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Pharmeuropa Bio Nov 2006European Pharmacopoeia (Ph. Eur.) general chapter 2.6.7. Mycoplasma requires for the culture test reference strains of mycoplasma field isolates with fewer than 15...
European Pharmacopoeia (Ph. Eur.) general chapter 2.6.7. Mycoplasma requires for the culture test reference strains of mycoplasma field isolates with fewer than 15 passages for validation and run control and in the test for inhibitory substances. Low passage field isolates of 5 mycoplasma strains (Mycoplasma hyorhinis, Mycoplasma synoviae, Mycoplasma fermentans, Mycoplasma orale and Acholeplasma laidlawii) have been prepared for this purpose and a small scale collaborative study involving European laboratories was carried out to confirm the suitability of the material for the intended purpose. Strains were prepared as 1 ml samples in frozen format and are stored below -60 degrees C. Each laboratory determined a titre for the material on their in-house media. A secondary part of the study also compared the growth of prediluted samples on the different culture media. Results of the study confirm that the material is suitable for use as a biological reference preparation (BRP) and an estimated titre has been provided for each strain based on the results of the study. It was noted that differences in the culture media used in the different laboratories did not have a detrimental effect on titre estimation. The estimated titre is intended as a guide for users to validate the use of the reference material in house. The candidate BRPs were adopted by the European Pharmacopoeia Commission on June 28, 2006 and are available for use from EDQM. A revision to chapter 2.6.7, including reference to the use of nucleic acid amplification techniques (NAT) was also adopted in June 2006 and will appear in the European Pharmacopoeia version 5.8 in January 2007 and come into force the 1st of July 2007. While it was not part of the study a number of participants also performed in-house NAT assays on the study material. Preliminary findings from these studies are presented.
Topics: Colony Count, Microbial; Culture Media; DNA, Bacterial; Europe; Mycoplasma; Nucleic Acid Amplification Techniques; Pharmacopoeias as Topic; Reference Standards
PubMed: 17270132
DOI: No ID Found -
Cancer Science Apr 2004We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas... (Comparative Study)
Comparative Study
We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas resemble Helicobacter pylori in terms of ammonia production and induction of inflammatory cytokines in immune and non-immune cells. The presence and identity of mycoplasmas were assessed by semi-nested PCR and sequencing, and the results were compared with pathologic data. Fifty-six samples collected from Korean chronic gastritis patients were used for this study. Twenty-three (41.1%) were positive for mycoplasmas. Eighteen sequenced samples contained a single human mycoplasma or two mycoplasmas, which were identified as Mycoplasma faucium (13/18), M. fermentans (3/18), M. orale (1/18), M. salivarium (2/18), and M. spermatophilum (1/18). Mycoplasma-infected chronic gastritis samples showed significantly more severe neutrophil infiltration than non-infected samples (P = 0.0135). Mycoplasma profiles in the oral cavity (M. salivarium is major) and stomach were different, and the presence of significant proinflammatory responses in mycoplasma-positive patients suggests that the mycoplasmas are not simply contaminants. Further studies are required to understand whether mycoplasmas play a role in gastric tumorigenesis.
Topics: Chronic Disease; DNA, Bacterial; Gastritis; Gastroscopy; Humans; Korea; Molecular Sequence Data; Mouth; Mycoplasma; Mycoplasma Infections; Mycoplasma fermentans; Mycoplasma salivarium; Organ Specificity; Pyloric Antrum; Sequence Analysis, DNA; Stomach; Stomach Neoplasms
PubMed: 15072588
DOI: 10.1111/j.1349-7006.2004.tb03208.x -
Microbial Pathogenesis Apr 1998We addressed the question whether the in vitro interaction of two Epstein-Barr virus (EBV)-genome-positive B cell lines (EB-3 and HilB-gamma) with either Mycoplasma...
We addressed the question whether the in vitro interaction of two Epstein-Barr virus (EBV)-genome-positive B cell lines (EB-3 and HilB-gamma) with either Mycoplasma pneumoniae or M. hominis, with the <
> mycoplasma species (M. fermentans, M. fermentans subsp. incognitus, M. penetrans, M. genitalium) or with mycoplasma species known to be mere commensals of the respiratory tract (M. orale and M. salivarium) would result in expression of mRNAs for IL-2, IL-2R, IL-4 and IL-6 as determined by reverse transcriptase (RT)-PCR after 4 and 24 h of cocultivation. The pattern of cytokine gene expression observed depended on (i) the origin of the transformed cell line, (ii) the pathogenicity of the Mycoplasma species, and (iii) the length of cocultivation. The EBV-immortalized lymphoblastoid cell line HilB-gamma showed mRNA expression for IL-2, IL-2-receptor, IL-4 and IL-6 peaking 24 h after stimulation with M. pneumoniae and all AIDS-related mycoplasma species tested. The Burkitt lymphoma cell line EB-3 showed a distinct and isolated strong II-2/IL-2 R-mRNA expression within 4 h after contact with the pathogenic and all of the AIDS related mycoplasma species. In neither EBV-containing cell line cytokine was gene expression detectable after stimulation with the commensal mycoplasma species, M. orale and M. salivarium, indicating species differences in the ability of mycoplasmas to interact with and stimulate B-cell lines. Our data suggest that some mcyoplasma species may act as immunomodulatory cofactors by eliciting inappropriate cytokine gene expression in B cells latently infected with EBV. Therefore, this cultivation model may prove useful in evaluating the pathogenetic potential of novel isolated mycoplasma species. Topics: B-Lymphocytes; Cell Line, Transformed; Gene Expression Regulation; Herpesvirus 4, Human; Humans; Interleukins; Mycoplasma; RNA, Messenger; Receptors, Interleukin-2
PubMed: 9533897
DOI: 10.1006/mpat.1997.0196 -
Applied and Environmental Microbiology Sep 2015Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the...
Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.
Topics: DNA, Bacterial; Laboratories; Mycoplasma; Nucleic Acid Amplification Techniques; World Health Organization
PubMed: 26070671
DOI: 10.1128/AEM.01150-15 -
Biochemistry Oct 1979We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis,...
We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.
Topics: DNA-Directed DNA Polymerase; Exonucleases; Molecular Weight; Mycoplasma; Spermidine; Spermine
PubMed: 497165
DOI: No ID Found -
FEMS Microbiology Letters Nov 2000The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of...
The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.
Topics: 5'-Nucleotidase; Animals; Arthritis, Rheumatoid; Colony Count, Microbial; Concanavalin A; Humans; Mycoplasma; Mycoplasma Infections; Nucleic Acid Hybridization; RNA, Bacterial; Rats; Synovial Membrane
PubMed: 11040429
DOI: 10.1111/j.1574-6968.2000.tb09359.x -
Cytotechnology 1994The polymerase chain reaction (PCR) has been used for the general detection of Mollicutes. 25 Mycoplasma and Acholeplasma species were detected including important...
The polymerase chain reaction (PCR) has been used for the general detection of Mollicutes. 25 Mycoplasma and Acholeplasma species were detected including important contaminants of cell cultures such as M. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1-2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.
Topics: Base Sequence; Genetic Code; Molecular Sequence Data; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Sensitivity and Specificity; Time Factors
PubMed: 7765790
DOI: 10.1007/BF00754609 -
The Journal of Experimental Medicine Feb 1983The immune response of experimentally infected hamsters and human patients to Mycoplasma pneumoniae was examined by radioimmunoprecipation in conjunction with gel...
The immune response of experimentally infected hamsters and human patients to Mycoplasma pneumoniae was examined by radioimmunoprecipation in conjunction with gel electrophoresis and fluorography. Both intrinsically and extrinsically labeled mycoplasma proteins were coincubated with acute and convalescent sera in a radioimmunoprecipitation assay. Two M. pneumoniae proteins were selectively precipitated by convalescent sera. These predominant immunogens were trypsin-sensitive, antibody-accessible surface proteins that co-migrate on polyacrylamide gels with proteins P1 and P2, which were previously implicated by us as mediators of cytadsorption. Anti-M. pneumoniae antiserum did not precipitate radiolabeled antigens derived from Mycoplasma orale or Mycoplasma salivarium. These data indicate that M. pneumoniae infection stimulates a specific and highly targeted host antibody response to key proteinaceous immunogens.
Topics: Acute Disease; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Chemical Precipitation; Cricetinae; Humans; Mycoplasma pneumoniae; Pneumonia, Mycoplasma; Trypsin
PubMed: 6401796
DOI: 10.1084/jem.157.2.502 -
PDA Journal of Pharmaceutical Science... 2018This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma cultivated in five...
UNLABELLED
This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma cultivated in five different cultivation media. The influence of relevant filtration process parameters, in particular transmembrane pressure and filtration temperature, on their respective retention was tested. The impact of the filtration temperature was further evaluated for the Gram-negative bacteria species , the Gram-positive bacteria species , the phage PP7, and the mycoplasma species The findings were correlated to the different mechanical properties of the particles, especially also with respect to the different bacterial cell envelopes found in those species. This study suggests that mycoplasma, surrounded by a flexible lipid bilayer, are significantly susceptible to changes in temperature, altering the stiffness of the cell envelope. Mycoplasma retention could thus be increased significantly by a decreased filtration temperature. In contrast, Gram-negative and Gram-positive bacteria species, with a cell wall containing a cross-linked peptidoglycan layer, as well as bacteriophages PP7 exhibiting a rigid protein capsid, did not show a temperature-dependent retention within the applied filtration temperatures between 2 and 35 °C. The trends of the retention of with increasing temperature and transmembrane pressure were independent of cultivation media. Data obtained with mycoplasma suggest that the trend of mycoplasma retention at different filtration temperatures is also independent of the membrane pore size and thus retention level. Media in biopharmaceutical processes are sterile-filtered to prevent them from bacterial contamination. Mycoplasma represent a relevant class of bacteria. In this publication it is shown that mycoplasma cell size depends on the media they are cultivated in. Membranes used for sterile filtration retain bacteria predominantly by size exclusion. Thus, an altered cell size can result in different retention values. Another characteristic of mycoplasma is the flexible lipid bilayer and the absence of a rigid cell wall. The lipid bilayer can undergo a phase transition from a gel to a liquid-crystal phase at a certain temperature, which makes it stiffer at lower temperatures. A higher stiffness can result in higher retention values during filtration, as the deformability of the mycoplasma cell is lower and the cell does not squeeze through the membrane pores.
ABBREVIATIONS
ALCM: culture medium; ASTM: American Society for Testing and Materials; ATCC: American Type Culture Collection; CFU/mL: colony-forming units per milliliter; DLS: Dynamic light scattering; LRV: Log reduction value; PES: Polyethersulfone; PFU/mL: Plaque-forming units per milliliter; PSD: Particle size distribution; PVP: Polyvinylpyrrolidone; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SLB: Saline lactose broth; TMP: Transmembrane pressure; TSB: Tryptic soy broth.
Topics: Acholeplasma laidlawii; Culture Media; Filtration; Mycoplasma; Sterilization; Temperature
PubMed: 29343618
DOI: 10.5731/pdajpst.2017.008102 -
Diagnostics (Basel, Switzerland) May 2021, , and sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections...
, , and sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the , , and sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of , , , , , , , , , , and . Limit of detection values varied between 125-300 and 50-100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from , , and in tested cell cultures.
PubMed: 34068904
DOI: 10.3390/diagnostics11050876