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Journal of Clinical Microbiology, June 2004, p. 2829-2832, Vol. 42, No. 6
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.6.2829-2832.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Microbiology Unit, Faculty of Medicine, University of Louvain, B-1200 Brussels, Belgium,1 Institute of Medical Microbiology, University Hospital RWTH Aachen, 52074 Aachen, Germany2
Received 13 October 2003/ Returned for modification 2 December 2003/ Accepted 2 March 2004
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Origin of the strains and clinical features. (i) Strains CF51, CF63, CF79, and CF91 were isolated from blood cultures of a patient with human immunodeficiency virus (HIV) during a 22-month period in November 1997, June 1998, March 1999, and August 1999, respectively. The patient had ambulatory treatment for his HIV infection, and the organisms were isolated during recurrent febrile episodes. Pulsed-field gel electrophoresis (3) performed on the four isolates resulted in identical profiles different from that obtained with a similar strain from another patient. Therefore, the four isolates were considered a single strain and only CF63 was selected for further studies. (ii) Strain CF52 was isolated on two consecutive days from several blood bottles from a young female neurological patient with a Hickman catheter. (iii) Strain CF105 (GH443) was isolated by G. Haase from the blood of a 33-month-old boy with acute myeloid leukemia. (iv) Strain CF127 was isolated from one blood bottle from a febrile hospitalized child. Only one blood culture was performed. (v) Strain CF110 was isolated from blood, but no other information was available. (vi) Strain DSM 9657 was identified by G. Funke and A. Carlotti as B. casei, isolated four times from peritoneal dialysate, and later deposited into the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (5).
Cellular fatty acids (CFA) of the strains, determined as described previously (16), were of the branched type, mainly anteiso 17:0 and anteiso 15:0, accounting for more than 75% of the total CFAs. Peptidoglycan analysis of strain CF63 was carried out at the DSMZ by N. Weiss according to the method of Schleifer and Kandler (15) and showed an A1
type, with the presence of meso-diaminopimelic acid as the diamino acid.
The 16S rRNA gene (rDNA) sequence determinations were conducted as described previously (18). The 16S rDNA sequence of strain CF63 displayed the highest similarity (98.8%) to the sequence of an unidentified bacterium (strain GH443) that had been submitted to GenBank in 1993 by A. Podbielski under accession number X76703. Its similarity to the sequence of B. casei DSM 20657T was only 97.7% (Table 1). When a new sequence determination of GH443 was performed at our laboratory along with that of CF63, a similarity of 99.9% was obtained for the two strains. The 16S rDNA sequence homologies of CF63 to the four other study strains were as follows: for CF52, 99.4%; for CF110, 99.8%; for CF127, 99.5%; and for DSM 9657, 99.9%.
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Most biochemical tests were carried out as outlined previously (6, 9, 17, 18). API 32 GN, API 50 CH, and API Biotype 100 strips (Biomerieux, Marcy l'Etoile, France) were used with AUX medium, incubated at 37°C, and read after 2 and 4 days. API ZYM strips (Biomerieux) were used according to the manufacturer's instructions. API CORYNE strips (Biomerieux) were incubated at 37°C and read after 1 and 2 days. Susceptibility to thallium acetate was tested, since this compound is known to inhibit several bacterial species and has been used as a selective agent (8). For this purpose paper disks were impregnated with 10 µl of a 0.5% (wt/vol) thallium acetate (Sigma-Aldrich, St. Louis, Mo.) solution and put onto blood agar. Assimilation-alkalinization of organic compounds was performed on Simmons agar base, replacing citrate by 0.2% (wt/vol) of the substrate according to the method of Martin et al. (11).
The six strains were nonmotile coryneform rods. They grew aerobically on blood agar at 37°C, and the colonies were grayish white and somewhat sticky. Catalase gave positive test results, and oxidase gave negative test results. The metabolism was oxidative, there was proteolytic but no saccharolytic activity, and production of methanethiol was positive. The code generated by the API CORYNE system was 6112004, which was similar to the code generated by B. casei. By conventional methods the phenotypic profile of the six strains was also closely related to that of B. casei (5); however, some differences allowed the study strains to be distinguished from the latter species. When paper disks containing 50 µg of thallium acetate were used on blood agar, B. casei was resistant, growing at the edge of the disk, whereas the new strains exhibited an inhibition zone of more than 25 mm. In the API Biotype 100 system, quinate was assimilated by the six strains but not by B. casei. Assimilation-alkalinization of quinate was also achieved by using Simmons agar base containing 0.2% (wt/vol) quinate (Sigma-Aldrich). On Simmons base containing 0.2% (wt/vol) tyramine hydrochloride (Sigma-Aldrich), similarly, this substrate was utilized by all the study strains but one (CF127) whereas none of the B. casei strains grew. As with other amines like putrescine or cadaverine, acidification, and not alkalinization, of the medium was observed when tyramine was assimilated. The main phenotypic characteristics that differentiate the new strains from Brevibacterium species encountered in humans are reported in Table 2.
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Phenotypic, taxonomic, and genetic properties suggest that the six strains belong to the genus Brevibacterium but constitute a new species related to B. casei for which the name B. sanguinis is proposed (Fig. 1).
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B. sanguinis is very similar to B. casei when tested by conventional methods or commercial identification sets. Their CFA compositions are similar and do not differentiate the two species. Therefore, it is likely that some strains previously reported as B. casei were misidentified. This is also suggested by the results seen with strain DSM 9657, which was deposited as B. casei but proved in this study to belong to the new species. A simple test, such as determination of susceptibility to thallium acetate, may help to differentiate the two species in the routine laboratory.
Description of B. sanguinis sp. nov.
Cells of B. sanguinis (san'-gui-nis, meaning blood, because most strains were isolated from blood) are gram-positive coryneform rods growing aerobically at 25 and 37°C. On blood agar, colonies are grayish white, opaque, somewhat sticky, and reach a diameter of 2 mm after 48 h at 37°C. Catalase gives positive test results, and oxidase gives negative test results. Gelatin, casein, tyrosine, and xanthine are hydrolyzed, but esculin is not. Methanethiol is produced. Strains are very susceptible to thallium acetate. Urease gives negative test results, pyrazinamidase gives positive test results, and nitrate reduction assays produce weak or negative results. Glucose and other carbohydrates are not acidified, but on API 50 CH and ID 32 GN strips glucose, D-arabinose, maltose, glycerol, N-acetylglucosamine, L-fucose, myo-inositol, sucrose, turanose, trehalose, and gluconate are assimilated. With API ZYM system tests, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, phosphoamidase, and 
-glucosidase give positive results. N-Acetyl-ß-D-glucosaminidase gives negative results by this method, but results are slightly positive when the nitrophenyl conjugate (Rosco, Taastrup, Denmark) is used. Pyrrolidone arylamidase gives variable results. Quinate and -aminobutyrate are assimilated and alkalinized on Simmons mineral base. Tyramine is assimilated and acidified on Simmons mineral base by most strains. Acid is produced from phenyl acetate. CFAs are of the branched type, with anteiso C15:0 and anteiso C17:0 being the major components. The diamino acid of the peptidoglycan is meso-diaminopimelic acid.
The organisms are isolated from human clinical samples; the type strain is CF63T (= DSM 15677T = CCUG 47857T). The G+C content of the DNA is 69.9 mol%. Pyrrolidone arylamidase gives weakly positive results, and tyramine is utilized. The strain was isolated from the blood of an HIV patient.
Nucleotide sequence accession numbers. The 16S rDNA sequences of the following strains were deposited in the EMBL (European Molecular Biology Laboratory) Nucleotide Sequence Database under the following accession numbers: for CF63, AJ564859; for CF52, AJ628351; and for DSM 9657, AJ628352. Two other strains with the following accession numbers have been deposited in the DSMZ and the Culture Collection, University of Göteborg (CCUG): CF105 (= GH443) = DSM 15679 = CCUG 47859 and CF52 = DSM 15678 = CCUG 47858.
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ACKNOWLEDGMENTS |
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