MYCOPLASMA and
Ureaplasma
Dr. R.K.Kalyan
Associate Professor
Microbiology Dept. KGMU
MYCOPLASMA
 Smallest free-living micro organisms, lack
cell wall.
 Size varies from spherical shape(125250nm to longer branching filaments 5001000 nm in size. Many can pass through a
bacterial filter.
 1st member of this group – isolated by
Nocard & Roux (1898) – caused bovine
pleuropneumonia.
 Later, many similar isolates were obtained
from animals, human beings, plants &
environmental sources – called as
“pleuropneumonia like organisms”(PPLO).
MYCOPLASMA
 Eaton (1944) first isolated the causative
agent of the disease in hamsters and
cotton rates.
 Also known as Eaton agent.
 1956- PPLO replaced by Mycoplasma.
– Myco : fungus like branching filaments
– Plasma : plasticity
 highly pleomorphic – no fixed shape or
size - Lack cell wall.
Morphology and Physiology
 Small genome size (M. pneumoniae is ~800 Kbp)
– Require complex media for growth
 Facultative anaerobes
– Except M. pneumoniae - strict aerobe
•
No cell wall means these are resistant to penicillins,
cephalosporins and vancomycin, etc.
•
Grow slowly by binary fission
 Doubling time can be as long as 16 hours, extended
incubation needed
Morphology and Physiology
cont’
• Require complex media for growth,
including sterols
•
Major antigenic determinants are
glycolipids and proteins, some cross
reaction with human tissues.
•
Requirements for growth allow one to
differentiate between species
Morphology and Physiology
cont’
• M. pneumoniae - glucose
• M. hominis - arginine
• U. urealyticum - urea (buffered media due
to growth inhibition by alkaline media)
• M. genitalium - difficult to culture
Mycoplasmas of Humans

1.
2.
3.

Parasitic
Established pathogens: M. pneumoniae
Presumed pathogens: M. hominis,
U. urealyticum
Non pathogenic: M. orale, M. buccale,
M. genitalium, M. fermentans
Saprophytic – present mainly on skin &
in mouth.
Pathogenicity

Produce surface infections – adhere to
the mucosa of respiratory,
gastrointestinal & genitourinary tracts
with the help of adhesin.

Two types of diseases:
Atypical Pneumonia
Genital infections
1.
2.
Pathogenicity cont’
 Adherence
– P1 pili (M. pneumoniae)
– Movement of cilia ceases (ciliostasis)
– Clearance mechanism stops resulting in cough
 Toxic metabolic products
– Peroxide and superoxide
– Inhibition of catalase
 Immunopathogenesis
– Activate macrophages
– Stimulate cytokine production
• Superantigen (M. pneumoniae)
Inflammatory cells migrate to infection and
release TNF-a then IL-1 and IL-6
Pathogenicity cont’
Mycoplasmal pneumonia
 Also called Primary Atypical Pneumonia/
Walking pneumonia.
 Seen in all ages
 Incubation period: 1-3 wks
 Transmission: airborne droplets of
nasopharyngeal secretions, close
contacts (families, military recruits).
Mycoplasmal pneumonia
 Gradual onset with fever, malaise, chills,
headache & sore throat.
 Severe cough with blood tinged sputum
(worsens at night)
 Complications: bullous myringitis & otitis,
meningitis, encephalitis, hemolytic anemia
Diseases Caused by
Mycoplasma

Organism

M. pneumoniae
Disease
Upper respiratory tract disease, tracheobronchitis
atypical pneumonia, (chronic asthma?)
M. hominis
Pyelonephritis, pelvic inflammatory disease,
postpartum fever

M. genitalium
Nongonococcal urethritis

U. urealyticum


Nongonococcal urethritis,
(pneumonia and chronic lung disease in
premature infants?)
Clinical Syndrome - M.
pneumoniae
 Incubation - 2-3 weeks
 Fever, headache and malaise
 Persistent, dry, non-productive cough
 Respiratory symptoms
– Patchy bronchopneumonia
– acute pharyngitis may be present
 Organisms persist
 Slow resolution
 Rarely fatal
 Note: Muscle pain and GI symptoms usually not present
Epidemiology - M. pneumoniae
 Occurs worldwide
 No seasonal variation
– Proportionally higher in summer and fall
 Epidemics occur every 4-8 year
 Spread by aerosol route (Confined
populations).
 Disease of the young (5-20 years),
although all ages are at risk
Laboratory Diagnosis - M.
pneumoniae
 Microscopy
– Difficult to stain
– This process can help eliminate other
organisms
 Culture (definitive diagnosis)
– Sputum (usually scant) or throat washings
– Special transport medium needed
• Must suspect M. pneumoniae
– May take 2-3 weeks or longer, 6 hour doubling
time with glucose and pH indicator included
– Incubation with antisera to look for inhibition.
Laboratory Diagnosis

Specimens – throat swabs,
respiratory secretions.

Microscopy –
Highly pleomorphic, varying from
small spherical shapes to longer
branching filaments.
1.
2.
Gram negative, but better stained
with Giemsa,Dienes’ stain, crystalfast violet, orcein or
fluorochroming with nucleic acid
stain as acredine orange
Laboratory Diagnosis
 Isolation of Mycoplasma (Culture) –
1. Semi solid enriched medium containing 20%
horse or human serum, yeast extract & DNA.
Penicillium & Thallium acetate are selective
agents.
(serum – source of cholesterol & other
lipids)
2. Incubate aerobically for 7 -12 days with 5–
10% CO2 at 35-37°C. (temp range 22- 41°C,
parasites 35- 37°C, saprophytes – lower
temp)
Laboratory Diagnosis
3. Typical “fried egg” appearance of
colonies - Central opaque granular area
of growth extending into the depth of
the medium, surrounded by a flat,
translucent peripheral zone.
4. Colonies best seen with a hand lens after
staining with Diene’s method.
5. Produce beta hemolytic colonies, can
agglutinate guinea pig erythrocytes.
Fried egg colonies
Dr Ekta,Microbiology, GMCA
Except for M. pneumoniae colonies which have a
granular appearance, described as being mulberry
shaped
Identification of Isolates
 Growth Inhibition Test – inhibition of
growth around discs impregnated with
specific antisera.
 Immunofluorescence on colonies
transferred to glass slides.
 Molecular diagnosis
– PCR-based tests are being developed and
these are expected to be the diagnostic test
of choice in the future.
– These should have good sensitivity and be
specific
Identification of Isolates
 Serological diagnosis
1. Specific tests – IF, HAI
2. Non specific serological tests – cold
agglutination tests (Abs agglutinate
human group O red cells at low
temperature, 4C).
1:32 titer or above is significant.
Ureaplasma urealyticum
 Strains of mycoplasma isolated from the
urogenital tract of human beings &
animals.
 Form very tiny colonies - hence called T
strain or T form of mycoplasmas.
 Hydrolyzes urea
Genital Infections
 Caused by M. hominis & U. urealyticum
 Transmitted by sexual contact
 Men - Nonspecific urethritis, proctitis,
balanoposthitis & Reiter’s syndrome
 Women – acute salpingitis, PID, cervicitis,
vaginitis
 Also associated with infertility, abortion,
postpartum fever, chorioamnionitis & low
birth weight infants
Mycoplasma & HIV infection
 Severe & prolonged infections in HIV
infected & other immunodeficient
individuals
Mycoplasma as cell culture
contaminants
 Contaminates continuous cell cultures
maintained in laboratories
 Interferes with the growth of viruses in
these cultures.
 Mistaken for viruses.
 Eradication from infected cells is difficult.
Treatment and Prevention
M. pneumoniae
 Treatment
– Tetracycline in adults (doxycycline) or
erythromycin (children)
• Newer fluoroquinolones (in adults)
– Resistant to cell wall synthesis inhibitors.
 Prevention
– Avoid close contact
– Isolation is not practical due to length of illness
– No vaccine, although attempted
Treatment
 Tetracycline, Erythromycin &
Clarithromycin – drug of choice
 Resistant to antibiotics which interfere
with bacterial cell wall synthesis.
 Newer macrolides & quinolones being
used now.
M. hominis, M. genitalium and
U. urealyticum
– Treatment
• Tetracycline or erythromycin
• U. urealyticum is resistant to tetracycline
• M. hominis is resistant to erythromycin and
sometimes to tetracyclin, Clindamycin for
these resistant strains
– Prevention
• Abstinence or barrier protection
• No vaccine
POINTS TO BE REMEMBER
 Mycoplasma
 No cell wall
 Pleomorphism
Cold agglutination test
Cell culture contamination
Ureaplasma hydrolysis of
urea
 Fried egg colonies
Primary atypical/ walking
pneumonia
 Diene’s stain
 Genital infections
Dienes stain
 Azure II
 Methylene blue
 Maltose
 Na2co3
 Benzoic acid
 DW
Standard solid media
PPLO agar base without crystal
violet ph 7.8
+Yeast extract +Horse serum
+Sodium deoxy ribonucleate
+Thallous acetate solution
+K2HPO4, Penicillin solution
Liquid medium
 PPLO agar base without crystal violet ph
7.8+Yeast extract +Horse serum +Sodium
deoxyribonucleate +Thallous acetate
solution+K2HPO4, Penicilin solution
+ Glucose serum + Phenol red +Methylene
blue.
Biphasic medium: 1. solid phase- Standard
solid medium
2. Liquid phase- Liquid medium.
MCQ
Q.1. Which of the following bacteria was named as Eaton
agent
a) Acholeplasma
b) Mycoplasma hominis
c) Mycoplasma pneumoniae
d) Ureaplasma urealyticum
Q.2. Dienes method is used to examine colonies of
a) Bordetella
b) Burkholderia
c) Mycoplasma
d) Helicobacter
Q.3.Which of the following bacteria is/are associated with
nongonococcal urethritis ?
a) Mycoplasma hominis
b) Ureaplasma urealyticum
c) Chlamydia trachomatis
d) All of the above
Q.4.Which is the causative agent of primary atypical pneumoniae
a) Influenza virus
b) Streptococcus Pneumoniae
c) Haemophilus influenzae
d) Mycoplasma pneumoniae
Q.5. Which of the following can hydrolyse urea
a) Mycoplasma
b) Acholeplasma
c) Ureaplasma
d) Escherichia
Q.6. Which of the following bacteria is/are also named T strain ?
a) Mycoplasma pneumoniae
b) Mycoplasma hominis
c) Ureaplasma urealyticum
d) Acholeplasma
Q.7.Postpartum fever due to Mycoplasma hominis is treated with
a) Penicillin G
b) A second generation Cephalosporins
c) Vancomycin
d) Tetracyclines
Q.8.A distinguishing feature of human mycoplasma species is that
they:
a) Stain well with Giemsa, but not by Gram stain
b) Contain no bacterial peptidoglycan
c) Are not immunogenic because they mimic host cell membrane
components
d) Cannot be cultivated in vitro
Q.9. which of the following tests can be used to identify Mycoplasma
pneumoniae ?
a) Haemadsorption test
b) Tetrazolium reduction test
c) Inhibition of growth by specific antisera
d) All of the above
Q.10. Which of the following bacteria shows fried egg colonies on
culture media ?
a) Helicobacter
b) Mycobacterium tuberculosis
c) Bordetella
d) Mycoplasma
ANSWERS OF MCQ
Q.1Q.2Q.3Q.4Q.5Q.6Q.7Q.8Q.9Q.10-
C
C
d
d
C
C
d
b
d
d
!
Thanks for attention !