Mycoplasma pneumoniae : Epidemiology , Pathogenesis , Laboratory Diagnosis and Treatment

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mycoplasma pneumoniae

Mycoplasma pneumoniae is the most common cause of upper respiratory tract disease. It has also been linked to walking pneumonia and primary atypical pneumonia.

Epidemiology of Mycoplasma pneumoniae

M. pneumoniae infection is found all over the world. The organism is responsible for up to 20% of community-acquired pneumonia cases that necessitate hospitalization. M. pneumoniae infection can be both epidemic and endemic. The condition is well-documented in both Europe and the United States. The developing world does not have precise information on M. pneumoniae infection. However, the findings of a few seroprevalence studies suggest that the disease may be endemic in many developing countries.

Habitat of M. pneumoniae

  • It only affects humans
  • It is usually found in the mucosa but can also be found in the upper respiratory tract of infected host.

Reservoir, host, and transmission of infection

  • Humans are the most common host of M. pneumoniae and thus serve as a significant reservoir of infection.
  • M. pneumoniae infection are contagious.
  • The pathogen is most commonly transmitted through nasal secretions through close contact.
  • The infection is spread through the inhalation of aerosolized droplets.
  • Person-to-person transmission is most common among college students and military recruits who live in close quarters.
  • M. pneumoniae is commonly associated with pneumonia, and the highest rate of infection is found in children aged 9 to 10 years old, as well as in young adults. Infection is common in school-aged children.

In recent years, the infection has become more common in people over the age of 65. M. pneumoniae is the second pathogen after Streptococcus pneumoniae as a cause of pneumonia in this elderly population, accounting for nearly 15% of community-acquired pneumonia.

Properties of Mycoplasma pneumoniae

Morphology

Mycoplasmas have the following morphological characteristics:

  • Mycoplasmas are very small bacteria that measure 150–250 nm in size. They lack a cell wall and typically have sterols in their cell membranes.
  • Because many mycoplasmas can pass through a 0.45 m filter, they were once thought to be viruses.
  • Because they lack a cell wall, Mycoplasma species were also thought to be L bacteria.
  • Mycoplasma species are characterized by pleomorphism and can be found as granular or filamentous structures of varying sizes. The filaments are long and slender, with true branching.
  • They multiply through binary fission. However, genomic replication and cell division are frequently asynchronous, resulting in the formation of multinucleate fragments and other body forms, as well as chains of beads.
  • Although they lack flagella and pili, some Mycoplasma species, including M. pneumoniae, exhibit gliding motility on liquid-covered surfaces.
  • Gram stain stains Mycoplasma organisms poorly and they are Gram negative. Giemsa and Diene stain work better on them.
Mycoplasma pneumoniae
Mycoplasma pneumoniae

Culture

  • Mycoplasmas are facultative anaerobes and aerobic bacteria.
  • M. pneumoniae, is strictly aerobe, is an exception.
  • They thrive at 37°C and pH levels ranging from 7.3 to 7.8.

A.   PPLO broth

Pleuropneumonia-like organism (PPLO) broth is a popular medium for mycoplasma isolation. This medium is fortified with 20% horse serum, 10% yeast extract, and glucose. As a pH indicator, phenol red is used. A high concentration of animal serum (horse serum) is used as an exogenous sterol source (cholesterol and other lipids). The addition of agar solidifies the medium. To inhibit the growth of contaminating bacteria, the medium is supplemented with penicillin, ampicillin, and polymyxin B, and amphotericin B is used to inhibit fungi contamination.

B.   PPLO agar

Agar is used to solidify the PPLO broth. Mycoplasmas are typically slow growers, with generation times ranging from 1–6 hours.

Mycoplasmas on PPLO agar form small colonies with a fried-egg appearance, with a central, opaque, granular area of growth surrounded by a flat, translucent, peripheral area. Initially, mycoplasmas multiply within the agar to form opaque, ball-shaped colonies that grow to the agar’s surface and then spread around it, forming a translucent peripheral zone. Colonies can be seen with a hand lens and are best studied after Diene’s staining.

The fried-egg appearance colonies of mycoplasma appear highly granular in this staining, with the center of the colonies stained dark blue and the periphery of the colonies stained light blue. In the medium, the agar appears clear or slightly violet. Mycoplasmas other than M. pneumoniae become colorless over time as the methylene blue is reduced.

M. pneumoniae, unlike other Mycoplasma species, grows slowly. They need 1–4 weeks to form colonies on agar.

M. pneumoniae does not produce fried-egg-like colonies, but rather mulberry-shaped colonies. Unlike fried-egg appearance colonies, these colonies do not have any thin hallow.

On blood agar, the majority of Mycoplasma colonies produce a zone of hemolysis. Mycoplasmas lack the ability to synthesize cholesterol and related sterols, so these must be obtained from outside sources in order for mycoplasmas to grow. They are also incapable of synthesizing purines and pyrimidines.

Biochemical reactions of Mycoplasma pneumoniae

Mycoplasmas exhibit the biochemical reactions listed below:

  • M. pneumoniae and other species (M. fermentans, M. genitalia, and M. agalactiae) rely heavily on glucose and other carbohydrates for energy.
  • M. salivarium and other species (M. orale, M. hominis, and M. fermentans) rely heavily on arginine for energy.
  • Mycoplasma are chemo-organotrophs with primarily fermentative metabolism.

The liquid culture medium used for the fermentation reaction contains glucose, arginine, and urea, as well as phenol red as an indicator. Mycoplasma species that ferment carbohydrates use glucose to produce lactic acid, resulting in an acidic pH. By metabolizing arginine, Mycoplasma species produce ammonia, CO2, and adenosine triphosphate. The production of ammonia causes the medium to become alkaline.

Physical and chemical susceptibility

Mycoplasmas are easily killed by heating at 56°C for 30 minutes. Antiseptic solutions, such as cycloheximide and cetrimide, inhibit the growth of the bacteria. Because they are resistant to UV light and the photodynamic action of methylene blue, M. pneumoniae can grow in agar with 0.002 percent methylene blue, whereas many other Mycoplasma species are inhibited at this concentration.

Cell Wall Components and Antigenic Structure

The main antigenic determinants of mycoplasmas are membrane glycolipids and proteins. Membrane glycolipid antigens react with human tissues as well as other bacteria. Complement fixation tests are used to identify these antigens.

Glycolipids with similar antigenic structures have been found in human brain neurons. Antibodies against M. pneumoniae glycolipid may cross-react with brain cells, causing neuronal cell damage. This cell damage may be to blame for the neurological manifestations of M. pneumoniae infection.

M. pneumoniae has two major surface proteins, one of which is the adhesion protein P1, which is responsible for bacterial attachment to cell structures. The enzyme-linked immunosorbent assay is used to identify these protein antigens (ELISA). The P1 protein stimulates the production of antibodies, which not only react with the P1 protein but also with antigenic determinants of RBCs, resulting in erythrocyte lysis in the autoimmune disease process.

Pathogenesis and Immunity of Mycoplasma pneumoniae

Mycoplasmas are extracellular pathogens that attach to the surface of ciliated and nonciliated epithelial cells.

Virulence factors

The adhesion protein P1 is the Mycoplasma’s main virulence factor. Bacteria do not usually infiltrate the bloodstream, resulting in systemic disease manifestation.

P1 antigen:

P1 antigen is a membrane-associated protein that aids mycoplasma adhesion to epithelial cells. This protein or adhesin binds to sialated glycoprotein receptors found at the base of cilia on the epithelial surface. This receptor is also found on the surface of erythrocytes. Antibodies to the P1 antigen can also act as an autoantibody against RBCs, causing them to clump together.

Pathogenesis of Mycoplasma infections

Mycoplasma causes direct damage to epithelial cells after attachment, destroying cilia and then ciliated epithelial cells. The loss of the cells disrupts the normal function of the upper respiratory tract. The lower respiratory tract becomes infected with microbes and mechanically irritated as a result. Mechanical irritation causes a persistent cough, which is common in patients with M. pneumoniae respiratory infections.

M. pneumoniae functions as a superantigen. This causes inflammatory cells to migrate to the site of infection and produces cytokines like tumor necrosis factor-alpha, interleukin-1, and interleukin-6. These cytokines aid in the elimination of bacteria and disease. Bacteria do not usually infiltrate the bloodstream, resulting in systemic disease manifestation.

Except in rare cases of immunosuppression or after instrumentation, Mycoplasma rarely penetrates the submucosa. They may infiltrate the bloodstream and cause infection in various organs of the body under these conditions.

Host immunity

Infection with M. pneumoniae does not result in protective immunity. Individuals who have been infected with M. pneumoniae are vulnerable to reinfection. Antibodies are produced in response to the P1 antigen. These antibodies are found in nearly half of M. pneumoniae-infected patients. The antibody against P1 antigen is an autoantibody that reacts with RBC antigen I and is therefore not protective.

Clinical Syndromes

M. pneumoniae is primarily responsible for respiratory infections in humans.

Respiratory infections

Upper respiratory tract infections: M. pneumoniae is commonly responsible for minor upper respiratory tract infections. The symptoms include a low-grade fever, malaise, and headache.

A nonproductive cough is a common symptom that appears 2–3 weeks after exposure. The cough is initially ineffective, but it may eventually produce small to moderate amounts of sputum, which may become mucopurulent and even blood tinged in more severe cases.

Lower respiratory tract infections include tracheobronchitis and bronchopneumonia. Primary bronchial infection with lymphocyte and plasma cell infiltration of bronchial epithelial cells characterizes the condition.

Walking pneumonia is another name for primary atypical pneumonia. The incubation period ranges between 2 and 3 weeks. Patients with atypical pneumonia usually do not appear to be ill. As a result, the illness is frequently referred to as “walking pneumonia.” The pharynx is affected and edematous, but there is no cervical adenopathy. The presence of patchy bronchopneumonia on a chest X-ray is associated with the condition. Infection is distinguished by a disparity between physical findings and radiological evidence of the chest. In most cases, the infection is self-limiting. Pleural effusion can occur in 5–20% of patients.

Extrapulmonary infections

Extrapulmonary manifestations aren’t uncommon. The most commonly reported extrapulmonary manifestations are cardiac abnormalities such as myocarditis and pericarditis. Neurological abnormalities, otitis media, and erythema multiforme (Stevens–Johnson syndrome) are some of the other manifestations. M. pneumoniae infections are much more likely to cause severe disease in:

  • children with immunosuppressive diseases;
  • children who have Down syndrome
  • individuals suffering from sickle cell anemia and functional asplenia
  • Subclinical infection can occur in up to 20% of adults.

Laboratory Diagnosis of Mycoplasma pneumoniae

The clinical diagnosis of M. pneumoniae infection is used to guide initial treatment. It usually takes 3–4 weeks to get a definitive diagnosis of the condition. As a result, treatment begins without waiting for the results of laboratory tests.

Specimens

  • Throat washings, bronchial washings, and expectorated sputum are examples of respiratory specimens.
  • Tracheal washings are more useful than sputum specimens because most patients with respiratory tract infections produce no sputum due to a dry and ineffective cough.
  • The specimens are immediately collected and transported to the laboratory.

If a delay is expected, they are usually inoculated in appropriate transport media, such as SP4 transport medium, to prevent desiccation. If the specimens cannot be sent to the laboratory immediately after collection, they can be stored at 70°C.

Microscopy

Microscopy is useless in the diagnosis of M. pneumoniae infections. Because mycoplasmas lack a cell wall, they stain poorly.

Direct antigen detection

With high sensitivity and specificity, antigen capture immunoassays have been used to detect M. pneumoniae in sputum specimens. Antigen detection in clinical specimens is accomplished using direct immunofluorescence, counter-current immunoelectrophoresis, and immunoblotting with monoclonal antibodies.

Culture

Bacterial culture has little practical value due to its fastidious growth requirements and 3–4 week culture time. M. pneumoniae isolation by culture from clinical specimens confirms the diagnosis of M. pneumoniae respiratory illness. The specimens are inoculated into mycoplasma medium, which includes PPLO agar supplemented with serum (a source of sterols), glucose, a pH indicator, yeast extract (a source of nucleic acid precursor), and antibiotics and antifungal agents (to inhibit bacteria and fungi). M. pneumoniae grows slowly on this medium, with colonies visible at 37°C. Incubation in 95% N2 and 5% CO2 promotes growth. The bacteria form small, homogeneous colonies that are commonly referred to as mulberry-shaped colonies.

Identification of Mycoplasma pneumoniae

The list below summarizes the distinguishing characteristics of M. pneumoniae colonies.

  • Very small bacteria that stain poorly with Gram stain but well with Diene stain.
  • A change in the color of phenol red from red to yellow.
  • Colonies stained directly with Diene stain
  • Colonies that passed the hemadsorption test.
  • Colonies that passed the tetrazolium reduction test.
  • The presence of the colonies is confirmed by inhibiting their growth with specific M. pneumoniae antisera.
  • Mycoplasmas produce colonies with a “fried egg” appearance on agar, with an opaque central zone of growth within the agar and a translucent peripheral zone on the surface.

The following tests are used to identify them.

Color change: Colonies are identified by observing a change in the color of phenol red from red to yellow caused by the fermentation of glucose, which results in the production of acid, which lowers the pH of the media.

Diene test: The Diene stain (diluted 1:10 with distilled water) is applied directly to the plate containing suspected Mycoplasma colonies in this method. To remove the stain, the plate is immediately rinsed with distilled water. The medium is then decolored by adding 1 mL of 95 percent ethanol and leaving it for 1 minute. The plate is rewashed with distilled water and dried. After drying, the colonies are examined under a low-power microscope.

The fried-egg appearance colonies of Mycoplasma appear highly granular when stained with Diene, with the center of the colonies stained dark blue and the periphery of the colonies stained light blue. In the medium, the agar appears clear or slightly violet. Mycoplasmas other than M. pneumoniae become colorless over time as the methylene blue is reduced. The presence of colonies is confirmed by inhibiting their growth with specific M. pneumoniae antisera.

Hemadsorption test: M. pneumoniae colonies grown on surface agar are flooded with 2 mL of 0.2–0.4 percent suspension of guinea pig erythrocytes in Mycoplasma growth medium. The plate is incubated at 35°C for 35 minutes before being washed with 3 mL of Mycoplasma growth medium and gently rotated. Aspiration with a pipette gently removes the washing fluid. Colonies of M. pneumoniae adsorb guinea pig erythrocytes, which is aided by 37°C. Under the microscope, M. pneumoniae colonies adsorb erythrocytes on their surface. The colonies are visible at a magnification of 40.

The tetrazolium reduction test is based on the principle that M. pneumoniae has the ability to convert triphenyl tetrazolium, a colorless compound, to formazan, a red compound. This test involves flooding M. pneumoniae colonies on agar with a solution of 2-p-iodophenyl3-nitrophenyl-5-phenyltetrazolium chloride and incubating them at 35°C for an hour. M. pneumoniae colonies appear reddish after 1 hour and may turn purple to black after 3–4 hours of incubation in a positive test.

Serodiagnosis

Serodiagnosis is based on the presence of specific antibodies in serum in response to Mycoplasma antigens. M. pneumoniae glycolipid antigen extracted with chloroform and methanol is also widely used for atypical pneumonia serodiagnosis. The most commonly used tests are the complement fixation test and ELISA.

Complement fixation Test: A fourfold increase in complement fixing antibody titer or a single titer of 1:64 or higher indicates a recent infection. Complement-fixing antibodies appear 7–10 days after infection and reach a peak after 4–6 weeks. Complement-fixing antibodies are present in approximately 80% of the cases.

Enzyme-linked immunosorbent assay: IgM ELISA is the most frequently used enzyme-linked immunosorbent assay. IgM ELISA is a technique for detecting specific IgM antibodies in a single serum specimen. This test has a 99 percent specificity and a 97 percent sensitivity. A quantitative, rapid, single-specimen membrane-based ELISA has recently been evaluated as a rapid diagnostic method for demonstrating IgM or IgG antibodies. The results of this test can be obtained within 30 minutes of the test being performed.

Nonspecific serological tests are so-called because they do not use specific Mycoplasma antigens, but rather cross-reacting and nonspecific antigens. The Streptococcus MG agglutination test and the cold agglutination test are two of these tests.

  • Streptococcus MG agglutination test: A heatkilled suspension of Streptococcus MG is used as antigen in this test. The antigen is combined with a series of dilutions of the patient’s unheated serum. After an overnight incubation at 37°C, agglutination is observed. An antibody titer of 1:20 or higher indicates M. pneumoniae infection.
  • Cold agglutination test: Human O group erythrocytes are used as antigen in this test. This is based on the fact that autoantibodies that agglutinate human O group cells at low temperatures are found in the majority of cases of atypical pneumonia. The test is carried out by drawing blood from the patient, which should never be refrigerated before serum separation because agglutinins are readily absorbed by homologous RBCs at low temperatures. The serum of the patient is mixed with an equal volume of 0.2 percent washed suspension of human O erythrocytes. The suspension is incubated at 4°C overnight and clumping is observed. At 37°C, the clumping dissociates. A titer of 1:32 or higher indicates M. pneumoniae infection.

Cold agglutinins typically appear in more than half of cases by the second week of infection and peak at 4–5 weeks. After that, the antibody titer drops rapidly, and the test becomes negative in about 5 months.

A fourfold increase in the cold agglutinin titer of the paired serum and convalescent sera, or a single titer of 1:32, indicates M. pneumoniae infection. The main disadvantage of this test is that it is nonspecific, as cold agglutinins are found in sera from other diseases such as rubella, infectious mononucleosis, adenovirus infections, psittacosis, tropical eosinophilia, trypanosomiasis, cirrhosis of the liver, and hemolytic anemia.

Treatment

Antibiotic therapy is usually not required to treat an upper respiratory tract infection caused by M. pneumoniae. However, antibiotic treatment may be beneficial in the management of Mycoplasma pneumonia because it shortens the duration of illness and reduces the number of Mycoplasma in clinical specimens. It also alleviates symptoms, improves pneumonia resolution, and speeds recovery from the disease. Pneumonia is usually a self-limiting disease that does not endanger the patient’s life.

M. pneumoniae is still susceptible to tetracyclines and erythromycin, which act on mycoplasmas by inhibiting protein synthesis. Tetracycline has the added benefit of being active against the majority of other mycoplasmas and chlamydiae, which are the most common causes of nongonococcal urethritis.

Penicillins and cephalosporins are resistant to mycoplasma organisms because these antibiotics act on the cell wall, which mycoplasmas lack.

Prevention and Control

Isolating patients infected with M. pneumoniae is the most effective way to keep the disease from spreading. Tetracycline or erythromycin prophylaxis is also beneficial. There is no vaccine available to protect against Mycoplasma infections.

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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