11 6/4/2016 Mycoplasmas Hugh B Fackrell

22 6/4/2016 Presentation Outline l Structure l Classification l Multiplication l Clinical manifestations l Epidemiology l Diagnosis l Control

33 6/4/2016 Pleuropneumonia organism l The mycoplasmas are essentially bacteria lacking a rigid cell wall during their entire life cycle, although they are also much smaller than bacteria. The first organism of this type was associated with pleuropneumonia of cattle, and was originally called the pleuropneumonia organism (PPO).

44 6/4/2016 General Characteristics l smallest known free-living organisms. l Because of the absence of cell walls, they do not stain with the Gram stain, and they are more pleomorphic and plastic than eubacteria. l Giemsa stain –they appear as tiny pleomorphic cocci, short rods, short spirals, and sometimes as hollow ring forms. Their diameter ranges from 0.15 u to 0.30 u.

55 6/4/2016 Mycoplasma, Ureaplasma l very small (0.2 x 0.8 um) – pass through a 0.45 um filter l No Cell wall: plasma membrane only –resistant to antibiotics that interfere with the integrity of cell wall; penicillins, cephalosporins, vancomycin, bacitracin l susceptible to tetracycline, erythromycin

66 6/4/2016 Structure l The cell is enclosed by a limiting membrane which is more similar to that of animal cells than that of bacterial cells because of sterols present in the membrane. l The cytoplasm contains ribosomes,but lacks mesosomes. There is no nuclear membrane. l In some strains, amorphous material on the outer surface of the membrane suggests the existence of a capsule.

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88 Mycoplasma l requires sterols for growth, can be grown on laboratory media l most are facultatively anaerobic –Exception M. pneumoniae l replication controversial – replication time 1-6 hours

99 6/4/2016 Mycoplasma pneumoniae l AKA Eaton’s agent – aerobic but very slow growing l extracellular pathogen:attaches to respiratory epithelium by an attachment factor called P1 l interacts with a glycoprotein receptor on the epithelial cell surface l ciliostasis is followed by epithelial cell destruction

10 6/4/2016 Clinical Syndrome l Pneumonia –walking pneumonia frequently confused with virus infection –primary atypical –clinical l Tracheobronchitis l Pharyngitis –differential diagnosis from Strep throat

11 6/4/2016 Children most susceptible

12 6/4/2016 No Seasonal Incidence

13 6/4/2016 Urethritis l 1/2 of urethral infections not caused by Chlamydia or N. gonorrhoeae. l Caused by – Mycoplasma hominus – Ureaplasma

14 6/4/2016 Infection of Tracheal ring Organ culture

15 6/4/2016 Destruction of host l due to release of hydrogen peroxide and superoxide anion.

16 6/4/2016 Laboratory diagnosis l Culture: –fried egg colonies on medium containing sterols –Most mycoplasmas require a rich medium containing a sterol and serum proteins for growth. l Serology: – Complement Fixation test, Hemagglutination

17 6/4/2016 Laboratory Diagnosis l Culture Mycoplasma from sputum, mucous membrane swabbings or other specimens l direct inoculation into liquid or solid media containing serum, yeast extract and penicillin to inhibit contaminating bacteria.

18 6/4/2016 Cultural Characteristics l Despite the lack of a cell wall, they do not require a medium of very high osmotic pressure. l On solid media, they form minute, transparent colonies. –looks like a fried egg. The different strains vary in their growth rate l may take from two days to several weeks to form a colony.

19 6/4/2016 Fried Egg Colonies

20 6/4/2016 Fried Egg Colonies l Stain intensely with neutral red or tetrazolium or methylene blue.

21 6/4/2016 serology: complement fixation l on acute and convalescent serum. l patient’s serum heated to 56C to eliminate complement l combine patient’s serum and known Mycoplasma antigen in presence of added complement. Mix. l Incubate - add indicator system –Red cells and anti-red cell antibody –hemolysis occurs if complement is unused.

22 6/4/2016 Hemagglutination l Cold agglutinins to human O erythrocytes. l hemabsorption & B-hemolysis of guinea pig red blood cells.

23 6/4/2016 Identification l conclusively identified by staining its colonies with fluorescein-labelled antibody.

24 6/4/2016 M. pneumoniae Nucleic Acid Probes l specific recombinants to oligonucleotide sequences that are only found in Mycoplasma pneumoniae.

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26 6/4/2016 L Forms l Some bacteria readily give rise spontaneously to variants that can replicate in the form of small filterable protoplasmic elements with defective or absent cell walls. l These organisms, called L-forms, can also be formed by many species when cell wall synthesis is impaired by antibiotic treatment or high salt concentration.

27 6/4/2016 L Forms vs Mycoplasma l contain a rigid cell wall, at least at one stage of their life cycle l no sterols in their cytoplasmic membrane.

28 6/4/2016 Pleuropneumonia-like organisms l Several organisms with similar morphological characteristics and cultural properties have been isolated. These are commonly referred to as pleuropneumonia- like organisms or PPLO. A certain group of mycoplasmas produce extremely tiny colonies on agar plates, and are called the T- strains.

29 6/4/2016 Metabolism l The parasitic mycoplasmas have truncated respiratory systems, lacking quinones and cytochromes. l Another indication for the simplicity of the electron transport chain is the finding that the reduced nicotinamide adenine dinucleotide (NADH) oxidase activity is cytoplasmic.

30 6/4/2016 Arginine dihydrolase Pathway l pathway Complex electron transport chains are usually membrane bound, since they depend on the spatial organization of their components. Ruling out oxidative phosphorylation as an ATP-generating system leaves only two proven ways of ATP generation, both based on substrate level phosphorylation. The major source for ATP is the arginine dihydrolase pathway.

31 6/4/2016 Metabolism l A few species derive their energy from the degradation of glucose or the hydrolysis of urea. l All species synthesize DNA, RNA, lipids and proteins. l Not known if they can synthesize amino acids. l Those species that require sterols incorporate these sterols (mainly cholesterol) into the cell membrane up to concentrations of 65%.

32 6/4/2016 Multiplication l In the absence of a rigid cell wall, the pattern of replication is quite different from that of typical bacteria, whose division starts with the formation of a well-defined septum.

33 6/4/2016 Life Cycle of PPLO Elementary body

34 6/4/2016 Fragmentation of filaments l mechanism of division in mycoplasmas is controversial, sequential microscopic observation suggests that new elementary particles arise by fragmentation of filamentous cells containing several discrete DNA components.

35 6/4/2016 DONE!!!