INTRODUCTION

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The seminal paper by Gilmour et al. (13) provided an accurate description of Bacterionema matruchotii and clearly distinguished it from members of the genus Leptotrichia and from other organisms with which it had been confused, including Cladothrix matruchotii, Leptothrix buccalis, Leptotrichia buccalis, and Leptotrichia dentium. Based on chemotaxonomic characteristics, Bacterionema matruchotii was assigned to the genus Corynebacterium by Collins et al. in 1982 (5).

When this laboratory evaluated the API Rapid CORYNE identification system (Biomérieux Vitek, Inc., Hazelwood, Mo.), it was discovered that reactions of Corynebacterium matruchotii had been omitted from its database (12). Since the database included most other recognized Corynebacterium species of human origin, we suspected that the developers had encountered some unresolvable discrepancies with representative strains of this species. To explore this possibility, we purchased all strains of C. matruchotii available at the American Type Culture Collection (ATCC). Because a large body of work has been published on the possible role of C. matruchotii in the pathogenesis of dental plaque, caries, and periodontitis, we also included two additional strains of C. matruchotii that were used in many of these studies, namely strain L2 and Richardson's strain 13 (22, 23).

The present study was designed to examine the taxonomic relatedness of commercially available and other reference strains of C. matruchotii. The question was addressed on the basis of whole-cell fatty acid analyses, DNA-DNA dot blot hybridizations, sequencing of two hypervariable regions of the 16S rRNA gene, and biochemical reactions. The results indicate that the seven ATCC strains deposited as C. matruchotii comprise three different taxa, including four true C. matruchotii strains, two strains of the recently proposed species Corynebacterium durum, and one representative of a novel taxon.

	MATERIALS AND METHODS

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Strains. Strains analyzed in the present study included seven strains deposited in the ATCC as C. matruchotii: ATCC 14266 (type strain), ATCC 14265, ATCC 33449, ATCC 33806, ATCC 33822, ATCC 43832, and ATCC 43833. C. matruchotii strains L2 and Richardson 13 were generously provided by I. Takazoe, Tokyo, Japan. Three strains, LCDC 81-379, LCDC 86-376, and LCDC 91-086, from the laboratory of K. A. Bernard, were also studied because they are similar to the C. matruchotii reference strains ATCC 33449 and ATCC 33822.

Media and biochemical tests. The broth culture medium was heart infusion broth (HIB; Difco Laboratories, Detroit, Mich.) supplemented with 0.2% Tween 80 (Difco Laboratories) (HIB-T). For strains that grew poorly in HIB-T broth, a half-strength recipe of HIB-T supplemented with 0.2% yeast extract (Difco Laboratories) and 0.2 µg of hemin/ml (Sigma Chemical Co., St. Louis, Mo.) (0.5 HIB-T/YH) was prepared as recommended by Marion Gilmour (personal communication). Heart infusion agar supplemented with 5% sheep blood (University Hospital Microbiology Media Room) was used for routine subcultures and for overnight cultures that were used to inoculate conventional biochemical tests. Trypticase soy agar supplemented with 5% sheep blood (TSBA; Prepared Media Labs, Tualatin, Oreg.) was used for all morphology studies.

GLC. Whole-cell fatty acid analyses were performed as previously described (7). The organisms were grown on TSBA. To obtain sufficient biomass of early-stationary-phase cells, one to three plates were inoculated and incubated at 35°C in an aerobic atmosphere with 5% CO₂ for up to 3 days, depending on each strain's growth rate. Whole-cell fatty acid composition was determined by gas-liquid chromatography (GLC) with the Microbial ID, Inc. (MIDI) system, and data were analyzed with the library generation software of MIDI, which is a program that provides two-dimensional cluster plots as well as dendrograms based on cluster analysis. The two-dimensional plots were based on principal component analysis. Principal Component 1 is the component responsible for the greatest degree of variability among the samples tested and is represented on the horizontal axis. Principal Component 2 is responsible for the second greatest degree of variability and is displayed on the vertical axis. The scale for both axes is the Euclidean distance.

DNA-DNA hybridization. Chromosomal DNA was prepared from cells that were incubated by shaking at 37°C for 48 h in 200 mL of HIB-T or the 0.5 HIB-T/YH broth. The DNA extraction procedure was described previously (6). The DNA-DNA hybridization method, using the NEBlot Phototope kit (New England Biolabs, Inc., Beverly, Mass.) and the PolarPlex Chemiluminescent Blotting kit (New England Biolabs, Inc.), was performed as described by Springer et al. (20) by using established conditions that allowed 26 to 27% mismatch for DNA with G+C content of 55 to 58 mol%. So that target DNAs immobilized on the membrane could be reused for subsequent DNA-DNA hybridizations, hybridized probe DNA was removed according to the manufacturer's instructions.

16S rRNA gene sequencing. In order to select hypervariable regions likely to be unique for each taxon, the 16S rRNA gene sequences in nine Corynebacterium species were compared using the Baylor College of Medicine search launcher ClustalW (version 1.6) alignment method. Two regions of over 40 bp in length were chosen and termed hypervariable region 1, corresponding to Escherichia coli 16S rRNA positions 177 to 218, and hypervariable region 2, corresponding to positions 988 to 1034.

Nucleotide sequence accession number. The full sequence of the 16S rRNA gene of strain ATCC 43833 has been deposited in GenBank with accession number AF262996.

	RESULTS

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Whole-cell fatty acid analyses. Fatty acid profiles of all strains were analyzed with the library generation software of MIDI, resulting in the two-dimensional plot shown in Fig. 1. The computerized cluster analysis demonstrated three defined clusters, which were assigned the designations of group A (ATCC 14266^T, ATCC 14265, ATCC 33806, ATCC 43832, L2, and Richardson 13), group B (ATCC 33449, ATCC 33822, LCDC 81-379, LCDC 86-376, and LCDC 91-086), and group C (ATCC 43833). A dental clinical isolate of C. matruchotii is included in the figure. The major fatty acid peaks of the group A (true C. matruchotii) strains and the group B (true C. durum) strains were palmitic, oleic, and stearic acid (Table 1). The remaining fatty acids detected by the MIDI system (not listed) are believed to be the products of mycolic acid degradation, which occurs at the high temperature in the system's injection port (11).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Two-dimensional plot generated by principal component analysis of fatty acid profiles showing the distribution of strains that are listed in Table 1. Circles, group A C. matruchotii; triangles, group B C. durum; squares, group C strain ATCC 43833. Data from repeat analyses are included. Strains: a, ATCC 14266; b, ATCC 14265; c, ATCC 33806; d, ATCC 43832; e, L2; f, Richardson's 13; g, ATCC 33449; h, ATCC 33822; i, LCDC 81-379; j, LCDC 86-376; k, LCDC 91-086; l, ATCC 43833; m, a dental isolate of C. matruchotii. Numbers on the axes indicate the Euclidean distance.

DNA-DNA hybridization. Representative lumigraphs of DNA dot blots for 11 of the 12 strains in this study are shown in Fig. 2. Because the DNA yield was low from strain ATCC 33806, it was excluded from the dot blot study. Whole-cell DNA probes for hybridization were selected on the basis of GLC groupings. DNA from strains in each GLC group hybridized only with the DNA probes from strains in their respective group. DNA from the single group C strain (ATCC 43833) hybridized only with autologous DNA. The DNA dot blot results were in complete agreement with the groupings indicated by the MIDI cluster analysis from cellular fatty acid profiles.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   DNA-DNA homology studies with solid-phase dot blot hybridizations. Probe and target DNAs are indicated.

16S rRNA sequencing. Sequencing of the two selected hypervariable regions confirmed the GLC and DNA group divisions (Fig. 3). The sequences from GLC group A strains correlated well with the published sequence of the C. matruchotii type strain. For four of the five strains in group B, the sequences of the two variable regions were almost identical to that published for C. durum (19). The sequence of variable region 1 in strain ATCC 33822 differed from that of C. durum by eight bases. The single strain in group C (ATCC 43833) has a unique sequence differing from any sequences accessible by BLAST searches of all nonredundant GenBank, EMBL (European Bioinformatics Institute), and DDBJ (DNA Data Bank of Japan) sequences.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Alignment of sequences of two variable regions in the 16S rRNA gene. GenBank sequences of C. matruchotii and C. durum were used as references and compared with those found in this study. Bold bases for C. durum indicate differences from the C. matruchotii sequence. The first and last nucleotides of regions 1 and 2 correspond to E. coli 16S rRNA positions 177 to 218 and 988 to 1034, respectively.

Morphology. When grown in air or a CO₂-enriched atmosphere for 48 h at 37°C on TSBA, all strains in this study produced colonies about 1 mm in diameter. Four of the six true C. matruchotii colonies were white, cream, or gray with a smooth matte surface and creamy texture when touched with a loop. Colonies of strains ATCC 33806 and Richardson's 13 had a crinkled surface and lifted as a single dry colony when touched by a wire loop. When grown in the presence of CO₂, these two strains produced distinct pits in the agar.

View larger version (147K): [in this window] [in a new window]   FIG. 4.   The Gram stain morphology of C. matruchotii after growth on blood agar in a CO2-enriched atmosphere at 37°C for 48 h. Arrows indicate whip handle morphology.

Biochemical tests. The results from conventional biochemical tests and the numerical profiles of the reactions from the Rapid CORYNE system are shown in Table 2. The production of pyrrolidonyl arylamidase was the only trait that consistently distinguished C. matruchotii strains from C. durum strains. The atypical strain, ATCC 43833, was unique in its failure to reduce nitrate and its production of alkaline phosphatase. The conventional esculin test was negative for all strains, whereas it was positive in the CORYNE strip for 9 of the 12 strains. In contrast, the CORYNE strip did not detect urease activity in two of the four C. durum strains that were urease positive in the conventional test. Two of the five C. durum isolates yielded both positive and negative reactions when tested three times in conventional mannitol broth. With the exception of one isolate, repeat testing of C. durum isolates for production of acid in galactose also yielded contradictory results.

	DISCUSSION

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Evidence from biochemical reactions, fatty acid analysis, DNA hybridization, and 16S rRNA hypervariable region sequencing shows that the seven strains deposited in the ATCC as C. matruchotii actually are diverse organisms including four true C. matruchotii strains, two C. durum strains, and a single unique strain that probably represents a novel taxon. The group corresponding to the C. matruchotii type strain was clearly and correctly described by Marion Gilmour and colleagues (13).

Gilmour and Turner described C. matruchotii as a pleomorphic organism whose variability in colonial morphology is a consequence of mode of reproduction, culture age, and cultivation conditions (14). They described a rough-to-intermediate-to-smooth morphological variation. When our biochemical results are examined, it is apparent that the true C. matruchotii strains have the biochemical reactions described in Bergey's Manual of Systematic Bacteriology (15). The API system, which does not include C. matruchotii, provides no identification for many of the strains in this study. Strains ATCC 33806, ATCC 43833, and Richardson's 13 are assigned to the genus Corynebacterium, and strain ATCC 33822 has a "good identification" as Listeria grayi.

Biochemical tests do not readily distinguish C. durum from C. matruchotii. The only API CORYNE test in which they consistently differ is the pyrrolidonyl arylamidase reaction, which occurred with the six C. matruchotii strains but with none of the five C. durum strains in this study. This difference is consistent with two earlier studies that tested a total of 63 C. durum isolates, none of which had pyrrolidonyl arylamidase activity (19, 24). The C. durum species is biochemically diverse. Within isolates from 58 cultures of healthy throats, von Graevenitz et al. found 8 API CORYNE profile numbers, none of which is identical to the four profile numbers in this study (24).

Riegel et al. noted that all five of their C. durum isolates produced acid from mannitol and galactose, which helped to distinguish them from C. matruchotii (19). However, when we tested our C. durum isolates on Hugh Leifson's oxidation-fermentation medium as used by Reigel, no acid was detected in mannitol or galactose. Using the in-house peptone water fermentation broth with Andrade indicator that the Centers for Disease Control and Prevention recommends for testing coryneforms (18), we did repeat tests with mannitol and galactose. Two of the C. durum strains were consistently positive with mannitol, one was negative, and results with two strains were not reproducible. Four of the C. durum strains also had inconsistent results in repeat galactose testing (Table 2). An additional study of these strains' reactions on both mannitol and galactose in peptone water broth from two providers revealed differences in the frequency of positive results (data not shown). The differences in urease activity and esculin hydrolysis observed between the Rapid CORYNE system and conventional tests might be attributed to the different basal media in these two systems.

It was surprising to find that our strains of C. durum did not fit the colonial morphology described by Riegel et al., who selected the species name to reflect its strong adherence when grown in an aerobic atmosphere on TSBA (19). When Riegel's strains were cultured on TSBA in the presence of CO₂, they were not adherent. Our C. durum strains on TSBA responded to the atmosphere in the reverse manner, with gummy adherence seen only in the presence of CO₂. The differences between our strains and Riegel's strains of C. durum might be attributed to the fact that our strains originally were deposited in the ATCC as C. matruchotii or were reported to closely resemble C. matruchotii, which might represent a unique subset of C. durum strains. Furthermore, the original specimens for our strains and the Riegel strains were quite different. In contrast to the five C. durum isolates described by Riegel et al., only one of the C. durum strains in the present study (LCDC 81-379) had been recovered from sputum. ATCC strains 33449 and 33822 were from a gingival margin and subgingival plaque, respectively, and LCDC strains 91-086 and 86-376 were isolated from blood and a foot abscess, respectively.

Within the true C. matruchotii strains, the sequences of hypervariable regions 1 and 2 plus flanking regions differed at only one position, in which the guanine of the type strain was replaced by an adenine in the other five strains. All but one of the C. durum strains also exhibited highly conserved sequences that varied in only two contiguous bases that were either two cytosines or two thymidines. However, the sequence of hypervariable region 1 in C. durum ATCC 33822 differed from that of the type strain by eight bases. Because this strain was consistent with the species C. durum in DNA dot blots, fatty acid analyses, and biochemical reactions, it seems likely that this is the correct identification. Intraspecies variation in the sequences of 16S rRNA has been reviewed by Clayton et al., who concluded that some of the sequence heterogeneity observed within species may be due to interoperon differences (4). We have notified the ATCC of the corrected identifications of C. durum strains ATCC 33449 and ATCC 33822.

C. matruchotii has been used by a number of researchers as a microbiologic model for intracellular calcification (1, 3, 8, 9, 21, 25). The organism has provided valuable information regarding calcification of bioprostheses (17), dental plaque (10), and the principles of membrane-mediated proteolipid-dependent calcification in vertebrate systems (2). It is important to note that the diversity found in this study does not affect the conclusions of pathogenesis studies with C. matruchotii. The vast majority of earlier studies have relied on results from one or more of four strains, namely ATCC 14265, ATCC 14266, L2, and Richardson 13, all of which we have confirmed to be true C. matruchotii strains.