Factors influencing microbiological assay of cell-culture mycoplasmas

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.     

Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines

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.     

Spread and control of mycoplasmal infection of cell cultures.

Environmental sampling was performed during trypsinization and passage of 3T-6 cell cultures that contained a mean of 4.3 X 10(7) colony forming units (CFU) per ml supernatant of A. laidlawii. The lip of the culture flask and the outside of the used pipet were always heavily contaminated. The outside of the culture flask (3/7), the work surface (8/12) and the outside of a pan of disinfectant (4/5) were regularly contaminated with mycoplasmas. Airborne mycoplasmas were detected eight of 32 times (25%) by settling plates; simultaneous forced-air samplers by two different methods were always negative. The technician's hands were contaminated two of 15 samples. When hands were contaminated, more contamination was detected in the environment. Droplets of A. laidlawii and M. orale inoculated onto work surfaces survived drying for a minimum of 3 days, even in laminar airflow cabinets. Twenty-five of 31 (80.6%) cell culture technicians carried M. salivarium in their throats; only two carried M. orale. It is concluded that mycoplasma-infected clltures are the most common source of further infection. Recommendations for prevention and control of mycoplasmal infection are listed.     

Comparison of Ciliostasis by Mycoplasmas in Mouse and Chicken Tracheal Organ Cultures

The cilia-stopping effect of mycoplasmas of human and various animal origin in mouse and chicken tracheal organ cultures was studied. From the results in mouse tracheal organ cultures, the mycoplasma strains tested were divided into three groups: Mycoplasma pulmonis m53, M. pulmonis JB, M. pulmonis OK, M. mycoides subsp. mycoides PG1 and M. gallisepticum S6 showed a strong cilia-stopping effect; M. pulmonis PG22, M. mycoides subsp. capri PG3, M. meleagridis 19729, M. neurolyticum Type A and M. arthritidis PG6 showed a mild effect; and M. pneumoniae FH, M. salivarium Hup, M. hominis type 1-C and M. orale N-C of human origin and Acholeplasma laidlawii PG8 showed a weak effect. On the other hand, in chicken tracheal organ cultures, only M. gallisepticum S6 showed a strong effect, M. meleagridis 19729 was affected to a lesser degree, and other mycoplasma strains showed a weak or no effect. The results indicate that some murine and poultry mycoplasmas showed a cilia-stopping tendency in mouse and chicken tracheal organ cultures, respectively, while human mycoplasmas showed weak or little effect in both organ cultures. In mouse tracheal organ cultures, M. pulmonis m53 treated with heat, trypsin or formaldehyde, and the sterile filtrate of an m53 broth culture showed no cilia-stopping effect. The relationship of the pathogenicity of mycoplasmas for their natural hosts to that for cultured respiratory cells is discussed.     

Elimination of Mycoplasma from various cell cultures

Elimination ofMycoplasma orale-I from chronically infected cell lines was achieved either by treatment with a mixture of antibiotics in a hypotonic solution, or with 10 vol % of anti -M.orale rabbit serum in tissue culture medium. The latter treatment was preferable in most cases, as it was practically harmless to the cells. Inactivation of this antiserum had no effect on its potency. The antibiotic-hypotonic treatment was rather destructive, but to a different degree for the various cell cultures. Both methods were equally useful for the treatment of a monkey kidney cell line contaminated with a mycoplasma strain related toM.hyorhinis. The available anti -M.hyorhinis rabbit serum was toxic for the monkey cells when not inactivated. The potency of the antiserum was rather low and even lower after inactivation. However, prolonged treatment successfully eliminated the mycoplasma. Pre-incubation of the inactivated anti -M.hyorhinis serum with tissue culture medium to which 10% non-inactivated calf serum had been added, favoured the elimination of the mycoplasma.During the treatment of contaminated cell cultures with single antibiotics a strain related toM.hyorhinis became resistant to chlortetracyclin.M.orale- I was found to be resistant to various single antibiotics.We are grateful to Professor Dr. A. Ch. Ruys (University of Amsterdam) and Dr. R. H. Leach (Mycoplasma Reference Laboratory, London) for helpful discussions and for identifying some of our mycoplasma strains; Dr. Leach also for kindly supplying us with his G. D. L.-strain. We thank Dr. H. Cohen and Dr. A. C. Hekker for their criticism and Mr. N. L. M. van Zwetselaar for his accurate technical assistance.     

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.     

PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

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.     

Detection of mycoplasma contaminations by the polymerase chain reaction

The polymerase chain reaction (PCR) has been used for the general detection ofMollicutes. 25Mycoplasma andAcholeplasma species were detected including important contaminants of cell cultures such asM. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1–2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.     

Mycoplasmal infection of lymphocyte cell cultures: Infection withM. salivarium

Summary Many conclusions concerning cell culture mycoplasmas are based on data from studies in fibroblast cultures. Some conclusions may not be valid in other types of differentiated cell cultures.M. salivarium was isolated from 35 human lymphocyte cultures (HLC), 34 from the same laboratory. The organism grew to more than 10⁸ colony forming units (CFU) per ml of lymphocyte suspensions and was readily detectable by microbiological culture, uridine phosphorylase, and uridine/uracil assays. Direct mycoplasmal assays on HLC by DNA fluorochrome staining and scanning electron microscopy (SEM) yielded artifacts that interfered with diagnosis. For DNA and SEM of HLC, inoculation into indicator cell cultures is recommended.M. salivarium infection of HLC did not produce any immediate difference in growth rates; however, infected cultures eventually died 14 to 29 passages after infection in contrast to uninfected controls. The same organism in 3T6 fibroblasts effected a 60% decrease in growth rate. AlthoughM. salivarium is a frequent isolate from the oral cavity, it is a rare cell culture isolate.M. salivarium was able to initiate growth over a wide pH range, grew as well in cell cultures as in cell-free media, and was resistant to 50 μg per ml of gentamycin, tylocine, kanamycin, and erythromycin. By C₀t_1/2 analysis,M. salivarium had a genomic molecular weight of 4.2×10⁸ daltons.M. salivarium did not increase chromosome aberrations in one HLC. Some of these results have application to infection of HLC by other mycoplasmal species. These studies were supported by contracts NO1-AG-82117 from the National Institute on Aging, NO1-GM-9-2101 from the National Institute of General Medical Sciences, and Grant RO1-A1-15748 from the National Institute of Allergy and Infectious Diseases.     

Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.     

Acholeplasma laidlawii PG8 ultramicroforms amplificate selectivelyrrnB nucleotide sequences

Mycoplasmas are frequent contaminants ofin vitro animal cell cultures. Despite a broad spectrum of modern methods, detection of mycoplasmas remains a serious problem. The situation is complicated by the fact that mycoplasmas may be presented in cell cultures or biological samples by viable but unculturable forms (ultramicroforms). We found that the DNA ofAcholeplasma laidlawii PG8 ultramicroforms showed selective amplification of therrnB nucleotide sequences while vegetative cells of the mycoplasma showed amplification both forrrnA andrrnB sequences. The role of enzyme deproteinization in PCR results was also shown. The results presented in this report indicate that the optimisation of primer sequences as well as PCR regime may be crucial steps in detection and differentiation of vegetative forms and ultramicroforms ofA. laidlawii.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institues of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749. from the National Institutes of Health.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata, andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749 from the National Institutes of Health.     

Differential Responses of Single Cells and Aggregates of Mycoplasmas to Ultraviolet Irradiation

Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.     

Detection of anaerobic mycoplasmas in cell cultures

Summary A commercially available anaerobic generator and incubation system that develops a low oxidation-reduction potential was used for the assay of cell cultures for mycoplasmal contamination. Mycoplasma broth and agar media supplemented with dextrose, yeast extract, and horse serum were used. This system supported growth of some mycoplasmas that failed to grow in incubators with 5% CO₂ in nitrogen previously used in culture of mycoplasmas in this laboratory. This work was supported by a grant from the John A. Hartford Foundation General Research Support Grant No. 5 SO1 RR05582-4 from the National Institutes of Health, and by a Grant-in-Aid Contract from the State of New Jersey.     

Cultivation of Mycoplasmas on Glass

Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.     

Detection of mycoplasmas infecting cell cultures by DNA hybridization

Summary Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10⁵ mycoplasmas. The study was supported by the U.S. Public Health Service Grant GM25286 awarded to G. G. and by a grant from the United States-Israel Binational Agricultural Research Development Fund (BARD) awarded to S. R.     

Endogenous HPRT activity in mycoplasmas isolated from cell cultures

Summary Five mycoplasma species most frequently isolated from cell cultures were tested for the presence of endogenous hypoxanthine phosphoribosyl-transferase (HPRT), activity. All of the five, cultured in cell-free medium, contained variable but significant levels of HPRT. Two strains ofM. hyorhinis exhibited a 13-fold difference in their specific HPRT activity. When infected with any of these mycoplasma species, HPRT-deficient mouse cell mutants rapidly acquired a cell-associated HPRT activity; however, the cells remained sensitive to HAT medium and resistant to 6-thioguanine. On the other hand, normal HPRT-positive cells deliberately infected with the mycoplasmas uniformly became sensitive to HAT medium. The apparent transfer of mycoplasma-specific HPRT activity to HPRT-deficient cells may be used as a sensitive measure of cell infection by these mycoplasma strains. The HPRT activities of mycoplasmas share several common properties so that they can be distinguished easily from the mammalian HPRT isozymes. Compared to the animal cell enzymes, the mycoplasmal HPRT activities are less heat stable, more strongly inhibited by 6-thioguanine, and in general migrate more slowly in electrophoresis at a neutral pH. This work was supported in part by PHS Research Grants 5 R01 GM21014 and 1 P03 GM19100 (Genetics Center Grant to Albert Einstein College of Medicine), and PHS Research Contracts N01 GM 6-2119 and N01-AG-4-2865 (to the Institute for Medical Research), from the National Institute of General Medical Sciences and National Institute on Aging. S. S. is a recipient of a Faculty Research Award from the American Cancer Society.     

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

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.     

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.