Comparison of commercially available media for detection and isolation of Ureaplasma urealyticum and Mycoplasma hominis.

The Mycotrim Triphasic flask system (Irvine Scientific, Irvine, Calif.) was compared with a system composed of Mycotrim GU broth (Irvine Scientific) and A7 or A8 agar (Remel, Lenexa, Kans.) for the ability to detect Ureaplasma urealyticum and Mycoplasma hominis from 129 genital specimens. Of the 64 specimens positive for U. urealyticum, 25, 98, and 100% were detected on Mycotrim Triphasic agar and A7 and A8 agars, respectively. All 18 specimens that grew M. hominis were detected by A7 and A8 agars, and 94% grew on Mycotrim Triphasic agar. Mycotrim GU broth detected all of the positive specimens, and Mycotrim Triphasic broth detected all but one. Mycotrim GU broth inoculated simultaneously with either A7 or A8 agar was found to be more sensitive and cost-effective than the Mycotrim Triphasic flask system.     

Evaluation of Mycotrim-GU for isolation of Mycoplasma species and Ureaplasma urealyticum.

The Mycotrim-GU (Hana Biologics, Berkeley, Calif.) biphasic culture system and a conventional system were compared for their ability to detect Ureaplasma urealyticum and Mycoplasma species in 100 clinical specimens. Both systems detected 18 Mycoplasma spp. isolates. The average colony detection time was 1.9 days with the Mycotrim-GU and 2.3 days with the conventional system. The Mycotrim-GU agar detected all 33 U. urealyticum isolates recovered in the study, and the conventional agar detected 31. In addition to the U. urealyticum isolates recovered from the agar, there were several specimens that, although they did not grow colonies on the agar, gave an alkaline broth change. Of these specimens, two were found with the conventional system and seven were found with the Mycotrim-GU. The average detection time of U. urealyticum colonies was 2.0 days for the conventional agar and 1.7 days for the Mycotrim-GU. The Mycotrim-GU offers several advantages over the conventional system: it is commercially available, consists of a one-flask system which is ready to use, has a significantly longer shelf life, and is cost competitive. This study showed the Mycotrim-GU to be an effective system for detecting the genital mycoplasmas.     

Evaluation of the Mycoplasma Duo kit for the detection of Mycoplasma hominis and Ureaplasma urealyticum from urogenital and placental specimens

This study compares the Mycoplasma Duo kit for the detection of genital mycoplasmas with conventional culture using A7 differential agar for the detection of Mycoplasma hominis and Ureaplasma urealyticum in clinical samples. Detection of the mycoplasmas is based on the specific metabolic properties of each organism to hydrolyse either arginine or urea. The Mycoplasma Duo test showed a significantly higher detection rate than did culture, although many of the culture-negative results may have been due to the presence of bacterial overgrowth.     

Comparison of media for isolation of Ureaplasma urealyticum and genital Mycoplasma species

A total of 484 frozen patient specimens originally positive for Ureaplasma urealyticum or Mycoplasma spp. or both were recultured, and the results were compared on the following media: Shepard's A7 agar, modified phenol red-urea, SP-4-urea, modified phenol red-arginine, and SP-4-arginine broths. Of 351 specimens positive for U. urealyticum, 30 (8.5%) were detected only in one or more of the broth media, whereas 117 (33%) were positive only on A7 agar. Separate use of the SP-4-urea broth or modified phenol red-urea broth isolated all but 1 and 2, respectively, of the negative A7 agar cultures. Of the 76 specimens positive for large colony Mycoplasma spp., 58 (76.3%) were not grown on the primary plating (A7) agar. Of 73 broth isolates, 73 (100%) grew in SP-4-arginine broth, and 64 (87.7%) grew in modified phenol red-arginine broth. Modified SP-4 broth appears to be a useful supplement to the A7 plating medium commonly used in the diagnostic laboratory for the isolation of U. urealyticum and Mycoplasma spp.     

In vitro activity of new quinolones against Mycoplasma pathogenic to humans

The in vitro activity of new quinolones was evaluated against Mycoplasma pneumoniae (10 strains) and Mycoplasma hominis (approximately equal to 70 strains) by agar dilution, and against Ureaplasma urealyticum (approximately equal to 115 strains) by broth dilution. The static effect of pefloxacin, ofloxacin, ciprofloxacin, enoxacin was investigated for all the strains. Rosoxacin was included in the tests for U. urealyticum and M. hominis. Pefloxacin, ofloxacin, ciprofloxacin and enoxacin were within the same range of sensitivity for M. pneumoniae; the minimal inhibitory concentrations (MICs) of the 10 strains were 1 mg/l for ciprofloxacin, 2 mg/l for pefloxacin, MICs range was (0.05-1 mg/l) for ofloxacin and (0.5-4 mg/l) for enoxacin. Ciprofloxacin was the most active compound against M. hominis; MICs range and mode MICs were respectively in mg/l: (0.1-1) 0.5 for ciprofloxacin, (0.2-2) 0.5 for ofloxacin, (0.5-2) 1 for pefloxacin, (0.5-8) 2 for enoxacin, (2-16) 2 for rosoxacin. Ofloxacin was the most active compound against U. urealyticum; MICs range and mode MICs were respectively in mg/l: (0.2-2) 1 for ofloxacin, (0.1-8) 2 for rosoxacin, (0.5-8) 4 for pefloxacin, (1-16) 4 for ciprofloxacin, (2-32) 8 for enoxacin. No difference could be observed between tetracycline sensitive or resistant strains.     

High rates of genital mycoplasma infection in the highlands of Papua New Guinea determined both by culture and by a commercial detection kit.

Duplicate vaginal swabs were collected from 100 women, and comparisons were made between an in-house broth-agar culture system and a commercially available kit, the Mycoplasma IST kit (bioMérieux), for the detection of Mycoplasma hominis and Ureaplasma urealyticum. There was good agreement between the two systems for detection of the genital mycoplasmas in terms of sensitivity, with values of > 92% being obtained. In terms of specificity, the mutual comparisons were less favorable, though specificity values of > 72% were obtained. Statistically there was no significant difference in the performance of the two tests (P < 0.1 for both M. hominis and U. urealyticum). While the broth-agar culture system was considerably less expensive than the kit, the Mycoplasma IST kit provided additional information on antibiotic susceptibilities and had the advantages of a shelf life of up to 12 months and not requiring the preparation of culture media. The prevalences of colonization obtained for M. hominis and U. urealyticum were extremely high in this randomly selected group of women from periurban and rural settlements in the Eastern Highlands of Papua New Guinea, being > or = 70% for M. hominis and > or = 78% for U. urealyticum. colonization with both genital mycoplasmas simultaneously was also very common, with > or = 60% of women being colonized by both M. hominis and U. urealyticum.     

Lack of association between genital mycoplasmas and infertility

We studied the relation between colonization with Mycoplasma hominis and Ureaplasma urealyticum, and the results of infertility studies in 205 women with involuntary infertility of at least one year's duration. Isolation of M. hominis (but not of U. urealyticum) was significantly (P = 0.002) more common in patients with a history of pelvic inflammatory disease. However, no relation could be shown between these genital mycoplasmas and any of the following: evidence of prior pelvic inflammatory disease as determined by hysterosalpingography and laparoscopy; cervical inflammation; numbers and motility of spermatozoa on postcoital test; pyosemia; quality of cervical mucus; whether the cause of infertility was related to male or female factors, both, or neither; and occurrence and outcome of subsequent pregnancy. Mycoplasmas were cultured from only 10 of 203 endometrial biopsy specimens (4.9 per cent), and in no instance was inflammation associated with this finding. Out studies do not support a role for genital mycoplasmas in the cause of infertility.     

Comparative evaluation of media for isolation of ureaplasma urealyticum and genital mycoplasma species.

Two media systems were compared for isolation of Ureaplasma urealyticum and genital Mycoplasma sp. System 1 (S-1) consisted of arginine agar and an arginine biphasic medium for isolation of Mycoplasma sp. and urea agar and urea broth for isolation of U. urealyticum. System 2 (S-2) utilized Boston broth, which is a urea-containing broth, and A7 agar, both of which support the growth of both species. Urine samples, some freshly collected and some known-positive frozen samples, were used as inocula for the two media systems. With S-1, U. urealyticum was recovered in 68% of U. urealyticum-positive cultures: 58% were detected in urea broth, and 60% were identified on urea agar. When the S-2 system was used for culture of the same samples. U. urealyticum was recovered in 98% of the cultures, with 94% detected in Boston broth and 92% identified on A7 agar. Mycoplasma sp. was recovered in S-1 and S-2 in 97 and 100% of Mycoplasma sp.-positive cultures, respectively. The S-1 arginine broth gave positive results for 89% of the cultures, and the arginine agar was positive for 97% of the cultures. The S-2 Boston broth and A7 agar gave positive results for 92 and 97% of the cultures, respectively. For the isolation of U. urealyticum, colony counts were higher, growth was seen sooner, and colonies were larger when the S-2 media system was used. Overall, the cost per test of the S-2 system was less both in technologist time and in reagent costs.     

Isolation of genital mycoplasmas from the blood of neonates and women with pelvic infection using conventional SPS-free blood culture media

Standard blood culture media used in our laboratory were tested for their ability to support the growth of Mycoplasma hominis and Ureaplasma urealyticum. Small inocula (approximately 10 colony forming units per ml) of both organisms grew in diphasic tryptone soya medium but not in any of several media containing sodium polyanetholesulphonate (SPS) including a modified Schaedler broth (RWH anaerobic medium) and two BACTEC media (6B and 7D). Both organisms were inhibited even by very low concentrations of SPS but grew well in the Royal Women's Hospital (RWH) anaerobic medium when SPS was omitted. During a 22-month period, routine "blind" plating of the aerobic blood cultures on to mycoplasma agar resulted in isolation of M. hominis or U. urealyticum from 12 women with postpartum or postoperative pelvic infection, and from 3 neonates. Genital mycoplasmas represented 35% of significant isolates from adult blood cultures.     

Antibiotic sensitivity of Mycoplasma pathogenic for man

Human pathogen mycoplasmas (Mycoplasma pneumoniae, Mycoplasma hominis, Ureaplasma urealyticum) are intrinsically resistant to antibiotics which inhibit the cell wall biosynthesis (beta-lactams, vancomycin, bacitracin), to polymyxins, rifamycins, sulfonamides, trimethoprim, 5-nitroimidazoles, nitrofurans and to presently available quinolones. These three species are moderately susceptible to aminoglycosides, susceptible to chloramphenicol and highly susceptible to tetracyclines. M. pneumoniae is susceptible to macrolides, lincosamins and streptogramins. M. hominis is resistant to early macrolides (erythromycin, oleandomycin, spiramycin) and susceptible to new macrolides (josamycin, midecamycin, rosaramicin), lincosamins and streptogramins. U. urealyticum is resistant to lincosamins and susceptible to macrolides and streptogramins. Discordant results from various reports can be explained by differences in methods and breakpoint concentration values. In M. pneumoniae species, two strains resistant to macrolides and lincosamins have been described. In M. hominis species, one strain resistant to tetracyclines and another one resistant to tetracyclines and chloramphenicol have been reported. Two to ten percent of U. urealyticum strains are resistant to tetracyclines. These resistances are likely to be plasmid-mediated.     

Experimental animal infections with Mycoplasma hominis and Ureaplasma urealyticum.

Subcutaneous tissue cavities in mice and guinea pigs were infected with human isolates of Ureaplasma urealyticum and Mycoplasma hominis. The minimal infective dose for M. hominis was as low as less than 10 color-changing units (CCU) for mice and 10(2) CCU for guinea pigs. The minimal infective dose for U. urealyticum was as low as less than 10 CCU for mice and 10(4) CCU for guinea pigs. Mouse infections with either U. urealyticum or M. hominis persisted for 1 day to greater than 4 months. Guinea pigs remained infected for up to 4 weeks. Two M. hominis isolates were similar in their ability to infect subcutaneous tissue cavities but two U. urealyticum isolates varied in their ability to infect the cavities. The histopathology of the M. hominis and U. urealyticum infections was similar: an initial intense polymorphonuclear response with giant cells, followed in 4 weeks by histiocytes and giant cells with some plasma cells and lymphocytes.     

Mycoplasma hominis and Ureaplasma urealyticum in different gynecologic diseases

The incidence of Mycoplasma hominis (M. hominis) y Ureaplasma urealyticum (U. urealyticum) was investigated in 113 endocervical samples obtained from women who were seen for different gynecological pathologies. Forty-seven (42%) patients were positive to these microorganisms; 26 cases (23%) were positive for M. hominis and 21 (19%; p = NS) for U. urealyticum. Average age was 32.1 +/- 7.7 years; the average number of sexual partners was 1.7 +/- 1.1. Eleven of 17 patients with 3 o more sexual partners were positive for Genital Mycoplasma (GM), and U. urealyticum was found more often in this group. A higher incidence of GM was found in women between 26 and 30 years (34%); 57.5% of the patients with positive cultures for GM had begun sexual activity before 20 years of age. M. Hominis was found in 61% of women with no parity and U. urealyticum in 71% of parous women. The cultures were positive in 10 of 14 patients with pelvic inflammatory diseases (PID). A cervical biopsy was taken from 52 cases and the diagnosis of cervical intraepithelial neoplasia (CIN) was made in 49 (94%) but only 24 of them were positive for GM (50%). Thirty-five patients suffered sterility, and 12 (34%) were positive for GM, however all positive cases consulted because of primary sterility. The conclusions obtained from this study are: 1) Near half of the patients was positive for GM and none of the species was predominant over the other. 2) The more sexual partners the higher was the incidence of GM, especially U. Urealyticum. 3) The lower the age of the first sexual intercourse the higher the probability of contamination with these microorganisms. 4) M. hominis was more common in nulliparous women and U. urealyticum was found more often in parous patients; the number of deliveries did not have influence in these findings. 5) A statistical significance between GM and PID was found (p = 0.03). 6) GM have no influence on spontaneous abortion. 7) No statistical significance was found between GM and the beginning and evolution of CIN. 8) No relation statistically significative was found between GM and sterility.     

Cytokine concentrations in seminal plasma from subfertile men are not indicative of the presence of Ureaplasma urealyticum or Mycoplasma hominis in the lower genital tract

The inflammatory response to the presence of Ureaplasma urealyticum or Mycoplasma hominis in the lower genital tract of subfertile men without any signs or symptoms of infection was investigated by measuring the concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in seminal plasma. Semen samples were collected from 30 culture-positive subfertile males and 23 culture-negative subfertile males. Enzyme-linked immunosorbent assays showed that IL-8 was present in relatively high concentrations (0.12-4.8 ng/ml) in all semen samples investigated. In contrast, the other cytokines were only detectable in 72% (IFN-gamma), 44% (IL-6) and 19% (TNF-gamma) of the samples and were present in relatively low concentrations (1-410 pg/ml). Seminal plasma cytokine concentrations were similar in samples from culture-positive and culture-negative males. These data strongly indicate that the presence of U. urealyticum or M. hominis in the lower genital tract of subfertile males reflects a silent colonisation rather than infection.     

Isolation of mycoplasmas from female genitalia

The author tested ways of collection, transport and storage of material for mycoplasmatological examination. The use of tampons on a stick during transport in urea substrate medium proved useful. The site of maximum occurrence of mycoplasmas was the posterior vaginal vault. On examination of the vaginal secretion of 804 women Mycoplasma hominis was isolated in 29.6% and Ureaplasma urealyticum in 65.2% of the cases. Concurrent isolation of Mycoplasma hominis and Ureaplasma urealyticum was recorded in 22.1% of the women. The results of the examination do not suggest the participation of mycoplasmas in the development of aminocolpitis. In pregnant women there is a greater probability of colonization of the vagina by U. urealyticum.     

Systematic screening tests for Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in urogenital specimens of infertile couples

We conducted a prospective study on 100 couples consulting for infertility at the teaching Hospital of Tours, with the scope to determine if there is a benefit for systematic screening of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum among genito-urinary specimen when exploring couples infertility. C. trachomatis was detected by PCR on sperm, endocervix and urine specimen. M. hominis and U. urealyticum were detected by culture on A7 agar medium and with minigaleries on sperm and endocervix specimen. Standard cultures were also performed on sperm, endocervix, vaginal and urine specimen. Only one specimen (sperm) was positive for C. trachomatis. Three percent of the specimen were positive for U. urealyticum (from which 2,5% of the sperm specimen). No specimen was positive for M. hominis. Our results show that screening of C. trachomatis, M. hominis and U. urealyticum is not systematically required for among check up of infertile couples, given the prevalence of chlamydiosis among the population studied. However, it would be interesting to perform it on a targeted population, according to anamnestic or clinical criteria. In addition, an important modification of vaginal flora was observed in 12% of cases, and 2 vaginosis were diagnosed; the putative consequences of this disequilibrium has to be further investigated.     

In vitro activity of sparfloxacin against mycoplasmas]

The in vitro activity of the new difluorinated quinolone sparfloxacin against mycoplasmas was studied comparatively with ofloxacin, doxycycline, and erythromycin. Minimal inhibitory concentrations (MICs) were determined by agar dilution for 21 strains of Mycoplasma pneumoniae (Mp), 20 strains of M. hominis (Mh), 7 strains of M. genitalium (Mg), and 3 strains of M. fermentans (Mf), and by broth dilution for 49 strains of Ureaplasma urealyticum (Uu). Sparfloxacin was very active against all tested mycoplasmas, with the following MICs (microgram/ml): 0.1 for Mp, 0.05-0.1 for Mg, less than or equal to 0.01 for Mh, less than or equal to 0.01-0.05 for Mf, and 0.1-0.5 for Uu. Minimal bactericidal concentration was 1 microgram/ml for Uu. Sparfloxacin was more active than ofloxacin against all the mycoplasmas tested and was the most active compound against Mh and Mf. Erythromycin had the lowest MICs against Mp and Mg. Sparfloxacin exhibited comparable effectiveness against doxycycline-susceptible and doxycycline-resistant strains. Sparfloxacin appears to be one of the most active agents in vitro against mycoplasmas.     

Mycoplasma hominis in pregnancy

One hundred and seventyone antenatal patients were examined for the presence of ;large colony' mycoplasmas in the vagina, and for complement-fixing antibody to Mycoplasma hominis. In 25 patients the findings before and after delivery were compared. In patients from whom M. hominis was grown, antibody was twice as common after delivery, and the development of antibody was sometimes associated with pyrexia and signs of genital tract infection.     

Evaluation of PPLO, A7B, E, and NYC agar media for the isolation of Ureaplasma urealyticum and Mycoplasma species from the genital tract

Four agar media (PPLO, NYC, A7B, and E) which are commonly used for the isolation of urogenital tract Mycoplasma species and Ureaplasma urealyticum were compared by culturing swabs of the endocervix of 334 pregnant women on all four media. To permit growth of both Mycoplasma and U. urealyticum, selective ingredients were omitted from the media tested. A7B and E agar were both satisfactory for the isolation of Mycoplasma species, recovering 92 and 82%, respectively, of all Mycoplasma species isolated. Only A7B agar was satisfactory for U. urealyticum isolation, recovering 96% of all isolates. Several modifications of PPLO, NYC, and E agar failed to significantly improve recovery of U. urealyticum on these media. A7B agar was clearly superior to all other media tested in terms of recovery rate, typical appearance of colonies, and ease of reading. A7B can be used for the isolation of both U. urealyticum and Mycoplasma species from urogenital sites.     

Isolation of genital mycoplasmas and Chlamydia trachomatis in stillborn and neonatal autopsy material

Chlamydia trachomatis and the genital mycoplasmas are significantly prevalent in sexually active women. How these organisms may affect the outcome of pregnancy and the neonate was the principal thrust of this investigation. Placenta, liver, and lung tissue were cultured from Mycoplasma hominis, Ureaplasma urealyticum, Chlamydia trachomatis, and aerobic as well as anaerobic bacteria in 432 stillborn and neonatal autopsies. Genital mycoplasmas were isolated from 36 cases (8.3%). Acute chorioamnionitis and funisitis were present significantly more often in cases with genital mycoplasma than in those without these organisms. Isolation of genital mycoplasmas was not associated with an increased incidence of intrauterine fetal death, villitis, hyaline membrane disease, congenital anomalies, or polymorphonuclear leukocytes in alveolar spaces. Chlamydia trachomatis was not found in any of the sites sampled.     

In vitro sensitivity to antibiotics of genital mycoplasmas isolated in Toulouse. Study of new molecules (macrolides and quinolones)]

The minimal metabolism-inhibiting concentrations (MMC) of 11 antibiotics were determined for 40 strains each of M. hominis and U. urealyticum using a terminal color change broth method. All strains were recovered in 1990. Resistance to tetracycline (MMC greater than 8 mg/l) was found for 12.5% of strains of M. hominis and U. urealyticum, as compared with 5% in 1985. Rokitamycin was the most active macrolide against M. hominis (MMC 90: 0.06 mg/l). U. urealyticum strains were susceptible to all the macrolides tested, with the greatest activities being seen for rokitamycin and clarithromycin (MMC 90: 0.06 mg/l and 0.12 mg/l respectively). Sparfloxacin was the most active quinolone against both species. Human clinical trials designed to evaluate these new molecules for the treatment of mycoplasmal and ureaplasmal genital infections are warranted.