Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 07 Dec 2018 09:31:37 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Oct. 1997, p. 952-957 Copyright 0 1997, International Union of Microbiological Societies 0020-7713/97/$04.00 +O Vol. 47, No. 4 Corynebacterium mucifaciens sp. nov., an Unusual Species from Human Clinical Material GUIDO FUNKE,l* PAUL A. LAWSON,2 AND MATTHEW D. COLLINS2 Microbiology,BBSRC Institute of Food Research, Reading RG6 6BZ, United Kingdom2 Department of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland, and Department of Eight strains of a previously undescribed coryneform bacterium had been isolated from human clinical material over a 5-year period. Colonies of the unknown coryneform bacterium had an unusual appearance as they were slightly yellowish and very mucoid. Biochemical and chemotaxonomic characterizationrevealed that the unknown coryneform bacterium belonged to the genus Corynebacterium. It could be readily differentiated from all previously described Corynebacterium species. Electron microscopy demonstrated the production of an extracellular substance causing connecting filaments between cells as a morphological correlate to the mucoid colonies. Comparative 16s rRNA gene sequence analysis revealed that the unknown coryneform bacterium represented a new subline within the genus Corynebacterium, for which the name Corynebacteriurn mucifaciens sp. nov. is proposed. The type strain is CCUG 36878 (= DSM 44265 = CIP 105129). The genus Corynebacterium is the largest genus in the coryneform group of bacteria (i.e., aerobically growing, as- porogenous, non-partially acid-fast, irregular gram-positive rods) and currently comprises more than 25 species (1 1). Dur- ing the past decade, 12 new Corynebacterium species have been established (9,ll). Interestingly, all of these were derived from human clinical specimens, whereas only 7 of the 17 Corynebac- terium species listed in Bergey's Manual of Systematic Bacteri- ology are considered medically relevant (4, 11). The present report deals with yet another novel Corynebacterium species isolated from human clinical specimens. Over a 5-year period, eight strains of a coryneform bacterium whose colonies were slightly yellowish and markedly mucoid were isolated from human clinical material. In a polyphasic approach including both phenotypic and molecular genetic methods, it was dem- onstrated that this coryneform bacterium represents a new Corynebacterium species, for which the name Corynebacterium rnucifaciens sp. nov. is proposed. MATERIALS AND METHODS Bacterial strains. The origins of the strains studied and their clinical sources are given in Table 1. Four strains were isolated by workers at the Department of Medical Microbiology, University of Zurich, Zurich, Switzerland, and four other isolates were received by the reference laboratory for coryneform bacteria of the same institution. Phenotypic characteristics. Strains were grown aerobically at 37°C in a 5% C0,-enriched atmosphere on Columbia agar (Difco, Detroit, Mich.) supple- mented with 5% sheep blood. The methods used for biochemical profiling of the bacteria have been outlined in detail previously (8). By using the API Coryne system (bioMCrieux, Marcy l'Etoile, France), enzymatic reactions were deter- mined after 24 h at 3TC, and acid production from carbohydrates was observed after 48 h of incubation. The API S0CH system (acid production from additional carbohydrates) was used in conjunction with 50CHE medium (both from bio- Merieux), and reactions were read after 96 h of incubation at 37°C. Electron microscopy. Cells were grown in brain heart infusion broth (Bccton Dickinson, Cockeysville, Md.) for 18 h at 37"C, harvested by centrifugation, and washed three times with 0.9% NaC1. Pelleted cells were fixed for 60 min at 4°C by using a solution of 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), washed with phosphate-buffered saline, and postfixed for 30 min with 2% os- mium tetroxide in cacodylate buffer. After two additional washes in water, the fixed pellets were incubated in a 2% uranyl acetate solution overnight. Cells were then dehydrated in 2,2-dimethoxypropanc and embedded in epon-araldite. Thin * Corresponding author. Mailing address: Department of Medical Microbiology, University of Zurich, Gloriastrasse 32, CH-8028 Zurich, Switzerland. Phone: 41-1-634-2700. Fax: 41-1-634-4906. E-mail: funke @immv.unizh.ch. sections were contrasted with lead citrate and examined with a Philips model EM400 transmission electron microscope. Chemotaxonomic investigations. The methods used for analyses of cellular fatty acids (gas-liquid chromatography [25]), cell wall diamino acids (thin-layer chromatography [S]), mycolic acids (thin-layer chromatography [S]), the acyl type of the muramic acid (tube test [23]), and the G + C content (high-performance liquid chromatography [7]) have been described previously. Antimicrobial susceptibility testing. MICs of 26 antimicrobial agents were determined by a microdilution method (Merlin Diagnostics, Bornheim-Hersel, Germany) as outlined previously (7). The results were interpreted by using National Committee for Clinical Laboratory Standards breakpoints (19). Determination of the 16s rRNA gene sequences and phylogenetic analyses. A large fragment of the 16s rRNA gene was amplified by PCR by using universal primers pA and pH* as previously described (15). The PCR products were purified by using a Prep-A-Gene kit (Bio-Rad, Hercules, Calif.) and were se- quenced by using a Tuq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif.) and a model 373A automatic sequencer (Applied Biosystems). Searches in the EMBWGenBank Data Library were performed with the program FASTA in order to establish the relatives of the clinical strains. Sequences of the close relatives were retrieved and aligned with the newly determined sequences by using the program PILEUP (5). The rRNA alignment was corrected manually, and approximately 100 bases at the 5' end of the molecule were omitted from further analyses because of alignment uncertainties due to highly variable region Vl. Levels of sequence similarity were calculated and corrected for substitution rates by using Kimura's parameters. A phyloge- netic tree was constructed by the neighbor-joining method (22), and the stability of relationships was assessed by using the programs SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE of the PHYLIP package (6). Nucleotide sequence accession number. The 16s rRNA gene sequence of strain DMMZ 227ST (= CCUG 3687gT = DSM 44265' = CIP 105125IT) has been deposited in the EMBL Data Library under accession no. Y11200. RESULTS AND DISCUSSION Six of the eight clinical strains were isolated from blood cultures (Table 1). The remaining strains originated from a wound swab and joint fluid. All eight strains grew in pure culture from clinical specimens. The patients from whom the isolates were recovered were epidemiologically not linked. Colonies of the strains were 1 to 1.5 mm in diameter after 24 h of incubation at 37°C in a 5% C02-enriched atmosphere. They were glistening, slightly yellowish, and very mucoid. A slimy consistency was also observed when colonies were sub- cultured for further testing, but colonies appeared to be less mucoid after 96 h of incubation. Initially, the mucoid colony appearance of the unknown bacterium somewhat resembled the appearance of Rhodococcus equi colonies (13). However, we did not observe a rod-coccus cycle (as seen in R. equi strains [13]) in the unknown bacteria when cells were grown on suitable media. Gram staining revealed typical diphtheroids which were 952