Mycoplasmas Hugh B Fackrell 11242020 1 Presentation Outline

Mycoplasmas Hugh B Fackrell 11/24/2020 1

Presentation Outline Structure l Classification l Multiplication l Clinical manifestations l Epidemiology l Diagnosis l Control l 11/24/2020 2

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). 11/24/2020 3

General Characteristics 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 l – 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. 11/24/2020 4

Mycoplasma, Ureaplasma l very small (0. 2 x 0. 8 um) – pass through a 0. 45 um filter 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 l 11/24/2020 5

Structure 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. l 11/24/2020 6

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Mycoplasma requires sterols for growth, can be grown on laboratory media l most are facultatively anaerobic l – Exception M. pneumoniae l replication controversial – replication time 1 -6 hours 11/24/2020 8

Mycoplasma pneumoniae l AKA Eaton’s agent – aerobic but very slow growing extracellular pathogen: attaches to respiratory epithelium by an attachment factor called P 1 l interacts with a glycoprotein receptor on the epithelial cell surface l ciliostasis is followed by epithelial cell destruction l 11/24/2020 9

Clinical Syndrome l Pneumonia – walking pneumonia frequently confused with virus infection – primary atypical – clinical Tracheobronchitis l Pharyngitis l – differential diagnosis from Strep throat 11/24/2020 10

Children most susceptible 11/24/2020 11

No Seasonal Incidence 11/24/2020 12

Urethritis 1/2 of urethral infections not caused by Chlamydia or N. gonorrhoeae. l Caused by l – Mycoplasma hominus – Ureaplasma 11/24/2020 13

Infection of Tracheal ring Organ culture 11/24/2020 14

Destruction of host l due to release of hydrogen peroxide and superoxide anion. 11/24/2020 15

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 11/24/2020 16

Laboratory Diagnosis 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. l 11/24/2020 17

Cultural Characteristics 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. l – 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. 11/24/2020 18

Fried Egg Colonies 11/24/2020 19

Fried Egg Colonies l Stain intensely with neutral red or tetrazolium or methylene blue. 11/24/2020 20

serology: complement fixation on acute and convalescent serum. l patient’s serum heated to 56 C to eliminate complement l combine patient’s serum and known Mycoplasma antigen in presence of added complement. Mix. l Incubate - add indicator system l – Red cells and anti-red cell antibody – hemolysis occurs if complement is unused. 11/24/2020 21

Hemagglutination Cold agglutinins to human O erythrocytes. l hemabsorption & B-hemolysis of guinea pig red blood cells. l 11/24/2020 22

Identification l conclusively identified by staining its colonies with fluorescein-labelled antibody. 11/24/2020 23

M. pneumoniae Nucleic Acid Probes l specific recombinants to oligonucleotide sequences that are only found in Mycoplasma pneumoniae. 11/24/2020 24

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L Forms 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. l 11/24/2020 26

L Forms vs Mycoplasma contain a rigid cell wall, at least at one stage of their life cycle l no sterols in their cytoplasmic membrane. l 11/24/2020 27

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. 11/24/2020 28

Metabolism 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. l 11/24/2020 29

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 ATPgenerating 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. 11/24/2020 30

Metabolism 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%. l 11/24/2020 31

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. 11/24/2020 32

Life Cycle of PPLO Elementary body 11/24/2020 33

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. 11/24/2020 34

DONE!!! 11/24/2020 35