MYCOPLASMA - THE SMALLEST BACTERIA SPECIES
1.1 INTRODUCTION
Cell culture simply refers to the process of removing cells from an animal or plant and their subsequent growth in an artificial medium totally different from the cell internal environment. Cell culture is one of the important tool in medical and biological research especially in industrial biotechnology where they are used in the production of biologically active pharmaceuticals such as antibiotics and vaccines.
Any handling of cell culture from always poses the risk of Mycoplasma contaminations, either with eukaryotic cells from other cell cultures or more frequently with microbiological organism including yeast, fungi and bacteria.
Mycoplasma are the smallest know, fastidious and self-replicating bacterium without a cell wall around their cell membrane. They are about 0.3-0.8µm in diameter.
One of the major advantages of cell culture is the ability to manipulate the physico-chemical (i.e. temperature, pH, osmotic pressure, oxygen) and the physiological environment (i.e. hormone and nutrient concentrations) in which the cells propagate with the exception of temperature, the cell environment is controlled by growth medium (Gong et al., 2005).
While the physiological environment of the culture is not as well defined as it physico-chemical environment, a better understanding of the component of serum, the identification of the growth factors necessary for proliferation, and a better understanding of the micro environment of the cells in culture (i.e. the cell to cell interactions, diffusion of gases, interactions with the matrix) now allow the culture of certain cell lines in serum-free medium (Drexler et al., 2003).
1.2 TYPES OF CELL CULTURE
Primary Cell Culture: This is the first stage of cell culture where the cell is isolated from the tissue and proliferated given the appropriate conditions until they finally utilizes all the substrate available in the medium. At this stage the cell is ready to be subcultured.
Primary can be divide into two, this are:
Adherent or Anchorage Depapendent Cells: This are cell cultured which are shown to require an attachment for growth.
Suspension or Non Anchorage Dependent Cells: This are cell culture which do not require an attachment for growth or do not attach to the surface of the culture vessels.
Secondary Cell Culture : This is a type of cell culture where a finite cell is subcultured and allow to divide indefinitely (Harline et al., 2008).
2.1 HISTORY OF MYCOPLASMA
Mycoplasma is a genus of bacteria that lack a cell wall around their cell culture membrane. Without a cell wall, they are resistance to many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprotrophic. Several species are pathogenic in humans, including M. pneumoniae, which is an important cause of s typical pneumonia and other respiratory disorders. M. genitalium, are also believed to be involved in pelvic inflammatory diseases. Mycoplasma are the smallest bacteria cells yet discovered, they are anaerobes and are typically about 0.1µm in diameter (Robinson et al., 1998).
2.1.1 ORIGIN OF THE NAME
The term Mycoplasma, is coined from the Greek mykes (fungus) and plasma (formed), was first used by Albert Bernhard Frank in 1889 to describe an altered state of plant cell cytoplasm resulting from infiltration by fungus-like microorganisms imagined to have both cellular and acellular stages in their life cycles, which could explain how they were visible with a microscope but passed through filters which are impermeable to bacteria (Bielanski et al., 2009).
2.2 SPECIES OF MYCOPLASMA
There are more than 20 species isolated from contaminated cell lines, detailed observation on the identity of the contaminating species shows that the largest portion of infections is caused by Mycoplasma species, about 90-95% of the contaminant were identified as either M. orale, M. hyorhinis, M. arginini, M. fermentans or M. hominis.
Common microplasma species includes:
M. fermentans, M. genitalium, M. orale, M. hominis, M. salivarium, M. galliseptium, M. hyopneumoniae, M. arginine, M. hyorhinis (McGarrity et al., 2010).
3.1. INCIDENCE OF MYCOPLASMA CONTAMINATION IN CELL CULTURE
Mycoplasma were first isolated from a contaminated cell culture in 1956. One Mycoplasma species can grow up to 1000000 CFU per ml within three to five days in an infected cell culture. Eukaryotic cell culture contaminated with Mycoplasma species have titers in the range 1000000 to 10000000 organisms per ml. it has been known that about 100 to 1000 Mycoplasma attached to each infected cell. Primary cell culture and cultures in early passage have been reported to be less frequent contaminated than continuous cell lines. Several large series on thousands of cell cultures analyzed over decades (1960s – 1980s) found an incidence of 15%. Recent studies documented significantly on a smaller series to be in the region 15-35%, but also as high as 65-80%. The expanding application of cell lines in research and biotechnology and the increasing use of certain antibiotics (mostly penicillin and streptomycin which merely serve to mask but do not remove Mycoplasma) in routine culture have presumably led to the increase in Mycoplasma contamination in cell culture (Drexler et al., 2003).
3.2 MOST COMMON CONTAMINATING MYCOPLASMA SPECIES
While more than 20 species have been isolated from contaminated Cell lines, detailed investigations on the identity of the contaminating species showed that the largest proportion of infections is caused by relatively small number of Mycoplasma spp where about 90 – 95% of the contaminant were identified as either M. orale, M. hyorhinis, M. fermentans, or M. hominis. Generally, M.orale which is the most common Mycoplasma spp in the oral cavity of clinically normal humans, also represent the single most common isolate accounting for about 20 – 40% of all Mycoplasma infection in cell cultures. Other non-pathogenic Mycoplasma spp from normal human microbial flora of the oropharynx which are seen in cell culture are M. fermentans and M. hominis (McGarrity et al., 2010).
The bovine group of Mycoplasma such as M. arginine account for about another third of all strains isolated from cell cultures. Here the most frequent infected are M. arginine and M. hgyorhinis. These two species have a relatively wide host range especially M. arginine which is isolated from cattle, sheep, goat and other ruminant animals. This cell culture contaminant is thought to be derived from bovine sources. Bovine sera were not routinely and as strictly screened for Mycoplasma contamination. M. hyorhinis a common inhabitant of the nasal cavity of the swine, also account for the high proportion of the infections (Barile et al., 2010).
3.3 SOURCES OF MYCOPLASMA CONTAMINATION OF CELL CULTURE
Sources of Mycoplasma in cell culture is generalized in all the equipment, cells, and materials used in cell culture. They can be grouped into three main categories, which are Swine sources, Bovine and Human sources. Tissues specimen used to initiate cell cultures do not appear to represent the major sources of Mycoplasma infection. The frequent of infection in primary cell culture is low, in order of 1% (Armstrong et al., 2010). The high incidence of bovine sources predominantly M. arginini. Implicates fetal or newborn bovine serum. Most lot of serum provided for use in cell culture if not properly screened are contaminated with Mycoplasma spp (M.arginini) which were isolated to be around 25.40%. however, there is an high decrease in bovine serum contamination in the last decades owing to the appropriate effort of the suppliers with regards to other prevention and control measures by laboratory personnel, despite this measures serum lots absolutely free from Mycoplasma contamination cannot be guaranteed.
This is because the largest proportion of Mycoplasma species found in cell culture are of human origin, one may assume that laboratory personnel account for the major sources of contamination. In laboratories with contaminated cells, most or all cultures are positive with the same Mycoplasma species (Armstrong et al., 2010).
Mycoplasma infected cell lines themselves the most important source for further spread of the contamination. This is made possible owing to the ease of droplet generation during handling of cell cultures, the high concentration of Mycoplasma in infected cultures, and the prolonged survival of dried Mycoplasma cells. Mycoplasmas are spread by various laboratory equipment’s such as incubators, laminar flow hood, liquid nitrogen, etc. media or reagents that have been contaminated by previous use in processing Mycoplasma infected cells (Coecke et al., 2005).
3.3.1. DIFFERENT SOURCES FOR THE SPREAD OF MYCOPLASMA IN THE LABORATORY
McGarrity designed a model to find out how Mycoplasma spread in a laminar flow hood during a routine subculturing procedure. He intentionally infected a cell culture with Mycoplasma. After trypsinization of the infected culture in a laminar flow hood, live Mycoplasmas were isolated by the technician outside the flask, a hemocytometer, the pipettor, and outside of the pipette discard pan. Live Mycoplasma could be successfully recovered from the surface of the laminar flow hood even four to six days later. A clean culture that was subcultured once a week in the same weeks. These results show how quickly and easily Mycoplasma can spread and also against the possibility of contamination of most if not all of the other cultures after the entry of a single Mycoplasma infected culture into the laboratory. (McGarrity et al., 2010).
Currently, the major source of Mycoplasma contamination is infected cultures obtained from other research laboratories of commercial suppliers. Some of the major sources of Mycoplasma contamination are listed below:
Media, Sera or Reagent Contaminated with Mycoplasma
Mycoplasmas can pass into the filter membrane used in sterilizing cell culture media, sera and other reagents since they are too small and pliable due to the absence of a cell wall. Therefore, cell culture media and animal products used in cell culture should be considered major routes for Mycoplasma contamination. In the 1960s and 1970s, sera products were a very important primary source of infection, with reported contamination rates of 18% to 40%. Today, sera media obtained from reputable manufacturers are rarely the source of Mycoplasma contamination. However, it is still the responsibility of the end user to verify that the products they purchase have been adequately filtered, tested and certified as mycoplasma-free (Bolske, 2003).
It is common in most cell culture laboratories to use single 0.2µm pore size filter membranes to filter media or other solutions. However, this method is relatively safe for solutions with low levels of Mycoplasma. It is not recommended to filter raw animal-derived sera or products since the Mycoplasma contamination could potentially be high in them. To remove Mycoplasma with filtration, the method of filtering plays an important role. Low pressure differential (5-10 psi) is less likely to face Mycoplasma through a membrane than filter systems using 20 psi or higher pressure. Filters with 0.1µm pore size should be used instead of 0.2µm ones in the case of dubious conditions (Bolske, 2003).
Nonserile Supplies, Media and Solutions
Improper sterilization is a major source of biological contaminants. Packing too much into an autoclave or dry heat oven will cause uneven heating, resulting in pockets of nonsterile supplies. Using too short a sterilization cycle, especially for autoclaving volumes of liquids greater than 500 ml per vessel or solutions containing solids or viscous materials such as agar or starches are other mistakes resulting in incorrect sterilization. To accomplish sterility, the size, mass, nature and volume of the materials for sterilization have to always be considered. Storing sterilized supplies and solutions in a dust-and insect-free area is an obligation to prevent recontamination (Uphoff et al., 2002).
Laboratory Personnel
Laboratory personnel are considered a major source of Mycoplasma contamination. M.orale a species commonly found colonizing the human oral cavity and oropharynx, has been the leading contaminant in study after study. Two other human Mycoplasma species M.fermantans and M. salavarium, are also detected in contaminated cultures but at a much lower rate. Improper laboratory practices by laboratory personnel such as talking, sneezing or the use of mouth pipetting are other sources of human contamination.
In 1976, the role of laboratory technicians in Mycoplasma contamination in cell culture was proved. It was shown that the majority (80.6%) of technicians were carriers of mycoplasma, primarily M.salivarium the modes of spreading mycoplasma were evaluated by collecting aerosols generated via talking and sneezing from known Mycoplasma carriers on culture plates. M. salivarium can be transmitted during talking and sneezing of technicians in 6.2% and 37.5%, respectively. Street clothes and dirty lab coats are the major source of dust and aerosols. Negligence in wearing a clean lab coat and gloves is a major cause of spreading particles during routine cell culture processing. Furthermore, talking and sneezing also generate a significant amount of aerosols. It is highly recommended to avoid working without gloves since frequent hand washing can cause dry and flaky skin which is one of the main sources of particles (Steube et al., 2005)
Incubators
Incubators equipped with fans and air currents are another route for spreading Mycoplasma-containing particles during closing and opening of the internal door of the incubator. ”Good laboratory practices” are essential to avoid diffusion of Mycoplasma in the incubator and other laboratory devices such as the pipetman, pip aid and laminar flow. After droplet dispersion an incubator, bacteria are spread by aerosols (Koshimizu et al., 2000).
Liquid Nitrogen
Liquid nitrogen is another cause for spreading Mycoplasma. It is significant that Mycoplasma can survive in liquid nitrogen even without cryopreservation. While Mycoplasma do not proliferate in liquid nitrogen, they are able to contaminate cell cultures stored in liquid nitrogen. Therefore, storing cryovials in the vapor phase of nitrogen tanks is highly recommended (Gong et al., 2005).
Airborne Particles and Aerosols
Airborne particles and aerosols generated during culture manipulations are the greatest sources of Mycoplasma contamination. The diameter of microbeladen particles is generally 4 to 28µm and they settle at a rate of almost one foot per minutes in still air. As a result, the air in a settled, draft-free room or laboratory is nearly free of biological contaminants. However, as soon as people enter the room, particles that have settled down will be easily resuspended.
Some equipment and activities such as pipetting devices, vacuum pumps and aspirators, centrifuges, blenders, sonicators, and heat sources such as radiators, ovens refrigerators and freezers generate microbial particles and aerosols. Another source of particles and aerosols is experimental animals whose house and care facilities should be kept far from cell culture areas (Bolske, 2003).
Overuse of Antibiotics
It is a common particles in research laboratories to use antibiotics in cell cultures to avoid microbial contamination. The consequence of overuse of antibiotics is concealment of the poor aseptic technique and it is a major cause for Mycoplasma contaminated cultures. Overuse of antibiotics can also lead to antibiotics resistance. Veterans of cell culture insist on doing cell culture without antibiotics to avoid the problems (McGarrity et al., 2010).
Improper Sealing of Culture Dishes
Another way of entering microbial contamination in flasks, plates and dishes is improper seal of cultures dishes. The route for microbial contamination is provided when the top and bottom side walls of dishes or flask and their caps become wet and microbes transfer by capillary action of the wet surface (Hay R. J. et al., 2002)
Other Mycoplasma Contamination Cell Cultures
A Mycoplasma-infected cell culture is a major source of Mycoplasma contamination of other cell cultures in the lab. To avoid Mycoplasma contamination in cell cultures, it is recommended to test the new cell lines which are obtained from an outside source. A single Mycoplasma contaminated cell culture is enough to endanger other cell culture in the lab. The contamination can spread by means of aerosols and particles generated during the handling of the Mycoplasma infected cell culture. So, working with only one cell culture at a time and preparing separate media and reagents for each individual cell lines can avert Mycoplasma contamination. A good cell culture practice and regular testing of all new cell culture can decrease the risk of Mycoplasma contamination (Uphoff C. C. et al., 2001).
3.4. EFFECTS OF MYCOPLASMA CONTAMINATION
Mycoplasma infections can have a myriad of different effects on the contaminated cell cultures. However, this multitude of different effects does not affect the various cells in the same manner and to the same degree. Many Mycoplasma species produce severe cytopathic effects while others may cause very little overt cytopathology. There can be qualitative and quantitative differences in the same parameter, depending on the infecting Mycoplasma species, the culture conditions, the type of the infected cell culture, the intensity and duration of the infection, an additional infection with viruses, and other parameters. Thus, contaminations can interfere with virtually every parameter measured in cell cultures during routine cultivation or in experimental investigations (Coecke et al., 2005)
Consequently, the mycoplasmas in these cultures cannot simply be ignored or regarded as harmless bystander organisms. Besides the loss of an important culture, in the worst case all experiments might be influenced by the infections and artefacts are produced. Because of the virtually unlimited number of reported Mycoplasma effects on cultured cells, only some of the most important parameters have been listed here in order to highlight the diversity of possible effects (Berile et al., 2010).
10,000x (Gong et al., 2005).
One of the main reasons for the more or less severe cytopathic effects on cell cultures is the consumption of nutrients and basic components of the cellular metabolism, e.g. nucleic acid precursors, amino acids, vitamins, lipids, cholesterole etc. by the Mycoplasmas. Due to their low metabolic capabilities, their unefficient energy gain, and the high number of Mycoplasmas in the cell culture, those compounds can be used up rapidly. The nonoxidative degradation of the compounds also leads to an alteration of the pH value in the culture medium. The pH can be decreased by the formation of acids by Mycoplasmas using the fermentative metabolic pathways. On the other hand, arginine-hydrolyzing Mycoplasma (e.g. M. arginini, M. hominis) can increase the pH value due to the production of ammonia, which is also a highly toxic agent inhibiting cell growth. Additionally, activity of Mycoplasma arginine deiminase as well as Mycoplasma uptake and depletion of the growth medium were shown to inhibit cell proliferation and to induce apoptosis in cell lines. As visible effects, the cells show an abnormal growth rate, a decreased viability, adherent cells sometimes detach from the cell culture vessel surface, and granules are formed in the cells (Gong et al., 2005).
The depletion of arginine might also be a reason for chromosomal aberrations, because this basic amino acid is a major component of the histones in the nucleus. Another cause of chromosomal and genetic alterations and growth inhibition might be the competition of Mycoplasma and eukaryotic cells for nucleic acid precursors. Chromosome breakage, multiple translocation events, and numerical chromosome changes were described in various cell cultures infected with different Mycoplasma species. Eukaryotic DNAs and RNAs are degraded by exo- and endonucleases, which are produced and exported by Mycoplasmas. Sokolova et al., (1998), showed for different lymphocyte and epithelial tumor cell lines that inhibition of proliferation and increased cell death, accompanied by DNA fragmentation and the morphological features of apoptosis was caused by mycoplasma infections. Similar DNA fragmentation and loss of chromosomal DNA was also observed by Rawadi et al., (1996) in M. fermentans-infected monocytic cell lines. The cytocidal effect was assigned to a nonlipid-associated protein fraction.
One of the nucleotide-transforming enzymes is the uridine phosphorylase which inactivates the artificial bromodeoxyuridine (BrdU). BrdU is toxic for eukaryotic cells and added as thymidine analogue for the selection of cells with a thymidine kinase (TK) defect. Cells with normal TK activity phosphorylate and incorporate BrdU and will die. Cells with a TK defect which are used for cell fusion experiments grow in the presence of BrdU. In the presence of mycoplasmas, BrdU is degraded and the eukaryotic cells survive even though they do not possess a TK defect (Sokolova et al., 1998).
Mycoplasma proteins alter a number of eukaryotic properties in different manners. Rawadi et al., (1996) showed that heat-inactivated Mycoplasma particles induced the inflammatory cytokines interleukin 1 (IL-1), IL-6, and tumor necrosis factor in monocytes and THP-1 cells (14). M. fermentans also induced IL-10 in human monocytes. The secretion of immunoglobulins was altered in B-cells, as well as the expression of various colony-stimulating activities (e.g. granulocyte-monocyte colony stimulating factor) and the induction of interferon expression.
Another example for the detrimental effects of Mycoplasma contaminations is the impact on virus propagation in cell cultures. The virus production can be decreased by suppression of metabolism and growth of the cells connected with partially severe cytopathic effects, and arginine depletion by arginine oxidizing Mycoplasmas. Decreased yields can be found with arginine requiring viruses, such as Herpes simplex, vaccinia, adeno-viruses and several others. Increased virus yields can be obtained due to interferon-α inhibition, leading to diminished cell resistance. On the other hand, interferon activity can also be induced or stimulated by Mycoplasma infection. For example Acholeplasma species lipoglycans have endotoxin-like activities that induce interferon activity leading to resistance against some viruses in vitro or in vivo (Rawadi et al., 1996).
The few examples out of the nearly endless array of possible effects of Mycoplasma infections on cell cultures can only give a percursory idea of the very complex relationship between Mycoplasma and eukaryotic cells. Thus, any experimental result from mycoplasma infected cell cultures may rise prima vista substantial doubts (Sokolova et al., 1998).
3.5 DETECTION OF MYCOPLASMA CONTAMINATION
Mycoplasma infections of cell cultures can be highly diverse and no universal effect can be observed which may serve as an indicator for a contamination. Thus, special techniques were developed to detect Mycoplasma in cell cultures. During the pre- PCR era many methods were developed based on microbiological culture, e.g. staining techniques, electron microscopy, biochemical and immunological tests, and recently some hybridization assays (Drexler et al., 2002).
Many of the assays are relatively elaborate and time consuming, applicable only to a portion of the contaminating mycoplasmas, exhibit a low sensitivity, or the interpretation is subjective and fault-prone, or special equipment is necessary.
One of the first and still one of the officially approved (European Pharmacopeia) assays is the microbiological culture method. In this test, an aliquot of the cell culture supernatant is added to rich liquid mycoplasma medium, cultivated for a few days and subsequently transferred to agar plates with the same medium components. The plates are incubated for up to two weeks aerobically at 37°C. In case of positive samples, typical small colonies (ca.100 – 400μm in diameter) often with a “fried eggs” appearance comprising a dense center and a brighter corona appear on the agar plates. The test is sensitive, reliable, and robust for monitoring cell culture contaminations. Nevertheless, some strains of M. hyorhinis grow poorly or not at all on those media. We found that a certain number of M. hyorhinis strains grow indeed on the media, but in a number of cases the growth is not supported (Drexler et al., 2002).
The dense growth and even confluence of colonies indicative of a high Mycoplasma titer. The colonies show the tell-tale “fried-egg” appearance. Original magnification 100x, (Drexler et al., 2002).
A second detection method recommended by the European Pharmacopeia is the DNA fluorochrome staining (4´,6-diamidino-2´-phenylindole-dihydrochloride [DAPI] and Hoechst 33258 stain). This assay is relatively easy and rapid to perform. But the results are sometimes difficult to interpret and some experience is definitely necessary. Especially when the cell culture is not in a good condition, mis-interpretations are frequent. The sensitivity and specificity of the direct DNA staining procedure can be highly increased by use of indicator cell lines. In this indirect DNA staining method, supernatant from the cell culture to be tested is added to a Mycoplasma-free adherent cell culture (e.g. Vero B4, NIH- 3T3 or 3T6 cell lines). The cells are grown in vessels containing sterile cover slips. After growth for several days to approximately half-confluency, the cover slips are washed and stained with the fluorochrome. Mycoplasma infections can be detected very efficiently, but again, the test is relatively-time consuming and Mycoplasmas are cultured in the laboratory, which may lead to further spread of contaminations (Drexler et al., 2002).
Nowadays, a number of assays are available, which can detect almost all Mycoplasma contaminations within at most two days, including one or more incubation steps over several hours. These techniques are all indirect tests, which determine or visualize Mycoplasma components or enzyme activities.
One of the most prevalent assays for the detection of Mycoplasma contaminations is the polymerase chain reaction (PCR) technique. The test is easy to perform, sensitive, specific, fast, reliable, and cost effective. Most of the 16S rRNA sequences of Mycoplasma are known and can be used to create primers for the amplification of specific DNA fragments. The primer design defines the specificity of the PCR reaction.
Oligonucleotides from variable 16S rRNA regions are usually specific for a limited number of Mycoplasma species. Sequences from the 16S-23S intergenic regions can be used for the detection of single Mycoplasma species. For the detection of Mycoplasma in cell cultures, the specificity of the primers needs to be broad enough to detect Mycoplasma as well as Acholeplasma species. On the other hand, the specificity should be narrow enough to exclude amplification of sequences from other common bacteria, which might be contaminations of the PCR reagents (Uphoff et al., 2003).
However, some more important general aspects should be considered when performing this technique.
The sensitivity of the procedure makes it susceptible to contaminations with the target DNA which is present in high amounts after the first amplification of Mycoplasma-specific DNA. Therefore, extreme care has to be taken to prevent carry-over of target DNA fragments. This is especially the case when a nested PCR is performed.
The PCR should be performed with extracted DNA and not with a crude lysate of the cell culture supernatant, because the cell culture components might contain inhibitors of the Taq polymerase.
The use of antibiotics in cell culture should be minimized and the cell cultures should be cultured without antibiotics for several passages or at least two weeks to allow the Mycoplasmas to grow to detectable amounts or to ensure that no residual Mycoplasma DNA is left in the culture medium.
It is of note that a positive result of the PCR does not necessarily indicate viable contaminants, especially after a mycoplasma elimination procedure using antibiotics against mollicutes.
Thus, the PCR method should be properly established and all assays should be performed with the utmost care (Uphoff et al., 2003).
The PCR can be performed with a single round of amplification or as nested PCR with two primer pairs. The second method increases the sensitivity and the specificity. But one of the drawbacks of the nested PCR is the possible generation of false positive results due to contamination with target DNA. For the routine cell culture technology, the PCR is satisfactory to detect Mycoplasma contaminations, because the titer of the Mycoplasmas in the cell cultures is sufficiently high to be detected by the PCR. Special conditions, e.g. after Mycoplasma elimination procedures or for the detection of Mycoplasma in cell culture products like FBS, the nested PCR might be of advantage. Another possibility to increase the sensitivity of the assay is to perform a reverse transcription PCR (RT-PCR) to detect ribosomal RNA which is more abundant in the cells than the rRNA-coding DNA. However, the latter option is clearly more labor-intensive. In summary, we would suggest to perform a single PCR with genomic DNA for routine cell culture and to test the cultures frequently for contaminations. Several PCR kits are commercially available, e.g. from ATCC, Minerva Biolabs, Roche, Stratagene, TaKaRa Bio, and detailed descriptions and positive and internal control DNAs for the establishment of a PCR can be obtained from the DSMZ (Uphoff et al., 2003)
Shown is an ethidium bromide-stained gel containing the reaction products following PCR amplification. Two paired PCR reactions were performed: one reaction containing an aliquot of the sample only and the second contained the sample under study plus a control DNA as internal standard. Cell line A is specifically positive for Mycoplasma and also for the internal control whereas cell line B is specifically negative for Mycoplasma being positive in the internal control (Uphoff et al., 2003).
There are also newly developped assays based on fluorescence in situ hybridization (FISH) and on ATP generation (Cambrex, UK) detected by fluorescence microscopy and luminometer, respectively. Until now, no published data are available concerning the sensitivity, specificity, and the accuracy of both assays applied in routine cell culture. But preliminary results are promising concerning the above mentioned parameters and in particular with regard to the speed of the assays. The FISH test takes about two to three hours and results from the luminescence test can be generated within 20 minutes.
All described methods may fail when cell cultures are tested which were treated with antibiotics. In general, all treated cell lines should be cultured for at least two weeks without any antibiotics before the cells are retested. Both, false negative as well as false positive results may occur. PCR and other assays depending on the determination of DNA or RNA can produce false positive results, because residual DNA or RNA is detected, in the absence of viable Mycoplasmas. False negative results are produced when the titers of the Mycoplasmas are below the detection levels of the assays. We recommend to perform two or even three independent assays for the detection of Mycoplasma in cell lines which newly arrive in the laboratory.The cells should be kept isolated in a quarantine laboratory until all tests show that the cells are free from Mycoplasma, if possible at all. During continuous culture one sensitive assay should be performed regularly to monitor the cell cultures (Harline et al., 2008).
3.6 ERADICATION OF MYCOPLASMA CONTAMINATION
Mycoplasmas cannot be regarded as harmless bystander organisms in cell cultures. Thus, the best way to get rid of the infections is to autoclave the culture and to replace it with a new and uncontaminated culture. Unfortunately, the contaminated cell culture may often be unique in some regards and may not be replaceable. In these cases, the Mycoplasmas have to be eliminated without affecting the eukaryotic cells. Over the years, a number of elimination methods had been developed, applying physical, chemical, immunological and chemotherapeutic treatments. The treatments are not restricted to cell cultures only, but also for surfaces, cell culture media and supplements. Methods include heat treatment, filtration, exposure to detergents, culture in the presence of 6 methylpurine deoxyriboside, passage through nude mice, antibiotic treatment, and others.Regarding the treatment of cell cultures, many of the methods are laborious or not efficient. Additionally, some of the elimination methods had been investigated only in experimentally infected cell cultures. This might not necessarily reflect the complex nature of a chronically infected culture and the occurrence of intracellular Mycoplasma also has to be considered. From our experience, treatment with several specific anti-Mycoplasma, antibiotics is the method of choice for infected cell cultures. Usually, the antibiotics are also active or even might be accumulated in the eukaryotic cells (Rawadi et al. 1995).
As Mycoplasmas are very unusual bacteria in many respects, this is manifested also in the susceptibility against chemotherapeutic agents. Many of the commonly applied antibiotics are not effective against Mycoplasma, due to the lack of the antibiotic target (e.g. penicillins, streptomycin, etc.). On the other hand, although not killing the Mycoplasmas, some antibiotics might suppress their growth and thus mask the presence of the infectants. Beside the enforcement of strictly sterile cell culture technique and the development of resistances, this is one reason not to apply antibiotics prophylactically in routine cell culture (Rawadi et al. 1995).
Until now, three groups of agents were shown to be highly active against Mycoplasmas: Macrolides, Tetracyclines, and Quinolones. Macrolides and tetracyclines both inhibit protein synthesis, but bind to different subunits of the ribosomes. The quinolones (also named fluoroquinolones) inhibit the bacterial gyrase, an enzyme which is essential for the DNA replication. The quinolones tested in cell cultures are: ciprofloxacin (brand name Ciprobay 100, Bayer, Germany), enrofloxacin (Baytril, Bayer), sparfloxacin (Aventis Pharma, Ireland), and an unpublished quinolone reagent available as Mycoplasma Removal Agent (MRA, ICN, Eschwege, Germany). The macrolide Tiamulin and the tetracycline Minocycline are available as BM-Cyclin from Roche (Mannheim, Germany) and are applied subsequently in one treatment (Rawadi et al. 1995).
Brand nameGeneric nameAntibiotic categoryBM-CyclinTiamulin
MinocyclineMacrolide
TetracyclineCiprobayCiprofloxacinFluoroquinoloneBaytrilEnrofloxacinFluoroquinoloneZagamSparfloxacinFluoroquinoloneMRAFluoroquinolonePlasmocinTetracycline
FluoroquinoloneSource: European Pharmacopeia (2002).
In our hands the curation efficiency of the antibiotic approaches varied between 66 and 85%, depending on the antibiotic used. But these numbers do not only reflect the killing of the Mycoplasmas, but also include the loss of the culture, due to growth inhibition of the eukaryotic cells. The loss of cultures is frequently seen when the cells are heavily infected and already in a very bad condition (3-11% of treated cultures, depending on the antibiotic) (Harline et al., 1993).
In these cases the antibiotics might be the last hit to kill the eukaryotic cells. On the other hand, resistances against one antibiotic (7-24% of treated cultures, depending on the antibiotic) can be overcome by application of antibiotics from another group. Another combination product developed for the eradication of Mycoplasma from cell cultures is Plasmocin (InvivoGen, San Diego, USA). It contains an unpublished antibiotic against protein synthesis (presumably one of the above mentioned) and a quinolone, which are used simultaneously. No published data are available for this treatment until now. Pretreatment of heavily infected cultures with other methods, e.g. exposure to hyperimmune antimycoplasma serum, co-culture with macrophages, or washing the cells with surfactincontaining solutions, might be helpful, because the bulk of the Mycoplasmas can be eliminated.
The more recently developed membrane-active peptides, e.g. alamethicin, dermaseptin B2, gramicidin S, and surfactin, are highly efficient in pure Mycoplasma cultures, but in the presence of serum, the activities are decreased. Thus, the concentrations and treatment times required for the elimination of Mycoplasmas from cell cultures are toxic to the eukaryotic cells (Harline et al., 1993).
3.7 PREVENTION OF MYCOPLASMA CONTAMINATION
The prevention of Mycoplasma contamination can be divided into three categories: cell culture facility, cell culture procedures, and operator technique. While the measures proposed will not automatically prevent any mycoplasma infection, they will significantly decrease the probability of its occurrence. Such efforts are also of great importance for the prevention of cross-contamination with other eukaryocytic cells which in the majority of cases appears to be the result of inadequate cell culturing as well (Uphoff et al., 2001).
Cell Culture Facility
Any sterile cell culture work should be performed in a vertical laminar-flow biohazard hood. It is important to disinfect all work surfaces before and after culture manipulations, including the various devices entering the laminar flow hood. Mycoplasmas are very sensitive to most disinfectants, but have shown extended survival in a dried state (Ben-Menachem et al., 2001).
Cell Culture Procedures
Cell culture laboratories should establish effective and regular Mycoplasma testing procedures in the form of a routine screening program for all forms of microbial contamination, including Mycoplasmas. For Mycoplasma screening, we recommend PCR analysis. Sera, media and supplements (and also cell lines whenever possible) should be purchased from reputable suppliers that adequately test for Mycoplasma contamination. All incoming cell lines should be quarantined until the contamination status is verified. Mycoplasma-free cultures should be segregated from infected cultures by time and place of handling. Reagents for the two sets of cultures should be separate. The general use of antibiotics is not recommended except in special applications and then only for short durations. Use of antibiotics may lead to lapses in aseptic technique, to selection of drug-resistant organisms, and to delayed detection of low-level infection by either Mycoplasmas or other bacteria. Master stocks of Mycoplasma-free cell lines should be frozen and stored to provide a continuous supply of cells should working stocks become contaminated. Actively growing Mycoplasma-infected cell lines should be discarded or treated with mycoplasmacidal measures as quickly as possible in order to prevent lateral spread (Koshimizu et al., 2000).
Operator Technique
Strict adherence of the cell culturist to general aseptic culture techniques is a fundamental aspect in mycoplasma control. Cell culturists should continually be aware of the possibility of contaminating clean cultures with aerosols from Mycoplasma-containing cultures which are handled in the same area. For example, the following procedures with liquid media generate droplets: pipetting, decanting, centrifuging, sonicating. These relatively large droplets settle into the immediate environment where they may remain viable for some time (Armstrong et al., 2010).
CHAPTER FOUR
4.1 CONCLUSION
Since it has been observed that the commonly used cell culture reagents and enzymes such as bovine, serum and porcine trpsin respectively are carriers of Mycoplasma species, hence culture reagent should be properly screened and test for contamination before they are employed in cell culture. However, experimental animals should be kept far form cell culture areas, since they are sources of Mycoplasma contamination.
Cell culture remains an important tool in biological and medical research because of their application in genetic engineering, biotechnology and medicine (especially in the treatment of infectious diseases). Hence, standard control measure should be taken to avoid the risk of Mycoplasma contamination.
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