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Journal of Bacteriology Nov 1967Mycoplasma strains (B1, B2, CS, and S1A) were isolated from the saliva of normal cats. These were compared with a strain (CO) isolated from the eye of a cat with severe...
Mycoplasma strains (B1, B2, CS, and S1A) were isolated from the saliva of normal cats. These were compared with a strain (CO) isolated from the eye of a cat with severe conjunctivitis. On the basis of morphology, biochemical reactions, and antigenic composition, two distinct species were recognizable. Strains CO, B1, and B2 were antigenically unrelated to the other species tested; strains CS and S1A possessed antigenic components in common with Mycoplasma arthritidis, M. salivarium, M. hominis, type 1, and M. orale, types 1 and 2. It was tentatively suggested that the two cat species be called M. felis and M. gateae, respectively.
Topics: Animals; Antigens; Cats; Chloramphenicol; Complement Fixation Tests; Conjunctivitis; Dihydrostreptomycin Sulfate; Erythromycin; Hemolysis; Immune Sera; Immunodiffusion; Lincomycin; Mycoplasma; Neomycin; Novobiocin; Rats; Saliva; Tetracycline
PubMed: 4168314
DOI: 10.1128/jb.94.5.1451-1458.1967 -
Applied and Environmental Microbiology May 2010Although mycoplasmas are generally considered to be harmless commensals, some mycoplasma species are able to cause infections in pediatric, geriatric, or...
Although mycoplasmas are generally considered to be harmless commensals, some mycoplasma species are able to cause infections in pediatric, geriatric, or immunocompromised patients. Thus, accidental contamination of biologics with mycoplasmas represents a potential risk for the health of individuals who receive cell-derived biological and pharmaceutical products. To assess the efficiency of inactivation of mycoplasmas by the agents used in the manufacture of egg-derived influenza vaccines, we carried out a series of experiments aimed at monitoring the viability of mycoplasmas spiked into both chicken allantoic fluid and protein-rich microbiological media and then treated with beta-propiolactone, formalin, cetyltrimethylammonium bromide, Triton X-100, and sodium deoxycholate, which are agents that are commonly used for virus inactivation and disruption of viral particles during influenza vaccine production. Twenty-two mycoplasma species (with one to four strains of each species) were exposed to these inactivating agents at different concentrations. The most efficient inactivation of the mycoplasmas evaluated was observed with either 0.5% Triton X-100 or 0.5% sodium deoxycholate. Cetyltrimethylammonium bromide at concentrations of >or=0.08% was also able to rapidly inactivate (in less than 30 min) all mycoplasmas tested. In contrast, negligible reductions in mycoplasma titers were observed with 0.0125 to 0.025% formaldehyde. However, increasing the concentration of formaldehyde to 0.1 to 0.2% improved the mycoplasmacidal effect. Incubation of mycoplasmas with 0.1% beta-propiolactone for 1 to 24 h had a marked mycoplasmacidal effect. A comparison of the mycoplasma inactivation profiles showed that strains of selected species (Mycoplasma synoviae, Mycoplasma gallisepticum, Mycoplasma orale, Mycoplasma pneumoniae, and Acholeplasma laidlawii) represent a set of strains that can be utilized to validate the effectiveness of mycoplasma clearance obtained by inactivation and viral purification processes used for the manufacture of an inactivated egg-based vaccine.
Topics: Acholeplasma laidlawii; Animals; Cetrimonium; Cetrimonium Compounds; Chickens; Deoxycholic Acid; Eggs; Formaldehyde; Microbial Viability; Models, Biological; Mycoplasma; Mycoplasma gallisepticum; Mycoplasma pneumoniae; Mycoplasma synoviae; Octoxynol; Propiolactone; Vaccines, Inactivated; Viral Vaccines
PubMed: 20228111
DOI: 10.1128/AEM.02776-09 -
Cytotechnology Dec 2009A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using...
A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.
PubMed: 20135349
DOI: 10.1007/s10616-010-9252-6 -
Applied and Environmental Microbiology Mar 2004We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini,...
We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.
Topics: Acholeplasma; Base Sequence; Cell Culture Techniques; DNA Primers; DNA, Ribosomal Spacer; Humans; Mycoplasma; Nucleic Acid Hybridization; Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Sensitivity and Specificity; Tenericutes; Ureaplasma
PubMed: 15006769
DOI: 10.1128/AEM.70.3.1483-1486.2004 -
Infection and Immunity Dec 1985Mycoplasma orale, maintained as a contaminant of a mouse hybrid cell line, induces an intense proliferation in short-term culture of lymphoid cells of inbred mice. Cell...
Mycoplasma orale, maintained as a contaminant of a mouse hybrid cell line, induces an intense proliferation in short-term culture of lymphoid cells of inbred mice. Cell division induced by the contaminated cell culture fluid reaches a maximum on day four and declines rapidly thereafter. Culture fluids from hybrid cells freed of contamination do not cause proliferation. Cells from the spleen, bone marrow, and thymus of each of several strains of inbred mice, including xid CBA/N, poorly responsive to lipopolysaccharide, are stimulated by the mitogen, as are cells from BALB/c nude mice. The characteristics of the stimulatory effect are analogous in several important aspects to those of naturally occurring T cell-derived growth factors. In the absence of detectable numbers of T cells, both small and large B lymphocytes undergo mitosis in the presence of contaminated cell culture fluid, and B cells stimulated to divide by lipopolysaccharide are sustained for further rounds of replication by M. orale-containing cell culture fluid. The fluid also augments the stimulatory effect on thymocytes of suboptimum concentrations of phytohemagglutinin mimicking the effect of interleukin-1. Unlike with most naturally occurring lymphoid cell mitogens, however, the dividing cells do not go on to immunoglobulin secretion.
Topics: Animals; Bone Marrow; Cell Division; Cells, Cultured; Culture Media; Interleukin-1; Lymphocyte Activation; Mice; Mice, Inbred Strains; Mycoplasma; Phytohemagglutinins; Spleen; Thymus Gland
PubMed: 3877689
DOI: 10.1128/iai.50.3.636-640.1985 -
Journal of Applied Microbiology Jan 2011To assess the limit of detection (LOD) and the feasibility of 16S rRNA-based reverse transcription-PCR (RT-PCR) assays for advanced detection of mycoplasma contamination...
AIMS
To assess the limit of detection (LOD) and the feasibility of 16S rRNA-based reverse transcription-PCR (RT-PCR) assays for advanced detection of mycoplasma contamination in cell substrates.
MATERIALS AND METHODS
The RT-PCR approach is based on detecting the 16S rRNA molecules that, in contrast to genomic bacterial DNA, are represented by multiple copies in mycoplasma cell. The number of 16S rRNA molecules in mycoplasma cells of five species i.e. Mycoplasma arginini, Myc. fermentans, Myc. hyorhinis, Myc. orale and Acholeplasma laidlawii, all known to be frequent cell line contaminants in industrial and research laboratories, was measured using molecular methods. The results of two independently prepared mycoplasma cultures harvested at the stationary phase of their growth showed that the 16S rRNA copy number per cell varied in the range from about 400 to 2000 copies, depending on species, but stayed close between different preparations of one species. The assessment of the LOD of the in-house 16S rRNA-based RT-PCR was performed using samples of MDCK cell culture spiked with different amounts of five aforementioned mycoplasma species. To minimize the bias in methods comparison, the LOD of the RT-PCR assay was expressed in terms of genome equivalents (GEs) and compared with that determined for highly optimized 16S rDNA-based mycoplasma testing methods previously described in scientific literature.
CONCLUSIONS
The results of the study showed that the in-house 16S rRNA-based RT-PCR assay was able to reliably detect the presence of less than one mycoplasma GE that is at least 10-fold higher of the LOD previously determined for well-optimized 16S rDNA-based assays developed and described by other researchers.
SIGNIFICANCE AND IMPACT OF THE STUDY
The results of the study showed that rapid RT-PCR methods based on the detection of bacterial 16S rRNA are able to expedite mycoplasma testing of cell cultures (1-2 days vs 28 days) and to ensure the limits of detection comparable to that of currently used culture-based mycoplasma testing methods.
Topics: Animals; Cell Line; Limit of Detection; Mycoplasma; RNA, Ribosomal, 16S; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 20854458
DOI: 10.1111/j.1365-2672.2010.04853.x -
Molecular and Cellular Biology Apr 1981HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these...
HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.
Topics: Chloramphenicol; Drug Resistance; Erythromycin; HeLa Cells; Humans; Mitochondria; Mutation; Mycoplasma; Phenotype; Protein Biosynthesis; Protein Synthesis Inhibitors
PubMed: 6965101
DOI: 10.1128/mcb.1.4.321-329.1981 -
Applied and Environmental Microbiology Sep 2008In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma...
In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28 degrees C to between 35 and 37 degrees C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.
Topics: Animals; Bacteriological Techniques; Cell Culture Techniques; Cell Line; Chlorocebus aethiops; Coculture Techniques; DNA, Bacterial; Dogs; Insecta; Mycoplasma; Mycoplasma Infections; Polymerase Chain Reaction; Sensitivity and Specificity; Vero Cells
PubMed: 18606798
DOI: 10.1128/AEM.00720-08 -
Journal of Clinical Microbiology Jun 1986Saliva samples collected from 393 subjects with and without oral diseases were examined for concentrations of mycoplasmas and Mycoplasma species. Mycoplasmas were...
Enumeration, isolation, and species identification of mycoplasmas in saliva sampled from the normal and pathological human oral cavity and antibody response to an oral mycoplasma (Mycoplasma salivarium).
Saliva samples collected from 393 subjects with and without oral diseases were examined for concentrations of mycoplasmas and Mycoplasma species. Mycoplasmas were isolated from 383 (97%) of the 393 subjects. Viable counts ranged from zero to 7.6 X 10(7) CFU/ml (median, 6.9 X 10(4)) and were significantly (P less than 0.01) higher in diseased subjects, except for those with arthrosis temporomandibularis, than in controls. Of 1,400 isolates, 897 (64%), 442 (30%), and 8 (1%) were identified as Mycoplasma salivarium, M. orale, and M. hominis, respectively, and the remaining 73 isolates (5%) were unidentifiable. More than two-thirds of the isolates from diseased subjects versus only half from controls were identified as M. salivarium. In diseased subjects other than those with ostitis (especially those with arthrosis temporomandibularis), the incidence of M. salivarium was higher than that of M. orale, whereas the former occurred about as frequently as the latter in the controls. Antibodies to M. salivarium were also measured in sera from some subjects by the metabolism inhibition test. Sera with metabolism inhibition titers of 16 or greater were rated positive. There was no significant difference in the prevalence of antibodies between diseased subjects (60%) and controls (40%), but the mean titers (97 to 220) of all positive sera from diseased subjects were two to four times those for sera from controls. In addition, a fourfold or greater rise or fall of antibody titers to the organism was shown in paired sera from some subjects. On the basis of these results, M. salivarium was strongly suggested to participate etiologically in some cases of oral infection.
Topics: Adolescent; Aged; Antibodies, Bacterial; Child; Female; Humans; Male; Middle Aged; Mouth Diseases; Mycoplasma; Osteomyelitis; Pericoronitis; Periodontal Diseases; Saliva; Temporomandibular Joint Disorders
PubMed: 3711294
DOI: 10.1128/jcm.23.6.1034-1038.1986 -
Nucleic Acids Research Mar 1980The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the... (Comparative Study)
Comparative Study
The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the methylated base characteristic of prokaryotic DNA. The extent of methylation of adenine residues in the mycoplasmal DNA ranged from 0.2% in Mycoplasma capricolum to about 2% in Mycoplasma arginini and Mycoplasma hyorhinis with intermediate methylation values for Mycoplasma orale and Acholeplasma laidlawii DNAs. About 5.8% of the cytosine residues in M. hyorhinis DNA were methylated also. Analysis of cell culture DNA for the presence of m6Ade as a means for detection of contamination by mycoplasmas, and the phylogenetic implications of the finding of methylated bases in mycoplasmal DNAs are discussed.
Topics: 5-Methylcytosine; Acholeplasma laidlawii; Adenine; Base Composition; Cytosine; DNA, Bacterial; Methylation; Mycoplasma
PubMed: 7433124
DOI: 10.1093/nar/8.6.1383