Lynn B. Duffy

Ureaplasma parvum or Mycoplasma hominis as Sole Pathogens Cause Chorioamnionitis, Preterm Delivery, and Fetal Pneumonia in Rhesus Macaques

The authors assess causal, cellular and inflammatory links between intraamniotic infection with Ureaplasma parvum or Mycoplasma hominis and preterm labor in a nonhuman primate model. Long-term catheterized rhesus monkeys received intraamniotic inoculations of clinical isolates of Ureaplasma parvum serovar 1, M hominis, media control or physiological saline. Genital mycoplasmas were quantified in amniotic fluid (AF) and documented in fetal tissues by culture and PCR. In association with elevated AF colony counts for U parvum or M hominis, there was a sequential upregulation of AF leukocytes, proinflammatory cytokines, prostaglandin E2 and F2a, metalloproteinase-9 and uterine activity ( P< .05). Fetal membranes and lung were uniformly positive for both microorganisms; fetal blood and cerebrospinal fluid cultures and PCR were more often positive for M hominis than U parvum. Histopathologic findings of chorioamnionitis, a systemic fetal inflammatory response and pneumonitis worsen with duration of in utero infection. U parvum or M hominis, as sole pathogens, elicit a robust proinflammatory response which contributes to preterm labor and fetal lung injury.

Elevated cytokine and chemokine levels and prolonged pulmonary airflow resistance in a murine Mycoplasma pneumoniae pneumonia model : a microbiologic, histologic, immunologic, and respiratory plethysmographic profile

ABSTRACT Because Mycoplasma pneumoniae is hypothesized to play an important role in reactive airway disease/asthma, a comprehensive murine model of M. pneumoniae lower respiratory infection was established. BALB/c mice were intranasally inoculated once withM. pneumoniae and sacrificed at 0 to 42 days postinoculation. All mice became infected and developed histologic evidence of acute pulmonary inflammation, which cleared by 28 days postinoculation. By contrast, M. pneumoniae persisted in the respiratory tract for the entire 42 days studied. Tumor necrosis factor alpha, gamma interferon, interleukin-6 (IL-6), KC (functional IL-8), MIP-1α, and MCP-1/JE concentrations were significantly elevated in bronchoalveolar lavage samples, whereas IL-4 and IL-10 concentrations were not significantly elevated. Pulmonary airflow resistance, as measured by plethysmography, was detected 1 day postinoculation and persisted even after pulmonary inflammation had resolved at day 28. Serum anti-M. pneumoniae immunoglobulin G titers were positive in all mice by 35 days. This mouse model provides a means to investigate the immunopathogenesis of M. pneumoniae infection and its possible role in reactive airway disease/asthma.

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.

Antimicrobial Susceptibility among Pathogens Collected from Hospitalized Patients in the United States and In Vitro Activity of Tigecycline, a New Glycylcycline Antimicrobial

ABSTRACT The activities of tigecycline and comparators against isolates collected from 76 U.S. centers between January 2004 and September 2005 were assessed. Tigecycline MIC90s were ≤2 μg/ml for Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Streptococcus agalactiae.

Comparative In Vitro Susceptibilities of Human Mycoplasmas and Ureaplasmas to a New Investigational Ketolide, CEM-101

ABSTRACT 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 ≤0.5 μg/ml, making CEM-101 the most potent compound tested.

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.

Detection and Characterization of Human Ureaplasma Species and Serovars by Real-Time PCR

ABSTRACT 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 × 10−2 CFU/μl PCR mixture for detecting U. parvum and 4.1 × 10−2 CFU/μl 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.

In Vitro Susceptibilities to and Bactericidal Activities of Garenoxacin (BMS-284756) and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas

ABSTRACT The in vitro susceptibilities to garenoxacin (BMS-284756), an investigational des-fluoroquinolone, and eight other agents were determined for 63 Mycoplasma pneumoniae, 45 Mycoplasma hominis, 15 Mycoplasma fermentans, and 68 Ureaplasma sp. isolates. Garenoxacin was the most active quinolone, inhibiting all isolates at ≤1 μg/ml. The garenoxacin MIC at which 90% of isolates are inhibited (MIC90s; ≤0.008 μg/ml) was at least 4-fold less than those of moxifloxacin and clindamycin, 8-fold less than that of sparfloxacin, and 64-fold less than those of levofloxacin and ciprofloxacin for M. pneumoniae. For M. hominis, the garenoxacin MIC90 (≤0.008 μg/ml) was 4-fold less than those of clindamycin and moxifloxacin, 8-fold less than that of sparfloxacin, and 64-fold less than those of levofloxacin and ciprofloxacin. All 15 M. fermentans isolates were inhibited by garenoxacin at concentrations ≤0.008 μg/ml, making it the most active drug tested against this organism. For Ureaplasma spp., the garenoxacin MIC90 (0.25 μg/ml) was equivalent to those of moxifloxacin and doxycycline, 4-fold less than those of levofloxacin and sparfloxacin, 8-fold less than that of azithromycin, and 32-fold less than that of ciprofloxacin. Garenoxacin and the other fluoroquinolones tested were demonstrated to have bactericidal activities against M. pneumoniae and M. hominis by measurement of minimal bactericidal activities and by time-kill studies. Further study of garenoxacin is required, as it has great potential for use in the treatment of infections due to mycoplasmas and ureaplasmas.

Mycoplasmal infections of cerebrospinal fluid in newborn infants from a community hospital population

Mycoplasma hominis or Ureaplasma urealyticum have previously been isolated from cerebrospinal fluid (CSF) in 13 of 100 newborn infants tested from a high risk university hospital populaton where the mothers were of predominantly lower income and socioeconomic status and had often received little or no prenatal care. We sought to determine whether such infections occur in neonates born to women cared for mainly through private obstetric practices and who delivered in 4 suburban community hospitals. CSF cultures where done in 318 infants during an 8-month period. M. hominis was isolated from 9 and U. urealyticum from 5 CSF cultures. Four infants infected with U. urealyticum and 3 infected with M. hominis were born at term. One infant infected with U. urealyticum had a birth weight of <1000 g. In 5 inftants clearance of the infecting organism was documented without specific treatment. Tweleve infants had good perinatal outcomes regardless of treatment and 2 died. One death in a 2240-g infant infected with M. hominis was associated with Haemophilus influenzae sepsis and pneumonia. The other death occurred 3 days after birth in a 630-g infant infected with U. urealyticum who had evidence of meningitis and intraventricular hemorrhage. Results of this study suggest that mycoplasmas are common causes of neonatal CSF infections, not only in high risk populations, but also in the general population.

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains

BackgroundUreaplasma 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.ResultsWe 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.ConclusionsComparative 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.