M. pneumoniae is the most important species causing upperrespiratory tract disease. It is also known for causing walking pneumonia or primary atypical pneumonia.
Mycoplasmas show the following morphological features:
Mycoplasmas are very small bacteria measuring 150–250 nm in dimension. They do not have a cell wall and typically their cell membranes contain sterols.
Many mycoplasma can pass through 0.45 mm filter, hence were once believed to be viruses. However, they differ from viruses in the following properties:
· They contain both ribonucleic acid (RNA) and deoxyribo-nucleic acid (DNA).
· They are able to grow on cell-free media in vitro.
· They show both intracellular and extracellular parasitism in vivo.
Mycoplasma species was also considered to be L form of bac-teria because they lack a cell wall. However, they differ from bacteria including L forms in the following properties:
· They have sterols in the cell membrane.
· They do not show any reversion to structure with cell walls.
· They do not share any DNA homology with other bacteria.
· They have genome with a low molecular weight.
· They have low guanine and cytosine contents.
· The absence of cell wall makes Mycoplasma resistant to penicillins, cephalosporins, vancomycin, and other anti-biotics that interfere with the synthesis of the cell wall.
Mycoplasma species typically show pleomorphism and occuras granular and filaments of various sizes. The filaments are slender of varying length and show true branching.
They multiply by binary fission. However, genomic repli-cation and cell division are often asynchronous, resulting in production of multinucleate fragments and other body forms and chains of beads.
They do not possess flagella or pili, but some Mycoplasma species including M. pneumoniae show gliding motility on liquid-covered surfaces.
Mycoplasma organisms stain poorly by Gram stain and areGram negative. They are better stained by Giemsa and Diene stain.
Mycoplasmas are aerobic and facultative anaerobes. M. pneu-moniae is an exception, which is a strict aerobe. They grow at37°C and at pH range of 7.3–7.8.
1. PPLO broth: Pleuropneumonia-like organism (PPLO)broth is a medium widely used for isolation of myco-plasma. This medium is supplemented with 20% horse serum, 10% yeast extract, and glucose. Phenol red is used as a pH indicator. The high concentration of animal serum (horse serum) is used as a source of exogenous sterols (cholesterol and other lipids). Addition of agar makes the medium solid. The medium is supplemented with penicil-lin, ampicillin, and polymyxin B to inhibit growth of con-taminating bacteria, and amphotericin B is used to inhibit contamination with fungi.
2. PPLO agar: The PPLO broth is solidified by addition ofagar. The mycoplasmas are typically slow growers with a generation time of 1–6 hours.
Mycoplasmas on PPLO agar produce small colonies, typically described as a fried-egg appearance, which consists of a central, opaque, granular area of growth surrounded by a flat, trans-lucent, peripheral area. Initially, the mycoplasmas multiply within the agar to produce opaque, ball-shaped colonies that grow up to the surface of the agar and then spread around it, forming a translucent peripheral zone. Colonies may be seen with a hand lens, which is best studied after staining by Diene’s method.
By this staining, the fried-egg appearance colonies of myco-plasma appear highly granular, with the center of the colonies stained dark blue and the periphery of the colonies staining light blue. The agar in the medium appears clear or a slightly violet. Mycoplasma other than M. pneumoniae becomes colorless after a period of time, because it reduces the methylene blue.
· M. pneumoniae unlike other Mycoplasma species are very slow-growing bacteria. They require 1–4 weeks to produce colonies on agar.
· M. pneumoniae does not produce fried-egg appearancecolonies but instead produces a colony known as mulberry-shaped colonies. These colonies do not show any thin hallow unlike that of fried-egg appearance colonies.
Most Mycoplasma colonies produce a zone of hemolysis on blood agar. Mycoplasmas do not have the capability to synthe-size cholesterol and related sterols; hence these are supplied from outside for growth of mycoplasmas. They also lack the ability to synthesize purines and pyrimidines.
Mycoplasmas show the following biochemical reactions:
· M. pneumoniae and other species (M. fermentans, M. genitalia,and M. agalactiae) utilize glucose and other carbohydrates as the major source of energy.
· M. salivarium and other species (M. orale, M. hominis, and
M. fermentans) utilize arginine as a major source of energy.
· Mycoplasma are chemo-organotrophs, the metabolism being mainly fermentative.
The liquid culture medium used for fermentation reaction is supplemented with glucose, arginine, and urea, and phenol red as an indicator. Mycoplasma species fermenting carbohydrate utilize glucose to produce lactic acid, resulting in formation of acidic pH. Arginine-utilizing Mycoplasma species produce ammonia, CO2, and adenosine triphosphate by metabolism of arginine. Ammonia production leads to change of the medium to alkaline.
Susceptibility to physical and chemical agents: Myco-plasmas are readily killed by heating at 56°C for 30 minutes. The bacteria are sensitive to antiseptic solutions, such as cyclo-heximide and cetrimide, which inhibit their growth. They are resistant to UV light and photodynamic action of methylene blue, hence M. pneumoniae can grow in presence of 0.002% of methylene blue in agar, while many other Mycoplasma species are inhibited at this concentration.