Antibiotic Susceptibility and Sequence Type Distribution of Ureaplasma Species Isolated from Genital Samples in Switzerland.
ABSTRACT: 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.
Project description:Objective. We compared laboratory developed real-time PCR assays for detection of Mycoplasma hominis and for detection and differentiation of Ureaplasma urealyticum and parvum to culture using genitourinary specimens submitted for M. hominis and Ureaplasma culture. Methods. 283 genitourinary specimens received in the clinical bacteriology laboratory for M. hominis and Ureaplasma species culture were evaluated. Nucleic acids were extracted using the Total Nucleic Acid Kit on the MagNA Pure 2.0. 5 μL of the extracts were combined with 15 μL of each of the two master mixes. Assays were performed on the LightCycler 480 II system. Culture was performed using routine methods. Results. M. hominis PCR detected 38/42 M. hominis culture-positive specimens, as well as 2 that were culture negative (sensitivity, 90.5%; specificity, 99.2%). Ureaplasma PCR detected 139/144 Ureaplasma culture-positive specimens, as well as 9 that were culture negative (sensitivity, 96.5%; specificity, 93.6%). Of the specimens that tested positive for Ureaplasma species, U. urealyticum alone was detected in 33, U. parvum alone in 109, and both in 6. Conclusion. The described PCR assays are rapid alternatives to culture for detection of M. hominis and Ureaplasma species, and, unlike culture, the Ureaplasma assay easily distinguishes U. urealyticum from parvum.
Project description:Ureaplasma urealyticum and Ureaplasma parvum are pathogens involved in urogenital tract and intrauterine infections and also in systemic diseases in newborns and immunosuppressed patients. There is limited information on the antimicrobial susceptibility and clonality of these species. In this study, we report the susceptibility of 250 contemporary isolates of Ureaplasma (202 U. parvum and 48 U. urealyticum isolates) recovered at Mayo Clinic, Rochester, MN. MICs of doxycycline, azithromycin, ciprofloxacin, tetracycline, erythromycin, and levofloxacin were determined by broth microdilution, with MICS of the last three interpreted according to CLSI guidelines. Levofloxacin resistance was found in 6.4% and 5.2% of U. parvum and U. urealyticum isolates, respectively, while 27.2% and 68.8% of isolates, respectively, showed ciprofloxacin MICs of ?4 ?g/ml. The resistance mechanism of levofloxacin-resistant isolates was due to mutations in parC, with the Ser83Leu substitution being most frequent, followed by Glu87Lys. No macrolide resistance was found among the 250 isolates studied; a single U. parvum isolate was tetracycline resistant. tet(M) was found in 10 U. parvum isolates, including the single tetracycline-resistant isolate, as well as in 9 isolates which had low tetracycline and doxycycline MICs. Multilocus sequence typing (MLST) performed on a selection of 46 isolates showed high diversity within the clinical Ureaplasma isolates studied, regardless of antimicrobial susceptibility. The present work extends previous knowledge regarding susceptibility to antimicrobial agents, resistance mechanisms, and clonality of Ureaplasma species in the United States.
Project description:Urinary tract infections (UTIs) affect nearly 20% of women age 15 to 29 and account for an estimated $3.5 billion in costs. Antibiotic resistance prolongs UTI treatment, and resistance profiles vary regionally. This regional variation is an important consideration in guiding empirical treatment selection. Regional studies in the United States have identified tetracycline resistance in over one-third of <i>Ureaplasma</i> species isolates, but no studies have evaluated antibiotic resistance levels in college-aged women with a first-time UTI. We tested a panel of antibiotics and determined the MICs of <i>Ureaplasma</i> species (60 <i>U. parvum</i> and 13 <i>U. urealyticum</i>) and 10 <i>Mycoplasma hominis</i> isolates obtained from urine from college-aged women with a first-time UTI. Low antibiotic resistance was found in this population of women with a first-time UTI. All <i>M. hominis</i> and <i>U. urealyticum</i> isolates were sensitive. However, two <i>U. parvum</i> isolates were resistant, with one to levofloxacin (MIC, 4 ?g/ml) and one to tetracycline (MIC, 8 ?g/ml). For the <i>Ureaplasma</i> spp., the MIC<sub>90</sub>s were highest against gentamicin (21 ?g/ml) and lowest against doxycycline (0.25 ?g/ml). In a comparison of MIC levels between <i>Ureaplasma</i> spp., <i>U. urealyticum</i> had significantly higher MICs against each antibiotic except doxycycline. For the resistant isolates, the genetic mechanisms of resistance were determined. PCR amplification identified <i>tetM</i> to be present in the tetracycline-resistant isolate and an S83W mutation within the <i>parC</i> gene of the quinolone-resistant isolate. To our knowledge, this study is the first to provide molecular and phenotypic evidence of the S83W <i>parC</i> mutation conferring levofloxacin resistance in <i>U. parvum</i> isolated from a patient in the United States.
Project description:Ureaplasma spp. cause several disorders, such as nongonococcal urethritis, miscarriage, and preterm delivery with lung infections in neonates, characterized by pathological chorioamnionitis in the placenta. Although reports on antibiotic resistance in Ureaplasma are on the rise, reports on quinolone-resistant Ureaplasma infections in Japan are limited. The purpose of this study was to determine susceptibilities to five quinolones of Ureaplasma urealyticum and Ureaplasma parvum isolated from perinatal samples in Japan and to characterize the quinolone resistance-determining regions in the gyrA, gyrB, parC, and parE genes. Out of 28 clinical Ureaplasma strains, we isolated 9 with high MICs of quinolones and found a single parC gene mutation, resulting in the change S83L. Among 158 samples, the ParC S83L mutation was found in 37 samples (23.4%), including 1 sample harboring a ParC S83L-GyrB P462S double mutant. Novel mutations of ureaplasmal ParC (S83W and S84P) were independently found in one of the samples. Homology modeling of the ParC S83W mutant suggested steric hindrance of the quinolone-binding pocket (QBP), and de novo prediction of peptide structures revealed that the ParC S84P may break/kink the formation of the ?4 helix in the QBP. Further investigations are required to unravel the extent and mechanism of antibiotic resistance of Ureaplasma spp. in Japan.
Project description: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.
Project description:We performed <i>in vitro</i> susceptibility testing for eravacycline in comparison to 4 other antimicrobials against 10 <i>Mycoplasma genitalium</i>, 40 <i>Mycoplasma hominis</i>, 44 <i>Mycoplasma pneumoniae</i>, 20 <i>Ureaplasma parvum</i>, and 20 <i>Ureaplasma urealyticum</i> isolates. All eravacycline MICs were??0.25??g/ml, except that for one isolate of <i>M. genitalium</i>, for which the MIC was 2??g/ml. Eravacycline was markedly more potent than tetracycline, azithromycin, moxifloxacin, and clindamycin against all isolates tested, which included 37 macrolide, tetracycline, and/or fluoroquinolone-resistant organisms.
Project description:<h4>Background</h4>The etiology of prostate cancer (PCa) is multiple and complex. Among the causes recently cited are chronic infections engendered by microorganisms that often go unnoticed. A typical illustration of such a case is infection due to mollicutes bacteria. Generally known by their lurking nature, urogenital mollicutes are the most incriminated in PCa. This study was thus carried out in an attempt to establish the presence of these mollicutes by PCR in biopsies of confirmed PCa patients and to evaluate their prevalence.<h4>Methods</h4>A total of 105 Formalin-Fixed Paraffin-Embedded prostate tissues collected from 50 patients suffering from PCa and 55 with benign prostate hyperplasia were subjected to PCR amplification targeting species-specific genes of 5 urogenital mollicutes species, Mycoplasma genitalium, M. hominis, M. fermentans, Ureaplasma parvum, and U. urealyticum. PCR products were then sequenced to confirm species identification. Results significance was statistically assessed using Chi-square and Odds ratio tests.<h4>Results</h4>PCR amplification showed no positive results for M. genitalium, M. hominis, and M. fermentans in all tested patients. Strikingly, Ureaplasma spp. were detected among 30% (15/50) of PCa patients. Nucleotide sequencing further confirmed the identified ureaplasma species, which were distributed as follows: 7 individuals with only U. parvum, 5 with only U. urealyticum, and 3 co-infection cases. Association of the two ureaplasma species with PCa cases proved statistically significant (P?<?0.05), and found to represent a risk factor. Of note, Ureaplasma spp. were mostly identified in patients aged 60 and above with prostatic specific antigen (PSA) level?>?4?ng/ml and an invasive malignant prostate tumor (Gleason score 8-10).<h4>Conclusions</h4>This study uncovered a significant association of Ureaplasma spp. with PCa arguing in favour of their potential involvement in this condition. Yet, this finding, though statistically supported, warrants a thorough investigation at a much larger scale.
Project description:BACKGROUND: Although Chlamydia trachomatis is the most commonly reported pathogen that causes urogenital infection such as urethritis or cervicitis, Ureaplasma parvum and Ureaplasma urealyticum, which are commensals in the genital tract, have also now been recognized as contributors to urogenital infection. However, whether the presence of either U. parvum or U. urealyticum is related to that of C. trachomatis in the urogenital tract remains unknown. We therefore attempted to estimate by PCR the prevalence of C. trachomatis, U. parvum and U. urealyticum in endocervical samples obtained from healthy women attending their first prenatal visit in Sapporo, Japan. METHODS: The samples were taken from 303 apparently healthy women, and the extracted DNAs (n = 280) were used for PCR detection targeting C. trachomatis, U. parvum and U. urealyticum. Statistical analysis of the data was performed by Fisher's exact test. RESULTS: PCR detection revealed that the prevalence of C. trachomatis, U. parvum and U. urealyticum was 14.3% (40/280), 41.7% (117/280) and 8.9% (25/280), respectively. C. trachomatis ompA genotype D was most frequently identified. Surprisingly, either C. trachomatis or Ureaplasma spp. was detected in almost half of the healthy women. Mixed infection of C. trachomatis with either U. parvum or U. urealyticum was also observed in 9.2% (26/280) of the women. There was a significant association between C. trachomatis and either U. parvum (p = 0.023) or Ureaplasma total (p = 0.013), but not U. urealyticum (p = 0.275). CONCLUSION: This study demonstrated that the presence of Ureaplasma had a significant effect on the presence of C. trachomatis in the genital tract of healthy women, suggesting that mixed infection is an important factor in bacterial pathogenesis in the genital tract.
Project description:This is to investigate the cervical cytological abnormalities associated with Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma urealyticum infections on routine screen. A total of 714 subjects who had undergone cervical Pap smears and concomitant analyses for cervical infections were included by a retrospective search. The frequencies of reactive cellular change (RCC) and squamous epithelial abnormalities were significantly higher in Chlamydia positive subjects than in uninfected subjects (P<0.001). Of the 124 subjects tested for M. hominis, M. genitalium, and U. urealyticum, 14 (11%) were positive for M. hominis and 29 (23%) were positive for U. urealyticum. Squamous abnormalities were more frequent in subjects with Ureaplasma infections than in uninfected subjects (24% versus 8%). Taking together these findings, C. trachomatis and U. urealyticum may have a causal role in the development of cervical epithelial changes, including RCC. Thus, extra awareness is warranted in cervical screening of women with Chlamydia or Ureaplasma infections.
Project description:BACKGROUND:Sexually transmitted infections (STIs) cause a major public health problem that affect both men and women in developing and developed countries. The aim of the study was to estimate the prevalence of 11 STIs among women who voluntarily participated in the study, while seeking gynecological checkup. The existence of an association between the presence of pathogens and symptoms and various sociodemographic risk factors was assessed. METHODS:A total of 505 vaginal and cervical specimens were collected from women above 18?years of age, with or without symptoms related to gynecological infections. Nucleic acid was extracted and samples were tested by real-time PCR for the following pathogens: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Urealplasma parvum, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma girerdii, Gardnerella vaginalis, Candida albicans and Human Papillomavirus (HPV). Positive HPV samples underwent genotyping using a microarray system. RESULTS:Of the 505 samples, 312 (62%) were screened positive for at least one pathogen. Of these, 36% were positive for Gardnerella vaginalis, 35% for Ureaplasma parvum, 8% for Candida albicans, 6.7% for HPV, 4.6% for Ureaplasma urealyticum, 3.6% for Mycoplasma hominis, 2% for Trichomonas vaginalis, 0.8% for Chlamydia trachomatis, 0.4% for Mycoplasma girerdii, 0.2% for Mycoplasma genitalium and 0.2% for Neisseria gonorrhoeae. Lack of symptoms was reported in 187 women (37%), among whom 61% were infected. Thirty-four samples were HPV positive, with 17 high risk HPV genotypes (HR-HPV); the highest rates being recorded for types 16 (38%), 18 (21%) and 51 (18%). Out of the 34 HPV positives, 29 participants had HR-HPV. Association with various risk factors were reported. CONCLUSIONS:This is the first study that presents data about the presence of STIs among women in Lebanon and the MENA region by simultaneous detection of 11 pathogens. In the absence of systematic STI surveillance in Lebanon, concurrent screening for HPV and PAP smear is warranted.