Fluorometric Quantitation of Broth-Cultured Mycoplasmas by Using Alkaline Ethidium Bromide

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1993, p /93/ $02.00/0 Copyright 1993, American Society for Microbiology Vol. 31, No. 5 Fluorometric Quantitation of Broth-Cultured Mycoplasmas by Using Alkaline Ethidium Bromide WARREN I. SCHAEFFER* AND ROBERT MELAMEDE Department of Microbiology and Molecular Genetics, University of Vermont, Given Building, Burlington, Vermont Received 10 December 1992/Accepted 29 January 1993 We developed a fluorometric system which does for broth-grown mycoplasmas what turbidimetric analysis does for broth-grown bacteria. It allows one to monitor the growth of broth-grown mycoplasmas at any interval desired. The entire procedure is quick, taking not more than 20 min. The fluorometric readings correlate with colonial growth on agar, making it possible, for the first time, to take readings which closely estimate the CFU present in the culture at a given moment in time. We show that this system can be used to assess the effectiveness of an antimycoplasmal antibiotic and to optimize medium components and that fluorometer readings taken during the logarithmic phase of growth correlate with the DNA content of the viable cells. Use of this methodology will permit investigators to know absolutely the phase of the growth cycle of the culture concomitant with the growth of the culture itself, and since fluorometer readings of culture aliquots can be converted to DNA equivalents, the standardization of mycoplasmal cultures within and between laboratories will be a possibility. Members of the genera Mycoplasma and Acholeplasma (hereinafter referred to as mycoplasmas) have a unique place in biology. They are the smallest (diameter, 0.2 to 2,um [5]) and the simplest free-living parasitic organisms known, and theypossess the smallest recorded genome in living cells (0.5 x 10 to 1.0 x 109 Da [13, 14]). They infect both animals and plants, causing or being implicated in the cause of a variety of diseases (1-4, 8-10, 12, 13, 16, 20). Therefore, because of their biological uniqueness and their emerging roles in disease as either an etiological agent, participant, or opportunist, it is important to study the biology of mycoplasmas. Many species of mycoplasmas can be grown axenically in broth medium containing undefined, exotic additives. However, the quantitation of broth-grown mycoplasmas has been a problem. Unlike broth cultures of larger bacteria, in which turbidity parallels growth and which therefore can be quantified turbidimetrically, cultures of many mycoplasmas have reached peak growth before turbidity is apparent. When ph changes occur in mycoplasmal cultures, a color change in the medium is often used to indicate peak growth. However, not all mycoplasmas cause a medium color change, and if they do, it does not always coincide with peak growth. Colonial growth on agar is one absolute method of quantitation, but the data are always obtained retrospectively because the original culture is gone by the time that colonies are of a size sufficient for quantitation. These difficulties have hampered the standardization of cultures within and between laboratories, the development of new and defined media for cultivation, the evaluation of antimycoplasmal agents, and the general understanding of mycoplasmal growth characteristics. The necessity of using complex and undefined media to grow mycoplasmas precludes any rapid development of mutants so important for the genetic analyses used in understanding mycoplasmal pathogenesis. We devised a system for the quantitation of mycoplasmal growth which is analogous to the turbidimetric system used with larger bacteria. That is, it is easy to use and can be * Corresponding author. performed quickly, produces data concomitant with growth, and correlates with agar growth, and the data can easily be converted to DNA equivalents. Since mycoplasmas are too small, even in large numbers, to refract sufficient light for quantitation spectrophotometrically, we developed a fluorometric assay system in which the mycoplasmal DNA is stained with the DNA-binding dye ethidium bromide (EtBr) and the results are determined with a fluorometer. What follows are descriptions of the fluorometric system and several experiments demonstrating the usefulness of the system. MATERIALS AND METHODS Fluorometric system. The fluorometric system is a modification of one published by Kowalski (7), which was originally devised as a differential fluorescence system for detecting nicked versus intact circular DNA. From it, we developed the fluorometric assay system for broth-grown mycoplasmas. A 0.5-ml sample was removed from a broth culture and was placed into a microcentrifuge tube along with 0.5 ml of Dulbecco's phosphate-buffered saline (PBS; GIBCO, Grand Island, N.Y.), which can, if desired, contain EDTA. The sample was centrifuged in a Sorvall refrigerated centrifuge with an SS-34 rotor at 15,000 rpm for 10 min at 4 C. The supernatant was removed, and the pellet was resuspended in 0.5 ml of PBS and was transferred to a small test tube. The microcentrifuge tube was then washed with another 0.5 ml of PBS, which was added to the resuspended pellet. A total of 2.0 ml of the EtBr solution (1.0,ug of EtBr per ml, 0.5 mm EDTA, 20 mm KH2PO4; final ph, 11.8 to 12.0, adjusted with 5 M KOH) was then added to the test tube. The fluorescence of the resulting solution was read in a model Ratio-2 System Filter Fluorometer (Optical Technology Devices, Inc.) against a medium blank which was processed as though it were inoculated. A 500-ng sample of DNA (which reads in the linear portion of a DNA standard curve) was used as an internal standard for the fluorometer. This permits conversion of fluorometer readings to DNA equivalents. Once the alkaline EtBr solution is added to the 1303

2 1304 SCHAEFFER AND MELAMEDE culture aliquot, the sample can be stored at 4 C, if required, but it should be protected from light. A standard curve of DNA was prepared by using purified salmon testis DNA (Sigma Chemical Co.) at concentrations ranging from 0 to 100,ug/ml. Mycoplasmal growth. Mycoplasma fermentans PG-18 or Mycoplasma hyorhinis (isolated by us from an infected cell culture) was grown in a modification of SP-4 medium originally described by Tully et al. (18) at 37 C and in 5% CO2. The yeast extract used was freshly prepared by the methodology of Kotani et al. (6). The medium was assembled by mixing the freshly prepared basal portion together with all of the additives, and the complete medium was then filter sterilized through a 0.22-p,m-pore-size filter. No part of the medium was autoclaved. Glucose in particular was always filter sterilized because of the potential for the generation of toxic products when glucose is autoclaved (17) in the presence of phosphates. Because fluorometric analysis was used, no additional phenol red was added to the medium beyond that which was present in the CMRL 1066 medium additive called for in the SP-4 recipe. For agar growth, a 10% Noble agar solution was prepared in distilled water, autoclaved, and dispensed into suitable aliquots so that when it was remelted it could be added to sufficient complete medium to yield 1% agar. Then 5 ml of the agar-medium mixture was poured into 60-mm petri plates. One-tenth of a milliliter of culture dilutions was spread over the surface of the agar before incubation at 37 C and 5% CO2. Titration of yeast extract. The fluorometric system was used to optimize the SP-4 yeast extract concentration for M. fermentans. The recipe for SP-4 calls for 2.5% yeast extract. We reduced the concentration by 75, 50, and 25% to 0.63, 1.3, and 1.9%, respectively. Determination of antimycoplasmal activity. The fluorometric system was used to examine the antimycoplasmal activity of chloramphenicol succinate, a water-soluble analog of chloramphenicol. The agent was serially twofold diluted from a final concentration of 100,ug/ml in the culture. The growth of M. hyorhinis was then monitored daily as described above. Study of nicked versus intact DNA. We and others have seen that, following inoculation of SP-4 broth with mycoplasmas, there is a very rapid period of growth within 24 to 48 h and then an equally rapid decline in viability. We wondered whether this might be due, in part, to the accumulation of DNA damage. The fluorometric system, as described by Kowalski (7) with purified DNA, could estimate the degree of nicking by comparing fluorometric readings taken before and after boiling the DNA samples in the alkaline EtBr solution. Therefore, following the initial reading in the fluorometer, the tube containing the alkaline EtBr-cells was boiled for 5 min, after which it was quickly cooled in an ice bath. It was then reread and the difference was recorded. To be certain that we were examining nicked DNA, we induced nicks in the mycoplasmal DNA by irradiating the mycoplasmal cells prior to fluorometric analysis. A total of 4.0 ml of a 24-h culture of M. fermentans was placed in a petri dish which was exposed to X-rays that were generated by using a Phillips X-ray generator with a beryllium window Machlett tube operated at 20 ma, yielding a dose rate of 80 kilorads/min. Cells were irradiated for various times from 20 to 160 s, after which aliquots were removed and treated as described above for fluorometric analysis pre- and postboiling and agar growth. 2,000 1,750 S* 1,500 0 ' 1,250 CU 1,000i E M 500 / DNA (jg/ml) FIG. 1. DNA standard curve with the alkaline EtBr system. RESULTS J. CLIN. MIICROBIOL. Since the method described here is based on the ability of EtBr to bind to DNA and since the ph of the solution excludes any contribution from the RNA, we decided to investigate the relationship of the DNA concentration to the fluorometer reading both for purified DNA (via a standard curve) and for mycoplasmal culture samples. Figure 1 illustrates the data received from one of several standard curves and indicates that there is linearity within the range of 0 to 20 jig/ml. There was a change in slope between 20 and 50,ug/ml, after which the curve became saturated. On the basis of the standard curve, a 500-ng/ml sample of DNA was always used during assays to standardize the fluorometer. In our hands, such a sample normally read between 20 and 30 fluorometer units, which was consistent with the reading for the standard curve. We then examined whether, during the earlier portion of the growth curve and before there is a significant ph change or medium precipitation, a given fluorometer reading from a culture sample is due primarily to the DNA present in the cells. The genome size of M. fermentans is reported to be 4.8 x 108 Da (15), and since 1 Da is the equivalent of 1.67 x g, the fenome of a single M. fernentans organism is x 10- jig. We sampled a 24-h culture and, in the fluorometer, received a reading of fluorometer units. The number of CFU present in this sample was 9.6 x 109/ml. Since one cell contains x 10-10,ug, 9.6 x 109 CFU would contain 7.8,ug of DNA. From the standard curve, 7.8,g of DNA should yield a fluorometer reading of 598, and as noted above, we actually received a reading of , which is certainly within the experimental error. A similar correlation existed for M. hyorhinis when it was examined similarly. Figure 2 demonstrates the consistency and reproducibility of the fluorometric system in determining growth kinetics. Three separate cultures of M. hyorhinis grown over a period of 16 days demonstrated essentially the same growth kinetics, peaking between days 2 and 4; this was followed by what appeared to be a protracted stationary phase. In order to determine whether the fluorometric readings would correlate with viability, we plated duplicate samples on agar. Figure 3 illustrates these data and shows that there is a high degree of correlation between fluorometer readings taken during the logarithmic phase of growth and the number of viable organisms present. The data indicate that, in the experiment whose results are shown in Fig. 3, there is a

3 VOL. 31, 1993 FLUOROMETRIC QUANTITATION OF BROTH-GROWN MYCOPLASMAS 1305 cn, LUJ En -j LL FIG. 2. DAYS OF GROWTH Reproducibility of the fluorometric method over time. Three successive passages of M. hyorhinis yielded the same results. rapid decline in the viability of the culture following peak growth and also that viability does not extend beyond the fifth day. These data further suggest that what appears to be a stationary phase is, in fact, an artifact. Since the original method described by Kowalski (7) is able to discriminate between intact and nicked DNA by determining the difference in fluorometric readings obtained before and after boiling the preparation, we next wanted to see if nicking played a determinative role in the rapid decline following peak growth. We induced DNA damage by exposing logarithmic-phase cells to X rays. Exposure of triplicate samples of M. fennentans to irradiation for 0, 20, 40, 80, and 160 s resulted in counts of ( ) x 1010, ( ) x 107, ( ) x 106, ( ) x 105, and ( ) x 103 CFU/ml, respectively (values are means ± standard deviations). Therefore, sequential irradiation led to a greater than 7-log-unit loss in viability over 2.5 min of irradiation, indicating that significant DNA damage had been induced. However, the data in Table 1 show that there was no significant change in the pre- and postboiling fluorometer -a- 4-, a, CA) En a) 0 ILL vo A : II.I I A / II 0 A I Days of Growth O p-3 * P-8 A P-9 FIG. 3. Correlation between the data received from the fluorometric system and the formation of M. hyorhinis colonies on agar. The correlation coefficient (r), which was derived from the data obtained on days 1 to 5, was , indicating a high degree of correlation between the fluorometer readings and the number of viable organisms prior to the onset of the tailing. -n c -.4 TABLE 1. Effect of boiling on fluorometer reading following irradiation of M. fermentans culturea Fluorometer reading Irradiation (mean ± SD) % Change time (s) Preboiling Postboiling ± ± ± ±0.6 45±2 36 a All samples were taken and treated in triplicate. readings as the radiation time increased. That is, there was a 34% change with no irradiation (nicking) and an average of 38.5% change thereafter. If nicking of the DNA were responsible, there would have been an increase in the difference between pre- and postboiling samples as the irradiation time increased. Thus, although there is a difference between preand postboiling samples during culture growth (as seen above with no irradiation), this difference, as well as the loss of viability, is not due to accumulated DNA damage. Figure 4 demonstrates the usefulness of the fluorometric method for evaluating the efficacy of an antibiotic against M. hyorhinis. The data show that chloramphenicol succinate is effective. A dose-response curve was obtained, and doses which were effective, intermediately effective, and ineffective were easily discerned. In Fig. 5, the fluorometric method was used to examine the titration of yeast extract. We found that it was possible to lower the amount of yeast extract by 75% and still achieve maximal growth when compared with the growth achieved with the complete medium. C) a} : 4-, 0L) E LL Days of Growth FIG. 4. The effectiveness of chloramphenicol succinate in inhibiting the growth of M. hyorhinis followed fluorometrically. A twosample t test comparing the control with the 12.5-p.g/ml curve, the with the 25-,ug/ml curve, the 25- with the 50-p,g/ml curve, and the 50- with the 100-p.g/ml curve yielded the following results. There was no significant difference between the control and 12.5-,ug/ml curves (P = ), there was a marginally significant difference between the and 25-p.g/ml curves (P = ), there was a significant difference between the 25- and the 50-p.g/ml curves (P = ), and there was no significant difference between the 50- and 100-pg/ml curves (P = ).

4 1306 SCHAEFFER AND MELAMEDE CM cc al) 4S C ō LL. juu 250-a SP-4 (2.5%) o 1.9% 100- A 1.3% * 0.63% Time in Hours FIG. 5. Optimization of the yeast extract concentration of SP-4 medium for M. fermentans. A two-sample t test comparing the growth in the presence of the quantity of yeast extract in the control (SP-4) with that in the presence of three other concentrations indicates that there was no significant difference between them. The significance was as follows: for the 2.5 versus the 1.9% concentrations, P ; for the 2.5 = versus the 1.3% concentrations, P = ; and for the 2.5 versus the 0.63% concentrations, P = DISCUSSION The data indicate that the fluorometric system described does for broth-grown mycoplasmas what turbidimetric analysis does for larger broth-grown bacteria. It conveniently monitors growth and it correlates with agar growth, but it has the added advantage that the fluorometer readings can be converted to DNA content as well. An additional advantage of this fluorometric system is the fact that once the alkaline EtBr solution is added to the cultural aliquot, the sample can be stored at 4 C, provided that it is protected from light. This will allow the accumulation of samples for fluorometry at a later time. When the resuspended cells are in the alkaline EtBr solution, the strong alkalinity causes the dissolution of the proteins of intact cells, releasing the nucleic acids. The alkalinity not only denatures any RNA present but also degrades it, thereby preventing hairpin looping and, thus, any contribution of RNA to EtBr binding. When bound to duplex DNA, the EtBr exhibits a 20- to 25-fold increase in its fluorescence compared with that when it is free in solution or in the presence of single-stranded DNA (11), and this property forms the basis for the fluorometric analysis system. The procedure is also a rapid one; the time involved in obtaining each individual reading, from removal of the sample from the culture to the reading in the fluorometer, is approximately 20 min. We found that the system is eminently suited for evaluation of the effectiveness of an antibiotic. We knew that the strain of M. hyorhinis, which we isolated from a contaminated cell culture, was susceptible to chloramphenicol succinate because we had successfully cured the cell culture in two successive passages in its presence. Thus, we were certain that the data received were genuine. Agar growth data confirmed the lethality (data not shown). Likewise, the system was suited to the task of titrating and evaluating the yeast extract component of SP-4. In that case, we learned that we could reduce this additive by 75% and still achieve maximal growth of M. fermentans. The system could easily be used to optimize other medium components in order to develop more defined media than are currently available. J. CLIN. MICROBIOL. Just as with the turbidimetric analysis, the fluorometric data are correlated with viability up to the late logarithmic phase of growth. It is also analogous in that we found the sensitivity of the fluorometric system to closely approximate that of the turbidimetric system used for larger bacteria. That is, when turbidity is first detectable in a bacterial culture, such as a culture of Escherichia coli, the culture has reached a cell population of approximately 107 organisms per ml (19). However, at that point, initial spectrophotometric readings are nonlinear. Similarly, by our fluorometric method, between 107 and 108 mycoplasmal cells were required before readings could be initiated. One difference between the turbidimetric analysis and the fluorometric method described here is the fact that while the turbidimetric analysis can easily be saturated, requiring that dilutions of the culture be made in order to achieve accurate readings, the fluorometric method is not saturated unless 50,g of DNA or more per ml is present. It would take up to 1011 cells per ml (approximately 78,ug of DNA per ml) before the system would be saturated and would require a dilution of the culture. A major difference between the mycoplasmal growth curve and those of larger bacteria, elucidated by the method described here, lies in the post-peak portion of the curve. Whereas the growth curves of all larger bacteria usually display a decline portion, which has been attributed to exhaustion of vital factors and/or the buildup of toxic products, it is usually preceded by a stationary phase of varying length. In our hands, however, we did not see a true stationary phase with mycoplasmas; we saw only a decline in viability following peak growth. We originally considered the tailing seen in Fig. 2 and 3 to be a form of the stationary phase of the growth curve. However, after the number of CFU was determined, it was obvious that what was present at this point was nonviable, but intact, mycoplasmal cells or precipitated medium components which were binding DNA. Either alternative is consistent with the methodology, because the initial step in the fluorometric method involves centrifugation, which would sediment both intact cells and precipitated medium components. However, the former alternative is the more likely since, under the latter alternative, DNA would have to remain intact in a rather hostile environment, given that the mycoplasmal cells would have had to lyse to release the DNA. In addition to M. hyorhinis and M. fermentans, we obtained essentially the same data for M. hominis, M. orale, and Acholeplasma laidlawii. In our hands, all of these organisms reached peak growth within 3 to 5 days, depending on the size of the inoculum used, and all lost viability following peak growth. With relation to the post-peak portion of the growth curve, we found that if a sample from this portion of the curve is boiled following the initial reading and is then reread, the initial reading will be reduced substantially (data not shown). Thus, the tailing which is observed in the latter part of the growth curve (and which contains no viable cells) is essentially eliminated. On the contrary, boiling of samples taken from earlier portions of the growth curve (i.e., pre-peak portions), where fluorometer readings and viability correlate highly, produces only a small change in the fluorometer reading, indicating that this procedure may, in some systems, prove useful in the assessment of viability. The data in Table 1 and Results, however, indicate that one must make this correlation with caution, because the same number of dead, but intact, cells can produce the same fluorometer reading postboiling. Therefore, each system needs to be

5 VOL. 31, 1993 FLUOROMETRIC QUANTITATION OF BROTH-GROWN MYCOPLASMAS 1307 evaluated individually if the boiling step is included in the fluorometric regimen. One of the major virtues of a system such as the one described here is that because the fluorometer readings are directly related to the DNA content of the sample, it will make possible the standardization of conditions for the growth of mycoplasmas within and between laboratories and for antigenic standardization for eventual vaccines. In addition, this system provides data, on a daily basis, concomitant with the growth of the culture itself, making it useful in assessing the effectiveness of antimycoplasmal antibiotics and developing new defined media for growing mycoplasmas. It will serve its greatest purpose in permitting one to know the precise status of a broth-grown mycoplasmal culture. ACKNOWLEDGMENTS We thank Lauren Cale, Stephen Simkins, and James Wilson for excellent technical assistance. REFERENCES 1. Brown, T. M., J. S. Bailey, and H. W. Clark Mycoplasma in pleural effusion of rheumatoid arthritis, p In International Congress on Rheumatology, vol. XIII. International Congress Series No Elsevier/Excerpta Medica, Amsterdam. 2. Brown, T. M., H. W. Clark, J. S. Bailey, and C. W. Gray A mechanistic approach to treatment of rheumatoid type arthritis naturally occurring in a gorilla. Trans. Am. Clin. Climatol. Assoc. 82: Chowdhury, M. I. H., T. Munakata, Y. Koyanagi, S. Kobayashi, S. Arai, and N. Yamamoto Mycoplasma can enhance HIV replication in vitro: a possible cofactor responsible for the progression of AIDS. Biochem. Biophys. Res. Commun. 170: Clark, H. W The potential role of mycoplasmas as autoantigens and immune complexes in chronic vascular pathogenesis. Am. J. Primatol. 24: Hay, R. J., M. L. Macy, and T. R. Chen Mycoplasma infection of cultured cells. Nature (London) 339:4. 6. Kotani, H., G. H. Butler, D. Tallarida, C. Cody, and G. J. McGarrity Microbiological cultivation of Mycoplasma hyorhinis from cell cultures. In Vitro Cell. Dev. Biol. 26: Kowalski, D A procedure for the quantitation of relaxed closed circular DNA in the presence of superhelical DNA: an improved fluorometric assay for nicking-closing enzyme. Anal. Biochem. 93: Kundsin, R. B., S. G. Driscoll, and P. A. Pelletier Ureaplasma urealyticum incriminated in perinatal morbidity and mortality. Science 213: Lemaitre, M., D. Guetard, Y. Henin, L. Montagnier, and A. Zerial Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells. Res. Virol. 141: LeMaitre, M., Y. Henin, F. Destouesse, C. Ferrieux, L. Montagnier, and A. Blanchard Role of mycoplasma infection in the cytopathic effect induced by human immunodeficiency virus type 1 in infected cell lines. Infect. Immun. 60: LePecq, J.-B., and C. Paoletti A fluorescent complex between ethidium bromide and nucleic acids: physical-chemical characterization. J. Mol. Biol. 27: Lo, S.-C., J. Shih, P. B. Newton, D. M. Wong, M. M. Hayes, J. R. Benish, D. J. Wear, and R. Wang The virus-like infectious agent (VLIA) is a novel pathogenic mycoplasma. Am. J. Trop. Med. Hyg. 41: Lo, S.-C., S. Tsai, J. R. Benish, J. W.-K. Shih, D. J. Wear, and D. M. Wong Enhancement of HIV-1 cytocidal effects in CD4+ lymphocytes by the AIDS-associated mycoplasma. Science 251: Razin, S Molecular biology and genetics of mycoplasmas (Mollicutes). Microbiol. Rev. 49: Razin, S., M. F. Barile, R. Harasawa, D. Amikam, and G. Glaser Characterization of the mycoplasma genome. Yale J. Biol. Med. 56: Saillard, C., P. Carle, J. M. Bove, C. Bebear, S. C. Lo, J. W. Shih, R. Y. H. Wang, D. L. Rose, and J. G. Tully Genetic and serologic relatedness between Mycoplasma fermentans and a mycoplasma recently identified in tissues of AIDS and non- AIDS patients. Res. Virol. 141: Stanier, R. Y Are there obligate cellulose-decomposing bacteria? Soil Sci. 53: Tully, J. G., R. F. Whitcomb, H. F. Clark, and D. L. Williamson Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma. Science 195: Umbreit, W. W Modem microbiology, p. 55. W. H. Freeman & Co., San Francisco. 20. Wright, K Mycoplasmas in the AIDS spotlight. Science Res. News 248: