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Clinical Microbiology and Infection :... Apr 2003Diagnosis of Mycoplasma pneumoniae infection is challenging due to the fastidious nature of the pathogen, the considerable seroprevalence, and the possibility of... (Review)
Review
Diagnosis of Mycoplasma pneumoniae infection is challenging due to the fastidious nature of the pathogen, the considerable seroprevalence, and the possibility of transient asymptomatic carriage. During recent years, various new techniques have been adapted for the diagnosis of M. pneumoniae infection, notably in the field of molecular biology. Standard polymerase chain reaction (PCR) is currently the method of choice for direct pathogen detection, but several PCR-related methods provide enhanced sensitivity or more convenient handling procedures, and have been successfully applied for research purposes. Among these techniques are real-time PCR, nested PCR, reverse transcriptase PCR (RT-PCR) and multiplex PCR. Generally, amplification-based methods have replaced hybridization assays and direct antigen detection. Serology, which is the basic strategy for mycoplasma diagnosis in routine clinical practice, has been improved by the widespread availability of sensitive assays for separate detection of different antibody classes. For the diagnosis of mycoplasma pneumonia, serology and direct pathogen detection should be combined. Extrapulmonary diseases may be diagnosed by direct pathogen detection alone, but the value of this diagnostic approach is limited by the probably immunologically mediated pathogenesis of some manifestations. This review summarizes the current state of Mycoplasma pneumoniae diagnosis, with special reference to molecular techniques. The value of different methods for routine diagnosis and research purposes is discussed.
Topics: Culture Media; Humans; Molecular Diagnostic Techniques; Mycoplasma pneumoniae; Nucleic Acid Amplification Techniques; Pneumonia, Mycoplasma; Polymerase Chain Reaction; Sensitivity and Specificity; Serologic Tests
PubMed: 12667235
DOI: 10.1046/j.1469-0691.2003.00590.x -
European Journal of Clinical... Oct 2019This study characterizes a large Mycoplasma pneumoniae outbreak observed in Kymenlaakso in Southeastern Finland during August 2017-January 2018. The first part of the...
This study characterizes a large Mycoplasma pneumoniae outbreak observed in Kymenlaakso in Southeastern Finland during August 2017-January 2018. The first part of the investigation included 327 patients, who sought healthcare consultation at local GPs or hospitals due to clinical symptoms, and were tested for M. pneumoniae antibodies (Patient cohort). The second part of the investigation, conducted approximately 4 weeks after the peak of the outbreak, consisted of school screening of pupils (N = 239) in three different school buildings by PCR on respiratory specimens and questionnaires (Screening cohort). PCR positive respiratory specimens were subsequently utilized for molecular typing. The outbreak peaked in late October 2017. Of the Patient cohort, 9/106 (8.5%) respiratory specimens were PCR positive. In contrast, 3/182 (1.6%) of the Screening cohort were PCR positive. Asymptomatic carriage was observed. Multiple-locus variable-number tandem-repeat analysis (MLVA) identified two distinct MLVA types. All typed M. pneumoniae strains belonged to P1 type 1. No mutations leading to macrolide resistance were observed. In total, 61/327 (19%) of the Patient cohort had a serological indication of recent infection. The IgM test reactivity at the time of a negative PCR test result varied from a completely non-reactive value up to very strong reactivity, highlighting the difficulty in a single specimen serodiagnosis.
Topics: Adolescent; Adult; Antibodies, Bacterial; Child; Clinical Laboratory Techniques; Cohort Studies; Disease Outbreaks; Female; Finland; Humans; Immunoassay; Immunoglobulin M; Male; Molecular Epidemiology; Molecular Typing; Mycoplasma pneumoniae; Pneumonia, Mycoplasma; Polymerase Chain Reaction; Young Adult
PubMed: 31263967
DOI: 10.1007/s10096-019-03619-7 -
Nucleosides, Nucleotides & Nucleic Acids 2014Mycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection...
Mycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection with Mycoplasmas compromises the efficacy of anticancer and antiviral nucleoside analog-based drugs due to the presence of Mycoplasma thymidine phosphorylase (TP). In this study, a TP-deficient strain of Mpn was generated in order to study the effect of Mpn TP in the metabolism of nucleoside analogs. Deficiency in TP activity led to increased uptake and incorporation of radiolabeled deoxyuridine and uracil but thymidine uptake was not affected. The activities of enzymes in the salvage of thymidine and deoxyuridine, e.g., thymidine kinase and uracil phosphoribosyltransferase were upregulated in the TP-deficient mutant, which may explain the increased uptake of deoxyuridine and uracil. Thirty FDA-approved anticancer and antiviral nucleoside and nucleobase analogs were used to screen their inhibitory activity toward the TP mutant and the wild type strain. Seven analogs were found to inhibit strongly the growth of both wild type and TP mutant. Differences in the inhibitory effect of several purine analogs between the two strains were observed. Further study is needed in order to understand the mechanism of inhibition caused by these analogs. Our results indicated that TP is not an essential gene for Mpn survival and TP deficiency affects other enzymes in Mpn nucleotide metabolism, and suggested that Mycoplasma nucleotide biosynthesis pathway enzymes are potential targets for future development of antibiotics.
Topics: Antineoplastic Agents; Antiviral Agents; Deoxyuridine; Mutation; Mycoplasma pneumoniae; Thymidine; Thymidine Phosphorylase
PubMed: 24940683
DOI: 10.1080/15257770.2013.853783 -
Proteomics Sep 2011This review covers progress in proteome research on Mycoplasma pneumoniae made over the last 5 years. This bacterium is one of the smallest known self-replicating... (Review)
Review
This review covers progress in proteome research on Mycoplasma pneumoniae made over the last 5 years. This bacterium is one of the smallest known self-replicating bacteria. With fewer than 700 proposed proteins, it is well suited to a comprehensive proteome analysis. While all of the proposed genes are transcribed, thus far 620 proteins, about 90% of the predicted proteome, have been identified experimentally. To study the proteome organization of M. pneumoniae, 178 soluble protein complexes were isolated under non-denaturing conditions by tandem affinity chromatography and their composition determined by SDS-PAGE and mass spectrometry. The 62 homomultimeric and 116 heteromultimeric protein complexes could be classified according to 12 different COG functional categories. The complexes interacted with each other to some extent, forming larger assemblies. Protein complexes that were large enough and had specific structures (e.g. ribosomes or DNA-dependent RNA polymerase) were visible and countable in their natural environment by cryo-electron tomography. In addition to characterization of the soluble complexes, the analysis of the Triton X-100 insoluble fraction has a major role in the elucidation of the cytoskeleton-like structure, because by analogy with eukaryotic cells, almost all of the structural proteins involved in its formation, and enriched sub-cellular structures, can be found in this fraction.
Topics: Bacterial Proteins; Chromosome Mapping; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Bacterial; Mycoplasma pneumoniae; Octoxynol; Phosphoproteins; Phosphorylation; Protein Interaction Mapping; Protein Processing, Post-Translational; Proteome; Solubility; Transcription, Genetic
PubMed: 21751371
DOI: 10.1002/pmic.201100076 -
Biomedical and Environmental Sciences :... Dec 2020The aim of this study is to investigate the macrolide resistance rate and molecular type with multiple-locus variable-number tandem-repeat analysis (MLVA) of of Beijing...
OBJECTIVE
The aim of this study is to investigate the macrolide resistance rate and molecular type with multiple-locus variable-number tandem-repeat analysis (MLVA) of of Beijing in 2016 in pediatric patients.
METHODS
Real-time quantitative polymerase chain reaction (PCR) was used to identify , and MLVA was performed. The domain V of the 23S rRNA was sequenced to detect macrolide-resistant point mutations. We also investigated the activities of antibiotics against isolates .
RESULTS
The PCR detection rate of in children in Beijing was 40%, and the macrolide resistance rate was 66%. The A2063G mutation in the 23S rRNA V region is the dominant mutation (137/146, 93.84%), whereas the A2064G mutation is rare (9/146, 6.16%). Seventy-three samples were typed successfully by MLVA typing, including 86.3% (63/73) were MLVA type 4-5-7-2, and 13.7% (10/73) were MLVA type 3-5-6-2. No other types were found. No strains were resistant to levofloxacin or tetracycline.
CONCLUSION
In 2016, a specific decrease in the macrolide resistance rate occurred in Beijing. The detection rate and macrolide resistance rate of outpatients are lower than those of inpatients. The A2063G mutants have high levels of resistance to erythromycin and azithromycin. The primary MLVA type is 4-5-7-2, followed by 3-5-6-2. No other MLVA types were detected. No strains resistant to tetracycline or levofloxacin were found .
Topics: Anti-Bacterial Agents; Beijing; Child; Drug Resistance, Bacterial; Genotype; Humans; Inpatients; Macrolides; Mutation; Mycoplasma pneumoniae; Outpatients; Polymerase Chain Reaction; RNA, Ribosomal, 23S; Respiratory Tract Infections
PubMed: 33472731
DOI: 10.3967/bes2020.125 -
International Journal of Medical... Sep 2004The interaction between Mycoplasma pneumoniae and its natural host, humans, cannot be studied directly for obvious reasons. Therefore, we used guinea pigs instead, which... (Review)
Review
The interaction between Mycoplasma pneumoniae and its natural host, humans, cannot be studied directly for obvious reasons. Therefore, we used guinea pigs instead, which had been recently introduced as an acceptable alternative host organism. The following experimental approaches were taken to study the pathogen-host relationship: characterization and subtyping of M. pneumoniae strains isolated from human patients, infection of guinea pigs with selected M. pneumoniae strains, and analysis of adaptation, preference and survival of individual strains in guinea pigs under competitive conditions. The results of our study indicated that the species M. pneumoniae is genetically very homogeneous. From 115 independently isolated strains two subtypes and one variant were found. The subtypes differed significantly in the amino acid composition of the P1 protein, the main adhesin of M. pneumoniae, while the variant showed only minor amino acid exchanges. Infection of guinea pigs indicated differences between the subtypes and the variant in their ability to colonize and survive in the animal. Preinfection of the host with a certain subtype or variant caused a subtype-specific immunity and had a strong influence on the type of surviving bacteria in superinfection experiments. The results of these studies explain the shift of subtypes frequently observed in epidemic outbreaks of M. pneumoniae infection appearing in intervals of 3-7 years.
Topics: Adaptation, Physiological; Adhesins, Bacterial; Amino Acid Sequence; Amino Acid Substitution; Animals; Bacterial Typing Techniques; Disease Models, Animal; Genetic Variation; Genotype; Guinea Pigs; Humans; Mycoplasma pneumoniae; Pneumonia, Mycoplasma
PubMed: 15493825
DOI: 10.1016/j.ijmm.2004.06.020 -
Nature Oct 2022Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here we use advances in cryo-electron tomography and...
Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here we use advances in cryo-electron tomography and sub-tomogram analysis to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.
Topics: Anti-Bacterial Agents; Cryoelectron Microscopy; Mycoplasma pneumoniae; Peptide Chain Elongation, Translational; Polyribosomes; Protein Biosynthesis; Ribosomal Proteins; Ribosomes
PubMed: 36171285
DOI: 10.1038/s41586-022-05255-2 -
Trends in Microbiology Jun 2021Mycoplasma genitalium (Mge) and Mycoplasma pneumoniae (Mpn) are two human pathogens associated with urogenital and respiratory tract infections, respectively. The recent...
Mycoplasma genitalium (Mge) and Mycoplasma pneumoniae (Mpn) are two human pathogens associated with urogenital and respiratory tract infections, respectively. The recent elucidation of the tridimensional structure of their major cytoadhesins by X-ray crystallography and cryo-electron microscopy/tomography, has provided important insights regarding the mechanics of infection and evasion of immune surveillance.
Topics: Adhesins, Bacterial; Cryoelectron Microscopy; Crystallography, X-Ray; Glycoproteins; Humans; Immune Evasion; Mycoplasma genitalium; Mycoplasma pneumoniae; N-Acetylneuraminic Acid
PubMed: 33593698
DOI: 10.1016/j.tim.2021.01.011 -
Journal of Medical Microbiology Jan 2021Infections with the respiratory pathogen are often chronic, recurrent and resistant, persisting after antibiotic treatment. grown on glass forms protective biofilms,...
Infections with the respiratory pathogen are often chronic, recurrent and resistant, persisting after antibiotic treatment. grown on glass forms protective biofilms, consistent with a role for biofilms in persistence. These biofilms consist of towers of bacteria interspersed with individual adherent cells. A tissue culture model for biofilms has not been described or evaluated to address whether growth, development and resistance properties are consistent with persistence in the host. Moreover, it is unclear whether the cells in the biofilm towers and individual bacterial cells have distinct roles in disease. We evaluated the properties of biofilms of grown on the immortalized human bronchial epithelial cell line BEAS-2B in relation to persistence in the host. We observed nucleation of biofilm towers and the disposition of individual cells in culture, leading to a model of how tower and individual cells contribute to infection and disease. With submerged BEAS-2B cells as a substrate, we evaluated growth and development of biofilms using scanning electron microscopy and confocal laser scanning microscopy. We characterized resistance to erythromycin and complement using minimum inhibitory concentration assays and quantification of colony forming units. We monitored biofilm tower formation using time-lapse microscopic analysis of host-cell-free cultures. Bacteria grown on host cells underwent similar development to those grown without host cells, including tower formation, rounding and incidence of individual cells outside towers. Erythromycin and complement significantly reduced growth of . Towers formed exclusively from pre-existing aggregates of bacteria. We discuss a model of the biofilm life cycle in which protective towers derive from pre-existing aggregates, and generate individual cytotoxic cells. can form protective biofilms in a tissue culture model, implicating biofilms in chronic infections, with aggregates of cells being important for establishing infections.
Topics: Anti-Bacterial Agents; Biofilms; Bronchi; Cell Line; Epithelial Cells; Humans; Microscopy, Electron, Scanning; Mycoplasma pneumoniae; Pneumonia, Mycoplasma
PubMed: 33170120
DOI: 10.1099/jmm.0.001266 -
Journal of Medical Microbiology Dec 2020Resistance against macrolide antibiotics in is becoming non-negligible in terms of both appropriate therapy and diagnostic stewardship. Molecular methods have...
Resistance against macrolide antibiotics in is becoming non-negligible in terms of both appropriate therapy and diagnostic stewardship. Molecular methods have attractive features for the identification of as well as its resistance-associated mutations of 23S ribosomal RNA (rRNA). The automated molecular diagnostic sytem can identify macrolide-resistant . To assess the performance of an automated molecular diagnostic system, GENECUBE Mycoplasma, in the detection of macrolide resistance-associated mutations. To evaluate whether the system can distinguish mutant from wild-type 23S rRNA, synthetic oligonucleotides mimicking known mutations (high-level macrolide resistance, mutation in positions 2063 and 2064; low-level macrolide resistance, mutation in position 2067) were assayed. To evaluate clinical oropharyngeal samples, purified nucleic acids were obtained from -positive samples by using the GENECUBE system from nine hospitals. After confirmation by re-evaluation of positivity, Sanger-based sequencing of 23S rRNA and mutant typing using GENECUBE Mycoplasma were performed. The system reproducibly identified all synthetic oligonucleotides associated with high-level macrolide resistance. Detection errors were only observed for A2067G (in 2 of the 10 measurements). The point mutation in 23S rRNA was detected in 67 (26.9 %) of 249 confirmed -positive clinical samples. The mutations at positions 2063, 2064 and 2617 were observed in 65 (97.0 %), 2 (3.0 %) and 0 (0.0 %) of the 67 samples, respectively. The mutations at positions 2063 and 2064 were A2063G and A2064G, respectively. The results from mutant typing using GENECUBE Mycoplasma were in full agreement with the results from sequence-based typing. GENECUBE Mycoplasma is a reliable test for the identification of clinically significant macrolide-resistant .
Topics: Anti-Bacterial Agents; Automation; DNA, Bacterial; Drug Resistance, Bacterial; Humans; Macrolides; Molecular Diagnostic Techniques; Mutation; Mycoplasma pneumoniae; Oligonucleotides; Pneumonia, Mycoplasma; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 33141009
DOI: 10.1099/jmm.0.001264