Mycopathologia vol. 58, 2, pag. 107 114, 1976

EX VIVO DETERMINATION OF POTENTIALLY VIRULENT SPOROTHRIX SCHENCKII John J. TAYLOR Department of Microbiology, University of Montana, Missoula 59801

Keywords: P y r o l y s i s - g a s - liquid chromatography, Sporothrix schenckii, Ophiostoma (Ascomyceteae), clinical mycology

Abstract Hyphae from 30 isolants of Sporothrix and Ophiostoma species were washed, dried and pyrolyzed at 350~ Pyrolysis products were separated on a Carbowax column heated 7.5~ to and maintained for 50 rain at 160~ Hydrogen flame detector responses were recorded graphically. Fifteen clinical isolants of S. schenckii from geographically separated sources produced qualitatively identical pyrograms. S. foliorum, 8 avirulent S. schenckii and other Sporothrix species isolants from soils, and Sporothrix states of 60phiostoma species yielded pyrograms readily distinguished from each other and from those of virulent S. schenckii. Taxonomic and clinical implications of the pyrograms are mentioned.

Introduction De Hoog (1) and Weijman & de Hoog (2) adequately characterized and differentiated morphologically several genera of sympodulosporous fungi, including the f o r m - genus Sporothrix Hektoen & Perkins. The descriptive data on the latter could be universally beneficial to clinical mycologists were it not that (a) some clinical isolants are not compatible with published discriptions, and (b) the clinical significance of the isolant may be equivocal where Sporothrix and morphologically related species are readily isolated from environmental sources. For example, not only is S. schenckii a morphologically, structurally, and antigenically heterogeneous f o r m - s p e c i e s w i t h both virulent and avirulent forms (3, 4, 6, 8, 12), but Sporothrix states of several Ophiostoma (Ascomyceteae) species may also infect man and other animals (7, 9, 11). Thus generic, specific and/or antigenic determinations of clinical isolants

cannot be considered tantamount per se to demonstrations of infectivity and may not be presumptive evidence of mycosis. In seeking, therefore, to correlate animal virulence with readily determinable in vitro aspects of young laboratory cultures of Sporothrix species, we found a modification of the p y r o l y s i s - g a s - liquid chromatography methods of Kulik & Vincent (5) and of Sprung & Wujek (10) experimentally and perhaps clinically useful.

Materials and Methods A Packard model 409 chromatograph was fitted with matched 244 • 0.216 cm stainless steel columns packed with Gas Chrome Q (Applied Science Laboratories, Inc., State College, Pa.) coated to 10% (w/w) with 20 M Carbowax. It was equipped for programmed control of column temperature. The pyrolysis furnace consisted of a heat - resistant glass tube about 1.5 • 20 cm, one - half of which was wrapped with electrical heating tape and encased in asbestos. The unheated portion included a sidearm through which nitrogen passed into the furnace, and a sealable port to admit samples (Fig. 1). Samples for pyrolysis were prepared from t h r i c e - washed hyphae sedimented from shake cultures grown for 5 days at 28~ in Czapek - Dox liquid medium containing 0.2% yeast extract. Suspended to 20% (v/v) in distilled water, the hyphae were fragmented for 3 min in a food blender at high speed, and a sufficient volume of the c e l l - w a l l - protoplasm mixture was added to aluminum combustion boats to yield 0.5 to 3 mg after drying at 22~ over anhydrous CaCI2 for 12 hr. After a sample was introduced into the unheated end of the pyrolysis unit, the port was closed and the unit purged with nitrogen. The sample was then moved with the aid of a 107

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Fig. 1. Diagrammatic instrumental layout used for this study. The heated tube leading from the furnace to the column prevented condensation of pyrolytic products. D = detector. magnetic couple to the heated portion of the unit and pyrolyzed at 350~ Nitrogen was allowed to flow through the pyrolysis chamber for 5 min to sweep pyrolysis products into the chromatograph column, then was diverted from the furnace directly into the column for fractionation of products. Column temperature was increased 7.5~ from an ambient 35 to 160~ at which temperature the columns remained isothermal for 50 rain. No additional pyrolysis products could be detected subsequently, and the columns were cooled. The total time elapsed for one complete run was 85 min.

Results From 15 clinical isolants ofS. schenckii received from Japan, India, Europe, Africa, South America, North America and New Zealand, three from the Center for Disease Control, Atlanta, Georgia, were chosen as reference standards. A pyrogram from one of these is reproduced in Fig. 2. Three highly volatile pyrolysis products were detected while the column was being loaded. The fourth pyrogram deflection Occurred at the beginning of the temperature program (column temperature = 40~ while the most abundant pyrolysis product(s) (at deflection 19) left the column immediately after the column reached isothermal 108

(160~ The last product left the column about 65 rain after pyrolysis. There were no qualitative differences among pyrograms of standards whether from replicate samples of a single reference isolant, or from individual samples of all three isolants. Pyrograms from the remaining clinical isolants, including forms from sources widely separated geographically, were also indistinguishable from each other and from pyrograms of standards (Figs. 3 and 4). Interestingly, virulent isolants of S. schenckii from New Zealand and from North Africa, both of which produced ascigerous states determined to be Ophiostoma stenoceras, produced pyrograms which were nearly identical with those from anascigerous isolants (Fig. 5 ) However, pyrolysis products from conidial O. stenoceras isolants included two slowly - moving components not found in entirely anascigerous forms. In contrast to the uniformity observed among pyrograms from clinical S. schenckii, avirulent S. schenckii and other Sporothrix species isolated from soils, as well as Sporothrix states of several Ophiostoma species other than O. stenoceras, produced pyrograms qualitatively different from each other and from those of the clinical isolants. For example, the pyrograms from two avirulent soil isolants orS. schenckii are reproduced in Figs. 6 and 7. Similarly, the

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Fig. 6. Pyrogram of an avirulent soil isolant of S. schenckii. Note accessory peak between peaks 7 and 8, and absence o~ l~r For details, see Fig. 1 and text.

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Fig. 8. Pyrogram ofO. gossypina. Small arrows mark qualitative differences from pyrograms ofS. schenckii and O. stelwceras. pyrogram from the Sporothrix state ofO. gossypina* (Fig. 8) differentiated it from S. schenckii and from O. stenoceras. An undetermined Sporothrix species which resembled S. schenckii in cultures, but which lacked virulence for laboratory animals, was also distinguished by its pyrogram (Fig. 9). Because some of the pyrogram deflections which permitted qualitative differentiation among isolants were weak (though reproducible), and in a further attempt to segregate clinical from non - clinical isolants on the basis of their pyrograms, peak - height ratios were also calculated (5) with peak 28 as reference. Corresponding ratios from all clinical isolants were pooled and the 't' test applied to determine whether ratios from each n o n - clinical isolant could be statistically excluded from the pool. All avirulent isolants pyrolyzed could be distinguished by five or more pyrogram peaks with p e a k - height ratios significantly different from ratios of corresponding peaks

from clinical strains. For example, statistical analysis of the pyrogram reproduced in fig. 7 revealed probabilities less than 0.001 that peak - height ratios for peaks 1, 3 through 8, 11, 13, 16, 17, 22 and 26 were comparable with analogous ratios from pyrograms of clinical isolants (Fig. 2-5). The pyrolysis products which differentiated among Sporothrix and Ophiostoma species and isolants have not yet been identified, although correlations of particular peaks with strain antigens and cell wall rhamnomannan type have been attempted. Products at some peaks are being examined currently. Too few cultures have been pyrolyzed to prepare pyrogram profiles for species other than S. schenckii. Our pyrograms do, however, provide further evidence for the heterogeneity of this f o r m - species, and may provide additional data for taxonomic and clinical clarification of the S. schenckii complex.

* Because Ophiostoma H. & P. Sydow has been differentiated from Ceratocystis Ellis & Halstead (1, 2), the following transfer is proposed: Ophiostoma gossypina (Davidson) J. Taylor comb. nov. -~ Ceratocystis gossypina Davidson, Mycologia 63: 12-13. 1971.

Conclusion

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From these studies with Sporothrix.and apparently related ascigerous species, it appears possible to remove and

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Fig. 9. Pyrogram of undetermined Sporothrix species isolant from soil. Note the distinctive deflection comprising peaks 27-29. and other qualitative differences (small arrows) from pyrograms of S. schenckii. For details, see Fig. 1 and text.

pyrolyze fungal hyphae directly from microcolonies or other young, n o n - sporulated laboratory cultures or natural substrates, fractionate the pyrolytic products by gas - liquid chromatography, and, through analysis of the resulting pyrograms, determine not only the probable identity of the fungus and its systematic relationships, but also its potential significance in clinical laboratory cultures. Further examination of the clinical applications of this procedure seems warranted.

References 1. Hoog, G.S. de. 1974. The genera Blastobotrys, Sporothrix, Calcarisporium and Calcarisporiella gen. nov. Stud. Mycol. (Baarn)7: 1-84. 2. Weijman, A.C.M. & G.S. de Hoog. 1975. On the subdivision of the genus Ceratocystis. Ant. van Leeuwen. 41: 353-360. 3. Howard, D.H. & G.F. Orr. 1963. Comparison of strains of Sporotrichum schenckii isolated from nature. J. Bact. 85: 816-821.

4. Ishizaki, H., R. Wheat & N.F. Conant. 1974. Serological cross reactivity between Sporothrix schenckii and Ceratocystis species. Abst. Ann. Mtg. Amer. Soc. Microbiol., p. 140. 5. Kulik, M. M. & P. G. Vincent 1973. Pyrolysis - gas liquid chromatography of fungi. Mycopathol Mycol. Appl. 51: 1-18; 251-265. 6. Mackinnon, J.E., I.A. Conti-Diaz, E. Gezuele, E. Civila & S. da Luz. 1969. Isolation of Sporothrix schenckii from nature and considerations of its pathogenicity and ecology. Sabouraudia 7: 38-45. 7. Nicot, J. & F. Mariat. 1973. Caracteres morphologiques et position systematique de Sporothrix schenckii, agent de la sporotrichose humaine. Mycopathol. Mycol. Appl. 49: 53q55. 8. Nishikawa, T., T. Harada, S. Harada & H. Hatano. 1975. Serologic differences in strains of Sporothrix schenckii. Sabouraudia 13: 285-290. 9. R u s h - Munro, F.M. 1971. Personal communication. 10. Sprung, D. G. & D. E. Wujek. 1971. Chemotaxonomic studies of Pleurastrum Chodat by means of pyroly-

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sis - gas - liquid chromatography. Phycol. 10: 251-254. 11. Taylor, J.J. 1970. A comparison of some Ceratocystis species with Sporothrix schenckii. Mycopathol. Mycol. Appl. 42: 233-240. 12. Travassos, L.R., P.A.J. Gorin & K.O. Lloyd. 1973. Comparison of the rhamnomannans from the human pathogen Sporothrix schenckii with those from the Ceratocystis species. Inf. Immun. 8: 686-693.

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