Mollicutes As Agents Of Human Disease

Classification and Cell Biology

Mycoplasmas and ureaplasmas are eubacteria classified in the class Mollicutes, which evolved from clostridium-like ancestors through the process of gene deletion. There are more than 150 species currently named in the class Mollicutes, 16 of which are known to have humans as their primary host. Mollicutes represent the smallest self-replicating organisms, both in cellular dimensions and genome, that are capable of cell-free existence. The extremely small genome, less than 600 kbp for the smallest mollicute Mycoplasma genitalium, and drastically limited biosynthetic capabilities explain the parasitic or saprophytic existence of these organisms, their sensitivity to environmental conditions, and their fastidious growth requirements. Mollicutes associated with humans range from coccoid cells of about 0.2-0.3 ^m diameter (e.g., ureaplasmas and Mycoplasma hominis) to tapered rods 1-2 ^m in length and 0.1-0.2 ^m in width in the case of Mycoplasma pneumoniae. All mollicutes totally lack a cell wall barrier, making them unique among prokaryotes. Lack of a cell wall also renders these organisms insensitive to the activity of P-lactam antimicrobials, prevents them from staining by Gram stain, and is largely responsible for their pleomorphic form.

The small cellular mass also means that mollicutes cannot be detected by light microscopy, and they do not produce visible turbidity in liquid growth media. Typical colonies require examination under a stereomicroscope to visualize their morphologic features. Mollicutes have never been found as freely living organisms in nature because they depend on a host cell to supply them with the things they need for their parasitic existence. Another characteristic of most members of the class Mollicutes is the requirement for sterols in artificial growth media, supplied by the addition of serum, to provide necessary components of the triple-layer cell membrane that gives structural support to the osmotically fragile organism. Maintenance of osmotic stability is especially important in these bacteria because of the lack of a rigid cell wall. Although mycoplasmas and ureaplasmas can flourish within an osmotically stable environment in their chosen eukaryotic host, they are extremely susceptible to desiccation, a fact that greatly impacts the need for proper handling of clinical specimens in which cultural isolation is to be attempted and the need for close contact for transmission of infection from person to person. The small size, complex and fastidious nutritional requirements of mollicutes makes them challenging for detection and characterization by the microbiologist and historically has greatly hampered the ability of diagnostic laboratories to provide reliable services for their detection and identification.

Mollicute Species Pathogenic in Humans

Most research in perinatal and neonatal pathology has focused on two organisms, M. hominis and the bacterium formerly known as Ureaplasma urealyticum, because they are clearly the most significant in terms of disease-producing potential in pregnant women and neonates. However, it is relevant to mention briefly three other pathogenic mycoplasmal species that may contribute to perinatal and neonatal conditions to a lesser extent.

M. pneumoniae is well known as a major respiratory tract pathogen in older children and adults and has been detected many times in infants younger than 1 year, but it is rarely considered to be of much importance in the perinatal or neonatal period and is believed to be uncommon. It was not detected by culture in an evaluation of more than 1500 neonates (5). Nonetheless, this mycoplasma is a common cause of respiratory infections in women of childbearing age; it has been transmitted transplacentally with subsequent detection in the nasopharynx from a neonate with congenital pneumonia (7) and is therefore worthy of further study as a potential pathogen in this setting. More sensitive diagnostic tests such as the polymerase chain reaction (PCR) assay might yield different results if the culture-based study mentioned above was repeated.

M. genitalium was first detected in men with urethritis, and it has since been estimated to occur in 9-20% of men with urethritis and in up to 20% of women with urethritis or cervicitis (8). This organism has the smallest genome known for any free-living micro-organism, grows very slowly, and cannot be readily detected by culture. The PCR assay has facilitated clinical studies of M. genitalium as evidence mounts for its role in male urethritis, pelvic inflammatory disease, and possibly cervicitis (8).

Blanchard et al. (9) did not detect M. genitalium by PCR or culture in 232 amniotic fluids. Three studies have found M. genitalium in a very small percentage of pregnant women (3.9-6.2%) but were unable to relate the presence of this mycoplasma to preterm birth or other adverse pregnancy outcomes (10-12). Vertical transmission of M. genitalium from mother to neonate has also been reported (13), but its significance in neonates is unknown. Thus far, compelling evidence for an important role in pregnancy outcome or neonatal disease for this mycoplasma is lacking, but studies addressing this topic have been somewhat limited, and it is inappropriate to completely discount its significance based on information available at the present time.

Mycoplasma fermentans may also be pathogenic for humans in some settings, with most recent attention given to its role as an opportunist in persons with human immunodeficiency virus infection and acquired immunodeficiency syndrome and to a possible association with chronic arthritic conditions. M. fermentans is known to inhabit the lower and upper urogenital tracts of some adults. Furthermore, this mycoplasma has been detected by culture in placental tissue and in amniotic fluid in the presence of inflammation, but no studies have been performed to evaluate its occurrence and significance in neonates (14).

Soon after ureaplasmas were first identified in the 1950s and were subsequently characterized, it became apparent that these organisms could be subclassified into several serotypes. Data obtained from 16S ribosomal ribonucleic acid sequencing has led to the further breakdown of the 14 serotypes into two biovars or clusters. The two biovars were designated as distinct species. Biovar 1 (parvo) became Ureaplasma parvum, whereas biovar 2 (T960) became U. urealyticum. Biovar 1 is the more common of the two biovars isolated in clinical specimens, especially pregnant women, but both species may occur simultaneously in the same person.

There has long been speculation that there may be differential pathogenicity of the various serotypes, biovars, and species. There was no conclusive evidence for this difference in pathogenicity for several years, and this was related to some degree to inefficient and imprecise methods for their accurate differentiation and the fact that many persons may harbor more than one serotype in their urogenital tract. The availability of the PCR assay has enabled a more rigorous assessment of whether one biovar or species is more pathogenic than the other. Kim et al. (15) found no difference in pregnancy outcome and magnitude of intra-amniotic inflammatory response, chorioamnionitis, birth weight, or gestational age at delivery or neonatal morbidity in 77 women whose amniotic fluid contained ureaplasmas detected by PCR according to biovar. Zheng (16) suggested that the property of invasiveness for ureaplasmas is likely not limited to one or a few particular serotypes, and that perhaps antigen variability and host factors may be more important determinants for Ureaplasma infections than different serotypes per se.

However, a few limited studies have identified some differences regarding pathogenicity between the two biovars. Abele-Horn et al. (17) found biovar 2 to be dominant in patients with pelvic inflammatory disease as well as in women who had had miscarriages, and it seemed to have more adverse effects on pregnancy outcome regarding birth weight, gestational age, and preterm delivery than biovar 1. Others have shown that biovar 2 can be isolated more frequently from patients with a history of recurrent miscarriages than from normal pregnant women (18).

Martinez (19) found no differences in antimicrobial susceptibilities or occurrence of the two ureaplasmal species in amniotic fluids of women with adverse pregnancy outcomes vs isolates from the lower urogenital tract of healthy pregnant women. In contrast, two other studies found more tetracycline resistance in biovar 2 than in biovar 1 (17,20). The apparent contradictory results of some of these studies suggest that differencenos in antimicrobial susceptibilities, when they are observed, may reflect the history of antimicrobial exposure, the population studied, and other local environmental and host factors rather than a different capacity of the organism to acquire the tet (M) transposon in some instances. Based on the modest, and somewhat contentious evidence for differential pathogenicity for the two ureaplasmal species available at the present time and limitations of widely available technology for organism identification, it is neither practical nor necessary to distinguish between the two Ureaplasma species for clinical purposes. Therefore, diagnostic laboratories should appropriately designate cultures as positive for Ureaplasma spp and leave it at that.

Table 1 summarizes the major conditions of adults and infants that have been purported to be associated with or caused by M. hominis, M. genitalium, and Ureaplasma spp and the relative strengths, based on published evidence, for their roles in these conditions. The discussion of the clinical importance of these organisms has been divided into two major categories: maternal aspects and neonatal aspects.

Routes of Transmission and Maternal Considerations

Following puberty, colonization of the male and female lower urogenital tract by M. hominis and Ureaplasma spp usually occurs as a result of sexual activity. Up to 80% of women may harbor ureaplasmas and more than 50% may harbor M. hominis. These organisms are also commonly found in the lower urogenital tract of pregnant women, and they usually persist throughout pregnancy, providing a reservoir for transmission to the developing fetus and neonate (5). In most healthy adults, mycoplasmas and ureaplasmas exist primarily as commensals, associated with the mucosal surfaces, and rarely cause serious invasive disease. However, in persons who are immunocompro-mised, especially if hypogammaglobulinemia invasion of extragenital sites can occur. If one considers pregnant women and preterm infants to have an altered immune status, it is not surprising that these organisms can cause invasive and destructive disease when given the right circumstances.

Ureaplasma spp can be transmitted from a colonized woman to her newborn infant in utero either transplacentally from the mother's blood or by an ascending route sec-

Table 1

Conditions Known To Be Associated With or Caused by Mycoplasmas and Ureaplasmas

Table 1

Conditions Known To Be Associated With or Caused by Mycoplasmas and Ureaplasmas

Disease

Ureaplasma spp

M. hominis

M. genitalium

Adults

Male urethritis

+

-

+

Chronic prostatitis

±

-

±

Epididymitis

±

-

-

Urinary calculi

+

-

-

Pyelonephritis

±

+

-

Bacterial vaginosis

±

±

-

Cervicitis

-

-

±

Pelvic inflammatory disease

-

+

+

Infertility

±

-

-

Chorioamnionitis

+

-

-

Spontaneous abortion

±

±

-

Extragenital disease (including arthritis)

+

+

+

Infants

Prematurity/low birth weight

+

-

-

Intrauterine growth retardation

±

-

-

Postpartum/postabortal fever

+

+

-

Congenital pneumonia

+

+

-

Chronic lung disease

±

-

-

Meningitis

+

+

-

Abscesses

+

+

-

-, no association or causal role demonstrated. In some conditions for M. genitalium, this may reflect the fact that no studies using appropriate techniques to detect this organism have been performed. +, causal role.

±, significant association and/or strong suggestive evidence, but causal role not proven. (Modified from ref. 29.)

-, no association or causal role demonstrated. In some conditions for M. genitalium, this may reflect the fact that no studies using appropriate techniques to detect this organism have been performed. +, causal role.

±, significant association and/or strong suggestive evidence, but causal role not proven. (Modified from ref. 29.)

ondary to colonization of the mother's urogenital tract, or at delivery by passage through a colonized birth canal. The rate of vertical transmission has been reported to range from 18 to 55% among full-term infants and from 29 to 55% among preterm infants (21). Ureaplasma spp and M. hominis can be isolated from neonates born to mothers with intact membranes and delivered by cesarean section and from amniotic fluid during early pregnancy (5,21). The rate of vertical transmission is not affected by method of delivery but is significantly increased when chorioamnionitis is present. The rate of colonization also appears to be higher in very low birth weight infants (21).

The first studies attempting to make a correlation of mycoplasmas and ureaplasmas with postpartum endometritis were based on cervicovaginal cultures and caused much confusion with their inconclusive results (5). However, both M. hominis and Ureaplasma spp can be detected in the bloodstream of some women with postpartum or postabortal fever, with M. hominis being the more common. This condition is usually self-limited, but some cases of dissemination to joints resulting in arthritis may occur.

Isolation of Ureaplasma spp, but not M. hominis, from the chorioamnion has been consistently associated with histologic chorioamnionitis and is inversely related to birth weight, even when adjusting for duration of labor, rupture of fetal membranes, and presence of other bacteria. These organisms can invade the amniotic cavity and persist for several weeks when fetal membranes are intact and initiate an intense inflammatory reaction in the absence of labor (22-24). Even though these conditions may be clinically silent, these findings are strongly supportive of a causal role for Ureaplasma spp in chorioamnionitis. M. hominis seems rarely to invade the chorioamnion and amniotic fluid in the absence of other micro-organisms, and data to support an independent role for this mycoplasma in either histologic or clinical amnionitis are modest at best. Chorioamnion colonization with Ureaplasma spp was associated with a threefold increased risk of post-cesarean delivery endometritis, an association that increased to eightfold in women in whom onset of labor was spontaneous (25). The extent to which genital mycoplasmas may produce clinical amnionitis is unclear because some women whose placentas show significant evidence of inflammation and from whom genital mycoplasmas can be isolated from chorioamnion or amniotic fluid may not have evidence of clinical amnionitis.

Intrauterine infection is believed to be a major cause of preterm labor and can be documented in approximately one-fourth of all preterm births. The earlier the gestation age at delivery, the higher the frequency of intra-amniotic infection (26). This relationship is believed to be related to the concept that uterine contractions may be induced by cytokines, prostaglandins, and phospholipases produced by micro-organisms (27). M. hominis and Ureaplasma spp can be isolated from endometrial tissue of healthy, non-pregnant women, indicating they may be present at the time of implantation and might therefore be involved in early pregnancy losses (5).

Studies of women from whom ureaplasmas and M. hominis were isolated from en-dometrium or placenta have shown a consistent association with spontaneous abortion, but this has not proven true for studies limited to sampling the lower genital tract (28). Isolation of Ureaplasma spp in pure culture from amniotic fluid obtained from women with intact fetal membranes who experienced subsequent fetal loss in the presence of histological chorioamnionitis has been documented by multiple investigators, indicating that in some cases the role of this organism is causal (22-24).

Other circumstantial evidence linking ureaplasmas to spontaneous abortion, low birth weight, intrauterine growth retardation, and preterm labor includes reports of successful pregnancies following antimicrobial treatment and serological studies (5). Underlying problems that complicate complete understanding of any potential role for genital mycoplasmas in low birth weight are that M. hominis and, to a lesser extent, Ureaplasma spp can be components of the varied flora that occur with bacterial vaginosis, and this condition is itself associated with low birth weight (28-31), problems in experimental study designs that failed to consider potential roles for organisms other than genital mycoplasmas, or use of control groups of uncertain comparability.

Neonatal Infections

M. hominis and Ureaplasma spp can be isolated from organs of aborted fetuses and stillborn infants in pure culture and in the presence of an inflammatory response (5). Ureaplasmas are more commonly detected in products of early abortions and midtrimester pregnancy losses than from induced abortions, and they are more commonly detected in endometrium of habitual aborters and from placentas of aborted fetuses than from controls (5). Several studies have reported an association between isolation of Ureaplasma spp from the chorioamnion and perinatal morbidity and mortality (5,32,33). Because these studies did not attempt to detect the organisms directly in the infants, it is uncertain whether the problems experienced by the infants were caused by infection or complications of prematurity. However, when investigations were designed to culture directly the lower respiratory tract, blood, and cerebrospinal fluid (CSF), it became evident that both Ureaplasma spp and M. hominis can cause a variety of clinically significant infections in neonates.

Retrospective as well as prospective studies and well-documented case reports indicated Ureaplasma spp can cause congenital pneumonia (5). Proof for causality includes isolation of the organism in pure culture from affected lungs of neonates and from the chorioamnion, demonstration of a specific immunoglobulin M response, presence of histologic pneumonia and chorioamnionitis, clinical manifestations of respiratory distress, and demonstration of the organisms in lung tissue by immunofluorescence and electron microscopy. In some instances, ureaplasmas have been detected from multiple sites in neonates before and after death. Although individual case reports suggested M. hominis may cause pneumonia in newborns, it has not been implicated as a common cause in prospective studies (5). No convincing evidence exists to support a significant role for Ureaplasma spp or M. hominis as common independent causes of pneumonia in otherwise healthy infants beyond the neonatal period.

In the late 1980s, bacteremia and progression to chronic lung disease of prematurity and death were described in very low birth weight infants who were infected with Ureaplasma spp in the lower respiratory tract (34-37). Presence of ureaplasmas in the lower respiratory tract has also been significantly associated with radiographic evidence of pneumonia when compared with uninfected infants, and precocious dysplas-tic changes were also significantly associated with the presence of these organisms

(38). Further studies have shown that infants from whom ureaplasmas were isolated from endotracheal secretions had significantly more neutrophils in endotracheal secretions, attesting to their inflammatory potential (39).

An explanation for the association of perinatal infections caused by Ureaplasma spp and development of chronic lung disease relates to intrauterine exposure to proinflammatory cytokines that are released in response to infection, which persists because antimicrobials commonly used are not active against this organism. Chronic inflammation then increases the requirement for supplementary oxygen, which can lead to dysplastic changes in the airways as a result of oxygen toxicity or a synergistic effect between the ureaplasmas and hyperoxia. It is also speculated that this cytokine cascade may induce both preterm labor and inflammation in the airways, which triggers the lung injury sequence before birth.

Several additional studies performed during the 1990s confirmed that there was a significant association between chronic lung disease of prematurity and the presence of Ureaplasma spp in the lower respiratory tract of preterm neonates (40). This association was also detected in studies performed after introduction of exogenous surfactant

(39). This relationship has not been shown consistently in all investigations performed to date (41-44), but two studies from Europe supported a role for Ureaplasma spp in chronic lung disease of prematurity even as technological advances in neonatology continue to improve survival of very low birth weight infants (45,46). A summary and review of all published studies on the role of ureaplasmas in neonatal lung disease through 2004 was published by Waites (47).

There are also data to suggest that infants with perinatal ureaplasmal infections have a significantly greater need for hospital care during the first year of postnatal life (48). Further evidence of the inflammatory potential for this organism comes from animal models that indicate Ureaplasma spp can induce pneumonia and chronic inflammation (49-51).

Despite numerous studies and considerable data supporting an association, demonstration of a cause-and-effect relationship between Ureaplasma spp and chronic lung disease or prematurity has not been conclusively proven. Even though treatment with intravenous erythromycin may eradicate ureaplasmas from the lower respiratory tract of neonates, at least temporarily (52), small randomized trials of erythromycin treatment initiated early in the neonatal period have failed to show a benefit in reducing chronic lung disease (47). A larger treatment trial would provide greater insights regarding whether targeted antimicrobial therapy can reduce the incidence of morbidity and mortality associated with chronic lung disease.

Both M. hominis and Ureaplasma spp have been isolated from maternal and umbilical cord blood and the blood of neonates. Both species can also invade the CSF (53,54), resulting in either mild, subclinical meningitis without sequelae or neurological damage with permanent handicaps. Mononuclear or polymorphonuclear pleocytosis and elevated protein have been reported, but in some cases inflammation is minimal or absent. CSF glucose concentrations are usually normal. Most reported cases have involved preterm infants; full-term infants with neurological defects, including menin-gomyelocele; or older children with ventriculoperitoneal shunts, but infections in otherwise normal full-term infants have also been described.

Cases of M. hominis CSF infection in infants are more numerous than those that can be attributed to Ureaplasma spp because the former is much more readily detected without specialized methods. There appears to be an association with hydrocephalus and intraventricular hemorrhage in preterm infants with Ureaplasma CSF infections (54,55). A report of a brain abscess in a neonate in which both M. hominis and Ureaplasma spp were isolated concomitantly proved these organisms can cause focal central nervous system infection as well as meningitis (56). Relatively little is known about the long-term prognosis and neurodevelopmental outcomes for infections of the central nervous system caused by M. hominis or Ureaplasma spp because most available information comes from individual case reports, many of which provided scant information beyond the immediate period following infection and short-term prospective studies.

In addition to the above examples, occasional cases have appeared in the literature describing other conditions that bear consideration by clinicians. These include infections of pericardial fluid causing cardiac tamponade (57) and subcutaneous abscesses associated with forceps delivery (58) or an internal heart monitor (59). Two studies have described isolation of Ureaplasma spp from the respiratory tract and blood in infants with persistent pulmonary hypertension of the newborn and suggested there may be a possible association or interaction between the ureaplasmas and the vascular events that characterize this syndrome (60,61). Other isolations of these organisms from urine and conjunctiva are more difficult to evaluate because of the uncertain contribution of these bacteria to illness in these cases.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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