1. MYCOPLASMA and Ureaplasma
Dr. R.K.Kalyan
Associate Professor
Microbiology Dept. KGMU

2. MYCOPLASMA Smallest free-living micro organisms, lack cell wall.
Size varies from spherical shape( nm to longer branching filaments 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).

3. 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.

4. 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

5. 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

6. 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

7. 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.

8. 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

9. 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

10. Pathogenicity cont’

11. 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).

12. 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

13. 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?)

14. Clinical Syndrome - M. pneumoniae
Incubation 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

15. 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

16. 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.

17. 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

18. 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 days with 5–10% CO2 at 35-37°C. (temp range °C, parasites °C, saprophytes – lower temp)

19. 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.

20. Dr Ekta,Microbiology, GMCA
Fried egg colonies
Dr Ekta,Microbiology, GMCA

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

22. 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

23. 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).
1:32 titer or above is significant.

24. 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

25. 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

26. Mycoplasma & HIV infection
Severe & prolonged infections in HIV infected & other immunodeficient individuals

27. 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.

28. Treatment and Prevention M. pneumoniae
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

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

30. 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

31. POINTS TO BE REMEMBER Cold agglutination test Mycoplasma
Cell culture contamination
Ureaplasma hydrolysis of urea
Primary atypical/ walking pneumonia
Genital infections
Mycoplasma
No cell wall
Pleomorphism
Fried egg colonies
Diene’s stain

32. Dienes stain
Azure II
Methylene blue
Maltose
Na2co3
Benzoic acid DW

33. 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

34. 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.

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. !
Thanks for attention !