J. Gen. Appl. Microbiol., 39, 135-214 (1993) NUMERICAL CLASSIFICATION OF CORYNEFORM BACTERIA AND RELATED TAXA PETER KAMPFER,* HERI3ERT SEILER,' AND WOLFGANG DOTT Fachgebiet Hygiene, Technische Universitat Berlin, Amrumerstr. 32, D-13353 Berlin, Federal Republic of Germany 'Suddeutsche Versuchs - and Forschungsanstalt fur Milchwirtschaft, Vottingerstr. 45, D-85354 Freising, Federal Republic of Germany (Received August 14, 1992) A numerical taxonomy of 604 strains of the genera Arthrobacter, Aureo- bacterium, Brevibacterium, Cellulomonas, Clavibacter, Corynebacterium, Curtobacterium, Microbacterium, Nocardia, Nocardioides, Rhodococcus, Terrabacter and Tsukamurella was undertaken based on 280 physiological characters with the aid of miniaturized tests. Clustering was by the unweighted pair group method (UPGMA) with 12 different measures of similarity. Test error, overlap between the phena and cophenetic correla- tion coefficients for the classifications obtained with the Jaccard coefficient (SJ), the Pearson coefficient (SP), the simple-matching coefficient (SsM), and the Dice coefficient (SE,) as measures of similarity were within acceptable limits. Clusters were defined at the 55.0 to 70.5% levels (SJ). The compositions of clusters corresponded largely the delineations of 81.1 to 93.5% (SSM),29.1 to 55.0% (Se), and 55.3 to 81.1% (SD). A total of 31 major clusters (containing five or more strains), 41 minor clusters and subclusters (containing less than five strains), and 54 single-member clusters were obtained in the UPGMA/SJ study. The following conclu- sions were reached: (i) A high degree of similarity between the genera Aureobacterium, Cellulomonas, Clavibacter, Curtobacterium and Microbac- terium found in phylogenetic-based studies could be shown also pheneti- cally. Strains belonging to these genera were found in SJ clusters 1 to 45, often representing closely related, or single species. (ii) Several strains of the plant pathogenic coryneform bacteria assigned to the genus Clavi- bacter and Curtobacterium flaccumfaciens were found to be within one SJ cluster, indicating the high similarity between these genera. The current classification of species within the genera Curtobacterium and Clavibacter * Address reprint requests to: Dr . Peter Kampfer, Fachgebiet Hygiene, Technische Universitat Berlin, Amrumerstr. 32, D-13353 Berlin, FRG. Abbreviations: OTU, operational taxonomic unit; pNA, p-nitroanilide; pNP, p-nitrophenyl. 135 136 KAMPFER, SEILER, and DOTT VOL. 39 is unsatisfactory; a close relationship to Microbacterium suggests a reclas- sification into a redefined genus. Subspecies of Clavibacter and pathovars of Curtobacterium flaccumfaciens should only be retained for practical purposes. (iii) Bacteria belonging to the genus Corynebacterium, includ- ing Corynebacterium glutamicum, C. ammoniagenes, C. minutissimum, C. striatum, C. variabilis, C. kutscheri, C. diphtheriae, C. pseudotuberculosis, and `C. ulcerans' formed a separate complex of the distinct, adjacent clusters 47 to 63. The physiologically inactive species C. mycetoides, C. pseudodiphtheriticum, C. xerosis, C. renale and C. pilosum were found in clusters 116 to 126. (iv) Differences between the Arthrobacter globiformis group and the Arthrobacter nicotianae group were reflected in the struc- ture of the phenogram, species differentiation being based on only a few characters. (v) Strains assigned to the four species of the genus Brevi- bacterium were grouped into two clusters; the taxonomic implications are discussed. (vi) The results of the study are largely in line with a previously published numerical survey and with chemotaxonomic and genetic data. Suggestions for an improved classification for some species is given in addition to an extensive data-base on physiological reactions for differentiation purposes. Taxonomy of the coryneform bacteria is largely based on chemotaxonomic data, including cell wall chemotypes, phospholipid types (74-76), cell sugar pat- terns and peptidoglycan types (89, 90), fatty acid patterns and menaquinone types (43, 70). These data have been summarized by various authors (42, 43, 45, 5 7, 76, 108). The application of chemotaxonomic methods to the actinobacteria and nocardioforms helped to improve the inconsistencies within the suprageneric groups of actinomycetes and within the genera (43). To establish a hierarchic classification system, comparative 5S and 16S ribosomal RNA (rRNA) sequence analyses have been carried out on representative strains of various genera within the Actinomycetales (40, 84,114). As stressed by Stackebrandt (109), both stability and practicality are prerequisites for a successful system, and the decision about the delineation of genera must be made by combining phylogenetic coherency with unifying phenotypic properties of taxonomic value. Furthermore, the use of molecular methods on the genus level to eliminate misclassified strains has been considered a main aim of molecular taxomomy (109). However, as pointed out by Sneath (103), a further main aim of taxonomy is to give a classification that is useful for varied scientific purposes, including identification and to produce the data-bases that summarize as much relevant information about organisms as possible. Numerical taxonomic studies are useful to produce phenetic groupings, which are often in line with the genomic groups, whether phylogenetic or phenetic. Therefore, numerical taxonomic studies have improved the classification of coryne- 1993 Classification of Coryneforms 137 form bacteria at both genus and species level (10,29,44,46,50,54,55,91-94,107). However, since the publication of the broader studies of Jones (54) and Seiler (92), no further study has been published to compare the results obtained by chemotaxo- nomic and genetic methods to those of numerical studies. This study was intended to provide additional information on the physiological properties of many strains belonging to the various genera of coryneform bacteria, and to compare the phenetic groupings obtained, with those from other numerical studies (7, 54, 92) . Furthermore, a comparison between this study and the genomic groupings (genera and species) defined by different methods, e.g. DNA-DNA hybridization studies (32,110,111, 114,119) "wall types" (108) and other chemo- taxonomic data, especially fatty acid data (2, 7, 49, 70, 121), is given. MATER [ALS AND METHODS Bacterial strains. 634 strains of the genera Arcanobacterium, Arthrobacter, Aureobacterium, Brevibacterium, Cellulomonas, Clavibacter, Corynebacterium, Cur- tobacterium, Erysipelothrix, Microbacterium, Nocardia, Nocardioides, Pimelobacter, Rhodococcus, Terrabacter and Tsukamurella were used in the study (Table 1). Most of them were obtained from the culture collection of Suddeutsche Versuchs- und Forschungsanstalt fur Milchwirtschaft, Weihenstephan, Freising, FRG. Strains of clinically important corynebacteria were obtained from the National Collection of Type Cultures, London, UK. With the exception of the clinically important corynebacteria and members of the genera Erysipelothrix and Arcanobac- terium, which were cultivated on sheep blood agar (OXOID), all strains were cultivated at 28°C on trypticase soy extract medium containing trypticase soy broth (3.0%, w/v), yeast extract (0.3%, w/v) and agar (1.5%, w/v). Biochemical tests. Each strain was examined for 329 unit characters includ- ing carbon source utilization tests, sugar fermentation and qualitative enzyme tests using chromogenic substrates (61,62). Tests were performed in standard micro- titration plates (Greiner, Nurtingen, FRG). The utilization of 240 carbon sources (62) was tested in a mineral medium containing the following constituents (g/l): K2HP04, 1.74; KH2P04, 1.36; NH4SO4, 5.0; MgS04 x 7H20, 0.5; CaC12x 2H20, 0.1; NaCI, 9.0; yeast extract (OXOID), 0.02; proteose peptone (DIFCO), 0.02; vitamin solution, 5 ml containing (mg/l): Ca-pantothenate, 0.1; nicotinic acid, 0.1; biotin, 0.005; cyanocobalamin, 0.005; folic acid, 0.1; pyridoxine, 0.1; p-aminobenzoic acid, 0.1; thiamine pyrophosphate, 0.1; inositol, 0.1; thiamine, 0.1; riboflavin, 0.1; trace element solution, 1 ml containing (mg/l): H3B04, 0.5; CuSO4 X 5H20, 0.04; KJ, 0.1; FeCl3 X 6H20, 0.2; MnS04 X H2O, 0.2; Na2Mo04 X 2H20, 0.2; ZnSO4 X 7H20, 0.4. After adding the different carbon sources at final concentrations of 0.2% (w/v)-for aromatic compounds concentrations of 0.05% (w/v) were used-the media were sterilized by filtration. Qualitative enzyme tests were done in filter-sterilized medium (pH 7.2) con- taining 0.05 M Tris-HC1 buffer and 0.05% (w/v) of yeast extract (OXOID) and 138 KAMPI 1 R, Si.ir.t~.x, and Do~i i Voi.. 39 0 C1) - U rU cs U U > ~ 0 0 a v I I aU v C .~ C C C C o`~b c o U C . 'C v v 0 C 0 •- C -o . E C v a~z Cti ~ U r C H 1993 Classification of Coryneforms 139 140 KAMPFER, SEIL,ER., and DOTT Voi.. 39 1993 Classification of Coryneforms 141 142 KAMPFER, SEILER, and DoTT Voi.. 39 1993 Classification of Coryneforms 143 144 KAMPFER, SEILE R, and DOTT VOL. 39 1993 Classification of Coryneforms 145 146 KAMPFER, SEILER, and DOTT VOL. 39 1993 Classification of Coryneforms 147 148 KAMPFER, SEILER, and DOTT VoL. 39 1993 Classification of Coryneforms 149 I50 KAMPFER, SEILER, and DoTT Vot.. 39 1993 Classification of Coryneforms 151 152 KAMPFER, SEILER, and DOTT VOL. 39 1993 Classification of Coryneforms 153 154 KAMPFER, SEILER, and DOTT VOL. 39 1993 C1assi fication of Coryneforms 155 I56 KAMPFER, SEILER, and DOTT VOL. 39 1993 CI asst fication of Coryneforms 157 158 KAMPFER, SEILE.R,