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(125-250nm to longer
branching filaments 500-1000 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
 Parasitic
1. Established pathogens: M. pneumoniae
2. Presumed pathogens: M. hominis,
U. urealyticum
3. 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:

1. Atypical Pneumonia
2. Genital infections
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 Disease

 M. pneumoniae 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 –
1. Highly pleomorphic, varying from
small spherical shapes to longer
branching filaments.

2. Gram negative, but better stained

with Giemsa,Dienes’ stain, crystal-
fast 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 Cold agglutination test

Cell culture contamination

 No cell wall
Ureaplasma hydrolysis of
 Pleomorphism urea

Primary atypical/ walking

 Fried egg colonies
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.1- C
Q.2- C
Q.3- d
Q.4- d
Q.5- C
Q.6- C
Q.7- d
Q.8- b
Q.9- d
Q.10- d
!

Thanks for attention !