Treatment of mycoplasma contamination in a large panel of cell cultures

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.     

Cytotoxic effect of a T-strain mycoplasma (Ureaplasma urealyticum) on cultured normal human cells (WI-38)

Normal human embryonic lung fibroblasts (WI-38) were infected with Ureaplasma urealyticum, a urea hydrolysing mycoplasma. It was possible to observe reduced rates of multiplication of infected cells and reduced plating efficiency as well as the morphological changes usually associated with mycoplasma infection of animal cells in vitro. The cytotoxic effect on multiplication was sensitive to aureomycin but not penicillin. It was not related to depletion of amino acids or nucleic acid precursors from the cell culture medium but appeared to require that the host cells be growing. Ureaplasmas could not be recovered from cell culture medium after 4 days post infection and their characteristic urease activity could not be demonstrated either in cell culture medium or associated with the cells after the first cell subcultivation. [³H]TdR was incorporated into the nuclei of infected cells and the percent labelled nuclei was reduced compared with uninfected cells. Nuclear labelling indices of infected cells increased as the cells were subcultivated by trypsinization suggesting that the ureaplasmas were removed from the host cell surface by this treatment. In general, the effects of ureaplasmas on WI-38 cells do not appear to be as pronounced as effects of other mycoplasmas on animal cells in culture. It is clear, nonetheless, that the urea hydrolysing mycoplasmas can infect cells in culture and cause discernible effects on the growth and metabolism of these cells.     

In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain

A simple, fast, and easily reproducible routine laboratory technique for detecting mycoplasma contamination in cell cultures is reported. Cells grown on a coverslip are fixed directly with Carnoy's, air-dried, stained with DNA-specific fluorescent Hoechst 33258, and examined microscopically. All cultures that were infected with mycoplasmas had readily discernible, small, morphologically uniform, bright fluorescent bodies in the extranuclear and intercellular space in contrast to the non-contaminated control cultures in which the extra-nuclear background appeared uniformly dark. To probe the degree of sensitivity to detect mycoplasmas, control cultures were infected with aliquots from serially diluted cells or media collected from Mycoplasma hyorhinus infected cultures. The lowest infection rate (0.40% by sampling 1 000 cells in average per culture 4–24 h after infection) scored presently, however, can easily be lowered by increasing sample size since a cell infected with even one mycoplasma can be discerned. These mycoplasmas resisted centrifugation at 2 500 rpm for 30 min and easily filtered through 0.22 μm pore-size filter membrane. Amazingly infection rate of 0.63% scored from 24 h post-infection incubation attained 100% contamination with several hundreds of mycoplasmas per host cell within 120 h.     

Effect of cell membrane-active antimicrobial agents on membrane potential and viability of mycoplasmas

The membrane potential ofMycoplasma mycoides subsp.capri has been determined to beE _M=−48 mV±10%, inside negative. In this study we investigated the influence of cell membrane-active antimicrobial agents, viz., valinomycin, gramicidin, polymyxin, and clotrimazole, on membrane potential and viability ofM. mycoides subsp.capri. Valinomycin, an ionophore with extreme potassium selectivity, induced a membrane hyperpolarization,E _M=−110 mV. Valinomycin was not cidal, but static to mycoplasmas. Obviously the potassium drain induced by valinomycin can be compensated for by the organisms. Gramicidin is an antibiotic forming cation conduction channels across membranes. It induced a rapid depolarization,E _M=+23 mV, of mycoplasma membranes. At low concentrations, gramicidin had a static effect, whereas at high concentrations it was cidal to mycoplasmas. The rapid permeation of cations through the stationary ion channels formed by gramicidin obviously exerts an inhibitory or even lethal effect on mycoplasma metabolism and growth. Polymyxin B induced a depolarization,E _M=−35 mV, of mycoplasma membranes only when the organisms had been pretreated and hyperpolarized with valinomycin. After treatment with both valinomycin and polymyxin B, a slight inhibition of mycoplasma growth was observed. Clotrimazole, a synthetic imidazole antimycotic, hyperpolarized mycoplasma membranes (E _M=−80 mV). At high concentrations clotrimazole was cidal, whereas at low concentrations it was static to mycoplasmas.     

Efficiency of Plasmocin™ on various mammalian cell lines infected by mollicutes in comparison with commonly used antibiotics in cell culture: a local experience

Mycoplasma contamination is a deleterious event for cell culture laboratories. Plasmocin™ is used to prevent and eradicate mycoplasma infections from cell. In this study, 80 different mammalian cell lines from various sources; human, monkey, mice, hamster and rat were used to study and evaluate plasmocin™ efficiency and compare it to commonly used antibiotics such as BM-cyclin, ciprofloxacin and mycoplasma removal agent (MRA). It was shown that mycoplasma infections were eradicated by plasmocin™, BM-cyclin, ciprofloxacin and MRA in 65%, 66.25%, 20%, and 31.25%, respectively, of infected cell cultures. However, re-infection with mycoplasmas after the period of 4 months occurred in 10–80% of the studied cell lines. Cell cytotoxicity and culture death was observed in 25, 17.5 and 10% of the treated cells, for plasmocin™, BM-cyclin and MRA, respectively. In this study, Plasmocin™ showed strong ability to eradicate mollicutes from our cell lines with minimal percentage of regrowth. However, due to its high cell cytotoxicity it should be used with caution especially when dealing with expensive or hard-to-obtain cell lines. Amongst the antibiotics tested, BM-cyclin was shown to remove mycoplasma with the highest efficiency.     

Comparative antibiotic eradication of mycoplasma infections from continuous cell lines

Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.     

Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention

The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mechanisms of Genetic Instability in Mollicutes (Mycoplasmas)

Mollicutes are unique microorganisms characterized by a great extent for the reduction in genetic material, which retained the capability of independent division on acellular nutrient media. Phenotypically mycoplasmas differed from other bacteria by their small size and lack of a cell wall (mollis, soft; cutis, skin). High dependence on metabolism components utilized in the cultivation medium and high metabolic plasticity due to the absence of many genome regulatory elements make mycoplasmas perfect parasites for cells of the eukaryotic origin. The ability of these microorganisms to pass through host cells and their assumed participation in AIDS activation facilitate the study of mycoplasma pathogenesis. Another important feature of mycoplasmas, which is expressed during their interaction with a macroorganism, is their ability to escape from the immune response of a host due to surface antigen variation. These adaptation capacities of mycoplasmas ensuring their life in various biological niches, given a limited genome and the direct metabolic dependence on an environment, cannot be adequately explained at present. In this review, we attempted to collect and systematize data that contribute to our understanding of the important feature of mycoplasmas, genetic instability, which may underlie many of their adaptive responses.     

New Assay Procedure for Separation of Mycoplasmas from Virus Pools and Tissue Culture Systems

Presence of mycoplasma organisms in tissue culture systems and virus pools was detected by titration of the contaminated material on agarose-suspended BHK21/13S cells. The use of this method permitted isolation of mycoplasmas which could not be detected by standard assay methods. Mycoplasma colonies at concentrations ranging from 10(4) to 10(6) colony-forming units/ml in agarose-BHK21/13S media could be distinguished from virus plaques, and the two populations of microorganisms could be easily disassociated either by electron microscopy or by biological methods. All isolated mycoplasmas were identified in growth inhibition tests as belonging to the GDL group. The growth inhibition test on agarose-BHK21/13S cell suspension plates could also be applied directly to those strains which could not be isolated by standard assay procedures.     

Elimination of mycoplasmas from cultured human cell lines utilizing a combination of two antibiotics, quinolone and a pleuromutilin derivative

Forty three cultured human cell lines were treated with a combination of 2 antibiotics to eliminate contaminant mycoplasmas. One course of treatment was composed of consecutive 3 or 4 cycles. Each cycle grew cells in BM-1 (pleuromutilin derivative; Boehringer Mannheim) containing medium (10 micrograms BM-1/ml culture) for 3 days, alternating with MC-210 (quinolone; Dainihon Pharmaceutical) containing medium (0.625 micrograms MC-210/ml culture) for 4 days. No treatment failure was encountered with this procedure. Before treatments, 18 (90%) of 20 cell line samples were contaminated with mycoplasma, as tested by DNA hybridization method (MYCOPLASMA T.C. RAPID DETECTION SYSTEM; Gen-Probe Inc.). Out of 43 cell lines treated, 7 were reduced in growth and dropped out. Among the other 36 cell lines, 27 became negative, 5 borderline and 4 slightly positive to the mycoplasma detection. All of the latter 9 cell lines, treated with one more similar course, found to be free from mycoplasma. Six of the dropout lines were cured of mycoplasma by a second treatment, under modified culture conditions. The last cell line (NATO) was successfully treated with another lot of FCS. Thus, the procedure proved successful even in treating promiscuously infected cell lines.     

Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats

Restriction and modification (R-M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase-variable R-M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R-M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony-forming units (cfu) lacked R-M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R-M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R-M activity of the mycoplasmal cell population. We propose that variations in the production of R-M activity and cell surface proteins are important for the survival of the mycoplasma within the host.     

Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis.

Surfactin, a cyclic lipopeptide antibiotic and biosurfactant produced by Bacillus subtilis, is well-known for its interactions with artificial and biomembrane systems (e.g., bacterial protoplasts or enveloped viruses). To assess the applicability of this antiviral and antibacterial drug, we determined the cytotoxicity of surfactin with a 50% cytotoxic concentration of 30 to 64 microM for a variety of human and animal cell lines in vitro. Concomitantly, we observed an improvement in proliferation rates and changes in the morphology of mycoplasma-contaminated mammalian cells after treatment with this drug. A single treatment over one passage led to complete removal of viable Mycoplasma hyorhinis cells from various adherent cell lines, and Mycoplasma orale was removed from nonadherent human T-lymphoid cell lines by double treatment. This effect was monitored by a DNA fluorescence test, an enzyme-linked immunosorbent assay, and two different PCR methods. Disintegration of the mycoplasma membranes as observed by electron microscopy indicated the mode of action of surfactin. Disintegration is obviously due to a physicochemical interaction of the membrane-active surfactant with the outer part of the lipid membrane bilayer, which causes permeability changes and at higher concentrations leads finally to disintegration of the mycoplasma membrane system by a detergent effect. The low cytotoxicity of surfactin for mammalian cells permits specific inactivation of mycoplasmas without significant deleterious effects on cell metabolism and the proliferation rate in cell culture. These results were used to develop a fast and simple method for complete and permanent inactivation of mycoplasmas in mammalian monolayer and suspension cell cultures.     

Conversion of glucose to fatty acids and methane: roles of two mycoplasmal agents.

Two species of obligately anaerobic mycoplasmas were the major components of a methanogenic glucose-limited enrichment culture. In pure culture, one of these organisms, tentatively named Anaeroplasma sp. strain London, was shown to be responsible for the fermentation of glucose to fatty acids, hydrogen, and carbon dioxide; the other mycoplasma was shown to produce methane from hydrogen and carbon dioxide and was named Methanoplasma elizabethii. This same methanogenic mycoplasma contained a low-molecular-weight fluorescent cofactor which had a maximum light absorbance at 430 nm. When both species of mycoplasmas were grown together on glucose, fermentation products included fatty acids and methane. For the first time, mycoplasmas are implicated as agents of anaerobic degradation and methanogenesis in a sewage sludge digester.     

Mycoplasma—virus contacts,budding and scars remaining in the membranes ofAcholeplasma laidlawii and its virus

Various stages of virus and mycoplasma budding indicated that both virus and, most probably some mycoplasma progeny developed by budding. Besides this alternative, binary fission was the mode of mycoplasma reproduction. Mycoplasma—virus and mycoplasma—mycoplasma connections by stems were observed. Circular scars, 40–80 nm in diameter, often in groups, were left in the membrane of mycoplasmas by the budding bodies. Cytoplasmic structures seen in cross-fracture are presented. A relatively small number of globular virus-like bodies, not identical with MV-Lg-L 172, were observed budding from mycoplasma cells in the non-infected host culture.     

Release of Hydrogen Peroxide from Human T Cell Lines and Normal Lymphocytes Co-Infected with Hiv-1 and Mycoplasma

Human T-cell lines and normal lymphocytes persistently or acutely co-infected with the human immunodeficiency virus type 1 (HIV-1) and mycoplasmas were found to release hydrogen peroxide (H₂O₂), a likely cause of oxidative stress in these cells. The spectrofluorometric measurement of H2O2 release from these cells, using the scopoletin fluorescence quenching technique, gave values of 16-84 p moles/10⁶ cells/min. In CEM cells, H₂O₂ was released only when acutely co-infected with HIV-1 and mycoplasmas, and not when infected with either organism alone. Anti-mycoplasmal antibiotics strongly reduced H₂O₂ release, and improved cell viability without blocking virus replication. These results suggest that the simultaneous infection by HIV-I and mycoplasma leads to the release of H₂O₂, a toxic and potentially lethal metabolite, which in vivo may contribute to HIV-1 pathogenicity.     

Surprisingly fast disappearance of beta-lactam selection pressure in cultivation as detected with novel biosensing approaches

Tetracycline and beta-lactam resistances among others are used as selection markers in the production of recombinant proteins. The beta-lactam resistance is based on degradation, i.e. the selection pressure gradually disappears from the culture, whereas tetracycline resistance is based on active efflux. We have studied the kinetics of the stability of antibiotic selection pressure in culture using a simple model system (pBR322 in Escherichia coli). Concentrations of ampicillin, carbenicillin and tetracycline were measured with novel sensor cells developed in our lab. These cells are specifically induced to produce light in the presence of the drugs and here their performance was shown to be excellent in monitoring antibiotic concentrations in cell culture. The sensor cells are cheap to produce and use and a high number of samples can be analysed simultaneously. To our surprise, ampicillin and carbenicillin were completely degraded after 2.5-3.0 h of culture, although it has been widely claimed that especially carbenicillin is a good selective agent, whereas tetracycline was stable in culture. beta-lactamase activity in culture was found to correlate with the kinetics of ampicillin degradation.     

Mycoplasmas, plants, insect vectors: a matrimonial triangle

Plant pathogenic mycoplasmas were discovered by electron microscopy, in 1967, long after the discovery and culture in 1898 of the first pathogenic mycoplasma of animal origin, Mycoplasma mycoides. Mycoplasmas are Eubacteria of the class Mollicutes, a group of organisms phylogenetically related to Gram-positive bacteria. Their more characteristic features reside in the small size of their genomes, the low guanine (G) plus cytosine (C) content of their genomic DNA and the lack of a cell wall. Plant pathogenic mycoplasmas are responsible for several hundred diseases and belong to two groups: the phytoplasmas and the spiroplasmas. The phytoplasmas (previously called MLOs, for mycoplasma like organisms) were discovered first; they are pleiomorphic, and have so far resisted in vitro cultivation. Phytoplasmas represent the largest group of plant pathogenic Mollicutes. Only three plant pathogenic spiroplasmas are known today. Spiroplasma citri, the agent of citrus stubborn was discovered and cultured in 1970 and shown to be helical and motile. S. kunkelii is the causal agent of corn stunt. S. phoeniceum, responsible for periwinkle yellows, was discovered in Syria. There are many other spiroplasmas associated with insects and ticks. Plant pathogenic mycoplasmas are restricted to the phloem sieve tubes in which circulates the photosynthetically-enriched sap, the food for many phloem-feeding insects (aphids, leafhoppers, psyllids, etc.). Interestingly, phytopathogenic mycoplasmas are very specifically transmitted by leafhoppers or psyllid species. In this paper, the most recent knowledge on phytopathogenic mycoplasmas in relation with their insect and plant habitats is presented as well as the experiments carried out to control plant mycoplasma diseases, by expression of mycoplasma-directed-antibodies in plants (plantibodies).     

Elimination ofMycoplasma hyorhinis infections from four cell lines

Summary Four monolayer mammalian cell lines were cured ofMycoplasma hyorhinis infections by cloning in microtiter dishes in the presence of tetracycline and kanamycin. During cloning, cultures were refed with fresh antibiotic containing medium every 2 or 3 d for 14 d and were then cultured without effective antibiotics for at least 21 d. From the four lines we recovered 29 clones, none of which were infected after treatment as judged by the lack of extranuclear fluorescence after staining with the fluorochrome Hoechst 33258, and by normal autoradiographic labeling of the cells by tritiated nucleosides. One clone from each line was tested further by attempted culture of mycoplasmas and was also judged to be uninfected. Infection has not reappeared in any of the clones after extensive culture in the absence of the effective antibiotics. This investigation was supported by Public Health Service Research Grant GM26137 from the National Institutes of General Medical Sciences, National Institutes of Health, Bethesda, MD.     

Development of Flouroquinolone (Ciprofloxacin) Resistance in Mycoplasma hominis in the Presence of HeLa Cells

The effect of cocultivation of eukaryotic HeLa cells and Mycoplasma hominis mycoplasma on the resistance of the latter to fluoroquinolones (ciprofloxacin) was examined. It was shown that cocultivation of the M. homonisand HeLa cells during 24 h with subsequent addition of ciprofloxacin resulted in an increase of the micoplasma resistance to this antimicrobial agent. In the M. hominis cells cultivated in the presence of HeLa cells and the increasing concentration of ciprofloxacin mutations in the parC gene were observed only at low concentrations of the antimicrobial agent, while mutations in the gyrA gene were never detected. A gradual elevation of ciprofloxacin concentration up to 10 g/ml resulted in the reversion of the parC mutations in mycoplasmas. Mycoplasma cells resistant to high flouroquinolone concentrations and isolated after cocultivation with the HeLa cells were characterized by the wild-type genotype in respect of the gyrA and parC genes. It was shown that infection of HeLa cells resulted in the appearance of genome rearrangements in M. hominis cells.     

Effects of mycoplasma infection on the host organism response via p53/NF-κB signaling

Mycoplasmas are bacteria lacking the cell wall, which is the major characteristic of this taxonomic class (Mollicutes). Among bacteria, mycoplasmas possess the smallest genome known for free-living organisms. This feature limits the autonomy of bacteria and makes them increasingly susceptible to changes in the host organism. Many mycoplasmas themselves cause pathological changes in the host organism, often complicated by immune disorders. Infection with certain strains of mycoplasma results in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells, which is the major mediator of the inflammatory response. Furthermore, mycoplasmas can inhibit p53-mediated checkpoint control of cell cycle and apoptosis. Collectively, these properties indicate that mycoplasmas might act as cancer-promoting factors. In this review, we summarize the information known to date on the role of mycoplasmas in the regulation of the host immune response and their functional interactions with p53.