Mutations in the gyrA, parC, and parE genes associated with fluoroquinolone resistance in clinical isolates of Mycoplasma hominis.
Five clinical isolates of Mycoplasma hominis from three different patients were examined for resistance to fluoroquinolones; some of these isolates were probably identical. All five isolates harbored amino acid substitutions in the quinolone resistance-determining regions of both DNA gyrase (GyrA) and topoisomerase IV (ParC or ParE). Furthermore, the novobiocin MIC for three isolates showed a significant increase. This is the first characterization of fluoroquinolone-resistant clinical mycoplasma isolates from humans. (+info)
Cyto-adherence studies of the adhesin P50 of Mycoplasma hominis.
Recombinant peptides of Mycoplasma hominis adhesin P50 were expressed in Escherichia coli to investigate which regions of the P50 molecule are responsible for cyto-adherence. The respective DNA fragments were obtained by PCR amplification of the p50 gene with the use of mutating oligonucleotides to change the TGA codons of mycoplasma to TGG codons, which are translated in E. coli as tryptophan. The resulting three clones (I, I+II and I+III) contained regions of P50 which closely represent the repeat regions A, A+B and A+C. After expression in E. coli, the polyhistidine-tagged recombinant peptides were purified by metal chelation chromatography. The three recombinant peptides were detected in Western blot analysis by a polyclonal antiserum directed against M. hominis FBG and two P50-specific monoclonal antibodies, BA10 and BG2. Each of the three recombinant peptides I, I+II and I+III was able to adhere to immobilised HeLa cells in an adhesion assay. The cyto-adhesion of the peptides could be inhibited by pre-incubation with the appropriate antibody. Therefore, it is suggested that adherence may be mediated by all regions of the P50 molecule. Attachment of the recombinant peptides to immobilised HeLa cells was inhibited by high mol. wt dextran sulphate (MW 500000), indicating that the respective P50 regions bind to sulphatides on the host cell membrane. (+info)
Evaluation of the Etest for susceptibility testing of Mycoplasma hominis and Ureaplasma urealyticum.
The Etest was used for antibiotic susceptibility testing of Mycoplasma hominis and Ureaplasma urealyticum isolates and the results were compared with those obtained with the broth microdilution method. For 50 clinical isolates of M. hominis the MICs of doxycycline, ofloxacin and ciprofloxacin agreed within +/- one dilution and +/- two dilutions in 82-98% and 98-100% of cases, respectively. The MICs of erythromycin, azithromycin, doxycycline, ofloxacin and ciprofloxacin were evaluated for 50 clinical isolates of U. urealyticum. The corresponding levels of agreement were 70-98% and 94-100%, respectively. Reference isolates M. hominis PG-21 and U. urealyticum T-960 were also used. The Etest seems to be an alternative method for determination of MICs of antibiotics with M. hominis and U. urealyticum. (+info)
Comparative in-vitro activity of levofloxacin, other fluoroquinolones, doxycycline and erythromycin against Ureaplasma urealyticum and Mycoplasma hominis.
The susceptibility of 56 Ureaplasma urealyticum and 57 Mycoplasma hominis strains to levofloxacin, ofloxacin, ciprofloxacin, fleroxacin, doxycycline and erythromycin was determined by an agar dilution method. The reference strain used was M. hominis PG 21. Agar plates containing serial dilutions of antibiotics (range 0.03-16 mg/L), and control plates (without antibiotics) were inoculated with bacteria suspended in modified Shepard's broth using a multipoint inoculator. Levofloxacin showed greater activity against all U. urealyticum and M. hominis strains compared with all other antibiotics tested. The MIC90 values for U. urealyticum were as follows: levofloxacin, 1 mg/L; ofloxacin, 2 mg/L; ciprofloxacin, 4 mg/L; fleroxacin, 4 mg/L; doxycycline, 1 mg/L; erythromycin, 8 mg/L. The MIC90s for M. hominis were: levofloxacin, 1 mg/L; ofloxacin, 2 mg/L; ciprofloxacin, 4 mg/L; fleroxacin, 4 mg/L; doxycycline, 4 mg/L; erythromycin, > or = 16 mg/L. In conclusion, the results of this study suggest that levofloxacin may be useful in the treatment of mycoplasma genital infections. (+info)
Relationship between Ureaplasma urealyticum vaginal colonization and polymorphism in the interleukin-1 receptor antagonist gene.
The relationship between polymorphisms in the interleukin-1 receptor antagonist (IL-1ra) gene and microbial vaginal colonization was examined in 88 asymptomatic women of reproductive age. Alleles of the intron 2 region of the IL-1ra gene were identified by polymerase chain reaction (PCR). PCR was also used to detect Ureaplasma urealyticum, Mycoplasma hominis, and Candida albicans; bacterial vaginosis (BV) was identified by clinical criteria. Among the 31 women with vaginal U. urealyticum, only 3 (9.7%) were homozygous for allele 2 of the IL-1ra gene; 21 (36.8%) of the 57 women who were negative for this organism were positive for allele 2 (P=.006). Only 7 women were positive for M. hominis; none were allele 2 homozygotes as opposed to 24 (29.6%) of the 81 women negative for M. hominis. There was no relation between C. albicans or BV and any IL-1ra allele. Reduced susceptibility to vaginal colonization with mycoplasmas may be associated with homozygosity of the 2 allele of the IL-1ra gene. (+info)
The adherence-associated lipoprotein P100, encoded by an opp operon structure, functions as the oligopeptide-binding domain OppA of a putative oligopeptide transport system in Mycoplasma hominis.
Mycoplasma hominis, a cell-wall-less prokaryote, was shown to be cytoadherent by the participation of a 100-kDa membrane protein (P100). To identify the gene encoding P100, peptides of P100 were partially sequenced to enable the synthesis of P100-specific oligonucleotides suitable as probes for the detection of the P100 gene. With this strategy, we identified a genomic region of about 10. 4 kb in M. hominis FBG carrying the P100 gene. Analysis of the complete deduced protein sequence suggests that P100 is expressed as a pre-lipoprotein with a structure in the N-terminal region common to peptide-binding proteins and an ATP- or GTP-binding P-loop structure in the C-terminal region. Downstream of the P100 gene, an additional four open reading frames putatively encoding the four core domains of an active transport system, OppBCDF, were localized. The organization of the P100 gene and oppBCDF in a transcriptionally active operon structure was demonstrated in Northern blot and reverse transcription-PCR analyses, as all gene-specific probes detected a common RNA of 9.5 kb. Primer extension analysis revealed that the transcriptional initiation site was localized 323 nucleotides upstream of the methionine-encoding ATG of the P100 gene. The peptide-binding character of the P100 protein was confirmed by fluorescence spectroscopy and strongly suggests that the cytoadherence-mediating lipoprotein P100 represents OppA, the substrate-binding domain of a peptide transport system in M. hominis. (+info)
Sparfloxacin selects gyrase mutations in first-step Mycoplasma hominis mutants, whereas ofloxacin selects topoisomerase IV mutations.
The role of mutations in the genes for GyrA and ParC in quinolone resistance in Mycoplasma hominis was studied. Selection with sparfloxacin gave mutations at GyrA83 (Ser-->Leu; Escherichia coli numbering) or GyrA87 (Glu-->Lys), and mutants had increased levels of resistance to sparfloxacin (8- to 16-fold) but not to ofloxacin. Selection with ofloxacin gave changes at ParC80 (Ser-->Ile) or ParC84 (Glu-->Lys), and mutants were four- to eightfold more resistant to ofloxacin but not to sparfloxacin. Selection of second-step mutants from strains with ParC mutations with either quinolone yielded double mutants with additional mutations at GyrA83 (Ser-->Trp or Ser-->Leu) or GyrA87 (Glu-->Lys). Second-step selection of GyrA mutants gave additional mutations at ParC80 (Ser-->Ile) or ParC84 (Glu-->Lys). Two-step mutants showed high levels of resistance to ofloxacin (MICs, 64 to 128 microg/ml) and moderate levels of resistance to sparfloxacin (MICs, 2 to 8 microg/ml). The primary target of ofloxacin in first-step mutants of Mycoplasma hominis was ParC, whereas that for sparfloxacin was GyrA. (+info)
In vitro susceptibilities of Mycoplasma hominis to six fluoroquinolones as determined by E test.
Twenty isolates of Mycoplasma hominis were tested for their susceptibility to six fluoroquinolones by the E test. The MICs at which 90% of the isolates were inhibited (in micrograms per milliliter) were as follows: sparfloxacin, 0.031; clinafloxacin, moxifloxacin, and trovafloxacin, 0.063; levofloxacin, 0.25; and ciprofloxacin, 0.5. Increasing the amount of inoculum or incubation in CO(2) elevated MICs by +info)