Azithromycin and Doxycycline Resistance Profiles of U.S. Mycoplasma genitalium Strains and Their Association with Treatment Outcomes.

Mycoplasma genitalium is a sexually transmitted bacterium associated with nongonococcal urethritis (NGU) in men and cervicitis, endometritis, and pelvic inflammatory disease in women. Effective treatment is challenging due to the inherent, and increasingly acquired, antibiotic resistance in this pathogen. In our treatment trial conducted from 2007 to 2011 in Seattle, WA, we demonstrated poor efficacy of azithromycin (AZM) and doxycycline (DOX) against M. genitalium among men with NGU. In the present study, we cultured M. genitalium from 74 of 80 (92.5%) PCR-positive men at enrollment (V-1) and defined the MICs of AZM (N = 56 isolates) of DOX (N = 62 isolates). Susceptibility to AZM was bimodal; MICs were >8 µg/ml (44.6%) and <0.004 µg/ml (55.4%) for these isolates. The association of MIC with treatment efficacy was determined for men initially treated with either AZM (N = 30) or DOX (N = 24). Men treated with AZM were more likely to experience microbiologic treatment failure (P < 0.001) if infected with isolates that had AZM MICs of >8 µg/ml (18/18 men) than those with isolates that had AZM MICs of <0.004 µg/ml (1/12 men). Clinical treatment failure also was more likely to occur (P = 0.002) with AZM MICs of >8 µg/ml (12/18 men) than with AZM MICs of <0.004 µg/ml (1/12 men). In contrast, DOX MICs ranged from <0.125 to 2 µg/ml and were not correlated with microbiologic (P = 0.71) or clinical treatment (P = 0.41) failure, demonstrating no relationship between DOX MICs and treatment efficacy. Given the rapid spread of AZM resistance and the emergence of quinolone resistance, the current second-line therapy, monitoring MICs and evaluating other potential treatments for M. genitalium will be critical.

Efficacy of standard therapies against Ureaplasma species and persistence among men with non-gonococcal urethritis enrolled in a randomised controlled trial.

OBJECTIVE: Ureaplasma urealyticum biovar 2 (UU-2), but not Ureaplasma parvum (UP), has been associated with non-gonococcal urethritis (NGU), but little is known about species-specific responses to standard therapies. We examined species-specific treatment outcomes and followed men with treatment failure for 9 weeks. METHODS: From May 2007 to July 2011, men aged ≥16 attending a sexually transmitted disease (STD) clinic in Seattle, Washington, with NGU (urethral discharge or urethral symptoms plus ≥5 polymorphonuclear leucocytes /high-powered field) enrolled in a double-blind, randomised trial. Participants received active azithromycin (1 g) + placebo doxycycline or active doxycycline (100 mg twice a day ×7 days) + placebo azithromycin. Ureaplasma were detected in culture followed by species-specific PCR. Outcomes were assessed at 3, 6 and 9 weeks. At 3 weeks, men with persistent Ureaplasma detection received 'reverse therapy' (e.g., active doxycycline if they first received active azithromycin). At 6 weeks, persistently positive men received moxifloxacin (400 mg×7 days). RESULTS: Of 490 men, 107 (22%) and 60 (12%) were infected with UU-2 and UP, respectively, and returned at 3 weeks. Persistent detection was similar for UU-2-infected men initially treated with azithromycin or doxycycline (25% vs. 31%; p=0.53), but differed somewhat for men with UP (45% vs. 24%; p=0.11). At 6 weeks, 57% of UU-2-infected and 63% of UP-infected men who received both drugs had persistent detection. Failure after moxifloxacin occurred in 30% and 36%, respectively. Persistent detection of UU-2 or UP was not associated with signs/symptoms of NGU. CONCLUSIONS: Persistent detection after treatment with doxycycline, azithromycin and moxifloxacin was common for UU and UP, but not associated with persistent urethritis. TRIAL REGISTRATION NUMBER: NCT00358462.

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.

Development and performance of a microwell-plate-based polymerase chain reaction assay for Mycoplasma genitalium.

BACKGROUND: Mycoplasma genitalium is associated with, and could be the cause of, idiopathic cases of urethritis, endometritis, and cervicitis. Further epidemiologic studies on this organism are needed, but currently used polymerase chain reaction (PCR) assays are labor-intensive and culture is insensitive. GOAL: The goal was to develop and evaluate a microwell-plate-based PCR assay for M. genitalium. STUDY DESIGN: We adapted an M. genitalium PCR assay targeting the MgPa gene to a 96-microwell plate format with colorimetric detection of PCR products and incorporation of an internal inhibition control to determine the limit of detection of this assay (termed MgPa-IMW) for M. genitalium DNA and evaluate its performance on cervical and male urine specimens. RESULTS: The MgPa-IMW PCR assay detected 1 and 17 genome copies of M. genitalium (with 27% and 95% confidence) and was able to detect specimens inhibited for amplification. This assay was 100% concordant (50 positive and 50 negative) with the Southern-blot-based PCR assay with cervical specimens. Similarly, this test was 89% concordant with the Southern-blot-based assay for 64 male urine specimens (25 positive, 32 negative, 7 discordant), 97% concordant after correcting for specimens no longer positive by the Southern blot-based assay after freezer storage. CONCLUSION: The MgPa-IMW assay is sensitive and specific for the detection of M. genitalium in patient specimens and should facilitate large-scale screening for this organism.

Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion.

Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae-integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.