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.

The relationship between the biovars and the antimicrobial resistance of Ureaplasma urealyticum in female patients with urogenital infections.

BACKGROUND: Ureaplasma urealyticum is considered as one of the main pathogens found in women with urogenital infection. This study aimed to investigate the relationship between the biovars, serovars, and their antimicrobial resistance against antibiotics in female patients with urogenital infection. METHODS: Two hundred and forty-six cervical secretion samples (125 female outpatients as the patient group, 121 healthy female subjects as the control group) were first collected and analyzed for U. urealyticum using the Mycoplasma Identification and Antimicrobial Susceptibility Testing; then polymerase chain reaction (PCR) was carried out to identify the biovars and serovars of U. urealyticum-positive samples. RESULTS: The prevalence of U. urealyticum in the patient group (57. 60%) was higher than that in the control group (24.79%, P<.01). The main biovar was biovar 1, and the main serovars were 1 (S1), 3 (S3), 6 (S6) in biovar 1. Mixed infection was observed in biovar 2. According to the results of Antimicrobial Susceptibility Testing in the patient group, biovar 1 shows more resistance to minocycline, doxycycline, and azithromycin than biovar 2 (P<.05). Serovars S1, S3 and S6 have the highest resistant rate to ofloxacin (84.38%), roxithromycin (84.62%), and azithromycin (90.90%), respectively. CONCLUSIONS: A high prevalence of U. urealyticum was observed in female patients with urogenital infections. And the biovar 1 and the serovars 1, 3, 6 were the main types of pathogens.

Clonality and distribution of clinical Ureaplasma isolates recovered from male patients and infertile couples in China.

Ureaplasma spp. have gained increasing recognition as pathogens in both adult and neonatal patients with multiple clinical presentations. However, the clonality of this organism in the male population and infertile couples in China is largely unknown. In this study, 96 (53 U. parvum and 43 U. urealyticum) of 103 Ureaplasma spp. strains recovered from genital specimens from male patients and 15 pairs of infertile couples were analyzed using multilocus sequence typing (MLST)/expanded multilocus sequence typing (eMLST) schemes. A total of 39 sequence types (STs) and 53 expanded sequence types (eSTs) were identified, with three predominant STs (ST1, ST9 and ST22) and eSTs (eST16, eST41 and eST82). Moreover, phylogenetic analysis revealed two distinct clusters that were highly congruent with the taxonomic differences between the two Ureaplasma species. We found significant differences in the distributions of both clusters and sub-groups between the male and female patients (P < 0.001). Moreover, 66.7% and 40.0% of the male and female partners of the infertile couples tested positive for Ureaplasma spp. The present study also attained excellent agreement of the identification of both Ureaplasma species between paired urine and semen specimens from the male partners (k > 0.80). However, this concordance was observed only for the detection of U. urealyticum within the infertile couples. In conclusion, the distributions of the clusters and sub-groups significantly differed between the male and female patients. U. urealyticum is more likely to transmit between infertile couples and be associated with clinical manifestations by the specific epidemic clonal lineages.

Antibiotic Susceptibility and Sequence Type Distribution of Ureaplasma Species Isolated from Genital Samples in Switzerland.

Antibiotic resistance in Ureaplasma urealyticum/Ureaplasma parvum and Mycoplasma hominis is an issue of increasing importance. However, data regarding the susceptibility and, more importantly, the clonality of these organisms are limited. We analyzed 140 genital samples obtained in Bern, Switzerland, in 2014. Identification and antimicrobial susceptibility tests were performed by using the Mycoplasma IST 2 kit and sequencing of 16S rRNA genes. MICs for ciprofloxacin and azithromycin were obtained in broth microdilution assays. Clonality was analyzed with PCR-based subtyping and multilocus sequence typing (MLST), whereas quinolone resistance and macrolide resistance were studied by sequencing gyrA, gyrB, parC, and parE genes, as well as 23S rRNA genes and genes encoding L4/L22 ribosomal proteins. A total of 103 samples were confirmed as positive for U. urealyticum/U. parvum, whereas 21 were positive for both U. urealyticum/U. parvum and M. hominis. According to the IST 2 kit, the rates of nonsusceptibility were highest for ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.9%), erythromycin (1.9%), and azithromycin (1%). However, inconsistent results between microdilution and IST 2 kit assays were recorded. Various sequence types (STs) observed previously in China (ST1, ST2, ST4, ST9, ST22, and ST47), as well as eight novel lineages, were detected. Only some quinolone-resistant isolates had amino acid substitutions in ParC (Ser83Leu in U. parvum of serovar 6) and ParE (Val417Thr in U. parvum of serovar 1 and the novel Thr417Val substitution in U. urealyticum). Isolates with mutations in 23S rRNA or substitutions in L4/L22 were not detected. This is the first study analyzing the susceptibility of U. urealyticum/U. parvum isolates in Switzerland and the clonality outside China. Resistance rates were low compared to those in other countries. We hypothesize that some hyperepidemic STs spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important issue.

Effects of biovar I and biovar II of ureaplasma urealyticum on sperm parameters, lipid peroxidation, and deoxyribonucleic acid damage in male infertility.

OBJECTIVE: To reveal the impact of 2 biovars of Ureaplasma urealyticum on the sperm of infertile men by producing reactive oxygen species (ROS). METHODS: A total of 223 infertile and 146 fertile men were recruited into the study. Standard semen analysis was performed. Culturing and real-time polymerase chain reaction were used to identify biovars of U urealyticum in the semen. Semen ROS, malondialdehyde, and total superoxide dismutase levels were measured. Sperm nuclear deoxyribonucleic acid (DNA) damage was assessed of by sperm chromatin structure assay and single-cell gel electrophoresis. RESULTS: Biovar II infection was more frequent in infertile men (P=.036). When compared with uninfected individuals, biovar II-infected men displayed decreases in spermatozoa concentration (P=.041); biovar II and mix-infected men displayed decreases in total motility (P=.015; P=.014, respectively) and increase in leukocyte counts (P=.004; P=.003, respectively). Except for total superoxide dismutase level, indicators for peroxide including ROS level, malondialdehyde level, DNA fragmentation index and high DNA stainable in sperm chromatin structure assay, and tail moment in single-cell gel electrophoresis exhibited the significant differences between both infected groups vs the uninfected group (P<05). CONCLUSION: Compared with biovar I, biovar II is more likely to cause male infertility. Increased leukocyte counts, ROS level elevation, and subsequent spermatozoa membrane and DNA damage may be involved in this pathogenesis.

[Distribution and pathogenicity of Ureaplasma urealyticum serotypes in female cervix].

OBJECTIVE: To explore the distribution of Ureaplasma urealyticum (UU) serotypes in cervix secretion and examine the relationship between serotype and host age, clinical phenotypes and vaginal Lactobacilli. METHODS: A total of 444 cervical secretion samples were collected from healthy subjects and 342 cervical secretion samples from females with genital diseases between October and December 2012 at Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. Biovar-typing was performed by PCR based on multiple-banded antigen (MBA) gene. And serotyping was performed by real-time PCR based on specific nucleotide sequences. RESULTS: The positive rate of UU in disease group (62.6%, 214/342) was significantly higher than that in healthy group (44.1%, 196/444, P < 0.05). The main serotypes in both groups were 1, 3, 6, 9 and mixed infection.No significant difference existed between host age and UU serotype (P > 0.05) . The distribution of serotype 3 in disease and Lactobacilli abnormal group was significantly higher than that in healthy and Lactobacilli normal groups (13.1% (28/214) vs 5.1% (10/196) , 12.2% (27/221) vs 5.8% (11/189) , both P < 0.05) whereas the distribution of serotype 6 in disease and Lactobacilli abnormal groups was significantly lower than that in healthy and Lactobacilli normal groups (14.0% (30/214) vs 30.1% (59/196) , 16.3% (36/221) vs 28.0% (53/189) , both P < 0.05). CONCLUSIONS: 1, 3, 6, 9 and mixed infection are the main UU serotypes in female cervix.Serotype 3 and mixed infection may be associated with female genital infection.

Molecular investigation of menstrual tissue for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis collected by women with a history of infertility.

AIM: At present, routine laboratory investigation of the infectious agents implicated in female genital infections is mainly based on culture/direct fluorescence antibody (DFA) (immunofluorescence antibody test) results of cervicovaginal secretions. In this study the use of the menstrual tissue is introduced for the molecular detection of pathogens which are implicated in female infertility. MATERIAL AND METHODS: Cervicovaginal secretions and menstrual tissue samples of 87 women (mean age 34.07 ± 5.17) experiencing infertility problems were screened for Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis presence using polymerase chain reaction (PCR, light cycler-PCR). Cervicovaginal secretions were also tested by the culture/DFA technique. The results were compared using the binomial test. RESULTS: In the overall study group, the prevalence of C. trachomatis was 25.3%, 18.3%, and 13.8%, the prevalence of U. urealyticum was 18.3%, 16.09% and 12.6% and the prevalence of M. hominis was 13.7%, 19.5% and 8.0% in the menstrual tissue, cervicovaginal secretions using PCR and cervicovaginal secretions culture/DFA, respectively. A statistically significant difference was revealed between the two methods for all three microbes and between menstrual tissue and cervicovaginal secretions PCR for chlamydia. CONCLUSIONS: The use of menstrual tissue along with the PCR method seems to be an effective and thus novel alternative for the investigation of the infectious agents lying in the genital tract. One of the main advantages of this technique compared to cervicovaginal secretions is that it is non-invasive and the sample can be collected at home, thus allowing the early detection and treatment of a condition that can otherwise lead to serious consequences, such as tubal obstruction, pelvic inflammatory disease, ectopic pregnancy, spontaneous abortions and unexplained infertility.

[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.

Investigation of Ureaplasma urealyticum biovars and their relationship with antimicrobial resistance.

PURPOSE: To develop Taqman fluorescence quantitative polymerase chain reaction (PCR) method for investigating the characteristics of the distributions of Ureaplasma urealyticum (UU) biovars and to explore the relationship between UU biovars and antimicrobial resistance. MATERIALS AND METHODS: By the method of culture, Ureaplasma species were detected. Taqman fluorescence quantitative PCR for detecting UU biovars were developed and UU clinical isolates were detected to distinguish biovars. The broth micro-dilution susceptibility testing methods were used to determine UU susceptibility. RESULTS: By Taqman PCR method, UU biovars was successfully detected. Of 126 samples, biovar 1 was found in 73 (57.94%). There was a statistical difference between genital-urinary tract infection group and asymptomatic group (P<0.05). In the region, UU biovar 1 to 9 kinds of agents kept higher susceptibility rates, but biovar 2 maintained higher susceptibility rates only to tetracyclines. Compared with biovar 1, UU biovar 2 resistance rates to 7 kinds of agents were higher (P<0.05). CONCLUSIONS: (1) Our new established Taqman PCR method is a useful tool for screening UU biovars. (2) UU biovar 1 predominated in asymptomatic population; whereas in genital-urinary tract infection population UU biovar 2 had a higher proportion. (3) The characteristics of drug resistance were different between UU biovars. Overall, both two biovars remained higher susceptibility rates to tetracyclines. A majority of biovor 1 strains were sensitive to macrolides and quinolones; while only a small number of biovar 2 strains kept sensitive to roxithromycin and quinolones, a large proportion of biovar 2 strains were found in intermediate ranges.

Genotypic characterization of ureaplasma serovars from clinical isolates by pulsed-field gel electrophoresis.

Genetic relationships within ureaplasma serovars were investigated by pulsed-field gel electrophoresis (PFGE). One hundred thirteen Ureaplasma parvum isolates and 78 Ureaplasma urealyticum isolates were different from their ATCC serovar type strains and different within the same serovars. The organisms were geographically widespread. No unique patterns were associated with invasive disease.

Extensive horizontal gene transfer in ureaplasmas from humans questions the utility of serotyping for diagnostic purposes.

Ureaplasma parvum and Ureaplasma urealyticum are sexually transmitted, opportunistic pathogens of the human urogenital tract. There are 14 known serovars distributed between the two species. For decades, it has been postulated based upon limited data that virulence is related to serotype specificity. The results were often inconclusive due to the small sample size and extensive cross-reactivity between certain serovars. We developed real-time quantitative PCRs that allow reliable differentiation of the two species and type strains of each of the 14 serovars. To investigate species and serovar distributions, we typed 1,061 clinical isolates of human ureaplasmas from diverse patient populations. There was only a tenuous association between individual Ureaplasma serovars and certain patient populations. This may in part be explained by the fact that almost 40% of the isolates were genetic mosaics, apparently arising from the recombination of multiple serovars. This explains the extensive cross-reactivity based upon serotyping and the lack of consistent association of given serotypes with disease.

Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism.

OBJECTIVES: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). MATERIALS AND METHODS: Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium) and a 559 bp fragment (U. urealyticum). Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. RESULTS: Of the 210 samples, a total of 100 (47.6%) samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%), and coinfections with both species were detected in four samples (1.9%). The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. CONCLUSION: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.

Novel multiplex real-time PCR system using the SNP technology for the simultaneous diagnosis of Chlamydia trachomatis, Ureaplasma parvum and Ureaplasma urealyticum and genetic typing of serovars of C. trachomatis and U. parvum in NGU.

To explore the possibilities of a novel multiplex real-time PCR system for rapid diagnosis, genetic typing of serovars and clinical application in NGU, we developed a multiplex real-time PCR system for the simultaneous diagnosis of Chlamydia trachomatis, Ureaplasma parvum and Ureaplasma urealyticum and molecular detection of serovars of C. trachomatis and U. parvum in NGU using the SNP technology and TaqMan-LNA probe. In 57 pathogen-positive clinical specimens, we identified the following C. trachomatis serovars: D (20.05%, 12/57), E (36.84%, 21/57), F (19.30%, 11/57), G (8.77%, 5/57), H (5.26%, 3/57), J (3.51%, 2/57), and K (5.26%, 3/57). In 115 pathogen-positive clinical specimens, we identified the following U. parvum serovars: 1 (0.87%, 2/115), 3 (55.65%, 64/115), 6 (20.87%, 24/115) and 14 (21.74%, 25/115). Our fast pathogen diagnosis and serotyping assay using real-time TaqMan-LNA PCR may improve our ability to study the pathogenesis and epidemiology of NGU.

Frequency of ureaplasma serovars in respiratory secretions of preterm infants at risk for bronchopulmonary dysplasia.

OBJECTIVE: Ureaplasma respiratory tract colonization is associated with bronchopulmonary dysplasia (BPD) in preterm infants. Whether the 4 Ureaplasma parvum and 10 Ureaplasma urealyticum serovars differ in virulence is unknown. This study was conducted to determine the distribution of Ureaplasma serovars in respiratory secretions of a prospective cohort of preterm infants and to assess whether any of the serovars are associated with BPD. METHODS: Serial endotracheal and/or nasopharyngeal aspirates were obtained for Ureaplasma culture and PCR from 136 infants of gestational age <33 weeks. All positive samples were speciated and serovars were determined by real-time PCR. RESULTS: A total of 51 (37.5%) infants were Ureaplasma-positive one or more times during the first month of life. Respiratory colonization was inversely related to gestational age. Sixty-five percent of infants <26 weeks compared with 31% infants ≥ 26 weeks were culture or PCR positive. U. parvum was more common (N = 32, 63%) than U. urealyticum (N = 17, 33%); both species were present in 2 samples. Serovars 3 and 6 alone and in combination accounted for 96% U. parvum isolates. U. urealyticum isolates were commonly a mixture of multiple serovars, with serovar 11 alone or combined with other serovars (10/17, 59%) being the most common serovar. No individual species or serovars or serovar mixtures were associated with moderate-to-severe BPD. CONCLUSIONS: U. parvum serovars 3 and 6 and U. urealyticum serovar 11 were the most common serovars detected in respiratory samples from a prospective cohort of preterm infants.

Prevalence of Ureaplasma urealyticum and Ureaplasma parvum in semen of infertile and healthy men.

Ureaplasma urealyticum is a causative agent of non-gonococcal urethritis and is implicated in the pathogenesis of prostatitis, epididymitis and infertility. The organism is more common in partners of infertile than fertile marriages. U. urealyticum infections not only jeopardize fertility but also pose a risk for infertility treatment and resulting pregnancies. The aim of this study was to determine the prevalence of U. urealyticum and Ureaplasma parvum in semen of infertile and healthy men by polymerase chain reaction (PCR). Semen samples were obtained from infertile patients and healthy controls and were subjected to the routine andrological analysis and PCR. DNA was extracted by the Cadieux method, and analysed by PCR protocol with specific primers for urease and multiple-banded antigen genes. Ureaplasmas were detected significantly by PCR in 12 of 100 (12%) semen specimens from infertile patients and in three of 100 (3%) healthy men. The volume of semen fluid, concentration of sperm cells, and sperm cell with normal morphology were significantly decreased in infertile men. In the group of infertile patients with PCR positive for Ureaplasmas, the volume, count and morphology of semen samples were lower than in the infertile patients with PCR-negative results. U. urealyticum species in semen of infertile men was found to be high (9%) than in healthy controls (1%). Detection rate for U. parvum was 3% in the infertile group and 2% in healthy men. The results indicate that U. urealyticum species is more common in specimens of infertile men. The percentage of normal sperm cells, the volume of semen and the percentage of sperm cells with motility in the PCR positive for U. urealyticum species group were lower than in the PCR positive for U. parvum group.

Ureaplasma urealyticum meningitis in an adult patient.

A 38-year-old patient developed meningitis after a complicated kidney transplantation and organ rejection. Ureaplasma urealyticum was identified as the etiological agent by molecular and microbiological analyses of the cerebrospinal fluid. The patient was successfully treated with doxycycline and chloramphenicol. This is the first report of Ureaplasma urealyticum meningitis in an adult.

Detection and biovar discrimination of Ureaplasma urealyticum in Indian patients with genital tract infections.

Prevalence of the 2 ureaplasma biovars was estimated in patients with genital tract infections by polymerase chain reaction targeting the urease and multiple-banded antigen gene. Ureaplasma was more frequently isolated in symptomatic than asymptomatic subjects (48% versus 22%) (P=0.001). Biovar 1 was predominant; however, there was no difference in the distribution of the 2 biovars between the symptomatic and asymptomatic subjects.

Two multiplex real-time TaqMan polymerase chain reaction systems for simultaneous detecting and serotyping of Ureaplasma parvum.

Studies of epidemiology and pathogenesis of infections with Ureaplasma have received much attention in recent years. Rapid molecular methods for serotyping of Ureaplasma parvum will be of great value in the studies. We developed 2 multiplex real-time TaqMan polymerase chain reaction systems that allow simultaneous detecting and serotyping of 4 serovars of U. parvum.

Intrarenal abscesses due to Ureaplasma urealyticum in a transplanted kidney.

A young woman developed multiple abscesses in her transplanted kidney. Amplification of the 16S rRNA gene with subsequent sequencing revealed Ureaplasma urealyticum as the infectious agent. Microbiological diagnosis and sensitivity testing led to therapy with levofloxacin, resulting in rapid recovery of the patient.