JOURNAL OF CLINICAL MICROBIOLOGY, June 1994, p. 1511-15180095-1 137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Vol. 32, No. 6

Human Infections Caused by Brevibacterium casei, formerlyCDC Groups B-1 and B-3

EVA GRUNER,I* ARNOLD G. STEIGERWALT,2 DANNIE G. HOLLIS,2 ROBBIN S. WEYANT 2ROBERT E. WEAVER,2 C. WAYNE MOSS,2 MARYAM DANESHVAR,2

JUNE M. BROWN,2 AND DON J. BRENNER2

Institute for Medical Microbiology, University of Zurich, 8028 Zurich, Switzerland,1 and Emerging Bacterial andMycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious

Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303332

Received 24 January 1994/Returned for modification 1 March 1994/Accepted 16 March 1994

Forty-one clinical strains of CDC coryneform groups B-1 and B-3 were compared biochemically, by analysisof cell wall sugars, amino acids, and cellular fatty acids, and by DNA relatedness to the type strains ofBrevibacterium casei, Brevibacterium epidermidis, and Brevibacterium linens. Twenty-two strains were shown to beB. casei, while five other strains formed a phenotypically inseparable genomospecies in the same genus. Theremaining isolates were genetically heterogeneous, and most are probably members of the genus Brevibacte-rium. They were not further identified, but they were biochemically distinguishable from B. casei. Eleven of theclinical strains of B. casei were isolated from blood, and two each were isolated from cerebrospinal fluid andfrom pleural fluid. At least five isolates were from multiple blood or cerebrospinal fluid cultures. To our

knowledge, these strains are the first described clinical isolates identified as B. casei, which was previouslyconsidered to be a nonpathogenic species.

Until recently, the genus Brevibacterium has been withoutany apparent relevance to the clinical microbiologist. Thegenus, as currently defined, was established in 1953 by Breed(6) and includes four species (17), Brevibacterium linens (thetype species), Brevibacterium epidermidis, Brevibacterium casei,and Brevibacterium iodinum. The habitat of brevibacteria isprimarily dairy milk products (13, 30), where the bacteriacontribute notably to the aroma and the color (orange-pig-mented B. linens) of surface-ripened cheeses (1, 24). Amongthe four species, only B. epidermidis (10, 28, 31), as suggestedby its name, has been isolated from human skin. However, onlya few reports mention it as a possible opportunistic pathogen(22, 27).Over a 30-year period, the Special Bacteriology Reference

Laboratory, Centers for Disease Control and Prevention(CDC), has received more than 50 patient isolates of nonfer-mentative coryneform bacteria that have been identified asCDC groups B-1 and B-3. These groups are composed ofnonmotile, diphtheroid-like, catalase-positive, gram-positiverods. Failure to oxidize carbohydrates differentiates group B-3from group B-1, which oxidizes glucose and sometimes alsomaltose and sucrose. Although a variety of clinical sources arerepresented, more than 60% of B-1 and B-3 isolates wereobtained from blood or cerebrospinal fluid (CSF).The purpose of the present investigation was to determine

the relationship between the CDC groups B-1 and B-3 andbrevibacteria. Forty-one CDC group B-1 and B-3 strains werestudied biochemically, by analysis of cellular fatty acids(CFAs), whole cell sugars, and amino acids, and by DNArelatedness. They were compared with the type strains of B.casei, B. epidermidis, and B. linens. B. iodinum was not includedin the study because of the ease of identification of this specieson the basis of the presence of crystals of iodinin (17).

* Corresponding author. Mailing address: Institute for MedicalMicrobiology, University of Zurich, Gloriastrasse 32, 8028 Zurich,Switzerland.

MATERIALS AND METHODS

Strains. The 41 clinical isolates and their sources are listedin Table 1. Except for three strains received from the NationalLaboratory for Bacteriology, Ottawa, Canada, and two strainsfrom the Institute of Medical Microbiology, Zurich, Switzer-land, all strains were obtained from the stock culture collectionmaintained by the Special Bacteriology Reference Laboratoryat the Emerging Bacterial and Mycotic Diseases Branch atCDC. They had been referred to this laboratory for identifi-cation over a 30-year period. The type strains of B. epidermidis,ATCC 35514T, and B. linens, ATCC 9172T, and B. linensATCC 9175 were obtained from the American Type CultureCollection (ATCC), Rockville, Md. The type strain of B. casei,ATCC 35513T, and reference strains B. epidermidis ATCC49089 and B. linens ATCC 19391 were provided by the GermanCollection of Microorganism and Cell Cultures in Braun-schweig, Germany, through the Institute of Medical Microbi-ology in Zurich, Switzerland. Strains were grown aerobically onheart infusion agar with 5% rabbit blood (BBL, BectonDickinson Microbiology Systems, Cockeysville, Md.). All B.linens strains were incubated at room temperature; all otherstrains were incubated at 35°C.

Biochemical tests. All strains were identified by conven-tional biochemical tests described previously (9). DNase pro-duction was tested on plates containing methyl green (33).Clearing of the medium within 1 week was read as a positiveresult. Use of serine, d-tartrate, or acetamide as a sole sourceof carbon was tested by the alkalinization technique (21).Methanethiol production from methionine was tested on 5'-dithiobis-2-nitrobenzoic acid (DTBN) agar as described byPitcher and Malnick (27). A positive reaction was the produc-tion of a bright yellow color; test results were read after 2, 4,and 24 h. Additional enzyme activities were tested by using theAPI ZYM system (API bioMerieux SA, La Balme-les-Grottes,France) according to the instructions of the manufacturer.CFAs. For CFA analysis, cells were grown on two or three

plates of rabbit blood agar and harvested after 24 h ofincubation. Cells from a second subculture were harvested 1

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J. CLIN. MICROBIOL.1512 GRUNER ET AL.

TABLE 1. Sources and backgrounds of clinical isolates used in this study

Stratin Source; diagnosis or Scx ae Geographic source,(CDC group) underlying conditioni S ag'1 yr received

Hybridization group I8183 (B-3)B 2341 (B-3)C 2409 (B-1)C 5383 (B-3)D 8146 (B-3)E 4046 (B-3)E 8294 (B-3)F 124 (B-3)F 3969 (B-3)F 4(152 (B-3)F 4157 (B-1)F 8007 (B-3)G 1995 (B-1)G 2002 (B-1)G 3657 (B-1)G 5048 (B-1)G 7635 (B-I)G 7814 (B-l)G 8419 (B-I)G 8504 (B-3)LCDC 79-419' (B-1)LCDC 81-1016' (B-1)

Flybridization group IIC 4514 (B-3)D 4556 (B-I)F 3333 (B-3)G 4871 (B-1)V07 18612" (B-3)

Hybridization group 111A 198t) (B-l)

B 2777 (B-1)E 8862 (B-3)F 2557 (B-3)F 3827 (B-I)F 7428 (B-1)F 7979 (B-3)G 2125 (B-1)G 2607 (B-3)G 3076 (B-3)G 4318 (B-3)G 6560 (B-3)LCDC 79-861' (B-3)V03 8717" (B-3)

BloodPleural fluidWoundPleural fluidSpleenJoint fluidBlood; septicemialCSF; head injury, possible meningitisBlood; bacteremia in cancerThroat and lochia; abortionShunt"; meningitisPeritoneal fluid; dialysis, lupus, peritonitisBlood"; chronic granulomatous disease with feverBlood"; broviac line infection in neuroblastomaBlood"; valvular heart disease, chronic renal failureCSF; meningitisBlood; cellulitisBlood"; bacteremiaBlood"; lymphomaSpleen; hantavirus infectionBloodBlood

CSFBloodCSF; ventriculitisBlood"; leukemia and feverCornea; perforated keratoplastic ulcus

Blood; impetigo, axillary lymphadenopathy andbacteremia

EarBone marrow; fever of unknown originBlood" neuroblastomaBlood"; septicemiaUrine; catheterBlood"; bone marrow transplantationBlood; bactercmiaBloodBloodBlood; bacteremiaToenailEar; otitis mcdia

MM, 24FFFFMM, IM, 65FM, 6 monthsF, 34M, 10FMF, newbornM, 30MFMFF, 46

MF, 3 monthsF, 56M, 79

F, 2

F, approx 80MMF, 4M, 3F, 71M, 9F, 2MFM, 9FF

W.Va., 1962Miss., 1969Nev., 1972Tex., 1973Miss., 1977Md., 1978S.C., 1980La., 1981Pa., 1982Hungary, 1983Calif., 1983Ind., 1986Minn., 1988Mich., 1988Nebr., 1989Colo., 1990Ind., 1992Ohio, 1992Ga., 1993N.Mex., 1993Montreal, Canada, 1979Ottawa, Canada, 1981

Calif., 1972Ariz., 1975P.R., 1982Ind., 1990Zurich, Switzerland, 1993

Tex., 1964

S.C., 1969Guam, 198(1Calif., 1982Calif., 1982Colo., 1985ColO., 1986Pa., 1989S.C., 1989Wash., 1989Del., 1990Bristol, United Kingdom, 1992Toronto, Canada, 1979Zurich, Switzerland, 1991

" M, matle; F, female. Agcs are in years uniless indicated otherwisc.' Isolated at least thrce times.' Obtained from the Laboratory Centre for Disease Control (LCDC), Ottawa, Ontario, Cainada.'" Ohtained from the Institute for Medical Microhiology, Zurich, Switzerland.

day later. Methods for saponification of cells, derivatization offatty acids, and analysis by gas-liquid chromatography andmass spectrometry have been described previously (23). My-colic acids were determined by high-resolution gas-liquid chro-matography and gas-liquid chromatography-mass spectrome-try. Cells of Coiynebacterilmn diphtheriae were used as a

positive control.Determination of m-DAP and whole cell sugar patterns.

Each strain was grown overnight in an aerobic atmosphere on

three plates of rabbit blood agar. After being harvested, cellswere washed twice with distilled water and then autoclaved tokill the organisms before being hydrolyzed overnight in ml of6 N HCI at 10()C (for determination of meso-diaminopimelic

acid [m-DAP]) or boiled for 2 h in 2 ml of 1.0 N H^SO4 (foranalysis of whole cell sugars). DAP and sugars were deter-mined by a paper chromatographic method as describedpreviously (3, 20). Ten microliters of DAP solution (2 mg/ml ofH,O) and 20 p.l of a mixture containing 0.1% each ofD-galactose, i)-glucose, D-mannose, L-arabinose, and D-riboseserved as chromatographic controls.DNA relatedness studies. For the preparation of unlabeled

DNA, strains were grown in up to 5 liters of brain heartinfusion broth (BHIB) for 2 to 4 days. All strains wereincubated at 37°C, except B. linens ATCC 9172T and ATCC9175, which were grown at room temperature. B. linens ATCC19391 and B. epidermidis ATCC 49089 were not included in the

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HUMAN INFECTIONS CAUSED BY B. CASEI 1513

molecular studies. Strains were sedimented by centrifugationat 4,200 x g for 35 min, and the pelleted cells were stored at4°C until use. The procedure for extraction of DNA is amodification of the methods of Brenner et al. (8) and Riegel etal. (29). The cells were suspended in a lysing solution (20ml/liter of BHIB) containing 0.2 M sucrose, 0.05 M Tris-HCl,and 2.5 mg of lysozyme per ml and were incubated in a waterbath at 37°C for 2 h. Lysis within a few minutes was accom-plished by the addition of sodium dodecyl sulfate to a concen-tration of 1%, followed immediately by the addition of anequal volume of phenol to prevent degradation of DNA. Forfurther purification of the DNA and for the use of hydroxyap-atite for DNA-DNA hybridizations, the protocol of Brenner etal. was followed (8). Labeling of DNA was done with[32P]dCTP by using a nick translation kit (Gibco BRL, Gaith-ersburg, Md.) as described by the manufacturer. All hybridiza-tion reactions were performed at 65°C (optimal reassociation)and at 80°C (stringent reassociation). The percentage ofunpaired bases within hybridized sequences (divergence) wascalculated by the decrease of thermal stability of a heterolo-gous reassociated DNA duplex compared with that of thehomologous DNA duplex. Each degree Celsius reduction ofthe melting point corresponds approximately to 1% of thedivergence within related sequences (5). All DNA-DNA hy-bridization reactions were done at least twice.The guanine-plus-cytosine (G+C) content of DNA from 10

CDC group B-1 or B-3 strains was determined spectrophoto-metrically by thermal denaturation. The type strains of B. casci,ATCC 35513r, and B. epidermidis, ATCC 35514T, were in-cluded as controls.

RESULTS

DNA relatedness studies. The G+C contents of 10 groupB-1 and B-3 strains and of the B. casei and B. epidermidis typestrains are given in Table 2. The values for the type strains ofB. casei and B. epidermidis correspond to the data from theliterature (B. casei, 66 to 67 mol%; B. epidermidis, 63 to 64mol% [17]). Since the amount of G+C is high, 63.3 to 67.0mol%, 65°C was chosen as the optimal incubation temperaturefor DNA reassociation and 80°C was chosen as the stringentincubation temperature.Twenty-two of the 41 CDC group B-I and CDC group B-3

strains and the type strain of B. casei formed a single DNAhybridization group (hybridization group I) with the labeledstrain F 3969. In 65°C reaction mixtures, hybridization group Istrains had an average relatedness of 82%, with 1.5% diver-gence in related sequences. Their average relatedness in 80°Creaction mixtures was 83%. Strains G 1995 and LCDC 81-1016exhibited only 63% relatedness to F 3969, but divergence waslow and relatedness remained high at 80°C (relatedness of G1995 to F 3969 was >70%). In reciprocal reactions (data notshown), labeled LCDC 81-1016 was 73% related to unlabeledF 3969 DNA at 65°C, with 0% divergence, and was 67%related to it at 80°C. These data agree with the definition of agenetic species, which should consist of strains whose DNAsare 70% or more related under optimum conditions (withrelatedness not dropping more than 15% in stringent condi-tions) and whose DNAs contain 5% or less divergence in theirrelated sequences (34).A second hybridization group contained strain F 3333 (the

source of labeled DNA) and four other strains. They were 74to 97% related (average 82%) at 65°C and 76 to 86% related(average of 81%) at 80°C. The average divergence in relatedsequences was 2.0% (Table 2). Hybridization group II was 42

to 55% related to F 3969, with 4.5 to 10% divergence in relatedsequences.The 14 remaining group B-1 and B-3 strains were 46% or

less related to hybridization group I or II. We will refer to themas "hybridization group III," with the realization that they aregenetically heterogeneous, containing at least five differenthybridization groups of two strains each. In 80°C reactionmixtures, only two of the B-1 and B-3 strains hybridized withlabeled DNAs from the type strains of B. epidermidis and B.linens at values higher than 21% (data not shown). Theexceptions were two orange strains, F 7979 and G 2607, whichwere 62% related to B. epidermidis at 65°C, with a divergenceof 3.5%. Their relatedness to B. epidermidis was 48% at 80°C.B. linens ATCC 9175 hybridized only slightly (0 to 7% at 80°C)with all hybridization groups.

Interrelatedness of Brevibacteriumn type strains is shown inTable 3. The type strains of B. epidermidis, B. linens, and B.iodinum were more closely related to each other than any ofthem was to B. casei.

Phenotypic characterization. The biochemical reactions ofall strains are listed in Table 4. Tests in which all strains werenegative or positive are listed in footnote b to Table 4. None ofthe strains grew anaerobically. They are short, gram-positive,nonmotile rods. Hybridization group I, including the typestrain of B. casei, and hybridization group II exhibited ahomogeneous colony morphology. These strains grew rapidly,with colonies reaching about 2 mm in diameter after 48 h.Colonies were dome shaped, with entire edges and a slightlymoist shiny surface. They were dirty white, and after 2 to 3 daysthey started to give off the typical sharp, ammonia-like odor ofan aging surface-ripened cheese (like Limburger or an oldCamembert). Of the 14 strains in hybridization group III, 8 hada characteristic pigment (lemon, darker yellow, or deep or-ange). Two more strains had a greenish tone, which intensifiedwith aging, and had a slight Pseudomonas aeruginosa-like odor.All the other strains in hybridization group III developed thesame cheese-like smell as the typical brevibacteria. Also, thefour noncolored isolates were morphologically indistinguish-able from B. casei.None of the 41 strains produced H,S in the butt of the triple

sugar iron agar slant, but all strains from hybridization groupsI and II typically produced a small amount of H,S on leadacetate test paper. None of the strains were 3-hemolytic;however, nearly all isolates presented a diffuse lysis pattern on

blood agar plates after 2 or more days.Reference strains B. linens ATCC 19391 and ATCC 9175

differed from the type strain in being nitrate, gelatin, andtartrate negative, and in several sugar reactions. Furthermore,ATCC 19391 was citrate negative. All B. linens strains devel-oped an orange pigment which was not light dependent. B.epidermidis ATCC 49089 did not differ from the type strain inany reaction.CFAs. All 41 isolates showed predominantly branched-chain

fatty acids, with 12-methyltetradecanoic (a-15:0) and 14-meth-ylhexadecanoic (a-17:0) acids as the main CFAs (Table 5). Insome instances, the relative amounts of CFAs differed notably(10%) in replicate analyses of the same strain grown underidentical conditions and for the same length of time.

Cell wall components. Except for two strains not included inhybridization groups I and II (A 1980 and F 2557), all isolatescontained m-DAP in whole-cell hydrolysates. Among thesugars tested, readily detectable amounts of D-glucose andD-ribose were found in all isolates, whereas the presence ofgalactose varied among the strains. Galactose was present in27% of the hybridization group I strains and in the type strainof B. casei; it was not found in any strain of the second

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TABLE 2. Percent DNA relatedness of CDC group B-1 and B-3 strains

Labeled DNA from CDC group B-3 strain:

Source of Mol% F 3969 F 3333unlabheled DNA G+C 6s% Relatedness D" % Relatedness % Relatedness D % Relatedness

at 65°C at 80°C at 650C at 80°C

Hybridization group IF 3969G 7814B 2341B. casei ATCC 35513TLCDC 79-419F 8007F 124E 8294G 8504G 5048D 8146E 4046F 4052C 5383G 84198183G 7635G 3657C 2409F 4157G 2002G 1995LCDC 81-1016

67.0 + 0.366.9 + 0.166.8 ±00.365.9 ±(0.3

10098939292919089878686848484837978767472726363

0.00.00.00.52.02.52.52.53.01.02.01.02.52.50.02.50.51.02.51.01.51.01.5

3510087829194869388878090858689868381807977757161

45

2825

Hybridization group IIC 4514G 4871F 3333V07 18612D 4556

66.7 + 0.2

55 6.554 4.552 10.04642 8.5

453632

30

97 2.5 7681 2.5 86100 0 10076 1.5 8074 1.0 81

Hybridization group IIIF 3827F 7428G 2607G 3076G 2125B 2777F 7979G 4318LCDC 79-861F 2557G 6560V03 8717E 8862A 1980B. linens ATCC 9175B. linens ATCC 91721B. epidermidis ATCC 35514T

a D, S'r divergence, calculated to the nearest 0.5%.

hybridization group but was detectable in 50% of the hybrid-ization group III strains (data not shown). Mycolic acids were

not detected in two strains of B. casei hybridization group Iwhich were analyzed in the same way as C. diphtheriae, whichshowed significant peaks of mycolic acids (30 to 32 carbonatoms).

DISCUSSION

We have shown that strains of CDC groups B-1 and B-3 are

genetically heterogeneous and that both groups contain strains

of the well-described species B. casei. The five strains ofhybridization group II belong to a new genomospecies (mean-ing a species defined by DNA relatedness [7]) in the genus

Brevibacterium. The species was not formally named because itcould not be distinguished phenotypically from B. casei andtherefore would be identified phenotypically as B. casei. Wewill provisionally refer to these strains as Brevibacterium geno-

mospecies 1. The reference strain of Brevibacterium genomo-

species I (F 3333) has been deposited in the ATCC as ATCC51396.

19

65.4 + 0.1

63.9 + 0.3

65.7 + 0.363.3 + 0.1

65.6 + 0.363.9 + 0.1

63.1 +0.1

9.59.512.0

11.5

4643413937302424222120202015402718

S

2735161831211714117779S

211514

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HUMAN INFECTIONS CAUSED BY B. CASEI 1515

TABLE 3. Percent relatedness among type strains of Brevibacteriunm species

Labeled DNA from:

Source of B. casei ATCC 35513 B. epidernidis ATCC 35514T B. iodinum ATCC 49514' B. linens ATCC 9172'unlabeled DNAe% Relatedness a

% Relatedness D % Relatedness D cl Relatednessat 65°C at 65°C D at 65°C at 65°C D

B. casei ATCC 35513' 100 0 35 13.5 24 9.4 37 13.5B. epidermidis ATCC 35514T 34 11.6 100 0 55 4.1 63 4.5B. iodinum ATCC 49514T 35 12.4 65 7.5 100 0 65 5.5B. linenis ATCC 9172T 22 13.5 50 6.1 62 4.1 100 0

D, %c divergence, calculated to the nearest t).5%.

Strains in hybridization groups I and II exhibited the same

phenotypic characteristics, except for the formation of acidfrom sugars, which varied among the strains and had been thebasis for the separation into the two distinctive CDC groups,

B-I and B-3. Utilization of sugars is not a species-definingcharacteristic, since strains that utilized sugars and those thatdid not were present in both hybridization groups I and II.

Significant characteristics were alkalinization of citrate, tar-trate, and serine within 2 days, hydrolysis of gelatin (within 7days) and casein (within 2 days), and the presence of DNase(within 3 days). The utilization of citrate contradicts a previousreport describing B. casei as citrate negative (30). None of thehybridization group I or II strains exhibited any insolublepigment, easily distinguishing them from the biochemicallysimilar but pigmented strains in hybridization group III. Thenonpigmented hybridization group III strains differed from theindividual hybridization group I and group II strains by one or

more biochemical characteristics on comparison of the bio-chemical profiles of the individual strains (Table 4).

Differentiation between the two species B. casei and B.epidermidis must remain cautiously based on a few biochemicaltraits. Our studies indicate that B. casei may be differentiatedfrom B. epidermidis on the basis of nitrate reduction, serineutilization, and, when negative, acetamide utilization. How-ever, since we only examined two strains of B. epidermidis, our

observations must be confirmed by testing additional strains.We have not been able to identify any of the 14 strains of

hybridization group III. A number of studies (2, 12, 19) havedescribed orange- and yellow-pigmented coryneform strainsisolated from various sources as being morphologically andphysiologically similar to brevibacteria (also similar with re-

spect to their cell walls, CFAs, and the formation of methane-thiol from methionine). Orange nonfermentative, gram-posi-tive rods have been found to be remotely related to B. linens byDNA-DNA hybridization studies (11, 13). Our strains were

genetically related to the type strains of B. casei, B. epidermidis,and B. linens, which confirms the previous data. The G+Ccontent of strains tested was within the same range as that ofthe different species of Brevibacterium. At present, all of our

strains, except for two not containing m-DAP in their cell wall,must be considered members of the genus Brevibacterium.However, they are biochemically different from the variousspecies incertae sedis assigned to the genus (17). The hetero-geneity of this genus is also indicated by a previous hybridiza-tion study that found B. linens ATCC 9175 to be only remotelyrelated (34% relatedness) to a hybridization group which

contained the type strain of B. linens, therefore possiblyconstituting an additional species of brevibacteria (14). Noneof the strains used in the present study hybridized significantlywith ATCC 9175 (0 to 8% in 80°C reaction mixtures). There-

fore, this strain should not be considered to be B. linens.

CFA analysis is useful for distinguishing Brevibacteriumspecies from all Corynebacterium species except "Cotynebacte-rium aquaticum" and CDC coryneform groups A-4 and A-5 (4,22a, 25). The overall CFA compositions of the species listed inTable 5 are essentially identical; they differ from Oerskoviaspecies by the absence of i-14:0, 14:0, and 15:0; from Rothiadentocariosa by the absence of 15:0, 18:2, and 18:0; and fromListeria species by the absence of 18:1 and 18:2 (4, 22a).However, identification of an unknown isolate as Brevibacte-rilini solely on the basis of these small quantitative differencesmust be considered tentative until confirmed by chemical andphysiologic tests or by DNA hybridization. The observation ofquantitative differences in the amounts of a-15:0 and a-17:0between the B. epiderrnidis-B. linens CFA pattern and that of B.casei, as previously reported (15), was not confirmed by thepresent study.

In terms of cell wall composition, brevibacteria differ fromother m-DAP-containing genera (Brachybacterium, Caseobac-ter, Corynebacteriuln, and Rhodococcus) by the absence ofL-arabinose and mycolic acids from the cell wall (16). Galac-tose may or may not be present, whereas glucose is uniformlypresent (14). For components that occur mainly or entirely inthe cell wall, such as m-DAP and neutral sugars, chromato-graphic analysis of whole cells instead of purified cell wallpreparations is sufficient and an easy approach to examininglarge numbers of cells. However, because ornithine and lysinemay be derived from protein and the pentose ribose may bederived from nucleic acid, their presence in the chromatogrammay lead to confusion when analyzing these spots (26). Indeed,a spot corresponding to ribose was found on all sugar chro-matograms, but it was ignored in the analysis of results. In ourcase, the analysis of the cell wall constituents confirmed thehomogeneity of the selected strains. Except for those of twostrains in hybridization group III, cell walls of all isolatescontained m-DAP, together with glucose and sometimes ga-lactose.

Brevibacteria appear to be involved in human infections.Most of our strains were isolated from normally sterile bodysites, with over 60% of them from blood or CSF. At least nineof the strains had been repeatedly isolated from blood or CSF.Except for strains discussed in two recent reports (15, 22), thegenus Brevibacterium has not been associated with humaninfections, and it was believed to represent a contaminationeither from the skin or the environment (27). It is interestingthat none of our strains were related to B. epidermidis at thespecies level. In earlier reports of isolation of B. epidermidisfrom human specimens (18, 22, 32), there is no indication thatmolecular confirmation (DNA-DNA hybridization or G+Ccontent) or determination of menaquinones as the only way ofspecies separation (17) was done. However, like B. epidermidis,

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TABLE 4. Biochemical characteristics" of clinical isolates and type strains of Brevibacterium

Result" for: %/C Positive results" and/or characteristic for hybridization group:

Test performed" B. casci B. epidermidis B. linents I II III (colored III (noncoloredATCC 355 AT4C (n = 22) (n = 5) strains, n = 10) strains, n = 4)

-~~ ,+n n r%nOxidase, Kovacs' methodPigment

Insoluble

SolubleTSI (S/B)"H,S (lead acetate paper)Action on blood

AcetamideSerineTartrateSimmons citrateUrea, ChristensenNitrate to nitriteGelatin hydrolysisLitmus milk

DNaseGrowth at:

250C350C420C

Methanethiol production(after 2/4/24 h)

Casein hydrolysisValine aminopeptidase'Cystine aminopeptidase'Phosphoamidaset'ot-GlucosidasefOF bases (after 3 weeks)

Acid from D-glucoseD-XyloseSucroseMaltoseD-MannitolGlycerolSalicinL-ArabinoseD-GalactoseFructoseD-MannoseL-RhamnoseTrehaloseRaffinoseD-Sorbitoli-InositolDextrini-ErythritolMelizitose

- - + 32 20 30 DU

or 0

B/N+

LG; ly (3days)

IR; B

+

+//

B/N+LG; ly (3

days)

+

IR; B

+

+

+//

+ +

+ +

+_

_ +

+ +

br 77 br; 18 tanB/N 86 B/N; 14 B/B+ 100ly (3 days) 55 LG; 9 gr; 91

ly (3 days)- 64w- 100+ 100+ 100

S

+ 0

+ 100NC 95 IR; 5 pep; 5

NC+ 100

+//

+

100100

986/86/100

10014145945

- 50- 14- 45- 36_ 0

+ 27_ 0

_ 0

+ 23_ 95- 23_ 0

- 14_ 5

_ 0

- 81_ S

_ 0

_ 6

0

80 tan; 20 br100 B/N10020 LG; 20 gr;

80 ly (3 days)80, 60 w1001001000

10060 IR; 80 B; 20pep

100

1001000

20/20/80

1000

6080

402040400

0

0

200

60400

400

0

800

0

40

50 yel; 30 or;20 p

40 br; 30 tan100 B/N4010 LG; 10 gr;

100 ly (3 days)60, 30 w804060401010040 IR; 40 pep;

50 B; 10 NC100

801001080/80/90

9(20405010

20)10

100

200

2040401040200

10202010

Abbreviations: yel, yellow; or, orange; p, greenish, pyocyanine-like pigment; br, brown; ,B, beta-hemolysis on rabbit blood agar; N, no change; LG, lavender green;ly, diffuse lysis; w, weak reaction; IR, inldicator reduction; B, basic; pep, peptonization; NC, no change; gr, greening.

" All strains gave negative reactions in tests for reduction of nitrite or formationl of gas from nitrate and hydrolysis of starch and esculin. With the API ZYM system,tests for lipase (C,4), chymotrypsin, a-galactosidase, [-galactosidase, [3-glucuronidase, [-glucosidase, [B-glucosaminidase, ax-mannosidase, and oa-fucosidase were alwaysnegative. The following sugars were not acidified: lactose, adonitol, dulcitol, cellobiose, inulin, glycogen, melibiose, and starch. All strains gave positive reactions forcatalase and growth in 6% NaCI nutrient broth and for the following tests with the API ZYM system: alkaline phosphatase, esterase (C4), esterase-lipase (Cs), leucineaminopeptidase, and acid phosphatase.

-, negative reaction; 4, positive reaction. Data in parentheses indicate time to achieve result." Percentage of positive results by 7 days. Data in parentheses indicate time to achieve result." TSI (S/B), triple sugar iron agar (slant/butt).J Done with the API ZYM system.

0

100 bar100 B/N10025 LG; 100 ly

(3 days)100, 50 w100100, 25 w75

1005050 pep; 50 IR;

25 B; 25 NC75

100752550/100/100

750010(50

1000505002525252510050252525025t00

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HUMAN INFECTIONS CAUSED BY B. CASEI 1517

TABLE 5. CFA composition of type strains of B. casei, B. epidermidis, and B. linens and Brevibacterium DNAhybridization groups I, II, and III

Value" for:

CFA" B. casei B. epidermidis B. linens Hybridization Hybridization Hybridization

ATCC 35513T ATCC 35514"' ATCC 9172 group I group II group III(n =22) (n =5) (ni = 14)

i-15:0 10 5 7 11 (2-18) 7(6-11) 11 (2-22)a-15:0 38 67 69 46 (33-60) 49 (34-66) 49 (32-75)i-16:0 4 4 3 4 (3-9) 4 (3-6) 5 (2-14)16:0 2 1 1 1 (0-2) T-2 2 (1-3)i-17:0 4 1 1 3 (1-5) 2 (1-3) 3 (1-7)a-17:0 39 22 17 35 (24-47) 36 (21-50) 28 (8-41)18:0 1 0 0 T-1 T-1 T-l

" The number to the left of the colon is the number of carbon atoms, and the number to the right is the number of double bonds. i, methyl group at the penultimate(iso) carbon atom; a, methyl group at the antepenultimate (anteiso) carbon atom.

' Values shown are percentages of total fatty acids and are arithmetic means rounded to the nearest whole percent. -, 0.0 to 0.29%c:; T, t).3 to 0.7%. The range isgiven in parentheses.

B. casei may also belong to the resident skin flora, and theseisolates might have been B. casei.

This is the first report of the isolation of B. casei from clinicalspecimens, although earlier, in 1976, Sharpe et al. expressedtheir suspicion of an origin other than dairy products (becauseof the halotolerance and preference for higher temperatures ofB. casei) (30). The recognition and the tentative identificationof B. casei in the clinical microbiological laboratory should befacilitated by using the tests, procedures, and informationpresented in this study.

ACKNOWLEDGMENTS

We thank K. Bernard, Laboratory Centre for Disease Control,Ottawa, Ontario, Canada, for providing strains LCDC 79-419, LCDC81-1016, and LCDC 79-861.

This study was supported in part by a grant from the Swiss NationalFoundation to E.G.

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