Chapter 25: Mycoplasmas and Cell Wall–Defective Bacteria

There are more than 200 known species in the class of Mollicutes (cell wall–free bacteria). At least 16 of these species are thought to be of human origin; others have been isolated from animals and plants. In humans, four species are of primary importance: Mycoplasma pneumoniae causes pneumonia and has been associated with joint and other infections. Mycoplasma hominis sometimes causes postpartum fever and has been found with other bacteria in uterine tube infections. Ureaplasma urealyticum is a cause of nongonococcal urethritis in men and is associated with lung disease in premature infants of low birth weight. Mycoplasma genitalium is closely related to M pneumoniae and has been associated with urethral and other urogenital infections. Other members of the genus Mycoplasma are pathogens of the respiratory and urogenital tracts and joints of humans and animals.

The smallest genome of known mycoplasmas, M genitalium, is little more than twice the genome size of certain large viruses. Mycoplasmas are the smallest organisms that can be free living in nature and self-replicating on laboratory media. They have the following characteristics: (1) the smallest mycoplasmas are 125–250 nm in size; (2) they are highly pleomorphic because they lack a rigid cell wall and instead are bounded by a triple-layered “unit membrane” that contains a sterol (mycoplasmas require the addition of serum or cholesterol to the medium to produce sterols for growth); (3) mycoplasmas are completely resistant to penicillin because they lack the cell wall structures at which penicillin acts, but they are inhibited by tetracycline or erythromycin; (4) mycoplasmas can reproduce in cell-free media; on agar, the center of the whole colony is characteristically embedded beneath the surface; (5) growth of mycoplasmas is inhibited by specific antibody; and (6) mycoplasmas have an affinity for mammalian cell membranes.

Morphology and Identification

A. Typical Organisms

Mycoplasmas cannot be studied by the usual bacteriologic methods because of the small size of their colonies and the plasticity and delicacy of their individual cells. Growth in fluid media gives rise to many different forms. Growth on solid media consists principally of protoplasmic masses of indefinite shape that are easily distorted. These structures vary greatly in size, ranging from 50 to 300 nm in diameter. The morphology appears different according to the method of examination (eg, dark field, immunofluorescence, Giemsa-stained films from solid or liquid media, and agar fixation).

B. Culture

Culture of mycoplasmas that cause disease in humans requires media with serum, a metabolic substrate such as glucose or urea, and growth factors such as yeast extract. There is no one medium that is optimal for all the species because of different properties and substrate requirements. After incubation at 37°C for 48–96 hours, there may be no turbidity in broth cultures; however, Giemsa stains of the centrifuged sediment show the characteristic pleomorphic structures, and subculture on appropriate solid media yields minute colonies.

After 2–6 days on biphasic (broth over agar) and agar medium incubated in a Petri dish that has been sealed to prevent evaporation, isolated colonies of the more rapidly growing mycoplasmas measuring 20–500 μm can be detected with a hand lens. These colonies are round, with a granular surface and a dark center typically buried in the agar. They can be subcultured by cutting out a small square of agar containing one or more colonies and streaking this material on a fresh plate or dropping it into liquid medium.

C. Growth Characteristics

Mycoplasmas are unique in microbiology because of (1) their lack of a call wall; (2) their extremely small size; and (3) their growth on complex but cell-free media.

Mycoplasmas pass through filters with 450-nm pore size and thus are comparable to chlamydiae or large viruses. However, parasitic mycoplasmas grow on cell-free media that contain lipoprotein and sterol. This sterol requirement for growth and membrane synthesis is unique.

Many mycoplasmas use glucose as a source of energy; ureaplasmas require urea.

Some human mycoplasmas produce peroxides and hemolyze red blood cells. In cell cultures and in vivo, mycoplasmas are observed predominantly at cell surfaces. Many established animal and human cell culture lines carry mycoplasmas as contaminants; often the mycoplasmas are intracellular as well.

D. Variation

The extreme pleomorphism of mycoplasmas is one of their principal characteristics.

Antigenic Structure

At least 16 antigenically distinct species can be identified from humans, including M hominis, M pneumoniae, M genitalium, and U urealyticum. Most Mycoplasma species have high evolved systems for variation of outer membrane antigens presumably for evading the host immune response during infection.

The species are classified by biochemical and serologic features. The complement fixation (CF) antigens of mycoplasmas are glycolipids. Antigens for enzyme-linked immunoassay (ELISA) tests are proteins. Some species have more than one serotype.

Pathogenesis

Many pathogenic mycoplasmas have flasklike or filamentous shapes and have specialized polar tip structures that mediate adherence to host cells. These structures are a complex group of interactive proteins, adhesins (eg, the P1 adhesin of M pneumoniae and the MgPa adhesin of M genitalium), and adherence-accessory proteins. The proteins are proline rich, which influence the protein folding and binding and are important in the adherence to cells. The mycoplasmas attach to the surfaces of ciliated and nonciliated cells, probably through the mucosal cell sialoglycoconjugates and sulfated glycolipids. Some mycoplasmas lack the distinct tip structures but use adhesin proteins or have alternative mechanisms to adhere to host cells. The subsequent events in infection are less well understood but may include several factors as follows: direct cytotoxicity through generation of hydrogen peroxide and superoxide radicals, cytolysis mediated by antigen–antibody reactions or by chemotaxis and action of mononuclear cells, and competition for and depletion of nutrients.

Mycoplasmal Infection

Mycoplasmas have been cultivated from human mucous membranes and tissues, particularly from the genital, urinary, and respiratory tracts. Mycoplasmas are part of the normal microbiota of the mouth and can be grown from normal saliva, oral mucous membranes, sputum, or tonsillar tissue. M hominis is found in the oropharynx of fewer than 5% of adults. M pneumoniae in the oropharynx is generally associated with disease (see below).

Some mycoplasmas are inhabitants of the genitourinary tract, particularly in women. In both men and women, genital carriage of mycoplasmas is directly related to the number of lifetime sex partners. M hominis can be cultured from 1–5% of asymptomatic men and 30–70% of asymptomatic women; the rates increase to 20% and more than 90% positive for men and women, respectively, in sexually transmitted disease clinics. U urealyticum is found in the genital tracts of 5–20% of sexually active men and 40–80% of sexually active women. Approximately 10% of women attending sexually transmitted disease clinics have M genitalium in their lower genital tracts. The presence of M genitalium in the male urethra is typically associated with disease, a syndrome termed nongonococcal urethritis. Other mycoplasmas also occur in the lower genital tract.

Diagnostic Laboratory Tests

A. Specimens

Specimens consist of throat swabs; sputum; inflammatory exudates; and respiratory, urethral, or genital secretions.

B. Microscopic Examination

Direct examination of a specimen for mycoplasmas is useless. Cultures are examined as described earlier.

C. Cultures

The material is inoculated into broth and onto special solid media depending on the organism sought. Agar media is best incubated at 37°C with 5–10% CO₂ (under microaerophilic conditions or even anaerobic conditions). Broths require incubation at 37°C under atmospheric (aerobic) conditions. The duration of incubation varies from 2–4 days for organisms such as M hominis and U urealyticum to up to 4 weeks for M pneumoniae.

One or two transfers of media may be necessary before growth appears that is suitable for microscopic examination by staining or immunofluorescence. Colonies of M hominis may have a typical “fried egg” appearance on agar, but those of M pneumoniae and M genitalium are smaller and may lack the typical appearance.

Specimens submitted for diagnosis of Ureaplasma species are usually inoculated to broth or agar media (eg, A8 agar) containing urea. Growth is signaled by a color change indicating hydrolysis of urea.

D. Serology

Antibodies develop in humans infected with mycoplasmas and can be demonstrated by several methods. CF tests can be performed with glycolipid antigens extracted with chloroform–methanol from cultured mycoplasmas. M pneumoniae and M genitalium are serologically cross-reactive using CF tests. HI tests can be applied to tanned red blood cells with adsorbed Mycoplasma antigens. Indirect immunofluorescence may be used. The test that measures growth inhibition by antibody is quite specific. Enzyme immunoassays (EIAs) are available in most laboratories, but sensitivity and specificity are quite variable depending on the assay. In general, EIAs are considered better than CF. With all of these serologic techniques, there is adequate specificity for different human Mycoplasma species, but a rising antibody titer is required for diagnostic significance because of the high incidence of positive serologic test results in normal individuals.

E. Nucleic Acid Amplification Tests

Molecular methods for the detection of the human mycoplasmas and ureaplasma are available in many reference laboratories, and a variety of primers and probes have been published. Very few assays are cleared by the U.S. Food and Drug Administration, although many platforms are in development and this situation will likely improve. Nucleic acid amplification tests (NAATs) are particularly useful for those organisms that are difficult to cultivate such as M pneumoniae and M genitalium and less useful for the more rapidly growing organisms. The difficulty arises when these test results are positive in the absence of corroborating clinical or other positive diagnostic test results. At this time, these assays are best used in combination with other traditional diagnostic methods such as serology until more clinical data become available.

Treatment

Many strains of mycoplasmas are inhibited by a variety of antimicrobial drugs, but most strains are resistant to penicillins, cephalosporins, and vancomycin. Tetracyclines and erythromycins are effective both in vitro and in vivo and are, at present, the drugs of choice in mycoplasmal pneumonia. Some ureaplasmas are resistant to tetracycline. Treatment of M genitalium urethritis in men is typically through a single dose of azithromycin administered in the clinic. This ensures compliance and reduces the likelihood of sexual transmission to other partners.

Epidemiology, Prevention, and Control

M pneumoniae behaves like a communicable viral respiratory pathogen (see later discussion) and is capable of causing both endemic and epidemic infections. The genital mycoplasmas and ureaplasma are spread by genital or oral–genital contact and may be transmitted along with other sexually acquired pathogens. Safe sexual practices should reduce spread. No vaccines are available to protect against any of these organisms.

M pneumoniae is a prominent cause of pneumonia, especially in persons 5–20 years of age.

Pathogenesis

M pneumoniae is transmitted from person to person by means of infected respiratory secretions. Infection is initiated by attachment of the organism’s tip to a receptor on the surface of respiratory epithelial cells (Figure 25-1). Attachment is mediated by a specific adhesin protein on the differentiated terminal structure of the organism. During infection, the organisms remain extracellular.

Clinical Findings

Mycoplasmal pneumonia is generally a mild disease. The clinical spectrum of M pneumoniae infection ranges from asymptomatic infection to serious pneumonitis, with occasional neurologic and hematologic (ie, hemolytic anemia) involvement and a variety of possible skin lesions. Bullous myringitis occurs in spontaneous cases and in experimentally inoculated volunteers.

The incubation period varies from 1 to 3 weeks. The onset is usually insidious, with malaise, fever, headache, sore throat, and cough. Initially, the cough is nonproductive, but it is occasionally paroxysmal. Later, there may be blood-streaked sputum and chest pain. Early in the course, the patient appears only moderately ill, and physical signs of pulmonary consolidation are often negligible compared with the striking consolidation seen on radiographs. Later, when the infiltration is at a peak, the illness may be severe. Resolution of pulmonary infiltration and clinical improvement occur slowly over 1–4 weeks. Although the course of the illness is exceedingly variable, death is very rare and is usually attributable to cardiac failure. Complications are uncommon, but hemolytic anemia may occur. The most common pathologic findings in complicated cases are interstitial with peribronchial pneumonitis and necrotizing bronchiolitis. Other diseases less commonly related to M pneumoniae include erythema multiforme; central nervous system involvement, including meningitis, meningoencephalitis, and mono- and polyneuritis; myocarditis; pericarditis; arthritis; and pancreatitis.

Common causes of community-acquired bacterial pneumonia, in addition to M pneumoniae, include Streptococcus pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Haemophilus influenzae. The clinical presentations of these infections can be very similar, and recognition of the subtleties of signs and symptoms is important. The causative organisms must be determined by sputum examination and culture, blood culture, and other tests.

Laboratory Tests

The diagnosis of M pneumoniae pneumonia is largely made by the clinical recognition of the syndrome. Laboratory tests are of secondary value. The white blood cell count may be slightly elevated. A sputum Gram stain is of value only to identify a nonmycoplasma bacterial pathogen (eg, S pneumoniae). The causative mycoplasmas can be recovered by culture from the pharynx and from sputum, but culture is a highly specialized test and is almost never done to diagnose M pneumoniae infection. Cold hemagglutinins for group O human erythrocytes appear in about 50% of untreated patients, in rising titer, with the maximum reached in the third or fourth week after onset. A titer of 1:64 or more supports the diagnosis of M pneumoniae infection. There is a rise in specific antibodies to M pneumoniae that is demonstrable by CF tests; acute and convalescent phase sera are necessary to demonstrate a fourfold rise in the CF antibodies. EIA to detect immunoglobulin M (IgM) and IgG antibodies can be highly sensitive and specific and are considered more sensitive than CF tests. Polymerase chain reaction (PCR) assays of specimens from throat swabs or other clinical material can be diagnostic (see earlier discussion).

Treatment

Tetracyclines, macrolides, or fluoroquinolones can produce clinical improvement but do not always eradicate M pneumoniae, possibly because of their ability to reside intracellularly as well as extracellularly.

Epidemiology, Prevention, and Control

M pneumoniae infections are endemic all over the world. In populations of children and young adults, where close contact prevails, and in families, the infection rate may be high (50–90%), but the incidence of pneumonitis is variable (3–30%). For every case of frank pneumonitis, there exist several cases of milder respiratory illness. M pneumoniae is apparently transmitted mainly by direct contact involving respiratory secretions. Second attacks are infrequent. The presence of antibodies to M pneumoniae has been associated with resistance to infection but may not be responsible for it. Cell-mediated immune reactions occur. The pneumonic process may be attributed in part to an immunologic response rather than only to infection.

M hominis has been associated with a variety of diseases but is a demonstrated cause in only a few of them. The evidence for a causal relationship in disease is from culture and serologic studies. M hominis can be cultured from the upper urinary tract in about 10% of patients with pyelonephritis. M hominis is strongly associated with infection of the uterine tubes (salpingitis) and tubo-ovarian abscesses; the organism can be isolated from the uterine tubes of about 10% of patients with salpingitis but not from women with no signs of disease. Women with salpingitis more commonly have antibodies against M hominis than women with no disease. M hominis has been isolated from the blood of about 10% of women who have postabortal or postpartum fever and occasionally from joint fluid cultures of patients with arthritis.

U urealyticum, like M hominis, has been associated with a variety of diseases but is a demonstrated cause in only a few of them. U urealyticum, which requires 10% urea for growth, causes nongonococcal, nonchlamydial urethritis in men. Recent data show that urethritis is associated with biovar 2 and not biovar 1 (Ureaplasma parvum). U urealyticum is common in the female genital tract, where the association with disease is weak. U urealyticum has been associated with lung disease in premature low-birth-weight infants who acquired the organism during birth, but a causal effect has not been clearly demonstrated. However, in a symptomatic neonate with radiographic abnormalities in the lung and the absence of another discernible cause for the pneumonia, treatment for Ureaplasma species and M hominis seems warranted. The evidence that U urealyticum is associated with involuntary infertility is at best marginal.

M genitalium was originally isolated from urethral cultures of two men with nongonococcal urethritis, but culture of M genitalium is difficult, and subsequent observations have been based on data obtained by using NAATs and serologic data. The data suggest that M genitalium in men is associated with some cases of acute as well as chronic nongonococcal urethritis. In women, M genitalium has been associated with a variety of infections such as cervicitis, endometritis, salpingitis, and infertility.

• The major pathogens of medical importance include M pneumoniae, the cause of endemic and epidemic respiratory infections, and the urogenital mycoplasmas, M hominis, M genitalium, and U urealyticum.

• M hominis and U urealyticum are easily cultivatable because of rapid growth and less stringent requirements; M genitalium and M pneumoniae require a much longer incubation.

• M pneumoniae is an important cause of community-acquired pneumonia. Infection is insidious and often protracted. Diagnosis is best made clinically and confirmed by serology (fourfold rise in IgG or IgM) or by NAATs or both.

• The urogenital Mycoplasmas have been associated with nonchlamydial, nongonococcal urethritis in men (U urealyticum). Both M hominis and U urealyticum may cause postpartum fever and respiratory infections in premature infants. M hominis is more prevalent in women with bacterial vaginosis than in healthy women.

• Mycoplasma and Ureaplasma infections do not respond to β-lactam antibiotics. Tetracyclines, macrolides, and quinolones are the agents of choice.