MYCOPLASMA and Ureaplasma

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MYCOPLASMA and Ureaplasma

• Dr. R.K.Kalyan
• Associate Professor
• Microbiology Dept. KGMU

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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).

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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.

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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

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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

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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

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Mycoplasmas of Humans

• 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.

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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

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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

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Pathogenicity cont
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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).

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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

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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?)

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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

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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

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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.

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Laboratory Diagnosis

• Specimens throat swabs, respiratory secretions.
• Microscopy
• 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

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Laboratory Diagnosis

• Isolation of Mycoplasma (Culture)
• 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
  510 CO2 at 35-37C. (temp range 22- 41C,
  parasites 35- 37C, saprophytes lower temp)

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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 Dienes method.
• 5. Produce beta hemolytic colonies, can
  agglutinate guinea pig erythrocytes.

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Fried egg colonies
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Except for M. pneumoniae colonies which have a
granular appearance, described as being mulberry
shaped
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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

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Identification of Isolates

• Serological diagnosis
• Specific tests IF, HAI
• 2. Non specific serological tests cold
  agglutination tests (Abs agglutinate human group
  O red cells at low temperature, 4?C).
• 132 titer or above is significant.

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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

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Genital Infections

• Caused by M. hominis U. urealyticum
• Transmitted by sexual contact
• Men - Nonspecific urethritis, proctitis,
  balanoposthitis Reiters syndrome
• Women acute salpingitis, PID, cervicitis,
  vaginitis
• Also associated with infertility, abortion,
  postpartum fever, chorioamnionitis low birth
  weight infants

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Mycoplasma HIV infection

• Severe prolonged infections in HIV infected
  other immunodeficient individuals

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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.

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Treatment and PreventionM. 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

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Treatment

• Tetracycline, Erythromycin Clarithromycin
  drug of choice
• Resistant to antibiotics which interfere with
  bacterial cell wall synthesis.
• Newer macrolides quinolones being used now.

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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

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POINTS TO BE REMEMBER

• Cold agglutination test
• Cell culture contamination
• Ureaplasma hydrolysis of urea
• Primary atypical/ walking pneumonia
• Genital infections

• Mycoplasma
• No cell wall
• Pleomorphism
• Fried egg colonies
• Dienes stain

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Dienes stain

• Azure II
• Methylene blue
• Maltose
• Na2co3
• Benzoic acid
• DW

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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

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Liquid medium

• PPLO agar base without crystal violet ph
  7.8Yeast extract Horse serum Sodium
  deoxyribonucleate Thallous acetate
  solutionK2HPO4, Penicilin solution
• Glucose serum Phenol red Methylene blue.
• Biphasic medium 1. solid phase- Standard solid
  medium
• 2. Liquid phase- Liquid medium.

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MCQ

• Q.1. Which of the following bacteria was named as
  Eaton agent
• Acholeplasma
• Mycoplasma hominis
• Mycoplasma pneumoniae
• Ureaplasma urealyticum
• Q.2. Dienes method is used to examine colonies of
• Bordetella
• Burkholderia
• Mycoplasma
• Helicobacter

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• Q.3.Which of the following bacteria is/are
  associated with nongonococcal urethritis ?
• Mycoplasma hominis
• Ureaplasma urealyticum
• Chlamydia trachomatis
• All of the above
• Q.4.Which is the causative agent of primary
  atypical pneumoniae
• Influenza virus
• Streptococcus Pneumoniae
• Haemophilus influenzae
• Mycoplasma pneumoniae

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• Q.5. Which of the following can hydrolyse urea
• Mycoplasma
• Acholeplasma
• Ureaplasma
• Escherichia
• Q.6. Which of the following bacteria is/are also
  named T strain ?
• Mycoplasma pneumoniae
• Mycoplasma hominis
• Ureaplasma urealyticum
• Acholeplasma

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• Q.7.Postpartum fever due to Mycoplasma hominis is
  treated with
• Penicillin G
• A second generation Cephalosporins
• Vancomycin
• Tetracyclines
• Q.8.A distinguishing feature of human mycoplasma
  species is that they
• Stain well with Giemsa, but not by Gram stain
• Contain no bacterial peptidoglycan
• Are not immunogenic because they mimic host cell
  membrane components
• Cannot be cultivated in vitro

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• Q.9. which of the following tests can be used to
  identify Mycoplasma pneumoniae ?
• Haemadsorption test
• Tetrazolium reduction test
• Inhibition of growth by specific antisera
• All of the above
• Q.10. Which of the following bacteria shows fried
  egg colonies on culture media ?
• Helicobacter
• Mycobacterium tuberculosis
• Bordetella
• Mycoplasma

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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

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!
Thanks for attention !