Tetracycline and fluoroquinolone resistance in clinical Ureaplasma spp. and Mycoplasma hominis isolates in France between 2010 and 2015.

Objectives: As information on Ureaplasma spp. and Mycoplasma hominis resistance is currently limited, the aim of this study was to investigate the susceptibility of Ureaplasma spp. and M. hominis to tetracyclines and fluoroquinolones in France. Methods: The susceptibility of 1014 clinical isolates obtained in Bordeaux University Hospital (Bordeaux, France) between 2010 and 2015 was evaluated using two commercial kits, S.I.R. Mycoplasma (Bio-Rad) from 1 January 2010 to 5 October 2012 and MYCOFAST RevolutioN kit (ELITech Group) from 6 October 2012 to 31 December 2015. The MICs of isolates designated as resistant were determined using the broth microdilution assay. Additionally, the tet(M) gene and fluoroquinolone resistance-associated mutations were identified. Results: Among 831 Ureaplasma spp. isolates, the tetracycline, levofloxacin and moxifloxacin resistance rates were 7.5%, 1.2% and 0.1%, respectively. Among 183 M. hominis isolates, the resistance rates were 14.8%, 2.7% and 1.6% for tetracycline, levofloxacin and moxifloxacin, respectively. Over the 6 year period, no significant change in resistance to tetracycline or fluoroquinolones was observed. The tet(M) gene was found in all tetracycline-resistant isolates. All levofloxacin-resistant isolates harboured a mutation in the parC or parE genes. Isolates that were also resistant to moxifloxacin harboured an additional mutation in the gyrA gene. The MYCOFAST RevolutioN kit significantly overestimated levofloxacin and moxifloxacin resistance in Ureaplasma spp. isolates. Conclusions: Resistance to tetracycline and fluoroquinolones is limited in France in mycoplasmas but compared with a previous report in 1999-2002, a significant increase in tetracycline resistance among Ureaplasma spp. was observed. Ongoing monitoring of the antibiotic susceptibility of these urogenital mycoplasmas remains necessary.

Interaction of Mycoplasma hominis PG21 with Human Dendritic Cells: Interleukin-23-Inducing Mycoplasmal Lipoproteins and Inflammasome Activation of the Cell.

Mycoplasma hominis lacks a cell wall, and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of M. hominis enriched with lipoproteins was shown to stimulate the production of interleukin-23 (IL-23) by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon M. hominis infection. We studied here the interaction between M. hominis PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell. IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two nondenaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and enzyme-linked immunosorbent assay (ELISA). We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular mass between 20 and 35 kDa and 19 with a molecular mass between 40 and 100 kDa. Among them, lipoprotein MHO_4720 was identified as potentially bioactive, and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1ß was observed. After 24 h of coincubation of hDCs with M. hominis, downregulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction of M. hominis and hDCs.IMPORTANCEMycoplasma hominis is a human urogenital pathogen involved in gynecologic and opportunistic infections. M. hominis lacks a cell wall, and its membrane contains many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on the interaction between M. hominis and human dendritic cells and examined both sides of the interaction, the mycoplasmal lipoproteins involved in the activation of the host cell and the immune response of the cell. On the mycoplasmal side, we showed for the first time that M. hominis lipoproteins with high molecular mass were potentially bioactive. On the cell side, we reported an activation of the inflammasome, which is involved in the innate immune response.

Molecular Epidemiology of Mycoplasma pneumoniae: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology.

Molecular typing of Mycoplasma pneumoniae is 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. pneumoniae strains, 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. pneumoniae clinical 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. pneumoniae typing 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.

Identification and subtyping of clinically relevant human and ruminant mycoplasmas by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-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 bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates 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.

Evaluation of the combination of the NucliSENS easyMAG and the EasyQ applications for the detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory tract specimens.

Mycoplasma pneumoniae and Chlamydia pneumoniae are respiratory tract pathogens frequently involved in community-acquired pneumonia, but are fastidious microorganisms. Their direct detection mainly requires molecular amplification techniques. A nucleic acid extraction system, NucliSENS easyMAG, and a real-time nucleic acid sequence-based amplification (NASBA) technique, NucliSENS EasyQ, were recently developed by bioMérieux to detect both bacteria. The aim of our study was to compare the easyMAG/EasyQ combination with our in-house combination, MagNA Pure extraction (Roche) and real-time polymerase chain reaction (PCR), to detect both bacteria in respiratory tract specimens. The analytical specificities of both combinations were similar. A higher analytical sensitivity was found for C. pneumoniae using the easyMAG/EasyQ combination, since the easyMAG/EasyQ system detected nucleic acid extracts 10(6) times more diluted than the in-house combination. Both combinations were equivalent when detecting M. pneumoniae in positive respiratory tract samples. Finally, the easyMAG/EasyQ combination is a potential useful tool for the detection of both bacteria regarding sensitivity, specificity, monitoring, and standardization of the procedure.

Development of multiple-locus variable-number tandem-repeat analysis for molecular typing of Mycoplasma pneumoniae.

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.

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.

Characterisation of in vitro-selected mutants of Ureaplasma parvum resistant to macrolides and related antibiotics.

Resistant mutants of Ureaplasma parvum were selected by serial passages of a susceptible strain in subinhibitory concentrations of different macrolides and related antibiotics (erythromycin, azithromycin, josamycin, quinupristin, quinupristin/dalfopristin, pristinamycin and telithromycin). Mechanisms of resistance were characterised by sequencing portions of genes encoding 23S rRNA and ribosomal proteins L4 and L22. Mutants with significantly increased minimum inhibitory concentrations could be selected with all the selector antibiotics, except quinupristin and pristinamycin. Mutants harboured mutations in domain V of the 23S rRNA gene at nucleotides G2056, G2057 or A2058 (Escherichia coli numbering) and in conserved portions of ribosomal proteins L4 and L22. Most of the mutations were associated with complete loss of macrolide and ketolide activity, whereas streptogramin combinations were less affected.

[Serologic diagnosis of chlamydial and Mycoplasma pneumoniae infections]. / Limites et perspectives du diagnostic sérologique à l'ère de l'amplification génique in vitro: infections génitales à Chlamydia trachomatis et infections respiratoires à Chlamydia pneumoniae et Mycoplasma pneumoniae.

The diagnosis of Chlamydia trachomatis infection can be based either on direct detection of the organism or its components or indirectly by measuring antibodies as markers of the individual's response to the infection. The latter is currently of limited value. Neither IgG or IgA antibodies can be used to diagnose current genital infection by Chlamydia trachomatis or to exclude such an infection. There is no solid ground as yet for the use of IgA antibodies as a marker of persistant or unresolved infection. Commercial tests in the Elisa format based on peptides from the MOMP of Chlamydia trachomatis are available and show good specificities and sensitivities. Hsp60 seems to have a unique role in the development of tubal scarring and antibodies to chsp60 could predict tubal factor infertility. Serology is the main diagnostic tool for the diagnosis of Mycoplasma pneumoniae infection. The serologic assays are the complement fixation test (CF), immunofluorescence, the microparticle agglutination and recently EIAs. The CF test is still used for serodiagnosis of Mycoplasma pneumoniae infection because of the sensitivity of 90%. Single titer of >or= 64 are considered to be indicative of recent infection. A number of commercial EIAs have been developped. The difficulty for IgG interpretation is a definition of a cutoff value for discriminating infected and healthy subjects. Most of the IgM assays show good diagnostic sensitivities and are valuable tools for the early diagnosis of Mycoplasma pneumoniae infection in children. There are no wholly satisfactory serological methods for diagnosis of Chlamydia pneumoniae infection. Problems arise from the high background of IgG antibody prevalence, the lack of standardized testing methods.

New developments in diagnostic and treatment of mycoplasma infections in humans.

Several methods can be used for the diagnosis of mycoplasmal human infections. Culture is not satisfactory for fastidious species, while serological procedures allow only a retrospective diagnosis. Recently, rapid methods have become available. Antigenic detection proposed for Mycoplasma pneumoniae lacks sensitivity. Hybridization based techniques include DNA probes and mainly DNA amplification. The main usefulness of the polymerase chain reaction (PCR) is the detection of fastidious organisms such as M. pneumoniae, M. genitalium, M. fermentans, M. penetrans, but PCR can also be used for characterization of the strains for epidemiological purposes, or for detection of antimicrobial resistance genes. The major advantage of PCR for detection is its very high sensitivity. However, until now, the major drawback of this technique has been the lack of commercial kits. When available, they should provide better standardization of the technique and, if available at a reasonable cost, become the major technique for the diagnosis of mycoplasma infections. The antibiotics used for the treatment of mycoplasmal infections belong to tetracyclines, macrolides-lincosamides and fluoroquinolones. These products are highly active in vitro against mycoplasmas. However, some of them have a differential activity according to the species, and acquired resistance has been reported, mainly in genital mycoplasmas. Most of mycoplasmal infections are cured by adapted antibiotics, but they may be difficult to cure in immunosuppressed patients.

Detection of Mycoplasma hominis antibodies by three different techniques, application to women with pelvic inflammatory disease.

Mycoplasma hominis, a mycoplasma potentially isolated from the genital tract of healthy women, seems to have some role in pelvic inflammatory diseases (PID). Three serologic techniques were developed to detect antibodies to M. hominis: ELISA (IgG, IgM), microimmunofluorescence (MIF) and Western blot. Rabbit and human control sera were used to establish the standard assay conditions. ELISA proved to be more sensitive but less specific than MIF. Cross-reactivity with antibodies to M. pneumoniae existed with ELISA but not with MIF. Twenty six and 60 kDa proteins were responsible for that cross-reactivity, as assessed by Western blot analysis. Among 33 patients with PID, 18 to 35 year old, 67% had antibodies to M. hominis by ELISA and 33% by MIF. Among 52 age comparable healthy women, 44 and 13% were positive for M. hominis by ELISA and MIF respectively. The Western blot analysis showed heterogeneity in the antibody response to M. hominis, but a 76 kDa protein reacted with half of the tested sera. Our study agrees with some role for M. hominis in PID. M. hominis was certainly the primary pathogen in one case, played a role that was probable in seven cases, and possible in three patients.

[Methods of studying mycoplasma infections]. / Méthodes d'exploration des infections à mycoplasmes.

Mycoplasmas are most often responsible for respiratory and genital infections. At present, diagnosis is carried out by serology for infections caused by M. pneumoniae and by culture for infections due to genital mycoplasmas. For M. pneumoniae, new prospects may lead to a rapid diagnosis, detection by molecular hybridization and immunological detection. Also, the research of specific antibodies should benefit from a better knowledge of the major antigens. Culture of the genital mycoplasmas, U. urealyticum and M. hominis is simple, but the interpretation of their presence is difficult because they may be recovered in a commensal condition. The envisaged advances should lead to a better assessment of their pathogenicity. The role of M. genitalium, a species related to M. pneumoniae recently discovered in respiratory specimens, should be better determined by sensitive techniques developed to distinguish it from M. pneumoniae.

Method comparison for molecular typing of French and Tunisian Mycoplasma genitalium-positive specimens.

In this study, 76 French and Tunisian urogenital specimens were subjected to molecular typing by using the two main Mycoplasma genitalium molecular typing methods, the mgpB single nucleotide polymorphism (SNP) typing method and the combination analysis of a variable-number tandem-repeat (VNTR) marker in MG309 and mgpB SNP. Furthermore, we tried to develop a multiple-locus VNTR analysis (MLVA) method. The genome of M. genitalium G37(T) was analysed for VNTRs and four VNTRs were used for an MLVA. The method, applied directly on clinical specimens, was based on a genescan analysis of VNTR loci labelled with fluorescent dyes by using multiplex PCR and capillary electrophoresis. This method had a 1.00 diversity index (DI) while the mgpB SNP typing and the combination of MG309 and mgpB SNPs had DIs of 0.853 and 0.989, respectively. However, among the sets of two concurrent specimens, taken at the same time from the urogenital tracts of 12 patients, only nine had matching MLVA profiles, while the two other methods gave identical profiles for all specimens amplified, except for one set. Moreover, eight new sequence types were described with the mgpB SNP typing method. The three molecular typing methods revealed a genetic heterogeneity, suggesting that M. genitalium was endemic in France and Tunisia and that the infections were not due to the clonal dissemination of one strain. Comparison of the typing results obtained with the three methods showed that the MLVA assay seemed too discriminatory to be used in future studies of sexual networks of M. genitalium infection. According to the discriminatory power and the feasibility of each mgpB-based method, we recommend that the mgpB analysis be used for general epidemiological studies and that the combination of MG309-STR and mgpB SNP methods should be used for sexual-network studies of M. genitalium infection.

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.

Detection and susceptibility testing of Mycoplasma amphoriforme isolates from patients with respiratory tract infections.

Three isolates of Mycoplasma amphoriforme, a new Mycoplasma species rarely described to date, were obtained from respiratory tract specimens from two children and one adult with respiratory tract infections. Molecular methods were required to distinguish them from Mycoplasma pneumoniae. MICs of macrolides, tetracyclines and fluoroquinolones were identical to those for M. pneumoniae, except for that of ciprofloxacin, which was slightly more potent against M. amphoriforme. M. amphoriforme could possibly have been involved in one case of severe respiratory infection with sepsis, but further studies are needed to specify its role as a potential respiratory tract pathogen.

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.

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.

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

Evaluation of moxalactam with the BD phoenix system for detection of methicillin resistance in coagulase-negative staphylococci.

The performance of moxalactam with the BD Phoenix system for the detection of methicillin resistance in coagulase-negative staphylococci was evaluated by use of a collection of 186 strains. Moxalactam was a better drug as an indicator of methicillin resistance for mecA-positive strains than oxacillin and cefoxitin were. For strains other than Staphylococcus saprophyticus, a moxalactam MIC >16 microg/ml was indicative of methicillin resistance.