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Microbiology and Immunology 1981The process of attachment of Mycoplasma hominis and M. orale to HAIN-55 cells, derived from normal embryonic human lung, was investigated quantitatively. The attachment...
The process of attachment of Mycoplasma hominis and M. orale to HAIN-55 cells, derived from normal embryonic human lung, was investigated quantitatively. The attachment reached its maximum within about 2-4 hr at 37 degrees C and increased linearly as a function of the number of organisms present in the system. The relative attachment efficiency of M. hominis was approximately 1% under our experimental conditions. Trypsin and EDTA were effective in detaching particles of M. hominis and M. orale from the surfaces of HAIN-55 cells. Therefore it was suggested that some proteinaceous substance and salt bridges might be involved in the attachment of these mycoplasmas to HAIN-55 cells.
Topics: Adhesiveness; Cell Division; Cell Line; Edetic Acid; Fibroblasts; Humans; Kinetics; Lung; Mycoplasma; Trypsin
PubMed: 6793814
DOI: 10.1111/j.1348-0421.1981.tb00078.x -
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 -
BMC Research Notes Jan 2012During the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem. When Mycoplasma contamination occurs in...
BACKGROUND
During the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem. When Mycoplasma contamination occurs in laboratories that study L. intracellularis, the cultures must be discarded for 4 reasons: 1) Mycoplasma is inevitably concentrated along with L. intracellularis during the passage of L. intracellularis; 2) Mycoplasma inhibits the growth of L. intracellularis; and 3) it is impossible to selectively eliminate Mycoplasma in L. intracellularis cultures. In this study, we observed the contamination of Mycoplasma species during L. intracellularis cultivation among multiple laboratories.
RESULTS
The presence of a Mycoplasma infection in the L. intracellularis cultures was verified using polymerase chain reaction (PCR), and a sequence analysis of the partial 16S rRNA and 23S rRNA genes was performed. A PCR-based assay using genus-specific universal primers revealed that 29 (85.3%) of the 34 cultures were contaminated with Mycoplasma, including 26 with M. hyorhinis (89.2%), 2 with M. orale (6.9%), and 1 with M. fermentans (3.4%). The Mycoplasma contamination was not the result of infection with material of pig origin. McCoy cells, which are required for the cultivation of L. intracellularis, were also ruled out as the source of the Mycoplasma contamination.
CONCLUSIONS
In this study, M. hyorhinis was identified as the most common mollicute that contaminated L. intracellularis cultures. Whether L. intracellularis enhances the biological properties of Mycoplasma to promote infection in McCoy cells is not known. Because the McCoy cell line stocks that were used simultaneously were all negative for Mycoplasma, and the same worker handled both the McCoy cells to maintain the bacteria and the L. intracellularis cultures, it is possible that the L. intracellularis cultures are more vulnerable to Mycoplasma contamination. Taken together, these results suggest that continuous cultures of L. intracellularis must be tested for Mycoplasma contamination at regular intervals.The GenBank accession numbers for the sequences reported in this paper are JN689375 to JN689377.
PubMed: 22284165
DOI: 10.1186/1756-0500-5-78 -
Transgenic Research Feb 2009Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed...
Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed reduced growth rate and viability (P < 0.0001) compared to control mESCs. Spectral karyotypic analysis of mycoplasma-infected mESCs showed a number of non-clonal chromosomal aberrations which increased with the duration of infection. The differentiation status of the infected mESCs was most affected at passage P13+6 where the infection was strongest and 46.3% of the mESCs expressed both POU5F1 and SSEA-1 markers whereas 84.8% of control mESCs expressed both markers. The percentage of germline chimeras from mycoplasma-infected mESCs was examined after blastocyst injection and embryo transfer to suitable recipients at different passages and, compared to the respective control group, was most affected at passage P13+5 (50% vs. 90%; P < 0.07). Further reductions were obtained at the same passage in the percentage of litters born (50% vs. 100%; P < 0.07) and in the percentage of pups born (22% vs. 45%; P < 0.001). Thirty three chimeras (39.8%) obtained from blastocyst injection with mycoplasma-infected mESCs showed reduced body weight (P < 0.0001), nasal discharge, osteoarthropathia, and cachexia. Flow cytometric analysis of plasma from chimeras produced with mycoplasma-infected mESCs revealed statistically significant differences in the proportions of T-cells and increased levels of IgG1 (P < 0.001), IgG2a (P < 0.05) and IgM (P < 0.05), anti-DNA antibodies (P < 0.05) and rheumatoid factor (P < 0.01). The present data indicate that mycoplasma contamination of mESCs affects various cell parameters, germline transmission, and postnatal development of the resulting chimeras.
Topics: Animals; Biomarkers; Blastocyst; Cell Differentiation; Cell Survival; Chimera; Embryo, Mammalian; Embryonic Stem Cells; Female; Fibroblasts; Germ Cells; Immunoglobulin G; Karyotyping; Male; Mice; Mycoplasma; Pregnancy
PubMed: 18819014
DOI: 10.1007/s11248-008-9218-z -
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 -
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 -
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 -
Zentralblatt Fur Bakteriologie,... Nov 1988A total of 1424 cell cultures was assayed for mycoplasmas by microbiological culture and fluorescent DNA staining. Of these cultures, 412 (29%) were infected with... (Comparative Study)
Comparative Study
A total of 1424 cell cultures was assayed for mycoplasmas by microbiological culture and fluorescent DNA staining. Of these cultures, 412 (29%) were infected with mycoplasmas. The most frequently occurring mycoplasma species were Mycoplasma orale (34%), M. hyorhinis (26%), M. arginini (21%), M. fermentans (13%) and Acholeplasma laidlawii (5%). A few isolates each of M. hominis, M. pulmonis and M. bovis were also detected. When detection methods were compared, microbiological culture produced false-negative results for 0.7% (3 of 412) of the infected cell cultures. DNA staining performed directly on the cells was falsely negative in 2.4% (5/207) of the mycoplasma-infected cultures that were compared, DNA staining performed on indicator cells was falsely negative in 3.1% (7/226). False positives appeared in direct DNA-staining in 1.8% (7/386) of the mycoplasma-free cultures and with DNA staining on indicator cells in 0.5% (3/620). For 11% of the cell cultures, the reading of the DNA staining was ambiguous. With DNA staining on indicator cells, 10% of the test results were ambiguous, but by further passage and staining on new indicator cells it was possible to get a definite diagnosis.
Topics: Animals; Cell Line; Cells, Cultured; Culture Media; DNA, Bacterial; False Negative Reactions; Fluorescent Antibody Technique; Mycoplasma; Purine-Nucleoside Phosphorylase; Vero Cells
PubMed: 3146169
DOI: 10.1016/s0176-6724(88)80176-7 -
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 -
European Journal of Cancer Aug 1968
[Ultrastructure of Mycoplasma salivarium and of Mycoplasma orale in tissue culture. The problem of the differentation because of the similarities in the cellular surfaces].
Topics: Cell Membrane; Culture Techniques; Diagnosis, Differential; Humans; Influenza, Human; Microscopy, Electron; Mouth; Mycoplasma; Mycoplasma Infections; Oncogenic Viruses; Surface Properties
PubMed: 4332479
DOI: 10.1016/0014-2964(68)90034-0