INTERNATIONALJOLJRNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1978, p. 209-216 VOL28, 2 0020-7713/78/0028-0209$02.00/0 N~. Copyright 0 1978 International Association of Microbiological Societies Printed in U.S. A. Corynebacterium pilosum and Corynebacterium cystitidis, Two New Species from Cows RYO YANAGAWA AND EIICHI HONDA Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan Two strains of corynebacteria were isolated from cows that showed signs of cystitis and pyelonephritis. According to the results of numerical-analysis and deoxyribonucleic acid homology studies, these strains differed from the known species of Corynebacterium parasitic on or pathogenic to humans and/or other animals. These strains are regarded as belonging to two new species, for which the names C. pilosum and C. cystitidis are proposed. The type strains of these species are 46 Hara (= ATCC 29592) and 42 Fukuya (= ATCC 29593), respec- tively. Three organisms, originally described as im- mentation of glucose; acid from 23 sugars and gas from munological types (I, 11, and 111) of Corynebac- glucose (serum broth-based sugars were used and were terium renale (19), were found not to be closely examined for 45 days); nitrate reduction (method 1); related when tested by deoxyribonucleic acid nitrite reduction; gelatin liquefaction (method 2); es- (DNA) hybridization (8) and, on the basis of a culin hydrolysis; hippurate hydrolysis; arginine hy- drolysis (method 1); starch hydrolysis (method 1); numerical analysis of their phenotypic charac- phosphatase test (method 1); catalase test (method 1); ters, were found to fall into three different phena urease activity (method 5) (cells grown on nutrient (18). Immunological type I (phenon 1) included agar were used for the two latter tests; with Coryne- the reference strain (American Type Culture bacterium pyogenes and C. haemolyticum, however, Collection [ATCC] no. 19412) of C. renale; types the cells were grown on serum agar, washed with saline I1 (strain 46 Hara and the strains that were by centrifugation, and then used); oxidase test similar to this strain and that belonged to C. (method 1); deoxyribonuclease activity (deoxyribonu- renale immunological type 11) and I11 (strain 42 clease test agar, Eiken Co., Tokyo, Japan, was used); Fukuya and the strains that were similar to this deamination of phenylalanine (method 1);malonate test (method 1); decarboxylase test (arginine, lysine, strain and that belonged to C. renale immuno- ornithine) (method 1); decomposition of tyrosine and logical type 111; phena 2 and 3, respectively) xanthine; production of indole (method l), hydrogen appeared to belong to two new species of the sulfide (method 31, and coagulase (method 1); Voges- genus Corynebacterium (18). The purpose of Proskauer reaction (method 1); methyl red reaction; this paper is to effect the valid publication of casein digestion; coagulation of milk; hemolytic zones names for these species. around colonies on blood agar (sheep, guinea pig, and rabbit bloods); susceptibility to antibiotics and other MATERIALS AND METHODS antibacterial substances was examined for 7 days (con- Bacterial strains. The strains used in this study centrations were expressed as parts per milliliter of are listed in Table 1. serum agar); growth within 7 days on MacConkey agar Maintenance of strains. All strains were stored as and Simmons citrate agar; and inhibition of growth freeze-dried cultures. Active cultures were maintained within 7 days on serum agar to which was added 6 or on a serum agar consisting of beef infusion (500 g of 8%of NaCl, 0.1%of sodium oleate, 2 or 4% of potassium beef muscle infused by heat in 1,OOO ml of water), 1% thiocyanate, 0.04% of tellurite, 0.05% of sodium azide, peptone (Polypeptone, Daigo Co., Tokyo, Japan), 0.5% 0.01% of tryphenyl tetrazolium, 0.01% of selenite, and NaC1, 1.5% agar (Shoei Co., Tokyo, Japan), and 5% 100 pg of hydroxylamine hydrochloride per ml; hy- calf serum, which was added after sterilizing by filtra- drolysis of Tweens 80,60,40, and 20; decomposition of tion through a Toyo 85 SB filter pad (Toyo Roshi Co., egg yolk protein (clear zones around colonies grown Tokyo, Japan). on serum agar with 10% egg yolk added; examined for Media. Serum agar and serum broth were the me- 14 days); decomposition of egg yolk lipid (turbid layer dia basically used. References to the compositions of formation around colonies grown on serum agar with the various media used for the biochemical and other 10% egg yolk added; examined for 14 days); lecithovi- tests are given below; these media contained 5% serum tellin test; salicylate degradation (the test organisms (exceptions are indicated in the text). For pigment were inoculated on serum agar containing 0.1%sodium production, serum agar containing 5% milk was used. salicylate and incubated at 37°C; a marked blackehing Biochemical and other tests. The following tests of the medium with growth after 7 days was recorded were performed as described by Cowan (1) (method as a positive reaction); and growth on nutrient agar numbers are given in parentheses): oxidation or fer- (without serum). 209 210 YANAGAWA AND HONDA INT. J. SYST.BACTERIOI,. Numerical analysis. Analysis of the results ob- RESULTS tained with the 17 strains studied was performed by Characteristics of strain 46 Hara. Strain the method of Lessel and Holt (14). C. pyogenes and C. haemolyticum were not used in numerical analysis 46 Hara contained gram-positive, nonmotile, because they differed profoundly from the other cor- densely piliated rods, 0.5 by 1.3 pm, occurring ynebacteria in several ways (15). After calculation of singly, in pairs (angular arrangements), and in similarity (S) values, the strains were clustered by masses; club-shaped forms with metachromatic single linkage in which each strain was admitted to a granules also were noted. group at the highest similarity level it had with any Colonies on nutrient agar and on serum agar other member of that group. were cream to pale yellow, entire, circular, Diagrammatic representation of the grouping of the opaque, and about 1 mm in diameter after 24 h strains was done by means of a dendrogram (Fig. l), of incubation at 37°C. No hemolysis was found which made apparent the affinities of the strains and the clusters of strains. around surface colonies on blood agar. In broth DNA homology test. Nutrient broth was used in and in serum broth, a pellicle and a granular the production of labeled cells of strains 46 Hara and sediment were formed, but there was no turbid- 42 Fukuya. Nutrient agar or nutrient agar mixed with ity. calf serum (10%)was used in the production of unla- Aerobic, facultatively anaerobic. Glucose was beled bacteria; all cells were grown at 37°C for 24 h, fermented. In the oxidation-fermentation test, with the exception of those of C. bovis, C. pyogenes, however, change of color was found only when and C. haemolyticum, which were grown at 37°C for oxidation-fermentation medium with more than 48 h. 4% of glucose was used. The biochemical and [3H]uridine (5.0 Ci/mmol, 20.6 mCi/mg) was pur- chased from the Radiochemical Centre ( Amersham, other characteristics of this strain are summa- England). [3H]adenine and E3H]thymidine were not rized in Table 2. The susceptibilities of this used because the former was not incorporated into the strain to various antibiotics and other antibac- DNA of C. renale to the extent that [3H]uridine was, terial substances are shown in Table 3. and the latter was not incorporated into the DNA of Characteristics of strain 42 Fukuya. Cul- strain 46 Hara due to the lack of thymidine kinase tures of strain 42 Fukuya contained gram-posi- activity (8). tive, nonmotile, piliated, straight to slightly DNA was extracted and purified by a modified curved rods, 0.5 by 2.6 pm, often occurring in phenol extraction procedure previously described (11). angular or palisade arrangements. Metachro- DNA labeled with tritium from C3H]uridine was ex- tracted from the cells, which were cultivated for 48 h matic granules were present. in the broth containing 1.5 pCi of [3H]uridine per ml. Colonies on nutrient agar and on serum agar The purified labeled DNA was further treated twice were white, entire, circular, semitranslucent, and with ribonuclease (EC 2.7.7.16). rather small in diameter, often not readily visible Immobilization of DNA on nitrocellulose filters and within 24 h of incubation at 37°C. In broth DNA-DNA hybridizations were performed as de- cultures, there was slight turbidity but no pelli- scribed previously (8). cle. Aerobic, facultatively anaerobic. Glucose was TABLE1. List of strains included in this study fermented. The biochemical and other reactions SI) e c ie s Source Desimation of this strain are summarized in Table 2. The susceptibilities of this strain to various antibiot- Corynebacterium bovis ..... ATCC 77 15 C. cystitidis ............... Author 42 FU- ics and other antibacterial substances are shown kuya" in Table 3. C. diphtheriae ............. ATCC 19409 Numerical taxonomic study. Twenty-two C. equi .................... ATCC 6939 characters (acid-fast reaction, motility, crenated C. haemolyticum ........... ATCC 9345 colony, growth in serum broth [pH 4.31, gas from C. hoagii .................. ATCC 7005 glucose, arginine hydrolysis, oxidase test, de- C. kutscheri ............... ATCC 15677 oxyribonuclease activity, deamination of phen- C. murisepticum ........... ATCC 21374 ylalanine, malonate test, decarboxylation of ar- C. nephridii ............... ATCC 11425 ginine, lysine, and ornithine, coagulation of milk, C. paurometabolum ........ ATCC 8368 decomposition of xanthine, production of indole C. pilosum ................ Author 46 Hara" C. pseudodiphtheriticum ... ATCC 10700 and coagulase, Voges-Proskauer reaction, C. pseudotuberculosis ...... ATCC 19410 growth on MacConkey agar and Simmons cit- C. pyogenes ............... ATCC 19411 rate agar, lecithovitellin test, and degradation of C. renale .................. ATCC 19412 sodium salicylate) which were negative for every C.