Chromosomal mutations responsible for fluoroquinolone resistance in Ureaplasma species in the United States.

We sequenced the full lengths of the gyrA, gyrB, parC, and parE genes in 13 fluoroquinolone-resistant Ureaplasma isolates (levofloxacin MICs, 4 to 32 µg/ml) and 10 susceptible isolates (MICs ≤ 2 µg/ml). Mutations were detected in all resistant isolates but in none of the susceptible isolates. The most prevalent mutation was the S83L substitution in the ParC protein. No plasmid-mediated fluoroquinolone resistance genes were detected.

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains.

BACKGROUND: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. RESULTS: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75-0.78 Mbp genomes and UUR serovars were 0.84-0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. CONCLUSIONS: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.

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.

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.

Comparison of bias and resolvability in single-cell and single-transcript methods.

Single-cell and single-transcript measurement methods have elevated our ability to understand and engineer biological systems. However, defining and comparing performance between methods remains a challenge, in part due to the confounding effects of experimental variability. Here, we propose a generalizable framework for performing multiple methods in parallel using split samples, so that experimental variability is shared between methods. We demonstrate the utility of this framework by performing 12 different methods in parallel to measure the same underlying reference system for cellular response. We compare method performance using quantitative evaluations of bias and resolvability. We attribute differences in method performance to steps along the measurement process such as sample preparation, signal detection, and choice of measurand. Finally, we demonstrate how this framework can be used to benchmark different methods for single-transcript detection. The framework we present here provides a practical way to compare performance of any methods.

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.

Genetic circuit design automation.

Computation can be performed in living cells by DNA-encoded circuits that process sensory information and control biological functions. Their construction is time-intensive, requiring manual part assembly and balancing of regulator expression. We describe a design environment, Cello, in which a user writes Verilog code that is automatically transformed into a DNA sequence. Algorithms build a circuit diagram, assign and connect gates, and simulate performance. Reliable circuit design requires the insulation of gates from genetic context, so that they function identically when used in different circuits. We used Cello to design 60 circuits forEscherichia coli(880,000 base pairs of DNA), for which each DNA sequence was built as predicted by the software with no additional tuning. Of these, 45 circuits performed correctly in every output state (up to 10 regulators and 55 parts), and across all circuits 92% of the output states functioned as predicted. Design automation simplifies the incorporation of genetic circuits into biotechnology projects that require decision-making, control, sensing, or spatial organization.

Molecular methods for the detection of Mycoplasma and ureaplasma infections in humans: a paper from the 2011 William Beaumont Hospital Symposium on molecular pathology.

Mycoplasma and Ureaplasma species are well-known human pathogens responsible for a broad array of inflammatory conditions involving the respiratory and urogenital tracts of neonates, children, and adults. Greater attention is being given to these organisms in diagnostic microbiology, largely as a result of improved methods for their laboratory detection, made possible by powerful molecular-based techniques that can be used for primary detection in clinical specimens. For slow-growing species, such as Mycoplasma pneumoniae and Mycoplasma genitalium, molecular-based detection is the only practical means for rapid microbiological diagnosis. Most molecular-based methods used for detection and characterization of conventional bacteria have been applied to these organisms. A complete genome sequence is available for one or more strains of all of the important human pathogens in the Mycoplasma and Ureaplasma genera. Information gained from genome analyses and improvements in efficiency of DNA sequencing are expected to significantly advance the field of molecular detection and genotyping during the next few years. This review provides a summary and critical review of methods suitable for detection and characterization of mycoplasmas and ureaplasmas of humans, with emphasis on molecular genotypic techniques.

Mutations in ribosomal proteins and ribosomal RNA confer macrolide resistance in human Ureaplasma spp.

Genetic mechanisms of macrolide resistance were investigated in six isolates of Ureaplasma spp. with erythromycin minimum inhibitory concentrations (MICs)≥ 8 µg/mL that were derived from 370 cultures obtained over a several year period. Point mutations in domain V of 23S rRNA and/or mutations in ribosomal protein L4 genes are likely to be responsible for this drug resistance. Overall, macrolide resistance was uncommon, in contrast to tetracycline resistance that was documented in 121 unique isolates (33%).