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  • Vol. 59, No. 6INFECTION AND IMMUNITY, June 1991, p. 2203-22060019-9567/91/062203-04$02.00/0Copyright C) 1991, American Society for Microbiology

    Hemadsorption by Colonies of Ureaplasma urealyticumJANET A. ROBERTSON* AND RICHARD SHERBURNE

    Department of Medical Microbiology and Infectious Diseases, University of Alberta,Edmonton, Alberta, Canada T6G 2H7

    Received 31 October 1990/Accepted 14 March 1991

    Hemadsorption by colonies of Ureaplasma urealyticum and Mycoplasma pneumoniae differed quantitativelyand qualitatively. Using standard methodology, few strains of U. urealyticum hemadsorbed; with a modifiedmethod, most strains hemadsorbed, indicating a second type of association. Scanning electron microscopy oftannin-osmium-stained preparations showed guinea pig erythrocytes embedded in ureaplasma colonies andcraters left when erythrocytes were dislodged.

    The genus Ureaplasma of the family Mycoplasmataceae,class Mollicutes, is composed of small procaryotes lacking acell wall and hydrolyzing urea (14). Ureaplasma urealyticumis a common inhabitant of the human urogenital tract whereit is an opportunistic pathogen (10). Although pathogenesis isnot understood, the ability to adhere to mucosal cells isbelieved to be requisite for infection.The phenomena of hemagglutination and hemadsorption

    have proved useful in studying the attachment of microor-ganisms to eucaryotic cells. Initial studies of adherence ofmembers of the class Mollicutes utilized the associationbetween erythrocytes (RBCs) and colonies cultivated onagar medium. These studies were primarily concerned withthe human pathogen Mycoplasma pneumoniae (3) and theanimal pathogens Mycoplasma gallisepticum and Myco-plasma pulmonis (7, 24). When it was found that the incor-poration of a zwitterion buffer into the agar medium gavelarger colonies of U. urealyticum and thus easier visualiza-tion, hemadsorption by colonies of this species was alsodemonstrated (8). Of the then eight- known serovars of U.urealyticum, only colonies of the serovar 3 standard (strain27) hemadsorbed RBCs; hemadsorption occurred with RBCsfrom guinea pigs but not from rabbits or from humans (1).Now at least 14 serovars of U. urealyticum have beendescribed (6, 20). In characterizing the standard strains ofthe members of the more commonly accepted scheme (20)and as a prelude to studies of U. urealyticum adhesion, wereexamined the interaction of both laboratory-adapted andclinical isolates of U. urealyticum with guinea pig RBCs.Mycoplasma cultures were incubated for 3 days on solid

    media (15, 28) under appropriate atmospheric conditions.Fresh blood was put into an acid-citrate-dextrose anticoag-ulant solution and washed three times, and the RBCs weresuspended in a dextrose-gelatin-Veronal buffer (2). Stocksolutions were kept at 5°C; working solutions had an absorb-ance of about 1.8 at 600 nm. We used a hemadsorptionmethod similar to one in widespread use for identifying M.pneumoniae (23). We flooded each petri plate (60 by 15 mm)with 5 ml of RBC working solution, incubated it at 36°C for20 to 30 min, removed the solution, and gently washed theagar surface three times with 5 ml of sterile Dulbeccophosphate-buffered saline (pH 7.2). Because washing mightdislodge RBCs from colonies of U. urealyticum 27, wemonitored, with an inverted microscope at x100, the num-ber of RBCs on the colonies after the last two washes. We

    * Corresponding author.

    counted adherent RBCs on 25 consecutive colonies andusually obtained a mean of between 10 and 30 per RBC.Strains with adherent RBCs on all colonies and a minimummean of five RBCs on each colony were judged positive.Both positive (M. pneumoniae ATCC 15531 and U. urealyti-cum 27, the serovar 3 standard) and negative (Mycoplasmahominis ATCC 14027 and U. urealyticum 7, the serovar 1standard) control strains gave the expected results.Given similar distribution and incubation time, colonies of

    U. urealyticum (Fig. 1K and L) were, as expected, smallerthan those of M. pneumoniae (Fig. 1M). The adherence ofRBCs to ureaplasma colonies differed quantitatively andqualitatively from adherence to colonies of M. pneumoniae.Figures 1L and M show typical hemadsorption patterns ofthese two species. On the basis of surface area, markedlymore RBCs were associated with M. pneumoniae (Fig. 1M)than with U. urealyticum (Fig. 1L). Because of this, RBCson the ureaplasma colonies appeared larger. Unlike M.pneumoniae, which eventually lyses RBCs by hydrogenperoxide activity (25), we noted no decrease in the numberof RBCs on positive ureaplasma colonies incubated for 24 h.For U. urealyticum to participate in hemadsorption, thecolonies as well as the RBCs had to be fresh; for M.pneumoniae, colonies could be stored for weeks and theRBCs for 1 to 2 months before loss of hemadsorbance.Unlike M. pneumoniae (24), hemadsorption by U. urealyti-cum was not blocked by pretreatment with homologous(anti-U. urealyticum 27) antiserum. To test this, we floodedthe plates with serum diluted 1:50 in 0.85% NaCl, incubatedthem for 2 h at 36°C, and then washed them twice beforetesting. Heterologous (anti-U. urealyticum T960 [CX8], theserovar 8 standard) antiserum and normal rabbit serum wereused as controls.We first compared the performance of RBCs from the

    three guinea pig strains available locally. All hemadsorbed toM. pneumoniae and were agglutinated by influenza virus towithin three doubling dilutions of the initial titer for at least2 months, the period over which measurements were made.Both M. pneumoniae hemadsorption and influenza virushemagglutination involve N-acetylneuraminic acid receptorson RBCs (4, 24). Attachment is mediated by the P1 proteinadhesion of M. pneumoniae (5) and the well-defined hemag-glutinin glycoprotein of influenza virus (4). With U. urealyti-cum 27, all RBC preparations gave positive results initially,but their ability to hemadsorb deteriorated at different rates.For example, after storage for 6 days at 5°C, RBCs from allHartley guinea pigs (five male and five female), all fivefemale but only two of the five male strain 13 guinea pigs,

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  • 2204 NOTES

    K

    L

    fe._

    FIG. 1. (A to C) Scanning electron micrographs of colonies of M. pneumoniae viewed from above (A) and laterally (B). Placing the agarplug into the fixative with colony facing upward (C) gave the RBCs a palisade effect around the periphery of a very small colony. Bars, 10~Lm. (D to G) Scanning electron microscopy of colonies of U. urealyticum 27 through the evolution of the preparatory method. Prints (D) werefound to b'e of two types (E and F) by advancing prefixation. The arrows in the x5 magnification (F) indicate RBCs; the brightly rimmed areasare the craters made by them. A lateral view of the same colony (G) suggests that the floors of the craters were molded by the RBCs. Arrows(F and G) also identify crevices between the surface of the colony and RBCs; these may be artifactual or indicative of lysis. Bars, 10 pLm. (Hto J) Further advancement of prefixation and placement of the colony face-downward at postfixation caught some RBCs detaching from thecolonies (H and I). Individual ureaplasma cells had coccoid morphology (H and J). Bars, 1 lim (H and J) and 10 [Lm (I). (K to M) Bright-fieldmicroscopy showed that the typical "fried-egg" colonies of U. urealyticum 27 without (K) or with (L) adherent RBCs were notably smallerthan colonies of M. pneumoniae (M). Differences in the patterns of RBC binding to these two species altered the apparent size of RBCs underthe same total magnification. Bar, 100 ~Lm (K to M).

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  • NOTES 2205

    and two of the three male Chase guinea pigs gave positiveresults. After 12 days, only 7 of the 10 Hartley RBCpreparations gave positive results. Hartley RBCs which hadbeen taken less than 1 week previously were used exclu-sively thereafter.We tested many strains of U. urealyticum. Of the 14

    serovars (20), only strain 27, the serovar 3 standard, showedhemadsorption. This confirmed that, unlike M. pneumoniae,hemadsorption by U. urealyticum was not species specific(1). We next examined other laboratory-adapted strains thatwe had found either by a modified metabolic inhibition test(19) or by the colony epifluorescence test (26) to carry theserovar 3 determinant: FT5 (from F. T. Black, Aarhus,Denmark); NIH3 (from M. F. Barile, Bethesda, Md.); strain113T (from G. J. Delisle, Queens University, Kingston,Ontario, Canada); the Boston T strain (from R. Kundsin,Peter Bent Brigham Hospital, Boston, Mass.); strains U9and U30 (strains from the serotyping scheme of J.-S. L. Lin[6], Channing Laboratory, Harvard University, Cambridge,Mass.); and four strains isolated locally. Only two of them,FB3 and NIH3, both higher passage numbers of the serovar3 standard, hemadsorbed. In case the ability to hemadsorbcould be lost by repeated in vitro transfer, we examined 118consecutive isolates of U. urealyticum obtained from malesattending a sexually transmitted disease clinic at this univer-sity. Of the 84 strains examined on primary plating, only 1was hemadsorption positive; all 34 isolates examined on3-day-old primary subcultures were negative. This strength-ened our observation that hemadsorbance was not linked tothe serovar 3 determinant. Admittedly, these particularclinic strains had not been serotyped. However, the type 3serovar determinant is one of the most commonly encoun-tered; it has been found in 29 to 50% of isolates in the largestsurveys conducted by ourselves (17, 21) or by others (12,22). In the present study, less than 1% of all wild-type strainshemadsorbed. Another strain of particular interest, 681/74,which lacks the serovar 3 determinant but has an extramem-branous layer (18), failed to hemadsorb.With the loan of a Hitachi S2500 scanning electron micro-

    scope, we examined these RBC-ureaplasma interactions ingreater detail at a magnification of x40. Agar plugs withhemadsorbing colonies were placed in 3.5% glutaraldehydefor 30 min at ambient temperature and washed twice in 0.1 Mcacodylate buffer (pH 7.2). Because the conductive metalcoating usually used to limit charging could obscure ordestroy morphological features, we used osmium-tannicacid-osmium staining (11, 27) instead. Obtaining high reso-lution at relatively low accelerating voltages reduced speci-men damage which could further obliterate detail. Ourprotocol was 1 h postfixation in 1%OS04, three washes, 1 hin 1% tannic acid, three washes, and a 1-h final fixation in 2%OSO4. The plugs were again washed, dehydrated in a gradedseries of alcohols, and dried in a critical point dryer.Viewed from above by scanning electron microscopy (Fig.

    1A), hemadsorption by M. pneumoniae presented an impres-sion similar to that given by bright-field microscopy (Fig.1M). One side view (Fig. 1B) emphasized the tight packing ofthe RBCs onto the surface of colonies of this species andanother (Fig. 1C) emphasized the earlier-described tenacityof the initial RBC-M. pneumoniae interaction (13). Ourinitial examination by scanning electron microscopy of hem-adsorption by colonies of U. urealyticum was less satisfac-tory. We saw only a few sparsely distributed "prints" on thesurface of the colonies (Fig. 1D). These prints had diametersof about 6 to 6.5,um, i.e., similar to the diameter of guineapig RBCs. Because we had noted the loss of some RBCs on

    cutting the plugs, we included a fixative step in situ, i.e., theaddition of a final concentration of 0.8% glutaraldehyde for30 min before excising the plugs. The prints were found to beof two types (Fig. 1E and F): one type consisted of individualRBCs and the other type represented craters in the colonysurface. A lateral view (Fig. 1G) confirmed these observa-tions. Sparsely distributed, flattened RBCs were embeddedin the colony and flush with its surface (Fig. 1G). The craterspresumably had been vacated by RBCs during the first wash,when the concentration of RBCs was too great to permit themonitoring of adherence. The markedly different relation-ship of the RBCs to the ureaplasma and M. pneumoniaecolonies and the reason for the discrepancy in apparent RBCsizes (Fig. 1L and M) was evident. Because of their heavierconcentration or binding pattern, RBCs were not so closelyopposed to the colonies of M. pneumoniae (Fig. 1B) as tothose of U. urealyticum. We further modified the methodused for the preparation for scanning electron microscopy;prefixation preceded the first wash. Few RBCs bound to theagar; more importantly, we had fixed some RBCs emergingfrom their craters (Fig. 1H and I). One can discern individualureaplasma cells. The craters were about two to four urea-plasmas or 1 to 1.5 ,um deep (Fig. 1H); cellular diameterswere relatively uniform at about 0.4 to 0.5 ,um (Fig. 1J).Companion preparations, coated with gold instead of receiv-ing conductive staining, were examined in the same micro-scope, but the topography of the colonies was obscured.Because of the fragility of the RBC-ureaplasma interac-

    tion, we questioned whether our negative test results withmost ureaplasma strains had resulted from the disturbance ofthe first wash. We retested the serovar standards but nowfixed them before any washes. When the RBCs were enu-merated, the positive ureaplasma control had a mean of 25RBCs per colony, within the upper range of counts obtainedin the initial tests. However, now, instead of being fewerthan 5 RBCs per colony, the mean counts of the negativecontrol and four other strains were between 10 and 20 RBCs,while those for six other strains were between 5 and 9 RBCsper colony, meeting our stated criteria for positivity. By thisapproach, 2 instead of 13 of the serotype standards weredeemed negative. This pattern was reproduced when fixingwith formaldehyde vapor instead of adding glutaraldehyde tothe test plates. By reducing the effects of manipulation, wehad identified a second, more tenuous association that existsbetween guinea pig RBCs and most U. urealyticum serovarstandards. Had this been the result of nonspecific interac-tions, we would have expected more RBCs on the surface ofthe strain 27 control and, perhaps, nonspecific binding to theagar surface. Because the Hitachi microscope was no longeravailable, the samples were examined in a Philips SEM 505.Viewed in this older instrument, not even the prints of RBCson colonies of strain 27 could be discerned in preparationsgiven conductive staining.

    Despite the considerable attention given to interactionsbetween RBCs and procaryotes, the adverse effect of RBCson a procaryotic organism is a novel observation. Becausevariation in the mechanisms of hemadsorption exist withinthe genus Mycoplasma (24), variation within the familyMycoplasmataceae cannot be considered unusual. The cra-ters could have resulted from ureaplasmal cells being com-pressed, displaced, or lysed by the RBCs. Rough measure-ments of ureaplasma cells in the craters (Fig. 1A) wereconsistent with earlier morphometric analysis (16) and thusdid not support compression. We ascertained that the bufferhad no deleterious effect on ureaplasma viability. A logarith-mic-phase culture of one of the two apparently nonhemad-

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    sorbing strains was diluted 1:10 in buffer and in 0.85%(wt/vol) NaCi at 36°C. Samples taken at 30 min and 3 h werediluted in 1:10 series in broth. At 18 h and 2 and 5 days ofincubation, the titers for test and control cultures wereidentical and at the expected levels. Because the guinea pigRBCs had been washed free of plasma and then stored underconditions unfavorable to the notoriously labile comple-ment, complement-mediated lysis is not a feasible explana-tion. Furthermore, exposure to homologous antiserum dur-ing the hemadsorption inhibition test did not reduceadherence. Evidently, U. urealyticum can lyse human andguinea pig RBCs under certain conditions (9). It may,indirectly, be able to initiate its own dissolution. Althoughcorrelation of this phenomenon with systemic ureaplasmainfections would be speculative, the fate of the ureaplasmacells is intriguing in its own right and will be given furtherattention.

    Able technical assistance was provided by the late DarleneMcAllister and by Jocelyne Kakulphimp. Ming H. Chen providedlight micrographs using equipment provided by the Alberta HeritageFoundation for Medical Research. Influenza virus PR8 was kindlyprovided by Linda Chui. We thank Nissei Sangyo Canada Inc. forthe loan of the Hitachi S2500 scanning electron microscope.

    This work was supported by a grant from the Medical ResearchCouncil, Ottawa, Ontario, Canada.

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    12. Naessens, A., W. Foulon, J. Breynaert, and S. Lauwers. 1988.Serotypes of Ureaplasma urealyticum isolated from normalpregnant women and patients with pregnancy complications. J.Clin. Microbiol. 26:319-322.

    13. Razin, S., M. Banal, H. Gamliel, A. Pollack, W. Bredt, and I.Kahane. 1980. Scanning electron microscopy of mycoplasmasadhering to erythrocytes. Infect. Immun. 30:538-546.

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    20. Robertson, J. A., and G. W. Stemke. 1982. Expanded serotypingscheme for Ureaplasma urealyticum strains isolated from hu-mans. J. Clin. Microbiol. 15:873-878.

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    26. Stemke, G. W., and J. A. Robertson. 1981. Modified colonyindirect epifluorescence test for serotyping Ureaplasma ure-alyticum and an adaption to detect a common antigenic speci-ficity. J. Clin. Microbiol. 14:852-854.

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Vol. 59, No. 6 INFECTION AND IMMUNITY, June 1991, p. 2203-2206 0019-9567/91/062203-04$02.00/0 Copyright C) 1991, American Society for Microbiology Hemadsorption by Colonies of Ureaplasma urealyticum JANET A. ROBERTSON* AND RICHARD SHERBURNE Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Received 31 October 1990/Accepted 14 March 1991 Hemadsorption by colonies of Ureaplasma urealyticum and Mycoplasma pneumoniae differed quantitatively and qualitatively. Using standard methodology, few strains of U. urealyticum hemadsorbed; with a modified method, most strains hemadsorbed, indicating a second type of association. Scanning electron microscopy of tannin-osmium-stained preparations showed guinea pig erythrocytes embedded in ureaplasma colonies and craters left when erythrocytes were dislodged. The genus Ureaplasma of the family Mycoplasmataceae, class Mollicutes, is composed of small procaryotes lacking a cell wall and hydrolyzing urea (14). Ureaplasma urealyticum is a common inhabitant of the human urogenital tract where it is an opportunistic pathogen (10). Although pathogenesis is not understood, the ability to adhere to mucosal cells is believed to be requisite for infection. The phenomena of hemagglutination and hemadsorption have proved useful in studying the attachment of microor- ganisms to eucaryotic cells. Initial studies of adherence of members of the class Mollicutes utilized the association between erythrocytes (RBCs) and colonies cultivated on agar medium. These studies were primarily concerned with the human pathogen Mycoplasma pneumoniae (3) and the animal pathogens Mycoplasma gallisepticum and Myco- plasma pulmonis (7, 24). When it was found that the incor- poration of a zwitterion buffer into the agar medium gave larger colonies of U. urealyticum and thus easier visualiza- tion, hemadsorption by colonies of this species was also demonstrated (8). Of the then eight- known serovars of U. urealyticum, only colonies of the serovar 3 standard (strain 27) hemadsorbed RBCs; hemadsorption occurred with RBCs from guinea pigs but not from rabbits or from humans (1). Now at least 14 serovars of U. urealyticum have been described (6, 20). In characterizing the standard strains of the members of the more commonly accepted scheme (20) and as a prelude to studies of U. urealyticum adhesion, we reexamined the interaction of both laboratory-adapted and clinical isolates of U. urealyticum with guinea pig RBCs. Mycoplasma cultures were incubated for 3 days on solid media (15, 28) under appropriate atmospheric conditions. Fresh blood was put into an acid-citrate-dextrose anticoag- ulant solution and washed three times, and the RBCs were suspended in a dextrose-gelatin-Veronal buffer (2). Stock solutions were kept at 5°C; working solutions had an absorb- ance of about 1.8 at 600 nm. We used a hemadsorption method similar to one in widespread use for identifying M. pneumoniae (23). We flooded each petri plate (60 by 15 mm) with 5 ml of RBC working solution, incubated it at 36°C for 20 to 30 min, removed the solution, and gently washed the agar surface three times with 5 ml of sterile Dulbecco phosphate-buffered saline (pH 7.2). Because washing might dislodge RBCs from colonies of U. urealyticum 27, we monitored, with an inverted microscope at x100, the num- ber of RBCs on the colonies after the last two washes. We * Corresponding author. counted adherent RBCs on 25 consecutive colonies and usually obtained a mean of between 10 and 30 per RBC. Strains with adherent RBCs on all colonies and a minimum mean of five RBCs on each colony were judged positive. Both positive (M. pneumoniae ATCC 15531 and U. urealyti- cum 27, the serovar 3 standard) and negative (Mycoplasma hominis ATCC 14027 and U. urealyticum 7, the serovar 1 standard) control strains gave the expected results. Given similar distribution and incubation time, colonies of U. urealyticum (Fig. 1K and L) were, as expected, smaller than those of M. pneumoniae (Fig. 1M). The adherence of RBCs to ureaplasma colonies differed quantitatively and qualitatively from adherence to colonies of M. pneumoniae. Figures 1L and M show typical hemadsorption patterns of these two species. On the basis of surface area, markedly more RBCs were associated with M. pneumoniae (Fig. 1M) than with U. urealyticum (Fig. 1L). Because of this, RBCs on the ureaplasma colonies appeared larger. Unlike M. pneumoniae, which eventually lyses RBCs by hydrogen peroxide activity (25), we noted no decrease in the number of RBCs on positive ureaplasma colonies incubated for 24 h. For U. urealyticum to participate in hemadsorption, the colonies as well as the RBCs had to be fresh; for M. pneumoniae, colonies could be stored for weeks and the RBCs for 1 to 2 months before loss of hemadsorbance. Unlike M. pneumoniae (24), hemadsorption by U. urealyti- cum was not blocked by pretreatment with homologous (anti-U. urealyticum 27) antiserum. To test this, we flooded the plates with serum diluted 1:50 in 0.85% NaCl, incubated them for 2 h at 36°C, and then washed them twice before testing. Heterologous (anti-U. urealyticum T960 [CX8], the serovar 8 standard) antiserum and normal rabbit serum were used as controls. We first compared the performance of RBCs from the three guinea pig strains available locally. All hemadsorbed to M. pneumoniae and were agglutinated by influenza virus to within three doubling dilutions of the initial titer for at least 2 months, the period over which measurements were made. Both M. pneumoniae hemadsorption and influenza virus hemagglutination involve N-acetylneuraminic acid receptors on RBCs (4, 24). Attachment is mediated by the P1 protein adhesion of M. pneumoniae (5) and the well-defined hemag- glutinin glycoprotein of influenza virus (4). With U. urealyti- cum 27, all RBC preparations gave positive results initially, but their ability to hemadsorb deteriorated at different rates. For example, after storage for 6 days at 5°C, RBCs from all Hartley guinea pigs (five male and five female), all five female but only two of the five male strain 13 guinea pigs, 2203 on April 1, 2021 by guest http://iai.asm.org/ Downloaded from
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