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Brazilian Journal of Microbiology :... 2014Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts...
Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts generally require two essential inputs, bovine serum and cells. The study herein aims to evaluate the mycoplasma concentrations that affect the growing of BHK21 and Vero cells. The species used were: Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis, cultivated in a SP4 media. Two contamination tests were performed with BHK21 and Vero cells and one of them applied different concentrations of mycoplasma. In the first one, mycoplasma was applied at the day zero and, in the second one, the contamination was performed after the monolayer establishment. The both cellular cultures presented cytopathic effects with mycoplasma contamination, but the Vero cells suffered more damages than the BHK21 ones. It was also observed that the severity of the cytopathic effect depended on the mycoplasma specie, on the concentration and on the time of contact with the cellular culture, which evidences the importance of controlling the presence of mycoplasma in biotechnological industries.
Topics: Animals; Cell Line; Chlorocebus aethiops; Coculture Techniques; Cricetinae; Culture Media; Epithelial Cells; Mycoplasma
PubMed: 25763061
DOI: 10.1590/s1517-83822014000400048 -
Israel Journal of Medical Sciences Sep 1984Following our previous demonstration that both viable and heat-killed Mycoplasma orale induce selective tumor cell killing by murine peritoneal macrophages, further...
Following our previous demonstration that both viable and heat-killed Mycoplasma orale induce selective tumor cell killing by murine peritoneal macrophages, further investigations reported here showed that also macrophages from a continuously proliferating cell line established from long-term cultures of murine bone marrow explants can effectively be induced by the heat-killed mycoplasmas to express cytolysis. The use of single-cell suspensions of M. orale from a 0.45-micron filtrate or following either sonication or treatment with DNase did not significantly affect the level of cytolysis. Minute quantities of M. orale acted synergistically with ineffectively low levels of either lymphokines (LK) or lipopolysaccharide (LPS) to produce killing. The exceptional resistance of M109 lung adenocarcinoma cells to macrophage-mediated killing induced by LK and LPS, as previously reported by us, could not be overcome by the addition of M. orale. These data appear to indicate a mechanism of macrophage activation by M. orale similar to that caused by LPS.
Topics: Animals; Cell Survival; Cells, Cultured; Lipopolysaccharides; Lymphokines; Macrophages; Mice; Mycoplasma; Neoplasms, Experimental
PubMed: 6511364
DOI: No ID Found -
Journal of Immunological Methods Jul 1992In the present report we describe a rapid and sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture...
In the present report we describe a rapid and sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture supernatants to modulate [3H]TdR incorporation by unstimulated mouse splenocytes. Several mycoplasma species (Mycoplasma orale, culturable and non-culturable strains of Mycoplasma hyorhinis) inhibited [3H]TdR incorporation and permitted the detection of some contaminated cell cultures that would otherwise have escaped detection in assays measuring [3H]TdR incorporation by mitogen-stimulated splenocytes. On the other hand, several other mycoplasma species (Mycoplasma arginini, Mycoplasma hominis) strongly enhanced [3H]TdR incorporation by unstimulated splenocytes. This enhancement was optimally detectable on day 2 after initiation of the cultures. The sensitivity of the assay was determined for a mycoplasma species (culturable M. hyorhinis) that inhibited as well as for one (M. arginini) that enhanced [3H]TdR incorporation. In both cases, the sensitivity was such that 1-3 x 10(2) mycoplasma colony-forming units (CFU) could be detected.
Topics: Animals; B-Lymphocytes; Cell Line; Concanavalin A; Female; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mycoplasma; Spleen; Thymidine; Tumor Cells, Cultured
PubMed: 1640109
DOI: 10.1016/0022-1759(92)90086-9 -
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 -
Cancer Letters Jul 1989Because mycoplasmas contaminate irreplaceable, established cell lines, several laboratories have been looking for effective procedures to eradicate the infection. This...
Because mycoplasmas contaminate irreplaceable, established cell lines, several laboratories have been looking for effective procedures to eradicate the infection. This report presents the experience of our laboratory in using the 5-bromouracil-fluorochrome method developed by Marcus et al. which turned out to be effective in eliminating Mycoplasma orale, M. hyorhinis and M. hominis from transformed cell lines of different origin. The metastatic potential of a highly metastatic murine fibroblastic cell line infected with M. orale was restored to its original level after the contamination was eliminated.
Topics: Animals; Benzimidazoles; Bisbenzimidazole; Bromouracil; Lung Neoplasms; Mice; Mycoplasma; Neoplasm Transplantation; Tumor Cells, Cultured
PubMed: 2473833
DOI: 10.1016/0304-3835(89)90016-5 -
Cytotherapy 2006Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia...
BACKGROUND
Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept.
METHODS
The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values.
RESULTS
The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale.
DISCUSSION
The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.
Topics: DNA, Bacterial; Humans; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Reproducibility of Results; Research Design; Self-Sustained Sequence Replication; Sensitivity and Specificity
PubMed: 16627346
DOI: 10.1080/14653240500518413 -
Journal of Applied Microbiology Oct 2011To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC)...
AIMS
To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)-based methods in comparison to the conventional microbiological methods.
METHODS AND RESULTS
A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤ 10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co-cultured with suspension of Chinese hamster ovary (CHO) cells.
CONCLUSIONS
Tested mycoplasma strains harvested at the exponential-early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study.
SIGNIFICANCE AND IMPACT OF THE STUDY
This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT-based mycoplasma testing methods.
Topics: Animals; Bacteriological Techniques; CHO Cells; Coculture Techniques; Colony Count, Microbial; Cricetinae; Cricetulus; DNA, Bacterial; Evaluation Studies as Topic; Gene Dosage; Limit of Detection; Mycoplasma; Polymerase Chain Reaction; Reference Standards; Validation Studies as Topic
PubMed: 21794032
DOI: 10.1111/j.1365-2672.2011.05108.x -
In Vitro Cellular & Developmental... Feb 2002Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma... (Comparative Study)
Comparative Study
Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.
Topics: Animals; Cattle; Cell Line; DNA, Bacterial; Humans; Mycoplasma; Nucleic Acid Hybridization; Polymerase Chain Reaction; RNA, Bacterial; Sensitivity and Specificity; Species Specificity; Swine
PubMed: 11928999
DOI: 10.1290/1071-2690(2002)038<0079:CPAFDO>2.0.CO;2 -
Journal of Virological Methods Jan 1994The efficacy of several antibiotic treatments to eliminate mycoplasma from Vero cells contaminated chronically with Mycoplasma orale II were tested. Minocyclin,...
The efficacy of several antibiotic treatments to eliminate mycoplasma from Vero cells contaminated chronically with Mycoplasma orale II were tested. Minocyclin, Kanamycin, Tylosine and Roxitromycin, at non cytotoxic concentrations, were assayed alone or in different combinations. Mycoplasma contamination was effectively eradicated without recurrence once the following regimen was applied: Incubation of contaminated cells with Tylosine (250 micrograms/ml) for 12 days followed by incubation with Minocycline (5 micrograms/ml) for 10 days. This treatment was not deleterious for cell growth, it was effective after only one application and it was successful to eradicate mycoplasma from other contaminated eukaryotic continuous cell lines.
Topics: Animals; Cells, Cultured; Chlorocebus aethiops; Cricetinae; Culture Techniques; Drug Resistance, Microbial; Drug Therapy, Combination; Eukaryotic Cells; Humans; Kanamycin; Minocycline; Mycoplasma; Roxithromycin; Tumor Cells, Cultured; Tylosin
PubMed: 8175949
DOI: 10.1016/0166-0934(94)90018-3 -
Vaccine Dec 1986Nine live virus veterinary vaccines from six sources were found to be contaminated with mycoplasma. The vaccines were for use in canine, feline and avian species, and 53...
Nine live virus veterinary vaccines from six sources were found to be contaminated with mycoplasma. The vaccines were for use in canine, feline and avian species, and 53 batches of the products were at fault. The isolates were identified as Mycoplasma hominis, M. arginini, M. orale, M. hyorhinis and M. gallinarum. Investigation of the contamination rate of other batches or other products from the same source in some cases helped to determine the source of infection. Mycoplasma contaminants can be considered important not only because of their role as pathogens but also because they may indicate that insufficient care has been taken during vaccine manufacture or quality control.
Topics: Animals; Drug Contamination; Mycoplasma; Viral Vaccines
PubMed: 3799018
DOI: 10.1016/0264-410x(86)90136-2