Organisms in this chapter belong to the class Mollicutes (Latin, meaning soft skin). This class comprises four orders, which, in turn, contain five families and eight genera (Figure 45-1). The mycoplasmas that colonize or infect humans belong to the family Mycoplasmataceae; this family comprises two genera, Mycoplasma and Ureaplasma. These organisms are highly fastidious, are slow growing, and most are facultative anaerobes that require nucleic acid precursor molecules, fatty acids, and sterols such as cholesterol for growth. These bacteria have a very small cell size (0.3 × 0.8 µm) and small genome. The Mollicutes appear most closely related to the gram-positive bacterial subgroup that includes bacilli, streptococci, and lactobacteria that diverged from the Streptococcus branch of gram-positive bacteria.

Figure 45-1 Taxonomy of the class Mollicutes.

Epidemiology and Pathogenesis

Mycoplasmas are part of the microbial flora of humans and are found mainly in the oropharynx, upper respiratory tract, and genitourinary tract. Besides those that are considered primarily as commensals, considerable evidence indicates the pathogenicity of some mycoplasmas; for others, a role in a particular disease is less clearly delineated.

Epidemiology

The mycoplasmas usually considered as commensals are listed in Table 45-1, along with their respective sites of colonization. These organisms may be transmitted by direct sexual contact, transplanted tissue from donor to recipient, or from mother to fetus during childbirth or in utero. M. pneumoniae may be transmitted by respiratory secretions. One species of Acholeplasma (these organisms are widely disseminated in animals), Acholeplasma laidlawii, has been isolated from the oral cavity of humans a limited number of times; however, the significance of these mycoplasmas and their colonization of humans remains uncertain.

TABLE 45-1

Mycoplasmas That Are Considered Normal Flora of the Oropharynx or Genital Tract

Organism	Site of Colonization
M. orale	Oropharynx
M. salivarium	Oropharynx, gingiva
M. amphoriforme	Oropharynx, respiratory tract?
M. buccale	Oropharynx
M. faucium	Oropharynx
M. fermentans	Oropharynx
M. fermentans	Genital tract
M. fermentans incognitus strain	Oropharynx
M. lipohilum	Oropharynx
M. penetrans	Genital tract
M. pirum	Unknown
M. primatum	Genital tract, oropharynx
M. spermatophilum	Genital tract
Acholeplasma laidlawii	Oropharynx

Of the other mycoplasmas that have been isolated from humans, the possible role that M. pirum, M. amphoriforme, M. fermentans, and M. penetrans might play in human disease is uncertain at this time. M. pirum, M. fermentans, and M. penetrans have been isolated from patients infected with the human immunodeficiency virus (HIV). It now appears that M. genitalium may account for as much as 15% to 20% of nongonococcal urethritis. M. genitalium is not associated with the presence of other mycoplasmas and ureaplasmas. In women, this organism may also cause cervicitis and endometritis. M. fermentans has been isolated from specimens such as bronchoalveolar lavage, bone marrow, peripheral blood, and the throats of children with pneumonia. The organism has been associated with infection in children and immunocompromised individuals. M. amphoriforme has been detected in the lower respiratory tract in patients with chronic respiratory disease and antibody deficiencies. The incidence of various Mycoplasma spp. infections in immunocompromised patients has been demonstrated following genital or respiratory tract colonization as well as medical procedures such as renal transplantation, genitourinary manipulations, or following trauma resulting in wound infections.

Finally, the remaining three species of mycoplasmas that have been isolated from humans—M. pneumoniae, U. urealyticum, and M. hominis—have well-established roles in human infections. Both U. urealyticum and M. hominis have been isolated from the genitourinary tract of humans, and M. pneumoniae has been isolated from the respiratory tract. Both Ureaplasma species have been isolated from the internal organs of stillborn, premature, and spontaneously aborted fetuses. However, the literature contains conflicting opinions as to the importance of U. urealyticum in comparison to U. parvum.

Infants are commonly colonized with U. urealyticum and M. hominis. Once an individual reaches puberty, colonization with these mycoplasmas can occur primarily as a result of sexual contact. In situations in which these agents cause disease in neonates, organisms are transmitted from a colonized mother to her newborn infant by an ascending route from colonization of the mother’s urogenital tract, by crossing the placenta from the mother’s blood, by delivery through a colonized birth canal, or postnatally from mother to infant.

M. pneumoniae is a cause of community-acquired atypical pneumonia, often referred to as walking pneumonia (see Chapter 69); infections caused by this agent are distributed worldwide, with an estimated 2 million cases per year in the United States. M. pneumoniae infection may also result in bronchitis or pharyngitis. M. pneumoniae may be transmitted person-to-person by respiratory secretions as previously stated or indirectly by inanimate objects contaminated with respiratory secretions (fomites). Infections can occur singly or as outbreaks in closed populations such as families and military recruit camps. Pneumonia caused by M. pneumoniae may present as asymptomatic to mild disease, with early nonspecific symptoms including malaise, fever, headache, sore throat, earache, and nonproductive cough. This differs significantly from the classic symptoms associated with pneumonia as a result of infection with Streptococcus pneumoniae (see Chapters 15 and 69). M. pneumoniae strongly attaches to the mucosal cells and may reside intracellularly within host cells, resulting in a chronic persistent infection that may last for months to years. The infections do not follow seasonal patterns as seen with influenzae and other respiratory pathogens. Besides respiratory infection, M. pneumoniae can cause extrapulmonary manifestations such as pericarditis, hemolytic anemia, arthritis, nephritis, Bell’s palsy, and meningoencephalitis resulting in various additional forms of paralysis.

Pathogenesis

In general, mycoplasmas colonize mucosal surfaces of the respiratory and urogenital tracts. Except for those mycoplasmas noted, most rarely produce invasive disease except in immunocompromised hosts or instrumentation. Of the mycoplasmas that are established as causes of human infections, these agents predominantly reside extracellularly, attaching with great affinity to ciliated and nonciliated epithelial cells. Recently, M. fermentans, M. penetrans, M. genitalium, and M. pneumoniae have been identified intracellularly. Intracellular invasion in bacterial infections is generally considered a means for immune evasion and may contribute to the persistent nature of infections and difficulties in cultivation or isolation of Mycoplasma spp. M. pneumoniae has a complex and specialized attachment organelle to accomplish this process that includes a P1 adhesin protein that primarily interacts with host cells. With respect to the mycoplasmas that are clearly able to cause disease, many of the disease processes are thought to be immunologically mediated. In addition to adherence properties and possibly immune-mediated injury, the ability to cause localized cell injury appears to contribute to their pathogenicity.

Of interest, the mycoplasmas associated with patients with HIV (M. fermentans, M. penetrans, and M. pirum) are all capable of invading human cells and modulating the immune system. Based on these findings, some investigators have proposed that these mycoplasmas might play a role in certain disease processes in these patients.

Spectrum of Disease

The clinical manifestations of infections caused by M. pneumoniae and the pathogenic genital mycoplasmas, U. urealyticum, U. parvum, M. hominis, and M. genitalium are summarized in Table 45-2.

TABLE 45-2

Clinical Manifestations of Mycoplasma Infections Caused by Mycoplasma pneumoniae, Ureaplasma urealyticum, Ureaplasma parvum, M. hominis, and M. genitalium

Organism	Clinical Manifestations
Mycoplasma pneumoniae	Asymptomatic infection
	Upper respiratory tract infection in school-aged children: mild, nonspecific symptoms including runny nose, pharyngitis, coryza (symptoms of a head cold, stuffy or runny nose, cough, aches), and cough; most without fever
	Lower respiratory tract infection in adolescents or young adults: typically mild illness with nonproductive cough, fever, malaise, pharyngitis, myalgias; approximately 33% of patients develop pneumonia; complications include rash, arthritis, encephalitis, myocarditis, pericarditis, and hemolytic anemia
	Occasionally the organism has been associated with infection in children < 5 years of age and elderly patients
Genital mycoplasmas: U. urealyticum and M. hominis	Systemic infections in neonates as a result of vertical transmission from the mother to the fetus in 18%-55% when the mother is colonized: meningitis, abscess, bacteremia, and pneumonia; U. urealyticum is also associated with the development of chronic lung disease
	Invasive disease in immunosuppressed patients: bacteremia, arthritis (particularly in patients with agammaglobulinemia), abscesses and other wound infections, pneumonia, peritonitis
	Urogenital tract infections: prostatitis, pelvic inflammatory disease (PID), amnionitis, nongonococcal urethritis, acute polynephritis
	These organisms proliferate in the urogenital tract of patients suffering with bacterial vaginosis (BV) caused by other microorganisms; some studies link M. hominis to the development of BV and may be associated with the development of additional disease such as PID
M. genitalium	Nongonococcal urethritis in men; possible cause of cervicitis and endometritis in females
M. genitalium	Vertical transmission from mother to fetus has been identified; however, the clinical significance is currently unknown
Ureaplasma spp.	Ureaplasma urealyticum and less frequently U. parvum have been isolated from the tissues of spontaneously aborted fetuses, stillborns, and premature infants, as well as full-term infants; the organisms may infect the chorioamnion

Data from Versalovic J: Manual of clinical microbiology, ed 10, Washington, DC, 2011, ASM Press.

Laboratory Diagnosis

The laboratory diagnosis of mycoplasma infections is extremely challenging because of complex and time-consuming culture requirements and the lack of reliable, widely available rapid diagnostic tests. Accurate, rapid diagnosis for M. pneumoniae is highly desired, because penicillin and other β-lactam agents are ineffective treatments. The laboratory diagnosis of the mycoplasmas well recognized as able to cause human disease (i.e., M. pneumoniae, U. urealyticum, M. hominis, and M. genitalium) is addressed.

Specimen Collection, Transport, and Processing

Various specimens are appropriate for the diagnosis of mycoplasma infections by culture or other means of detection. Acceptable specimens include body fluids (e.g., blood, joint fluid, amniotic fluid, urine, prostatic secretions, semen, pleural secretions, sputum, bronchoalveolar lavage specimens), tissues, wound aspirates and swabs of wounds, the throat, nasopharynx, urethra, cervix, or vagina. Blood for culture of genital mycoplasmas should be collected without anticoagulants and immediately inoculated into an appropriate broth culture medium. Mycoplasmas are inhibited by sodium polyanethol sulfonate (SPS), the anticoagulant typically found in commercial blood culture media. This may be overcome by the addition of 1% wt/vol of gelatin; however, commercial blood culture media and automated instruments are not adequate for the detection of Mycoplasma spp. Swab specimens should be obtained without the application of any disinfectants, analgesics, or lubricant; Dacron or polyester swabs on aluminum or plastic shafts should be used. Care must be taken to collect urine samples to avoid contamination with lubricants and antiseptics used during gynecologic examination.

Because mycoplasmas have no cell wall, they are highly susceptible to drying; therefore, transport media are necessary, particularly when specimens are collected on swabs. Liquid specimens such as body fluids do not require transport media if inoculated to appropriate media within 1 hour of collection. Tissues should be kept moist; if a delay in processing is anticipated, they should also be placed in transport media. Specific media for the isolation of Mycoplasma spp. include those containing 10% heat-inactivated calf serum containing 0.2 M sucrose in a 0.02 M phosphate buffer, pH 7.2, such as SP4, Shepard’s 10B broth or 2 SP. Additional commercial media available for cultivation of these organisms include Stuart’s medium, trypticase soy broth supplemented with 0.5% bovine serum albumin, Mycotrans (Irvine Scientific, Irvine, California), and A3B broth (Remel, Inc.). Excessive delays in processing can result in decreased viability and recovery of organisms from clinical specimens. If the storage time is expected to exceed 24 hours prior to cultivation, the samples should be placed in transport media and frozen at −80°C. Frozen samples should be thawed in a hot water bath at 37°C. Transport and storage conditions of various types of specimens are summarized in Table 45-3.

TABLE 45-3

Transport and Storage Conditions for Mycoplasma pneumoniae, Ureaplasma urealyticum, and M. hominis

Specimen Type	Transport Conditions	Transport Media (examples)^§	Storage	Processing
Body fluid or liquid specimens*	Within 1 hr of collection on ice or at 4° C	Not required	4° C up to 24 hr^†	Concentrate by high-speed centrifugation and dilute (1 : 10 to 1 : 1000) in broth culture media to remove inhibitory substances and contaminating bacteria; urine should be filtered through a 0.45-µm pore size filter
Swabs	Place immediately into transport media	0.5% albumin in trypticase soy broth modified Stuart’s	4°C up to 24 hr^†	None
		2SP (sugar-phosphate medium with 10% heat-inactivated fetal calf serum)
		Shepard’s 10B broth for ureaplasmas
		SP-4 broth for other mycoplasmas and M. pneumoniae^‡
		Mycoplasma transport medium (trypticase phosphate broth, 10% bovine serum albumin, 100,000 U of penicillin/milliliter and universal transport media [Copan, Murrieta, CA])
Tissue	Within 1 hr of collection on ice or at 4° C	Not required as long as prevented from drying out	4° C up to 24 hr^†	Mince (not ground) and dilute (1 : 10 and 1 : 100) in transport media

^*Except blood (see text).

^†Can be stored indefinitely at -80° C if diluted in transport media following centrifugation.

^‡SP-4 broth: sucrose phosphate buffer, 20% horse serum, Mycoplasma base, and neutral red.

^§Not a complete list. A variety of commercial media is available.

Direct Detection Methods

At present, no direct methods for identifying M. pneumoniae, Ureaplasmas spp., or other Mycoplasma spp. in clinical samples are recommended, although some methods have been described, such as immunoblotting and indirect immunofluorescence. Direct detection by gram staining may rule out the presence of other infectious organisms, but it will not stain cell wall-deficient mycoplasmas and ureaplasmas. Acridine orange or a flurochrome stain may be useful to visualize organisms. However, these are nonspecific stains that will stain nucleic acids in bacteria as well as human cells.

Molecular Diagnostics

Several amplification methods, such as polymerase chain reaction (PCR), have been developed for the detection of the clinically relevant Mycoplasma and Ureaplasma species. Various targets including 16srRNA sequences, insertion sequences, and organism specific genes have been used in the development of these assays. As a result of the fast turnaround time, specificity, and lack of need to cultivate fastidious organisms, PCR amplification for the diagnosis of these organisms is particularly attractive. When considering the use of molecular amplification methods for the detection of infectious diseases, it is important to note that although an organism is detectable, the patient’s signs and symptoms must be correlated with the identified agent. It is possible to detect an organism by one method and not another—in other words, a patient may be PCR positive but culture negative or serologically negative for a Mycoplasma based on the patient’s response to infection and current disease manifestation. Chapter 8 provides a more detailed description of the advantages, limitations, and methods used in the development of amplification assays. Multiplexed real-time PCR assays that detect M. pneumoniae as well as other atypical respiratory tract pathogens such as Chlamydophila pneumoniae and Legionella pneumoniae have been developed.

Because there is no reliable medium for its isolation, M. genitalium has been directly detected by PCR targeting its attachment protein in urine and urethral swabs in men. In women, vaginal or cervical swabs are used.

Cultivation

In general, the medium for mycoplasma isolation contains a beef or soybean protein with serum, fresh yeast extract, and other factors. As a result of the slow growth of these organisms, the medium must be selective to prevent overgrowth of faster-growing organisms that may be present in a clinical sample. Culture media and incubation conditions for these organisms are summarized in Table 45-4. Culture methods for M. pneumoniae, U. urealyticum, and M. hominis are provided on the Evolve site in Procedures 45-1, 45-2, and 45-3, respectively. The quality control of the growth media with a fastidious isolate is of great importance.

TABLE 45-4

Cultivation of Mycoplasma pneumoniae, Ureaplasma spp., and M. hominis

Organism	Media (examples)	Incubation Conditions
M. pneumoniae	Biphasic SP-4 (pH 7.4) Triphasic system (Mycotrim RS, Irvine Scientific, Irvine, California) PPLO broth or agar with yeast extract and horse serum Modified New York City medium

Broths: 37° C, ambient air for up to 4 wk

Agars: 37° C, ambient air supplemented with 5 to 10% CO₂ or anaerobically in 95% N₂ plus 5% CO₂.

All cultures should be retained for 4 weeks before reporting as negative.

U. urealyticum/U. parvum*/M. hominis^†

A7 or A8 agar medium (Remel, Lenexa, Kansas); penicillin should be included to minimize bacterial overgrowth ^‡

New York City medium

Modified New York City medium

SP-4 glucose broth with arginine ^§

SP-4 glucose broth with urea ^||

Triphasic system (Mycotrim GU, Irvine Scientific)

Shepard’s 10B broth (or Ureaplasma 10C broth)^||

Broths: 37° C, ambient air for up to 7 days

Agars: 37° C in 5 to 10% CO₂ or anaerobically in 95% N₂ plus 5% CO₂ for 2 to 5 days.

Genital cultures should be retained for 7 days before reporting as negative.

^*Utilizes urea and requires acidic medium.

^†Converts arginine to ornithine and grows over a broad pH range.

^‡Commercially available.

^§For M. hominis isolation.

^||For U. urealyticum isolation.

Procedure 45-1 Isolation of Mycoplasma Pneumoniae

Principle

Mycoplasma pneumoniae is primarily a respiratory tract pathogen that is extremely fastidious. Because this bacterial agent lacks a cell wall and has strict nutritional requirements, the recovery of this organism from clinical specimens requires special conditions in terms of both culture media and incubation conditions.

Method

1. Prepare biphasic SP-4 culture media as follows:

A Combine the ingredients for the media base.

Mycoplasma broth base (BD), 3.5 g

Tryptone (Difco), 10 g

Bacto-Peptone (Difco), 5 g

(50% aqueous solution, filter sterilized), 10 mL

Distilled water, 615 mL

B Stir the solids into boiling water to dissolve them and adjust the pH to 7.4 to 7.6. Autoclave according to the manufacturer’s instructions. Cool to 56° C before adding the following supplements for each 625 mL of base to make a final volume of 1 L:

CMRL 1066 tissue culture medium with glutamine, 10× (GIBCO), 50 mL

Aqueous yeast extract (prepared as described in New York City Medium), 35 mL

Yeastolate (Difco; 2% solution), 100 mL

Fetal bovine serum (heat inactivated at 56° C for 30 minutes), 170 mL

Penicillin G sodium, 1000 1U/mL

Amphotericin B, 0.5 g

Polymyxin B, 500,000 U

C To prepare the agar necessary for making biphasic media, add 8.5 g Noble agar (Difco) to the basal medium ingredients before adding the supplements.

D Dispense 1 mL of SP-4 agar aseptically into the bottom of sterile 4-mL screw-capped vials. Allow the agar to set and dispense 2 mL of SP-4 broth above the agar layer in each vial. Seal caps tightly and store at –20° C.

2. Place 0.1 to 0.2 mL of liquid specimen or dip and twirl a specimen received on a swab in a vial of biphasic SP-4 culture medium. After expressing as much fluid as possible from the swab, remove the swab to prevent contamination.

3. Seal the vial tightly and incubate in air at 35° C for up to 3 weeks.

4. Inspect the vial daily. During the first 5 days, a change in pH, indicated by a color shift from orange to yellow or violet, or increased turbidity is a sign that the culture is contaminated and should be discarded.

5. If either a slight acid pH shift (yellow color) with no increase in turbidity or no change occurs after 7 days’ incubation, subculture several drops of the broth culture to agar. Continue to incubate the original broth.

6. If broth that exhibited no changes at 7 days shows a slight acid pH shift at any time, subculture to agar as noted earlier. Broths that show no change at 3 weeks are blindly subcultured to agar.

7. Incubate the agar plates in a very moist atmosphere with 5% to 10% CO₂ at 35° C for 7 days.

8. Observe the agar surface under 20 to 60× magnification using a stereomicroscope after 5 days for colonies, which appear as spherical, grainy, yellowish forms, embedded in the agar, with a thin outer layer (see Figure 45-2).

9. Definitive identification of M. pneumoniae is accomplished by overlaying agar plates showing suspicious colonies with 5% sheep or guinea pig erythrocytes in 1% agar prepared in physiologic saline (0.85% NaCl) instead of water. The 1% agar is melted and cooled to 50° C, the blood cells are added, and a thin layer is poured over the original agar surface.

10. Reincubate the plate for 24 hours, and observe for beta hemolysis around colonies of M. pneumoniae caused by production of hydrogen peroxide. Additional incubation at room temperature overnight enhances the hemolysis. No other species of Mycoplasma produces this reaction.

Procedure 45-2 Isolation of Ureaplasma urealyticum

Principle

Method

1. Inoculate one Ureaplasma agar plate and one Ureaplasma broth each with 0.1 mL specimen from transport medium.

2. Incubate broth in tightly sealed test tubes for 5 days. Observe twice daily for a color change in the broth to red, with no increase in turbidity. If color change occurs, immediately transfer one loopful to a Ureaplasma agar plate and streak for isolated colonies.

3. Agar plates are incubated in a candle jar or, optimally, in an anaerobic jar at 35° C. Colonies appear on agar within 48 hours. Plates are inspected in the same way as described for M. pneumoniae (see Procedure 45-1). Ureaplasma colonies appear as small, granular, yellowish spheres.

4. To identify definitively colonies on Ureaplasma agar after 48 hours’ incubation, pour a solution of 1% urea and 0.8% MnCl₂ in distilled water over the agar surface. U. urealyticum stains dark brown because of production of urease (see Figure 45-3).

Procedure 45-3 Isolation of Mycoplasma hominis

Principle

Method A

1. Inoculate one M. hominis agar plate and two M. hominis broth tubes, one broth containing phenol red indicator and one without the possibly inhibitory phenol red, each with a 0.1-mL specimen from transport media.

2. Incubate broths in tightly sealed test tubes for 5 days. If the phenol red–containing broth changes color to red or violet, both broths are subcultured to M. hominis agar. After 48 hours of incubation, transfer 0.1 mL or a loopful of broth from tubes that exhibited no change or only a slight increase in turbidity to M. hominis agar and streak for isolated colonies.

3. M. hominis agar plates are incubated in the same manner as Ureaplasma cultures. Plates should be observed daily for up to 5 days for colonies.

Method B (Alternative Method)

1. Inoculate specimen onto a prereduced colistin-nalidixic acid (CNA) sheep blood agar or anaerobic blood plate and incubate anaerobically for 48 to 72 hours.

2. Examine for pinpoint colonies that show no bacteria on Gram stain.

3. Streak suspicious colonies to M. hominis agar and incubate as in step 3 above.

For the most part, the different metabolic activity of the mycoplasmas for different substrates is used to detect their growth. Glucose (dextrose) is incorporated into media selective for M. pneumoniae, because this mycoplasma ferments glucose to lactic acid; the resulting pH change is then detected by a color change in a dye indicator. Similarly, urea or arginine can be incorporated into media to detect U. urealyticum and M. hominis, respectively (Table 45-5). If a color change—that is, a pH change—is detected, a 0.1- to 0.2-mL aliquot is immediately subcultured to fresh broth and agar media.

TABLE 45-5

Basic Biochemical Differentiation of the Major Mycoplasma spp. and Ureaplasma urealyticum

Organism	Glucose Metabolism	Arginine Metabolism	Urease
M. fermentans	Positive	Positive	Negative
M. genitalium	Positive	Negative	Negative
M. hominis	Negative	Positive	Negative
M. pneumoniae	Positive	Negative	Negative
U. urealyticum	Negative	Negative	Positive

In some clinical situations, it may be necessary to provide quantitative information regarding the numbers of genital mycoplasmas in a clinical specimen. For example, quantitation of specimens taken at different stages during urination or after prostatic massage can help determine the location of mycoplasmal infection in the genitourinary tract.

Approach to Identification

On agar, M. pneumoniae will appear as spherical, grainy, yellowish forms that are embedded in the agar, with a thin outer layer similar to those shown in Figure 45-2. The agar surface is examined under 20 to 60× magnification using a stereomicroscope daily for Ureaplasma spp., at 24 to 72 hours for M. hominis, and every 3 to 5 days for M. pneumoniae and other slow-growing species. Because only M. pneumoniae and one serovar of U. urealyticum hemadsorb, M. pneumoniae is definitively identified by overlaying suspicious colonies with 0.5% guinea pig erythrocytes in phosphate-buffered saline instead of water. After 20 to 30 minutes at room temperature, colonies are observed for adherence of red blood cells.