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Research in Microbiology 1989The DNA polymerase activity of different members of Mollicutes was studied. A single DNA polymerase was found in Mycoplasma mycoides and Ureaplasma urealyticum, type... (Comparative Study)
Comparative Study
The DNA polymerase activity of different members of Mollicutes was studied. A single DNA polymerase was found in Mycoplasma mycoides and Ureaplasma urealyticum, type species of the genera Mycoplasma and Ureaplasma, and was compared with the previously described Mycoplasma orale enzyme. Most of their properties were comparable; an immunological relationship was demonstrated between M. orale and M. mycoides enzymes by immunoblotting. In contrast to these results, three different DNA polymerases were purified in Acholeplasma laidlawii, type species of the genus Acholeplasma which, in this aspect, resembles the genus Spiroplasma. A 3'-5' exonuclease activity was found in the different purified preparations. In M. mycoides, M. orale and one of the three A. laidlawii preparations, the 3'-5' exonuclease could be separated from the DNA polymerase by non-denaturing PAGE. The presence of a single DNA polymerase seems to be a typical feature of the Mycoplasmataceae, which include the genera Mycoplasma and Ureaplasma, in contrast to the occurrence of three enzymes within the Acholeplasmataceae and Spiroplasmataceae. These results are in agreement with the phylogenetic tree of Mollicutes proposed from their 5 S and 16 S rRNA sequence comparisons, in which the evolution of Acholeplasma and Spiroplasma branches led, by genome reductions, to Mycoplasma and Ureaplasma species.
Topics: Acholeplasma laidlawii; DNA-Directed DNA Polymerase; Exodeoxyribonuclease V; Exodeoxyribonucleases; Immunochemistry; Mycoplasma mycoides; Mycoplasmatales; Species Specificity; Ureaplasma
PubMed: 2694245
DOI: 10.1016/0923-2508(89)90075-2 -
Journal of Bacteriology Sep 1968Monkey, rat, and chicken tracheal epithelial cells, as well as monkey, rat, guinea pig, and chicken erythrocytes, adsorbed firmly to colonies of Mycoplasma pneumoniae...
Monkey, rat, and chicken tracheal epithelial cells, as well as monkey, rat, guinea pig, and chicken erythrocytes, adsorbed firmly to colonies of Mycoplasma pneumoniae and M. gallisepticum. Colonies of M. pulmonis also adsorbed erythrocytes but with less avidity than M. pneumoniae or M. gallisepticum; unlike the latter organisms, M. pulmonis did not adsorb tracheal epithelial cells. Colonies of M. orale type 1 and M. orale type 3 adsorbed only chicken red cells. Other mycoplasma species tested, including four of human origin and one of animal origin, did not adsorb red cells or epithelial cells. M. pneumoniae and M. gallisepticum appeared to attach to erythrocytes or tracheal epithelial cells by neuraminic acid receptors on these cells, whereas M. orale types 1 and 3 and M. pulmonis seemed to utilize another type or other types of receptors. Pretreatment of red cells or tracheal epithelial cells with receptor-destroying enzyme, neuraminidase, or influenza B virus removed the adsorption receptors for M. pneumoniae. Similarly, pretreatment of M. pneumoniae colonies with neuraminic acid-containing materials prevented adsorption of erythrocytes or respiratory tract cells. The adsorption sites on M. pneumoniae were specifically blocked by homologous but not heterologous antisera. This property made it possible to study the nature of the mycoplasma adsorption sites by testing the capacity of different fractions of the organism to block the action of adsorption-inhibiting antibodies. Such studies suggested that the mycoplasma binding sites were probably lipid or lipoprotein in nature. The glycerophospholipid hapten was implicated as one such site, since this serologically active hapten blocked the action of hemadsorption-inhibiting antibodies in M. pneumoniae rabbit antiserum. The affinity of M. pneumoniae for respiratory tract epithelium, unique among the mycoplasmas that infect man, may play a role in virulence, since this type of attachment provides an unusual opportunity for peroxide, secreted by the organism, to attack the tissue cell membrane without being rapidly destroyed by catalase or peroxidase present in extracellular body fluids.
Topics: Adsorption; Animals; Binding Sites; Chickens; Chromatography, Gel; Complement Fixation Tests; Epithelium; Erythrocytes; Guinea Pigs; Haplorhini; Immune Sera; Lipids; Mycoplasma; Neuraminic Acids; Neuraminidase; Orthomyxoviridae; Phospholipids; Rats; Respirovirus; Trachea; Trypsin; Virulence; gamma-Globulins
PubMed: 4183967
DOI: 10.1128/jb.96.3.695-705.1968 -
Journal of Microbiology (Seoul, Korea) Feb 2006In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M....
In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the diagnosis of mycoplasmal contamination in cell culture systems.
Topics: Animals; Bacterial Typing Techniques; Base Sequence; DNA Primers; DNA, Bacterial; DNA, Ribosomal Spacer; Humans; Molecular Sequence Data; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Sensitivity and Specificity; Species Specificity
PubMed: 16554716
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 -
Brazilian Journal of Medical and... Jul 2006A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell...
A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.
Topics: Base Sequence; Cells, Cultured; DNA, Bacterial; Electrophoresis, Agar Gel; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Tenericutes
PubMed: 16862282
DOI: 10.1590/s0100-879x2006000700009 -
Research in Microbiology Apr 2013The presence of foreign contamination, especially of mycoplasmas, is a major hindrance in long term in vitro cultivation of Plasmodium falciparum and may be a source of...
The presence of foreign contamination, especially of mycoplasmas, is a major hindrance in long term in vitro cultivation of Plasmodium falciparum and may be a source of false-positive results. Efforts have been made to control mycoplasma contamination by trypsinization of P. falciparum culture. Samples of accidentally contaminated cultures were used for this study. The presence of Mycoplasma orale in contaminated culture was ascertained by a species-specific PCR-based mycoplasma detection kit (Takara; Cat. No.6601). Trypsinization was carried out using trypsin-EDTA and the growth profile of P. falciparum was monitored for more than three weeks post-trypsinization. The studies were carried out with four different P. falciparum strains, various serum supplements and human erythrocytes belonging to different blood groups. It was interesting to observe that, irrespective of the different strains of P. falciparum and the variety of serum supplements and erythrocytes, mycoplasma contamination can successfully be removed from P. falciparum culture by trypsinization. No antibiotic except gentamicin, which is routinely used, was added to the medium. Results of this study indicate that the frequent appearance of mycoplasma in continuous long-term cultures of P. falciparum can be managed by trypsinization.
Topics: Anti-Bacterial Agents; Cell Culture Techniques; Culture Media; Erythrocytes; Gentamicins; Humans; Mycoplasma orale; Plasmodium falciparum; Trypsin
PubMed: 23277231
DOI: 10.1016/j.resmic.2012.12.010 -
Journal of Oral Pathology & Medicine :... Feb 2015Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia...
BACKGROUND
Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue.
OBJECTIVE
Our aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry.
DESIGN
We produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry.
RESULTS
We obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivarium DNA in the epithelial cells of leukoplakia.
CONCLUSION
Intracellular M. salivarium was identified in the epithelial cells of leukoplakia.
Topics: Adult; Aged; Aged, 80 and over; Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Antibody Specificity; Bacteriological Techniques; Chlorocebus aethiops; Epithelial Cells; Female; Fluorescent Antibody Technique; Freund's Adjuvant; Humans; Immunohistochemistry; Intracellular Space; Leukoplakia, Oral; Male; Microscopy, Immunoelectron; Middle Aged; Mouth Mucosa; Mycoplasma salivarium; Polymerase Chain Reaction; Rabbits; Rats; Vero Cells; Young Adult
PubMed: 25065471
DOI: 10.1111/jop.12215 -
Applied and Environmental Microbiology Mar 1994A fast and simple method to detect mycoplasmal contamination in simulated samples of animal sera by using a PCR was developed. The following five mycoplasma species that...
A fast and simple method to detect mycoplasmal contamination in simulated samples of animal sera by using a PCR was developed. The following five mycoplasma species that are major cell culture contaminants belonging to the class Mollicutes were investigated: Mycoplasma arginini, Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, and Mycoplasma fermentans. After a concentration step involving seeded sera, genus-specific primers were used to amplify a 717-bp DNA fragment within the 16S rRNA gene of mycoplasmas. In a second step, the universal PCR was followed by amplification of variable regions of the 16S rRNA gene by using species-specific primers, which allowed identification of contaminant mycoplasmas. With this method, 10 fg of purified DNA and 1 to 10 color-changing units of mycoplasmas could be detected. Since the sensitivity of the assay was increased 10-fold when the amplification products were hybridized with an internal mycoplasma-specific 32P-labelled oligonucleotide probe, a detection limit of 1 to 10 genome copies per PCR sample was obtained. This highly sensitive, specific, and simple assay may be a useful alternative to methods currently used to detect mycoplasmas in animal sera.
Topics: Animals; Base Sequence; Blood Specimen Collection; Cattle; Horses; Molecular Sequence Data; Polymerase Chain Reaction; Sensitivity and Specificity; Species Specificity; Tenericutes; Time Factors
PubMed: 8161186
DOI: 10.1128/aem.60.3.953-959.1994 -
Applied Microbiology Sep 1969Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M....
Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.
Topics: Cell Aggregation; Cold Temperature; Culture Media; Filtration; Humans; Mouth; Mycoplasma; Pharynx; Radiation Effects; Ultraviolet Rays; Vibration
PubMed: 5373673
DOI: 10.1128/am.18.3.360-364.1969 -
Infection and Immunity Mar 1991Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins...
Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.
Topics: Animals; Bacterial Proteins; Blotting, Western; Chromatography, High Pressure Liquid; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Hydrolases; Immunoenzyme Techniques; Lipid Metabolism; Male; Mycoplasma; Prions; Rabbits; Serine Endopeptidases
PubMed: 1997407
DOI: 10.1128/iai.59.3.1037-1042.1991