Mycoplasma complete
41 likes11,212 views
This document discusses Mycoplasma, a class of bacteria that lack cell walls. It describes their general characteristics, examples of pathogenic species and diseases they cause, laboratory techniques for isolation and identification of Mycoplasma pneumoniae and genital Mycoplasmas, and serological tests for diagnosis of Mycoplasma infections. Key points are that Mycoplasma are the smallest free-living organisms, are difficult to culture, and cause respiratory and genital tract infections in humans.
Mycoplasma complete
- 1. Mycoplasma Dr. Kanwal Deep Singh Lyall
- 2. • Nocard and Roux (1898) • Resemblence to organisms causing benign pleruopnemonia • Pleuropnemonia like organisms or PPLO • Fungus like form of branching filaments • Class-mollicutes (mollies-soft, Kutis-skin)
- 4. Mycoplasma pneumoniae pneumonia, brochiolitis, croup, etc. Mycoplasma orale no disease association Mycoplasma salivarum no disease association Mycoplasma buccale no disease association Mycoplasma faucium no disease association Mycoplasma lipophilum no disease association Mycoplasma spermatophilum no disease association Mycoplasma primatum no disease association Mycoplasma Hominis septicemia, GU tract infections, organ transplant & wound infections Mycoplasma genitalum NGU, RTI etc. Mycoplasma fermentans associated with HIV Mycoplasma penetrans associated with HIV Mycoplasma pirum associated with HIV Ureaplasma urealyticum LRTI, septicemia, meningitis, NGU Ureaplasma parvum GU tract infections Mycoplasma & Ureaplasma Isolated From Humans
- 5. General Characteristics • Lack a rigid cell wall • Extremely pleomorphic • Surrounded by a single triple layered membrane containing cholesterol
- 6. • Smallest free living organisms • Gram –ve, but better stained with Giemsa stain • Non-sporing, non-flagellated • Non-motile, may show gliding motility
- 7. • Reproduce by fission & budding • Spherical (125-250nm) • Branching filaments (500-1000nm)
- 8. • Killed by heating at 56° C X 30 mnts • Lysis by surface active agents & lipolytic agents • Cannot synthesize cholesterol • Need incorporation of sterol in medium • Resistant to cell wall active antibiotics • Contain both RNA & DNA • Can reproduce in cell free medium • Ureaplasma hydrolyze urea
- 9. Virulence Factors • Attachment organelle • P1 protein • Additional accessory proteins ( HMW1-4, P65, P40,30,P116, P50, P100) • Vaa (variable adherence associated) antigen • A1,A2, C (phospholipases) • Induction of inflammatory cytokines (TNFα, IL6) • Induction of apoptosis in human epithelial cells & macrophages • IgA proteases • Arginine deaminases • Nucleases • Ability to invade & survive intracellularly
- 10. Pathogenesis & Clinical Picture • M.pneumoniae causes atypical pneumonia & spread is maimly by droplets, close families & military recruits • M.hominis & others causes non-gonococcal urethritis , postpartum urethritis, proctitis, acute salpingitis,PID etc. • Transmitted by sexual contact
- 11. Lab Diagnosis
- 12. Specimen Collection • Preferably inoculated soon after collection • Media dispensed in small vials for swabs • Respiratory samples homogenized before inoculation • Sputolysins & other chemicals used for liquification are toxic for mycoplasmas • Swabs with wooden shafts not recommended as wood itself is toxic for mycoplasmas • Contact with antiseptics, creams & jellies should be avoided • Swabs should not be dried • Saline should not be used
- 13. specimens Upper & lower respiratory tract specimens • Throat swabs • Nasophyrangeal swabs • Throat washings • Sputum • Tracheal & transtracheal aspirates • BAL • Lung tissues etc. Genital tract specimens • Urethral, cervical & vaginal swabs • Prostatic secretions • Semen, urine, etc • Others • CSF • Blood • Amniotic fluid • Synovial & pericardial fluid etc.
- 14. Transport Media • Tripticase-soy broth with 0.5% bovine serum albumin • 2SP broth (10% heat inactivated fetal calf serum albumin with 0.2M sucrose in 0.02M phosphate buffer. pH 7.2) • Penicillin, polymyxin B, amphotericin B etc may be added • Transported immediately or • Held at 4° C x 24 hrs • Urine-centr.-sediment 1:1 dil with transport media & freezed
- 15. Media Used • Methylene blue-glucose diphasic medium (PPLO broth+agar+yeast extract+serum supplements+glucose+methylene blue+phenol red+tahllium acetate solution+penicillin) • Mycoplasma glucose agar medium (mycoplasma agar, yeast extract, agar, glucose solution, thallium acetate sol., penicillin) • SP4 medium • H-broth, H-agar for M.hominis • U-broth, U-agar for U. urealyticum (made selective by adding lincomycin)
- 16. Isolation & identification of M. pneumoniae
- 17. specimen
- 18. specimen Methylene blue-glucose diphasic medium
- 19. specimen Methylene blue-glucose diphasic medium Incubate at 35 C
- 20. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation No change after 4 weeks
- 21. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days No change after 4 weeks Culture negative
- 22. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth No change after 4 weeks Culture negative
- 23. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth Confirm with hemabsorption &/or tetrazolium reduction test No change after 4 weeks Culture negative
- 24. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth Confirm with hemabsorption &/or tetrazolium reduction test No change after 4 weeks Culture negative • M.pneumoniae –absorbs RBC • Flood plate with 1ml of RBC suspension • Incubate x 35 °C x 30mnts & rotate occasionally to prevent RBC from settling out • Wash plate thrice with 3ml of mycoplasma broth by gently rotating & remove • Examine colonies at 50 to 100x magnification under dissecting microscope • positive=colonies studded with RBCs
- 25. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth Confirm with hemabsorption &/or tetrazolium reduction test No change after 4 weeks Culture negative • Ability to reduce colorless tetrazolium (2-p- iodophenyl-3-nitrophenyl-5-phenyl tetrazolium chloride) to red colored formozan • flood colonies with 2ml of tetrazolium reagent • Incubate x 35 ° C for 30-60mnts • Examine under 40-60 x magnification with a dissecting microscope • Positive = colonies darken, become red to purple after 60mnts, on prolonged incubation turn black • Negative = no color change in 60 mnts • Can be performeb after hemabsorbtion test
- 26. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth Confirm with hemabsorption &/or tetrazolium reduction test Positive confirmatory tests Epifluorescence & growth inhibition tests No change after 4 weeks Culture negative
- 27. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth Confirm with hemabsorption &/or tetrazolium reduction test No change after 4 weeks Culture negative • Colonies flooded with M.pneumoniae- specific antibodies conjugated with fluorescein isothiocyanate • Washed to remove conjugate • Examined with microscope equipped with epifluorescence procedures Positive confirmatory tests Epifluorescence & growth inhibition tests
- 28. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth Confirm with hemabsorption &/or tetrazolium reduction test No change after 4 weeks Culture negative • Lawn culture from broth suspenesion • Piece of filter paper impregnated with anti-M.pneumoniae antibodies placed on agar surface • incubation—inhibition of growth around paper Positive confirmatory tests Epifluorescence & growth inhibition tests
- 29. specimen Methylene blue-glucose diphasic medium Incubate at 35 C Shift to slightly acidic pH after 8-15 days incubation s/c broth to mycoplasma glucose agar medium Incubate at 35 C x 5-7 days Growth Confirm with hemabsorption &/or tetrazolium reduction test Positive confirmatory tests Epifluorescence & growth inhibition tests No change after 4 weeks Culture negative M.Pneumoniae isolated
- 30. Non-cultural Detection Of M.Pneumoniae • Slow growth-direct detection • Antigen capture assay • Direct immunoflorescence assay • Immunoblot analysis • Nucleic acid probe methods • Lack sufficient sensitivity & specificity • PCR assays
- 31. Isolation & Identification Of Genital Mycoplasmas • M.hominis • M.genitalum • M.fermentans • U.urealyticum • M.hominis & U.urealyticum-easily cultiviable, growth within 1-5days • M.genitalum & M.fermentans grow slowly & difficult to cultivate
- 32. Specimen M broth M agar U broth U agar (contain arginine & phenol red) (contain urea & phenol red) (incubate at 35 °C, broth in air, agar in CO2 incubator or in candle jar) 5-7 days x daily inspection Alkaline change Alkaline changeInspect 100x Inspect 100x s/c to H agar No growth NG Colonies with red haloes Colonies with red haloes Contd.. s/c to U agar Or A7/A8 agar
- 33. Incubate as above NG Typical large colonies, Fried egg morphology (Diene’s stain) NG Incubate as above Typical small colonies, Golden yellow on A7/A8 agar M.Hominis Flood U agar with MnCl2 urea Golden brown colonies Ureaplasma urealyticum Contd.. NG
- 34. Diene’s stain • Methylene blue, azure II, maltose, Na2CO3, DW • Flood agar plate with 1ml of stain solution • Immediately rinse with distilled water • Decolorize with 1ml of 95% ethanol, leave for 1mnt and remove. Repeat for second time • Rinse with DW, dry • Observe under low power microscope
- 35. Fried Egg Colonies With Dark Blue Centre, Light Blue Periphery (Highly Granular)
- 36. Incubate as above NG Typical large colonies, Fried egg morphology (Diene’s stain) NG Incubate as above Typical small colonies, Golden yellow on A7/A8 agar M.Hominis Flood U agar with MnCl2 urea Golden brown colonies Ureaplasma urealyticum Contd.. NG • U.urealyticum hydrolyses urea to ammonia • Flood colonies with 2-3ml of manganous chloride- urea reagent • Leave for 5mnts at room t° • Examine under 50-100x with dissecting microscope • Colonies of U.urealyticum stain dark brown • Others remain unstained
- 37. Non-cultural Detection Of Genital Mycoplasmas • IFA(more sensitive than culture) • DNA probes • PCR (highly sensitive)
- 38. Serological Tests For Genital Mycoplasmas • LAMPs (lipid associated membrane proteins) • Antibodies to LAMPs are highly species specific • LAMPs are used as target antigens in EIA • EIA with purified organism sonicate s as solid phase antigen • Wetsern immunoblot techniques
- 39. Commercial Mycoplasma Culture Systems • Mycotrim RS system-M.pneumoniae -a layer of enriched glucose agar medium containing phenol red, on one side & a broth of similar compound -30 mnts before inoculation disks of antibiotics (cefoperazone, nystatin, thallous acetate) are added & allowed to diffue -incubated with agar side up x 2 weeks x 37° C -reinoculation x 3 days -Broth washes the agar -inspect colonies with or without Diens’s stain
- 40. • Mycotrim GU broth -contains arginine, urea, phenol red -0.1ml of specimen & incubate x 37 C Color change -s/c on solid agar (A7 or A8)
- 41. • Mycotrim triphasic flask -diphasic system contains A8 differential agar with CaCl & broth with arginine, urea, phenol red -disks added (cefoperazone, nystatin) -0.10ml of specimen added -incubate x 35 C, reinoclate after 24 hrs -color change -examine
- 42. Serological Tests For Diagnosis Of M.Pnumoniae Infections • Limited availability of culture systems • Technical difficulties & long time period • Diagnosis=clinical picture + serology • Most widely used is CFT • Antigen used is glycolipid from the organism extracted by chloroform-methanol • Measures predominantly IgM & to lesser extent IgG
- 43. • IgM appears during 3rd week & then declines • IgG rise slowly & peak in 5 weeks & remain elevated for 3-4 years • Compliment fixing antibodies persist for years • Greatest diagnositic accuracy when acute & convalescent sera demonstrate a 4 fold rise • Or single convalescent titres of 32 or more highly suggestive
- 44. • Mycolplasma antibody test system – Indirect IF assay-detects IgG & IgM • EIA detection of antobody • Microtitre & cassete ELISA • Microparticulate agglutination assays
- 45. Cold Agglutination Assay • Macroglobulin antibodies-agglutinate human O grup erytrocytes at low t° • Serial dilutions of serum mixed with washed human O erythrocytes x 4 °C overnight • Clumping dissociated at 37 °C • Titre of ≥1:32 is suggestive • Rising titre in paired sera more reliable
- 46. Streptococcus MG test • Serial dilutions of patients unheated serum • Mixed with killed suspension of streptococcus MG • Incubated x 37 C x overnight • Agglutination observed • Titre of 1:20 or more is suggestive
- 47. AST & Treatment • Because of difficulty in culturing, slow growth & lack of readily available methods AST is neither necessary nor appropriate • Generally susceptible to tetracycline & erythromycin • Susceptible to qunilones(ciprofloxacin, levofloxacin, ofloxacin etc.), clindamycin, lincomycin, doxycycline
- 48. • Are frankly resistant to penicillins, cephalosporins, nalidixic acid & rifampicin • Most M. hominis strains are resistant to erythromycin & some are resistant to tetracycline & clindamycin
- 49. L-forms • Kleinberger (1935) found PPL forms in culture of Streptobacillus moniliformis • Named L forms (after Lister institute, London) • Many bacteria either spontaneously or induced (penicillins etc.), lost all or part of their cell wall & develop into L forms
- 50. Differences Between L-forms & Mycoplasmas • Unstable forms revert back • Stable cell wall deficient permanently but resemble parent bacteria both biochemically & antigenically • Remnants of cell wall can be demonstrated • Do not require sterol for growth • No filterable • Don’t initiate infection, play role in chronic infections during antibiotic therapy
- 51. HIV Associated Mycoplasmas • M. fermentans(incognitus strain), M. penetrans & M. pirum • Disease in both healthy & patients with AIDS • Act as OI & co-factors in pathogenesis • Specific activation of cellular immune system • Production superantigens • Generation of free radicals (cause oxidative stress) • Use glucose and hydrolyse arginine
- 52. X