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' 111,1 11,11 :'1:‘ , 11111.11. "1-111.1.“11"'.'.a"1'"..1'.!"."."1 111111 1' 1 11 1|! 1111'1'1'11'1'1'111111'1" 1"11'1'11"'1 ,11, '1111 1111111 1"""" 1311.11”??? 1,111"."'1f{1,«1'1'1"1$ 11,11 1,111 11111111111‘1'1111 111,1'111111'1,'1""11 ”1111'1"11"'111"'11,'1|(1'111'1",1G'1111 '1 1,11111' "11111;”;1'15‘1'” “111.11 111111'11'1111111'H '1 "1'1111 1111111111111 111111111111 11111' ' "11111111111111.1111M1. LIBRARY Michigan State University This is to certify that the thesis entitled STUDIES ON THE TAXONOMY, PHYSIOLOGY, AND NUTRITION 0F CORYNEBACTERIUM SUIS presented by Jon G. Wegienek has been accepted towards fulfillment of the requirements for Master of Science degree in Microbioiogy Major professor Date //// 7/5)] 0-7639 MSU LIBRARIES \— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wil] be charged if book is returned after the date stamped beiow. STUDIES ON THE TAXONOMY, PHYSIOLOGY, AND NUTRITION 0F CORYNEBACTERIUM SUIS By Jon Geraldine Negienek A THESIS Submitted to Michigan State University in partia] fulfiiiment of the requirements for the degree of MASTER OF SCIENCE Department of Microbioiogy and PubTic HeaIth 1981 ABSTRACT STUDIES ON THE TAXONOMY, PHYSIOLOGY. AND NUTRITION OF CORYNEBACTERIUM SUIS BY Jon 6. Negienek Studies on the taxonomy, metabolism and nutritional requirements of Strain 50052 of Corynebacterium suis were performed. 9, suis, an anaerobic, Gram-positive, nonsporeforming rod.fermented maltose, starch, and glycogen and produced formate, acetate, and ethanol, but not propionate, as the major metabolic products. It was urease-posi- tive, but negative for other biochemical tests. Rhamnose and lysine were the major cell wall components of this organism. Major amounts of b- type cytochrome and minor amounts of c-type cytochrome were demonstrated in the cell extracts. Guanine and cytosine content was 55%. Based on these data it was proposed that Q. suis be transferred to the genus Eubacterium as E. suis. Nutritional studies showed that a fermentable carbohydrate, ribo- flavin, nicotinic acid, pyridoxal, adenine, and uracil were required for optimal growth. CO2 was stimulatory but not required for growth. The results further suggested a possible peptide requirement for growth of Strain 50052. ACKNOWLEDGEMENTS I would like to thank Dr. C. A. Reddy for his help and guidance and for the opportunity to learn about research in his laboratory. Dr. Maria Patterson and Dr. Gordon Carter, members of the guidance committee, gave helpful advice and moral support over the years, and I extend my appreciation to both of them. Excellent technical assistance from C. P. Cornell and Jean Fraser is gratefully acknowledged. Thanks are also due to the Department of Microbiology and Public Health for providing finan- cial assistance during a portion of this study. Finally, my special thanks go to C. P. Cornell, Larry Forney, Cathy Potrikus, Pamela Salvas, and to the staff of the laboratory at Olin Health Center for their unflagging interest and support. True friends, all. 11' TABLE OF CONTENTS List of Tables .......................... List of Figures .............. . ........... I Introduction. . . . . . . . . . . ..... . . . ........ Literature Review ......................... Genus Corynebacterium. . . . ........... . ...... Aerobic to Facultatively Anaerobic Corynebacteria ........ Anaerobic Corynebacteria .................... Corynebacterium suis ...................... Disease and Epidemiology ............. . ..... Pathogenicity . . . ......... . . . . . . . . . . . . Morphological, Biochemical, and Serological Characteristics. . Literature Cited ...... . . . ..... . . . . . . ..... Chapter 1, Article l - Taxonomic Study of Corynebacterium suis Soltys and Spratling: Proposal of Eubacterium suis (Nomen Revictum) Comb. Nov. Abstract ............... . ........ . . . . Introduction ......................... Materials and Methods . . . .................. Results ............................ Discussion .......................... Literature Cited ........................ Chapter 2, Article 2 - Certain Nutritional and Metabolic Studies on Eubacterium suis Abstract ............................ Page v vi AOkONMOOU 10 ll 15 21 22 24 29 41 48 52 Introduction ........................... 53 Materials and Methods ....................... 55 Results. . . .I .......................... 59 Discussion ........... ' ................. 70 _Literature Cited ............. . ...... ; ..... 74 iv Table LIST OF TABLES Page Literature Review Selected characteristics of corynebacteria pathogenic to animals and man. . . ......... . ....... . 5 Biochemical characteristics of different.strains of £0 SU‘SOO O O O O O O 000000 I O O O O O 0000000 12 Article 1_ Effect of pH on growth of Soltys 50052 strain in PYM medium. . . . ..................... 34 Selected characteristics of Soltys 50052 strain ...... 36 Characteristics that differentiate Soltys 50052 from nonbutyrate producing species of the genus Eubacterium ..................... 44 Article _2_ Fermentation balance for g, suis grown with maltose or starch as the energy source ................ 61 Effect of certain single and double deletions from PYS on growth of E, suis .................... 63 Effect of certain deletions from a modified PYS medium on growth of E, suis .................... 66 Figure 1.A. 1.3. 1.C-D. 2.A-C. «th LIST OF FIGURES . Page Article _1_ Phase - contrast photomicrograph of’Soltys 50052 grown in PYM medium for 24 h. . . . . . . . . . ...... 30 Thin section electron micrographs of Soltys 50052 grown for 36 h ....................... 30 Thin section electron micrographs of SOltys 50052 grown for 36 h ....................... 31 Thin section electron micrographs of Soltys 50052 grown for 36 h ....................... 32 Dithionite-reduced vs air-oxidized difference spectrum and air-oxidized vs air-oxidized dif- ference spectrum of a cell extract of Soltys 50052 containing 15 mg of protein/ml ............... 38 Dithionite-reduced vs air-oxidized difference spectrum and air-oxidized vs air-oxidized difference spectrum of a pyridine hemochrome from acid extract of Soltys 50052. 39 Dithionite-reduced vs air-oxidized difference spectrum and air-oxidized vs air-oxidized difference spectrum of pyridine hemochrome from acid-acetone residue of Soltys 50052 cell extract ................. 40 Article.g Growth of E. suis in PYS and PYM .............. 60 Effect of single deletions from PYS on growth of_§. suis. . 62 Effect of vitamins on growth of E. suis .......... 67 Effect of purine and pyrimidine bases on growth of g. suis. 68 vi INTRODUCTION Infectious cystitis and pyelonephritis as disease entities of pigs have been recognized since the beginning of this century (18). Earliest investigators isolated various diphtheroid bacteria, E. 9911, streptococci, and staphylococci from the urine and kidneys of affected animals (18, 41, 73). It was suggested that the predominant diphtheroid bacterium isolated from a majority of the cases be named Corynebacterium polymorphum §u_i_§ (18, 73). In 1957 Soltys and Spratling further investigated cases of cysti- tis and pyelonephritis in pigs on several farms in England, isolated an obligately anaerobic bacterium in pure culture from the bladders of six animals, and named the organism Corynebacterium suis (67). These results plus the uniformity of lesions and the distribution of coryneform bacteria in histological sections suggested a specific infectious disease. These workers were unable to establish whether or not this organism was identi- cal to g, polymorphum suis described earlier because the latter organism was not isolated in pure culture. In 1961 Soltys reported isolating 10 more strains of Q, suis from the kidneys of sows showing clinical and pathological signs of pyelonephritis and cystitis and 9 others from the semen of normal boars (66). Since then, g, suj§_infections in pigs have been reported from other parts of the world. Aalvik (1), Percy (58), Frijlink (23), and Munro and Wong (55) described the isolation of the or- ganism from individual cases occurring in Norway, Canada, the Netherlands, and Hong Kong, respectively. Larsen (40) and Dijkstra (19) described 1 several strains isolated in Denmark. In the largest survey reported, Narucka and Nestendorp from the Netherlands examined 57 sows with cysti- tis and pyelonephritis and found C, suis as the causative agent in 38 of them (56, 57). According to Blood and Henderson (8), the disease is also well-recognized in swine practice today in the U.S., but case re- ports are lacking. ‘These early investigators used various anaerobic methodologies, all of which predated the use of more efficient modern anaerobic techniques (35, 47, 70), in characterizing their isolates. None of them undertook any detailed taxonomic, physiological or metabolic studies of this organ- ism. As such 9, suis remains an inadequately described species and is not recognized in the 8th edition of Bergey's Manual. Since 9, suis is the only known obligately anaerobic corynebacter- ium which is pathogenic to animals, we sought to answer the following questions: 1) What is the relationship of this organism to aerobic and facultatively anaerobic animal pathogenic corynebacteria?; 2) Is it re- lated to human pathogenic anaerobic coryneforms, which have been recently reclassified as propionibacteria on the basis of metabolic end products and cell wall composition (37, 51)?; 3) What are the nutritional require- ments of g, suis}; and 4) What are the products of metabolism of starch and glucose by this organism? LITERATURE REVIEW Genus Corynebacterium The genus Corynebacterium as described by Lehmann and Neumann in 1896 consisted of a group of Gram-positive, nonsporing, rod-shaped bac- teria, most of which were aerobic, nonmotile, showed varying degrees of pleomorphism, and were pathogenic for animals and man (44). Eventually, however, the terms "coryneform" and "diphtheroid" were used to describe almost any Gram-positive rod which was club-shaped or had irregularly staining segments or granules, so that at present this group also contains many saprophytic species and plant pathogens (63). It has been shown that individual species vary considerably in biochemical characteristics, motility, acid end products, cell wall composition, and deoxyribonucleic acid base ratios (3, 4, 14, 15, 16, 27, 36, 37, 39, 43, 45, 59, 61, 62, 63, 72, 75). Aerobic to FacultativelyiAnaerobic Corynebacteria According to the 8th edition of Bergey's Manual, genus Qggyng- bacterium (63) includes seven species of human and animal pathogenic coryneform organisms which are aerobic or facultatively anaerobic, non- sporeforming, and characterized by cell walls containing arabinose and galactose as the major sugar components and mesa-diaminopimelic acid (meso~DAP) as the major diamino acid. .9. diphtheriae is the etiological agent of diphtheria in humans (3, 63) but reportedly infects animals as well (30). Contagious bovine pyelonephritis, caused by C, renale, is 3 a disease which affects cows more often than bulls, with clinical cases showing a high mortality rate (8). g, bgvis has been found as a com- mensal on cows' udders (63) but has been implicated in bovine mastitis (12). g, kutscheri is a frequent parasite in mice and rats (7, 24). It can, however, cause pseudotuberculous lesions in animals whose resis- tance to infection has been lowered (63). 9,,gseudotuberculosis is an important pathogen primarily causing caseous lymphadenitis in sheep and ulcerative lymphahgitis in horses (5, 10, 63). g, gggj was originally isolated from pneumonia in foals, but has also been found in the genital tract of mares and submaxillary lymph glands of swine (9, 13, 63). IQ. pyogenes is reported to be the most frequently isolated organism from sup- purative infections in a wide variety of domestic animals (10, 60). It has also been implicated in serious infections in man (2, 11, 22, 42, 46, 71). . ' Some selected biochemical characteristics of these organisms are shown in Table 1. All except 9, pyogenes are catalase-positive and lac- tose-negative and do not hydrolyze gelatin. All except 9, gggi ferment glucose; the fermentation of other sugars varies among species. Several species are urease-positive. Only 9. pyogenes produces acid coagulation and peptonization of litmus milk; 9. regal; produces an alkaline reac- tion in the same medium. It has recently been shown that the human and animal pathogenic corynebacteria are metabolically heterogeneous and can be distinguished from one another on the basis of metabolic end products of carbohydrate fermentation (61). This study is the first to have examined such pro- ducts formed by aerobic to facultatively anaerobic pathogenic corynebac- teria; these results are also shown in Table 1. All strains of g, 9123: theriae and g, pseudotuberculosis studied produced major amounts of . .mcwmxp .mzp memo: isms; .mgc monouapm .apm "mmoccae .cms .mmouompmo .Pmm momocwamea .mcm mvwom uwpwspaocwsm_u-0mms .aFuamm= .- “covuuomg xamz .3 "mgmmsm see; ovum co m>_u_moa .+u .mcpocum Fpm x5 vmuavocn no: mummsucmcma :_ muuzvoga .Aps ooH\cms “Hwy muuzuoca so=_s acmmmcawc mgouump ammo -333 .2... 8:35 HM v 3269:. Lone... 2323.. 9333 8333: .38 3583 .m 3.8m 0.53.3.8 £8 mu_om o_=oraogq .g muFum uwumom—mxo .mo “upon o_uoap .4 mwpom opacoe .g muwua upcuszm .am ”crow upumom .oa am :Pm .cme .Pam .mcm .m :owuoaumc mumcpwz apoc:_ mmmpmumo owned: mm: :Fumpmw mmopxx mmopmsmgh mmogoaw socmum :_owpmm mmocezoomm mmocamzx + + + + l to \ H I + + + + + + + + + + + + mmoccez Fouwccmz mmoupmz mmopa>m4 mmouomg pocmoxaw mmouzpo amouompmw mmoaozgm :_gaxma «mo:_nmc< xawpvuoz mmcoam m>wa_moausmeu o_aogmmc< “my aaomxnpa Afiv AHV “NHV Aumv Hy Amy A_V x=_pn_cu .Pu um agovcmumoz co: :mmcmb xp>pm< xoema can mxuzcmz ace accoz Amv weapon «Amy acpppmeam was manpom ovumpcmuoeceso mean .0 mo wavegum “cosmeepu we mopumvcmuomeego _uowsmzoowm .N m—nmh 13 .a=__ax_a spo;m_Fm .xm .mcwpmx—m .xv .uocwmem m:_mcum eo o>PHFmoa m:_mcum mo cmnszzo .:o_umn:u=w eo mane «H Levee copuomman .xvzum use c_ umu:_u=_ m:_mcum we coaszzm .A.e.o=oov N opaae 14 Antimicrobial susceptibility testing was performed by Soltys and Spratling using dilution methods (67). Various quantities of penicillin, streptomycin, chloramphenicol, and different tetracycline drugs were added individually to tubes of nutrient broth containing 10% serum. Only chloramphenicol and oxytetracycline prevented growth of C, suj§_at lower concentrations than that needed to inhibit growth of Staphylococcus aurgus in the control tubes. The investigators suggested that these two drugs might be the best agents to use against the disease. None of the other reports dealt with susceptibility testing. Serological testing of the organism was later performed by Soltys (66). Antisera were prepared to five strains of g, sgj§_and to one of 9,.rena1e by intravenous injection of formalized suspensions into rabbits. He reported that the results of precipitin tests suggested no antigenic relationship between the two organisms, although no data were presented to substantiate the claim. I Larsen attempted growing the organism in tissue culture in order to study its ability to establish itself intracellularly. He hypothe- sized that kidney epithelial cells and those of the reticuloendothelial system might act as "microniches" for the bacteria, thereby protecting them from antimicrobial drugs (40). The success achieved using this technique was limited due to the problems attendant to growing an anaero- bic organism in tissue culture; 10. 11. 12. 13. LITERATURE CITED Aalvik, B. 1968. Corynebacterium suis isolert fra et tilfelle av pyelonefritt hos purke. Nord. Vet. Med. 20;319-320. Ballard, 0. 0., A. E. Upsher, and D. E. Seeley. 1947. Infection ggtg Cogygebacterium pyogenes in man. Am. J. Clin. Pathol. 09 1 Barksdale, L. 1970. Corynebacterium diphtheriae and its relatives. Bacteriol. Rev. 343378-422. Barksdale, W. L., K. Li, C. S. Cummins, and H. Harris. 1957. The mutation of Carynebacterium pyogenes to Corynebacterium haemo— _, lyticum. J. Gen. Microbiol. 165749-758. Benham, C. L, A. Seaman, and M. Woodbine. 1962. Corynebacterium pseudotuberculosis and its role in diseases of animals. Vet. Bull. (London) 32: 645- 657. Bergey, D. H., et al. 1923. Bergey's manual of determinative bac- teriology.’ lst ed. The Williams & Wilkins Co., Baltimore. Bicks, V. A. 1957. Infection of laboratory mice with Corynebac- terium murium. Aust. J. Vet. Sci. 29520-22. Blood, 0. C., and J. A. Henderson. 1974. Diseases caused by Cor- ypebacterium sp., p. 300-307. In Veterinary Medicine, 4th Ed— The Williams & Wilkins Co., Baltimore. Bruner, D. W., and P. R. Edwards. 1941. Classification of Coryne- bacterium equi. Ky. Agr. Exp. Sta. Bull. 44592-107. Bruner, D. W., and J. H. Gillespie. 1973. Hagan's infectious di- seases of domestic animals, p. 308-323. Cornell University Press, Ithaca, N.Y. Chlosta, E. M., G. K. Richards, E. Wagner, and J. F. Holland. 1970. An opportunistic infection with Corynebacterium pyogenes produc- ing empyema. Am. J. Clin. Pathol. §§;167-170. Cobb, R. W., and J. K. Walley. 1962. Corynebacterium bovis as a probable cause of bovine mastitis. Vet. Rec. 145101-102. Cotchin, E. 1943. Corynebacterium eqpi in the submaxillary lymph nodes of swine. J. Comp. Pathol. .53:298-309. 15 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 16 Cummins, C. S. 1962. Chemical composition and antigenic struc- ture of cell walls of Corynebacterium, Mycobacterium, Nocardia, Actinomycetes, and Arthrobacter. J. Gen. Microbiol. 23535-42. Cummins, C. 5., and H. Harris. 1956. The chemical composition of the cell wall in some Gram-positive bacteria and its possible value as a taxonomic character. J. Gen. Microbiol. 143583-600. Cummins, C. S., and H. Harris. 1958. Studies on the cell wall composition and taxonomy of Actinomycetales and related groups. J. Gen. Microbiol. 183173-189. Davis, G. H., and K. G. Newton. 1969. Numerical taxonomy of some named coryneform bacteria. J. Gen. Microbiol. §§3195-214. Degen, K. 1907. Untersuchungen uber die hematogene eitrige neph- ritis des schweines. Diss, Giesen. Dijkstra, R. G. 1969. Cysto-pyelonephritis bij varkens veroar- zaakt door Corynebacterium suis. Tijdschr. Diergeneesk. 24; 393-394. Douglas, H. C., and S. E. Gunter. 1946. The taxonomic position of Corynebacterium acnes. J. Bacteriol. .§;:15-23. Fleming, Alexander. 1909. On the etiology of acne vulgaris and its treatment by vaccines. Lancet 131035-1038. . Forgeot, 9., P. Halbron, and M. Levy-Bruhl. 1940. Pyobacillase generalisée martelle chez un berger. Ann. Inst. Pasteur Paris 65:326-335. Frijlink, G. P. A., J. E. van Dijk, and J. Goudswaard. 1969. Een hemarrhagische-necrotiserende cysto-pyelonefritis bij een drach- tige zeug, veroarzaakt door Corynebacterium suis. Tijdschr. Diergeneesk. 245389-393. Giddens, W. E., Jr., K. K. Keahey, G. R. Carter, and C. K., White- hair. 1968. Pneumonia in rats due to infection with Corynebac- terium kutcheri. Pathol. Vet. 53227-237. Gilchrist, T. C. 1900. A bacteriological and microscopical study of over 300 vesicular and pustular lesions of the skin, with a research upon the etiology of acne vulgaris. Johns Hopkins Hosp. Rept..g:409-430. Gilchrist, R. C. 1903. The etiology of acne vulgaris. J. Cutaneous Dis. 213107-120. Goodfellow, M., and G. Alderson. 1977. The actinamycete genus Rhodococcus: a home for the rhodocharous complex. J. Gen. Microbial. 198599-122. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 17 Goodfellow, M., M. 0. Collins, and D. E. Minnikin. 1976. Thin- layer chromatographic analysis of mycolic acids and other long chain components in whole-organism methanalysates of coryneform and related taxa. J. Gen. Microbial. .263351-358. Gordon, R. E. 1966. Some strains in search of a genus - Coryne- bacterium, Mycobacterium, Nocardia, or what? J. Gen. Microbiol. 43:329-343. Greathead, M. M., and P. J. N. R. Bisschap. 1963. A report on the occurrence of g. diphtheriae in dairy cattle. S. Afr. Med. J.‘3Z:1261-1262. Gutierrez, J. 1953. Numbers and characteristics of lactate uti- lizing organisms in the rumen of cattle. J. Bacteriol. 44:123- 128. Halle, J., and A. Civatte. .1907. Contribution a la bacteriologie des glandes sebacées. Ann. dermatol. syphilig., 4 Serie 83 184-188. Harrington, B. J. 1966. A numberical taxonomic study of some cory- nebacteria and related organisms. J. Gen. Microbiol. 4§331-40. Hartwell, H. F., and E. C. Streeter. 1909. Bacillus of acne - B3 acnes. Boston Med. Surg. J. 4643882. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977. Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, Va. Jensen, H. L. 1952. The corynebacteria. Annu. Rev. Microbiol. 6:77-79. Johnson, J. L., and C. S. Cummins. 1972. Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryne- forms, classical propionibacteria and strains of Arachnia pro- pionica. J. Bacteriol. 49231047-1066. Jones, D. 1975. A numerical taxonomic study of coryneform and re- lated bacteria. J. Gen. Microbiol. 423229-252. Keddie, R. M., and B. L. Cure. 1977. The cell wall composition and distribution of free mycolic acids in named strains of cor- ynebacteria and in isolates from various natural sources. J. Appl. Bacterial. 423229-252. Larsen, J. L. 1970. Corynebacterium suis infectioner hos svin. Nord. Vet. Med. 223422-431. Larsen, N. 8., and E. Tondering. 1954. Nephritis interstitialis leucalymfocyturia hos svin. Nord. Vet. Med. 6335-46. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 18 Laufe, L. E. 1954. Acute ulcerative vulvovaginitis. Obstet. Gynecol. 3346-49. Lazar, I. 1968. Comparative morphological, cultural and biochemi- cal investigations on species of Corynebacterium from plants, animals and man. Rev. Roum. Biol. .433221-226. Lehmann, K. B., and R. O. Neumann. 1896. Bakteriologische Diag- nostik. Lehman verlag, Munich. Lelliott, R. A. 1966. The plant pathogenic coryneform bacteria. J. Appl. Bacterial. 293114-118. Maclean, P. 0., A. L. Averill, and A. A. Rosenberg. 1946. A hema- lytic carynebacterium resembling Corynebacterium ovis and Coryne- bacterium ngg§fl§_ 1n man J. Infect. Dis. 79. 69- 90. Mandle, R. J. and T. J. Wade. 1976. Use of gas chromatography for the identification of microorganisms, p. 163- 180. In J. E. Prier, J. T. Bartola, and H. Friedman (ed. ), Modern- methods in microbiology - systems and trends. University Park Press, Bal- timore. Molesworth, E. H. 1910. The cultural characteristics of the micro- bacillus of acne. Br. Med. J. 4:1227-1229. Moore, W. E. C. 1970. Relationship of metabolic products to tax- onomy of anaerobic bacteria. Int. J. Syst. Bacteriol. 29:535- 538. Moore, W. E. C. , and E. P. Cato. 1963. Validity of Pro ionibac- terium acnes (Gilchrist) Douglas and Gunter comb. nov. 3. Bac- terial. 85,:879- -874. Moore, W. E. C. , and L. V. Holdeman. 1974. Propionibacterium, p. 633-641. In R. E. Buchanan and N. E. Gibbons (ed. ), Bergey' 5 manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co. , Baltimore. Moss, C. W., and W. B. Cherry. 1967. Characterization of C 5 branched-chain fatty acids of Corynebacterium acnes by gag chromatography. J. Bacteriol. [253241-242. Moss, C. W., V. R. Dowell, Jr., D. Farshtchi, L. J. Raines, and W. 8. Cherry. 1969. Cultural characteristics and fatty acid composition of propionibacteria. J. Bacteriol. 923561-570. Moss, C. W., V. R. Dowell, Jr., V. J. Lewis, and M. A. Schekter. 1967. Cultural characteristics and fatty acid composition of Corynebacterium acnes. J. Bacteriol. 9431300-1305. Munro, R., and F. Wong. 1972. First isolation of'Corynebacterium Suis in Hong Kong. Br. Vet. J. 428329-32. 56. 57. 58. 59. 60. 61. . 62. 63. 64. 65. 66. 67. 68. 69. 19 Narucka, U., and J. F. Westendorp. 1971. Corynebacterium suis bij het varken. Tidjschr. Diergeneesk. .g§:399-404. Narucka, U., and J. F. Westendorp. 1972. Car nebacterium suis bij het varken II. 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CHAPTER 1 ARTICLE 1 TAXONOMIC STUDY OF CORYNEBACTERIUM SUIS SOLTYS AND SPRATLING: PROPOSAL OF EUBACTERIUM SUIS (NOMEN REVICTUM) COMB. NOV.1 J. Wegienek and C. Adinarayana Reddy Department of Microbiology and Public Health Michigan State University East Lansing, MI 48824 Running Title: Eubacterium suis (namen revictum) comb. nov. 1Journal article No. 9148 from the Michigan Agricultural Experiment Station. This paper has been submitted for publication in the International Journal of Systematic Bacteriology. ABSTRACT The name Corynebacterium suis (Soltys and Spratling 1957, 500) has not been included in the 1980 List of Bacterial Names and has no current taxonomic standing. This commonly occurring swine pathogen was studied to determine its taxonomic status. It is an anaerobic, Gram-positive, catalase-negative, nonmotile, nonsporulating, short to medium-sized rod and grows optimally at pH 7.0-8.0 and at 37°C. 0f 27 substrates tested,' it fermented only maltose, glycogen and starch. It is urease-positive, but is negative for other commonly employed biochemical tests. Growth in peptone-yeast extract-maltose medium is not enhanced by Tween 80, heme or menadione and is inhibited by bile. Rhamnose and lysine are the major components while mannose, glutamic acid and alanine are the minor components in cell walls of this organism. Acetate, ethanol and formate are the major metabolic products from maltose fermentation. Na detect- able levels of propionate are produced. Major amounts of b-type cyto- chrome and minor amounts of c-type cytochrome appear to be present in cell extracts. It has a guanine + cytosine content of 55% and is sensi- tive to penicillin-G, ampicillin, erythromycin, tetracycline, cephalo- thin and clindamycin. The above data indicate that this organism does not belong in the genus Corynebacterium. It is recommended that this or- ganism be transferred to the genus Eubacterium. Eubacterium suis (nomen revictum) comb. nov. is proposed and Soltys 50052 (ATCC 33144) is desig- nated the type strain. 21 INTRODUCTION Soltys and Spratling (42) in 1957 isolated an anaerobic diphtheroid bacterium associated with cases of cystitis and pyelonephritis in pigs, primarily sows (41), and proposed the name Corynebacterium suis for this organism. The genus designation was apparently based almost exclusively on the diphtheroid morphology of the organism, a common practice at the time. Neither the name Corynebacterium suis proposed by Soltys and Spratling nor the name Corynebacterium suis, Hauduroy et al., 1937, 167 . (14), was included in the 1980 Approved List of Bacterial Names (39) and has no current taxonomic standing. In the past few years cases of cys- titis and pyelonephritis in pigs due to this organism have been reported from Hong Kong (32), Canada (36), and other parts of the world (1, 9, 22, 33, 34). Also, 9, §gfi§_infections are apparently "well-recognized in swine practice" today in the U.S. (3). It, therefore, appeared impor- tant to obtain a better understanding of the taxonomic status of this swine pathogen utilizing modern anaerobic techniques (15). Using these techniques, Moore et al. (29) confirmed the earlier findings of Douglas and Gunter (10) that Corynebacterium acnes and other anaerobic coryne- bacteria belang in the genus Propionibacterium (30) since they produce major amounts of propionic and acetic acids, a feature characteristic of the latter genus. Furthermore, Cummins (6,7) showed that cell wall composition of anaerobic coryneforms was similar to that of propionic acid bacteria but was different from that of classical corynebacteria (38). However, the relatedness of 23.441; to anaerobic or aerobic to facultatively anaerobic coryneforms was not known. The objective of this study, therefore, was to determine the morphological, cultural and 22 23 biochemical characteristics (including metabolic end products, cytochrome content and cell wall composition) of Q. 4414 to obtain a better under- standing of its taxonomic status. The results of this study show that g, .441; belongs in the genus Eubacterium and g, 441; (nomen revictum) comb. nov. is proposed. [This work was presented in part at the 77th Annual Meeting of the American Society for Microbiology, 8-13 May, 1977, New Orleans, LA.] 1 24 MATERIALS AND METHODS Bacteria. A strain of Q. 441;, Soltys 50052, was obtained from M. A. Soltys, Ontario Veterinary College, Guelph, Ontario. M3413. Basal medium used in this study was modified prereduced-anaero- bically sterilized-peptone-yeast extract (PRAS-PY) medium described by Holdeman et al. (15). Minerals 1 and 2 (4 ml each) of Caldwell and Bryant (4) were used in place of the salts solution and the vitamin K- hemin solution was deleted. Carbohydrates or other substrates used in biochemical studies were added to this basal medium in concentrations recommended by Holdeman et al. (15), prior to the adjustment of pH. An oxygen-free CO2 gas phase was used, unless specified otherwise. L- cysteine-HCl hydrate was added to the medium immediately prior to heat- ing. The mixture was boiled under C02 in a round-bottomed flask (until the resazurin was decolorized), stappered, allowed to cool, and sterile, COZ-equilibrated 8% (W/V) sodium carbonate solution added (5 ml/IOO ml, of medium). The medium was then tubed in screw-capped Hungate tubes (Bellco, 3.5 ml/tube) or 18 x 150 mm rubber-stoppered test tubes (10 ml/ tube) and autoclaved (15 lb/inz for 15 min at 121°C). Solid medium was prepared by the incorporation of 2% agar (Difca) into the above medium. The composition of all other media was exactly as previously described (15). All incubations were at 37°C, except where indicated otherwise. The pH of cultures was determined using a pH meter and a combination elec- trode. Growth in liquid medium was estimated by determining the absor- bance at 600 nm (A600) in 18 x 150 mm tubes using a Bausch & Lamb Spec- tronic 20 spectrophotometer. 25 Culture maintenance. Slants of PRAS-PY maltose (PYM) medium were inocu- lated with a loapful of a suspension of the lyophilized organism in ap- proximately 0.5 ml of PYM broth, incubated at 37°C for 24 h, and stored at 4°C. Subcultures were made in the same slant medium once every 4 weeks. Purity was checked periodically by examination of Gram-stained smears and of wet mounts under a Zeiss phase contrast microscope, and by streaking plates of PRAS-supplemented Brain-heart infusion (BHI) agar or Trypticase soy agar plates, each supplemented with 5% sheep blood. BHI plates were incubated anaerobically in a GasPak jar (BBL) under C02’ and TSA plates were incubated aerobically. Morphological studies. Wet mounts of PYM broth cultures as well as the water of syneresis from PYM slant cultures were observed for the deter- mination of motility, using a Zeiss phase contrast microscope. For elec- tron microscopic studies, cells were fixed in 2.5% glutaraldehyde and 1% 0504, dehydrated, and embedded in Epon 812. Cell preparations were ex- amined with a Philips 300 electron microscope. Biochemical testipg_and gas chromatographic analyses. An actively grow- ing culture in PYM (A600 = 0.5) was used for inoculating various liquid and solid biochemical test media. All were incubated for at least 48 h after inoculation. Volatile and non-volatile acid end products in PYM were determined at the end of 48 h of incubation as described previously (37). A model 15C-3 Dohrmann gas chromatograph equipped with a reso- flex column was used. The column temperature was 120°C and the helium (carrier gas) flow rate was 120 cc/min. A 50 ml PYM culture was distilled according to the procedures of Neish (35) for the determination of alco- holic end products. The latter were analyzed by a Varian model 2440 gas chromatograph with an H2 flame ionization detector, and Porapak 0 column packing. The temperature of the column was 170°C, and the N2 (carrier 26 gas) flow rate was 30 ml/min. Determination of optimal temperature. For this experiment PYM broth was prepared as previously described except that phosphate buffer (P04; pH 7) was added to the medium, in place of sodium carbonate, to a final concentration of 0.02 M prior to the adjustment of pH. Also, N2 gas phase was used instead of 002. All tubes were inoculated with one loop- ful of a young culture (A600 = 0.2), and incubated at 25, 30, 37, and 43°C. Growth was recorded after 72 h. Mean A600 of triplicate tubes was used in reporting growth at each temperature. Determination of optimal pH. Ten ml aliquots of PYM buffered at pH 5, 6, 7, 8, 8.2, and 8.5 were used to determine the optimal pH for growth. Acetate, P04 or tris(hydroxymethyl)aminomethane-HCl (Tris) buffers were added as appropriate, in place of sodium carbonate, to give a final con- centration of 0.01 M, 0.02 M and 0.025 M, respectively. N2, instead of C02, gas phase was used. Media were inoculated with one loapful of a young culture (A600 - 0.2) and A600 was recorded every 12 h for 72 h. Cell wall analysis and DNA base composition. The cell wall composition of this organism was determined by C. S. Cummins of the VPI Anaerobe Lab- oratory, Blacksburg, VA., according to procedures previously described (8). John Johnson, also of the VPI Anaerobe Laboratory, determined the mole percent guanine plus cytosine (G + C) in deoxyribonucleic acid (DNA) by the thermal melting point (Tm) method (26, 20). Escherichia coli B DNA was used as the standard. Cytochromes. For cytochrome analyses, the organism was grown in 3 1 of PRAS-PY starch (PYS) medium supplemented with 0.25 mg hemin and 5 mg ferrous sulfate per 100 ml of medium. About 100 ml of a culture grown in PYS for 24 h was used as the inoculum. After incubation for 72 h, the bacteria were harvested by centrifugation at 16,300 x g for 15 min at 27 4°C, washed six times with four volumes of 0.02M P04 buffer, pH 7, re- suspended in the above buffer (1.0 g wet wt/ml) and a few crystals of Ribonuclease-A (Sigma) and Deoxyribonuclease 1 (Sigma) were added. This suspension was then disrupted by sonic treatment for 10 min, and recen- trifuged as above. The protein content of the supernatant (cell extract) was determined by the Lowry method (23), using crystalline bovine serum albumin (Sigma) as the standard. Cell extracts were stored at 4°C under C02 until analyzed. The presence of cytochromes in cell extracts was determined based on their difference spectra (5, 44). 'In this procedure, a few crystals of sodium dithionite were added to reduce the extract in the sample cu- vette and compared with air-oxidized extract in the reference cuvette. All spectral analyses were performed in 1 ml cuvettes (10 mm light path) at room temperature using a Varian model 634$ double beam recording spec- trophotometer connected to a Sargent-Welch model SR recorder. Modified procedures of Jacobs and Wolin (19) were used for heme extraction and characterization. Lyophilized cell extract containing at least 140 mg of protein was thoroughly mixed with 40 ml cold acetone in a homogenizer and centrifuged at 15,000 x g for 15 min at 4°C. This acetone wash was repeated once more and the protoheme in the washed pell let was extracted with 40 ml cold acetone containing 1% (V/V) of 2.4 N HC1. This extraction was repeated once more, and the extracts were pooled and dried under vacuum. The dried residue was then suspended in 3.5 ml pyridine plus 3.5 ml 0.2 N KOH and the difference spectrum of the alka- line pyridine hemochrome was obtained as described above. In order to detect the heme of cytochrome c, the pellet remaining after acid-acetone extraction was mixed as above with pyridine and KOH and this suspension was then spectrally analyzed. 28 Antimicrobial susceptibility testing. The disc-diffusion method of Wil- kins et al.(45) and the broth-disc method of Wilkins and Thiel (46) were used for antimicrobial susceptibility testing. 29 RESULTS Morphology and staining characteristics. Cells of Strain 50052 were Gram-positive although they easily decolorized, especially in old cul- tures, and often had a beaded appearance. Cells were not acid-fast. No spores were observed and the organism did not survive heating at 80°C for 10 min. Cells were non-motile, slender, pleomorphic, and rod-shaped ranging in size from 1 - 3/um x 0.5,um (Fig. 1.A). Cells were often found in clumps, palisades or at angles to each other (arrows in Fig. 1.A). Ultrastructural studies. Resolution of cell membrane, cell wall and fringe- like outer coat layers is clearly seen in thin section electron micro- graphs (Fig. 1.8, D and 2.A). Cytoplasmic membrane was tightly associ- ated with the cytoplasm as is typical of most Gram-positive organisms. At high magnifications (Fig. 2.C) cell walls of many cells had a multi- layered appearance as previously reported for Actinomyces and Eubacterium species (2,11). Large electron opaque areas were seen in many cells es- pecially at the poles. The nature of these structures is not known. Many rudimentary branching cells were seen. For example, in Fig. 1.8 two rounded budding cells are seen at an angle to the long hroizontal cell. Although the septa in this figure are not at right angles to the longitudinal axis of the cell and the completed septa are curved, the most frequent type of cell division appeared to be by the formation of septa at right angles to the longitudinal axis of the cell (Fig. 2.8). In this case septum formation was initiated by invagination of a portion of the cytoplasmic membrane at the division plane on either side of the cell and these invaginations become transformed into round or oval mem- branous structures resembling mesosomes (Fig. 1.0). Ingrowth of these structures and joining of the opposing mesosomes (Fig. 2.A) seemed to 3O .memce macoao cocuompo u oenn< .Et~.o mucmmocnwc Lem .; cm Low czocm «mocm mappom we :amemogowe :oguoopw co_pumm cwgh .m.fi wczmwm “-11 a, ,1. -11- .5 am com E=_uos z>m =_ cxocm Nmoom mxupom mo gqmcmoco_eouo;a umocacou-mmmga .<.~ «gnaw; 31 .m.~ me=m_u cw we mponexm .; on do» czoem Nmoom mxupom mo sneemoeu_s cocuuopm :owpomm :_;H .o.H meamwm .mmezaozeum maococasms umcw»mucz op some; mzogc< ;=«~.o mucmmwea .8; com .5 on Low czocu Nmoom maupom we gaoemocows coguompm :o_uumm zen» .u.H wgsmwm 32 Thin section electron micrographs of Soltys 50052 grown for 36 h. Symbols as in Figure 1.8. Figure 2.A-C. 33 result in complete septum formation (Fig. 2.8). Mesosome-like membranous organelles were often seen in the cells at sites other than division plane (Fig. 1.8 and 2.8) also. These intracytoplasmic membranous ele- ments appeared to be continuous with the cytoplasmic membrane. Undefined membranous structures, apparently formed by invagination of the cytoplas- mic membrane, were also occasionally seen in the cytoplasm (arrows, Fig. 1.6). Cultural characteristics. Colonies on blood agar plates, incubated an- aerobically for 40 h, were white, circular, and granular with entire to slightly irregular margins. A dense, slightly raised region in the cen- ter of each colony lent them a "fried-egg" appearance. After incuba- tion for 3 days at 37°C, an indefinite "hazy" beta hemolysis around the colonies was apparent, and most colonies were 0.5 to 2.0 mm in diameter. After one week, colonies often attained a diameter of 3 to 5 mm, were flatter, and the central raised region seen earlier became nearly indis- tinguishable from the rest of the colony. Colonies an anaerobic PYM agar were similar to those described above, although these appeared smoother than those obtained on blood agar plates. No growth was ob- tained, initially, an aerobic cultivation. After numerous subcultures, however, barely discernable pin-point colonies were detected after in- cubation in air or under 6% CO2 for 7 days. PYM broth supported moderately good growth of this organism (A600 = 0.6). Growth in PYM was not enhanced by the addition of Tween 80, hemin or menadione, and bile was inhibitory. Effect of pH and temperature on growth. This organism appeared to have a broad pH optimum between pH 7 and 8, while growth decreased substan- tially at pH less than 7 and greater than 8 (Table 1). 34 Table 1. Effect of pH on growth of Soltys 50052 strain in PYM medium .- ' Buffer pH System Growthi 5.0 Acetate 0.00 6.0 Phosphate 0.29 7.0 Phosphate I 0.59 8.0 Tris 0.56 8.2 Tris 0.38 8.5 Tris 0.05 'QGrowth expressed as mean absorbance at 600 nm of triplicate 18 X 100 mm tubes. 35 Optimal temperature for growth was 37°C (A600 = 0.58), although good growth was also obtained at 30°C (A600 = 0.52) and 43°C (A600 = 0.46). Growth was not detected at room temperature after 72 h. Biochemical characteristics. Soltys 50052 fermented only maltose, gly- cogen, and starch during 48 h of incubation, while the other sugars and sugar alcohols tested were not (Table 2). Xylose and ribose were attacked after 14 days of incubation. Lactate, pyruvate, and threonine were not metabolized. This organism was strongly urease-positive but was negative for the other commonly tested biochemical characteristics (Table 2). These results were confirmed for us by L. V. Holdeman at the VPI Anaerobe Laboratory. The present results are in agreement with those of Soltys (41) who performed a limited number of tests and showed that maltose was fermented by all strains of 23‘4415, and that lactose, salicin, mannitol, levulose and galatose wererunzfermented by any; furthermore, the strains tested were positive for urease but failed to produce indole or liquefy gelatin. However, Soltys reported an alkaline reaction after 7 days in- cubation in litmus milk but we did not see any change in the milk medium. Since metabolic products were shown to be important in classify- ing anaerobic bacteria (15, 28), fermentation products of this organism were determined. Ethanol, formate, and acetate were the major products (Table 2); lactate or succinate were only found in trace amounts; pro- pionate, butyrate or butanol were not found. Cell wall composition. The major sugar component of g, §gj§_cell walls was rhamnose; a small amount of glucosamine and a trace amount of man- nose were also present. The major diamino acid found was lysine; small amounts of glutamic acid and alanine were also found. 36 Table 2. Selected characteristics of Soltys 50052 strain Positive Reactions Negative Reactions Acid from: Maltose Glycogen Starch Hydrolysis of starch Growth at: 30°C 37°C 43°C Urease reaction Products from PYM: Acetateé Ethanolfl Formateé Sensitivity to: Penicillin G (2 U/ml) Erythromycin 3 ,ug Tetracycline 6 g Chloramphenico 12 [a Clindamycin 1.6 ,ug Ampicillin 4 F9 Cephalothin 6 pg Acid from: Amygdalin Arabinose Cellobiose Erythritol Fructose Galactose Glucose Inositol Lactose Mannitol Mannose Melezitose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Sucrose Trehalose Xylose Motility Spore production Growth at 25°C Gelatin liquefaction Digestion of meat Acid, curd or digestion of milk Nitrate reduction Indole production Catalase reaction Growth stimulation by: Hemin Menadione Bile (20%) Lecithinase Lipase Hemolysis Utilization of Pyruvate Lactate Threonine Production of: Acetylmethylcarbinol Hydrogen sulfide Ammonia Products from PYM: Propionate Succinate Lactate Butyrate Butanol 2Major products (a 1 meq/100 ml). 37 Cytochrome analyses. Earlier work by Meyer and Jones (27) indicated that cytochromes are relatively important in bacterial taxonomy. Dithionite- reduced vs. air-oxidized difference spectrum (Fig. 3) of cell extracts of Soltys 50052 grown in PYS supplemented with hemin (0.002% W/V) showed 4, p and 1 absorption maxima at 562, 530 and 430 nm, respectively, a pattern characteristic of a b-type cytochrome (18). The absorption maxi- ma of the pyridine hemochrome derivative of the protoheme were at 558, 525, and 423 nm, confirming the presence of a b-type cytochrome (Fig. 4). In contrast, the pyridine hemochrome of the residue left after acid- acetone extraction showed absorption maxima at 552, 523, and 417 nm (Fig. 5), a pattern characteristic of a c-type cytochrome. The results indicate that a b-type cytochrome is the major cytochrome in Q3‘ggig and that it completely masks a c-type cytochrome present in crude cell extracts. The presence of the c-type cytochrome became obvious only when the pyridine hemochrome of the mesoheme in acid acetone residue was examined. G + C content of DNA. The DNA base composition of this organism is 55 moles percent G + C. Antiobiotic susceptibility. This organism was sensitive to penicillin, ampicillin, erythromycin, tetracycline, cephalothin and Clindamycin when tested by the disc-diffusion method and broth-disc method (Table 2). These results were later confirmed by L. V. Holdeman at the VPI Anaerobe Laboratory. 38 8 Figure 3. Dithionite-reduced vs air-oxidized difference spectrum (solid line) and air-oxidized vs air-oxidized difference spectrum (broken line) of a cell extract of Soltys 50052 containing 15 mg of protein/ ml. 39 ABSORBANCE I l l l ’1 400 450 500 ON) 600 653 WAVELENGTH (nm) Figure 4. Dithionite-reduced vs air-oxidized difference spectrum (solid line) and air-oxidized vs air-oxidized difference spectrum (broken line) of a pyridine hemochrome from acid extract of Soltys 50052. The acid acetone extract from 140 mg of cell protein was resus- pended in 7 ml of pyridine-KOH. 4O NW ABSORBANCE I l I F 400 450 500 550 600’ 650 WAVELENGTH (nm) ' | 1 L__.v.. .--_~_ ,_ __L___ Figure 5. Dithionite-reduced vs air-oxidized difference spectrum (solid line) and air-oxidized vs air-oxidized difference spectrum (broken line) of pyridine hemochrome from acid-acetone residue of Soltys 50052 cell extract. The residue of 140 mg acid-acetone extracted cell protein was resuspended in 7 ml of pyridine-KOH. 41 DISCUSSION At the time Soltys and Spratling (42) named g, suis, it was a com- mon practice to arbitrarily assign any Gram-positive, nonsporeforming diphtheroid organism to the genus Corynebacterium. According to the 8th edition of Bergey's Manual, genus Corynebacterium (38) includes those coryneform organisms of human or animal origin which are aerobic or facul- tatively anaerobic, nonsporeforming, and characterized by cell walls containing arabinose and galactose as the major sugar components and meso-diaminopimelic acid (meso-DAP) as the major diamino acid. Further- more, it has recently been shown that strains of Q3 diphtheriae, the type species, and a closely related species, 9, pseudotuberculosis, produce major amounts of acetate, propionate, and formate and variable amounts of other acids as products of carbohydrate metabolism (37). In contrast, Soltys 50052 is anaerobic, has rhamnose and lysine as its major cell wall components and produces acetate, formate and ethanol, but not pro- pionate, as major and products of carbohydrate metabolism. It, therefore, appears that this organism does not belong in the genus Corynebacterium. Haemophilus vaginalis (21% recently reclassified as Gardnerella vaginalis (13), is a Gram-positive, coccoid rod which carries out fer- mentative metabolism of sugars and produces acetic acid as a major pro- duct; lactic and formic acids are also often produced. Although this organism is generally believed to be facultatively anaerobic, Malone et al. (25) recently isolated some obligately anaerobic strains of this or- ganism which are biochemically similar or identical to the facultatively anaerobic strains. Most strains ferment maltose and starch with acid but not gas. Cell walls of this organism contain lysine, but not 42 diaminopimelic acid as the major diamino acid, and contain 6-deoxytalose rather than arabinose. g, sgfl§_is similar to G. vaginalis in most of the characteristics described above except that it contains rhamnose as the major cell wall sugar (instead of 6-deoxytalose), produces ethanol in addition to acetate and formate as a major metabolic product, and does not ferment glucose. Furthermore, unlike 93 vaginali , Soltys 50052 grows well at pH 8.0, is not stimulated by Tween-80, shows no hemolysis or only a faint hemolysis, does not ferment fructose and arabinose and is negative for lipase production. Also, the moles percent G + C of g. vaginalis is 42 t 1 whereas that of Soltys 50052 is 55%. Thus, it is apparent that 231441; is different from 43 vaginalis. ' Douglas and Gunter (10) and Moore and Cato (29) showed that the hua man pathogen g3 gggg§_and related "anaerobic coryneforms" produced major amounts of propionate and acetate as end products of carbohydrate meta- bolism, and therefore actually belong in the genus Propionibacterium (30). Furthermore, cell walls of propionibacteria typically contain LL- DAP and galactose as the major diamino acid and sugar components, respec- tively (20). It can be seen that Soltys 50052 is quite different from propionibacteria in metabolic and products, in cell wall composition, and biochemical characteristics, and, therefore, cannot be classified as a member of the genus Propionibacterium. The results of this study show that Soltys 50052 does not belong in the genera Actinomyces,Bifidobacterium or Clostridium (15). Unlike bifidobacteria, it does not produce lactic acid as a major product. It is similar to Actinomyces in being Gram-positive, anaerobic, nonmotile, non-acid fast, havingdiphtheroid morphology. containing lysine in cell walls, and in containing cytochromes (7, 40, 43). However, in contrast to Actinomyces, it does not produce succinic acid (in the presence of 43 002) or lactic acid as major products, and products do not change in the presence of C02 (Wegienek and Reddy, unpublished data). It is also quite different from Actinomyces in biochemical tests (40). Members of the genus Clostridium are generally Gram-positive sporeforming rods, . "usually motile", and are not known to contain cytochromes, except in rare instances (12), and therefore are different from Soltys 50052. The genus Eubacterium (16) includes a rather diverse group of Gram- positive, obligately anaerobic (oxygen sensitivity varies between spe- cies and strains within a species), nonsporeforming rods, uniform or pleomorphic, motile or nonmotile, saccharoclastic or nonsaccharoclastic chemoorganotrophs which do not produce major amounts of propionate, lac- tate or succinate (singly or in combination ), but instead produce var- ied mixtures of other organic acids from carbohydrates or peptones. Based<fllthe above mentioned characteristics, Soltys 50052 appears to be- long to the genus Eubacterium. In addition, similar to most eubacteria, it is catala,se-negative and grows optimally at pH 7.0 and 37°C. Based on existing data, Soltys 50052 can be differentiated from the 37 other currently recognized species of the genus (15, 16). Certain characteristics helpful in differentiating Soltys 50052 from eight eubac- terial species which do not produce butyrate are given in Table 3. Un- like most of the species listed, Soltys 50052 ferments glycogen and starch but not glucose or fructose, and does not curdle or digest milk. It is also strongly urease-positive, a characteristic shared by only two other currently recognized members of the genus. It contains rhamnose as a major sugar component of the cell wall, a characteristic also re- ported for one species, 2. saburreum; however, information on the cell wall sugar component(s) of other eubacteria is not available. Similarly, while nine species have been shown to contain meso-DAP as the major cell 44 1 1 1 on on nu 10+ 0 to xpwz + 1 1 1+ 1 1 1 1 +1 mpmapocu»; zueeum + 1 1 13 1 31 1 31 31 :ueeum + 1 1 1 1 , 1 1 1 1 :mmooxpo 1 1 + 31 + 1+ + + +3 mmoozpw 1 1+ 11 1 1 1 1 1 1 ooeeewz 1 1 ,e 1 +1. 1 1 3+ 1 mmocsozm 1 1 + 1; a 1 a 1+ 31 emeeoaed 1 1 1 1 1 1 1 1 m+. mpoucm A_meev ANHmv Asa.m_ev 33a.em_v Away ANHmV A>,a_em_v ~L< L< < -L< ~eee we mu3weemge3=m .eepmemwe .e "ease .o ”xpwz mao.m e» o.m 33v ePee xee3 .3 mo.c.n we :3 Peewe 3e ce_peemc e>puemee .1 meezep 3e m.m we :3 Fee?» 3e cepueeeg e>wpwme3 .+ umpeeszmw .mcveepm Peceemeeee An Apce eeeeee33 wee memezueece3 :_ meeeee33 ._e:e;ae .N Newsweeem .m mew>=3>3 .33 muvcem3eg3 .3 mupueep .4 3e33e_e>em. .>H Newcaueeemw .mm me_53em .3 mopexuee .m muwueee .< ._E ooH\eeE fl v..meeuump Ppesm "guess FE ooH\e_ee ems Hmw maee_ecp meeuuep _eu_3eu .emeapes >3 seee eecvseepee e3e3 mmoom mxupem ee mueeeec3 .cueee emeeapo >3 see» eeepsceuee e3e3 mueeeec3 _pe .Nmoom emu—em gee u3mexmm .Aamv enseepe: ecu eemcsee .eeeez sece epemm .Asfiv egeez eee eeseepez Eece eueam .AoHV eceez eee :eaeepe: See» macaw .A.e.oeeo3 .m o.eae 46- wall diamino acid, this information is lacking for the rest of the species. In contrast, Soltys 50052 contained lysine as the major diamino acid. DNA base ratios of only a few species of this genus have been de- termined; these range from 31 to 45 moles percent G + C, values which are considerably lower than the 55 moles percent G + C found for this or- ganism. Since only a small number of organisms have been examined for males percent G + C, the importance of this characteristic in the tax- onomy of Eubacterium remains to be determined. Based on the results of this investigation, it is proposed that Corynebacterium suis Soltys and Spratling (42) be transferred to the ge- nus Eubacterium. The name Corynebacterium suis originally proposed for this organism was not included in the 1980 List of Bacterial Names (39) and has no current taxonomic standing. It is therefore proposed that g. 3141:; be transferred to the genus Eubacterium as _E_. _syfi (nomen re- victum) comb. nov. The type strain is Soltys 50052 (ATCC 33144). gpbacterium suis (nomen revictum) comb. nov. Slender, nonmotile, pleomorphic rods ‘1-3 by 0.5 pm; arranged in singles, pairs (often found at an angle to each other or in palisades) or small clusters. Gram-positive, but rather easily decolorized, especially in old cultures. Not acid-fast and non- sporulating; does not survive heating at 80°C for 10 min. Capsules not observed by capsule-staining; however, a fringe-like outer coat, exter- nal to the cell wall, is seen in thin section electron micrographs. Colonies an anaerobic blood agar plates are 0.5-3.0 mm at 48 h, white, circular, and granular with entire to slightly irregular margins. Colonies often show slightly raised centers, giving a "fried egg" ap- pearance. After one week, colonies are 3-5 mm and flatter. Growth is barely discernible after incubation for seven days under 6% 002 or in air. 47 Peptone-yeast extract-starch broth supports excellent growth. Optimal pH 7-8; no growth <.pH 5.0. Optimal temperature 37°C; range 30- 43°C; no growth at 22-23°C. Anaerobic; metabolism strictly fermentative. Maltose, starch and glycogen fermented. Acetate, ethanol, and formate are main products from maltose fermentation. Does not ferment: adonitol, amygdalin, ara- binose, cellobiose, dulcitol, erythritol, esculin, fructose, galactose, glucose, glycerol, inositol, inulin, lactose, mannitol, mannose, melezi- tose, melibiose, raffinose, rhamnose, salicin, sorbitol, sucrose, tre- halose, lactate, pyruvate and threonine. Strongly urease-positive. Does not: produce catalase, indole, acetylmethylcarbinol, hydrogen sul- fide, lipase, or lecithinase; ammonia from peptone; hydrolyze esculin or gelatin; digest meat or milk; or reduce nitrate. Major amounts of b-type cytochrome and minor amounts of c-type cytochrome synthesized. I Cell wall sugars are rhamnose and mannose; diamino acid of pepti- doglycan is lysine. The %G + C content of the DNA is 55%. Originally isolated from cases of cystitis and pyelonephritis and of metritis in pregnant sows (41, 42). Not isolated from healthy sows, but frequently recovered from urine and semen of apparently healthy boars. Saws can be artificially infected by intrarenal injection of live organisms plus 5% saponin (41). No demonstrable exotoxin produced. Type Strain: 50052 (ATCC 33144). IO. 11. 12. LITERATURE CITED Aalvik, B. 1968. Corynebacterium suis isolert fra et tilfelle av pyelonefritt hos purke. Nord. Vet. Med. 223319-320. Bladen, H. A., M. U. Nylen, and R. J. Fitzgerald. 1964. Internal structure of a EubacteriUm sp. demonstratedTby the negative staining technique. J. Bacteriol. 223763-770. Blood, 0. C., and J. A. Henderson. 1974. Cystitis and pyelone- phritis of pigs. In veterinary Medicine, 4th ed. Williams & Wilkins Company, BETtimore, p. 302-303. Caldwell, D. R., and M. P. Bryant. 1966. Medium without rumen. fluid for nonselective enumeration and isolation of rumen bac- teria. Appl. Microbiol. 443794-801. Chance, 8. 1954. Spectrophotometry of intracellular respiratory pigments. Science. 4293767-776. Cummins, C. S. 1962. Chemical composition and antigenic structure of cell walls of Corypebacterium, Mycobacterium, Nocardia, Act- inomyces and Arthrobacter. J. Gen. Microbiol. 22335-50. 22mmins, C. S., and H. Harris. 1956. The chemical composition of the cell waTTTin some Gram-positive bacteria and its possible value as a taxonomic character. J. Gen. Microbiol. 443583- 600. Cummins,,C. S., and J. L. Johnson. 1971. Taxonomy of the clas- tridia: Wall composition and DNA homologies in Clostridimm butyrium and other butyric acid producing clostridia. J. Gen. Microbiol. 24333-46. Dijkstra, R. G. 1969. Cysto-pyelonefritis bij varkens veroarzaakt doorCErynebacterium suis. Tijdschr. Diergeneesk. 243393- 394. Dogglas,H. C., and S. E. Gunter. 1946. The taxonomic position of Corynebacterium acnes. J. Bacteriol. .22:15-23. Duda, J. J.,,and J. M. Slack. 1972. Ultrastructural studies on the genus Actinomyces. J. Gen. Microbiol. 44363-68. Gottwa44,M4, J. R. Andreesen, J. LeGall, and L. G. Ljungdahl. 1975. Presence ofcytochrome and menaquinone in Clostridium formicgaceticum and Clostridium thermoaceticum. J. Bacteriol. 422:325-328. ' 48 '13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 49 Greenwood, J. R. and J. M. Pickett. 1980. Transfer of Haemophilus va inalis Gardner and Dukes to a new genus, Gardnerellag. 2. va inalis (Gardner and Dukes) comb. nov. Int. J. Syst. Bacter- {Joly—T. _3_:170-178. Hauduroy, P., G. Ehringer, A. Urbain, G. Guikot, and J. Magrou. 1937. Dictionnaire des bacteries pathogenes, Masson and Co., Paris, p. 1-597. Holdeman, L. V., E. P. Cato, and W. E. C, Moore (ed.). Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, VA. Holdeman, L. V. and W. E. C. Moore. 1974. Eubacterium, p. 641- 657. In R.1E} Buchanan and N. E. Gibbons (ed.), Bergey's man- ual of‘determinative bacteriology, 8th ed. The Williams a Wilkins Co., Baltimore. 'Holdeman, L. V., and W. E. C. Moore. 1974. New genus, Coprococcus, twelve new species, and emended descriptions of four previously described species of bacteria from human feces. Int. J. Syst. Bacteriol. 243260-277. International Union of Pure and Applied Chemistry and the Inter- pgtional Union of Biochemistry. 1973. RecommendationsT1972). Cytochromes. p. 34-38. .42 Enzyme nomenclature. Elsevier Scientific Publishing Co., Amsterdam. Jacobs, N. J., and M. J. Wolin. 1963. Electron-transport system of Vibrio succinogenes. I. Enzymes and cytochromes of the electron transport system. Biochem. Biophys. Acta. 22318-28. Johnson,,J. L,, and C. S. Cummins. 1972. Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria and strains of Arachnia propionica. J. Bacteriol. 42231047-1066. Lapage, S. P. 1974. Haemophilus va inalis, p. 368-370. 4g_R. E. Buc anan and N. E. Gibbons (ed.), Bergey's manual of determina- tive bacteriology. 8th ed. The Williams and Wilkins Co., Balti- more. Larsen, J. 1970. Car nebacterium suis infektioner hos svin. Nord. Veterinaermed. 22:422-431. Lowry, 0. H.,,N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 4223265-275. Macy, J., I. Probst,_and G. Gottschalk. 1975. Evidence for cyto- chrome involvement in fumarate reduction and adenosine 5'- triphosphate synthesis by Bacteroides fragilis grown in the pre- sence of hemin. J. Bacteriol. 4223436-442. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. SO Malone,48. H. M. Schreiber, N. J. Schneider, and L. V. Holdeman. 1975. Obligately anaerobic strains of Car nebacteriumv vaginale (Haemophilus vaginalis). J. Clin. Microb1ol. :27 - 75. Marmur, J., and P. Doty, 1962. Determination of the base compo- sition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 23109-118. Meyer,_0. J., and C. W. Jones. 1973. Distribution of cytochromes in bacteria: réTationship to general physiology. Int. J. Syst. Bacteriol. 23: 459-467. Moore, W. E. C. 1970. Relationship of metabolic products to taxon- omy of anaerobic bacteria. Int. J. Syst. Bacteriol. .22:535- 538. ' Moore,li. E. C, and E. P. Cato. 1963. Validity of Propionibacter- ium acnes (Gilchrist) Douglas and Gunter comb. nov. J. Bacter- 131. §_5: ___-870 874. Moore, W. E. C.,,and L V. Holdeman. 1974. Propionibacterium, p. 633- 641. In R. E. Buchanan and N. E. Gibbons (ed. )2 Bergey' 5 manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. Moore,_W. E. C., J. L. Johnson, and L. V. Holdeman. 1976. Emenda- tion of Bacteroidaceae and Butyr1vibr1o and descriptions of Desulfomonas gen. nov. and ten new species in the genera De- sulfomonas, Butyrivibrio, Eubacterium, Clostridium and Rumina- coccus. Int. J. Syst. Bacteriol. 26: 238-252. Munro, R., and F. Wong. 1972. First isolation of Corynebacterium suis in Hong Kong. Br. Vet. J. 422329-32. Narucka, U., and J. F. Westendorp. 1971. Corynebacterium suis bij het varken. Tidjschr. Diergeneesk. 223399-404. Narucka,,U., and J. F. Westendorp.1972.Cor nebacterium suis bij het varken II. Tidjschr. Diergeneesk. 97: 647-652.“ Neish, A. C. 1952. Analytical methods for bacterial fermentations. NRCC Report No. 46-8-3. p. 18. Perpy, D. H., H. L. Ruhnke,_and M. A. Soltys. 1966. A case of in- fectious cystitis and pyelonephritis of swine caused by Coryne- bacterium suis. Can. Vet. J. 43291-292. Reddy, C. A., and M. Kao. 1978. Value of metabolic products in identification of certain corynebacteria. J. Clin. Microbiol. 43428-433. Rogosa, M, C. S. Cummins,,R. A. Lelliott, and R. M. Keddie. 1974. Coryneform group of bacteria, p. 599- 632. In R. E. Buchanan and N. E. Gibbons (ed. ), Bergey' 5 manual of determinative bac- teriology, 8th ed. The Williams & Wilkins Co. , Baltimore. 39. 40. 41. 42. 43. 44. 45. 46. 51 Skerman,,V. B. D., V. McGowan, and P. H. A.,§neath. 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 393225-420. Slack, J. M. 1974. Actinomycetaceae, p. 659-681. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bac- teriology, 8th ed. The Williams 8 Wilkins Co., Baltimore. Soltys, M. A. 1961. Corynebacterium suis associated with a speci- fic cystitis and pyelonephritis in pigs. J. Pathol. Bacterial. 813441-446. Soltys, M. A., and F. R. Spratling. 1957. Infectious cystitus and pyelonephritis of pigs: a preliminary communication. Vet. Rec. 623500-504. Taptykova, S. D., and L. V. Kalakoutski. 1973. Low temperature cytochrome spectra of anaerobic actinamycetes. Int. J. Syst. Bacteriol. .g33468-471. White, D. C., M. P. Bryant, and D. R. Caldwell. 1962. Cytochrome- linked fermentation in Bacteroides rumincola. J. Bacteriol. 843822-828. Wilkins,AT. D.,,L. V. Holdeman, I. J. Abramsan, and W. E. C. Moore. 1972. A standardized single-disc method for antibiotic suscep- tibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. .1z451-459. Wilkins, T. D., and T. Thiel. 1973. A modified broth-disc method for testing the antibiotic susceptibility of anaerobic bacteria. Antimicrob. Agents Chemother. ‘3:350-356. CHAPTER 2 ARTICLE 2 Certain Nutritional and Metabolic Studies on Eubacterium suis J. Wegienek and C. Adinarayana Reddy Department of Microbiology and Public Health Michigan State University East Lansing, MI 48824 ABSTRACT Selected nutritional and metabolic studies on Eubacterium suis, an anaerobic animal pathogen, were performed. A medium (PYS) contain- ing Trypticase (Try), yeast extract (YE), starch, minerals, cysteine, and sodium carbonate was shown to support excellent growth of this or- ganism (A600=1.8). Growth was considerably'less'(A600 = 0.6) when starch in the latter medium was replaced by maltose (PYM). Fermen- tation products produced in both the media were similar and included for- mate, acetate, and ethanol. Deletion of starch or YE from PYS resulted in approximately four and 25% respectively, of the growth seen in the complete medium. Total growth decreased only about 20% in PYS from which 002 was rigorously excluded. Simultaneous deletion of YE and Try resulted in negligible growth. When YE in PYS was replaced by a defined mixture of purine and pyrimidine bases, vitamins, and amino acids, growth was 2 80% of that observed in PYS. Comparable growth was obtained on sub- stitution of adenine and uracil for the mixture of purine and pyrimidine bases, and pyridoxal, riboflavin, and nicotinic acid for the vitamin mixture. Substitution of a mixture of 20 amino acids for Try did not support detectable growth of the organism, indicating a possible peptide requirement for growth of E3 suis. The nutritional requirements of E3 suis reflect its successful adaptation to its natural niche by its having done away with certain unnecessary biosynthetic capabilities. 52 INTRODUCTION Soltys and Spratling were the first to isolate an obligately anaero- bic, Gram-positive, nonsporeforming bacterium from cases of infectious cystitis and pyelonephritis in pigs (27). They assigned this organism to the genus Corynebacterium primarily on the basis of its diphtheroid morphology, and proposed the name Q. suis. Cases of cystitis and pye- lonephritis due to g, suj§_have since been described by several other in- vestigators (1, 6, 8, 15-18, 20, 26) and the disease syndrome is appar- ently well-recognized in swine practice today (2). However, little was published about the taxonomy, physiology, and nutritional characteristics of this organism. Consequently Q. suis remains an inadequately described species and is not recognized in the 8th edition of Bergey's manual. Recent studies by Wegienek and Reddy showed that of the many sub- strates tested, 93 suj§_fermented only maltose, starch and glycogen, and produced acetate, formate and ethanol, but not propionate, as major end products of carbohydrate metabolism (31). Furthermore, it contained rhamnose and lysine as major cell wall components. These data suggested that Q3‘§31§.does not belong in either the genus Corynebacterium or in the genus Propionibacterium. It was proposed that the organism be trans- ferred to the genus Eubacterium as E. §!i§_(31), To date there have been no nutritional studies on E3‘sgis. Further- more, the nature of the metabolism of starch or glucose by this organism remains unknown. In this paper we have presented the results of our re- cent investigation on the nutrition and metabolism of g, suis. (This 53 54 work was presented in part at the 80th Annual Meeting of the American Society for Microbiology, 11-16 May, 1980, Miami, FL.) 55 MATERIALS AND METHODS Bacteria. g, suis, Strain Soltys 50052, was obtained from M. A. Soltys, Ontario Veterinary College, Guelph, Ontario. Mggia3 Basal medium (PY) used in most of these studies was modified pre- reduced-anaerobically sterilized-peptone-yeast extract (PRAS-PY) medium described by Holdeman et al. (12). The vitamin K-hemin solution was de- leted and 4 ml each of minerals 1 and 2 of Caldwell and Bryant (3) were added in place of the salts solution to give a final concn. of 0.48 mg (NH4)ZSO4 per ml of medium. Trypticase and yeast extract (10 mg/ml each) were added. In different experiments, either maltose or starch was added (10 mg/ml) as the primary energy source to the (PY) medium. These media are subsequently referred to as PYM and PYS, respectively. The pH of the media was adjusted to 6.7 with 10% NaOH. L-cysteine-HCl hydrate was added to media immediately prior to heating. All anaerobic procedures were carried out under 100% oxygen-free C02 gas phase except where mentioned otherwise. Media were prepared (31) and dispensed anaerobically (10 ml/ 18X150 mm tube), stappered, and autoclaved in a press (15 lb/in2 for 15 min at 121°C). A modified PYS medium containing charcoal-treated Trypticase (see below), and a defined mixture of purine and pyrimidine bases and vitamins (described below), in place of yeast extract, was used for determining purine, pyrimidine, and vitamin requirements. Oxygen-free N2 was the gas phase. To determine vitamin requirements, single vitamin solutions were added individually or in various combinations, at the concentrations indicated, to modified PYS without the vitamin mixture. Similarly, for determining purine and pyrimidine requirements, one or more individual stock solutions of these bases were added to modified PYS without the 56 purine P105 pyrimidine mixture. To determine nitrogen requirements for growth, Trypticase in modified PYS was replaced by a defined mixture of amino acids. Preparation of charcoal-treated Trypticase. In some experiments Trypti- 'case was charcoal-treated to remove vitamins and certain organic sub- stances. Charcoal (10 g) was added to a solution of Trypticase (10 g per 90 ml double distilled water) adjusted to pH 3.5 with glacial acetic acid. The solution was stirred for 1 h, filtered throughWhatman #1 filter paper, and its pH readjusted to 6.5 with 10 M NaOH. The solution was again mixed with charcoal (5 g), stirred for 1 h, refiltered, and its final volume adjusted to 200 ml. 20 ml of this Trypticase solution was added per 100 ml of medium. Vitamins. Individual aqueous stock solutions of different vitamins in double distilled water contained (mg/ml): Thiamine-H01, 20; PYridoxal- HCl, 5; calcium-D-pantothenate, 20; riboflavin, 20; nicotinic acid, 10; p-aminobenzoic acid, 1; biotin, 0.5; pyridoxine-HCl, 10; pyridoxamine-HCl, 5; and nicotinamide, 10. Vitamin solutions were filter-sterilized and stored in the dark at 4°C in acid-cleaned, sterile test tubes. One or more of these stocks were added (1% vol/vol) as needed aseptically and anaerobically to autoclaved, tubed media just prior to inoculation. All of the above vitamins were added to modified PYS for studying purine and pyrimidine requirements. Amino acids. The stock solution of L-amino acids in water contained (mg/100 ml): Alanine, 220; arginine, 180; asparagine, 100; aspartate, 250; glutamate, 940; glycine, 20; histidine, 80; hydroxyproline, 20; isoleucine, 260; leucine, 380; lysine, 300; methionine, 140; phenyla- lanine, ZOO; proline, 320; serine, 300; threonine, 160; tryptophan, 40; tyrosine, 260; and valine, 260. The solution was stored frozen and was 57 added (10% vol/vol) as needed to media prior to autoclaving. Purine and pyrimidine bases. Individual aqueous stock solutions (2 mg/ml) of adenine sulfate, guanine HCl'2H20, uracil, xanthine, and thymine were prepared. Guanine was solubilized by the addition of 10% HCl and xanthine was solubilized with 10% NaOH. The stocks were autoclaved and stored at 4°C in the dark in acid-cleaned sterile tubes. These solutions were added (1% vol/vol) as needed to autoclaved, tubed media just prior to inocu- lation. All the above bases were added to modified PYS for studying vitamin requirements. Preparation of inoculum for nutritional studies and growth conditions. Cells from a mid-log culture of g. suis grown in 3.5 ml PYS were asepti- cally harvested by centrifugation, washed twice with 3.5 ml of sterile sodium phosphate buffer (0.02 M; pH 7), and resuspended in the same buffer to give an absorbance of approximately 0.5 at 600 nm. One drop of this washed culture (v0.05 ml) from a sterile Pasteur pipette was used to inocu- late 10 ml of experimental medium. The organism was subcultured a few times in each experimental medium to minimize the effect of nutrient carryover from the original inoculum. All cultures were incubated at 37°C and growth was monitored with a Bausch & Lomb Spectronic 20 spectro- photometer by measuring the absorbance at 600 nm. Absorbance values given represent the mean of the values for triplicate tubes. Fermentation balance procedure and analyses. Cells were grown in 250 ml rubber-stoppered, round-bottomed flasks containing 100 ml of FY, PYM, or PYS. Each flask was inoculated with 5 ml of a mid-log culture grown in the respective medium. Uninoculated flasks of the above media served as negative controls. All flasks were incubated at 37°C for 72 h. The total amount of gas produced was calculated as previously described (22). 58 The proportion of H2 and C02 in the gas phase was assayed as previously described (28) with a Varian model 1400 gas chromatograph equipped with a thermal conductivity detector. A stainless steel column (3.2 mm x 1.8 m) packed with Porapak R (80/100 mesh) was used for CO2 determina- tions. Helium was the carrier gas (25 ml/min). A similar column packed with Molecular Sieve 5A (60/80 mesh) was used for H2 determinations; nitrogen was the carrier gas. Dissolved CO2 in the medium was estimated as previously described (22). At the end of incubation, the media were acidified with 1 ml of 50% H2S04 (vol/vol) and clarified by centrifugation. Portions of the clarified fermentation liquor (CFL) were assayed directly for 2,3 butan- ediol (5), acetoin and diacetyl (2), and total carbohydrate (13). A maltose standard curve was used for determining the total carbohydrate concentration (CHO) in PYM and PY, while a starch standard curve was used for determining CHO in PYS. Organic acids were extracted from CFL with ether (19) and were identified and quantified (12) by a Varian model 1400 gas chromatograph equipped with a thermal conductivity detector and a column packed with 15% SP 1220 - 1% H3PO4 on Chromosorb H AH (100/120 mesh). Helium was used as the carrier gas (25 ml/min). The column was maintained at 135°C; injector at 175°C; and detector at 195°C. Volatile distillates (19) of CFL were assayed for alcohol end products using a Varian model 2400 gas chromatograph with an H2 flame ionization detector. The column was packed with Porapak Q and was maintained at 170°C; H2 flow was 30 ml/min, and nitrogen (carrier gas) flow was 30 ml/min. Fermentation balance calculations were made according to the method of Hood (33). 59 RESULTS Fermentation Balances for Growth on Maltose and Starch. Growth in PYM was less than half that obtained in PYS (Fig. 1); however, fermentation products produced in PYM and PYS were qualitatively similar. The main metabolic products from starch and maltose included formate, acetate and ethanol (Table 1). Products not detected included diacetyl, acetoin, 2,3-butanediol, H2, 002, propionate, lactate, succinate, propanol, and butanol. C1 recovery ratios and OR balance values for both PYM and PYS suggest that an oxidized one-carbon compound (possibly formate) was mis- sing (Table 1). Formate may have been underestimated since the accuracy of the chromatographic method used for its determination diminished at high levels of formate. If it were assumed that formate was the missing product, then the Clzcz ratio for growth on PYM would be 1.0, the OR balance would be 1.08, and the carbon recovery value, 101%. A similar correction for growth on PYS would yield an OR balance of 1.33 and a car- bon recovery of 117. These somewhat elevated values could be attributed to the fact that the analytical procedure used for starch estimations was considerably less precise and underestimates starch values as compared to that for maltose. Effects of different components of PYS on growth. Excellent growth (A600 = 1.7) was obtained in PYS when the Trypticase level was modified to con- tain 0.0, 0.1, 0.2, 0:4, or 0.6 g per 100 ml of medium (data not shown). Although growth in PYS minus Trypticase was comparable to that in PYS (Table 2 and Fig. 2), it took considerably longer (81 h) for maximal absorbance to be reached in the former medium,indicating that Trypticase is stimulatory but not required for growth in this paricular medium. Growth was comparable in PYS or in PYS mOdified to. contain only 0.1% 2.0 1.0 5:’. (11 ABSORBANCE — 600 nm :3 '0 en .01 20 60 - #4", PYS PYM 4O 60 80 100 TIME-h Figure l. Growth curves for g, suis in PYS and PYM. Growth is expressed as in Table 2. 61 Table 1. Fermentation balance for E. suis grown with maltose or starch as the energy source PYMa vaa Product , (mmol/100 mmol hexose equiva- lent) Formate 156.9 155.2 Acetate 98.8 146.8 Ethanol . 83.0 ' 87.1 Carbon recovery (%) 96 ' 104 on balanceb 0.95 0.89 . C ° CI‘CZ . 0.86 0.66 aResults are the mean of the values obtained from at least two separate experiments. All values were corrected for products present in the un- inoculated medium and in inoculated PY medium. bOxidation-reduction balance. cRatio of one-carbon to two-carbon products. 62 ABSORBANCE - 600 nm 0 20 40 60 80 100 120 TIME-h Figure 2. Effect of single deletions from PYS on growth of g, suis. A, complete PYS; 8, minus Na CO3; C, minus Trypticase; 0, minus yeast extract; E, minus stargh. 63 Table 2. Effect of certain single and double deletions from PYS on growth of E. suis Deletion Growtha None 1.87 (41) Salts 1.80 (57) Cysteine (Cys) . 1.80 (41) Trypticase (Try) 1.70 (81) Na2003, 002 1.30 (41) Yeast Extract (YE) 0.70 (58) Starch 0.07 (61) Try + Cys 1.70 (58) YE + Cys 0.49 (63) Try + YE 0.03 (41) aGrowth is expressed as the mean of maximal absorbance in three 18 x 150 mm tubes at 600 nm. Numbers in parentheses refer to h of incubation required for maximal growth. 64 (wt/vol) yeast extract (data not shown). However, complete deletion of yeast extract from PYS resulted in only about one third as much growth as that observed in complete PYS (Table 2 and Fig. 2). These results show that neither Trypticase nor yeast extract alone serves as optimal nitrogen sources for growth in PYS medium. Thus, both yeast extract and Trypticase appear to contain nutrients required for optimal growth of g, §u1§_and either of these two substances could only partially replace the requirement for the other. In contrast, when both yeast extract and Trypticase were deleted from PYS, negligible growth resulted (Table 2). This suggests that E. suis is unable to use the ammonium sulfate present in the medium as a sole source 0f nitrogen. Deletion of soluble starch from PYS resulted in about 95% decrease in growth. This low level of growth was consistently seen even after several serial transfers in this medium. The small amount of growth seen in this medium may have been due to trace amounts of carbohydrate known to be present in Trypticase and yeast extract. To determine the nutritional requirement, if any, for CO2 for growth, 002 was rigorously excluded from PYS as previously described by Dehority (4) and N2 gas phase was used. Total growth in this medium decreased by about 20% (Table 2 and Fig. 2) but the rate of growth was not affected in comparison to that observed in PYS. This indicated that CO2 is stimu- latory but not required for growth. Growth was comparable in PYS and in PYS minus salts or cysteine. Interestingly, growth was substantially faster when both Trypticase and cysteine were deleted than when Trypticase alone was omitted from PYS (Table 2). Deletion of yeast extract decreased growth by about 70%, while simultaneous deletion of both yeast extract and cysteine further decreased growth, suggesting that cysteine at least partially satisfies 65 the organism's nitrogen requirement for growth in PYS minus yeast extract medium (Table 2 and Fig. 2). V In modified PYS medium containing a defined mixture of 20 amino acids, growth was 80% of that observed in PYS (Table 3). When Trypticase was then deleted from this medium, there was negligible growth when compared to that in PYS. In contrast, deletion of amino acids, instead of Trypticase, from modified PYS medium resulted in no decrease in growth; in fact, "slightly better growth (A600 = l.8) was observed in this medium as compared to that in modified PYS without deletions (A600 = 1.43; Table 3). This dramatic requirement for Trypticase for growth in a medium containing a full complement of amino acids and ammonium sulfate was rather surprising and strongly suggests a possible peptide requirement for the growth of this organisn. Vitamin Requirements. The growth response of E, suis to various additions of vitamins was tested in modified PYS minus vitamins as described in Methods. Growth was negligible when no vitamins were added (Fig. 3). Single additions of pyridoxal, riboflavin or nicotinic acid resulted in only marginal growth. Slight stimulation of growth was observed on additions of pyridoxal plus riboflavin, or pyridoxal plus nicotinic acid, but not by riboflavin plus nicotinic acid (data not shown). A combination of pyridoxal, riboflavin and nicotinic acid supported growth comparable to that obtained in the medium with the full complement of all ten vitamins. Purine-Pyrimidine Requirements. In modified PYS without the defined mixture of purines and pyrimidines or with only uracil present, growth was negligible (Fig. 4). The single addition of adenine resulted in a slight increase in growth. Addition of both adenine and uracil gave growth comparable to that obtained in the medium with the full complement of purine and 66 Table 3. Effect of certain deletions from a modified PYS mediun? on growth of _E_. suis Deletion Growthb None . 1.43 (41) Trypticase 0.04 (82) Amino acids 1.80 (87) aModified PYS is similar to PYS except that: i. yeast extract was replaced with a defined mixture of amino acids, vitamins and purine and pyrimidine bases as described in Methods; and ii. Trypticase was charcoal-treated. bGrowth is expressed as described in Table 2. 67 205 o o co I LLI 0 Z < 01 m D: O (0.0 m < .01 0 20 40 60 80 100 120 140 TIME - h Figure 3. Effect of vitamins on growth of E. suis. PYS medium was modified to contain charcoal-treated'Trypticase and a defined mixture of purine and pyrimidine bases in place of yeast extract. Single vitamin solutions were added individually or in various combinations to this modified PYS as described in Materials and Methods. A, the full complement of vitamins; B, pyridoxal, riboflavin and nicotinic acid; C, nicotinic acid; 0, pyridoxal; E, riboflavin; F, no vitamins. 68 ABgORBANCE - 600 nm .01 o 20 4o 60 80 100 120 140 TIME - h Figure 4. Effect of purine and pyrimidine bases on growth of g, suis. PYS medium was modified to contain charcoal-treated Trypticase and a defined mixture of vitamins in place of yeast extract. Stock solutions of individual purine and pyrimidine bases were added singly or in various combinations to the modified basal medium as described in Materials and Methods. A, the full com- plement of purines and pyrimidines; B, adenine and uracil; C, adenine; D, uracil; E, no purines or pyrimidines. 69 pyrimidine bases. Combined addition of adenine, guanine, thymine and xanthine gave growth no better than that obtained on single addition of adenine (data not shown). 70 DISCUSSION We previously reported that E, guis fermented only maltose, starch and glycogen, but not gluCose or many other carbohydrate and non-carbo- hydrate substrates tested (31). The results of this study show that growth is more than two-fold greater in PYS than in PYM and that a fer- mentable carbohydrate appears to be required as an energy source for growth (Table 2). The results show that formate, acetate and ethanol are the main products produced from fermentation of starch or maltose by §:.§!i§- Numerous attempts to detect H2 and C02 as possible products of carbohy- drate metabolism in this organism failed. These results are in agree- ment with earlier observations by Soltys (26) who did not find evidence of gas formation in media containing maltose. The results suggest that pyruvate generated from carbohydrate catabolism undergoes an E. _c_9_]_i- type clastic reaction to produce formate and perhaps acetyl CoA; acetyl CoA, in turn, could be converted to either acetate or to ethanol, utiliz- ing the reducing equivalents generated during carbohydrate oxidation to the pyruvate level. while approximately equimolar amounts of ethanol and ace- tate were produced in PYM, almost twice as much acetate as ethanol was generated in PYS. Since the conversion of acetyl CoA to acetate via acetyl phosphate results in the synthesis of an additional molecule of ATP (9),itiaPPears that more ATP might be generated from fermentation of starch than that of maltose. This may at least partially account for the higher level of growth attained with starch as the energy source. Althought it would seem reasonable to expect that NH4+, presumably readily available to the organism in vivo due to its urease activity, might satisfy the nitrogen requirements of this strain, our studies 71' indicated that an organic nitrogen source was required, at least in 11359. Furthermore, in the absence of Trypticase, NH4+ and/or a full complement of free amino acids could nbt satisfy the nitrogen requirements of the organism; addition of Trypticase to the medium, however, drama- tically restored growth. These data suggest that Trypticase serves as a source of peptides (and perhaps other nutrients) required for the growth of E. ggjg, It is possible that the organism has a lesion in the transport system for free amino acids (14). The requirement for peptides even in the presence of a full complement of free amino acids has been observed in a few other organisms (14, 21). Riboflavin, nicotinic acid and pyridoxal were required for optimal growth of E, 331;. Riboflavin and nicotinic acid are precursors of co- factors used in oxidation-reduction reactions. These cofactors play a key role in carbohydrate metabolism and in various biosynthetic reactions; without them, the organism's capacity for energy production and growth would be seriously limited. Pyridoxal is a precursor of pyridoxal phos- phate which plays an important role in various transamination and de- amination reactions. A strong requirement for both adenine and uracil was found for E. E213. The requirement for adenine may be due to a defect in the synthe- sis of adenylate from inosinic acid. The requirement for uracil might possibly be due to a lesion in the pathway' leading to the formation of UTP and, therefore, CTP. We have previously shown that E, ggig differs from other members of the genus Corynebacterium on the basis of cell wall composition, fer- mentation end products, and biochemical characteristics (31). The data presented here show that it differs from other animal pathogenic coryne- bacteria in its nutritional requirements as well. For example, almost 72 all strains of the type species E, diphtheriae require pimelic acid, ni- cotinic acid, and fiB-alanine (24). Van Eseltine et al. (30) have raported that E, renale, the etiologic agent of infectious pyelonephritis of cattle, can use ammonium ion as the sole source of nitrogen, although other workers have indicated that amino acids in addition to NH4+ are re- quired (10, 11). .§,.Eg!i§_was also shown to utilize ammonium ion as its sole nitrogen source and to require unsaturated long chain fatty acids for growth. Some strains of E, ngi§_were shown to require "ICOtIOIC acid (24). Little is known about the nutritional requirements of other species of animal pathogenic corynebacteria including E, pseudotuberculosis and E, kutscheri (24). More recent studies showed that E, pyogenes re- quired adenine, uracil, riboflavin and thiamine and that a defined mix- ture of amino acids could serve as a source of nitrogen only in the pre- sence of inositol (C.A. Reddy and A. McClellan, 1981, unpublished data). In this investigation E. suig was not tested for growth in medium contain- ing free amino acids plus inositol. The nutritional requirements of E, égj§_also appear to differ from those of the anaerobic coryneforms (propionibacteria). Ferguson and Cummins (7) reported that these organisms grew well when a basal salts medium was supplemented with glucose, pantothenate, biotin, thiamine, and many amino acids. Nicotinamide was required for optimal growth of Propionibacterium acnes strainsonly. The vitamin requirements of these species reflect the need for those cofactors which are essential for the propionic acid fermentation pathway. Since E, £315 does not utilize this pathway, it is not surprising that its vitamin requirements differ. In addition, Smith (25), Ushijima (29), and Ferguson and Cummins (7) have shown that the purines guanine and/or adenine were stimulatory but not required for the growth of the anaerobic coryneforms. E, suis required 73 both adenine and uracil for maximal growth. E, éflié produces pyelonephritis almost exclusively in female animals, often in association with pregnancy and parturition (26). Perhaps in its natural milieu the organism utilizes cellular stores of glycogen, the chief reserve carbohydrate of animals, as the main energy source. It is unlikely the organism will have access to maltose or starch _i_n_s_igu. Filterable peptides, vitamins, and nucleic acid bases would also be avail- able to the organism in its natural niche. The accumulation of suitable nutrients in the sow kidney may occur as a result of the obstrUction of tubular drainage, a condition frequently brought about by pregnancy (26). The nutritional requirements of E. ggig. therefore, reflect its adapt- ability to its niche, since it has done away with biosynthetic abilities that it does not need. 10. 11. 12. LITERATURE CITED Aalvik, B. 1968. Corynebacterium suis isolert fra et tilfelle av pyelonefritt hos purke. Nord. Vet. Med. EQ;319-320. Blood, 0. C., and J. A. Henderson. 1974. Diseases caused by Coryne- bacterium sp., p. 300-307. In Veterinary Medicine, 4th ed. The HilIiams & Wilkins Co., Baltimore. Caldwell, D.R., and M.P. Bryant. 1966. Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 145794-801. Dehority, B.A. 1971. Carbon dioxide requirement of various species ' of rumen bacteria. J. Bacteriol. EQ§;70-76. Desnuelle, P., and M. Naudet. 1945. 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