A novel bioreactor for the stable growth of Ureaplasma parvum and Ureaplasma urealyticum.

Ureaplasma species, including Ureaplasma parvum and Ureaplasma urealyticum, are challenging to culture and maintain. Here, we describe a novel bioreactor for growing high-titer liquid Ureaplasma cultures in a stable manner.

Antimicrobial activity of enacyloxin IIa and gladiolin against the urogenital pathogens Neisseria gonorrhoeae and Ureaplasma spp.

AIMS: To determine the antimicrobial activity of enacyloxin IIa and gladiolin against Neisseria gonorrhoeae and Ureaplasma spp. METHODS AND RESULTS: The Burkholderia polyketide antibiotics enacyloxin IIa and gladiolin were tested against 14 N. gonorrhoeae and 10 Ureaplasma spp. isolates including multidrug-resistant N. gonorrhoeae isolates WHO V, WHO X and WHO Z as well as macrolide, tetracycline and ciprofloxacin-resistant ureaplasmas. Susceptibility testing of N. gonorrhoeae was carried out by agar dilution, whereas broth micro-dilution and growth kinetic assays were used for Ureaplasma spp. The MIC range for enacyloxin IIa and gladiolin against N. gonorrhoeae was 0·015-0·06 mg l-1 and 1-2 mg l-1 respectively. The presence of resistance to front line antibiotics had no effect on MIC values. The MIC range for enacyloxin IIa against Ureaplasma spp. was 4-32 mg l-1 with a clear dose-dependent effect when observed using a growth kinetic assay. Gladiolin had no antimicrobial activity on Ureaplasma spp. at 32 mg l-1 and limited impact on growth kinetics. CONCLUSIONS: Enacyloxin IIa and gladiolin antibiotics have antimicrobial activity against a range of antibiotic susceptible and resistant N. gonorrhoeae and Ureaplasma isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the potential for a new class of antimicrobial against pathogens in which limited antibiotics are available. Development of these compounds warrants further investigation in the face of emerging extensively drug-resistant strains.

In-vitro activity of lipoic acid against Ureaplasma urealyticum and Ureaplasma parvum isolated from women with infections of the urogenital tract. A pilot study.

Several species of Ureaplasma bacteria are known to be present in the urogenital tract of humans, in both healthy individuals and symptomatic patients. These pathogens are associated with urogenital tract infections, infertility problems and spontaneous abortion in humans. The present study involved 77 strains of Ureaplasma species (Ureaplasma spp.), including 21 Ureaplasma urealyticum (U. urealyticum) strains and 56 Ureaplasma parvum (U. parvum) strains. Lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) are synthesized in all prokaryotic and eukaryotic cells. Research of recent years increasingly points to therapeutic properties of exogenously supplemented LA. In our study, we examined for the first time the effect of LA on the bacteria multiplication and its bactericidal activity against U. urealyticum and U. parvum. The LA concentrations used were: 1200 µg/ml, 120 µg/ml, and 12 µg/ml. The titer for each strain of Ureaplasma spp. was estimated using the color changing units (CCU) assay. For CCU measurements, a series of 10-fold dilutions of each cell culture in 0.9% NaCl (titration) was prepared and 1 CCU/ml was defined as the highest dilution of cells at which color change was detected. The strongest bacteriostatic and bactericidal effect of LA was observed at a concentration of 1200 µg/ml. In contrast, at lower LA concentrations, stimulation of the bacteria multiplication was noted for 14% of the total number of strains tested. Taken together, the current data provide novel findings about potential beneficial antimicrobial effects of LA.

Adverse pregnancy and neonatal outcomes associated with Neisseria gonorrhoeae, Mycoplasma genitalium, M. hominis, Ureaplasma urealyticum and U. parvum: a systematic review and meta-analysis protocol.

INTRODUCTION: Several bacterial sexually transmitted and genital mycoplasma infections during pregnancy have been associated with poor pregnancy and perinatal outcomes. Comprehensive and systematic information about associations between sexually transmitted infections (STI) and genital infections in pregnancy and adverse perinatal outcomes is needed to improve understanding about the evidence for causal associations between these infections and adverse pregnancy and neonatal outcomes. Our primary objective is to systematically review the literature about associations between: (1) Neisseria gonorrhoeae in pregnancy and preterm birth; (2) Mycoplasma genitalium in pregnancy and preterm birth; (3) M. hominis, Ureaplasma urealyticum and/or U. parvum in pregnancy and preterm birth. METHODS AND ANALYSIS: We will undertake a systematic search of Medline, Excerpta Medica database and the Cochrane Library and Cumulative Index to Nursing and Allied Health Literature. Following an initial screening of titles by one reviewer, abstracts will be independently assessed by two reviewers before screening of full-text articles. To exclude a manuscript, both reviewers need to agree on the decision. Any discrepancies will be resolved by discussion, or the adjudication of a third reviewer. Studies will be included if they report testing for one or more of N. gonorrhoeae, M. genitalium, M. hominis, U. urealyticum and/or U. parvum during pregnancy and report pregnancy and/or birth outcomes. In this review, the primary outcome is preterm birth. Secondary outcomes are premature rupture of membranes, low birth weight, spontaneous abortion, stillbirth, neonatal mortality and ophthalmia neonatorum. We will use standard definitions, or definitions reported by study authors. We will examine associations between exposure and outcome in forest plots, using the I2 statistic to examine between study heterogeneity. Where appropriate, we will use meta-analysis to combine results of individual studies. ETHICS AND DISSEMINATION: This systematic review of published literature does not require ethical committee approval. Results of this review will be published in a peer reviewed, open access journal. PROSPERO REGISTRATION NUMBER: CRD42016050962.

Ureaplasma parvum causes hyperammonemia in a pharmacologically immunocompromised murine model.

A relationship between hyperammonemia and Ureaplasma infection has been shown in lung transplant recipients. We have demonstrated that Ureaplasma urealyticum causes hyperammonemia in a novel immunocompromised murine model. Herein, we determined whether Ureaplasma parvum can do the same. Male C3H mice were given mycophenolate mofetil, tacrolimus, and prednisone for 7 days, and then challenged with U. parvum intratracheally (IT) and/or intraperitoneally (IP), while continuing immunosuppression over 6 days. Plasma ammonia concentrations were determined and compared using Wilcoxon rank-sum tests. Plasma ammonia concentrations of immunosuppressed mice challenged IT/IP with spent broth (median, 188 µmol/L; range, 102-340 µmol/L) were similar to those of normal (median, 226 µmol/L; range, 154-284 µmol/L, p > 0.05), uninfected immunosuppressed (median, 231 µmol/L; range, 122-340 µmol/L, p > 0.05), and U. parvum IT/IP challenged immunocompetent (median, 226 µmol/L; range, 130-330 µmol/L, p > 0.05) mice. Immunosuppressed mice challenged with U. parvum IT/IP (median 343 µmol/L; range 136-1,000 µmol/L) or IP (median 307 µmol/L; range 132-692 µmol/L) had higher plasma ammonia concentrations than those challenged IT/IP with spent broth (p < 0.001). U. parvum can cause hyperammonemia in pharmacologically immunocompromised mice.

Antibiotic Resistance among Clinical Ureaplasma Isolates Recovered from Neonates in England and Wales between 2007 and 2013.

Ureaplasma spp. are associated with numerous clinical sequelae with treatment options being limited due to patient and pathogen factors. This report examines the prevalence and mechanisms of antibiotic resistance among clinical strains isolated from 95 neonates, 32 women attending a sexual health clinic, and 3 patients under investigation for immunological disorders, between 2007 and 2013 in England and Wales. MICs were determined by using broth microdilution assays, and a subset of isolates were compared using the broth microdilution method and the Mycoplasma IST2 assay. The underlying molecular mechanisms for resistance were determined for all resistant isolates. Three isolates carried the tet(M) tetracycline resistance gene (2.3%; confidence interval [CI], 0.49 to 6.86%); two isolates were ciprofloxacin resistant (1.5%; CI, 0.07 to 5.79%) but sensitive to levofloxacin and moxifloxacin, while no resistance was seen to any macrolides tested. The MIC values for chloramphenicol were universally low (2 µg/ml), while inherently high-level MIC values for gentamicin were seen (44 to 66 µg/ml). The Mycoplasma IST2 assay identified a number of false positives for ciprofloxacin resistance, as the method does not conform to international testing guidelines. While antibiotic resistance among Ureaplasma isolates remains low, continued surveillance is essential to monitor trends and threats from importation of resistant clones.

[PERSISTENCE OF MYCOPLASMA DURING UROLITHIASIS].

AIM: Study the frequency of detection of mycoplasma and ureaplasma in clinical material from urolithiasis patients. MATERIALS AND METHODS: Clinical material samples (blood sera, urine, uroliths) from 31 urolithiasis patients were obtained during operations of urolith-removal. Cultural method, LAR and PCR were used in the study. RESULTS: The study of clinical material from 31 patients by PCR has shown, that in 25 individuals. (80.6%) DNA of mycoplasma and ureaplasma was detected, and mycoplasma DNA was more frequently detected in uroliths and less--in-blood sera. Mycoplasma hominis DNA was detected in clinical material of a significantly largerninmber of patients. 23 cultures were isolated from 8 patients by a cultural method, that were identified by PCR as M. hominis. All the isolates have grown as "mini colonies". Even after multiple passages in agar medium, reversion of "mini-colonies" into colonies with a classic morphology was not obtained. CONCLUSION: A high frequency of detection of mycoplasma and ureaplasma in clinical material of patients with urolithiasis was established. The isolated M. hominis cultures have only grown as "mini-colonies". The phenomenon discovered could give evidence on high variability of mycoplasma and a possibility of existence of previously unknown form of their persistence in human organism.

A simple agar plate preparation for effective transfer of Ureaplasma colonies onto nitrocellulose membranes for colony immunoblotting.

A simple method for preparing agar plates is presented, which allows an efficient transfer of Ureaplasma colonies to nitrocellulose membranes for subsequent immunological detection. This simple and reproducible procedure was used to demonstrate antigenic variation in the phase-variable mba-locus of Ureaplasma parvum serovar 3.

[Measurement of genital mycoplasma concentration by using a microbiological and molecular-biological methods].

AIM: Establishment of ratios that would allow to execute recalculation of mycoplasma concentration from CFU/ml and/or CCU/ml into units obtained during PCR analysis--geq/ml. MATERIALS AND METHODS: Pure cultures of Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum were studied by cultural and molecular-biological methods with quantitative evaluation. Studies of initial cultures as well as series of 10-fold dilutions were carried out. 32 experiments in total were carried out. RESULTS: Ratio between geq/ml and CFU/ml for M. hominis was 3.5; geq/ ml and CCU/ml ratio--4.4. Ratio between geq/ml and CCU/ml for U. parvum was 7.1; for U. urealyticum--11.2. CONCLUSION: Ratios between indexes obtained during quantitative study of pure genital micoplasma cultures by using 2 methods were established.

Treatment of genital mycoplasma in colonized pregnant women in late pregnancy is associated with a lower rate of premature labour and neonatal complications.

Mycoplasma hominis and Ureaplasma spp. may colonize the human genital tract and have been associated with adverse pregnancy outcomes such as preterm labour and preterm premature rupture of membranes. However, as these bacteria can reside in the normal vaginal flora, there are controversies regarding their true role during pregnancy and so the need to treat these organisms. We therefore conducted a retrospective analysis to evaluate the treatment of genital mycoplasma in 5377 pregnant patients showing symptoms of potential obstetric complications at 25-37 weeks of gestation. Women presenting with symptoms were routinely screened by culture for the presence of these bacteria and treated with clindamycin when positive. Compared with uninfected untreated patients, women treated for genital mycoplasma demonstrated lower rates of premature labour. Indeed preterm birth rates were, respectively, 40.9% and 37.7% in women colonized with Ureaplasma spp. and M. hominis, compared with 44.1% in uncolonized women (Ureaplasma spp., p 0.024; M. hominis, p 0.001). Moreover, a reduction of neonatal complications rates was observed, with 10.9% of newborns developing respiratory diseases in case of Ureaplasma spp. colonization and 5.9% in the presence of M. hominis, compared with 12.8% in the absence of those bacteria (Ureaplasma spp., p 0.050; M. hominis, p <0.001). Microbiological screening of Ureaplasma spp. and/or M. hominis and pre-emptive antibiotic therapy of symptomatic pregnant women in late pregnancy might represent a beneficial strategy to reduce premature labour and neonatal complications.

[Dynamics of change of ureaplasma laboratory strain titers and quantity of their DNA in transport medium at varying temperature].

AIM: Study of preservation dynamics of ureaplasma laboratory strain live cultures and their DNA in transport medium at varying temperature. MATERIALS AND METHODS: The study was carried out in laboratory strains Ureaplasma urealyticum serotype 8 and Ureaplasma parvum serotype 1. The quantity of live ureaplasmas was determined by method of tenfold dilutions in liquid medium. The growth of ureaplasmas was registered by changes in the color of the cultivation medium due to its alkalization by metabolism products and expressed in CCU/ml. DNA quantity in samples was determined by real time PCR performed by using Florocenosis-micoplasmas-FL test system produced by ILS. RESULTS: Live ureaplasmas wer shown to be preserved in transport medium at 4 degrees C for 12 - 29 days, at 18 - 22 degrees C--for 9 - 20 days and at 37 degrees C--for only 2 days. In samples incubated at 37 degrees C the quantity of live ureaplasmas increased and then sharply decreased to 0, at lower temperature titers of the cells decreased smoothly. The quantity of ureaplasma DNA in the process of their incubation did not change significantly. CONCLUSION: Fundamental differences in the duration of survival of U. urealyticum strain and U. parvum strain in transport medium at varying temperature were not detected. Based on the studies performed a practical conclusion can be drawn that in cases of emergency when clinical material transportation is necessary its storage in transport medium for several days is acceptable.

Ureaplasma parvum serovar 3 multiple banded antigen size variation after chronic intra-amniotic infection/colonization.

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2 × 10(7) colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n = 8; C69, n = 4); day 117 (7d Up, n = 8; C7, n = 2); and day 121 (3d Up, n = 8; C3, n = 2) of gestation (term = 145-150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation.

Azithromycin to prevent bronchopulmonary dysplasia in ureaplasma-infected preterm infants: pharmacokinetics, safety, microbial response, and clinical outcomes with a 20-milligram-per-kilogram single intravenous dose.

Ureaplasma respiratory tract colonization is associated with bronchopulmonary dysplasia (BPD) in preterm infants. Previously, we demonstrated that a single intravenous (i.v.) dose of azithromycin (10 mg/kg of body weight) is safe but inadequate to eradicate Ureaplasma spp. in preterm infants. We performed a nonrandomized, single-arm open-label study of the pharmacokinetics (PK) and safety of intravenous 20-mg/kg single-dose azithromycin in 13 mechanically ventilated neonates with a gestational age between 24 weeks 0 days and 28 weeks 6 days. Pharmacokinetic data from 25 neonates (12 dosed with 10 mg/kg i.v. and 13 dosed with 20 mg/kg i.v.) were analyzed using a population modeling approach. Using a two-compartment model with allometric scaling of parameters on body weight (WT), the population PK parameter estimates were as follows: clearance, 0.21 liter/h × WT(kg)(0.75) [WT(kg)(0.75) indicates that clearance was allometrically scaled on body weight (in kilograms) with a fixed exponent of 0.75]; intercompartmental clearance, 2.1 liters/h × WT(kg)(0.75); central volume of distribution (V), 1.97 liters × WT (kg); and peripheral V, 17.9 liters × WT (kg). There was no evidence of departure from dose proportionality in azithromycin exposure over the tested dose range. The calculated area under the concentration-time curve over 24 h in the steady state divided by the MIC90 (AUC24/MIC90) for the single dose of azithromycin (20 mg/kg) was 7.5 h. Simulations suggest that 20 mg/kg for 3 days will maintain azithromycin concentrations of >MIC50 of 1 µg/ml for this group of Ureaplasma isolates for ≥ 96 h after the first dose. Azithromycin was well tolerated with no drug-related adverse events. One of seven (14%) Ureaplasma-positive subjects and three of six (50%) Ureaplasma-negative subjects developed physiologic BPD. Ureaplasma was eradicated in all treated Ureaplasma-positive subjects. Simulations suggest that a multiple-dose regimen may be efficacious for microbial clearance, but the effect on BPD remains to be determined.

Appropriate antibiotic therapy improves Ureaplasma sepsis outcome in the neonatal mouse.

BACKGROUND: Ureaplasma causes sepsis in human neonates. Although erythromycin has been the standard treatment, it is not always effective. No published reports have evaluated Ureaplasma sepsis in a neonatal model. We hypothesized that appropriate antibiotic treatment improves Ureaplasma sepsis in a neonatal mouse model. METHODS: Two ATCC strains and two clinical strains of Ureaplasma were evaluated in vitro for antibiotic minimum inhibitory concentration (MIC). In addition, FVB albino mice pups infected with Ureaplasma were randomly assigned to saline, erythromycin, or azithromycin therapy and survival, quantitative blood culture, and growth were evaluated. RESULTS: MICs ranged from 0.125 to 62.5 µg/ml and 0.25 to 1.0 µg/ml for erythromycin and azithromycin, respectively. The infecting strain and antibiotic selected for treatment appeared to affect survival and bacteremia, but only the infecting strain affected growth. Azithromycin improved survival and bacteremia against each strain, whereas erythromycin was effective against only one of four strains. CONCLUSION: We have established a neonatal model of Ureaplasma sepsis and observed that treatment outcome is related to infecting strain and antibiotic treatment. We speculate that appropriate antibiotic selection and dosing are required for effective treatment of Ureaplasma sepsis in neonates, and this model could be used to further evaluate these relationships.

[Microbiological diagnosis of mycoplasma infections]. / Diagnóstico microbiológico de las infecciones por Mycoplasma.

The microbiological diagnosis of mycoplasma and ureaplasma infections has always been limited due to the fastidious growth of these microorganisms, as well as the lack of commercially prepared growth media, absence of rapid diagnostic procedures, and the clinical perception that these organisms are less significant in the infectious diseases setting. During the last few years, this situation has substantially improved due to the commercial availability of culture media, the development of rapid serological techniques, and, in particular, to the introduction of nucleic acid amplification assays, commercially available or "in-house" preparations. Despite the lack of proper standardisation and validation of the molecular and serological techniques, methodological advances have led to an increased detection of these microorganisms and, consequently, a greater appreciation of their clinical relevance.

The role of the multiple banded antigen of Ureaplasma parvum in intra-amniotic infection: major virulence factor or decoy?

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32-170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1ß, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.

Maternal administration of erythromycin fails to eradicate intrauterine ureaplasma infection in an ovine model.

Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. during pregnancy; however, maternally administered erythromycin may be ineffective at eliminating intra-amniotic ureaplasma infections. We examined whether erythromycin would eradicate intra-amniotic ureaplasma infections in pregnant sheep. At Gestational Day (GD) 50 (term, GD 150), pregnant ewes received intra-amniotic injections of erythromycin-sensitive Ureaplasma parvum serovar 3 (n = 16) or 10B medium (n = 16). At GD 100, amniocentesis was performed; five fetal losses (ureaplasma group, n = 4; 10B group, n = 1) had occurred by this time. Remaining ewes were allocated into treatment subgroups: medium only (n = 7), medium and erythromycin (n = 8), ureaplasma only (Up; n = 6), or ureaplasma and erythromycin (Up/E; n = 6). Erythromycin was administered intramuscularly (500 mg) every 8 h for 4 days (GDs 100-104). Amniotic fluid samples were collected at GD 105. At GD 125, preterm fetuses were surgically delivered, and specimens were collected for culture and histology. Erythromycin was quantified in amniotic fluid by liquid chromatography-mass spectrometry. Ureaplasmas were isolated from the amniotic fluid, chorioamnion, and fetal lung of animals from the Up and Up/E groups, however, the numbers of U. parvum recovered were not different between these groups. Inflammation in the chorioamnion, cord, and fetal lung was increased in ureaplasma-exposed animals compared to controls but was not different between the Up and Up/E groups. Erythromycin was detected in amniotic fluid samples, although concentrations were low (<10-76 ng/ml). This study demonstrates that maternally administered erythromycin does not eradicate chronic, intra-amniotic ureaplasma infections or improve fetal outcomes in an ovine model, potentially because of the poor placental passage of erythromycin.

Detection and characterization of human Ureaplasma species and serovars by real-time PCR.

We designed primers and probes for the detection and discrimination of Ureaplasma parvum and U. urealyticum and their 14 serovars by real-time PCR. The analytical sensitivity and specificity of the multiplex species-specific PCR were determined by testing corresponding American Type Culture Collection (ATCC) type strains, 47 other microbial species, and human genomic DNA. The limits of the multiplex PCR were 2.8 x 10(-2) CFU/microl PCR mixture for detecting U. parvum and 4.1 x 10(-2) CFU/microl PCR mixture for detecting U. urealyticum. Clinical specificity and sensitivity were proven by comparison with culture and traditional PCR. For the detection of any Ureaplasma species, the clinical sensitivity and specificity of real-time PCR were 96.9% and 79.0%, respectively, using culture as a reference. Multiplex real-time PCR was also more sensitive than traditional PCR in discriminating the two Ureaplasma species in culture-positive subcultures. Each of the 14 monoplex serovar-specific PCR assays was specific for the corresponding ATCC type strain serovar. This new species identification PCR is specific and sensitive in the detection of Ureaplasma species in clinical specimens, and the serovar-specific PCR assays are the first set of complete genotypic assays to differentiate all 14 known Ureaplasma serovars. These assays provide quick and reliable means for investigating the epidemiology and pathogenicity of ureaplasmas at the serovar level.

Concurrent titration and determination of antibiotic resistance in ureaplasma species with identification of novel point mutations in genes associated with resistance.

Antibiotic resistance determination of Ureaplasma spp. (Ureaplasma parvum and Ureaplasma urealyticum) usually requires predetermination of bacterial titer, followed by antibiotic interrogation using a set bacterial input. This 96-well method allows simultaneous quantification of bacteria in the presence and absence of antibiotics. A method for determining precise MICs and a method for screening against multiple antibiotics using breakpoint thresholds are detailed. Of the 61 Ureaplasma-positive clinical isolates screened, one (1.6%) was resistant to erythromycin (MIC, >64 mg/liter) and clarithromycin (MIC, 4 mg/liter), one to ciprofloxacin (1.6%), and one to tetracycline/doxycycline (1.6%). Five isolates were also consistently found to have an elevated MIC of 8 mg/liter for erythromycin, but this may not represent true antibiotic resistance, as no mutations were found in the 23S rRNA operons or ribosome-associated L4 and L22 proteins for these strains. However, two amino acids (R66Q67) were deleted from the L4 protein of the erythromycin-/clarithromycin-resistant strain. The tetM genetic element was detected in the tetracycline-resistant clinical isolate as well as in the positive control Vancouver strain serotype 9. The tetM gene was also found in a fully tetracycline-susceptible Ureaplasma clinical isolate, and no mutations were found in the coding region that would explain its failure to mediate tetracycline resistance. An amino acid substitution (D82N) was found in the ParC subunit of the ciprofloxacin-resistant isolate, adjacent to the S83L mutation reported by other investigators in many ciprofloxacin-resistant Ureaplasma isolates. It is now possible to detect antibiotic resistance in Ureaplasma within 48 h of positive culture without prior knowledge of bacterial load, identifying them for further molecular analysis.

Ureaplasma colonization of amniotic fluid and efficacy of antenatal corticosteroids for preterm lung maturation in sheep.

OBJECTIVE: The objective of the study was to assess the efficacy of maternal betamethasone for improving preterm lung function in the presence of inflammation induced by amniotic fluid Ureaplasma colonization. STUDY DESIGN: Ewes bearing single fetuses were randomized to receive an intraamniotic injection of Ureaplasma parvum (serovar 6; 2 x 10(7) colony-forming units) or vehicle at 86 +/- 2 days of pregnancy (mean +/- SD: term is 150 days), followed by maternal intramuscular betamethasone (0.5 mg/kg) or saline, either 2 or 7 days before delivery of lambs at 123 +/- 1 d. RESULTS: Amniotic fluid interleukin-8 was elevated by ureaplasmas (P = .049) but unaffected by betamethasone. Lung inflammation induced by ureaplasmas was not affected by betamethasone. Lung compliance was increased by Ureaplasma colonization (P = .009) and betamethasone (P = .042), and effects were additive. Lung surfactant was increased by Ureaplasma colonization (P < .001) and betamethasone 7 days (P = .001), but not 2 days, before delivery. CONCLUSION: Inflammation improves preterm lung function because of increases in surfactant. Antenatal corticosteroids further augment lung function through an apparently independent mechanism.