacteria are gram-positive and non-acid fast.
 Non-motile rods with irregularly stained segments, and sometimes granules.
 They frequently show club shaped swellings and hence the name
corynebacteria (from coryne, meaning club).
 The terms ‘diphtheroid’ and ‘coryneform’ have been used to describe
other corynebacteria that do not commonly cause human infection and
were considered less significant.
  Corynebacteria are closely related to mycobacteria and nocardiae.
 These three groups collectively may be referred as CMN group.

CORYNEBACTERIUM DIPHTHERIAE

 The diphtheria bacillus was first observed and described by

Klebs (1883) but was first cultivated by Löffler (1884).
 It is hence known as the Klebs-Löffler bacillus.
 Löffler studied the effect of the bacillus in experimental
animals and concluded that the disease was due to some
diffusible product of the bacillus.
 Roux and Yersin (1888) discovered the diphtheria exotoxin and
established its pathogenic effect.
 The antitoxin was described by von Behring (1890).
 C. diphtheriae owes its pathogenicity to the production of a
potent exotoxin active on a range of tissues, including heart
muscle and peripheral nerves.

DISEASES: -

 The major disease caused by C. diphtheriae is diphtheria (Greek,

diphtheria, “leathery skin”, referring to the pseudomembrane that initially
forms on the pharynx).
 Bretonneau, a French army surgeon, described in 1821 the
unique clinical characteristics of the disease and used the term
‘diphtherie’ to signify the leathery membrane that occurs in the
oropharynx, or sometimes the nasopharynx, and which is the hallmark
of the disease.
 Occasionally, the organism is implicated in wound and chronic skin
infections. Non-toxigenic strains have been associated with endocarditis,
meningitis, cerebral abscess and osteoarthritis throughout the world.

MORPHOLOGY:-
 They are thin, slender gram-positive bacilli but are easily decolorized,
particularly in old cultures, measuring approximately 3-6 μm × 0.6-0.8
μm.
 They have a tendency to clubbing at one or both ends.
 They are highly pleomorphic.
 Cells often show septa, and branching is infrequently observed.
 They are non-motile, non-spore forming, and non-acid fast.
 The bacilli are arranged in a characteristic fashion in smears.
 They are usually seen in pairs, palisades (resembling stakes of a fence) or
small groups or as individual cells lying at sharp angles to another,
resembling the letters V or L.
 This particular arrangement with C. diphtheriae has been called the
Chinese letter or cuneiform arrangement (Fig. 28.1).
 This is due to the incomplete separation of the daughter cells after binary
fission.
 This organism has granular and uneven staining.
 When stained with methylene blue or toluidine blue, the granules in the cell
stain metachromatically reddish purple.
 These granules are known as metachromatic granules, volutin granules
or Babes-Ernst granules.
 They are often situated at the poles of the bacilli and are called polar
bodies.
 Special stains, such as Albert’s Neisser’s and Ponder’s have been
devised for
demonstrating the granules clearly.
 With Albert’s stain, the granules stain bluish black and the protoplasm
green.
 The granules represent accumulation of polymerized polyphosphates.
 The granules formation is best seen on Löffler’s serum slope.
 It seems that the represent storage depots for materials needed to form
high- energy phosphate bonds.
Cultural Characteristics:-
 C. diphtheriae is an aerobe and facultative anaerobe; the optimum
temperature for growth is 37°C (range 15-40°C) and optimum pH 7.2.
 Complex media are required for primary isolation and characterization.
 It can grow on ordinary nutrient agar, but its growth is improved by the
presence of animal proteins such as blood or serum.
 Two media are useful for this purpose:
1. 1. Löffler’s serum slope.
2. 2. Blood agar containing fresh, lysed or heated blood.

3. Löffler’s serum slope:-

 Diphtheria bacilli grow on Löffler’s serum slope very rapidly and colonies can
be
seen in 6-8 hours, long before other bacteria grow.
 Colonies are at first small, circular white opaque disks but enlarge on
continued incubation and may acquire a distinct yellow tint.
 Löffler’s medium is also useful because it does not support growth of
streptococci and pneumococci that may be present in the clinical specimen
and inhibits the growth of most oral commensals and retards the growth of
others such as Candida albicans and Staphylococcus aureus, acting as a
selective agent but has little effect on diphtheria bacilli.

Fig: Loeffler medium

Tellurite Blood Agar

 The addition of potassium tellurite (0.03-0.04%) makes the medium
selective for corynebacteria by inhibiting most other pathogenic and
commensal bacteria.
 On this medium, C. diphtheriae give grey/black, shiny or dull black colonies
because the tellurite ion passes through the cell wall and membrane into the
cytoplasm, where it is reduced to the metal tellurium and precipitated inside
the cells.
  Some other corynebacteria, staphylococci, and yeasts reduce the tellurite
salts producing characteristic grey to black colonies.
 The addition of cystine to a tellurite containing medium (Tinsdale’s medium)
has
greatly helped the isolation of diphtheria bacilli.
 The growth of diphtheria bacilli may be delayed on the tellurite medium
and colonies may take two days to appear.
 Based on colonial morphology on the tellurite medium and other
properties, McLeod and Anderson described three different biotypes:
gravis, intermedius
and mitis.
 The names were originally proposed to relate to the clinical severity of the
disease produced by the three types of gravis, causing the most serious,
and mitis the mildest variety, with intermedius being responsible for
disease of intermediate severity.
 However, this association is not constant.
 In general, biotype mitis is predominant in endemic areas, while
intermedius and gravis tend to be epidemic.

Fig: Tellurite Blood Agar

Biochemical Reactions:-
 C. diphtheriae ferments glucose and maltose with the production of acid (but
no gas) but not lactose, mannitol, trehalose or sucrose.
 Starch and glycogen are used for biochemical differentiation of three biotypes
of
C. diphtheriae.
 Gravis strains utilize glycogen and starch, while mitis and intermedius do
not. Fermentation of sugars are usually done in Hiss’s serum peptone
water medium.
 C. diphtheriae is H2S positive and reduces nitrate to nitrite.
 It does not liquefy gelatine or hydrolyse urea or form phosphatase.

Pyrazinamidase (PYZ) Test

  In pyrazinamidase (PYZ) test, pyrazinamide is converted into pyrazinoic acid
by the organisms which produce pyrazinamidase (PYZ).
 This test is helpful to distinguish C. diphtheriae (PYZ-negative) from other
corynebacterium species (mostly PYZ-positive) except C. ulcerans and
C. pseudotuberculosis which are also PYZ-negative, but they are urease
test positive which differentiate them from C. diphtheriae (urease
negative).

Fig: Pyrazinamidase (PYZ) Test

Toxin:-
 Toxigenic strains of C. diphtheriae produce a very powerful exotoxin.
 The toxicity observed in diphtheria is directly attributed to the toxin secreted
by the bacteria at the site of infection.

Synthesis
 Almost all strains of gravis, 95-99% of intermedius and 80-85% of mitis
produce this toxin.
 Strains of all three types are invariably virulent when isolated from acute
cases. Avirulent strains are common among convalescents, contacts and
carriers, particularly in those with extrafaucial infection.
 There is considerable variation in the amount of toxin produced by the
different strains, some strains producing it abundantly and others only
poorly.
 However, toxin produced by different biotypes are antigenically similar.
 The strain almost universally used for toxin production is the ‘Park Williams 8’
strain, which has been variously described as a mitis (Toplev and Wilson) and
an intermedius strain (Cruickshank).
 The classic Park-Williams strain (PW8) of C. diphtheriae isolated in 1896, is
still used as a source of toxin for preparation of diphtheria toxoid (vaccine).

Lysogeny and Toxin Production

 The toxigenicity of the diphtheria bacillus depends on the presence in it
of corynephages (tox+), which act as the genetic determinant controlling
toxin production.
  Nontoxigenic strains can be converted to tox+ by infection with the
appropriate bacteriophage.
 This is known as lysogenic or phage conversion.
 The bacillus loses the toxigenicity when it is cured of its phage, as by growing
it in the presence of antiphage serum.

Iron for Toxin Production

 Toxin production is also influenced by the concentration of iron in the
medium.
 The optimum level of iron for toxin production is 0.1 mg per liter,
while a concentration of 0.5 mg per liter inhibits the formation of
toxin.
 The toxin is released in significant amounts only when the available iron in
the culture medium is exhausted.

Properties of Toxin
 Diphtheria toxin is an iron-free, crystalline, heat labile protein.
 It is extremely potent and is lethal for humans in amounts of 130 ng per kg
of body weight.
 In highly susceptible species (guinea pig, rabbit), the lethal dose of
diphtheria
toxin is 0.1 µg/kg or less.
 The diphtheria toxin is a protein and has a molecular weight of about
62,000. Fragment A has all the enzymatic activity whereas fragment B is
responsible for
binding the toxin to the cells.
 The toxin is heat labile.
 It is extremely potent (0.0001 mg kills a guinea pig of 250 gm weight).
 The toxicity of the toxin is due to its ability to block protein
synthesis in eukaryotic cells.
 The toxin consists of two fragments A (active) and B (binding) of MW 24,000
and 38,000, respectively.
 Both fragments are necessary for the toxic effect.It has a special affinity
for certain tissues such as the myocardium, adrenals and nerve endings.
 Prolonged storage, incubation at 37ºC for 4-6 weeks, treatment with 0.2-
0.4 percent formalin or acid pH converts it to toxoid.
 Toxoid is toxin that has lost its toxicity but not its antigenicity.
 It is capable of inducing antitoxin and reacting specifically with it.
Mode of Action
 The toxin is secreted by the bacterial cell and is nontoxic until exposed
to trypsin.
 The trypsinization results in two polypeptide fragments, A and B, which
are linked together by a disulfide bridge.
 Fragment A is responsible for the cytotoxicity; fragment B binds to receptors
on the eukaryotic cells and mediates the entry of fragment A into the
cytoplasm.
 The antibody to fragment B is protective by preventing the binding of the
toxin to
the cells. The diphtheria toxin acts by inhibiting protein synthesis.
 Specifically, fragment A splits nicotinamide adenosine dinucleotide (NAD)
to form nicotinamide and adenosine diphosphoribose (ADPR).
 ADPR binds to and inactivates elongation factor 2 (EF-2), an enzyme required
for elongation of polypeptide chains on ribosomes.
 Inhibition of protein synthesis is probably responsible for both the necrotic
and neurotoxic effects of the toxin.
 The reaction can be summarized as follows:
 NAD+ + EF-2 = ADPR-EF-2 + nicotinamide + H+
Active Inactive
Resistance:-
 C. diphtheriae is more resistant to the action of light, desiccation, and
freezing than are most non-spore forming bacilli.
 On dried fragments of pseudomembranes, organisms survive for at least
14 weeks.
 They are readily killed, however, by a 1 minute exposure to 100°C or a 10
minute exposure to 58°C.
 They are susceptible to most of the routinely used disinfectants.
 It dies rapidly in 0.85% NaCl solution, but remains alive for weeks in dust and
on fomites when dry and protected from sunlight.
 It is susceptible to penicillin, erythromycin and broad spectrum antibiotics.

Antigenic Structure
 Diphtheria bacilli are antigenically heterogeneous.
 They possess three distinct antigens:
1. A deep-seated antigen found in all corynebacterial species as well as
in Mycobacterium tuberculosis
2. A heat-labile protein (K antigen).
3. A heat-stable polysaccharide (O antigen).

Serotypes
 On the basis of agglutination reaction, biotypes gravis, intermedius and
mitis have been divided into 13, 4and 40 serotypes, respectively.
 No connection has been established between type specificity and
other characters.

Bacteriophage Typing
 The susceptibility of C. diphtheriae to bacteriophage strains has been
most comprehensively studied in Romania; 22 phages were used to type
the gravis strains into 14 types, the intermedius into 3 and the mitis into 4.
 An additional set of 33 phages has also been used.
 The only other corynebacteria reported as susceptible to the diphtheria
typing phages are C.ulcerans and C. pseudotuberculosis.

Bacteriocin Typing
 With the help of nine indicator strains of C. diphtheriae, Gibson and
Colman (1973) demonstrated 10 patterns of bacteriocin types amongst
Australian strains.
  Other methods of typing include bacterial polypeptide analysis, DNA
restriction patterns and hybridization with DNA probes.

Pathogenesis:-
 In the upper respiratory tract, diphtheria bacilli elicit an inflammatory
exudate and cause necrosis of the cells of the faucial mucosa.
 The diphtheria toxin possibly assists colonization of the throat or skin by
killing epithelial cells or neutrophils.
 In nature, C diphtheriae occurs in the respiratory tract, in wounds, or on the
skin of infected persons or normal carriers.
 It is spread by droplets or by contact to susceptible individuals; the bacilli then
grow on mucous membranes or in skin abrasions, and those that are toxigenic
start producing toxin.
 Diphtheria toxin is the major virulence factor of C. diphtheria and is a heat-
labile, single-chain, three domain polypeptide (62 kDa) that can be lethal in a
dose of 0.1 μg/kg body weight.
 The tox gene that codes for the exotoxin is introduced into strains of C.
diphtheriae by a lysogenic bacteriophage, β-phage.
 Two processing steps are necessary for the active gene product to be
secreted: (1) proteolytic cleavage of the leader sequence from the Tox protein
during secretion from the bacterial cell and (2) cleavage of the toxin molecule
into two polypeptides (A and B) that remain attached by a disulfide bond.
 Three functional regions exist on the toxin molecule: a catalytic region on the
A subunit and a receptor-binding region and a translocation region on the B
subunit.
 The receptor for the toxin is heparin-binding epidermal growth factor, which
is
present on the surface of many eukaryotic cells, particularly heart and nerve
cell.
 The binding of the receptor domain to host cell membrane proteins CD-9 and
heparin-binding epidermal growth factor (HB-EGF) triggers the entry of the
toxin into the cell through receptor-mediated endocytosis.
 Acidification of the translocation domain within a developing endosome leads
to creation of a protein channel that facilitates movement of Fragment A into
the host cell cytoplasm.
 The A subunit then terminates host cell protein synthesis by inactivating
elongation factor-2 (EF-2) which is required for translocation of polypeptidyl-
transfer RNA from the acceptor to the donor site on the eukaryotic ribosome.
  Toxin Fragment A inactivates EF-2 by catalyzing a reaction that yields free
nicotinamide plus an inactive adenosine diphosphate-ribose-EF-2 complex
(ADP-ribosylation).
 Because the turnover of EF-2 is very slow and approximately only one
molecule per ribosome is present in a cell, it has been estimated that one
exotoxin molecule can inactivate the entire EF-2 content in a cell, completely
terminating host cell protein synthesis.
 It is assumed that the abrupt arrest of protein synthesis is responsible for the
necrotizing and neurotoxic effects of diphtheria toxin.
 Toxin synthesis by lysogenized C diptheriae is regulated by a chromosomally
encoded element, diphtheria toxin repressor (DTxR) which is activated in the
presence of high iron concentrations, and can bind to the toxin gene operator
and prevent toxin production.
 Low iron concentration and other factors such as osmolarity, amino acid
concentrations and pH, however enhance the production of toxin.

Clinical Diseases:-
 The organism is carried in the upper respiratory tract and spread by
droplet infection or hand-to-mouth contact.
 The incubation period of diphtheria is 2-5 days, with a range of 1-10
days.
 Diphtheria, which occurs in two forms (respiratory and cutaneous), is
found worldwide but is uncommon in North America and Western Europe.

A. Respiratory Diphtheria
  The illness begins gradually and is characterized by low-grade fever,
malaise, and a mild sore throat.
 The most common site of infection is the tonsils or pharynx.
 The organisms rapidly multiply on the epithelial cells, and the toxigenic
strains of
C. diphtheriae produce toxin locally, causing tissue necrosis and
exudate formation triggering an inflammatory reaction.
 This combination of cell necrosis and exudate forms a tough grey to
white
pseudo membrane, which attaches to the tissues-commonly over the
tonsils, pharynx, or larynx.
 Any attempt to remove the pseudomembrane results in bleeding.
 In nasopharyngeal infection, the pseudomembrane may involve nasal
mucosa, the pharyngeal wall and the soft palate.
 In this form, edema involving the cervical lymph glands may occur in the
anterior
tissues of the neck, a condition known as bullneck diphtheria.
 Laryngeal involvement leads to obstruction of the larynx and lower airways.

B. Systemic Effects
 The toxin also is absorbed and can produce a variety of systemic effects
involving the kidneys, heart, and nervous system, although all tissues
possess the receptor for the toxin and may be affected.
 Intoxication takes the form of myocarditis and peripheral neuritis, and may
be associated with thrombocytopenia.
 Visual disturbance, difficulty in swallowing and paralysis of the arms and
legs also occur but usually resolve spontaneously.
 Complete heart block may result from myocarditis.
 Death is most commonly due to congestive heart failure and
cardiac arrhythmias.

C. Complication
The common complications are:

1. Asphyxia due to mechanical obstruction of the respiratory passage by

the pseudo membrane for which an emergency tracheostomy may
become necessary.
2. Acute circulatory failure, which may be peripheral or cardiac.
3. Post diphtheritic paralysis, which typically occurs in the third or fourth week
of the disease; palatine and ciliary but not pupillary paralysis is
characteristic, and spontaneous recovery is the rule.
4. Septic, such as pneumonia and otitis media.

D. Cutaneous Diphtheria
  In the cutaneous diphtheria, which is prevalent in the tropics, the toxin also is
absorbed systemically, but systemic complications are less common than
from upper respiratory infections with C. diphtheriae.

Laboratory Diagnosis:-
 Diagnostic laboratory tests serve to confirm the clinical impression and
are of epidemiologic significance but not for the treatment of individual
cases. Specific treatment should be instituted immediately on suspicion of
diphtheria without waiting for laboratory tests.
 Any delay may be fatal. Laboratory diagnosis consists of isolation of
the diphtheria bacillus and demonstration of its toxicity.

1. Specimens
 Swabs from the nose, throat, or other suspected lesions must be
obtained before antimicrobial drugs are administered.
 In suspected cases, whether of faucial or nasal diphtheria, swabs should
be taken both from the throat and from the nose, and preferably two
swabs from the site most affected.
 Swabs should also be taken from skin lesions and wounds where
diphtheritic infection is suspected, and both throat and nose swabs should
be taken from suspected carriers.

2. Microscopy
 Direct microscopy of a smear is unreliable since C. diphtheriae
is morphologically similar to other coryneforms.
 Smears stained with alkaline methylene blue or Gram stain show beaded rods
in typical arrangement.
 Hence smear examination alone is not sufficient for diagnosing diphtheria but
is
important in identifying Vincent’s angina.
 For this, a Gram or Leishman stained smear is examined for Vincent’s
spirochetes and fusiform bacilli. Toxigenic diphtheria bacilli may be identified
in smears by immunofluorescence.

3. Culture
  The swab should be inoculated on Löffler’s serum slope, tellurite blood agar,
and blood agar.
 The cultures should be incubated aerobically at 37°C.
 Unless the swab can be inoculated promptly, it should be kept moistened
with sterile horse serum so the bacilli will remain viable.

i. Löffler’s Serum Slope

 After incubation for 6 hours or overnight, make a smear of growth from all
parts of the slope mixed in the condensation water, stain by the Albert-
Laybourn method and look for the presence of slender green-stained bacilli
containing the purple-black granules characteristic of C. diphtheriae. In 12-18
hours, the Löffler slant may yield organisms of typical “diphtheria-like”
morphology.

ii. Tellurite Blood Agar

 Blood tellurite agar is examined after 24 hours and after 48 hours, as growth
may sometimes be delayed.

iii.Blood Agar

 It is used for differentiating streptococcal or staphylococcal pharyngitis,

which may simulate diphtheria.

4. Identification Tests
 Identification is based on carbohydrate fermentation reactions and
enzymatic activities.
 C. diphtheriae ferments glucose and maltose, producing acid but not gas, and
is catalase positive.
 It reduces nitrate to nitrite and is nonmotile.
 Commercial kits such as the API Coryne strip provide a reliable identification.

5. Virulence Tests
 Any diphtheria-like organism cultured must be submitted to a “virulence” test
before the bacteriologic diagnosis
 of diphtheria is definite. Such tests are really tests for toxigenicity of
an isolated diphtheria-like organism.
Diagnosis of diphtheria depends on showing that the isolate produces
diphtheria toxin.
 Virulence testing may be by in vivo or in vitro methods.
 In vivo testing is rarely done because the in vitro methods are reliable,
less expensive, and free from the need to use animals.
A. In Vivo Tests
1. Subcutaneous test.
2. Intracutaneous test.

B. In Vitro Test
1. i. Precipitation test.
2. ii. Tissue culture test.
3. iii. Enzyme-linked immunosorbent assays.
4. iv. Polymerase chain reaction (PCR).

A. In Vivo Tests

1. Subcutaneous Test
 The growth from an overnight culture on Löffler’s slope is emulsified in 2-4 ml
broth and 1 ml of the emulsion injected subcutaneously into two guinea pigs
or rabbits, one of which has been protected with the diphtheria antitoxin (500
-1000 units) 18-24 hours previously and was used as control.
 If the strain is virulent, the unprotected animal will die within four days.
 Postmortem examination would show haemorrhage at the site of injection
and injected blood vessels, with typically haemorrhagic adrenal necrosis.
 Simple and reliable subcutaneous toxigenicity tests in rabbits or larger
guinea pigs were used in the past, at a time when laboratories had many
isolates each
day.
 The method is not usually employed as it is wasteful of animals.

2. Intracutaneous (Intradermal) Test

 The broth emulsion of the culture is inoculated intracutaneously into two

guinea pigs (or rabbits) so that each receives 0.1 ml in two different sites.
 One animal acts as the control and should receive antitoxin (500 units)
the previous day.
 After four hours of the skin test, the other is given 50 units of
antitoxin intraperitoneally in order to prevent death.
 In the test animal, toxigenicity is indicated by inflammatory reaction at the
site of injection, progressing to necrosis in 48-72 hours and in the control
animal, no change.

Advantages of Intracutaneous Test

  The animals do not die.
 As many as ten strains can be tested at a time on a
rabbit.

B. In Vitro Test
1. Elek’s Gel Precipitation Test
 The in vitro diphtheria toxin detection procedure is an immunodiffusion test
first described by Elek.
 In the Elek test, organisms (controls and unknowns) are streaked on media
of low iron content to optimize toxin production.
 Procedure: A rectangular strip of filter paper impregnated with diphtheria
antitoxin (1000 units/ml) is placed on the surface of a 20% normal horse
serum
agar in a petridish while the medium is still fluid.
 Sheep or rabbit serum may be used, if horse serum is not available. When
the agar has set, the surface is dried.
 The plate should be streaked with the test strain as well as the control
positive and negative strains at right angles to the strip in a single straight
line parallel to each other and 10 mm apart.
 The plate is incubated at 37°C and examined after 24 and 48 hours.
  Interpretation: Toxins produced by the bacterial growth will diffuse in the agar
and where it meets the antitoxin at optimum concentration will produce a
line of precipitation.
 If an isolate is positive for toxin production, and it is placed next to the
positive control, the toxin line of the positive control should join the toxin
line of the positive unknown to form an arch of identity.
 A negative control should be free of any line.
 No precipitate will form in the case of nontoxigenic strains.

2. Tissue Culture Test

 The toxigenicity of diphtheria bacilli can be demonstrated by incorporating
the strains in the agar overlay of cell culture monolayers.
 The toxin produced diffuses into the cells below and kills them.

3. Enzyme-linked Immunosorbent Assays (ELISA)

 Rapid, enzyme-linked immunosorbent assays and
immunochromatographic strip assays are also available for the detection
of diphtheria toxin.

4. Polymerase Chain Reaction (PCR)

 In addition, procedures for detecting the C. diphtheriae
 tax gene by the polymerase chain reaction (PCR) have
 been developed.
 The PCR assay can also be applied directly to clinical specimens.

Schick Test
 Schick (1913) introduced an intradermal test (Schick test) for
distinguishing between susceptible and immune persons.

Principle
  This test depends upon the principle of toxin-antitoxin neutralization, in vivo,
and the test is carried out by injecting one Schick test dose of diphtheria toxin
(0.2 ml containing l/50 MLD) intradermally on the anterior surface of the left
forearm and a control injection in the right forearm contains a heat-
inactivated dose (70°C for 30 minutes) or preferably, purified diphtheria
toxoid.
 Readings are taken after 1, 4 and 7 days.
 Four types of reactions may occur:

i. Positive Reaction
 In the test arm there appears erythema and swelling at the site of
inoculation in 24-36 hours, reaching its maximums (1-5 cm) by the 4th to 7th
days and then fading with superficial scaling and persistent brownish
pigmentation.
 On the control arm there is no reaction.
 A positive Schick test indicates that the individual is susceptible to
diphtheria and little or no antitoxin (less than 0.01 unit/ml) is present.
 The subject is not immune and should be immunized.

ii. Negative Reaction

 There is no reaction of any kind in either arm.
 This indicates that the toxin has been neutralized by the circulating antitoxin
and the person is immune to diphtheria and does not need immunization.
 In quantitative terms, the test will be negative if the blood serum contains
the antitoxin concentration of 0.0 I unit or more/ml and is not sensitive to
any of the antigens in the toxin or toxoid and does not need immunization.

iii.Pseudoreaction
 There is erythema occurring within 6-24 hours and dis
 appearing within four days.
 The reaction is the same on both arms.
 This indicates that the individual is immune to diphtheria and also that
he is hypersensitive to one or more antigens in the toxin preparation.
 This individual does not need immunization.

iv. Combined Reaction

 Here the initial picture is that of pseudo reaction, but while the erythema in
the control arm fades, within four days, it progresses in the test arm to a
typical positive reaction.
  This indicates that the individual is susceptible to diphtheria and is sensitive
to one or more antigens in the toxin preparation making immunization
necessary but likely to induce reaction.
 Doses of the vaccine should be greatly induced, and the number of
injections increased.

Epidemiology:-
 Diphtheria is a disease found worldwide, particularly in poor urban
areas where there is crowding and the protective level of vaccine-induced
immunity is low.
 C. diphtheriae is maintained in the population by asymptomatic carriage in
the oropharynx or on the skin of immune people.
 Infection is confined to man and usu ally involves contact with a diphtheria
case or a carrier.
 Humans are the only known reservoir, with carriage in oropharynx or on skin
surface.Diphtheria is primarily a pediatric disease, but the highest incidence
has
shifted toward older age groups in areas where there are active
immunization programs for children.
 Acquired immunity to diphtheria is due primarily to toxin-neutralizing
antibody
(antitoxin) Passive immunity in utero is acquired transplacentally and can
last for 1 or 2 years after birth.
 Skin infection with toxigenic C. diphtheriae (cutaneous diphtheria) also
occurs.

Prophylaxis:-
 The methods of immunization available are active, passive or combined.
 Of these only active immunization can provide herd immunity and lead
to eradication of the disease.
 Passive and combined immunization can only provide emergency protection
to susceptible individual's exposed to risk.

A. Active Immunization
The preparations used for active immunization are as follows:
1. Toxin-antitoxin mixture: It is not without hazards.
2. Single vaccines: It is less frequently used.
3. Combined preparations.
Combined Preparations
 DPT (diphtheria-pertussis-tetanus) vaccine
 DT (diphtheria-tetanus toxoid)
 DT (diphtheria-tetanus, adult type).

DPT Vaccine
 Diphtheria toxoid is usually given in children as a trivalent preparation
containing tetanus toxoid and pertussis vaccine is known also as the DTP, DPT
or triple vaccine.
 A quadruple vaccine containing in addition the inactivated polio vaccine is
also available.
 The WHO recommends that only adjuvant DPT preparations be utilized
in immunization programs.Schedule of primary immunization:
 The schedule of primary immunization of infants and children consists of
DPT given at the age of 6 weeks, 10 weeks, 14 weeks and 16-24 months
followed by booster dose DT at the age of 5-6 years (school entry).

B. Passive Immunization
 This is an emergency measure to be employed where susceptibles
(nonimmunized) are exposed to infection, as when a case of diphtheria
is admitted to general pediatric wards.
 It consists of the subcutaneous administration of 500-1000 units of
antitoxin (antidiphtheritic serum, ADS).
 As this is a horse serum, precaution against hypersensitivity should be
observed.

C. Combined Immunization
 This consists of administration of the first dose of adsorbed toxoid, while ADS
is given on the other arm, to be continued by the full course of active
immunization since protection conferred by passive immununization is of
short duration. Ideally, all cases that receive ADS prophylactically should
receive combined immunization.

Treatment:-
 Specific treatment of diphtheria consists of antitoxic and antibiotic therapy.
 Antitoxin should be given immediately as soon as clinical diagnosis is made to
neutralize the toxin being produced, because antitoxin is ineffective if given
after the toxin is bound to cell receptorsites.
 The dosage recommended is 20,000 units intramuscularly for moderate cases
and 50,000 to 100,000 units for serious cases, half the dose being
given intravenously.
  C. diphtheriae is sensitive to most antibiotics, including penicillin and
erythromycin and are used for the treatment of patients as well as
carriers.
 The antibiotics do not neutralize circulating toxin.
 They prevent further toxin production by killing diphtheria bacilli.
 Penicillin sensitive individuals can be given erythromycin.
 Erythromycin is more active than penicillin in the treatment of carriers.

OTHER MEDICALLY IMPORTANT CORYNEBACTERIA:-

 The nondiphtheria corynebacteria are diverse; usually isolated from
the environment and commensals of the skin and mucous
membranes.
 The principal species involved and the main clinical syndromes associated
with infection are shown in table

Corynebacterium ulcerans
 A veterinary pathogen causing mastitis in cattle and other domestic and wild
animals, C. ulcerans has been isolated from patients with diphtheria-like
illness.
 It resembles gravis type of C. diphtheriae but it liquefies gelatin,
ferments trehalose slowly and does not reduce nitrate to nitrite.
 It is PYZ negative and urease positive.
 It commonly produces diphtheria toxin as well as the separatetoxin
produced by
C. pseudotuberculosis.
 It is pathogenic to guinea pigs.
 The lesions produced resembling those caused by C. diphtheriae.
 It has been isolated from raw milk and can cause mastitis in Cattle and
man exposed to infected animals or milk may develop infection.
 Man to man transmission has not been reported.
 In man, infection usually takes the form of acute pharyngitis with
pseudomembranes, and cardiac or neurological complications may
occur.

Corynebacterium pseudotuberculosis (C. ovis)

 Like C. ulcerans, C. pseudotuberculosis (Preisz-Nocard bacillus) is primarily
an animal pathogen and rarely infects man.
 Human infections mainly occur in patients with animal (sheep) contact.
 It causes caseous lymphadenitis in sheep and goats and abscesses
or ulcerative lymphangitis in horses.
 Rarely, it may cause subacute and chronic lymphadenitis involving axillary
or cervical lymph nodes in those with prolonged exposure to sheep and
horses.

Corynebacterium minutissimum
  It is a localized infection of the stratum corneum which produces reddish-
brown scaly patches in the intertriginous sites.
 Lesions usually involve the groin, toe, and axillae.
 duces reddish-brown scaly patches in the intertriginous sites.
 Lesions usually involve the groin, toe, and axillae.

Corynebacterium jeikeium
 Cornyebacterium jeikeium is named after Johnson and Kaye who first linked
this organism with human infections.
 Infections have been limited to patients who are immune compromised,
have undergone invasive procedures, or have a history of intravenous drug
abuse.
 It is the most common cause of diphtheroid prosthetic valve endocarditis
in adults.
 C. jeikeium has been reported to be resistant to a wide range of
antimicrobials.
 Most strains are susceptible to vancomycin.

Corynebacterium xerosis
 C. xerosis is commonly found on skin and mucocutaneous sites.
 Human infection with C. xerosis is rare, and affected patients are
invariably immunosuppressed.

Corynebacterium bovis
 C. bovis, commensal of cow’s uddar, which may cause bovine mastitis.
 Many of them cause infections in immunocompromised patients.
 less commonly, systemic infections such as septicemia and endocarditis.
 Infections can be treated with penicillin or erythromycin.
OTHER CORYNEFORM GENERA:-
 Other genera of irregularly shaped, gram-positive bacilli are
Arcanobacterium, Brevibacterium, Oerskovia and Turicella.
 They have been found to colonize humans and cause disease.

Arcanobacterium
 Arcanobacterium can cause pharyngitis with a “scarlet fever-like” rash,
polymicrobic wound infections, and, ess commonly, systemic infections such
as septicemia and endocarditis.
 Infections can be treated with penicillin or erythromycin.

Brevibacterium

 Brevibacterium colonize the skin surface and have been blamed for
malodorous feet in some colonized people.
 It causes septicemia, osteomyelitis, and foreign body infections.
Oerskovia

 Oerskovia is an environmental organism found in the soil and decaying

organic matter.
 This has been associated with septicemia, endocarditis, meningitis, soft
tissue infections, and infections in the presence of foreign bodies.
Turicella
 Turicella (Turicella otitidis is the only species) has been isolated in the ears of
healthy and infected individuals.

REFERENCE:-
 Textbook of Microbiology - Surinder Kumar
 https://microbenotes.com/corynebacterium-diphtheriae/#pathogenesis-of-
corynebacterium-diphtheriae