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E L K E F L E C K E N S T E I N A N D H A N S G . D R E X L E R * F r o m t h e G e r m a n C o l l e c t i o n o f M i c r o o r g a n i s m s a n d C e l l C u l t u r e sD e p a r t m e n t o f H u m a n a n d A n i m a l C e l l C u l t u r e s , B r a u n s c h w e i g , G e r m a n y* Corresponding author: Email: Mycoplasma contamination remains a
These antibiotic treatments had a high efficiency of permanent cure: MRA 64%, ciprofloxacin 77%, BM-Cyclin 84%. Resistance to mycoplasma eradication was observed significant impediment to the culture of
in some cell cultures: BM-Cyclin 5%, ciprofloxacin 14%, MRA 22%. Nearly all resistantcontaminants that could be identified belonged to the species M. arginini and M. orale. eukaryotic cells. For certain cultures,
Detrimental effects of the antibiotics were seen in form of culture death caused by cyto-toxicity (in 9-13% of the cultures). Alterations of the cellular phenotypic features or selec- attempts to eliminate the infection are
tive clonal outgrowth might represent further untoward side-effects of exposure to theseantibiotics. feasible alternatives to the normally recom-
Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reli- able, and simple method: 269/366 (73%) chronically and heavily contaminated cultures mended disposal of the contaminated culture.
were cured, while 97/366 (27%) cultures could not be cleansed and were either lost orremained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas Here three antibiotic regimens for mycoplas-
are amenable to antibiotic treatment and that a cure rate of about three-quarters is a reasonable expectation.
mal decontamination were compared in a
large panel of naturally infected cultures: (i) a
Mycoplasma contamination of cell culture systems continues to present major prob- lems for basic research as well as for the manufacturing of bioproducts (1,2). Mycoplasmas one-week treatment with the fluoroquinolone
affect virtually every parameter within the cell culture system (3). As mycoplasmal infec-tion of cell cultures might often persist for long periods of time without apparent cell dam- Mycoplasma Removal Agent (MRA), (ii) a two-
age (4), it is important to use one or several efficient detection methods (1,3,5,6).
Mycoplasma-positive cell cultures are themselves the major source of infection (7). Thus, week treatment with the fluoroquinolone
it was generally recommended that positive cultures should be discarded and replaced inorder to prevent the spreading of the contaminant (2,3). If the culture is considered irre- ciprofloxacin, and (iii) three rounds of a
placeable, it is possible to effectively eliminate the contaminant(s). Therefore, treatment ofmycoplasma-positive cell cultures has become a practicable option (4).
sequential one-week treatment with BM-
A number of methods have been employed in recent years with mixed results.
Procedures for elimination have included use of antibiotics, complement, and heterologous Cyclin, which contains a pleuromutilin and a
antisera; passage in nude mice; exposure to mouse macrophages; treatment with trypsin, S-bromouracil, and Hoechst 33258; culture in soft agar; and several other techniques tetracycline derivative.
B I O C H E M I C A ■ N O . 1 [ 1 9 9 6 ] F E A T U R E S
(2–4,8–16). However, few of these tech- niques produced satisfactory and consistent results. It became apparent that elimination biotics. None of the cell lines were deliber- (Boehringer Mannheim) using specific anti- of mycoplasmas from infected cell cultures represented chronically contaminated cul- Mycoplasma elimination
unsuccessful exercise posing the risk of sec- tures. Cultures were passaged according to ondary infection to other cultures (4).
Mycoplasma detection
the manufacturer’s instructions, using tamination should be simple, have minimal effect on cell growth, and not lead to loss of mycoplasmas with standard tests (5,6).
BM-Cyclin 2 for four days; this alternating Thus, the cultures were tested before and cycle was performed three times. The final simple technique appears to be antibiotic rescence staining and the microbiological treatment. Here, in particular, it is impor- broth-agar colony assay. Most treated cul- tetracycline derivative) were 10 ␮g/ml and tant to closely monitor effectiveness of the 5 ␮g/ml, respectively. Ciprofloxacin was used for 14 days at 10 ␮g/ml. Mycoplasma tion and eukaryotic cytotoxicity (2). It has an ELISA with specific polyclonal antisera (e.g., Mycoplasma Detection Kit, Boehringer medium for 8 days at a final concentration approach is to identify the contaminant by species and determine the antibiotic sensi- tivity of the infectant; then the mycoplasma- antibiotic-free medium (also without peni- positive eukaryotic cell culture is exposed using a mix of outer and inner primers (e.g., to several consecutive treatments with an antibiotic cocktail (17). However, for most Mannheim, Germany). All assays were weeks prior to testing for residual scientists, cell culturing is only the means to previously described in detail (5,6,24).
patience, or interest required for cumber-some trials. Therefore, new products to be marketed specifically for these purposes.
these studies examined only limited num-bers of treated cultures; furthermore, few comparative data are available. We sum-marize here our experience with antibiotic large number of positive cell cultures.
Materials and Methods
Cell culture
formed in cell cultures from cell lines that Outcome of treatment of mycoplasma-positive cell cultures with either
BM-Cyclin, ciprofloxacin, or MRA. According to the reagent manufacturers’ instructions, cells
were originally submitted to the Deutsche were treated for 1 week with MRA, for 2 weeks with ciprofloxacin, or for 3 weeks with BM-Cyclin, and
then cultured in antibiotic-free medium (also without penicillin or streptomycin) for at least another 2
weeks prior to testing for residual mycoplasma contamination. All cultures were tested with DAPI DNA
fluorescence staining and a broth-agar colony assay; 80% of the cultures were also investigated with a
that were found to contain mycoplasmas.
third method, either DNA/RNA hybridization (Mycoplasma Detection Kit), ELISA, monoclonal antibody
CCM-2, or a polymerase chain reaction (Mycoplasma PCR ELISA). The results are shown as

Cells were cultured under standard condi- percent of cultures that were either cured or that remained mycoplasma contaminated (due to resis-
tance) or that were lost during the treatment period (due to cytotoxicity). The number of cultures ana-
lyzed in each category is indicated (N).
B I O C H E M I C A ■ N O . 1 [ 1 9 9 6 ] F E A T U R E S
Results and Discussion
cell lines. For such low-level persistent In this study we set out to determine (i) infection, the introduction of the PCR tech- suggested that it might be preferable to per- form appropriate antibiotic sensitivity tests lines could be efficiently cleansed using the battery of available mycoplasma detec- antibiotic treatment, and (ii) whether the unnecessary on a routine basis as they are plasmas over long-term culturing, (i.e., type of outcome of the antibiotic treatment was loss of the culture, which occurred in eradicated or only suppressed). The results ment, is illustrated by the percentages of successful outcomes: 64–84% of the cul- effects of the reagents. Although previous studies on mycoplasma-negative cell lines Figure 1; mycoplasma infection was elimi- did not provide any evidence of antibiotic the literature shows several similar results cytotoxicity on the eukaryotic cells (18), the 77%, and by BM-Cyclin in 84% of the situation is certainly different in chronically cultures studied. Furthermore, the decon- and heavily contaminated cultures, such as With regard to the last point, the possi- tamination was total and permanent, as 14 the ones treated here, because of the ble adverse effects of the treatment on the days after treatment, no mycoplasmas were cell culture, three unwanted developments detected in cultures deemed to be cured.
reduced viability. Ciprofloxacin has been must be considered: (i) cytotoxicity; (ii) reported to have an effect on intracellularly loss of special cellular characteristics; and (iii) clonal selection of treated cells. A cultures remained unequivocally negative.
however, in that study, ciprofloxacin was cytotoxic to all 9 cell lines treated (23).
used at significantly higher concentrations of the three different regimens are evalu- (14–15 times the concentration used here) plasma immediately at the end of the treat- ated: (i) There was no selection on the (27). Other reports described inhibitory ment period, all cultures were ultimately cultures that were treated; attempts to elim- effects of ciprofloxacin on hematopoietic lost due to poor growth and extensive cell cell growth (28–30). The long-term cocul- death (23). These latter data stand in clear positive cultures submitted to the cell bank contrast to the 11% culture death seen in from 1990–1995, using at least one of the might lead to a sort of symbiosis whereby our series (Figure 1). In another report, no an abrupt termination might be detrimental to the cells. Further studies are required to Cyclin treatment of 11 cell lines (22).
ments in parallel (see below); (ii) All elaborate the reason(s) why some cell lines cultures were treated under the same con- are more susceptible to the cytotoxic effects loss of cell culture in two series of 9 and 26 ditions, (e.g., incubation, concentration of of the antibiotics than others treated at the antibiotics, treatment protocol). Thus, our lished on cytotoxic effects of MRA during apoptosis might also play some as yet unde- specific cellular characteristics and/or acci- dental clonal selection have not yet been floxacin or MRA were similar (Figure 1).
method should be simple, efficient, and not studied systematically in cultures exposed Although the species of contaminants were have any adverse effects on the cell culture not identified in all cases, our data demon- (4). The simplicity of antibiotic eradication analysis is, however, urgently needed since strate clearly that M. arginini and M. orale account for the vast majority of resistant lines were cultured under the same condi- tions during the treatment period as prior cross-resistance to ciprofloxacin and MRA; to decontamination, only the reagents were of the consequences of the unsolicited rou- these cell lines contained mostly M. arginini added to the culture media. We noted that tine application of anti-mycoplasma anti- or M. orale. This cross-resistance is not sur- it is advantageous to increase the FCS con- biotics in the daily cell culture work: the prising because ciprofloxacin and MRA are centration and to incubate the cells at high- emergence of resistant mycoplasma strains both fluoroquinolones of similar structure er densities. However, it must be pointed can surely be expected, and alterations of (25). It should be added that consideration out that antibiotic mycoplasma decontami- the eukaryotic phenotype in the long term should be given to the possibility that a low are quite possible as well. It is further level infection could persist undetected by suming: the duration of the treatment plus standard methods. The possibility of recur- detection methods (6) at regular intervals in order to examine cleared cultures for B I O C H E M I C A ■ N O . 1 [ 1 9 9 6 ] F E A T U R E S
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Pack Size
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Also Available
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