Mycoplasma pneumoniae causes community-acquired respiratory tract infections, particularly in school-aged children and young adults. These infections occur both endemically and epidemically worldwide. M. pneumoniae lacks cell wall and is subsequently resistant to beta-lactams and to all antimicrobials targeting the cell wall. This mycoplasma is intrinsically susceptible to macrolides and related antibiotics, to tetracyclines and to fluoroquinolones. Macrolides and related antibiotics are the first-line treatment of M. pneumoniae respiratory tract infections mainly because of their low MIC against the bacteria, their low toxicity and the absence of contraindication in young children. The newer macrolides are now the preferred agents with a 7-to-14 day course of oral clarithromycin or a 5-day course of oral azithromycin for treatment of community-acquired pneumonia due to M. pneumoniae, according to the different guidelines worldwide. However, macrolide resistance has been spreading for 15 years worldwide, with prevalence now ranging between 0 and 15% in Europe and the USA, approximately 30% in Israel and up to 90–100% in Asia. This resistance is associated with point mutations in the peptidyl-transferase loop of the 23S rRNA and leads to high-level resistance to macrolides. Macrolide resistance-associated mutations can be detected using several molecular methods applicable directly from respiratory specimens. Because this resistance has clinical outcomes such as longer duration of fever, cough and hospital stay, alternative antibiotic treatment can be required, including tetracyclines such as doxycycline and minocycline or fluoroquinolones, primarily levofloxacin, during 7–14 days, even though fluoroquinolones and tetracyclines are contraindicated in all children and in children < 8 year-old, respectively. Acquired resistance to tetracyclines and fluoroquinolones has never been reported in M. pneumoniae clinical isolates but reduced susceptibility was reported in in vitro selected mutants. This article focuses on M. pneumoniae antibiotic susceptibility and on the development and the evolution of acquired resistance. Molecular detection of resistant mutants and therapeutic options in case of macrolide resistance will also be assessed.

Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes , strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes . Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.

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...

Mycoplasma pneumoniae is a pathogenic mycoplasma responsible for respiratory tract infections in humans, which occurs worldwide in children and adults. This article focuses on its antibiotic susceptibility profile and on the development of acquired resistance in this microorganism. The lack of a cell wall in mycoplasmas makes them intrinsically resistant to β-lactams and to all antimicrobials that target the cell wall. M. pneumoniae is susceptible to macrolides and related antibiotics, tetracyclines and fluoroquinolones. Macrolides and related antibiotics are the first-line treatment for respiratory infections caused by M. pneumoniae. However, strains with acquired resistance to macrolides have recently emerged worldwide and have been spreading in Europe, USA and A sia especially, with more than 90% of Chinese isolates resistant to erythromycin and azithromycin. This acquired resistance can be detected by PCR methods directly from respiratory specimens and is related to 23S rRNA mutations.

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...

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...