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1.
Vahid Molla Kazemiha Mohammad Ali Shokrgozar Mohammad Reza Arabestani Morteza Shojaei Moghadam Shahram Azari Susan Maleki Amir Amanzadeh Mahmood Jeddi Tehrani Fazel Shokri 《Cytotechnology》2009,61(3):117-124
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. 相似文献
2.
Several antibiotics were examined for their potential to eliminate mycoplasmas from contaminated cell cultures. Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma hyorhinis and Mycoplasma orale were effectively eliminated from experimentally contaminated mouse fibroblasts and mink epithelial cells by the use of the antibiotics minocycline and tiamutin. An elimination procedure was established, which involved the consecutive treatment of the cultures over a period of 3 weeks, followed by cell cloning. This procedure was effective when applied to cell lines which had been contaminated with unidentified and partially non-cultivable strains of mycoplasmas. 相似文献
3.
A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied. 相似文献
4.
Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody
Ellen F. Freiberg Gerald K. Masover 《In vitro cellular & developmental biology. Plant》1990,26(6):585-588
Summary Conditions are presented for application of both bisbenzamide (Hoechst 33258) stain and a specific fluoresceinated anti-Mycoplasma hyorhinis IgG to a single cell culture preparation. This allows the same field on a slide to be viewed for presumptive diagnosis of
any cell culture contaminant mycoplasma by bisbenzamide staining and for definitive diagnosis ofM. hyorhinis strains using fluoresceinated antibody. The use of this method plus a cultural procedure will permit identification of the
“noncultivable”M. hyorhinis strain DBS 1050. 相似文献
5.
Gerard J. McGarrity Judi Sarama Veronica Vanaman 《In vitro cellular & developmental biology. Plant》1979,15(2):73-81
Summary A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar
and broth media. Mycoplasmas were detected in 375 cultures (5.8%).M. orale andA. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aerobic incubation detected 45.8%.
Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static
antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains ofM. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas.
These studies were supported in part by Contracts N01-AG-4-2865 and N01-AG-8-2117 from the National Institute on Aging and
N01-GM-6-2119 from the National Institute of General Medical Sciences. 相似文献
6.
Dmitriy V. Volokhov Hyesuk Kong Joseph George Christine Anderson Vladimir E. Chizhikov 《Applied microbiology》2008,74(17):5383-5391
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°C to between 35 and 37°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. 相似文献
7.
Susan E. Ditty BingJie Li Shimin Zhang Nianxiang Zou Shyh-Ching Lo 《Current microbiology》2003,46(4):0302-0306
In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in
the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L)
structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding
has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two
different mycoplasma species.
Received: 17 April 2002 / Accepted: 9 July 2002 相似文献
8.
Mycoplasma infection in human and its contamination in cell cultures are worldwide problems. The drugs currently available for preventing or treating mycoplasma infection suffer from low sensitivity, strong resistance and high toxicity. Our previous work showed that Mycoplasma hyorhinis (M. hyorhinis) infection was mediated by the interaction between p37 of M. hyorhinis and Annexin A2 (ANXA2) of host cells, however the translational value of this mechanism was unknown. Herein, we synthesized the N-terminal of ANXA2 polypeptide (A2PP) and found that A2PP could decrease the infection of M. hyorhinis to gastric cancer cells and block M. hyorhinis infection-induced cell migration. Furthermore, we found that A2PP could reduce M. hyorhinis contamination of passage cells. Moreover, compared with the commercial antibiotics commonly used in cell culture to prevent M. hyorhinis infection, A2PP demonstrated a more effectiveness but a low toxicity on cell growth. Thus, our study for the first time revealed A2PP’s potential for the treatment and prevention of M. hyorhinis infection. 相似文献
9.
G. Darai L. Zöller R. M. Flügel H. Gelderblom 《In vitro cellular & developmental biology. Plant》1983,19(1):7-15
Summary In an extensive host range study ofM. hyorhinis mink lung cells (MvlLu, ATCC CCL 64) were found to be the cells of choice for the propagation of this mycoplasm, which otherwise
is often difficult to grow in a cell-free medium. Furthermore, rapid plaque assay and plaque purification procedures were
developed forM. hyorhinis. The titer ofM. hyorhinis grew to 1×107 to 1×108 pfu/ml within three d postinoculation on mink lung cells. DNA restriction enzyme analysis of the genome ofM. hyorhinis was performed. Endonucleases Bst EII and Xho I are the most suitable enzymes for cleavingM. hyorhinis DNA into distinct fragment patterns. Thus, the use of the combination mink lung cells for mycoplasma growth with subsequent
restriction enzyme analysis leads to an unamibiguous detection and identification toM. hyorhinis strains even in minute amounts. 相似文献
10.
Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and
potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma
contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples.
Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 101 CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial
titers (101 and 106 CFU/ml) after storage at 4° C and −30° C were analyzed. During storage, viable organisms were found with little or no reduction
in titers after storage for 8 wk at −30° C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred
after 4 wk storage for high-dose cultures (106 CFU/ml) at 4° C. The titers of viable organisms were diminished over 8 wk at 4° C under aerobic and anaerobic conditions. 相似文献
11.
Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:
- 1. 1. When cells are labelled with [8-3H]guanosine or [5-3H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-3H]guanosine or [5-3H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.
- 2. 2. [5-3H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M1 and 23S and 16S rRNA.
- 3. 3. [3H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.
- 4. 4. With [32P]orthophosphate or [3H]adenosine as precursors, the labelling is primarily in the host RNA.
12.
Hans G. Drexler Suzanne M. Gignac Zhen-Bo Hu Anne Hopert Elke Fleckenstein Maren Voges Cord C. Uphoff 《In vitro cellular & developmental biology. Animal》1994,30(5):344-347
Summary Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts
to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here,
three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a
1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin,
and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments
had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was
observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be
identified belonged to the speciesMycoplasma arginini andM. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the
cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward
side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive,
reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures
could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing
mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation. 相似文献
13.
14.
Johan Vande Voorde Suna Sabuncuo?lu Sam Noppen Anders Hofer Farahnaz Ranjbarian Steffen Fieuws Jan Balzarini Sandra Liekens 《The Journal of biological chemistry》2014,289(19):13054-13065
The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2′,2′-difluoro-2′-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2′,2′-difluoro-2′-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2′-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors. 相似文献
15.
Background
Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo.Principal Findings
We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture.Conclusions
This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination. 相似文献16.
Growth on axenic agar medium is one of several characters by which mycoplasmas are defined. In apparent contradiction of the
definition, DBS 1050 and other noncultivable strains ofMycoplasma hyorhinis do not grow on axenic medium but grow in cell culture. Our results show that BHK-21 cell extracts support DBS 1050 growth
in appropriate medium. An inhibition assay, based on a virus neutralization format, shows that a variety of common medium
ingredients inhibit DBS 1050 growth. The most potent activity was found in yeast extract. All other noncultivable strains
ofM. hyorhinis tested have a yeast extract sensitivity, while cultivable strains do not. The apparent cell dependence of DBS 1050 can be
attributed to growth inhibition due to factors present in a wide variety of peptones and extracts commonly used in medium;
preferential growth in cell cultures is due to the absence of effective levels of these factors. Data are not available to
determine if cell cultures provide growth factors not found in standard medium. The infraspecific taxon,M. hyorhinis cultivar α, is proposed for formerly noncultivable strains ofM. hyorhinis. 相似文献
17.
C. W. Long R. DelGiudice R. S. Gardella M. Hatanaka 《In vitro cellular & developmental biology. Plant》1977,13(7):429-433
Summary Human H. Ep-2 and mouse 3T6 cells infected byMycoplasma hyorhinis showed an increase in [3H]uracil uptake and a more than 20-fold increase in the activity of uracil phosphoribosyltransferase (UraPRT). Uninfected
cell cultures gave background levels of this enzyme activity. A survey of 16 strains of mycoplasma showed 13 to possess UraPRT
activity. Rabbit kidney cells (RK13) were infected with eight different strains of four mycoplasma species known to be common
cell culture contaminants. Seven of the eight cell cultures showed elevated UraPRT activities four days after infection. This
enzyme activity may be of value in monitoring cell cultures for mycoplasma and aid in classification.
This work was supported by Contract NO1-CP-53530 with the National Cancer Institute, National Institutes of Health, and Contract
FDA 74-41 of the Food and Drug Administration, Bethesda, Maryland 20014. 相似文献
18.
Taylor-Robinson, David (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Otakar Sobeslavsky, and Robert M. Chanock. Relationship of Mycoplasma pneumoniae to other human Mycoplasma species studied by gel diffusion. J. Bacteriol. 90:1432-1437. 1965.-Conditions are presented for the production of four lines of precipitate between Mycoplasma pneumoniae antigen and homologous hyperimmune rabbit serum in double diffusion in agar. The specificity of the reaction was shown by the fact that M. pneumoniae antigen did not react with antisera to the other human mycoplasma species, nor did M. pneumoniae antiserum produce lines with antigens prepared from the other human mycoplasmas. In addition, there was no reduction in the number or intensity of precipitation lines after absorption of M. pneumoniae antiserum with heterotypic mycoplasma antigens, or after absorption of heterotypic mycoplasma antisera with M. pneumoniae antigen. These findings indicate that, of the human mycoplasma species so far studied, M. pneumoniae is antigenically the most distinct. 相似文献
19.
Hitoshi Kotani Gary H. Butler Diane Tallarida Christine Cody Gerard J. McGarrity 《In vitro cellular & developmental biology. Plant》1990,26(1):91-96
The failure of many cell culture isolates ofMycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly
prepared yeast extract in mycoplasmal media together with incubation in 5% CO2/air successfully detectedM. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic
incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures.
This study was supported by grant AI 15748 from NIH. We thank Ronnie Vanaman and Judi Sarama for their technical assistance. 相似文献
20.
Göran Bölske Marie-Louise Strandberg Katrin Bergström Karl-Erik Johansson 《Current microbiology》1987,15(4):233-239
Cell proteins from the porcine mycoplasmasMycoplasma hyorhinis, M. hyopneumoniae, andM. flocculare have been analyzed by SDS-gel electrophoresis and immunoblotting. The protein profiles ofM. hyopneumoniae andM. flocculare were similar, but the protein profile ofM. hyorhinis was quite different from the others. Antisera prepared against whole cells of the above three mycoplasmas were used in immunoblotting of electrophoretically separated antigens and in enzyme-linked immunosorbent assay. One major antigen, which had a molecular weight of 73 k, was found to be common to all three mycoplasmas. Another major antigen, with a molecular weight of 41 k, was common toM. hyopneumoniae andM. flocculare and may also be present inM. hyorhinis. Several antigens of comparatively high molecular weights (108 k, 102 k, 93 k, 89 k, and 87 k) seemed to be specific forM. hyopneumoniae. Three antisera prepared by immunization of rabbits with immunoprecipitates obtained by crossed immunoelectrophoresis ofM. hyopneumoniae were also used in blotting experiments. One of these antisera was found to be directed against the 73 k antigen common to the three porcine mycoplasmas investigated. The other two antisera were directed againstM. hyopneumoniae-specific antigens with molecular weights of 74 k, 58 k, 45 k, 44 k, and 38 k. 相似文献