In Vitro Activities of Eravacycline and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas.

We performed in vitro susceptibility testing for eravacycline in comparison to 4 other antimicrobials against 10 Mycoplasma genitalium, 40 Mycoplasma hominis, 44 Mycoplasma pneumoniae, 20 Ureaplasma parvum, and 20 Ureaplasma urealyticum isolates. All eravacycline MICs were ≤0.25 µg/ml, except that for one isolate of M. genitalium, for which the MIC was 2 µg/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.

Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae.

We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P < 0.05). Specificities of all assays were 99.5 to 100%. The Resistance Plus MP assay detected macrolide resistance in 27/33 specimens, resulting in a sensitivity of 81.8%. This study provides the first large-scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of various test performances on patient outcome.

In Vitro Activities of Gepotidacin (GSK2140944) and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas.

Gepotidacin, a novel first-in-class triazaacenaphthylene topoisomerase II inhibitor, was tested against 85 type strains and clinical isolates of Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum in comparison to levofloxacin, moxifloxacin, azithromycin or clindamycin, and tetracycline. Gepotidacin MIC90s (µg/ml) were 0.125 (M. pneumoniae), 0.032 (M. genitalium), 2 (M. hominis), and 8 (Ureaplasma species). Gepotidacin activity was not affected by resistance to fluoroquinolones, tetracyclines, or macrolides in the strains tested. Gepotidacin merits further study for treating infections caused by these organisms.

In Vitro Activities of Lefamulin and Other Antimicrobial Agents against Macrolide-Susceptible and Macrolide-Resistant Mycoplasma pneumoniae from the United States, Europe, and China.

Lefamulin, an investigational pleuromutilin, was tested against a collection of 18 macrolide-susceptible and 42 macrolide-resistant Mycoplasma pneumoniae strains, and the results were compared with those of azithromycin, erythromycin, tetracycline, doxycycline, and moxifloxacin testing. Lefamulin was highly active against all strains tested, with all MICs at ≤0.008 µg/ml. The lefamulin MIC90 (0.002 µg/ml) for macrolide-resistant strains was the lowest among all drugs tested. Minimum bactericidal concentrations were within 2 dilutions of the MIC values, indicating a bactericidal effect.

In Vitro Activities of Omadacycline (PTK 0796) and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas.

In vitro activities of omadacycline, a new aminomethylcycline, were determined for Mycoplasma and Ureaplasma spp. and compared with those of azithromycin, clindamycin, moxifloxacin, tetracycline, and doxycycline. All omadacycline MICs were <2 µg/ml. MIC90s were 0.063 µg/ml for Mycoplasma hominis, 0.25 µg/ml for Mycoplasma pneumoniae, and 2 µg/ml for Ureaplasma spp. Omadacycline had the lowest MIC90 among all drugs tested against M. hominis Omadacycline activity was not affected by macrolide, tetracycline, or fluoroquinolone resistance.

In vitro antibacterial activity of AZD0914 against human Mycoplasmas and Ureaplasmas.

In this study, susceptibilities were determined for AZD0914, a spiropyrimidinetrione DNA gyrase inhibitor, azithromycin, doxycycline, and levofloxacin against Mycoplasma and Ureaplasma species. The activity of AZD0914 was comparable to that of levofloxacin and doxycycline against Mycoplasma genitalium and Mycoplasma pneumoniae. The AZD0914 MIC90 against Mycoplasma hominis was 8-fold greater than that for levofloxacin. The AZD0914 MIC90 against Ureaplasma species was 4-fold less than that for azithromycin and 8-fold less than that for levofloxacin and doxycycline.

Comparison of the illumigene Mycoplasma DNA amplification assay and culture for detection of Mycoplasma pneumoniae.

A loop-mediated isothermal amplification (LAMP) system, the illumigene Mycoplasma DNA amplification assay (Meridian Bioscience, Inc., Cincinnati, OH) was evaluated to determine its analytical sensitivity, specificity, and clinical application in comparison to historic culture in a collection of archived respiratory specimens. The illumigene limit of detection was ≤88 CFU/reaction for 10 Mycoplasma pneumoniae reference strains. This assay correctly identified 36 M. pneumoniae reference strains and clinical isolates from various geographic origins, including both of the main subtypes. No cross-reactions were detected with other mycoplasmas, ureaplasmas, other bacterial species, viruses, yeasts, or human DNA. Among 214 respiratory specimens previously cultured for M. pneumoniae, when real-time PCR with bidirectional sequencing of the PCR products was used to resolve discrepancies, the sensitivity was 22 of 22 (100%) and the specificity was 190 of 192 (99%). This commercial LAMP assay is a useful rapid method for detecting M. pneumoniae in clinical specimens. Additional prospective clinical trials with direct comparison to culture and PCR are warranted.

Transplacental transfer of Azithromycin and its use for eradicating intra-amniotic ureaplasma infection in a primate model.

BACKGROUND: Our goals were to describe azithromycin (AZI) pharmacokinetics in maternal plasma (MP), fetal plasma (FP), and amniotic fluid (AF) following intra-amniotic infection (IAI) with Ureaplasma in pregnant rhesus monkeys and to explore concentration-response relationships. METHODS: Following intra-amniotic inoculation of Ureaplasma parvum, rhesus monkeys received AZI (12.5 mg/kg every 12 hours intravenously for 10 days; n = 10). Intensive pharmacokinetic sampling of MP, FP, and AF was scheduled following the first (ie, single) dose and the last (ie, multiple) dose. Noncompartmental and pharmacokinetic modeling methods were used. RESULTS: The AF area under the concentration-time curve at 12 hours was 0.22 µg×h/mL following a single dose and 6.3 µg×h/mL at day 10. MP and AF accumulation indices were 8.4 and 19, respectively. AZI AF half-life following the single dose and multiple dose were 156 and 129 hours, respectively. The median MP:FP ratio in concomitantly drawn samples was 3.2 (range, 1.3-9.6; n = 9). Eradication of U. parvum occurred at 6.6 days, with a 95% effective concentration (EC95) of 39 ng/mL for the maximum AZI AF concentration. CONCLUSIONS: Our study demonstrates that a maternal multiple-dose AZI regimen is effective in eradicating U. parvum IAI by virtue of intra-amniotic accumulation and suggests that antenatal therapy has the potential to mitigate complications associated with U. parvum infection in pregnancy, such as preterm labor and fetal sequelae.

Maternal azithromycin therapy for Ureaplasma intraamniotic infection delays preterm delivery and reduces fetal lung injury in a primate model.

OBJECTIVE: We assessed the efficacy of a maternal multidose azithromycin (AZI) regimen, with and without antiinflammatory agents to delay preterm birth and to mitigate fetal lung injury associated with Ureaplasma parvum intraamniotic infection. STUDY DESIGN: Long-term catheterized rhesus monkeys (n = 16) received intraamniotic inoculation of U parvum (10(7) colony-forming U/mL, serovar 1). After contraction onset, rhesus monkeys received no treatment (n = 6); AZI (12.5 mg/kg, every 12 h, intravenous for 10 days; n = 5); or AZI plus dexamethasone and indomethacin (n = 5). Outcomes included amniotic fluid proinflammatory mediators, U parvum cultures and polymerase chain reaction, AZI pharmacokinetics, and the extent of fetal lung inflammation. RESULTS: Maternal AZI therapy eradicated U parvum intraamniotic infection from the amniotic fluid within 4 days. Placenta and fetal tissues were 90% culture negative at delivery. AZI therapy significantly delayed preterm delivery and prevented advanced fetal lung injury, although residual acute chorioamnionitis persisted. CONCLUSION: Specific maternal antibiotic therapy can eradicate U parvum from the amniotic fluid and key fetal organs, with subsequent prolongation of pregnancy, which provides a therapeutic window of opportunity to effectively reduce the severity of fetal lung injury.

Standardized methods and quality control limits for agar and broth microdilution susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum.

An international multilaboratory collaborative study was conducted to develop standard media and consensus methods for the performance and quality control of antimicrobial susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum using broth microdilution and agar dilution techniques. A reference strain from the American Type Culture Collection was designated for each species, which was to be used for quality control purposes. Repeat testing of replicate samples of each reference strain by participating laboratories utilizing both methods and different lots of media enabled a 3- to 4-dilution MIC range to be established for drugs in several different classes, including tetracyclines, macrolides, ketolides, lincosamides, and fluoroquinolones. This represents the first multilaboratory collaboration to standardize susceptibility testing methods and to designate quality control parameters to ensure accurate and reliable assay results for mycoplasmas and ureaplasmas that infect humans.

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.

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.

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.

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%).

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.

Critical role of macrophages and their activation via MyD88-NFκB signaling in lung innate immunity to Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp), a common cause of pneumonia, is associated with asthma; however, the mechanisms underlying this association remain unclear. We investigated the cellular immune response to Mp in mice. Intranasal inoculation with Mp elicited infiltration of the lungs with neutrophils, monocytes and macrophages. Systemic depletion of macrophages, but not neutrophils, resulted in impaired clearance of Mp from the lungs. Accumulation and activation of macrophages were decreased in the lungs of MyD88(-/-) mice and clearance of Mp was impaired, indicating that MyD88 is a key signaling protein in the anti-Mp response. MyD88-dependent signaling was also required for the Mp-induced activation of NFκB, which was essential for macrophages to eliminate the microbe in vitro. Thus, MyD88-NFκB signaling in macrophages is essential for clearance of Mp from the lungs.

Emerging macrolide resistance in Mycoplasma pneumoniae in children: detection and characterization of resistant isolates.

BACKGROUND: Epidemiologic data from Asia have documented the rapid and extensive emergence of macrolide resistance in Mycoplasma pneumoniae. This drug resistance has also been documented in Europe and recently in the United States, but there is very little information currently available on its prevalence. A rapid technique to identify macrolide-resistant M. pneumoniae is needed to guide management of patients with community-acquired respiratory infections. METHODS: Culture and Minimum Inhibitory Concentration testing identified macrolide-resistant M. pneumoniae infection in 2 seriously ill hospitalized children with community-acquired pneumonia. A portion of the 23S ribosomal RNA gene from 2 macrolide-resistant M. pneumoniae isolates from these children as well as 4 laboratory-induced macrolide-resistant strains was amplified by PCR, and the PCR products were sequenced to identify mutations associated with macrolide resistance. A real-time PCR assay was designed to identify 3 known mutations in the 23S rRNA gene associated with macrolide resistance and applied to the clinical specimens from which these isolates were obtained and to the bacterial isolates. RESULTS: : Macrolide-resistant M. pneumoniae from both children were found to carry an A2063G transition in the 23S rRNA gene previously identified in resistant isolates from China, Japan, France, and recently in an encephalitis outbreak in Rhode Island. Three laboratory-induced mutant strains had an A2064G mutation whereas the other one had an A2063G mutation. A real-time PCR assay successfully detected the macrolide-resistant M. pneumoniae directly in clinical specimens and discriminated them from wild-type isolates. CONCLUSIONS: Macrolide-resistant M. pneumoniae can be associated with prolonged severe respiratory infection in children. Real-time PCR offers a rapid method of diagnosing macrolide resistance in community-acquired respiratory infections due to M. pneumoniae.

Deficient immune response to Mycoplasma pneumoniae in childhood asthma.

Prospective studies have suggested that some individuals have a persistent IgM response to Mycoplasma pneumoniae infection with relatively little IgG production over many months. Persistence of the organism in patients with an allergic phenotype might predispose to the development of asthma. This study was designed to analyze the prevalence of M. pneumoniae infection and the immune response to that infection among children with asthma compared with controls. A prospective study was performed in 82 children with physician-diagnosed asthma and 98 nonasthmatic controls over a 5-year period comparing them for evidence of current or prior infection by M. pneumoniae using serology (IgG and IgM), culture, and polymerase chain reaction (PCR), and in vitro cellular responses to M. pneumoniae antigen. Similar numbers of controls (9/98) and asthmatic children (6/82) were PCR(+) for M. pneumoniae at some time during the study. IgM antibody to M. pneumoniae was detected in similar numbers of controls (21/98) and asthmatic children (18/82), but positive IgG antibody titers were detected in significantly more controls (13/98) than asthmatic children (3/82; p = 0.03). Similar numbers from each group were IgM(+) on more than one annual visit (9/98 controls and 7/82 asthmatic children). Antigen-driven proliferation and interferon (IFN) gamma production by mononuclear cells from IgM(+) controls were significantly greater than that of IgM(-) controls, but there was no difference in proliferation and IFN-gamma production by cells from IgM(+) and IgM(-) asthmatic children. These results suggest that asthmatic children have deficient cellular and humoral responses to M. pneumoniae infection compared with nonasthmatic controls.

Comparative in vitro susceptibilities of human mycoplasmas and ureaplasmas to a new investigational ketolide, CEM-101.

MICs were determined for an investigational ketolide, CEM-101, and azithromycin, telithromycin, doxycycline, levofloxacin, clindamycin, and linezolid against 36 Mycoplasma pneumoniae, 5 Mycoplasma genitalium, 13 Mycoplasma hominis, 15 Mycoplasma fermentans, and 20 Ureaplasma isolates. All isolates, including two macrolide-resistant M. pneumoniae isolates, were inhibited by CEM-101 at < or = 0.5 microg/ml, making CEM-101 the most potent compound tested.