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 PCRrestriction 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.Mycoplasma pneumoniae represents one of the most common etiological agents of community-acquired respiratory tract infections, with the clinical courses ranging from mild forms of pharyngitis and tracheobronchitis to severe cases of interstitial pneumonia. M. pneumoniae infections are responsible for 20% or more of all cases of community-acquired pneumonia (22) and occur endemically, with epidemic peaks occurring every 4 to 7 years. School-age children and young adults are the populations that are the most affected.Strain subtyping by molecular methods is a powerful tool for surveillance and outbreak investigation. However, molecular typing is hampered by the fact that M. pneumoniae is a genetically homogeneous species and isolates are poorly differentiated by PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene, the most common genotyping method. PCR-RFLP analysis of the gene encoding the P1 protein, a major adhesin that induces a strong humoral immune response during infection, enables the separation of isolates into two types, types 1 and 2 (1, 2, 17). More recent studies used repetitive regions, RepMp2/3 and RepMp4, which can be found in the P1 gene, for molecular typing. Besides the subtype classification, those methods allow the identification of variants of subtypes 1 and 2 (3,5,8).In addition to these molecular typing methods that target only one gene, other methods based on the study of the whole genome have been adapted to M. pneumoniae, such as pulsedfield electrophoresis (1) and multilocus sequence typing (MLST) (4). Pulsed-field electrophoresis is able to differentiate strains into two groups (groups 1 and 2), like P1 gene PCR-RFLP analysis can, and can subdivide group 2 into two subgroups, subgroups 2a and 2b (1). Due t...

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.

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.

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 ( 111 IPRA 114 ) 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 g/ml, respectively) on their mutants selected after 50 passages.Mycoplasma pneumoniae is a common etiological agent of community-acquired respiratory tract infections in children and young adults. Macrolide and related antibiotics are the drugs of choice for the treatment of these infections, and strains with acquired resistance to macrolides have been rarely described. However, resistant strains of M. pneumoniae have been obtained in vitro, by selection in the presence of erythromycin A (20,22,27), and few macrolide-resistant M. pneumoniae isolates have been reported from patients treated with these antibiotics (8,22,27).Macrolide, lincosamide, streptogramin, and ketolide antibiotics (MLSKs) inhibit protein synthesis by binding to domain V of 23S rRNA, and domain II is in the vicinity of this binding site (1,12,17,26). Three main mechanisms of resistance have been reported: drug inactivation, active efflux, and modification of the target sites by methylation or mutation (29, 34). Mutations in domains II and V of 23S rRNA and in ribosomal proteins L4 and L22 were involved in resistance to MLSKs, first in bacteria with a small number of rrn operons and more recently in bacteria like Streptococcus pneumoniae and Staphylococcus aureus with four and six rrn operons, respectively (25,29,32). In mycoplasmas, gram-positive related bacteria which possess one or two copies of 23S rRNA, only resistance by point muta...

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 [ 14 C]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.Human mycoplasmas are responsible for urogenital and respiratory tract infections. Macrolide, lincosamide, and streptogramin antibiotics (MLSs) are a class of antimicrobials commonly used for the treatment of these infections. The MICs of ketolides, a new class of antimicrobials derived from erythromycin, are low for these microorganisms (3). The MLSs and ketolide antibiotics (MLSKs) inhibit protein synthesis by binding to domain V and domain II of 23S rRNA (13, 21). Three main mechanisms of resistance have been reported: drug inactivation, active efflux, and modification of the target sites by methylation or mutation (46, 49). Resistance by ribosomal mutations in domain II and domain V of 23S rRNA has been described, and recently, mutations in ribosomal proteins L4 and L22 were also associated with resistance to MLSKs (7,30,33,42,43).Mycoplasmas present different phenotypes of intrinsic resistance to macrolides. Mycoplasma pneumoniae, a respiratory mycoplasma, is susceptible to all MLSKs. In contrast, M. hominis, a genital species, is naturally resistant to 14-and 15-membered macrolides and ketolides but is susceptible to josamycin, a 16-membered macrolide, and lincosamides. Intrinsic resistance to 14-membered macrolides has been observed in other mycoplasma s...

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.Mycoplasma pneumoniae is a common pathogen responsible for community-acquired respiratory tract infections, particularly in school-aged children and young adults. Epidemics occur periodically at 4-to 7-year intervals (22). The 170-kDa P1 protein is a major adhesin protein that induces a strong immunological response (1,10,22). Only one copy of a functional full-length P1 gene is present in the M. pneumoniae genome (9). This gene is composed by two repetitive regions, RepMP4 located at the 5Ј end of the coding region and RepMP2/3 located at the 3Ј end of the coding region (16). Eight to 10 closely related but not identical copies of both repetitive regions are dispersed through the fully sequenced genome of the M. pneumoniae M129 strain (16). Based on the sequence of the P1 gene, two types, 1 and 2, have been reported (3,20). M. pneumoniae M129 is a type 1 prototype, while M. pneumoniae FH, Mac, and 1842 strains belong to type 2. Moreover, one type 1 variant and two type 2 variants that showed sequence variations in the RepMP2/3 but not the RepMP4 element of the P1 gene have been described (6,8,11).Previous studies found that one or the other of the two types tended to predominate among clinical isolates in specific geographical regions and that the predominant type changed over time (3,7,17,20). These changes in the P1 adhesin type may play a role in the development of outbreaks. In France, the 1987 and 1992 epidemics were due to strains belonging to type 2 and 1, respectively (3).Macrolides are the drug of choice for the treatment of M. pneumoniae infections...

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.Human mycoplasmas, Mycoplasma pneumoniae and Mycoplasma hominis, are etiological agents of respiratory and genitourinary tract infections, respectively, for which fluoroquinolones offer the potential for empirical treatment. Newer compounds of this class have activity against mycoplasmas that is improved over that of older fluoroquinolones (1). To date, acquired resistance to fluoroquinolones has been reported among human mycoplasma clinical isolates only for the genital species, M. hominis, M. genitalium, and Ureaplasma spp. (3-5). In vivo target alterations located in the quinolone resistancedetermining regions (QRDRs) of both DNA gyrase and topoisomerase IV subunits were confirmed by in vitro quinolone resistance studies with M. hominis (2,8).The purpose of this study was to determine and compare the abilities of six older and newer fluoroquinolones to select for fluoroquinolone-resistant mutants of M. pneumoniae. Furthermore, our previous data on fluoroquinolone resistance in M. hominis were completed by selecting for mutants of this mycoplasma that were resistant to the newer compounds levofloxacin, moxifloxacin, gemifloxacin, and gatifloxacin.Growth conditions and antibiotic susceptibility testing of the mycoplasma strains have been previously described (12). MICs of fluoroquinolones were determined in the presence and absence of reserpine (20 g/ml) as described previously (11). Two selection methods, with either broth or agar medium, were used for M. pneumoniae FH (ATCC 15531), while only the agar-based selection was done for M. hominis PG21 (ATCC 23114). Broth-selected mutants were obtained by serial transfers of M. pneumoniae FH in Hayflick modified broth medium containing subinhibitory concentrations of each fluoroquinolone, as previously described (10). For the first passage, the reference strain M. pneumoniae FH was inoculated in Hayflick modified medium with increasing twofold dilutions of each antibiotic. The MIC was determined as the lowest concentration of antimicrobial agent that prevented a color change in the medium at the time when the drug-free growth control first showed a color change (after about 5 days of incubation at 37°C). The culture containing the highest antibiotic concentration with visible growth (subinhibitory concentration) was used to inoculate another antibiotic dilution panel for the following passage. Fifteen passages were performed for each selector antibiotic except for gatifloxacin, and two of the five clones subcultured from passages 5, 7, 10, and 15 were studied. With gatifloxacin, the characterization of passage 2 was added and the selection was conducted up to 10 passages. S...

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.