H. Renaudin

Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis

OBJECTIVES Mycoplasma pneumoniae is a common aetiological agent of community-acquired respiratory tract infections for which macrolides are the treatment of choice. In France, only two macrolide-resistant isolates were reported in 1999. In contrast, several recent data reported that macrolide-resistant M. pneumoniae isolates have been spreading since 2000 in Japan. Mutations A2058G (Escherichia coli numbering), A2058C, A2059G, A2062G, C2611A and C2611G in domain V of the 23S rRNA gene were associated in vivo or in vitro with this resistance. The aim of this study was to determine whether macrolide resistance of M. pneumoniae is emerging in France. PATIENTS AND METHODS We developed a duplex real-time PCR for the detection of the six 23S rRNA mutations associated with macrolide resistance in M. pneumoniae and a simplex real-time PCR for the identification of the A2058G mutation, the most common one. Both methods rely on fluorescence resonance energy transfer coupled to melting curve analysis and are directly applicable to clinical samples. The duplex real-time PCR assay, first validated on 40 genetically characterized M. pneumoniae strains, was then applied directly on 248 French respiratory tract clinical samples. RESULTS Among M. pneumoniae-positive specimens collected before 2005, no macrolide-resistant M. pneumoniae isolate was detected. In contrast, among 51 samples collected between 2005 and 2007, five (9.8%) yielded a resistant genotype, suggesting a recent increase in macrolide-resistant M. pneumoniae isolates in France. CONCLUSIONS The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas.

Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs)). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden–Meyerhoff–Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine) and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes.

In Vitro Selection and Characterization of Resistance to Macrolides and Related Antibiotics in Mycoplasma pneumoniae

ABSTRACT Macrolide-resistant mutants of Mycoplasma pneumoniae were selected in vitro from the susceptible reference strain M129, by 23 to 50 serial passages in subinhibitory concentrations of macrolides and related antibiotics, erythromycin A, azithromycin, josamycin, clindamycin, quinupristin, quinupristin-dalfopristin, pristinamycin, and telithromycin. Mutants for which the MICs are increased could be selected with all antibiotics except the streptogramin B quinupristin. Portions of genes encoding 23S rRNA (domains II and V) and ribosomal proteins L4 and L22 of mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible strain M129. No mutation could be detected in domain II of 23S rRNA. Two point mutations in domain V of 23S rRNA, C2611A and A2062G, were selected in the presence of erythromycin A, azithromycin, josamycin, quinupristin-dalfopristin, and telithromycin. Mutants selected in the presence of clindamycin and telithromycin harbored a single amino acid change (H70R or H70L, respectively) in ribosomal protein L4, whereas insertions of one, two, or three adjacent glycines at position 60 (M. pneumoniae numbering) were selected in the presence of both streptogramin combinations. Telithromycin was the sole antibiotic that selected for substitutions (P112R and A114T) and deletions (111IPRA114) in ribosomal protein L22. Three sequential mutational events in 23S rRNA and in both ribosomal proteins were required to categorize the strain as resistant to the ketolide. Azithromycin and erythromycin A were the only selector antibiotics that remained active (MICs, 0.06 and 1 μg/ml, respectively) on their mutants selected after 50 passages.

Molecular typing of Mycoplasma pneumoniae strains by PCR-based methods and pulsed-field gel electrophoresis. Application to French and Danish isolates.

Restriction fragment length polymorphism (RFLP) analysis of the amplified P1 gene was used to type 153 strains of Mycoplasma pneumoniae isolated in France between 1977 and 1994, and in Denmark between 1962 and 1994, and an additional group of 28 strains isolated from Belgium and Germany between 1990 and 1993. Random amplified polymorphic DNA (RAPD) analysis was tested on French, Belgian and German strains. Both methods separated the strains into two groups corresponding to the two reference strains M129 (group I) and FH (group II), and gave concordant results. When 75 selected strains of different geographical origin were analysed by pulsed-field gel electrophoresis (PFGE), strains of group II fell into two closely related subgroups, subgroup IIa corresponding to the reference strain FH, and subgroup IIb. Most of the strains isolated in Denmark in the period 1962-86 belonged to group I. Almost all strains isolated in France and Denmark between 1987 and 1988 were from group II, the two subgroups being present. In 1991-3, almost all strains from France as well as Denmark, Germany and Belgium belonged to group I.

Mutations in 23S rRNA Account for Intrinsic Resistance to Macrolides in Mycoplasma hominis and Mycoplasma fermentans and for Acquired Resistance to Macrolides in M. hominis

ABSTRACT The mechanisms of intrinsic resistance of Mycoplasma hominis to 14- and 15-membered macrolides were investigated in comparison with those of M. pneumoniae, which is naturally susceptible to macrolides. Radiolabeled erythromycin was not accumulated by M. hominis PG21, but addition of an ABC transporter inhibitor increased the level of erythromycin uptake more than two times, suggesting the existence of an active efflux process. The affinity of [14C]erythromycin to ribosomes isolated from M. hominis was dramatically reduced relative to that to ribosomes isolated from M. pneumoniae. The nucleotide sequences of 23S rRNA of both ribosomal operons rrnA and rrnB and ribosomal proteins L4 and L22 of M. hominis were obtained. Compared to the sequence of M. pneumoniae, M. hominis harbored a G2057A transition in its 23S rRNA sequence, as did M. fermentans, another mycoplasma that is erythromycin resistant. An additional C2610U change was also found in the sequence of M. hominis. Moreover, two M. hominis clinical isolates with acquired resistance to 16-membered macrolides were examined for mutations in domain II and domain V of 23S rRNA and in ribosomal proteins L4 and L22. Compared to the sequence of reference strain PG21, one isolate harbored a A2059G transition and a C2611U transition in one of the two rrn operons, while the other one was mutated only at position 2059, also on the same operon. No mutation was found in the two ribosomal protein sequences. Overall, the present study is an exhaustive characterization of the intrinsic resistance of M. hominis to 14- and 15-membered macrolides and the first description of mycoplasma clinical isolates resistant to macrolide, lincosamide, and streptogramin antibiotics harboring a mutation at position 2611 in the 23S rRNA.

In Vitro Development of Resistance to Six and Four Fluoroquinolones in Mycoplasma pneumoniae and Mycoplasma hominis, Respectively

ABSTRACT Selection of resistant mutants in sequential subcultures with increasing concentrations of six and four different fluoroquinolones was studied for one reference strain each of Mycoplasma pneumoniae and Mycoplasma hominis, respectively. All fluoroquinolones tested selected for resistance, with alterations affecting the quinolone resistance-determining regions of the four target topoisomerase genes.

Activity of moxifloxacin against the urogenital mycoplasmas Ureaplasma spp., Mycoplasma hominis and Mycoplasma genitalium and Chlamydia trachomatis

The activity of moxifloxacin was compared with that of other antimicrobial agents against 54 strains of Ureaplasma spp., 54 strains of Mycoplasma hominis, 14 strains of Mycoplasma genitalium, and 44 strains of Chlamydia trachomatis. Moxifloxacin inhibited 90% of all isolates at a concentration </=1 mg/L, being the most active compound against C. trachomatis and sharing the highest activity with garenoxacin and gemifloxacin against mycoplasmas. Moxifloxacin killed the 30 mycoplasma isolates tested at a concentration </=1 mg/L, except those resistant to fluoroquinolone. Thus, moxifloxacin has attracted interest as a potential therapy for mycoplasmal or chlamydial urogenital infections.

DNA Gyrase and Topoisomerase IV Mutations in Clinical Isolates of Ureaplasma spp. and Mycoplasma hominis Resistant to Fluoroquinolones

ABSTRACT Twelve clinical isolates of Ureaplasma spp. and one isolate of Mycoplasma hominis were examined for resistance to fluoroquinolones. Previously described mutations at positions 83 and 95 in GyrA (Escherichia coli numbering) and positions 80 and 87 in ParC were found. Unusual alterations were described at positions ParC 123 and 134.

Tetracycline Resistance in Ureaplasma spp. and Mycoplasma hominis: Prevalence in Bordeaux, France, from 1999 to 2002 and Description of Two tet(M)-Positive Isolates of M. hominis Susceptible to Tetracyclines

ABSTRACT Twenty-four of 128 clinical isolates of Mycoplasma hominis and 6 of 276 clinical isolates of Ureaplasma spp. from Bordeaux, France (1999 to 2002), were resistant to tetracycline and harbored the tet(M) gene. For M. hominis, we also found an increase in tetracycline resistance and two tet(M)-positive isolates that were susceptible to tetracyclines.

Development of a real‐time PCR targeting the yidC gene for the detection of Mycoplasma hominis and comparison with quantitative culture

Mycoplasma hominis is an opportunistic human mycoplasma species that can be either commensal or pathogenic. Its detection by culture is considered to comprise the reference technique. Previously reported PCR techniques target the 16S rRNA or the gap gene, although sequence variations among clinical isolates may lead to variations in clinical sensitivity. The present study aimed to develop a specific TaqMan quantitative real-time PCR assay, targeting a gene conserved in all M. hominis isolates, and to compare it with quantitative culture. With the knowledge of the M. hominis PG21 genome sequence, the yidC gene, encoding a membrane protein translocase, was chosen as target. Its intraspecies heterogeneity was checked at the nucleotide level using 31 reference or clinical strains. The limit of detection, the analytical specificity and the reproducibility of the assay were assessed. Moreover, PCR and culture results were compared using 153 urogenital specimens. The limit of detection was seven copies/μL. The analytical specificity was 100%, with good inter- and intra-assay reproducibility. Among the 153 urogenital specimens, the yidC PCR and culture allowed detection of 55 and 45 M. hominis-positive samples, respectively. Comparison of the bacterial load among the 45 specimens found to be M. hominis-positive by both techniques revealed a statistically significant association between the quantitative results obtained. In conclusion, we developed a specific, sensitive and reproducible real-time PCR to detect all M. hominis clinical isolates. This PCR was shown to have higher sensitivity than culture, although both methods were correlated for quantification of M. hominis loads in urogenital specimens.