Vol. 32, No. 11JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1994, p. 2686-26910095-1137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Primary Identification of Aureobacterium spp. Isolated fromClinical Specimens as "Corynebacterium aquaticum"

GUIDO FUNKE,1* ALEXANDER VON GRAEVENITZ,1 AND NORBERT WEISS2Department of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland,1 and Deutsche Sammlung

von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig, Germany2

Received 24 May 1994/Returned for modification 19 July 1994/Accepted 8 August 1994

Over a 6-year period 11 yellow-pigmented gram-positive rods (GPRs) with an oxidative carbohydratemetabolism were isolated from clinical specimens or were received as reference cultures and tentativelyidentified as "Corynebacterium aquaticum" according to the guide of Hollis and Weaver for the differentiationofGPRs (D. G. Hollis and R. E. Weaver, Gram-Positive Organisms: a Guide to Identification, 1981). Because theseisolates seemed to be rather heterogeneous, comparative analyses with the type strain of "C. aquaticum" as wellas six type strains of species belonging to the genus Aureobacterium were performed by biochemical andchemotaxonomic methods. Only four clinical strains were found to be "C. aquaticum," whereas seven strainswere found to belong to the genus Aureobacterium. Discriminative phenotypic reactions between "C. aquaticum"and Aureobacterium spp. included hydrolysis of gelatin and casein (both reactions negative for "C. aquaticum"strains but positive for most Aureobacterium strains). Moreover, peptidoglycan analysis provided a reliablemeans of differentiating yellow-pigmented GPRs at the genus level (diaminobutyric acid as the interpeptidebridge in "C. aquaticum" and glycine-ornithine as the interpeptide bridge in Aureobacterium spp.). Antimicro-bial susceptibility testing revealed that vancomycin showed an intermediate MIC for three of the four clinical"C. aquaticum" isolates, whereas all Aureobacterium strains were susceptible to vancomycin. To our knowledge,this is the first report outlining the isolation of Aureobacterium spp. from clinical specimens. However,Aureobacterium isolates could not be identified to the species level by the tests used in the study.

In 1962 Leifson (16) described gram-positive rods (GPRs)isolated from distilled water that had many characteristics ofmotile plant-pathogenic corynebacteria; he named these rods"Corynebacterium aquaticum." Subsequent taxonomic investi-gations revealing the presence of 2,4-diaminobutyric acid(DAB) within the cell wall suggested that "C. aquaticum" didnot belong to the genus Corynebacterium sensu strictu (8, 24).In the guide of Hollis and Weaver for the identification ofGPRs (11), "C. aquaticum" ranks third (after Corynebacteriumdiphtheriae and Listeria monocytogenes) with regard to thenumber of isolates tested for each species of GPR. In contrast,"C. aquaticum" has rarely been described as a cause of diseasein humans, e.g., as an etiologic agent of bacteremia, endocar-ditis, meningitis, peritonitis, and urinary tract infection (1, 3,12, 13, 17, 27, 30), or as a cause of pseudobacteremia (22).Between 1988 and 1993 the Department of Medical Micro-

biology, University of Zurich, isolated or received 11 isolatesinitially identified as "C. aquaticum" on the basis of the schemeof Hollis and Weaver (11). Since these isolates seemed to berather heterogeneous biochemically, we questioned their pre-liminary identifications and performed further phenotypic aswell as chemotaxonomic investigations in order to determinethe precise phylogenetic positions of the isolates. Surprisingly,we found that only 4 of the 11 strains were "C. aquaticum" andthat 7 isolates were, in fact, Aureobacterium spp. The genusAureobacterium was defined by Collins et al. (5) in 1983 byreclassifying members of six taxa that were previously thoughtto belong to the genera Microbacterium, Arthrobacter, Coryne-bacterium, and Curtobacterium. To the best of our knowledge,

* Corresponding author. Mailing address: Department of MedicalMicrobiology, University of Zurich, Gloriastrasse 32, CH-8028 Zurich,Switzerland. Phone: 41-1-257-2700. Fax: 41-1-252-8107.

this is the first report regarding the isolation ofAureobacteriumspp. from clinical samples.

MATERIALS AND METHODS

Strains, media, and growth conditions. Clinical material wascultured at 37°C with 5% CO2 on Columbia agar (BectonDickinson Microbiology Systems [BBL], Cockeysville, Md.)with 5% sheep blood (SBA) and on other media as describedpreviously (10).The following strains were obtained from the Deutsche

Sammlung von Mikroorganismen (DSM), Braunschweig, Ger-many: "C. aquaticum" (DSM 20146, ATCC 14665T) ,A. lique-faciens (DSM 20638T, ATCC 43647T), A. terregens (DSM20449T, ATCC 13345T),A. flavescens (DSM 20643T, ATCC13348T), A. barkeri (DSM 20145T, ATCC 15954T), A. testa-ceum (DSM 20166T, ATCC 15829T), and A. saperdae (DSM20169 , ATCC 19272T). All but the following three referencestrains were also grown on SBA at 37°C in 5% CO2; A.liquefaciens, A. fiavescens, and A. saperdae were always incu-bated at 30°C because of their lower optimal growth temper-ature (5).

Biochemical profiles. Preparation of traditional media wasdone as described by Nash and Krenz (18). Oxidation of sugarswas tested in Cystine Trypticase Agar medium (Becton Dick-inson) containing 1% carbohydrates. Assimilation of carbohy-drates was tested by using the AUX medium (api bioMerieuxSA, Marcy l'Etoile, France) within the API 50CH gallery (apibioMerieux) as outlined before (9). Enzyme activities weretested by using the API ZYM system (api bioMerieux) accord-ing to the guidelines provided by the manufacturer. DNaseproduction was tested with DNase test agar with methyl green(Difco, Detroit, Mich.) at 37°C. Gelatin hydrolysis was testedby immersing film strips (Diagnostics Pasteur, Marnes-la-Coquette, France) in bacterial suspensions and incubating

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IDENTIFICATION OF AUREOBACTERIUM SPP. AS C. AQUATICUM 2687

TABLE 1. Origins of strains studied

Strain no. Identification Source or diagnosis Patient's sex,age (yr)"

Reference strains1 "C. aquaticum" DSM 20146T, ATCC 14665T Distilled water2 A. liquefaciens DSM 20638T, ATCC 43647T Milk3 A. terregens DSM 20449T, ATCC 13345T Soil4 A. flavescens DSM 20643T, ATCC 13348T Soil5 A. barkeri DSM 20145T, ATCC 15954T Raw domestic sewage6 A. testaceum DSM 20166T/ATCC 15829T Rice7 A. saperdae DSM 20169T/ATCC 19272T Elm borer

Clinical strains8 "C. aquaticum" Blood culture M, 739 "C. aquaticum" Blood culture, fever of unknown origin NK10 "C. aquaticum" Maxillary sinus M, 5211 "C. aquaticum" Blood culture, fever of unknown origin NK12 Aureobactenium sp. Easy flow drainage, polytrauma M, 2513 Aureobactenum sp. Blood culture, fever of unknown origin F, 3514 Aureobactenium sp. Cerebrospinal fluid NK15 Aureobacterium sp. Peritonitis, chronic ambulatory peritoneal dialysis NK16 Aureobactenum sp. Abdominal deep wound, kidney/pancreas transplantation M, 4817 Aureobactenium sp. Epidural abscess NK18 Aureobacterium sp. Soft tissue infection M, 47

a_, not applicable; M, male; F, female; NK, not known.

them for up to 10 days. Hydrolysis of casein, tyrosine, andxanthine meant clearing of the medium around the coloniesafter incubation for up to 1 week.

Susceptibilities to antimicrobial agents. The MICs of eightantimicrobial agents used in the treatment of GPRs weredetermined and interpreted by following the guidelines of theNational Committee for Clinical Laboratory Standards (19,20). The agar dilution procedure (Mueller-Hinton agar sup-plemented with 5% sheep blood) was used.CFA profiles. Cultures of all but the following three strains

were grown for 48 h at 37°C with 5% CO2 on Trypticase SoyAgar with 5% sheep blood; cultures of the type strains of A.liquefaciens, A. flavescens, and A. saperdae were incubated at30°C. The cellular fatty acid (CFA) profiles were analyzed bythe MIDI system (Microbial ID, Inc., Newark, Del.) as de-scribed previously (28).

Peptidoglycan analysis. Preparation of cell walls and deter-mination of their peptidoglycan structures were carried out bythe methods described by Schleifer and Kandler (23, 24), withthe modification that thin-layer chromatography on cellulosesheets was used instead of paper chromatography. One milli-gram of freeze-dried cell walls was hydrolyzed in 0.2 ml of 4 NHCl at 100°C for 16 h (total hydrolysate) and 45 min (partialhydrolysate). Ornithine and DAB were identified from totalhydrolysate by one-dimensional chromatography in the solventsystem methanol-pyridine-water-10 N HCl (320:40:70:10; vol!vol/vol/vol). Amino acids and peptides from partial hydroly-sates were identified after two-dimensional chromatography inthe systems given by Schleifer and Kandler (24) by theirmobilities and staining characteristics with ninhydrin spray.The resulting fingerprints were compared with those of knownpeptidoglycan structures.

RESULTS

Over a 6-year period the Department of Medical Microbi-ology, University of Zurich, has isolated or received 11 strainsthat were tentatively identified as "C. aquaticum" (Table 1).The isolates came from patients who had been treated in eight

different hospitals on different wards. All 11 isolates grew inpure culture and were associated with marked leukocyticreactions (as revealed by Gram stains) in six of the specimens.GPRs were seen in direct smears of four of six specimens. Noother cultures were available from the patients. In five of sixpatients antimicrobial chemotherapy was initiated or the in-fected focus was removed surgically. All patients recoveredaccording to the records. No specific predisposing factors orany particular source for the organisms could be demonstrated.

All 11 clinical isolates, which grew on aerobically incubatedplates only, exhibited a yellow pigment which appeared morerapidly (within 36 h) in Aureobacterium spp. than in "C.aquaticum." Colonies were about 1 mm in size after 24 h ofincubation in 5% CO2 at 37°C (except for A. liquefaciens, A.flavescens, and A. saperdae, which were grown at 30°C [seeabove]), convex, and circular with entire edges for both "C.aquaticum" and Aureobacterium spp. The biochemical featuresof all strains tested are listed in Table 2. All "C. aquaticum"and Aureobacterium strains were characterized by weak oxida-tive acid production from carbohydrates. By using the API50CH system we observed assimilation reactions for 49 carbo-hydrates and found that all strains utilized galactose, fructose,and maltose. None of the 18 strains tested was able to utilizeerythritol, L-xylose, adonitol, dulcitol, D-fucose, D-arabitol, orL-arabitol. Only the utilization of D-arabinose, L-fucose, and,-methyl-xyloside was found to be of some value for differen-tiating between "C. aquaticum" and Aureobacterium spp. (Ta-ble 2). For the 19 enzymatic reactions covered by the APIZYM system, we observed that all strains exhibited esterase(C4), leucine arylamidase, and a-glucosidase activities. Incontrast, trypsin and 3-glucuronidase activities could not bedetected in any of the strains. Valine arylamidase, cystinearylamidase, and oa-fucosidase were found to have some dis-criminative value (Table 2). DNase production became positiveafter 2 days (mean) in "C. aquaticum" strains and after 4 days(mean) in 10 of 13 Aureobactenum strains, whereas DNaseproduction was not observed in 3 of 13 Aureobacterium strains.Furthermore, hydrolysis of gelatin and casein differentiatedbetween "C. aquaticum" and Aureobacterium strains: all five

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IDENTIFICATION OF AUREOBACTERIUM SPP. AS C. AQUATICUM 2689

TABLE 3. Antimicrobial susceptibility patterns of strains studied

Organism (no. of isolates) MIC (,ug/ml)and antimicrobial agent Range 50% 90%

"C. aquaticum" (5)Ciprofloxacin 1-8 2 2Clindamycin 1-8 4 8Erythromycin <0.125-0.125 <0.125 <0.125Gentamicin 4-16 4 8Penicillin G 1-16 8 8Rifampin <0.125-8 1 2Tetracycline 0.5-4 1 1Vancomycin 0.5-8 8 8

Aureobacteriumn spp. (13)Ciprofloxacin 0.5-32 2 8Clindamycin 0.25-16 4 8Erythromycin <0.125-16 <0.125 0.5Gentamicin 1-64 8 64Penicillin G <0.125-16 0.5 1Rifampin <0.125-32 1 8Tetracycline 0.125-2 0.5 2Vancomycin <0.125-4 0.5 4

a50% and 90%, MICs for 50 and 90% of strains tested, respectively.

"C. aquaticum" strains were unable to hydrolyze gelatin within10 days, whereas all Aureobacterium strains, with the exceptionof A. saperdae, hydrolyzed this substrate. None of the "C.aquaticum" strains was able to hydrolyze casein, whereas thisreaction was positive for 6 of 13 Aureobacterium strains tested.Tyrosine and xanthine hydrolysis did not differentiate between"C. aquaticum" and Aureobacterium spp.

Tetracycline was found to be the only antimicrobial agent towhich all strains tested were fully susceptible (Table 3). OnlyA.barkeri was resistant to erythromycin, whereas all other strainswere susceptible to this drug. The MIC of vancomycin for 3 "C.aquaticum" strains was intermediate (MIC, 8 ig/ml), but all 15other strains examined remained susceptible to vancomycin.The MICs of ciprofloxacin and clindamycin for 50% of strainstested were greater than the susceptibility ranges for both "C.aquaticum" and Aureobacterium strains, indicating the limited

TABLE 4. CFA profiles of strains studied

% of total fatty acidStrain no.a, taxon

Ci 15:0 Caiso5: Ci 16:0 C16:0) Ci 17 0 Cai 17:0

1, "C. aquaticum" 4 36 14 1 3 412, A. liquefaciens 1 47 8 4 353,A. terregens 17 57 2 5 1 104, A. flavescens 5 57 6 5 5 175,A. barkeri 10 31 9 6 6 346,A. testaceum 4 29 13 3 5 437,A. saperdae 2 50 20 4 1 198, "C. aquaticum" 3 22 17 1 4 529, "C. aquaticum" 3 31 11 1 2 5210, "C. aquaticum" 4 28 9 2 3 5411, "C. aquaticum" 3 22 15 1 4 5312,Aureobacterium sp. 6 38 16 3 4 3113, Aureobacterium sp. 2 34 25 4 1 3414,Aureobactenum sp. 6 44 11 2 3 3415,Aureobactenium sp. 6 44 10 3 4 3316,Aureobacterium sp. 5 51 18 3 3 2017,Aureobacterium sp. 4 26 11 4 8 4218,Aureobactenium sp. 6 34 6 2 8 30

a Strain number according to Table 1.

A - MurNAc - G1cNAc -

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D-Glu - o Gly ,N5L-Hsr D-Orn D-AlaIL . J +

D-Ala L-Hsr

.D-Glu

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FIG. 1. Primary structures of the peptidoglycans of "C. aquaticum"(A) and Aureobacterium spp. (B). Abbreviations: MurNAc, N-acetyl-muramic acid; GIcNAc, N-acetylglucosamine; Gly, glycine; D-Glu,D-glutamic acid; L-Dab, L-diaminobutyric acid; D-Ala, D-alanine;D-Dab, D-diaminobutyric acid; L-Hsr, L-homoserine; D-Orn, D-orni-thine. The interpeptide bridge is enclosed by dashed lines.

potentials of these drugs. When categories for staphylococciwere applied (20), all strains except one Aureobacterium strainwere resistant to penicillin G.

For all 18 strains tested 12-methyltetradecanoic (Cji15:0) and14-methylhexadecanoic (Caji7:0) fatty acid methyl ester werethe major CFA components (Table 4). There was no clear-cutdistinction between "C. aquaticum" and Aureobacterium spp.by CFA profiling.The definitive assignment to a genus was established by

analysis of the peptidoglycan. Strains with glycine-ornithine asthe interpeptide bridge were assigned to the genus Aureobac-terium, whereas strains containing DAB as the interpeptidebridge were designated "C. aquaticuin" (Fig. 1). The assign-ment to "C. aquaticum" was further established by menaqui-none (MK) analysis, with MK-10 and MK-1l being the majorMKs (data not shown).

DISCUSSION

Of the 11 clinical isolates initially identified as "C. aquati-cum," only 4 were found to be true "C. aquaticum" strains,whereas the majority of strains (7 of 11) turned out to bemembers of the genus Aureobacterium. We were not able toidentify these seven isolates to the species level because noneof the phenotypic tests applied was found to be species specificwithin the genus Aureobacterium. Some of the seven Aureobac-

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terium isolates may belong to one of the seven Aureobacteriumspecies defined recently (31). However, by using the pheno-typic data presented by Yokota et al. (31), our strains could notbe assigned to any of these Aureobacterium species. Theproblem with Aureobacterium strains is still the very limitednumber of strains of each species available (mostly not morethan one) (5, 31), and, therefore, no reliable data base for thedifferentiation of Aureobacterium species can yet be created.Moreover, the phenotypic data presented in the literature (5,14) are in some way incomplete because not all reactions (e.g.,motility, nitrate reduction, gelatin hydrolysis, and casein hy-drolysis) have been reported for every Aureobacterium species.Furthermore, some of our results were in contrast to thepublished data (5, 14), including the ability of A. terregens togrow at 37°C and to hydrolyze gelatin as well as the inability ofA. flavescens to reduce nitrate. Overall, the correct speciesidentification of our isolates would be established only byquantitative DNA-DNA hybridizations (29).Even with the limited number of strains, we propose that

hydrolysis of gelatin and casein be used to differentiate be-tween "C. aquaticum" and Aureobacterium spp. (see above). Itis most likely that some strains included as "C. aquaticum" inthe tables of Hollis and Weaver (11) are, in fact, Aureobacte-rium spp. Their table lists only 66% of the "C. aquaticum"strains as motile and 26% as able to hydrolyze gelatin. Incontrast, Leifson (16) defined "C. aquaticum" as strictly motileand unable to hydrolyze gelatin.Although CFA analysis has been demonstrated to be a

diagnostic tool for the differentiation of GPRs (2, 28), the CFApatterns observed for the "C. aquaticum" and Aureobacteriumstrains were neither genus nor species specific.The definitive genus identifications of GPRs can be obtained

by using chemotaxonomic methods. Glycine-ornithine as theinterpeptide bridge so far has been found only in Aureobacte-rium spp. (14). Another closely related genus with a B-typepeptidoglycan is Curtobacterium, with ornithine as the inter-peptide bridge (15). Curtobacterium spp. can also be separatedfrom Aureobacterium (MK-11, MK-12) by having MK-9 as themajor MK within the cell membrane (15). No report regardingthe isolation of Curtobacterium spp. from clinical specimenshas been found in the literature.

Surprisingly, we observed that for three of our four clinical"C. aquaticum" isolates the vancomycin MIC was intermedi-ate, and these isolates were most likely not epidemiologicallylinked. In contrast, in previous publications on "C. aquaticum"(3, 17, 25) all strains tested were found to be fully susceptibleto vancomycin. However, decreased susceptibility to vancomy-cin is not unusual for a GPR since it has been described forLactobacillus spp. and Erysipelothrix rhusiopathiae (21). Atpresent, it remains unclear whether this elevated level ofvancomycin susceptibility in "C. aquaticum" strains is intrinsicor acquired.The routine laboratory should be aware of Aureobacterium

spp. when yellow-pigmented GPRs are isolated. The differen-tial diagnosis includes Oerskovia spp. (25), Microbacterium spp.(6), Cellulomonas spp. (26), and CDC coryneform group Abacteria (11), but of all these taxa are fermentative; again,Microbacterium spp. and Cellulomonas spp. have not beenidentified in clinical specimens.

Analysis of the peptidoglycan structure must be reserved forthe reference laboratory. However, it is an invaluable tool fortaxonomic investigations within GPRs. Furthermore, for thedifferentiation of "C. aquaticum" from other DAB-containinggenera (Agromyces [4], Clavibacter [7], and Rathayibacter [32]),MK analysis is needed, because MK-10 and MK-11 are char-acteristic major MKs of "C. aquaticum" only.

To the best of our knowledge, this is the first report outliningthe isolation of Aureobacterium spp. in clinical specimens. Wecan only speculate that some undefined environmental sourcemay be the reservoir for Aureobacterium strains (Table 1).Although Aureobacterium spp. are not expected to be isolatedfrequently from clinical specimens, we believe that additionalcase reports will make the clinical microbiologist alert toencountering Aureobacterium spp. in clinical materials.

ACKNOWLEDGMENTS

V. Punter provided excellent assistance.This work was supported by grants from the Hartmann-Muller-

Stiftung, Zurich, Switzerland, and the EMDO-Stiftung, Zurich, Swit-zerland.

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