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Atypical Infections:
Mycoplasma, Mycobacterium,
Chlamydia
Chijioke Onejeme
[email protected]
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Objectives
 To identify Mycoplasma virulence factors
 To identify the infectious diseases caused by Mycoplasma species
 To identify the unique biological and biochemical properties of
Mycoplasma species
 Discuss the biological, physiological and biochemical characteristics
of chlamydial agent.
 Discuss the major chlamydial human pathogens from the standpoint
of:
A.
B.
C.
D.
E.
Human diseases produced
Clinical features of disease
Mechanism of pathogenesis of spirochetal pathogen inducing disease.
Clinical diagnosis of disease.
Treatment and prevention of disease induced by those agents.
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Mycoplasma





Lack peptidoglycan
Pleomorphic (takes on many different shapes)
Cell membranes contains sterols
Facultative anaerobe
Smallest genome (minimal amount of nucleic acid to
form cell)
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Mycoplasma
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Mycoplasma pneumoniae




Primary atypical pneumonia (typical due to strep)
Transmission via coughing or sneezing
Incubation up to 3 weeks before symptoms
Symptoms
 Fever, headache, non-productive cough (strep produce a
lot of material)
 Treatment (macrolides)
 Erythromycin, Tetracycline ; Penicillin is ineffective
 Mechanism of Pathogenicity
 Virulence Factor – P1 adhesion
 Binding of M. pneumoniae to respiratory cells via P1 protein.
 Damage of respiratory cells
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Other Mycoplasma
 Ureaplasma urealyticum
 Implicated in non gonococcal urethritis (NSU)
 Mycoplasma hominis
 Implicated in pelvic inflammatory disease
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Endotoxin
 Present in the outer membrane
 Released in the extracellular space
 Mediates the release of cytokines (it alone)
 TNF, Il-1, Il-6
 Activation of disseminated intra vascular
coagulation (DIC) (release of clotting factors)
 Vascular damage, increased vascular leakage
 Septic shock
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M. tuberculosis
General Features
 They are obligate aerobes and
grows very slowly with a generation
time of 12-15 hours.
 On solid media the colonies are
raised and rough with a wrinkled
surface.
 M. tuberculosis cells grow either as
discrete rods or as aggregates.
 Virulent strains tend to grow as an
aggregated long arrangement
called serpentine cord. Cord factor
is a derivative of mycolic acids,
trehalose 6'-dimycolate.
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- Unique structures:
arabinogalactan (AG),
mycosides, sulfatide, and
lipoarabinomannan (LAM).
- Unique component of lipids is
mycolic acid.
- Waxes, account for 60% of cell
weight, are composed of lipids
and some polypeptides.
- It can be visualized by acidfastness stains.
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Acid Fastness Stain
(Ziehl-Neelsen stain)
 flood the slide with basic fuchsin
(a red dye) in 5% phenol as a
mordant.
 heat gently for few minutes to
melt the wax. (so red dye can
penetrate outer cell - mycolic)
 wash with 3% HCl in ethanol.
(wash out colors except those
inside cell)
 counter-stain with methylene
blue.
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Host Immune Response
(Review)
 Humoral immune response
antibodies, cytokines (TNF, IL)
 Cell-mediated immune response
T cells, macrophages
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Phagocytosis of Invading Microbes
1. Oxygen radicals
2. Lysosomal
hydrolytic
enzymes
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Survival of M. tuberculosis in Macrophages
 Reduction of reactive oxygen
radicals production.
 Resistance to oxidative killing.
 AG and LAM absorb oxygen
radicals.
 No phagosome and lysosme
fusion.
M. tuberculosis survives and
multiplies inside the
macrophages.
 Modify the surface structure of
phagosome with AG and LAM.
 Keep the phagosome pH at
neutral.
 Prevent the fusion of phagosome
and lysomes, therefore no
lysosomal enzymes digestion.
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Multinucleated giant cells  tubercles, granulomas
Cell mediated immune response
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Symptoms:
Activation of macrophages ->
cytokine secretion,
IL-1: fever,
TNF: lipid metabolism,
weight loss, tissue necrosis
(mucus production; deep cough;
bloody sputum).
Oxygen radicals: tissue
damages
Tissue necrosis -> inflammation ->
mucous secretion, destruction of
blood vessels -> frequent cough and
bloody sputum
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Primary TB
Tuberculin test
Miliary TB
Ghon complex
(latent)
Reactivated TB
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Tuberculin Skin Test
 Tuberculin is a mixture known as purified protein
derivatives (PPD) from TB bacilli.
 It is a test for delayed type hypersensitivity (cell
mediated immune response). Positive reaction,
reddening and thickening (> 5mm) at the site of
injection after 2-3 days, indicates cellular immunity
to tubercle bacilli.
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acetyl-CoA
Oxaloacetate
Fatty acid
Citrate
acetyl-CoA
malate
cis-aconitate
MS
glyoxylate ICL
fumarate
isocitrate
succinate
a-ketoglutarate
ICL: isocitrate lyase
MS: malate synthase
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Factors for TB Crisis
1. An increased population of AIDS patients.
 HIV-infected persons are more susceptible to the TB bacilli and
develop miliary TB.
 HIV infection can reactivate latent TB.
 AIDS patients could be tuberculin test negative. It relies on the
cultivation of organism.
 Nosocomial transmission of TB is becoming serious.
2. An increased population of the homeless and in the
prisons.
3. Increased immigration from countries where TB is
common.
4. Occurrence of multi-drug resistant strains of M.
tuberculosis.
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Laboratory Diagnosis
for M. tuberculosis
 A rapid diagnostic approach includes
- a careful patient history,
- tuberculin test,
- chest X-ray, (for Ghon complex)
- direct microscopic examination of sputum,
- PCR reaction, (fast)
- cultivation of microorganism. (takes long time)
 Time consuming for cultivation and antibiotic
susceptibility assay.
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Treatment and Prevention for Tuberculosis
 Drug Treatment
 Vaccination
BCG is an attenuated M. bovis used as a
mycobacterial vaccine. BCG is an abbreviation of
bacillus Calmette- Guerrin, after its French
discoverers.
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Drug Treatment for Tuberculosis
treated with a combination of multiple drugs for a long period of
time: rifampin, isoniazid (INH), pyrazinamide, ethambutol, and
streptomycin.
 Standard 9 month regimens
a.
INH (300mg) + RMP (600mg) + PZA (30 mg/kg)
+ STM
(1 g) or EMB (25 mg/kg), orally, daily, for 2 months,
INH (300mg) + RMP (600mg), orally, daily for 7 months.
b.
INH (300 mg) + RMP (600mg), orally, daily for 2
months,
INH (900 mg) + RMP (600 mg), twice weekly for
7 months.
 Six month regimens
INH-RMP-STM-PZA for 2 months + INH-RMP for 4
months
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Antimycobacterium agents
 Rifampin - inhibits DNA-dependent RNA polymerase, kills
intracellular and extracellular M.tb.
 Streptomycin - aminoglycoside, inhibits protein synthesis
kills extracellular M.tb.
 Isoniazid (INH) - antimetabolite, inhibits mycolic acid
synthesis, requires katG (catalase) for activity, kills
intracellular and extracellular M.tb.
 Ethambutol - antimetabolite, inhibits RNA synthesis, kills
intracellular and extracellular
 Pyrazinamide - disrupts membrane transport and
energetics, kills intracellular M.tb.
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M. leprae Infection
It is exclusively a human parasite.
It primarily proliferates inside macrophages.
M. leprae has a predilection for skin and peripheral nerve.
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Leprosy
Leprosy is distinguished by its chronic slow progress and by its mutilating
and disfiguring lesions.
1. Tuberculoid leprosy.
- It is usually benign and frequently self-limited. Only segments of peripheral
nerves are involved, leading to localized patches of anesthesia.
- It is lepromin test positive.
2. Lepromatous leprosy
- It is a malignant form of disease: formation of large firm nodules on
the skin, loss of sensation in fingers and toes.
- It is lepromin test negative.
3. Borderline leprosy.
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Tuberculoid leprosy
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Lepromatous leprosy
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Leprosy Treatment and Prevention
 Drugs used: dapsone, rifampin, and clofazimine.
 No vaccine.
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Previously, the family consisted of one genus, Chlamydia, with four species. Now
the family has been divided into two genera, Chlamydia and Chlamydophila.
The family Chlamydiaceae was revised extensively on the basis of genomic studies
of these organisms
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Differentiation of Chlamydiaceae That Cause Human Disease
Chlamydia trachomatis
Chlamydophila
pneumoniae
-
-
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Chlamydophila psittaci
The Chlamydiaceae were once considered viruses because
they are
• Small enough to pass through 0.45-μm filters
• Obligate intracellular parasites.
The organisms have the following properties of bacteria:
(1) possess inner and outer membranes similar to those of
gram-negative bacteria;
(2) contain both deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA);
(3) possess prokaryotic ribosomes;
(4) synthesize their own proteins, nucleic acids, and lipids; and
(5) are susceptible to numerous antibacterial antibiotics.
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Unlike other bacteria, the Chlamydiaceae have a unique developmental
cycle, forming metabolically inactive infectious forms (elementary bodies
[EBs]) and metabolically active, noninfectious forms (reticulate bodies
[RBs]).
Microvilli
cytoplasmic phagosomes
fusion of cellular lysosomes with the
EB-containing phagosome and
subsequent intracellular killing is
inhibited.
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PHYSIOLOGY AND STRUCTURE
EBs
• Are resistant to many harsh environmental factors, non replicative infectious form.
• Lack the rigid peptidoglycan layer found in most other bacteria.
• Their outer membrane proteins are extensively cross-linked by disulfide bonds
between
cysteine residues.
RBs
• Are the metabolically active, replicating chlamydial form.
• The extensive cross-linked proteins are absent in RBs, this form is osmotically fragile;
• However, RBs are protected by their intracellular location.
Other important structural components of Chlamydiaceae are a genus-specific
lipopolysaccharide (LPS)
Specific LPS Can be detected by complement fixation (CF) test and species specific
outer membrane proteins.
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COMPLEMENT FIXATION TEST
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Chlamydia trachomatis
Subdivided into three biovars,
1.Trachoma.
2.LGV (lymphogranuloma venereum): sexually transmitted disease caused by the
invasive serovars L1, L2, L2a, or L3
3.mouse pneumonitis.
Table 47-2. Clinical Spectrum of Chlamydia trachomatis
Infections.
antigenic differences in the major outer membrane protein (MOMP)
Serovars
Site of Infection
A, B, Ba, C
Primarily conjunctiva
D-K
Primarily urogenital tract
L1, L2, L2a, L3
inguinal lymph nodes
Biovar is a variant prokaryotic strain that differs physiologically and/or biochemically from other strains in a particular species.
Serovars are those strains that have antigenic properties that differ from other strains.
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PATHOGENESIS AND IMMUNITY
Receptors for EBs are primarily restricted to non ciliated
columnar, cuboidal, and transitional epithelial cells, which are
found on the mucous membranes of the urethra, endocervix,
endometrium, fallopian tubes, anorectum, respiratory tract,
and conjunctiva.
The LGV biovar replicates in mononuclear phagocytes
present in the lymphatic system.
The clinical manifestations of chlamydial infections are
caused by
(1)the direct destruction of cells during replication and
(2)the host inflammatory response.
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Cont,
In LGV, the lesions form in the lymph nodes. Granuloma
formation is characteristic. The lesions may become necrotic,
attract polymorphonuclear leukocytes, and cause the
inflammatory process to spread to surrounding tissues.
Subsequent rupture of the lymph node leads to formation of
abscesses or sinus tracts.
Non-LGV serovars of C. trachomatis stimulates a severe
inflammatory response consisting of neutrophils, lymphocytes,
and plasma cells.
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Cont,
Infection does not confer long-lasting immunity.
Rather, reinfection characteristically induces a vigorous inflammatory response with
subsequent tissue damage.
This response produces the vision loss in patients with chronic ocular infections, and
scarring with sterility and sexual dysfunction in patients with genital infections.
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CLINICAL DISEASES
1. Trachoma: caused by serovars A, B, Ba, and C.
Initially, patients have a follicular conjunctivitis with
diffuse inflammation that involves the entire conjunctiva.
The conjunctivae become scarred as the disease
progresses, causing the patient's eyelids to turn inward.
The turned-in eyelashes abrade the cornea,
eventually resulting in corneal Scarring
, and loss of vision.
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2. Adult Inclusion Conjunctivitis:
• An acute follicular conjunctivitis
• Caused by the C. trachomatis strains associated with genital infections (serovars
A, B,
Ba, D to K)
• In sexually active adults.
• The infection is characterized by
-Mucopurulent discharge
-Keratitis
-Corneal scarring has been observed in patients with chronic infection.
3. Neonatal Conjunctivitis: Eye infections can also develop in infants exposed to C.
trachomatis at birth.
4. Urogenital Infections :
genital infections in men are symptomatic.
The clinical manifestations include, cervicitis,
endometritis, perihepatitis, salpingitis,
and urethritis.
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5. Reiter's syndrome (urethritis, conjunctivitis, polyarthritis, and
mucocutaneous lesions) is believed to be initiated by genital infection
with C. trachomatis
6. Lymphogranuloma Venereum :
• Incubation of 1 to 4 weeks.
• Stage1: A primary lesion appears at the site of infection. Painless.
• Stage2: inflammation and swelling of the lymph nodes
draining the site of initial infection.
•Stage3: Systemic manifestations include fever, chills, anorexia, headache, and arthralgia.
Mucopurulent cervicitis caused by Chlamydia trachomatis.
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LABORATORY DIAGNOSIS
(1) on the basis of cytologic, serologic, or culture findings.
Examination of Giemsa-stained cell scrapings for the presence of
inclusions. Not sensitive.
Culture is the most specific method of diagnosing C. trachomatis infections
The bacteria infect a restricted range of cell lines in vitro
e.g.,
HeLa
McCoy
HEp-2
Derived from human cervical cancer cells.
Derived from the synovial fluid in the knee joint of a patient .
Derived from rat tumors.
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Chlamydia trachomatis is grown in cell
cultures and detected by staining inclusion
bodies (arrows) with either iodine or specific
fluorescein-labeled antibodies.
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(2) Direct detection of antigen in clinical specimens.
a. Immunofluorescence staining with
fluorescein-conjugated monoclonal antibodies.
b. ELISA: Enzyme Linked Immunosorbent Assay
IN BOTH TECHNIQUES: antibodies are used that have been prepared against either
the chlamydial MOMP or the cell wall LPS
(3) Molecular probes.
Specific but insensitive for the detection of small numbers of chlamydiae.
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TREATMENT, PREVENTION, AND CONTROL
Penicillin is ineffective against Chlamydia because they lack peptidoglycan in their
cell wall.
LGV: treated with a tetracycline (e.g., doxycycline) for 21 days.
Children younger than 9 years, pregnant women, and patients unable to
tolerate tetracyclines.
macrolide (e.g., erythromycin, azithromycin)
or sulfisoxazole is recommended.
It is difficult to prevent C. trachomatis infections because the population with endemic
disease commonly has limited access to medical care.
The blindness associated with advanced stages of trachoma can be prevented only
by prompt treatment of early disease and the prevention of reexposure.
Improving sanitary conditions.
Chlamydia conjunctivitis and genital infections are prevented through the use of safe
sex practices and the prompt treatment of symptomatic patients and their sexual
partners.
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Special Thanks to Paula for compiling the slides
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