Observations on experimental colonisation of mice by ureaplasmas of human origin.

Three serovars (5, 8 and 10) of Ureaplasma urealyticum were inoculated intravaginally into groups of oestradiol-treated young adult BALB/c strain mice. Hormone treatment was essential for vaginal colonisation. The proportion of mice colonised initially and the persistence of colonisation were different with the three serovars; half of those given serovar 8 were still colonised after 84 days. A strain of serovar 5 after a further 50 subcultures in vitro was a little less persistent than it was before such subculture, but not in a way to suggest that subculturing was the main reason for differences in the behaviour of the serovars. At autopsy of six mice that were still colonised vaginally 158 days after inoculation of serovar 8, spread to the upper genital tract was shown to have occurred in three of them and dissemination to the liver and kidney in one. Compared with immunocompetent mice of strain CB20, such dissemination was not a feature in genetically related mice with severe combined immunodeficiency. This is not in keeping with the situation in hypogammaglobulinaemic patients in whom ureaplasmas and other mycoplasmas are known to disseminate. However, differences in the proportion of immunocompetent mice colonised or in ureaplasmal persistence with different serovars may act as a marker for differences in human pathogenicity and is worthy of further study.

Failure of Mycoplasma pneumoniae infection to confer protection against Mycoplasma genitalium: observations from a mouse model.

Mycoplasma pneumoniae and M. genitalium are genomically distinct but share antigens that induce some serological cross-reactivity. Therefore, the possibility that M. pneumoniae infection of the human respiratory tract might provide immunity to M. genitalium infection of the genital tract was considered. Because of the difficulty of assessing this proposition in man, it was evaluated experimentally in a mouse model. Female BALB/c mice were susceptible to infection of the vagina with M. pneumoniae, whereas those infected previously in the oropharynx with M. pneumoniae were completely immune to infection of the vagina with this mycoplasma. However, all mice with such a respiratory tract infection were susceptible to infection of the vagina with M. genitalium. The findings suggest that an M. pneumoniae infection of the human respiratory tract is unlikely to influence infection of the genital tract by M. genitalium.

Observations on the antibiotic treatment of experimentally induced mycoplasmal infections in mice.

Antibiotics were given subcutaneously to female mice, colonized by mycoplasmas in the vagina and by others in the oropharynx. Mycoplasma pulmonis was eradicated from the vagina of 36 of 42 immunocompetent TO or BALB/c mice with oxytetracycline, and from 17 of 18 TO mice with lymecycline. Mycoplasma hominis was eradicated from the vagina of all of 18 immunocompetent BALB/c mice with oxytetracycline or tetracycline. Regarding oropharyngeal organisms, M. pulmonis was eradicated from only 20 of 42 TO or BALB/c mice with oxytetracycline and from none of 15 mice with lymecycline. However, Mycoplasma pneumoniae was eliminated from the oropharynx of eight of nine BALB/c mice with oxytetracycline. In contrast to the success in eradicating mycoplasmas from the vagina or oropharynx of immunocompetent BALB/c mice with oxytetracycline, no such effect was seen in nude BALB/c mice, indicating the importance of a competent immune system in conjunction with antibiotic treatment for eradication of these organisms.

Genital mycoplasma infections.

Since 1937, 13 Mycoplasma species, two Acholeplasma species, and one Ureaplasma species have been isolated from humans. Six of these have the urogenital tract as the primary site of colonisation but others, which have the oropharynx and respiratory tract as the primary site, are found occasionally in the urogenital tract because of orogenital contact. Mycoplasma hominis was the first to be isolated and is most strongly associated with bacterial vaginosis (BV), together with a variety of other bacteria. Its involvement in pelvic inflammatory disease (PID) and other conditions may be as part of BV, although when isolated in pure culture from the blood of women who have postpartum or postabortal fever there is no reason to suspect its aetiological role. There is evidence for an aetiological role for Ureaplasma urealyticum organisms (ureaplasmas) in acute non-gonococcal urethritis (NGU) and particularly chronic NGU in men, but they rank third to Chlamydia trachomatis and M. genitalium. Whether the association of ureaplasmas with miscarriage and preterm labour is in the context of BV is not clear. Of no doubt, however, is the ability of ureaplasmas to cause septic arthritis in hypogammaglobulinaemic patients and there is evidence that they may cause some cases of sexually acquired reactive arthritis. The advent of polymerase chain reaction technology has seen an advance in the understanding of the role of M. genitalium; there is strong evidence that it is one of the causes of both acute and chronic NGU independent of C. trachomatis. There is some support for the role of M. genitalium in PID, but this needs to be substantiated. Other mycoplasmas, for example M. fermentans, M. pivum, M. primatum, M. penetrans, M. spermatophilum and even M. pneumoniae have the capacity to cause urogenital tract disease but there is no evidence to indicate that they do so.

Mycoplasmal arthritis in patients with primary immunoglobulin deficiency: clinical features and outcome in 18 patients.

A survey of 358 patients with primary antibody deficiency shows that mycoplasmal infection is the commonest cause of severe chronic erosive arthritis. We review our experience with 18 patients with confirmed or probable mycoplasmal arthritis. There was a broad spectrum of severity from a monoarthritis rapidly responding to tetracyclines to severe debilitating polyarthritis, sometimes with antibiotic-resistant organisms which in two cases were eliminated following hyperimmune animal serum therapy. Most patients had very low serum 1gG levels at the onset of arthritis, suggesting that maintaining levels within the normal range with immunoglobulin replacement may prevent infection. The unique susceptibility of these patients to mycoplasmal arthritis shows that antibodies play a crucial role in protection against these organisms.

Common variable immunodeficiency presenting as a Mycoplasma hominis septic arthritis.

A case is reported of common variable immunodeficiency (CVID) presenting as an acute septic arthritis due to Mycoplasma hominis. The diagnosis was not considered until the hypogammaglobulinaemia was discovered and the synovial fluid cultured specifically for mycoplasmas. The importance of diagnosing immunodeficiency states and searching for mycoplasmas in 'bacteriologically culture negative' cases is emphasized.

Site of localization of Mycoplasma pulmonis and Mycoplasma hominis in the genital tract of female mice demonstrated by culture and scanning and immuno-electron microscopy.

Thirty young adult mice, of strain BALB/c, treated previously with progesterone, were inoculated intravaginally (10 mice) or directly into the uterus (10 mice) with Mycoplasma pulmonis and 10 mice remained uninoculated. Ten mice not treated with the hormone were also inoculated intrauterinely with M. pulmonis. The same numbers of mice treated with oestradiol were inoculated in the same ways with M. hominis. Vaginal swab specimens were obtained from all mice 7, 14 and 28 days after inoculation and samples of genital tract tissue were collected from pairs of mice at the same time intervals. Large numbers of M. pulmonis and M. hominis organisms were isolated from the vagina throughout the course of the experiments and they were cultured also from the cervix and uterine horns. Mycoplasma-like bodies were demonstrated by scanning electron microscopy in the cervix and in the uterus, but neither mycoplasmal species was found attached to vaginal epithelium. The results of silver-enhanced immunogold labelling in conjunction with scanning microscopy provided assurance that the mycoplasma-like bodies were, in fact, mycoplasmas. The importance of hormone treatment was indicated by the diminished susceptibility of untreated mice to M. pulmonis and the almost complete insusceptibility to M. hominis, shown by culture and scanning electron microscopy.

Protection of mice against vaginal colonisation by Mycoplasma pulmonis.

Resistance against vaginal colonisation by Mycoplasma pulmonis in strain TO mice after exposure to the mycoplasma was investigated. Eighteen mice from which M. pulmonis had been eliminated from the vagina, either naturally or by antibiotic therapy, were resistant to vaginal recolonisation. Specific antibody was measured by an indirect microimmunofluorescence technique and the geometric mean titre (GMT) for each group of mice is presented. Almost all of 31 mice that had developed circulating antibody (GMT 83) or local antibody (GMT 40), or both, after vaginal exposure were resistant to re-colonisation, as were those in which antibodies could not be detected. Seven other mice which had been colonised only in the oropharynx previously and which possessed antibody--circulating (GMT 64) or local (GMT 30), or both--were resistant to vaginal colonisation, but 13 mice with little or no antibody after lack of colonisation at either anatomical site were susceptible. All of 15 mice given killed M. pulmonis organisms intravenously, despite developing circulating antibody in high titre (GMT 122), were susceptible to vaginal colonisation, as were 14 of 15 mice that developed circulating (GMT 15) and local antibodies after being given killed organisms intravaginally. However, 25 mice with high titres of circulating (GMT 154-170) or local (GMT 20) antibody, or both, after receiving live organisms intravenously, were less susceptible to vaginal colonisation (17 becoming colonised) than were 21 non-immunised mice (all becoming colonised) and the organisms were eradicated more rapidly from the former. Despite this, the mice that were colonised following intravenous inoculation of live organisms had pre-challenge antibody titres that were as great as those that were not colonised.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycoplasmas and ureaplasmas in patients with hypogammaglobulinaemia and their role in arthritis: microbiological observations over twenty years.

OBJECTIVES: To study the occurrence of mycoplasmas and ureaplasmas in patients with hypogammaglobulinaemia and the relationship of these micro-organisms to septic arthritis. METHODS: Over a period of about 20 years, 53 men and 38 women with hypogammaglobulinaemia, most of whom were less than 50 years old, were examined clinically and microbiologically. Mycoplasmas and ureaplasmas were sought in the throat, urogenital tract and joints by standard cultural methods, although not consistently in the three sites of all patients. RESULTS: Arginine-hydrolysing mycoplasmas and ureaplasmas occurred with similar frequency in the sputum/throat of the hypogammaglobulinaemic patients, but no more often than might be expected in immunocompetent patients. Ureaplasmas, however, dominated in the urogenital tracts of both men and women, being found in 75% of vaginal specimens. Arginine-hydrolysing mycoplasmas occurred two to six times more frequently and ureaplasmas two to three times more frequently in urine specimens from hypogammaglobulinaemic patients than they did in such specimens from sex- and age-matched non-venereal disease, hospital patients or healthy subjects; these differences were statistically significant (p < 0.05). Enhanced mucosal colonisation probably increases the chance of spread to distant sites, such as the joints. Of the 91 patients, 21 (23%) had septic arthritis involving one or more joints. Mycoplasmas and/or ureaplasmas, but not bacteria, were isolated from the joints of eight (38%) of these patients. However, dissemination to joints apparently had not occurred in some despite the opportunity by virtue of mycoplasmal or ureaplasmal colonisation at a mucosal site. Sometimes antibiotic therapy failed clinically, and microbiologically and recommendations for management are outlined. CONCLUSIONS: Hypogammaglobulinaemic patients appear to be more susceptible to colonisation of mucous membranes, especially of the urogenital tract, with mycoplasmas and ureaplasmas than are immunocompetent individuals. These micro-organisms are responsible for about two fifths of the septic arthritides occurring in these patients.

Mycoplasma pulmonis infection of the murine oropharynx protects against subsequent vaginal colonization.

Intranasal inoculation of 12 young adult mice (strain TO) with Mycoplasma pulmonis protected all of them against vaginal colonization when they were challenged intravaginally 60 days later with the same mycoplasmal strain. In contrast, all 15 mice without a respiratory infection became colonized vaginally (geometric mean titre [GMT] 4.6 x 10(6) colour-changing units [c.c.u.]) when challenged in the same way. The GMT of serum antibody, measured by a microimmunofluorescence technique, prior to challenge was 200 and 8 for the oropharyngeally infected and unexposed mice, respectively. The GMT of antibody in vaginal washings from the two groups was 6 and 3, respectively. All four nude BALB/c mice were susceptible to vaginal colonization (GMT 5.6 x 10(6) c.c.u.) after oropharyngeal infection (GMT 5.1 x 10(4) c.c.u.) resulting from intranasal inoculation, as were all six nude mice (vaginal GMT 1.4 x 10(7) c.c.u.) that had not been inoculated intranasally. In contrast, all ten of their immunocompetent counterparts were resistant to vaginal colonization after oropharyngeal infection (GMT 1.3 x 10(3) c.c.u.), whereas all nine such mice that had not been infected oropharyngeally were susceptible to vaginal colonization (GMT 7.6 x 10(6) c.c.u.). These results show the important role that a respiratory infection has in protecting the vagina against colonization and that protection is dependent on a functioning T-lymphocyte system.

Mycoplasma fermentans--HeLa cell interactions.

A survey of previous evidence for the intracellular localization of mycoplasmas within nonphagocytic cells indicated that it was insufficient to conclude unequivocally that such localization occurred. Illustrations of the seemingly intracellular existence of Mycoplasma fermentans in the tissues of patients with AIDS and other patients rekindled interest in the topic of mycoplasmal entrance into epithelial cells. Accordingly, the "incognitus" and PG18 strains of M. fermentans were sought and demonstrated within HeLa cells by electron microscopy following treatment of the cell cultures with gold-labeled antiserum specific to the mycoplasma, together with ruthenium red staining. The phenomenon is not unique to this mycoplasma, being demonstrated also with Mycoplasma hominis. In hindsight, some of the earlier investigators who hinted at intracellular localization were probably correct in doing so.

Models of infection due to mycoplasmas, including Mycoplasma fermentans, in the genital tract and other sites in mice.

Progesterone induces susceptibility in murine genital tracts to Mycoplasma pulmonis, Mycoplasma pneumoniae, and Mycoplasma genitalium when each is given intravaginally. In contrast, estradiol, but not progesterone, induced such susceptibility to Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma fermentans, the "incognitus" and PG18 strains of the latter behaving similarly. None of the mice that had been given M. fermentans intravaginally died, whereas others that had been given large numbers of M. fermentans organisms intraperitoneally died within a few days. M. pulmonis spreads from the respiratory tract to other sites more often in T cell-depleted mice than in immunocompetent mice. In addition, the "incognitus" strain of M. fermentans given intravenously killed BALB/c nude mice but did not kill immunocompetent control mice of the same strain or CBA nude mice. The effect is not unique to M. fermentans since M. hominis inoculated intravenously also killed CBA nude mice or made them ill. Such observations raise the possibility that immunosuppression caused by the human immunodeficiency virus could allow mycoplasmas to flourish and exacerbate disease.

The contrasting effects of progesterone and oestrogen on the susceptibility of mice to genital infection with Mycoplasma pulmonis.

The genital tract of young female mice was rendered susceptible to colonisation with Mycoplasma pulmonis by pre-treating them with progesterone (usually 2.5 mg) given subcutaneously at weekly intervals for 4 weeks. Colonisation was influenced by the size of the inoculum and by the dose of progesterone; at least 2.5 x 10(4) organisms and at least 0.5 mg of hormone (administered on four occasions) were required. The duration of colonisation was related also to the size of the inoculum and the dose of progesterone. Similar results were obtained in TO, BALB/c and CBA strains of mice. Progesterone treatment induced the dioestrous stage of the reproductive cycle within 5 days and the cycle of the majority of untreated, mycoplasma-susceptible mice was also at this stage. However, mice, particularly of the CBA strain, were far less susceptible when not given progesterone and the mycoplasmas tended to persist for a shorter time. Mice treated with oestradiol, even in small doses, became completely refractory to infection with M. pulmonis. In vitro, progesterone inhibited the growth of M. pulmonis, as did oestradiol, but vaginal washings from progesterone-treated mice were no more inhibitory than those from untreated mice. Thus, the success of progesterone in enhancing colonisation could not be attributed to a direct stimulatory effect of the hormone at the mucosal surface and we suggest that it may be due to a greater availability of progesterone-induced receptors for mycoplasmas in the dioestrous phase of the reproductive cycle than in the oestrous phase.

Factors influencing the ability of different mycoplasmas to colonize the genital tract of hormone-treated female mice.

Colonization of the genital tract of female mice, mainly BALB/c, by Mycoplasma genitalium, M. pneumoniae and M. pulmonis was enhanced by pretreatment of the mice with progesterone. M. fermentans, M. hominis and M. salivarium, and three serotypes and two untyped strains of Ureaplasma urealyticum colonized under the influence of oestradiol but not progesterone. Mycoplasmas dependent on progesterone were glucose-metabolizing, with strong haemadsorptive and other attachment properties, and possessed a terminal structure. Mycoplasmas dependent on oestradiol were arginine or arginine/glucose metabolizing. Ureaplasmas also required oestradiol. The oestradiol-requiring group appeared to be less cytadsorptive and devoid of a morphological terminal structure. Mycoplasmas that had had multiple passes in media were less able to colonize. This may be one, but not the only, reason for the failure of seven mycoplasmas to colonize under the influence of either hormone. The observations suggest the existence of a receptor mechanism for colonization, progesterone-requiring mycoplasmas being exposed to, and needing, genital tract cells different from those exposed to oestradiol-requiring mycoplasmas.

Intracellular location of mycoplasmas in cultured cells demonstrated by immunocytochemistry and electron microscopy.

Mycoplasma fermentans (strain 'incognitus') was incubated with HeLa cells for up to 96 h. After 24 h, mycoplasma organisms were demonstrated intracellularly by immunocytochemistry using mule anti-M. fermentans antiserum and gold labelling on ultrathin sections of both Lowicryl K4M and Araldite-embedded HeLa cells, the latter being treated with hydrogen peroxide. The Araldite-embedded cells were fixed with glutaraldehyde and osmium tetroxide in the presence of ruthenium red to stain the mucopolysaccharide surface components of both the procaryotic and eucaryotic cells. Intracellular localization of some M. fermentans organisms was confirmed by exclusion of ruthenium red from their membranes. Various numbers of mycoplasma organisms were seen per cell and occasionally some were within vacuoles, the membranes of which were also unstained by ruthenium red. The PG18 strain of M. fermentans and a strain of M. hominis were also detected intracellularly using similar methodology and homologous mule or rabbit antisera. The occasional presence of both apparently normal and some denser degenerate mycoplasmas in the same cell may indicate gradual degradation by phagolysosomal digestion.

Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium.

The polymerase chain reaction (PCR) was developed to detect Mycoplasma genitalium. Oligonucleotide primers were used to amplify a 374 bp region of the attachment protein of the mycoplasma. DNA from three strains of M. genitalium tested gave a characteristic PCR product which was not seen with DNA from any other source. As little as 10(-15) g of M. genitalium DNA could be detected and it was found in the vagina of progesterone-treated BALB/c mice inoculated with M. genitalium organisms later than they could be cultured from this site, but not in mice that never became colonised vaginally.

Neisseria gonorrhoeae colonises the genital tract of oestradiol-treated germ-free female mice.

Germ-free BALB/c mice treated with oestradiol and inoculated intravaginally with a serum-resistant strain or a freshly isolated, piliated strain of Neisseria gonorrhoeae were colonised vaginally. The organisms were recovered intermittently for a month or longer and there was evidence that they could reach the upper genital tract. Mice given progesterone and those not treated with either hormone did not become colonised. This is the first evidence of sustained mucosal colonisation in animals other than chimpanzees.