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ABSTRACTMolecular typing of Mycoplasma pneumoniaeis an important tool for identifying grouped cases and investigating outbreaks. In the present study, we developed a new genotyping method based on single nucleotide polymorphisms (SNPs) selected from the whole-genome sequencing of eight M. pneumoniaestrains, using the SNaPshot minisequencing assay. Eight SNPs, localized in housekeeping genes, predicted lipoproteins, and adhesin P1 genes were selected for genotyping. These SNPs were evaluated on 140 M. pneumoniaeclinical isolates previously genotyped by multilocus variable-number tandem-repeat analysis (MLVA-5) and adhesin P1 typing. This method was also adapted for direct use with clinical samples and evaluated on 51 clinical specimens. The analysis of the clinical isolates using the SNP typing method showed nine distinct SNP types with a Hunter and Gaston diversity index (HGDI) of 0.836, which is higher than the HGDI of 0.583 retrieved for the MLVA-4 typing method, where the nonstable Mpn1 marker was removed. A strong correlation with the P1 adhesin gene typing results was observed. The congruence was poor between MLVA-5 and SNP typing, indicating distinct genotyping schemes. Combining the results increased the discriminatory power. This new typing method based on SNPs and the SNaPshot technology is a method for rapid M. pneumoniaetyping directly from clinical specimens, which does not require any sequencing step. This method is based on stable markers and provides information distinct from but complementary to MLVA typing. The combined use of SNPs and MLVA typing provides powerful discrimination of strains.

ABSTRACTMatrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of =1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovisand Mycoplasma agalactiaeisolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniaeisolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.

ABSTRACTMycoplasma pneumoniaeisolates 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. pneumoniaecollected 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. pneumoniaechromosome. 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 colinumbering) 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. pneumoniaein France, but at this time, there is no sign of recent diffusion of resistant strains.

Objectives: Mycoplasma hominis is intrinsically resistant to 14- and 15-membered macrolides and to the ketolide telithromycin but is susceptible to josamycin, a 16-membered macrolide, and lincosamides. The aim of our study was to investigate the in vitro development of macrolide resistance in M. hominis and to study the impact of ribosomal mutations on MICs of various macrolides and related antibiotics.Methods: Selection of macrolide-resistant mutants was performed by serial passages of M. hominis PG21 in broth medium containing subinhibitory concentrations of clindamycin, pristinamycin, quinupristin/dalfopristin and telithromycin. Stepwise selection of josamycin-resistant mutants was performed onto agar medium containing increasing inhibitory concentrations of josamycin. Resistant mutants were characterized by PCR amplification and DNA sequencing of 23S rRNA, L4 and L22 ribosomal protein genes.Results: Various mutations in domain II or V of 23S rRNA were selected in the presence of each selector antibiotic and were associated with several resistance phenotypes. Josamycin was the sole antibiotic that selected for single amino acid changes in ribosomal proteins L4 and L22. Unexpectedly, the C2611U transition selected in the presence of clindamycin and the quinupristin/dalfopristin combination was associated with decreased MICs of erythromycin, azithromycin and telithromycin, leading to a loss of the intrinsic resistance of M. hominis to erythromycin and azithromycin.Conclusions: Ribosomal mutations were associated with resistance to macrolides and related antibiotics in M. hominis. Some mutants showed a loss of the intrinsic resistance to erythromycin and azithromycin.

Mycoplasma pneumoniae is a pathogenic mycoplasma responsible for respiratory tract infections in humans, occurring worldwide in children and adults. This review briefly focuses on its antibiotic susceptibility profile and on the development of acquired resistance for this microorganism. The lack of a cell wall in mycoplasmas makes them intrinsically resistant to β-lactams and to all antimicrobials which target the cell wall. Intrinsic resistance related to specific mycoplasma species concerns essentially the macrolide-lincosamide-streptogramin-ketolide (MLSK) antibiotic group. M. pneumoniae is susceptible to all MLSK antibiotics, except to lincomycin. Among the three antibiotic classes used for the treatment of mycoplasmal infections including tetracyclines, MLSK group, and fluoroquinolones, macrolides and related antibiotics are the drug of choice for respiratory infections caused by M. pneumoniae. Both target alterations and efflux mechanisms implicated in acquired antibiotic resistance have been described in mycoplasmas either by genetic mutation or transfer of new genes carried by transposons. At present, M. pneumoniae remains greatly susceptible to antibiotics, but as this mycoplasma is difficult to isolate, the number of clinical strains tested is limited and the occurrence of acquired resistance not well documented. However some strains having acquired resistance to MLSK have been decribed in vivo and erythromycin-resistant isolates are spreading now in Japan. To date, no clinical isolates resistant to fluoroquinolones or tetracyclines have been described in the literature, but some strains having acquired resistance to both classes have been selected in vitro. Molecular diagnosis of this acquired resistance has been related to target alterations, in ribosome for macrolides and tetracyclines, or in topoisomerase II genes for fluoroquinolones.

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

ABSTRACTThe activities of garenoxacin, gatifloxacin, and gemifloxacin were compared with those of four fluoroquinolones against human mycoplasmas and ureaplasmas, including fluoroquinolone-resistant genetically characterized strains. Garenoxacin exhibited the highest activity, followed by gemifloxacin, moxifloxacin, and gatifloxacin. The minimal bactericidal activities of these three compounds were lower than those of the four fluoroquinolones.

ABSTRACTMacrolide-resistant mutants of Mycoplasma pneumoniaewere 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. pneumoniaenumbering) 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.

ABSTRACTThe mechanisms of intrinsic resistance of Mycoplasma hoministo 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. hominisPG21, 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. hominiswas dramatically reduced relative to that to ribosomes isolated from M. pneumoniae.The nucleotide sequences of 23S rRNA of both ribosomal operons rrnAand rrnBand ribosomal proteins L4 and L22 of M. hominiswere obtained. Compared to the sequence of M. pneumoniae, M. hominisharbored 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. hominisclinical 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 rrnoperons, 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. hoministo 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.

Les complications vasculaires de la fièvre Q sont maintenant bien décrites, mais ces infections sont quasiment toujours monomicrobiennes, contrairement aux infections de prothèses vasculaires et endovasculaires. Lorsque les hémocultures et/ou les prélèvements locaux sont positifs en culture, il n’y a actuellement pas de recommandations pour rechercher systématiquement des micro-organismes intracellulaires. A notre connaissance, seuls cinq cas de fièvre Q surinfectée ont été décrits dans la littérature à ce jour.

Le diagnostic étiologique de l’aortite est souvent difficile, la distinction entre causes infectieuses et inflammatoires étant parfois complexe. L’objectif de cette étude était de comparer les caractéristiques étiologiques et évolutives des aortites infectieuses (AI) et non infectieuses (ANI).

La population carcérale est une population vulnérable à risque d’infections sexuellement transmissibles (IST). Mycoplasma genitalium (Mg) est un agent d’IST dont le traitement repose en 1religne sur les macrolides. Leur efficacité diminue du fait d’une augmentation récente des résistances. Le but de cette étude est d’estimer la prévalence de Mg en milieu carcéral (pas de données dans la littérature), de définir les caractéristiques médico-sociales et les manifestations cliniques des patients infectés, d’évaluer le taux de résistance aux macrolides et la prise en charge thérapeutique.

In order to develop a potential tool for SPET examination of the serotonin transporter (5-HTT) in the human brain, we report the synthesis and radiolabelling of 2β-carbomethoxy-3β-(3'-iodo-4'-isopropylphenyl) nortropane (LBT-44). We also report ex vivo autoradiography performed in the rat brain. The radiosynthesis of [¹²⁵I]LBT-44 was accomplished by iododestannylation of a trimethyltin precursor using [¹²⁵I]sodium iodide and peracetic acid or choramine-T as oxidant. After purification by reverse phase HPLC, [¹²⁵I]LBT-44 was obtained in a radiochemical yield higher than 75% and a radiochemical purity greater than 95%. Ex vivo autoradiographic studies revealed a marked accumulation of [¹²⁵I]LBT-44 in brain areas rich in 5-HTT whereas no accumulation was observed in the striatum, which is rich in dopamine transporters. These results are in favour of specific binding of [¹²⁵I]LBT-44 to 5-HTT that is required for SPET exploration in human. Copyright © 1999 John Wiley & Sons, Ltd.

ABSTRACTThe performances of five commercial TaqMan real-time PCR assays for the detection of Mycoplasma pneumoniaein respiratory tract specimens were evaluated in comparison with an in-house real-time PCR. All kits allowed prompt and specific results, validated by the use of an internal control. The Nanogen kit showed the best clinical sensitivity.

ABSTRACTTwelve clinical isolates of Ureaplasmaspp. and one isolate of Mycoplasma hominiswere examined for resistance to fluoroquinolones. Previously described mutations at positions 83 and 95 in GyrA (Escherichia colinumbering) and positions 80 and 87 in ParC were found. Unusual alterations were described at positions ParC 123 and 134.

ABSTRACTWe report on the case of a 54-year-old woman diagnosed as having culture-negative endocarditis (clinical and histopathologic evidence compatible with a recent episode of endocarditis). The responsibility of Chlamydia pneumoniaein this episode of endocarditis was suggested by a serological study and was then confirmed by the positive results of PCR and in situ hybridization tests with aortic and mitral valves tissues. To our knowledge, this is the first case of endocarditis due to C. pneumoniaeconfirmed by molecular biology-based techniques.

Depuis quelques années, Mycoplasma genitaliumest une cause reconnue d’urétrite sexuellement transmissible chez l’homme. Chez la femme, M. genitaliumest en train de devenir une cause émergente d’infection sexuellement transmissible, on parle de « nouveau chlamydia ». De nombreuses études ont montré une association positive entre M. genitaliumet la survenue de cervicites muco-purulentes mais aussi d’infections génitales hautes à type d’endométrites et de salpingites. Cependant, il n’a jamais été établi d’association claire entre la présence de M. genitaliumet la survenue de conséquences obstétricales péjoratives de type menace d’accouchement prématuré ou prématurité. L’objectif de cette étude est d’évaluer la prévalence et les conséquences obstétricales des infections à M. genitaliumchez les femmes enceintes suivies au CHU de Bordeaux.

Depuis quelques années, Mycoplasma genitaliumest une cause reconnue d’urétrite sexuellement transmissible chez l’homme. Chez la femme, M. genitaliumest en train de devenir une cause émergente d’infection sexuellement transmissible, on parle de « nouveau chlamydia ». De nombreuses études ont montré une association positive entre M. genitaliumet la survenue de cervicites muco-purulentes mais aussi d’infections génitales hautes à type d’endométrites et de salpingites. Cependant, il n’a jamais été établi d’association claire entre la présence de M. genitaliumet la survenue de conséquences obstétricales péjoratives de type menace d’accouchement prématuré ou prématurité. L’objectif de cette étude est d’évaluer la prévalence et les conséquences obstétricales des infections à M. genitaliumchez les femmes enceintes suivies au CHU de Bordeaux.

Les infections de prothèses vasculaires (IPV) sont associées à des taux de mortalité élevés et de diagnostic difficile. Différentes études évaluant les performances de la TEP au 18-FDG (TEP) ont été publiées utilisant cependant souvent des critères d’interprétation différents. De plus, ces échelles visuelles d’interprétation semblent présenter des insuffisances dans cette pathologie. Nous avons donc comparé, à partir d’une étude prospective, les performances de 3 échelles visuelles d’interprétation pour le diagnostic d’IPV.

Les infections de prothèses vasculaires (IPV) sont associées à des taux de mortalité élevés. Le diagnostic est souvent difficile, surtout dans les infections latentes. Pour améliorer le résultat du traitement médical et chirurgical, un diagnostic précoce et précis est primordial et les critères de diagnostic réels sont souvent insuffisants. Si la tomographie par émission de positons marquée 18F-fludéoxyglucose (TEP) et la scintigraphie aux leucocytes radiomarqués (SLM) ont de bonnes performances dans cette indication, une seule étude et sur une petite série de patients a, à ce jour, comparé les deux examens. Notre étude a pour but de comparer de façon prospective, sur une plus large série de patient, les performances de la TEP et de la SLM pour le diagnostic d’IPV.