South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange Electronic Theses and Dissertations 1969 Cell-wall Analyses of Diphtheroids Isolated from Cattle and Swine Everett E. Scheetz Follow this and additional works at: https://openprairie.sdstate.edu/etd Recommended Citation Scheetz, Everett E., "Cell-wall Analyses of Diphtheroids Isolated from Cattle and Swine" (1969). Electronic Theses and Dissertations. 3598. https://openprairie.sdstate.edu/etd/3598 This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. ,f'I CELL-WALL ANALYSES OF DIPIITHEROIDS ISOLATED FROM CATTLE AND SWINE BY EVERETTE. SCHEETZ '\ A thesis submitted in partial fulfillment of the requirements for the degree Master of Science, Major in Bacteriology, South Dakota State University 1969 SOUTH DAKOTA STATE UNIVERSITY LIBR -RY CE LL-WALL ANALYSES OF DIPHTHEROIDS ISOLATED FROM CATTLE AND SW INE This thesis is approved as a creditable and independent investigqtion by a candidate for the degree, Master of Science, and is acceptable as meeting the thesis requirements for this degree, but without implying that the conclusions reached by the candidate are necessarily the conclusions of the major department. Thesis Advisor "'Date , uate ACKNOWLEDGEMENTS I wish to express my sincere apprec�ation to Dr. G. W. Robertstad for his assistance and guidance as my major professor on this study, to Dr. R. M. Pengra for his help with the thin-layer chromatography work, analysis of <lat�, and proofreading, to Dr. P. R. Midd�ugh for his assistance in analysing the results and proofreading, to Dr. S. G. Jensen and Lewis Nelson at the Northern Grain Insects La_boratory for their help with the gas-chromatography work and use of equipment, to Dr. E. C. Berry for his counseling and guidance as a friend, and to my fellow graduate students who have helped to make this possible. I wish to thank the South Dakota Experiment Station for financial support during the course of this study. I wish to express very special appreciation to my wife, Eva, and my children for their encouragement and understanding during the writing of this thesis. EES TABLE OF CONTENTS Page INTRODUCTION••••••.•••• · ••••.•••••••.•••••••••••••••••••••• 1 LITERA.TURE REVIEW••.••••••••••. •...•••••••••••••••••••.•••.• •••• 3 21 MATER IALS AND METHODS ••••..••••••••.••••••••••••••••• o • • • • • • • • • Biochemical tests. 21 Catalase Production. 21 Fermentations ..••..••••.• • 22 Chroma tog r a ph y . • . •. • . .. • . •. • . • . • • 2 3 Preparation of cells·..... ............................ 23 Thin-layer chromatography. 28 Gas chromatography. 29 RES ULTS AND DIS CUS SIONS . 31 CONCLUS IONS . 45 LITER A.TUR E CITED ••.••••••••• 46 LIST OF TABLES Table Page 1. Cell-wall composition of Actinomyces ·spp. and unknown strains . 7 2. Classification of organisms with FA technique .12 3. Cell-wall composition of various groups of streptococci .15 4. Cell-wall composition of Corynebacterium spp. .16 5. Cell-wall composition of Lactubacillus spp .. ..17 6. Cell-wall composition of various cocci ... .18 7. Classification of actinomycetes, based on cell-wall composition ......· .... ..19 8. Sources of reference organisms. .24 9. Clinical sources of unknown organisms . .25 10. Catalase reactions of referc: �� organisms and unidentified diphtheroids. .32 11. Fermentation reactions of reference organisms .33 12. Fermentation reactions'of unidentified diphtheroids . .34 13. Sugar content of cell-wall hydrolysates of reference organisms . ..35 14. Sugar content of cell-wall hydrolysates of unidentified diphtheroids. 36 15. Amino acid content of cell-wall hydrolysates of reference organisms . .37 16. Amino acid content of cell-wall hydrolysates of unidentified diphtheroids. .38 17. Percentage of similarity of reference organisms and unidentified diphtheroids based upon catalase test and cell-wall constituents. ......... ....40 18 . Grouping of diphtheroidal isolates from cattle and swine ..43 INTRODUCTION Biever (2), working in this laboratory, isolated numerous diphtheroidal organisms from cattle and swine sources. These anae·robic diphtheroids were .morphologically similar to organisms in the genera Corynebacterium and Actinomyces. King and Meyer (25) found that isolates of anaerobic diphtheroids from human sources were more closely related to the Corynebacterium based upon metabolic and serologic differentiation. Their work consisted of separating!!:.• bovis from several isolates of anaerobic diphtheroids on the basis of the catalase test, litmus milk reactions, and the fermentation of xylose, salicin, and raffinose. However, Grasser (16) reported that the fermentation reactions are variable and cannot be used as definite criteria for positive identification. The catalase test, which in the past was considered a specific test for differ­ entiating Actinomyces spp. from anaerobic diphtheroids and Corynebacterium spp., is no longer as useful because some of the more recently described Actinomyces spp. are catalase positive (36). Slack et al. (44) defines anaerobic diphtheroids as being widely used to designate "organisms having morphological characteristics of a Corynebacterium but which cannot or are not designated by a given species name.'' Smith and Bodily (45) stated that the anaerobic diphtheroids may resemble or be confused with Actinomyces spp. as well as with the anaerobic corynebacteria. Many of the tests performed by this group of workers gave variable results. However, the isolates studied 2 were provisionally identified as Corynebacterium acnes on the basis of selected biochemical tests together with serological methods. Cummins and Harris (8) used paper chromatography for cell-wall analysis and concluded that the cell-wall composition could be used as a taxonomic characteristic in identifying organisms. Obviously, much confusion exists in classifying the anaerobic diphtheroids and differentiating Actinomyces spp. and Corynebacterium spp.· In this study thin-�ayer (TLC) and gas chromatographic methods were used to determine the cell-wall constituents of known Coryne­ bacterium spp. , Actinomyces spp., and a selected number of anaerobic diphtheroids isolated by Biever (2). 3 LITERATURE REVIEW Morris (cited by Batty, 1) defined the genus Actinomyces as: Gram-positive, non-acid-fast, non-motile organisms which in young cultures appear as rods. These rods grow out to form a septate branching mycelium which produces terminal swollen cells . These break off and germinate to produce an irregular branching, non-septate mycelium. Spores are produced on short stalks from the non-septate filaments. No aerial mycelium is produced . Type: Actinomyces bovis . In 1877 Harz (18) isolated arr organism from lumpy jaw in cattle, which was similar to the organism described by Bollinger (3) as A. bovis . Several years later Ponfick (cited by Frost, 13) demonstrated that similar lesions occurred in humans . Cutler and Gross (11) stated that 60% of the cases of actinomycosis in humans occurred in the face, jaw or neck. The next most frequent site of infection was.the abdominal cavity where 20% of the infections appeared. About 15% of the infections were present in the bronchial tree, pulmonary tissues, pleura or chest wall . The skin and other viscera accounted for the remaining 5% of the infections . Cutler and Gross identified the causative agent in these human cases as A. bovis . Shortly after Bollinger (3) and Harz (18) had identified actinomyces in cattle, Israel (23) described lesions in man, which resembled those observed in bovine diseases (13). Wolff and Israel (52) successfully cultured an anaerobic actinomycete from cases of human actinornycosis . Their classical 4 description of the morphology and physiology of the organism enabled subsequent workers to identify and establish the organism as a cause of human actinomycosis. In 1900, Silberschmidt (37) first successfully cultured an ·anaerobic actinomycete from lacrimal concrements. A year later he isolated a strain from maxillary actinomycosis of a cow and compared it with strains isolated from lacrimal infections in man (38). All strains were considered to be identical . Breed and Conn (4) reviewed the literature pertaining to the taxonomy and concluded that the name Actinomyces was the most valid name for the organism according to the rules of nomenclature and usage. As a result of these previous studies, the name A. bovis was applied to the anaerobic organism causing human and bovine actinomy­ cosis . However, in 1940, Erikson (12) compared isolates of Actinomyces from both human and cattle sources. On the basis of colony morphology, growth characteristics, and serology, she concluded that there were sufficient differences between the strains to warrant species separation. As a result she restricted the name Actinomyces israelii to those strains isolated from humans which grew slowly to form granular or rough colonies in liquid and on solid media. Actinomyces bovis was then used to denote those organisms which, when isolated from bovine sources, grew rapidly and formed smooth, circular, soft colonies on solid media or formed turbid growth in liquid