A. Charron

First report of macrolide-resistant strains and description of a novel nucleotide sequence variation in the P1 adhesin gene in Mycoplasma pneumoniae clinical strains isolated in France over 12 years.

ABSTRACT Mycoplasma pneumoniae isolates are divided in two types based on the sequence variations in the P1 adhesin gene. The type of P1 adhesin gene of 155 clinical isolates of M. pneumoniae collected in France between 1994 and 2006 was determined by a PCR-restriction fragment length polymorphism method. Until 1995, all strains belonged to type 1. In 1996 and 1997, type 1 was still predominant, but type 2 increased. Finally, since 1998, both types were present in about the same proportion. In our study, a novel sequence of the P1 adhesin gene was described in one strain. This strain could not be classified into type 1 or 2 because of variability in both P1 gene repeat elements, RepMP4 and RepMP2/3. This new sequence was certainly issued from recombination with repetitive sequences localized outside of the P1 gene in the M. pneumoniae chromosome. Moreover, MICs of erythromycin, tetracycline, and ciprofloxacin were determined for the 155 isolates. All isolates remained susceptible to tetracycline and ciprofloxacin, but two macrolide-resistant strains, isolated from two children in 1999, were identified. They harbored an A-to-G substitution at position 2058 or 2059 (Escherichia coli numbering) in domain V of 23S rRNA, associated with resistance to macrolides, lincosamides, and ketolides. To our knowledge, this is the first description of macrolide-resistant isolates of M. pneumoniae in France, but at this time, there is no sign of recent diffusion of resistant strains.

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.

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for Molecular Typing of Mycoplasma pneumoniae

ABSTRACT In this study we report on the development of a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The genomic content of M. pneumoniae M129 was analyzed for VNTRs, and 5 of the 17 VNTRs identified were selected for use in an MLVA assay. The method was based on a GeneScan analysis of VNTR loci labeled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This approach was applied to a collection of 265 isolates from various European countries, Japan, and Tunisia; and 26 distinct VNTR types were found. The VNTR assay was compared to the P1 adhesin PCR-restriction fragment length polymorphism (RFLP) typing method and showed a far better resolution than the P1 PCR-RFLP method. The discriminatory power of MLVA (Hunter-Gaston diversity index [HGDI], 0.915) for the 265 isolates was significantly higher than that of the P1 PCR-RFLP method (HGDI, 0.511). However, there was a correlation between the typing results obtained by MLVA and the P1 gene PCR-RFLP method. The potential value of MLVA of M. pneumoniae as an epidemiological tool is discussed, and the use of the VNTR markers in further investigations of the potential use of MLVA in outbreaks of M. pneumoniae infections is proposed.

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.

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.

The spread of Mycoplasma pneumoniae is polyclonal in both an endemic setting in France and in an epidemic setting in Israel.

Mycoplasma pneumoniae infections occur both endemically and epidemically, and macrolide resistance has been spreading for 10 years worldwide. A substantial increased incidence of M. pneumoniae infections has been reported in several countries since 2010. Whether this increased incidence is attributed to different or to the same M. pneumoniae genotype is unknown. We have developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for the molecular typing of M. pneumoniae isolates. In this study, the MLVA typing method was modified and validated to be applicable directly to respiratory tract specimens without culture. This method was applied to 34 M. pneumoniae-positive specimens received at the Bordeaux Hospital, France, between 2007 and 2010 in an endemic setting, and to 63 M. pneumoniae-positive specimens collected during an epidemic surge of M. pneumoniae infections in 2010 in Jerusalem, Israel. The M. pneumoniae endemic spread was shown to be polyclonal in France, with 15 MLVA types identified. Strikingly, the Israeli epidemic surge was also a multi-clonal phenomenon, with 18 circulating MLVA types. The macrolide resistance-associated substitution, A2058G, was found in 22% of the Israeli patients. Macrolide-resistant M. pneumoniae belonged to four MLVA types, the MLVA type Z being the most frequent one. An association between the MLVA type Z and macrolide resistance might exist since macrolide resistance was present or generated during the course of illness in all patients infected with this MLVA type. In conclusion, the discriminatory power of the MLVA showed that the spread of M. pneumoniae strains in France in an endemic setting was polyclonal as well as the surge of M. pneumoniae infections in Israel in 2010.

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.