MEDICAL MICROBIOLOGY II

Lesson 10
Chlamydia and Mycoplasma

Chlamydia
The genus Chlamydia consists of small obligate
intracellular microorganisms - cause infections
in avian and mammalian species (including
human)
They resemble both bacteria and viruses
Like bacteria, they possess both DNA and RNA
and are sensitive to antibiotics
Also, they have cell wall but it lacks muramic
acid and peptidoglycan

Chlamydia
They resemble viruses in being able to
reproduce only inside a living cell and depend
on ATP produced by the host cell for their
synthetic process
Chlamydiae occur in2 forms:
1. Elementary bodies
2. Initial or reticulate bodies

Chlamydia

Chlamydia
The elementary body is a spherical particle,
200 - 300 nm in diameter, and it is the
extracellular infective form
Upon entering a suitable host cell, it enlarges
to form the initial body
The initial body is 600 - 1200 nm in diameter
and it is the metabolically active, non-infective
fragment form into which the elementary
body changes during the multiplication cycle

Chlamydia

Chlamydia
The initial body multiplies by binary fission to
form new smaller initial or reticulate bodies
When released as elementary bodies by the
rupture of host cell, they become infective
Thus, the elementary body survives extracelullarly while the initial body is adapted for
intracellular growth

Classification and Species

Chlamydiae were formerly classified into subgroup A and sub-group B
Sub-group A members: cause
lymphogranuloma venereum (LGV), trachoma
and inclusion conjunctivitis; form compact
inclusions with glycogen matrix; and are
susceptible to sulphonamides and Dcycloserine

Classification and Species

Sub-group B members: cause psittacosis;
produce diffuse inclusion with no glycogen
matrix; and are resistant to sulphonamides
and D-cycloserine
Now, sub-group A is C. trachomatis and subgroup B is C. psittaci
C. trachomatis is subdivided into 15 serotypes:
A-K including Ba and, L1, L2 and L3

Classification and Species

C. psittaci contains many serotypes some of
which are not fully identified
C. pneumoniae is the recently recognised
member of chlamydiae and it belongs to subgroup B

Characteristics
Chlamydia are Gram negative but are better
stained with Giemsa, Castaneda or
immunofluorescence methods
They produce basophilic inclusion bodies in the
infected cell
They are heat-labile and can be inactivated at
56 C within minutes
They are susceptible to ethanol, ether and low
concentrations of formalin and phenol
Chlamydiae can be preserved at -70 C

Chlamydia Infection
Chlamydia infection is a common sexually
transmitted disease (STD) caused by the
bacterium, Chlamydia trachomatis, which can
damage a woman's reproductive organs
Even though symptoms of Chlamydia infection
are usually mild or absent
However, serious complications that cause
irreversible damage, including infertility, can
occur silently before a woman ever recognizes
a problem

Chlamydia Infection
Chlamydia also can cause discharge from the
penis of an infected man
Chlamydia can be transmitted during vaginal,
anal, or oral sex
Chlamydia can also be passed from an infected
mother to her baby during vaginal childbirth
Any sexually active person can be infected with
chlamydia
The greater the number of sex partners, the
greater the risk of infection

Chlamydia Infection
The cervix (opening to the uterus) of teenage
girls and young women is not fully matured
and is probably more susceptible to infection
Therefore, they are at particularly high risk for
infection if sexually active
Since Chlamydia can be transmitted by oral or
anal sex, men who have sex with men are also
at risk for chlamydial infection

Chlamydia Infection
Chlamydia is known as a silent disease because
the majority of infected people have no
symptoms
If symptoms do occur, they usually appear
within 1 to 3 weeks after exposure
In women, the bacteria initially infect the cervix
and the urethra (urine canal)
Women who have symptoms might have an
abnormal vaginal discharge or a burning
sensation when urinating

Chlamydia Infection
If the infection spreads from the cervix to the
fallopian tubes, some women still have no
signs or symptoms; others have lower
abdominal pain, low back pain, nausea, fever,
pain during intercourse, or bleeding between
menstrual periods
Chlamydial infection of the cervix can spread
to the rectum

Chlamydia Infection

Chlamydia Cervicitis

Chlamydia Infection
Men with signs or symptoms might have a
discharge from their penis or a burning
sensation when urinating
Men might also have burning and itching
around the opening of the penis
Pain and swelling in the testicles are
uncommon

Chlamydia Infection
Men or women who have receptive anal
intercourse may acquire chlamydial infection
in the rectum, which can cause rectal pain,
discharge, or bleeding
Chlamydia can also be found in the throats of
women and men having oral sex with an
infected partner

Chlamydia Infection
If untreated, chlamydial infections can progress
to serious reproductive and other health
problems with both short-term and long-term
consequences
Like the disease itself, the damage that
Chlamydia causes is often silent
In women, untreated infection can spread into
the uterus or fallopian tubes and cause pelvic
inflammatory disease (PID)
This happens in about 10 - 15% of women with
untreated Chlamydia infection

Chlamydia Infection
Chlamydia can also cause fallopian tube infection
without any symptoms
PID and silent infection in the upper genital
tract can cause permanent damage to the
fallopian tubes, uterus, and surrounding tissues
The damage can lead to chronic pelvic pain,
infertility, and potentially fatal ectopic pregnancy
Chlamydia may also increase the chances of
becoming infected with HIV, if exposed

Chlamydia Infection
Complications among men are rare
Infection sometimes spreads to the
epididymis, causing pain, fever, and, rarely,
sterility
Rarely, genital chlamydial infection can cause
arthritis that can be accompanied by skin
lesions and inflammation of the eye and
urethra (Reiter's syndrome)

Chlamydia Infection
In pregnant women, there is some evidence
that untreated chlamydial infections can lead
to premature delivery
Babies who are born to infected mothers can
get chlamydial infections in their eyes and
respiratory tracts
Chlamydia is a leading cause of early infant
pneumonia and conjunctivitis (pink eye) in
newborns

Habitat
C. trachomatis is found in the urogenital tract
of human
C. psittaci is found in many wild and domestic
birds
Infection in man is by inhaling the organism in
the dried droppings of birds

Pathogenicity
1. C. trachomatis
i. C. trachomatis, serotypes A, B, Ba, and C
Cause endemic blinding trachoma - a severe
eye infection of the conjunctiva and cornea
Untreated cases result in marked
inflammation and scarring
The cornea is usually damaged, painful and
watering; there is discomfort in light; opacity
and eventually blindness (can be prevented if
the case is diagnosed and treated early)

Pathogenicity
Infection can be transmitted from eye to eye
by fingers or formites e.g. handkerchiefs and
towels
Flies may also transmit the infection
Poor conditions of living help in the spread of
the disease
It is endemic in some parts of Africa, Middle
and Far East, India and South America

Pathogenicity
ii. C. trachomatis serotypes D-K cause
a) Inclusion conjunctivitis (IC), a low grade eye
infection that is contracted by self-inoculation
after touching infected genital area or infected
eye. Infection can also be contracted by
exposure to contaminated water in swimming
pools. TRIC (trachoma-inclusion conjunctivitis)
agent is the term often used to describe C.
trachomatis strains that cause eye infections

Pathogenicity
b) Congenital inclusion conjunctivitis is an
infection of new born babies by mothers with
C. trachomatis. The infection occurs during
delivery and the baby develops conjunctivitis
which is often accompanied by neonatal
pneumonia

Pathogenicity
c) Non-gonococcal urethritis (NGU) is a
condition which was also referred to as nonspecific urethritis caused by C. trachomatis in
about 40% of cases in men. It is associated
with pelvic inflammatory disease (PID) and
cervicitis in women.

Pathogenicity
iii. C. trachomatis serotypes L1, L2 and L3
These strains of C. trachomatis cause
lymphogranuloma venereum (LGV)
This condition is sexually transmitted and is
common in tropical countries
A small ulcer develops at the site of infection
on the external genitalia following a 3 - 14 days
incubation period
In males, there is inflammation and swelling of
the inguinal lymph nodes (inguinal adenitis)

Pathogenicity
The swellings are known as buboes
It may lead to genital strictures and may
become more generalised, causing pain, fever
and chills
The buboes may enlarge, suppurate and burst
to form sinuses discharging pus
In females, the intrapelvic lymph nodes are
involved
It may lead to elephantiasis of the vulva
following lymphatic blockage

Pathogenicity
2. C. psittaci
It causes psittacosis which is a disease of
parrots and other psittacine birds
It is a pneumonia or influenza-like illness
The chlamydiae are shed in the droppings or
nasal discharge and are released in the form
of aerosols

Pathogenicity
The disease is characterised by pneumonia,
septicaemia, and occasionally
meningoencephalitis, endocarditis and
pericarditis
Inhalation of infected aerosols by man leads
to atypical pneumonia

Pathogenicity
3. C. pneumoniae
Associated with atypical pneumonia which is
transmitted from man to man
Unlike C. psittaci, there is no known bird or
animal reservoir
It is a mild infection which may be associated
with pharyngitis, sinusitis and sore throat

Laboratory Diagnosis
Specimens: conjunctival scraping, blood, sputum,
pus (aspirated from the buboes), and swabs of
urethra, endocervix, conjunctiva and nasopharynx
Microscopy: direct smears stained with Giemsa
(standard method, but less sensitive than IF), with
iodine or by immunofluorescence (IF) to detect
inclusion bodies. IF is the most sensitive method
but it requires expensive reagents and equipment

Laboratory Diagnosis
1. Microscopy:
Giemsa preparation: under oil immersion,
chlamydia inclusion bodies in epithelial cells
appear as distinct intracytoplasmic masses of
particles ranging from small (300 nm) redpurple stained clumps of elementary body
inclusions, to larger (1 m) blue-purple stained
initial body inclusions with relatively less dense
particles. These two types of particles are
frequently present in the same inclusion

Laboratory Diagnosis

Laboratory Diagnosis
Several structures including bacteria may
resemble C. trachomatis inclusion and lead to
error in the interpretation of smears
Pigment: e.g. melanin granules (black or blackgreen in Giemsa stained smears). Clumps of the
pigment granules cover the nucleus of the cell
resembling trachoma inclusion
Nuclear extrusions appear as irregular masses
in the cytoplasm but they are similar to the
nucleus in colour and texture

Laboratory Diagnosis
Goblet cell granules may appear in clusters, are
lighter in colour and not as distinct as the
elementary bodies
Bacteria such as Neisseria, Haemophilus,
Moraxella and diphtheroids all stain blue and
appear in the cytoplasm and so can be
mistaken for inclusion bodies. It is best to
examine a Gram stained smear as well as a
Giemsa stained smear

Laboratory Diagnosis
Iodine preparation: C. trachomatis inclusion
bodies contain glycogen which stain brown
with iodine. This method can be used to screen
for C. trachomatis inclusion bodies. It is not a
permanent preparation. It is more suited for
materials from neonatal inclusion
conjunctivitis and also useful in detecting
inclusions in cell culture

Laboratory Diagnosis
IF staining: relies on the detection of inclusions
by IF with polyclonal antiserum from
hyperimmunised animals or inclusion and
elementary bodies with monoclonal
antibodies. Commercial kits are available. A
quick method for diagnosing chlamydial
infection. The elementary bodies fluoresce
green-yellow and appear small and round

Laboratory Diagnosis
2. Culture
Only performed in lab with facilities for tissue
culture
Two general method: egg (grown in yolk sac,
time consuming method) and tissue culture
(relatively fast method, uses monolayer cells
such as McCoy cells)
Refer to WHO guidelines

Laboratory Diagnosis
3. Serodiagnosis
Used to serotype unknown isolate of C.
trachomatis and to measure type specific
antibodies in human sera
Methods: micro-IF (most commonly used),
inclusion IF, ELISA, and complement fixation
test (CFT)
The antigens used consists of organisms
propagated in the yolk sac of fertile chick
embryos and their specific antisera are
prepared in mice

Laboratory Diagnosis
4. Antibiotic susceptibility
C. trachomatis is sensitive to tetracycline,
sulphonamides, chloramphenicol and
rifampicin
Topical preparations may also be prepared

Mycoplasma
The genus Mycoplasma consists of
Mycoplasma, Acholeplasma, and Ureaplasma
Mycoplasma are the smallest free living
microorganisms
Very pleomorphic
0.1 - 2 m
Occur as cocci or long filaments which may
appear branched
True bacteria but resemble viruses in their
filterable size

Mycoplasma

Mycoplasma
They do not have rigid cell wall
Composed of a small unit of cytoplasm
encased in a 3-layered membrane that
contains cholesterol or carotenol in addition to
the usual phospholipids
Do not produce their own cholesterol but
require it for growth
Inhibited by antibiotics, but not by penicillin
Filterable due to their small size and non-rigid
cell wall

Mycoplasma
Poorly Gram stained negative, other staining
techniques are used
Species of medical importance: M. pneumoniae,
M. hominis, Ureaplasma urealyticum
Part of human normal flora: M. salivarium, M.
buccale, M. faucium, M. fermentans and M.
genitalium
Mycoplasma are found in soil, plants, sewage
and mucous surfaces of animal body
M. hominis and U. urealyticum: urogenital tract
and URT; M. pneumoniae: respiratory tract

Pathogenicity
M. pneumoniae
causes pharyngitis, sinusitis, inflammation of
the ear, and febrile bronchitis or pneumonia
The infection is accompanied by the formation
of cold haemagglutinins, Streptococcus MG
agglutinins or anti-tissue antibodies
Complication include extra pulmonary
conditions such as arthritis, hepatitis, CNS
involvement (meningoencephalitis, cerebral
ataxia), muscle inflammation, skin rashes,
myocarditis, and GI disorders

Pathogenicity
M. hominis, M. fermentans and U. urealyticum
Cause pelvic inflammatory diseases (PID),
salpingitis, tubo-ovarian abscess, pelvic
abscess, septic abortion and puerperal fever
M. hominis is reported to cause pyelonephritis

U.urealyticum and M. genitalium

Cause non-gonococcal urethritis (NGU) or post
gonococcal urethritis in men, and cerviticitis
and PID in women
U.urealyticum also causes UTIs and arthritis

Laboratory Diagnosis
Specimens: respiratory tract samples (sputum,
throat swabs and tracheal aspirates),
urogenital tract samples (high vaginal swab,
urethral swab, cervical swab, and urine), eye
swab, warts, synovial fluid, blood, placental
membranes, and foetal tissues

Laboratory Diagnosis
1. Microscopy
Direct microscopy on stained preparation - not
useful (Mycoplasma are poorly Gram stained)
They are better observe when stained by
Giemsa, IF or with Dienes stain

2. Culture
Grow on solid or liquid media enriched with
20% horse or human serum and yeast extracts
Media are made selective with penicillin and
thallium acetate

Laboratory Diagnosis
Colonies appear after 2 - 3 days aerobic
incubation at 35 - 37 C
They show the typical fried-egg appearance
of a central opaque granular area of growth
extending into the depth of the medium and
surrounded by a flat translucent peripheral
zone
Incubation may continue for up to 3 weeks
before culture is reported as negative

Typical Fried-egg Appearance

Laboratory Diagnosis
3. Identification tests
Mycoplasma isolates are identified by
biochemical, biological and serological
methods
The isolates are separated into fermenters and
non-fermenters on the basis of their
biochemical (colour) reaction in the standard
growth medium (contain phenol red indicator)
Production of acid will result in change of
colour to yellow

Laboratory Diagnosis
4. Haemolysis and haemadsorption tests
Isolate must be grown on complete
Mycoplasma agar and young colonies are used
for test
Haemolysis test: a clear beta haemolysis
around the colonies is produced by M.
pneumoniae due to production of haemolysin
(aerobic environment). Other human
mycoplasmas produce smaller and rather
greenish zones that take longer time to appear

Laboratory Diagnosis
Haemadsorption test: positive colonies are seen
surrounded by erythrocytes - M. pneumoniae
colonies are positive

5. Serological tests
Most reliable for identifying and classifying
mycoplasmas
The usual tests are growth inhibition test,
metallic inhibition test and IF test
Other test such as CFT, indirect IF, agglutination
and haemagglutination test (not practical)

Laboratory Diagnosis
Growth inhibition test: simplest and most
practical, highly specific
Metabolic inhibition test: based on the fact
that when mycoplasmas utilise their
substrate medium, they give off a by-product
that changes the pH of the medium, resulting
in a colour change of the indicator used in
the medium. A specific antiserum against
mycoplasmas will inhibit its metabolic activity
and so there will be no growth - no colour
change in the medium

Laboratory Diagnosis
IF test: rapid, sensitive and specific. It makes
use of an agar block from the culture plate of
test organism.
Cold agglutinins: diagnosis of M. pneumoniae
can be made based on the production of IgM
autoantibodies that agglutinate patients own
red cells and other adult human group O Rh
negative red cells at 4 C (cold agglutinins).
The autoantibodies present in 35 - 76% of
patients with M. pneumoniae infections and
50% of normal healthy individuals

Laboratory Diagnosis
6. Antibiotic sensitivity
Usually resistant to penicillin and
cephalosporins
U. urealyticum is resistant to tetracycline
Some are sensitive to erythromycin

THE END