INTRODUCTION

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Eubacterium lentum is a gram-positive, non-spore-forming obligate anaerobe isolated from normal human feces and infections in humans (8) which comprises 5 to 10% of anaerobic isolates in hospitals (3). The microorganism is characterized by a few positive biochemical reactions: namely, it is asaccharolytic, it reduces nitrate, and its growth is greatly enhanced by arginine (13). Recently E. lentum has been demonstrated to show orange to red fluorescence under UV light; this feature may represent a rapid tool for its laboratory identification (9). However, investigators have noted a heterogeneity among isolates of E. lentum. MacDonald et al. (7) identified two groups, i.e., E. lentum and phenotypically similar organisms, on the basis of production of two steroid-metabolizing enzymes (bile acid 3-hydroxysteroid dehydrogenase and bile acid 12-hydroxysteroid dehydrogenase) with minimal overlapping of biochemical characteristics. In fact, the synthesis of the steroid enzymes was positively correlated with stimulation by arginine, catalase activity, and H₂S production. These latter biochemical characteristics were also correlated with the production of red fluorescence (9). Moreover, Verhulst et al. (14) demonstrated that although they had similar G+C contents ranging between 63.7 and 69.1 mol%, steroid-producing and non-steroid-producing strains of E. lentum were characterized by different patterns of long-chain fatty acids.

Gas chromatographic analysis of cellular fatty acids and examination of protein patterns by polyacrylamide gel electrophoresis have been used taxonomically to distinguish among strains within a species and among organisms within a genus or family (2).

The aim of the present study was to better delineate the taxonomic relatedness of strains of E. lentum by examining soluble-protein contents, cellular fatty acid profiles, and antimicrobial resistance patterns to ascertain whether differences in these characteristics could be correlated with the previously described differences in biochemical activities.

	MATERIALS AND METHODS

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Bacterial strains and their characterization. Strains used were from the culture collection of the Veterans Administration Wadsworth Anaerobic Bacteriology Laboratory, Los Angeles, Calif. At the time of isolation all strains were identified as E. lentum or "most likely E. lentum" by the criteria of Holdeman et al. (6). Reference strains of E. lentum (ATCC 25559 and ATCC 34055) were also included in the study. All the isolates have previously been examined by Mosca et al. (9) for the production of red fluorescence; 21 of 29 clinical strains as well as the type strains were red fluorescent. Among the eight isolates that did not exhibit red fluorescence, six were different from all the other strains with regard to colony morphology, cellular morphology, catalase and H₂S production, and arginine stimulation. The colony morphology of these six strains was punctiform and translucent in contrast to that of the other strains, which had circular, grey, speckled colonies. On Gram staining, these isolates appeared as coccobacilli, while the others were pleomorphic bacilli (Table 1).

Gas-liquid chromatography of cellular fatty acids. Each organism was subcultured in 10 ml of PRAS-PYG broth (Carr-Scarborough Microbiologicals, Stone Mountain, Ga.) supplemented with arginine and incubated overnight at 35°C. Each of the six strains producing only scant turbidity was inoculated in four tubes to obtain a satisfactory cell pellet. Lysis of cells through saponification, methylation of fatty acids, and extraction of the methyl esters into the organic phase were achieved as previously described (4, 5). Samples were processed on a Hewlett-Packard 5890A gas chromatograph with a Hewlett-Packard 7673A automatic sampler and integrator. The chromatography unit was coupled to a computer with Microbial Identification System (MIS) automated software (5). For identification, the Virginia Polytechnic Institute broth-based anaerobe library was employed.

Polyacrylamide gel electrophoresis of cellular proteins. E. lentum strains were inoculated in 100 ml of prereduced brucella broth supplemented with arginine and incubated overnight at 35°C. The cultures were centrifuged and washed in Tris buffer (100 mM Tris, 10 mM MgSO₄, pH 7.4). The bacterial pellets were resuspended in 1.5 ml of Tris buffer, and cells were broken in a French pressure cell. The suspensions were centrifuged at 13,000 × g for 1 min to remove whole cells. The supernatants were transferred to microcentrifuge tubes, Triton X-100 medium (20% Triton X-100, 0.1 M HEPES, 0.1 M MgCl₂, pH 7.4) was added, and the tubes were placed in ice for 15 min. The tubes were then centrifuged, washed in Tris buffer, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% separating gels. Samples were then resuspended in sample buffer (0.5 M Tris, glycerol, 10% sodium dodecyl sulfate, 2% mercaptoethanol, 0.05% bromophenol blue), loaded onto gels, and electrophoresed at 200 V for about 4 h.

Antimicrobial susceptibility testing. Ampicillin, cefotetan, cefotaxime, ceftriaxone, clindamycin, piperacillin, cefoxitin, metronidazole, imipenem, and chloramphenicol were selected either as representative of a class of compounds or as drugs for which MICs for quality control strains were published. MICs were determined by the National Committee for Clinical Laboratory Standards reference agar dilution method (10). Briefly, the antibiotic powders were reconstituted according to the manufacturer's instructions, and serial dilutions ranging from 256 to 0.015 µg/ml were prepared in brucella agar supplemented with vitamin K, hemin, and 5% laked sheep blood. The inoculum was prepared in an anaerobic chamber by suspending several colonies from a 72-h culture plate in brucella broth to achieve the visual turbidity of a 0.5 MacFarland standard. A Steers replicator (Craft Machine Co. Inc., Chester, Pa.) was used to apply the organisms to the plates for a final inoculum of 10⁵ CFU per spot. Plates were incubated in an anaerobic atmosphere for 48 h. The MIC was defined as the lowest concentration of antimicrobial that resulted in no growth.

	RESULTS

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Cellular fatty acid analysis. The MIS is the first commercial system that takes into account the presence or absence of fatty acids, the nature of each acid, and their ratio for the classification of anaerobic organisms (1, 4, 12).

Soluble-protein patterns. Cellular proteins were studied by electrophoretic separation. Each strain was characterized by a pattern containing about 30 discrete bands, most of which were relatively weakly stained. Although some qualitative and quantitative differences were observed, the grouping of the strains was difficult since considerable homogeneity in the protein profiles was present (data not shown).

Susceptibility testing. The strains of E. lentum showed two different antimicrobial susceptibility patterns (Table 3). In particular, group B strains were susceptible to ampicillin, cefotetan, cefotaxime, and ceftriaxone, in contrast to the strains belonging to group A. Although all the strains were susceptible to clindamycin, piperacillin, and imipenem, MICs for group B strains were lower than those for group A. No difference was observed when metronidazole and chloramphenicol were tested.

	DISCUSSION

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Early reports described gram-positive non-spore-forming bacilli similar to E. lentum but with some different phenotypic and genetic properties (7, 9, 14). In our study of 29 isolates of E. lentum, we could identify 6 (group B) that were different from all the others. These strains in fact were coccobacilli with translucent colonies, catalase and H₂S negative, not fluorescent, and susceptible to beta-lactam drugs, and their growth was not stimulated by arginine. The fatty acid profile revealed the presence of straight-chain fatty acids identical to those observed for steroid-inactive E. lentum strains (14). The remainder of the strains of E. lentum (group A), including the type species, were pleomorphic bacilli with speckled colonies, catalase and H₂S positive, and all (except for two strains) fluorescent, and their growth was greatly stimulated by arginine. Moreover, these strains were more resistant to beta-lactam drugs, and their cellular fatty acid pattern was characterized by saturated branched-chain fatty acids, with 15:0 as the most abundant.

Electrophoretic separation of cellular proteins represents a highly sensitive analysis providing distinctive phenotypic evidence of the similarity of strains (2). Although considerable homogeneity was observed in the protein profile, some qualitative and quantitative differences were detected in some species belonging to group B. Another method that has been used to analyze even minor genomic variation between strains is the restriction endonuclease analysis of bacterial chromosomal DNA. The pattern of DNA bands obtained with a given enzyme is a reproducible feature of a DNA and therefore has potential for typing closely related bacteria (11). We tested some endonucleases (HindIII, MspI, and BsxII), and only with MspI did we obtain complete digestion exclusively for the strains of group B. Taken together, our data support the notion that E. lentum should be further differentiated by the sequence analysis of DNA in order to resolve the appropriate taxonomic status.