Mycoplasma

Mycoplasmas differ from other bacteria because of their small size and absence of a cell wall. In taxonomy, mycoplasmas are distinguished from other bacteria in the Mollicutes class by the absence of cell walls (mollis, soft; cutis, skin, in Latin). Mycoplasma mycoides ssp. mycoides, the first to be identified, was isolated from pleuropneumonia in cattle by Nocard and Roux in 1898. Pleuropneumonia-like organisms (PPLO) were named after a group of pathogenic and saprophytic isolates gathered from veterinary and human sources.

Classification of Mycoplasmas

Mycoplasmas belong to the Mollicutes class (mollis, soft; kutis, skin) and the Mycoplasmatales order. Mollicutes is divided into five families.

  • Family Mycoplasmataceae: It’s split into two genera: 1) Mycoplasma uses glucose or arginine as a source of energy, but does not break urea. There are more than 110 recognized species on the island. 2) Ureaplasma is a genus of bacteria that hydrolyzes urea. It is made up of six different species.
  • Family Acholeplasmataceae: There is just one genus in this family. Acholeplasma is a genus of bacteria that includes at least 14 species, the first of which being Acholeplasma laidlawii.
  • Family Spiroplasmataceae: It comprises the spiroplasma genus which infects both plants and arthropods.
  • Family Anaeroplasmataceae: Anaeroplasma is a genus of bacteria found in the rumen of cattle and sheep.
  • Family Entomoplasmataceae: It includes the Entomoplasma and Mesoplasma genera, which are found in insects and plants.

Mycoplasma causes illness in a wide range of mammalian, insect, and plant hosts. There are more than 60 species of Mycoplasma. Around 16 species are present in humans belonging to three groups.

Characteristics

Mycoplasmas are unique among bacteria in that they lack a solid cell wall and are therefore extremely pleomorphic, with no fixed shape or size. Many of the mycoplasmas’ unusual characteristics, such as sensitivity to osmotic stress and detergents, resistance to penicillin, and the development of peculiar fried-egg shaped colonies, are explained by their complete lack of a cell wall. A soft trilaminar unit membrane containing sterols surrounds these organisms. Furthermore cell wall precursors like muramic acid and diaminopimelic acid are missing.

Morphology

Mycoplasmas are the tiniest free-living microbes on the planet. They’re able to get through bacterial filters. They lack a cell wall but are surrounded by an 8–10 nm thick trilaminar membrane rich in cholesterol and other lipids. They are tiny pleomorphic cells that range in size from 0.2 to 0.8 mm in diameter and come in a variety of shapes and sizes, including spherical, coccoid, coccobacillary, ring, and dumbbell forms, as well as short and long branching, beaded, and segmented filaments. The filaments are long and thin with genuine branching. Mycoplasmas are gram-negative, however Giemsa stain works better on them. Binary fission is used for replication.

The majority of mycoplasmas are nonmotile and lack flagella. On liquid-covered surfaces, however, several flask-shaped Mycoplasma species, such as M. pneumoniae, M. genitalium, M. gallisepticum, M. pulmonis, and M. mobile, display gliding movement.

Culture Characteristics

Only a small percentage of the mycoplasmas found in nature have been grown thus far. Some cultivable mycoplasmas, such as M. pneumoniae, the human respiratory pathogen, grow slowly (2–3 weeks incubation at 370C), especially on first isolation. Others, such as U. urealyticum, grow rapidly in vitro but plateau at 106–107 colony-forming units (cfu) per milliliter, compared to 109–1010 cfu/ml in logarithmic cultures of well-growing mycoplasmas.

Although most mycoplasmas are facultative anaerobes, because organisms from primary tissue specimens usually grow exclusively under anaerobic circumstances, a primary isolation environment of 95 percent nitrogen and 5% carbon dioxide is recommended. They thrive in a temperature range of 22–41°C, with parasitic species flourishing at 35–37 °C and saprophytes flourishing at lower temperatures.

Mycoplasma culture media are supplemented with 20% horse or human serum and yeast extract. As a supply of cholesterol and other lipids, serum with a high content is required. Mycoplasmas may be grown in both liquid and solid mediums. These bacteria can thrive on artificial medium, but they need nucleic acid precursors and other components, which blood can provide. The most ideal medium for growing M. pneumoniae and M. hominis are probably SP4 glucose broth and agar, but arginine must be added to isolate the latter bacterium.

With the inclusion of A8 as the solid medium, Shepard’s 10B urea broth (pH 6.0) may be utilized to separate M. hominis and Urealyticum. There are also commercially available biphasic systems that use a broth and agar mix. The broths should be kept at 37°C and the agar plates should be kept at 5–10% CO2.

Colonies on agar are generally biphasic, with an opaque center zone of growth within the agar and a transparent outer zone on the top, giving them a fried egg look. With platinum loops, colonies can’t be selected. Cutting an agar block with colonies and rubbing it on new plates is how subculture is done. Mycoplasma colonies that are hemolytic are the most common.

Pathogenicity

Infections of the urogenital tract are linked to M. hominis and U. urealyticum. However, because these organisms can be isolated from the majority of asymptomatic, sexually active people, their involvement in illness remains a matter of debate. The majority of human illnesses do not develop to pneumonia. When pneumonia strikes, it usually develops gradually and presents as a mild to moderately severe sickness, with early symptoms relating to the lower respiratory tract.

M. pneumoniae infection usually results in moderate upper respiratory tract infection. Less than 10% of individuals have a more serious illness with symptoms in the lower respiratory tract. Although M. pneumoniae is well recognized for causing “walking or atypical pneumonia,” the most common clinical condition is tracheobronchitis or bronchiolitis, which is frequently accompanied by upper respiratory tract symptoms. Hoarseness, fever, cough that is initially nonproductive but subsequently produces small to moderate quantities of non-bloody sputum, sore throat, headache, chills, coryza, and overall malaise are common problems that can last weeks to months.

M. Urealyticum and M. hominis are the bacteria that cause genital infections. They can cause urethritis, proctitis, balanoposthitis, and Reiter’s syndrome in men and acute salphingitis, pelvic inflammatory disease, cervicitis, and vaginitis in women. They’ve also been linked to infertility, abortion, postpartum fever, chorioamnionitis, and babies with low birth weight.

Adhesion to host cells

Most mycoplasmas in humans and animals cling to the epithelial linings of the respiratory and urogenital tracts, seldom penetrating tissues. As a result, they might be classified as surface parasites. Mollicutes must adhere to host cells in order for the parasite to colonize and infect them. Infectivity is lost when adhesion ability is lost due to mutation, and reversion to the cytadhering phenotype is followed with a return of infectivity and virulence.

Molecular basis of mycoplasma pathogenicity

Mycoplasmas have not been linked to potent toxins. Hydrogen peroxide and superoxide radicals, which are moderately hazardous byproducts of Mycoplasma metabolism, have been implicated in oxidative damage to host cell membranes.

Laboratory Diagnosis

The traditional bacteriological techniques, such as morphology, cultural features, physiological, and serological properties, have been used to identify mycoplasmas and diagnose mycoplasmal infections in the laboratory. While these tests continue to play an important role in mycoplasma diagnosis, novel tests based on molecular analysis of genomic DNA, ribosomal RNAs, cell proteins, and lipids appear to be pushing them aside, and it is possible that molecular tests may soon overtake the classic assays.

Specimens

Throat swabs, nasopharyngeal swabs, sputum, throat washings, bronchoalveolar lavage, tracheal aspirate, and lung tissue specimens can all be used to diagnose M. pneumoniae.

Culture

If inoculation is not possible right away, the material can be kept at 4°C for up to 24 hours in the laboratory. If there will be a delay of more than 24 hours, the material should be frozen at – 70°C. Because mycoplasmas do not generate turbidity in broth media, a commonly used isolation medium comprises bovine heart infusion (PPLO broth) with fresh yeast extract and horse serum supplemented with penicillin (to kill other bacteria), thallium acetate, glucose, and phenol red as a pH indicator.

The ability of M. pneumoniae to generate acid from glucose in clinical specimens is used to identify the organism’s growth. Methylene blue-glucose diphasic medium is one of the most used broth media. With the lids tightened, broth cultures are incubated at 35°C. Color changes (from salmon to yellow) in the medium are checked daily in the tubes. A true-positive culture is identified by a small, progressive change in the pH indicator over 8–15 days without significant turbidity. As soon as color changes in the media become visible, the broth must be subcultured to suitable agar medium. The organisms will appear as tiny colonies on the agar surface when examined under a low power microscope.

Mycoplasma hominis on A7 agar. As a result of the organisms developing into the agar at the center of the colony and superficially on the periphery, M. hominis colonies exhibit a distinctive “fried egg” look. The colonies appear in 1 to 5 days and have a diameter of 50 to 300 meters. The arginine positive of M. hominis and its characteristic colony shape are typically enough to identify it. Specific antisera can be used to limit the organism’s growth for definitive identification.

Colonies: M. pneumoniae colonies can take up to 21 days to grow, but M. hominis colonies can form in as little as 2–4 days. M. pneumoniae colonies are tiny, betahemolytic, and have an uniform granular appearance (“mulberry shaped”), as opposed to other mycoplasmas fried-egg morphology. Mycoplasma-like colonies are stained with Dienes or methylene blue stains since they do not stain well with gram or acridine orange stains. M. hominis colonies give the appearance of a big “fried egg.”

Other serological tests

A wide range of serological tests have been used, notably in the identification of mycoplasma species and strains. The traditional suggested tests include antisera that impede growth and metabolism, as well as direct and indirect immunofluorescence assays on mycoplasma colonies.

Complement fixation, metabolic inhibition, inhibition of tetrazolium reduction, immunofluorescence on sections of chick embryo lung, direct or antibody capture enzyme- immunosorbent assays (EIA), or agglutination of antigen-coated erythrocytes, latex, or glycerol can all be used to measure the development of antibody to M. pneumoniae by infected subjects.

Polymerase Chain Reaction (PCR) Amplification

Polymerase chain reaction (PCR) amplification of a specific sequence in M. pneumoniae genome is used to detect it in respiratory exudates or secretions.

Treatment

The medicines of choice for treating Mycoplasma and Ureaplasma infections include tetracyclines and erythromycin. Patients with NGU should be treated with one of the tetracyclines, and ureaplasmasresistant to tetracyclines should be treated with erythromycin, which is effective against the majority of tetracycline-resistant ureaplasmas.

Mycoplasmas and L forms of bacteria

Kleineberger (1935) discovered pleuropneumonia-like forms in Streptobacillus moniliformis cultures and named them L forms after the Lister Institute in London, where the discovery was made. It was later discovered that many bacteria lose part or all of their cell wall and evolve into L forms, either naturally or as a result of specific drugs like penicillin. Such L forms can be either ‘unstable’ (reverting to their normal morphology) or ‘stable’ (remaining in the cell wall deficient condition indefinitely).

Differenecs between L-forms and Mycoplasma

Mycoplasmas may constitute stable L forms of bacteria, according to certain theories. L-forms, like Mycoplasma, generate fried egg colonies, but they differ in the following ways:

  • L-forms cannot be filtered and don’t require sterols for their growth.
  • Even though L-forms do not have cell walls, traces of cell wall components may be seen in them.
  • The stable L-forms remain cell wall-deficient forever, but are biochemically and antigenically similar to progenitor bacteria.
  • L-forms play an important role in persistence of chronic infection during antibiotic therapy and subsequent recurrence of the infection but may not initiate disease.

References

  1. Topley Wilson’s Microbiology
  2. Clinical Infectious diseases
  3. Essentials of Microbiology
  4. Color Atlas of Microbiology
  5. Diagnostic Microbiology

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