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Differentiation of Mycoplasma genitalium from Mycoplasma pneumoniae by immunofluorescence
B9 DIFFERENTIATION OF Mycoplasma genitalium FROM Mycoplasma pneumoniae BY IMMUNOFLUORESCENCE Joseph G. Tully
General Introduction Strains of Mycoplasma genitalium were first isolated from the urethra of men with nongonoccocal urethritis in 1980. Although the organism was clearly established in early studies as a distinct species, it was found to share a number of important biologic and serologic properties with strains of M. pneumoniae (Tully et aL, 1981; Taylor-Robinson et al., 1983; Lind et al, 1984). Thus, the two mycoplasmas have similar organized attachment structures (Tully et aL, 1983) and exhibit sequence homology between their adhesin genes (Dallo et aL, 1989), as well as sharing common epitopes among their adhesin and membrane proteins (Morrison-Plummer et aL, 1987), and they contain similar membrane glycolipids (Taylor-Robinson et aL, 1983; Lind et aL, 1984). Since the respiratory tract was obviously established as the primary site of M. pneumoniae colonization, the interactions with M. genitalium were not thought at the time to complicate the delineation of the role of M. genitalium in human genital tract infections. However, the subsequent discovery of M. genitaliumlM. pneumoniae mixtures in nasopharyngeal throat specimens of patients with acute respiratory disease (Baseman et aL, 1988) not only contributed new concepts about the host distribution of M. genitalium, but prompted important questions about the poten169 Molecular and Diagnostic Procedures in Mycoplasmology, Vol. II
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
1 70
Joseph G. Tully
tial pathogenicity of the organism and its interrelationship with M. pneumoniae (Tully and Baseman, 1991). A synovial fluid isolate from a patient with polyarthritis following an acute respiratory disease was found to contain both M. genitalium and M. pneumoniae (Tully et al, 1995). With the use of conventional laboratory serologic techniques for mollicutes (growth inhibition and agar plate immunofluorescence tests), each Mycoplasma species was identified within the mixed culture, and individual strains were recovered and their identity was reconfirmed. The applications of these diagnostic techniques are described in detail here. Rapid developments have occurred in the application of DNA amplification procedures (polymerase chain reaction; PCR) to the detection of M. genitalium in clinical specimens, as well as the ability of selected primers to differentiate this organism from M. pneumoniae (de Barbeyrac et al, 1993; Homer et al., 1993; Jensen et al., 1991; Palmer et al, 1991a,b) (see also Section A, this volume). These procedures will obviously become increasingly important in the identification of such fastidious and difficult to cultivate mollicutes as M. genitalium. Likewise, immunoblotting, with mycoplasma cell suspensions and monoclonal antibodies specific either to the adhesin protein of M. pneumoniae (the 168-kDa PI) or M. genitalium (the 140-kDa MgPa), has also successfully been applied to the identification of mixed populations of the two organisms (Baseman ^f«/., 1988).
Materials M. genitalium and M. pneumoniae type or representative strains, grown in SP-4 broth medium (see Chapter A2 in Vol. I.) Agar plates, SP-4 formulation Polyclonal antiserum to type strains of M. genitalium and M. pneumoniae, preferably prepared in rabbits Fluorescein-conjugated antiserum to type strains of M. genitalium andM. pneumoniae. Freeze at -20°C. [If conjugated antiserum is not available, polyclonal rabbit antiserum can be used in an indirect immunofluorescence test, employing fluorescein-conjugated, anti-rabbit (IgG, IgA, IgM) goat serum (Cappel No. 55652, Organon Teknika Corp., Durham, N C ] Phosphate-buffered saHne (PBS), pH 7.8 A fluorescence microscope system, such as the Zeiss standard fluorescence model (with quartz halogen lamp for both incident and transmitted illumination and Zeiss 48-77-05 filter system) (Gardella et al, 1983) Screw-cap glass vials (1-dram or about 4-ml size), sterile Glass or plastic petri dishes (90-mm diameter)
B9 Differentiation of M. genitalium
171
GasPak incubation system (BBL Microbiology Systems, Cockeysville, MD), with carbon dioxide generation envelopes
Procedure CONTROL DIRECT IMMUNOFLUORESCENCE TESTS WITH ESTABLISHED MYCOPLASMA STRAINS
1. Prior to agar plate immunofluorescence tests on the candidate culture, it is necessary to establish a number of critical parameters related to the responses of established strains of the two mycoplasmas, to variations in the potency of the individual conjugates, to the selection of appropriate dilutions of these conjugates, and to possible variations in the results obtained with different fluorescence microscope systems. 2. Appropriate dilutions of SP-4 broth cultures of the type or representative strains of M. genitalium and M. pneumoniae are plated to individual SP-4 agar and agar plates incubated in the GasPak system in a carbon dioxide atmosphere. Plates selected for testing should contain about 100-200 individual and wellspaced colonies. 3. Thaw frozen fluorescein-conjugated antiserum to each mycoplasma and prepare a series of eight two-fold dilutions (starting a 1:8 and ending at 1:1024) in a final volume of about 2 ml PBS. 4. With a flamed spatula or wire loop, cut eight individual 1-cm-square agar pieces from the plate of M. genitalium colonies and place each piece on an individual glass microscope slide. Label each slide with the appropriate conjugate dilution to be tested. To prevent drying, place slides in a covered petri dish containing two glass rods. Place a filter paper circle in the bottom of the dish and wet with water. 5. With individual capillary pipettes, add 1-2 drops of the appropriate conjugate dilution to the surface of each agar piece, cover, and incubate for 15 minutes at room temperature. Examine each slide and add further conjugate to each agar piece, if necessary, and continue incubation for an additional 15 minutes. 6. At the end of the incubation, gently wash the conjugate from the agar piece with PBS and allow agar to dry for 5-10 minutes. The colonies are embedded in the agar and should not, under most circumstances, become removed by the washing procedure. 7. Examine each agar piece in the fluorescence microscope, using a magnification of about 160x (16x objective and 10x eyepiece). Colonies should first be located on the agar with transmitted white light and then with incident ultraviolet light from the quartz halogen lamp. 8. M. genitalium colonies treated with low dilutions (1:8) of homologous
1 72
Joseph G. Tully TABLE I EPI-IMMUNOFLUORESCENCE TESTS FOR DIFFERENTIATION OF AGAR COLONIES
OF Mycoplasma genitalium FROM Mycoplasma pneumoniae"^ Fluorescence of agar colonies when treated with indicated dilutions of each specific conjugate M. genitalium[
M. pneumoniae
Antigens (agar colonies)
1:16
1:64
1:128
1:32
1:256
1:1024
M. genitalium M. pneumoniae Strain UTMB-10 (primary isolate) Strain UTMB-lOG (cloned M. genitalium) Strain UTMB-lOP (cloned M. pneumoniae)
4+ 3+ 3+
1+ Neg ±_
±_
3+ 4+ 4+
Neg 2+
Neg
Neg Neg
2+
±
4+
1+
+
3+
Neg
Neg
4+
Neg
Neg
4+
2+ to 3+
1+
+
"Reproduced from Tully et al., 1995, with permission of the American Society of Microbiology.
conjugate should exhibit strong fluorescence (4-I-), and fluorescence should fade as agar colonies are exposed to more dilute conjugates. The end point dilution of each conjugate is that dilution which still produces a 1+ fluoresence, whereas the working dilution is that which yields a 3-4+ fluorescence. Record the end point and working dilution of the M. genitalium conjugate tested. Repeat this procedure with M. genitalium colonies treated with the M. pneumoniae conjugate. 9. In a similar manner, test M. pneumoniae colonies with a series of M. pneumoniae conjugate dilutions, recording the end point and working dilutions of the specific conjugate. As described earlier, the M. pneumoniae colonies are then tested with the M. genitalium conjugate, and similar end point and working dilutions are recorded. 10. These control tests should establish the dilution of each conjugate which will clearly induce fluorescence in colonies of the homologous organism but will not yield fluorescence in colonies of the other mycoplasma. Table I shows the results with established strains of M. genitalium andM. pneumoniae and several selected dilutions of two conjugated antisera.
Cultivation of Test Isolate in Broth 1. The isolate to be tested for mixed infection should be examined immediately after primary isolation from the host and, if possible, with no more than two or three passages on artificial media. The isolate is then grown on SP-4
B9 Differentiation of M. genitalium
1 73
broth, preferably in a plastic T-25 tissue culture flask containing 10 ml of broth. The culture flask is incubated horizontally at 37°C until there is evidence of attached growth to the plastic surface under the broth layer, as well as some slight color change (toward yellow) in the phenol red indicator in the medium. 2. Pour the supernatant fluid into a discard container for decontamination and add 3-4 ml of fresh SP-4 broth to the flask. Using a sterile tissue cell scraper or rubber policeman, rub the plastic surface to remove the adherent mycoplasmas. Place the flask in an upright position for a few minutes to drain the fluid and then remove the cell suspension with a sterile pipette. Divide the suspension into about two equal volumes in small screw-cap glass vials (4-ml size) and freeze at -70°C.
Preparation and Inoculation of Agar Plates of Test Isolate
1. Prepare a series of five glass vials containing 1:8 ml of SP-4 broth. Thaw a vial of the frozen mycoplasma suspension to be tested and add 0.2 ml to the first vial in the series (final 1:10 dilution). Continue serial 10-fold dilutions of the inoculum by passage of 0.2-ml volumes until the culture is diluted through the last vial (1:10-^). Refreeze the remaining cell suspension material at -70°C. 2. Plate 0.2-ml volume from each individual dilution vial onto two fresh SP-4 agar plates. Mark on the plates the respective dilution factor of the mycoplasma cell suspension. After the inoculum on each agar plate has dried, place the plates in the GasPak jar and incubate in a carbon dioxide atmosphere at 37°C. 3. At 4- to 5-day intervals, remove plates from the incubator and examine microscopically for the appearance and number of mycoplasma colonies on the agar surface. Plates selected for immunofluorescence testing should have numerous but well-spaced individual colonies. Plates containing confluent growth or those with only 10-20 colonies should not be used for subsequent tests. 4. If the initial dilution series yields an unsatisfactory agar colony population for testing, the number of colonies on the plates should indicate whether the culture dilution should be increased or decreased. The frozen mycoplasma suspension can then be thawed, diluted, and again used to prepare a second series of plates, with two agar plates for each dilution.
Immunofluorescence Tests on Agar Colonies of Test Isolate
1. Wet the agar colonies by adding about 5 ml of PBS to each of the two agar plates containing appropriate numbers of colonies of the test organism. Incubate for 5-10 minutes, and then discard the fluids in a container of disinfectant. Tip the dish slightly to drain off excess fluid and remove any remaining fluid with a capillary pipette.
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Joseph G. Tully
2. Add about 1-2 ml of a freshly prepared dilution of the selected conjugate of M. genitalium to one plate and an appropriate dilution of the M. pneumoniae conjugate to the second plate. Mark plates with respective conjugate and dilution. Incubate plates for 30 minutes at room temperature. Pour off conjugate into disinfectant solution, wash plates several times with about 5 ml of PBS, and discard wash fluid in disinfectant. Tip plates slightly to drain excess fluid and remove with a capillary pipette. 3. Examine each agar plate in the fluorescence microscope and observe the number and size of colonies showing fluorescence with either conjugate. M. genitalium colonies in a mixture with M. pneumoniae will usually show a smaller size colony, and the numbers may be only 1-5% of the total number of M. pneumoniae colonies.
Separation/Cloning of Isolates from M, genitalium/ M. pneumoniae Mixtures 1. Primary clinical isolates exhibiting mixed populations of M. genitalium and M. pneumoniae frequently have low numbers of M. genitalium. Since it is usually very difficult to select and separate the few individual M. genitalium colonies in this type of mixed population, some effort is necessary during early passages of the isolate to shift the population of M. genitalium colonies toward a 50-50 ratio. 2. The earliest available passage of the mixed culture is grown to the logarithmic phase of growth in SP-4 broth. Four dilutions (1:50, 1:100, 1:150, and 1:200) of the broth culture are plated onto SP-4 agar, using about 0.2 ml per plate. The remaining broth culture is frozen at -70°C. After the inoculum has dried, four 6-mm filter paper disks, saturated with polyclonal antiserum to M. pneumoniae, are placed on the surface of each 60-mm SP-4 agar plate. The disks should be located about halfway from the center to the periphery of the plate surface and about an equal distance from each other. The position of the disks should leave a clear zone of about 2.0 to 2.5 cm^ in the center of the plate. The agar plates are then incubated in the GasPak jar as outlined earlier. 3. At 4- to 5-day intervals, the plates are removed from the incubator and examined under low power magnification (60x). Colonies appearing in the center of the agar surface, in the zone where the M. pneumoniae antiserum has diffused, are selected for subculture to broth. At least one of the plates receiving the diluted test organism should show a moderate number of individual organisms growing within the central area. 4. Aseptically remove agar pieces from the central area of as many of the plates as possible, transferring each to individual screw-cap vials containing about 2.5 ml each of SP-4 broth. Incubate the vials at 37°C and examine in 5-10 days for turbidity or pH changes. When such changes occur, plate the broth
B9 Differentiation of M. genitalium
1 75
cultures, with appropriate dilutions, to SP-4 agar and incubate plates anaerobically. 5. Agar plates containing colonies selected from the central zone containing M. pneumoniae antiserum should then be tested in the immunofluorescence test. Individual agar pieces are removed to slides and are stained with conjugates to both M. pneumoniae and M. genitalium. These tests should indicate whether significant shifts in the population of M. genitalium have occured and should show the differences in the colony morphology of the two Mycoplasma species. M. genitalium colonies are usually smaller in size than M. pneumoniae colonies on SP-4 agar. 6. The continued selection of agar colony types and immunofluorescence testing with appropriate dilutions of the two conjugates should provide for the eventual isolation of each individual Mycoplasma species in the mixture. Such isolates should then be subjected to conventional filtration cloning techniques (Tully, 1983) and to reconfirmation by immunofluorescence tests with conjugates employed earlier (Table I).
Discussion Although only few isolates of M. genitalium have been made from humans, experience indicates that the organism is extremely fastidious and has an exceedingly slow growth rate. In those instances where the organism has occurred along with M. pneumoniae, it has been obvious that continued passage of the mixture will not favor the survival of M. genitalium. Where the two organisms were found in a synovial fluid isolate, the culture had been passaged about five times. When examined by the differential immunofluorescence test outlined in this chapter, the number of M. genitalium colonies on the initial plating of the synovial isolate was less than 1% of the total number of agar colonies obtained. Detection procedures for M. genitalium, regardless of whether the immunofluorescence or immunoblotting techniques are utilized, should be performed only on early passages of clinical specimens. It should be emphasized that each conjugated antiserum employed in the test must be evaluated in standard immunofluorescence tests against the homologous mycoplasma strain before such conjugates can be applied to potentially mixed cultures. Likewise, if the indirect immunofluorescence method is employed, a checkerboard program must be performed with agar colonies of the representative mycoplasma. In this program, various dilutions of the unlabeled, polyclonal antiserum are compared to various dilutions of the labeled, anti-rabbit conjugate, with the view of selecting the optimum level of conjugate necessary for the mycoplasma antiserum. The eventual delineation of the role of M. genitalium in human disease,
1 76
Joseph G. Tully
involving either the urogenital or respiratory tracts, or other tissue sites, will depend on the further application of rapid diagnostic techniques, laboratory isolation, and identification by conventional methodology outlined here, and a serologic analysis of host immune responses by specific and differential techniques.
References Baseman, J. B., Dallo, S. F., Tully, J. G., and Rose, D. L. (1988). Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. J. Clin. Microbiol. 26, 22662269. Dallo, S. F., Chavoya, A., Su, C.-J., and Baseman, J. B. (1989). DNA and protein sequence homologies between the adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. Infect. Immun. 57, 1059-1065. de Barbeyrac, B., Bemet-Poggi, C , Febrer, F., Renaudin, H., Dupon, M., and Bebear, C. (1993). Detection of Mycoplasma pneumoniae and Mycoplasma genitalium in clinical samples by polymerase chain reaction. Clin. Infect. Dis. 17(Suppl. 1), S83-S89. Gardella, R. S., Del Giudice, R. A., and Tully, J. G. (1983). Immunofluorescence. In "Methods in Mycoplasmology" (S. Razin and J. G. Tully, eds.). Vol. 1, pp. 431-439. Academic Press, New York. Homer, P. J., Gilroy, C. B., Thomas, B. J., Naidoo, R. O. M., and Taylor-Robinson, D. (1993). Association of Mycoplasma genitalium with acute non-gonococcal urethritis. Lancet Ml, 582585. Jensen, J. S., Uldum, S. A., S0ndergard-Anderson, J., Vuust, J., and Lind, K. (1991). Polymerase chain reaction for detection of Mycoplasma genitalium. J. Clin. Microbiol. 29, 46-50. Lind, K., Lindhardt, B. O., Schutten, H. J., Blom, J., and Christiansen, C. (1984). Serological cross-reactions bQiwccn Mycoplasma genitalium and Mycoplasma pneumoniae. J. Clin. Microbiol. 20, 1036-1043. Morrison-Plummer, J., Lazzell, A., and Baseman, J. B. (1987). Shared epitopes between Mycoplasma pneumoniae major adhesin protein PI and a 140-kilodalton protein of Mycoplasma genitalium. Infect. Immun. 55, 49-56. Palmer, H. M., Gilroy, C. B., Claydon, E. J., and Taylor-Robinson, D. (1991a). Detection of Mycoplasma genitalium in the genitourinary tract of women by the polymerase chain reaction. Int. J. STD AIDS 2, 261-263. Palmer, H. M., Gilroy, C. B., Furr, P. M., and Taylor-Robinson, D. (1991b). Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium. FEMS Microbiol. Lett. 77, 199-204. Taylor-Robinson, D., Furr, P. M., and Tully, J. G. (1983). Serological cross-reactions between Mycoplasma genitalium and M. pneumoniae. Lancet 1, 527. Tully, J. G. (1983). Cloning and filtration techniques for mycoplasmas. In "Methods in Mycoplasmology" (S. Razin and J. G. Tully, eds.). Vol. 1, pp. 173-177. Academic Press, New York. Tully, J. G., and Baseman, J. B. (1991). Mycoplasma. Lancet 337, 1296. Tully, J. G., Taylor-Robinson, D., Cole, R. M., and Rose, D. L. (1981). A newly discovered mycoplasma in the human urogenital tract. Lancet 1, 1288-1291. Tully, J. G., Taylor-Robinson, D., Rose, D. L., Cole, R. M., and Bove, J. M. (1983). Mycoplasma
B9 Differentiation of M. genitalium
M7
genitalium, a new species from the human urogenital tract. Int. J. Syst. Bacteriol. 33, 387396. Tully, J. G., Rose, D. L., Baseman, J. B., Dallo, S. F., Lazzell, A. L., and Davis, C. P. (1995). Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate. J. Clin. Microbiol. 33, 1851-1855.
General Introduction Strains of Mycoplasma genitalium were first isolated from the urethra of men with nongonoccocal urethritis in 1980. Although the organism was clearly established in early studies as a distinct species, it was found to share a number of important biologic and serologic properties with strains of M. pneumoniae (Tully et aL, 1981; Taylor-Robinson et al., 1983; Lind et al, 1984). Thus, the two mycoplasmas have similar organized attachment structures (Tully et aL, 1983) and exhibit sequence homology between their adhesin genes (Dallo et aL, 1989), as well as sharing common epitopes among their adhesin and membrane proteins (Morrison-Plummer et aL, 1987), and they contain similar membrane glycolipids (Taylor-Robinson et aL, 1983; Lind et aL, 1984). Since the respiratory tract was obviously established as the primary site of M. pneumoniae colonization, the interactions with M. genitalium were not thought at the time to complicate the delineation of the role of M. genitalium in human genital tract infections. However, the subsequent discovery of M. genitaliumlM. pneumoniae mixtures in nasopharyngeal throat specimens of patients with acute respiratory disease (Baseman et aL, 1988) not only contributed new concepts about the host distribution of M. genitalium, but prompted important questions about the poten169 Molecular and Diagnostic Procedures in Mycoplasmology, Vol. II
Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
1 70
Joseph G. Tully
tial pathogenicity of the organism and its interrelationship with M. pneumoniae (Tully and Baseman, 1991). A synovial fluid isolate from a patient with polyarthritis following an acute respiratory disease was found to contain both M. genitalium and M. pneumoniae (Tully et al, 1995). With the use of conventional laboratory serologic techniques for mollicutes (growth inhibition and agar plate immunofluorescence tests), each Mycoplasma species was identified within the mixed culture, and individual strains were recovered and their identity was reconfirmed. The applications of these diagnostic techniques are described in detail here. Rapid developments have occurred in the application of DNA amplification procedures (polymerase chain reaction; PCR) to the detection of M. genitalium in clinical specimens, as well as the ability of selected primers to differentiate this organism from M. pneumoniae (de Barbeyrac et al, 1993; Homer et al., 1993; Jensen et al., 1991; Palmer et al, 1991a,b) (see also Section A, this volume). These procedures will obviously become increasingly important in the identification of such fastidious and difficult to cultivate mollicutes as M. genitalium. Likewise, immunoblotting, with mycoplasma cell suspensions and monoclonal antibodies specific either to the adhesin protein of M. pneumoniae (the 168-kDa PI) or M. genitalium (the 140-kDa MgPa), has also successfully been applied to the identification of mixed populations of the two organisms (Baseman ^f«/., 1988).
Materials M. genitalium and M. pneumoniae type or representative strains, grown in SP-4 broth medium (see Chapter A2 in Vol. I.) Agar plates, SP-4 formulation Polyclonal antiserum to type strains of M. genitalium and M. pneumoniae, preferably prepared in rabbits Fluorescein-conjugated antiserum to type strains of M. genitalium andM. pneumoniae. Freeze at -20°C. [If conjugated antiserum is not available, polyclonal rabbit antiserum can be used in an indirect immunofluorescence test, employing fluorescein-conjugated, anti-rabbit (IgG, IgA, IgM) goat serum (Cappel No. 55652, Organon Teknika Corp., Durham, N C ] Phosphate-buffered saHne (PBS), pH 7.8 A fluorescence microscope system, such as the Zeiss standard fluorescence model (with quartz halogen lamp for both incident and transmitted illumination and Zeiss 48-77-05 filter system) (Gardella et al, 1983) Screw-cap glass vials (1-dram or about 4-ml size), sterile Glass or plastic petri dishes (90-mm diameter)
B9 Differentiation of M. genitalium
171
GasPak incubation system (BBL Microbiology Systems, Cockeysville, MD), with carbon dioxide generation envelopes
Procedure CONTROL DIRECT IMMUNOFLUORESCENCE TESTS WITH ESTABLISHED MYCOPLASMA STRAINS
1. Prior to agar plate immunofluorescence tests on the candidate culture, it is necessary to establish a number of critical parameters related to the responses of established strains of the two mycoplasmas, to variations in the potency of the individual conjugates, to the selection of appropriate dilutions of these conjugates, and to possible variations in the results obtained with different fluorescence microscope systems. 2. Appropriate dilutions of SP-4 broth cultures of the type or representative strains of M. genitalium and M. pneumoniae are plated to individual SP-4 agar and agar plates incubated in the GasPak system in a carbon dioxide atmosphere. Plates selected for testing should contain about 100-200 individual and wellspaced colonies. 3. Thaw frozen fluorescein-conjugated antiserum to each mycoplasma and prepare a series of eight two-fold dilutions (starting a 1:8 and ending at 1:1024) in a final volume of about 2 ml PBS. 4. With a flamed spatula or wire loop, cut eight individual 1-cm-square agar pieces from the plate of M. genitalium colonies and place each piece on an individual glass microscope slide. Label each slide with the appropriate conjugate dilution to be tested. To prevent drying, place slides in a covered petri dish containing two glass rods. Place a filter paper circle in the bottom of the dish and wet with water. 5. With individual capillary pipettes, add 1-2 drops of the appropriate conjugate dilution to the surface of each agar piece, cover, and incubate for 15 minutes at room temperature. Examine each slide and add further conjugate to each agar piece, if necessary, and continue incubation for an additional 15 minutes. 6. At the end of the incubation, gently wash the conjugate from the agar piece with PBS and allow agar to dry for 5-10 minutes. The colonies are embedded in the agar and should not, under most circumstances, become removed by the washing procedure. 7. Examine each agar piece in the fluorescence microscope, using a magnification of about 160x (16x objective and 10x eyepiece). Colonies should first be located on the agar with transmitted white light and then with incident ultraviolet light from the quartz halogen lamp. 8. M. genitalium colonies treated with low dilutions (1:8) of homologous
1 72
Joseph G. Tully TABLE I EPI-IMMUNOFLUORESCENCE TESTS FOR DIFFERENTIATION OF AGAR COLONIES
OF Mycoplasma genitalium FROM Mycoplasma pneumoniae"^ Fluorescence of agar colonies when treated with indicated dilutions of each specific conjugate M. genitalium[
M. pneumoniae
Antigens (agar colonies)
1:16
1:64
1:128
1:32
1:256
1:1024
M. genitalium M. pneumoniae Strain UTMB-10 (primary isolate) Strain UTMB-lOG (cloned M. genitalium) Strain UTMB-lOP (cloned M. pneumoniae)
4+ 3+ 3+
1+ Neg ±_
±_
3+ 4+ 4+
Neg 2+
Neg
Neg Neg
2+
±
4+
1+
+
3+
Neg
Neg
4+
Neg
Neg
4+
2+ to 3+
1+
+
"Reproduced from Tully et al., 1995, with permission of the American Society of Microbiology.
conjugate should exhibit strong fluorescence (4-I-), and fluorescence should fade as agar colonies are exposed to more dilute conjugates. The end point dilution of each conjugate is that dilution which still produces a 1+ fluoresence, whereas the working dilution is that which yields a 3-4+ fluorescence. Record the end point and working dilution of the M. genitalium conjugate tested. Repeat this procedure with M. genitalium colonies treated with the M. pneumoniae conjugate. 9. In a similar manner, test M. pneumoniae colonies with a series of M. pneumoniae conjugate dilutions, recording the end point and working dilutions of the specific conjugate. As described earlier, the M. pneumoniae colonies are then tested with the M. genitalium conjugate, and similar end point and working dilutions are recorded. 10. These control tests should establish the dilution of each conjugate which will clearly induce fluorescence in colonies of the homologous organism but will not yield fluorescence in colonies of the other mycoplasma. Table I shows the results with established strains of M. genitalium andM. pneumoniae and several selected dilutions of two conjugated antisera.
Cultivation of Test Isolate in Broth 1. The isolate to be tested for mixed infection should be examined immediately after primary isolation from the host and, if possible, with no more than two or three passages on artificial media. The isolate is then grown on SP-4
B9 Differentiation of M. genitalium
1 73
broth, preferably in a plastic T-25 tissue culture flask containing 10 ml of broth. The culture flask is incubated horizontally at 37°C until there is evidence of attached growth to the plastic surface under the broth layer, as well as some slight color change (toward yellow) in the phenol red indicator in the medium. 2. Pour the supernatant fluid into a discard container for decontamination and add 3-4 ml of fresh SP-4 broth to the flask. Using a sterile tissue cell scraper or rubber policeman, rub the plastic surface to remove the adherent mycoplasmas. Place the flask in an upright position for a few minutes to drain the fluid and then remove the cell suspension with a sterile pipette. Divide the suspension into about two equal volumes in small screw-cap glass vials (4-ml size) and freeze at -70°C.
Preparation and Inoculation of Agar Plates of Test Isolate
1. Prepare a series of five glass vials containing 1:8 ml of SP-4 broth. Thaw a vial of the frozen mycoplasma suspension to be tested and add 0.2 ml to the first vial in the series (final 1:10 dilution). Continue serial 10-fold dilutions of the inoculum by passage of 0.2-ml volumes until the culture is diluted through the last vial (1:10-^). Refreeze the remaining cell suspension material at -70°C. 2. Plate 0.2-ml volume from each individual dilution vial onto two fresh SP-4 agar plates. Mark on the plates the respective dilution factor of the mycoplasma cell suspension. After the inoculum on each agar plate has dried, place the plates in the GasPak jar and incubate in a carbon dioxide atmosphere at 37°C. 3. At 4- to 5-day intervals, remove plates from the incubator and examine microscopically for the appearance and number of mycoplasma colonies on the agar surface. Plates selected for immunofluorescence testing should have numerous but well-spaced individual colonies. Plates containing confluent growth or those with only 10-20 colonies should not be used for subsequent tests. 4. If the initial dilution series yields an unsatisfactory agar colony population for testing, the number of colonies on the plates should indicate whether the culture dilution should be increased or decreased. The frozen mycoplasma suspension can then be thawed, diluted, and again used to prepare a second series of plates, with two agar plates for each dilution.
Immunofluorescence Tests on Agar Colonies of Test Isolate
1. Wet the agar colonies by adding about 5 ml of PBS to each of the two agar plates containing appropriate numbers of colonies of the test organism. Incubate for 5-10 minutes, and then discard the fluids in a container of disinfectant. Tip the dish slightly to drain off excess fluid and remove any remaining fluid with a capillary pipette.
1 74
Joseph G. Tully
2. Add about 1-2 ml of a freshly prepared dilution of the selected conjugate of M. genitalium to one plate and an appropriate dilution of the M. pneumoniae conjugate to the second plate. Mark plates with respective conjugate and dilution. Incubate plates for 30 minutes at room temperature. Pour off conjugate into disinfectant solution, wash plates several times with about 5 ml of PBS, and discard wash fluid in disinfectant. Tip plates slightly to drain excess fluid and remove with a capillary pipette. 3. Examine each agar plate in the fluorescence microscope and observe the number and size of colonies showing fluorescence with either conjugate. M. genitalium colonies in a mixture with M. pneumoniae will usually show a smaller size colony, and the numbers may be only 1-5% of the total number of M. pneumoniae colonies.
Separation/Cloning of Isolates from M, genitalium/ M. pneumoniae Mixtures 1. Primary clinical isolates exhibiting mixed populations of M. genitalium and M. pneumoniae frequently have low numbers of M. genitalium. Since it is usually very difficult to select and separate the few individual M. genitalium colonies in this type of mixed population, some effort is necessary during early passages of the isolate to shift the population of M. genitalium colonies toward a 50-50 ratio. 2. The earliest available passage of the mixed culture is grown to the logarithmic phase of growth in SP-4 broth. Four dilutions (1:50, 1:100, 1:150, and 1:200) of the broth culture are plated onto SP-4 agar, using about 0.2 ml per plate. The remaining broth culture is frozen at -70°C. After the inoculum has dried, four 6-mm filter paper disks, saturated with polyclonal antiserum to M. pneumoniae, are placed on the surface of each 60-mm SP-4 agar plate. The disks should be located about halfway from the center to the periphery of the plate surface and about an equal distance from each other. The position of the disks should leave a clear zone of about 2.0 to 2.5 cm^ in the center of the plate. The agar plates are then incubated in the GasPak jar as outlined earlier. 3. At 4- to 5-day intervals, the plates are removed from the incubator and examined under low power magnification (60x). Colonies appearing in the center of the agar surface, in the zone where the M. pneumoniae antiserum has diffused, are selected for subculture to broth. At least one of the plates receiving the diluted test organism should show a moderate number of individual organisms growing within the central area. 4. Aseptically remove agar pieces from the central area of as many of the plates as possible, transferring each to individual screw-cap vials containing about 2.5 ml each of SP-4 broth. Incubate the vials at 37°C and examine in 5-10 days for turbidity or pH changes. When such changes occur, plate the broth
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cultures, with appropriate dilutions, to SP-4 agar and incubate plates anaerobically. 5. Agar plates containing colonies selected from the central zone containing M. pneumoniae antiserum should then be tested in the immunofluorescence test. Individual agar pieces are removed to slides and are stained with conjugates to both M. pneumoniae and M. genitalium. These tests should indicate whether significant shifts in the population of M. genitalium have occured and should show the differences in the colony morphology of the two Mycoplasma species. M. genitalium colonies are usually smaller in size than M. pneumoniae colonies on SP-4 agar. 6. The continued selection of agar colony types and immunofluorescence testing with appropriate dilutions of the two conjugates should provide for the eventual isolation of each individual Mycoplasma species in the mixture. Such isolates should then be subjected to conventional filtration cloning techniques (Tully, 1983) and to reconfirmation by immunofluorescence tests with conjugates employed earlier (Table I).
Discussion Although only few isolates of M. genitalium have been made from humans, experience indicates that the organism is extremely fastidious and has an exceedingly slow growth rate. In those instances where the organism has occurred along with M. pneumoniae, it has been obvious that continued passage of the mixture will not favor the survival of M. genitalium. Where the two organisms were found in a synovial fluid isolate, the culture had been passaged about five times. When examined by the differential immunofluorescence test outlined in this chapter, the number of M. genitalium colonies on the initial plating of the synovial isolate was less than 1% of the total number of agar colonies obtained. Detection procedures for M. genitalium, regardless of whether the immunofluorescence or immunoblotting techniques are utilized, should be performed only on early passages of clinical specimens. It should be emphasized that each conjugated antiserum employed in the test must be evaluated in standard immunofluorescence tests against the homologous mycoplasma strain before such conjugates can be applied to potentially mixed cultures. Likewise, if the indirect immunofluorescence method is employed, a checkerboard program must be performed with agar colonies of the representative mycoplasma. In this program, various dilutions of the unlabeled, polyclonal antiserum are compared to various dilutions of the labeled, anti-rabbit conjugate, with the view of selecting the optimum level of conjugate necessary for the mycoplasma antiserum. The eventual delineation of the role of M. genitalium in human disease,
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involving either the urogenital or respiratory tracts, or other tissue sites, will depend on the further application of rapid diagnostic techniques, laboratory isolation, and identification by conventional methodology outlined here, and a serologic analysis of host immune responses by specific and differential techniques.
References Baseman, J. B., Dallo, S. F., Tully, J. G., and Rose, D. L. (1988). Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. J. Clin. Microbiol. 26, 22662269. Dallo, S. F., Chavoya, A., Su, C.-J., and Baseman, J. B. (1989). DNA and protein sequence homologies between the adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. Infect. Immun. 57, 1059-1065. de Barbeyrac, B., Bemet-Poggi, C , Febrer, F., Renaudin, H., Dupon, M., and Bebear, C. (1993). Detection of Mycoplasma pneumoniae and Mycoplasma genitalium in clinical samples by polymerase chain reaction. Clin. Infect. Dis. 17(Suppl. 1), S83-S89. Gardella, R. S., Del Giudice, R. A., and Tully, J. G. (1983). Immunofluorescence. In "Methods in Mycoplasmology" (S. Razin and J. G. Tully, eds.). Vol. 1, pp. 431-439. Academic Press, New York. Homer, P. J., Gilroy, C. B., Thomas, B. J., Naidoo, R. O. M., and Taylor-Robinson, D. (1993). Association of Mycoplasma genitalium with acute non-gonococcal urethritis. Lancet Ml, 582585. Jensen, J. S., Uldum, S. A., S0ndergard-Anderson, J., Vuust, J., and Lind, K. (1991). Polymerase chain reaction for detection of Mycoplasma genitalium. J. Clin. Microbiol. 29, 46-50. Lind, K., Lindhardt, B. O., Schutten, H. J., Blom, J., and Christiansen, C. (1984). Serological cross-reactions bQiwccn Mycoplasma genitalium and Mycoplasma pneumoniae. J. Clin. Microbiol. 20, 1036-1043. Morrison-Plummer, J., Lazzell, A., and Baseman, J. B. (1987). Shared epitopes between Mycoplasma pneumoniae major adhesin protein PI and a 140-kilodalton protein of Mycoplasma genitalium. Infect. Immun. 55, 49-56. Palmer, H. M., Gilroy, C. B., Claydon, E. J., and Taylor-Robinson, D. (1991a). Detection of Mycoplasma genitalium in the genitourinary tract of women by the polymerase chain reaction. Int. J. STD AIDS 2, 261-263. Palmer, H. M., Gilroy, C. B., Furr, P. M., and Taylor-Robinson, D. (1991b). Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium. FEMS Microbiol. Lett. 77, 199-204. Taylor-Robinson, D., Furr, P. M., and Tully, J. G. (1983). Serological cross-reactions between Mycoplasma genitalium and M. pneumoniae. Lancet 1, 527. Tully, J. G. (1983). Cloning and filtration techniques for mycoplasmas. In "Methods in Mycoplasmology" (S. Razin and J. G. Tully, eds.). Vol. 1, pp. 173-177. Academic Press, New York. Tully, J. G., and Baseman, J. B. (1991). Mycoplasma. Lancet 337, 1296. Tully, J. G., Taylor-Robinson, D., Cole, R. M., and Rose, D. L. (1981). A newly discovered mycoplasma in the human urogenital tract. Lancet 1, 1288-1291. Tully, J. G., Taylor-Robinson, D., Rose, D. L., Cole, R. M., and Bove, J. M. (1983). Mycoplasma
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genitalium, a new species from the human urogenital tract. Int. J. Syst. Bacteriol. 33, 387396. Tully, J. G., Rose, D. L., Baseman, J. B., Dallo, S. F., Lazzell, A. L., and Davis, C. P. (1995). Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate. J. Clin. Microbiol. 33, 1851-1855.