Lecture 4 ................................................................... .

Mycoplasma

Mycoplasmas & Cell Wall-Defective Bacteria

MYCOPLASMAS There are over 150 species in the class of cell wall-free bacteria. At least 15 of these species are thought to be of human origin, while 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 infections. Other members of the genus mycoplasma are pathogens of the respiratory and urogenital tracts and joints of animals. The identity of the evolutionary ancestors of mycoplasmas is unclear. The smallest genome of mycoplasmas 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 125250 nm in size. (2) They are highly pleomorphic because they lack a rigid cell wall and instead are bounded by a triplelayered 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. (6) Mycoplasmas have an affinity for mammalian cell membranes. Morphology & 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 (owing to the lack of a rigid cell wall). Growth in fluid media gives rise to many different forms. Growth on solid media consists principally of plastic 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, darkfield, immunofluorescence, Giemsa-stained films from solid or liquid media, agar fixation). B. CULTURE

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Many strains of mycoplasmas grow in heart infusion peptone broth with 2% agar (pH 7.8) to which about 30% human ascitic fluid or animal serum (horse, rabbit) has been added. Following incubation at 37 C for 4896 hours, there may be no turbidity; however, Giemsa stains of the centrifuged sediment show the characteristic pleomorphic structures, and subculture on solid media yields minute colonies. After 26 days on diphasic (broth over agar) and agar medium incubated in a Petri dish that has been sealed to prevent evaporation, isolated colonies measuring 20500 m 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. The organisms can be stained for microscopic study by placing a similar square on a slide and covering the colony with a cover-glass onto which an alcoholic solution of methylene blue and azure has been poured and then evaporated (agar fixation). Such slides can also be stained with specific fluorescent antibody. C. GROWTH CHARACTERISTICS Mycoplasmas are unique in microbiology because of (1) their extremely small size and (2) 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. The sterol requirement for growth and membrane synthesis is unique. Mycoplasmas are resistant to thallium acetate, which can be used to inhibit bacteria. 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 develop predominantly at cell surfaces. Many established animal and human cell culture lines carry mycoplasmas as contaminants: often mycoplasmas are intracellular. D. VARIATION The extreme pleomorphism of mycoplasmas is one of their principal characteristics. Antigenic Structure Many antigenically distinct species of mycoplasmas have been isolated from animals (eg, mice, chickens, turkeys). In humans, at least 14 species can be identified, including M hominis, Mycoplasma salivarium, Mycoplasma orale, Mycoplasma fermentans, M pneumoniae, M genitalium, U urealyticum , and others. The species are classified by biochemical and serologic features. The CF antigens of mycoplasmas are glycolipids. Antigens for ELISA tests are proteins. Some species have more than one serotype.

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Pathogenesis Many pathogenic mycoplasmas have flask-like 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, and adherence-accessory proteins. The proteins are proline-rich, which influences the protein folding and binding and is 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 The mycoplasmas appear to be host-specific, being communicable and potentially pathogenic only within a single host species. In animals, mycoplasmas appear to be intracellular parasites with a predilection for mesothelial cells (pleura, peritoneum, synovia of joints). Several extracellular products can be elaborated (eg, hemolysins). A. INFECTION OF HUMANS Mycoplasmas have been cultivated from human mucous membranes and tissues, particularly from the genital, urinary, and respiratory tracts. Mycoplasmas are part of the normal flora of the mouth and can be grown from normal saliva, oral mucous membranes, sputum, or tonsillar tissue. M salivarium, M orale, and other mycoplasmas can be recovered from the oral cavities of many healthy adults, but an association with clinical disease is uncertain. M hominis is found in the oropharynx of less 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 females. 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 15% of asymptomatic men and 3070% of asymptomatic women; the rates increase to 20% and over 90% positive for men and women, respectively, in sexually transmitted disease clinics. U urealyticum is found in the genital tracts of 520% of sexually active men and 4080% of sexually active women. Approximately 20% of women attending sexually transmitted disease clinics have M genitalium in their lower genital tracts. Other mycoplasmas also occur in the lower genital tract. B. INFECTION OF ANIMALS

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Bovine pleuropneumonia is a contagious, occasionally lethal disease of cattle associated with pneumonia and pleural effusion. The disease probably has an airborne spread. Mycoplasmas are found in inflammatory exudates. Agalactia of sheep and goats in the Mediterranean area is a generalized infection with local lesions in the skin, eyes, joints, udder, and scrotum; it leads to atrophy of lactating glands in females. Mycoplasmas are present in blood early and in milk and exudates later. In poultry, several economically important respiratory diseases are caused by mycoplasmas. The organisms can be transmitted from hen to egg to chick. Swine, dogs, rats, mice, and other species harbor mycoplasmas that can produce infection involving particularly the pleura, peritoneum, joints, respiratory tract, and eye. In mice, a mycoplasma of spiral shape (spiroplasma) can induce cataracts. C. INFECTION OF PLANTS Aster yellows, corn stunt, and other plant diseases appear to be caused by mycoplasmas. They are transmitted by insects and can be suppressed by tetracyclines. 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 above. C. CULTURES The material is inoculated onto special solid media and incubated for 310 days at 37 C with 5% CO2 (under microaerophilic conditions), or into special broth and incubated aerobically. One or two transfers of media may be necessary before growth appears that is suitable for microscopic examination by staining or immunofluorescence. Colonies may have a fried egg appearance on agar. 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. HI tests can be applied to tanned red cells with adsorbed mycoplasma antigens. Indirect immunofluorescence may be used. The test that measures growth inhibition by antibody is quite specific. With all 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 tests in normal individuals. M pneumoniae and M genitalium are serologically cross-reactive.

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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. Epidemiology, Prevention, & Control Isolation of infected livestock will control the highly contagious pleuropneumonia and agalactia. No vaccines are available. Mycoplasmal pneumonia behaves like a communicable viral respiratory disease (see below).

MYCOPLASMA PNEUMONIAE & ATYPICAL PNEUMONIAS

Mycoplasma pneumoniae is a prominent cause of pneumonia, especially in persons 520 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. 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 lassitude, 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 to the striking consolidation seen on x-rays. Later, when the infiltration is at a peak, the illness may be severe. Resolution of pulmonary infiltration and clinical improvement occur slowly over 14 weeks. Although the

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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 are interstitial and peribronchial pneumonitis and necrotizing bronchiolitis. Other diseases possibly 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 Mycoplasma pneumoniae, include Streptococcus pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Haemophilus influenzae . 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 cell count may be slightly elevated. A sputum Gram stain is of value in not suggesting some other bacterial pathogen (eg, Streptococcus 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 complement fixation (CF) tests; acute and convalescent phase sera are necessary to demonstrate a fourfold rise in the CF antibodies. EIA to detect antibodies against M pneumoniae can be highly sensitive and specific but may not be readily available. PCR assay of specimens from throat swabs or other clinical material can be diagnostic, but is generally performed only in reference laboratories. Treatment Tetracyclines or erythromycins can produce clinical improvement but do not eradicate the mycoplasmas. Epidemiology, Prevention, & 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 (5090%), but the incidence of pneumonitis is variable (330%). 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

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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 by mycoplasmas.

MYCOPLASMA HOMINIS
Mycoplasma 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.

UREAPLASMA UREALYTICUM
Ureaplasma 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, probably causes nongonococcal urethritis in some men, but a majority of cases of nongonococcal urethritis are caused by Chlamydia trachomatis. 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. The evidence that U urealyticum is associated with involuntary infertility is at best marginal.

MYCOPLASMA GENITALIUM
Mycoplasma 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 the polymerase chain reaction, molecular probes, and serologic tests. The data suggest that M genitalium is associated with some cases of acute as well as chronic nongonococcal urethritis.

CELL WALL-DEFECTIVE BACTERIA

L phase variants (L forms) are wall-defective microbial forms that can replicate serially as nonrigid cells and produce colonies on solid media. Some L phase variants are stable; others are unstable and revert to

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bacterial parental forms. Wall-defective forms are not genetically related to mycoplasmas. They can result from spontaneous mutation or from the effects of chemicals. Treatment of eubacteria with cell wall-inhibiting drugs or lysozyme can produce cell wall-defective microbial forms. Protoplasts are such forms usually derived from gram-positive organisms; they are osmotically fragile, with external surfaces free of cell wall constituents. Spheroplasts are cell wall-defective forms usually derived from gram-negative bacteria; they retain some outer membrane material. There is no genetic relationship between mycoplasmas and cell wall-defective microbial forms or their parent bacteria. The characteristics of the cell wall-defective forms are similar to those of mycoplasmas, but by definition, mycoplasmas do not revert to parental bacterial forms or originate from them. Cell wall-defective forms continue to synthesize some antigens that are normally located in the cell wall of the parent bacteria (eg, streptococcal L forms produce M protein and capsular polysaccharide). Reversion of L forms to the parental bacterial form is enhanced by growth in the presence of 1530% gelatin or 2.5% agar. Reversion is inhibited by inhibitors of protein synthesis. It is uncertain whether cell wall-defective microbial forms cause tissue reactions resulting in disease. They may be important for the persistence of microorganisms in tissue and recurrence of infection after antimicrobial treatment, as in rare cases of endocarditis.

Dr. Shama M.J Saadaldin Al-Shadidi 3rd grade / College of Medicine Mustansiryah University 1/4/2012 Updated 2012