Evaluation of methods for molecular typing and identi¢cation ofmembers of the genus Brevibacterium and other related species

Artur Alves, Orlando Santos 1, Isabel Henriques, Anto¤nio Correia �

Centro de Biologia Celular, Campus Universita¤rio de Santiago, Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal

Received 9 April 2002; received in revised form 7 June 2002; accepted 14 June 2002

First published online 12 July 2002

Abstract

The genus Brevibacterium includes pleomorphic Gram-positive bacteria with a high mol% G+C content. Species in the genus aredifficult to identify by classical methods. The discriminatory power of DNA-based methods is assessed. Strains representing the four wellestablished Brevibacterium species, and other related bacteria, were compared by amplified ribosomal DNA restriction analysis(ARDRA), repetitive-sequence-based PCR (rep-PCR) and ribotyping. Fingerprinting by rep-PCR and ribotyping provided complexgenomic profiles with the highest discriminatory potential for molecular typing at the strain level, whereas ARDRA showeddifferentiation from the genus to the species levels. A high degree of heterogeneity within the genus Brevibacterium is apparent, thusindicating that the taxonomy of the genus should be further studied. 7 2002 Federation of European Microbiological Societies.Published by Elsevier Science B.V. All rights reserved.

Keywords: Brevibacterium ; Ampli¢ed ribosomal DNA restriction analysis ; Repetitive-sequence-based polymerase chain reaction; Ribotyping;Brevibacterium linens

1. Introduction

The genus Brevibacterium includes species of industrial,biotechnological and environmental interest, as well asspecies of clinical signi¢cance [1,3]. Brevibacterium linensis by far the best studied species of the genus. Some phys-iological characteristics of B. linens strains emphasize theirimportance [1]. Brevibacterium species are pleomorphic,Gram-positive bacteria, and are di⁄cult to identify dueto their morphological and physiological similarity tomembers of genera such as Arthrobacter, Caseobacter,Corynebacterium and Rhodococcus [1]. Identi¢cation atthe genus level is based mainly on chemotaxonomic fea-tures [2]. At present, the genus contains four validly de-scribed species with a mol% G+C of the DNA in the rangeof 60^67%. They are B. linens (the type species), B. casei,B. iodinum and B. epidermidis [2]. The distinction between

the four species is essentially based on pigment production(yellow to orange in B. linens, iodinin in B. iodinum), orhabitat for the non-pigmented species (dairy products forB. casei and human skin for B. epidermidis). Di¡erentia-tion of the two non-pigmented species relies heavily ondetermination of DNA^DNA reassociation levels.Since discrimination of Brevibacterium at species and

strain level is based mainly on phenotypic characteristics,which are frequently inconclusive, it is important to estab-lish methods that contribute to a better understanding ofthe classi¢cation of the genus, and allow an easier, fasterand more reliable identi¢cation of new isolates. DNA ¢n-gerprinting methods have proven useful in the identi¢ca-tion and discrimination at species and strain level of di¡er-ent types of bacteria [20]. Ampli¢ed ribosomal DNArestriction analysis (ARDRA) has been shown to be asuitable method for di¡erentiation of Brevibacterium [7]and Corynebacterium [8] strains at species level and forisolate identi¢cation. Other PCR-based methods take ad-vantage of the presence of repetitive sequences that areinterspersed throughout the genome of diverse bacterialspecies [9]. They include the 35^40-bp repetitive extragenicpalindromic (REP) sequence and the 154-bp BOX ele-ment. Ribotyping is used for the characterization of therestriction fragment length polymorphism (RFLP) inside

0378-1097 / 02 / $22.00 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 1 8 - 2

* Corresponding author. Tel : +351 (234) 370778;Fax: +351 (234) 426408.E-mail address: acorreia@bio.ua.pt (A. Correia).

1 Present address: SOQUIFA Medicamentos S.A., Av. ImaculadaConceicOa‹o, 4700 Braga, Portugal.

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the rRNA operon and/or the genomic regions surroundingthe rRNA operons [10].In this study, the discriminatory power of ARDRA,

repetitive-sequence-based PCR (rep-PCR), and conven-tional ribotyping was evaluated for a set of well-charac-terized strains deposited in di¡erent culture collections.The study was performed on strains classi¢ed as belongingto the true Brevibacterium species and, for comparativepurposes, other strains that were previously classi¢ed inthe Brevibacterium genus, such as ‘B. vitarumen’, ‘B. lacto-fermentum’ and ‘B. liquefaciens’. Also, the type strain ofthe related genus Corynebacterium, C. glutamicum ATCC13032, was included.

2. Materials and methods

2.1. Bacterial strains

The bacterial strains used in this study are listed inTable 1. All strains were grown aerobically in tryptic soybroth (Difco) at 28^30‡C.

2.2. DNA isolation

Genomic DNA was isolated from 2 ml of liquid culturesharvested at late exponential phase, with the GenomicDNA Puri¢cation Kit (MBI Fermentas, Vilnius, Lithua-nia). To improve lysis a previous step of incubation withlysozyme 1 mg ml31 was included. Puri¢ed DNA wasaliquoted in TE bu¡er (10 mM Tris^HCl, 1 mM EDTA,pH 8.0). DNA concentrations were estimated spectropho-tometrically.

2.3. PCR protocols

The primers were synthesized by Gibco BRL (Eggen-stein, Germany). Taq polymerase, nucleotide mix andbu¡ers were from MBI Fermentas. A Perkin-Elmer 2400thermal cycler was used. PCR reactions were performed in50-Wl reaction mixtures containing 1UPCR bu¡er (PCRbu¡er without MgCl2 :PCR bu¡er with (NH4)2SO4, 1:1),3 mM MgCl2, 5% dimethylsulfoxide, 1 mg ml31 bovineserum albumin, 200 WM each nucleotide, 0.3 WM eachprimer, 1 U Taq polymerase and 50^100 ng puri¢ed tem-plate DNA.Primers fD1 (5P-AGAGTTTGATCCTGGCTCAG-3P)

and rD1 (5P-AAGGAGGTGATCCAGCC-3P) were usedto amplify nearly full-length 16S rRNA genes [11]. Thetemperature pro¢le was as follows: initial denaturation(94‡C for 9 min); 30 cycles of denaturation (94‡C for30 s), annealing (56‡C for 30 s), and extension (72‡c for90 s); and a ¢nal extension (72‡C for 10 min). The ampli-cons were puri¢ed with the Concert Rapid PCR Puri¢ca-tion System (Gibco BRL, Eggenstein, Germany), and di-gested with one of the following restriction endonucleases:

MboI, TaqI, SmaI and NciI (Gibco BRL), as recom-mended by the manufacturer.The primers REP1R (5P-IIIICGICGICATCIGGC-3P)

and REP2I (5P-ICGICTTATCIGGCCTAC-3P) [12] andBOXA1R (5P-CTACGGCAAGGCGACGCTGACG-3P)[13] were used to generate REP- and BOX-PCR pro¢les.The ampli¢cation cycles were as described previously [19] :initial denaturation (95‡C for 7 min); 30 cycles of dena-turation (94‡C for 1 min), annealing (40‡C for REP prim-ers and 53‡C for BOX primer, for 1 min), and extension(65‡C for 8 min); and a ¢nal extension (65‡C for 16 min).

2.4. Gel electrophoresis of DNA

DNA fragments were electrophoretically separated inagarose gels using Tris^acetate^EDTA as bu¡er. Separa-tion of ARDRA fragments was made on 2% agarose gelsand rep-PCR products were separated on 1% agarose gels.As molecular mass markers, GeneRuler 1-kb and Gene-Ruler 100-bp DNA Ladders (MBI Fermentas) were used.Restriction digests of genomic DNA were resolved on0.7% agarose gels, with V DNA digested with HindIII assize marker. The gels were stained with ethidium bromide,visualized on a UV transilluminator and photographedwith Polaroid T667 ¢lm.

2.5. Labeling of DNA probes

V DNA digested with HindIII was digoxigenin-labeledby a random priming procedure. For introduction of dig-oxigenin label in 16S rDNA from B. linens ATCC 9172,PCR DIG Labeling Mix (Roche, Germany) was includedin the PCR reaction instead of dNTPs.

2.6. Ribotyping

Genomic DNA (5 Wg) from each bacterial strain wasdigested with the restriction endonucleases EcoRI andBamHI (Pharmacia Biotech, Uppsala, Sweden), as recom-mended by the manufacturer. Electrophoretically sepa-rated DNA fragments were transferred by vacuum to ny-lon membranes (Roche, Germany), and UV cross-linkedto the membranes. The blots were probed with digoxi-genin-labeled V/HindIII and 16S rDNA from B. linensATCC 9172. The probes were boiled for 10 min prior touse. Membranes were prehybridized for 2 h at 42‡C inhybridization bu¡er (50% formamide, 5USSC, 0.1% N-lauroylsarcosine, 0.02% sodium dodecylsulfate (SDS), 1%Blocking Reagent), and hybridized for 12^16 h at 42‡C inthe same bu¡er. Two 5-min washes in 2USSC, 0.1% SDSat room temperature were followed by two more 15-minwashes at 42‡C, in 0.5USSC, 0.1% SDS [14]. Detectionwas carried out by incubation with anti-digoxigenin anti-bodies coupled to alkaline phosphatase, followed by treat-ment with the colorimetric substrate NBT/BCIP (X-phos-phate) at room temperature.

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2.7. Computer-assisted analysis

Photographs and membranes were scanned on a GS-710Imaging Densitometer (Bio-Rad) and recorded as TIFFimages. The banding patterns were analyzed with the soft-ware Quantity One (Bio-Rad) and converted to a two-dimensional binary matrix (1, presence of a band; 0, ab-sence of a band). Similarity matrices were calculated withthe Dice coe⁄cient [15]. Cluster analysis of similarity ma-trices was performed by the unweighted pair group meth-od using arithmetic averages (UPGMA). The copheneticcorrelation coe⁄cient (r) was calculated to assess thegoodness of the clustering method. Computer-assistedanalysis was performed with the NTSYSpc2 program forWindows [16].

3. Results and discussion

3.1. Analysis of 16S rDNA RFLPs

Nearly full-length 16S rDNAs of 16 strains (Table 1)were ampli¢ed via PCR with universal primers fD1 andrD1. The PCR products were restricted with enzymesMboI, TaqI, SmaI and NciI. These enzymes were chosenafter checking restriction sites present on 16S nucleotidesequences deposited in the GenBank database and belong-ing to the following species : B. linens (X77451), B. casei(X76564), B. liquefaciens (AJ251417) and C. glutamicum

(X84257). The choice was directed to enzymes hydrolyzing16S rDNA in a small number of fragments (less than ¢ve)allowing the comparison of strains in a short run agarosegel. The individual RFLP patterns are shown in Fig. 1.The pro¢les obtained revealed that the strains classi¢ed

as B. linens do not have a common genotype. Among B.linens strains, two genotypes are apparent: genotype A,including strains ATCC 9172 and ATCC 19391, and ge-notype B represented by the strains CCUG 12168, CCUG23846, CCUG 23896 and LMG 3915. On the other hand,‘B. lactofermentum’ strains (ATCC 13869 and DSM20412) formed a very consistent group with C. glutamicumATCC 13032, exhibiting pro¢les that clearly distinguishthe group by SmaI, MboI or NciI digestions of the ampli-¢ed rDNA.Irrespective of the enzyme used, B. casei could not be

distinguished from B. linens genotype B strains. The twoother ‘true’ Brevibacterium species, B. iodinum and B. epi-dermidis, are characterized by MboI and TaqI restrictionpro¢les identical to B. linens strains from genotype A, butcan be distinguished from those strains by NciI and SmaIdigestions. It should also be noted that the enzymes useddid not discriminate between B. iodinum and B. epidermi-dis. The ARDRA patterns readily distinguished non-pig-mented strains of Brevibacterium (B. casei and B. epider-midis). Other authors [7] have previously reported similarresults. Although B. iodinum could not be di¡erentiatedfrom B. epidermidis and B. casei from B. linens genotypeB strains based on ARDRA patterns, these species can be

Table 1List of bacterial strains used in this study and summary of ¢ngerprinting results

Species Strain Characteristics/source

ARDRA Rep-PCR Ribotyping Cluster Taxonomic outcome

MboI TaqI NciI SmaI BOX REP EcoRI BamHI

B. linens ATCC 9172T Harzercase cheese M1 T1 N1 S1 B1 R1 E1 Bm1 I B. linens genotypeA

ATCC 19391 producer ofL-lysine

M1 T1 N1 S1 B2 R2 E2 Bm2 I B. linens genotypeA

CCUG 12168 M T2 N1 S1 B3 R3 E3 Bm3 II B. linens genotype BCCUG 23846 poultry litter M2 T2 N1 S1 B4 R4 E4 Bm4 II B. linens genotype BCCUG 23896 M2 T2 N1 S1 B3 R3 E5 Bm3 II B. linens genotype BLMG 3915 M2 T2 N1 S1 B3 R3 E5 Bm3 II B. linens genotype B

B. casei DSM 20657T Cheddar cheese M2 T2 N1 S1 B5 R5 E6 Bm5 IIIDSM 20658 raw milk M2 T2 N1 S1 B5 R5 E6 Bm5 III

B. iodinum DSM 20626T milk M1 T1 N2 S2 B6 R6 E7 Bm6 IV closerLMG 2201 milk M1 T1 N2 S2 B6 R6 E7 Bm6 IV to

B. epidermidis DSM 20660T human skin M1 T1 N2 S2 B8 R8 E9 Bm8 V Corynebacterium?‘B. vitarumen’ DSM 20294T M3 T3 N3 S2 B7 R7 E8 Bm7 VI C. vitaruminis [5]‘B. liquefaciens’ LMG 16159T sewage M4 T2 N1 S1 B9 R9 E10 Bm9 VII Arthrobacter?‘B. lactofermen-tum’

ATCC 13869 L-glutamic acidproducer

M3 T4 N4 S3 B10 R10 E11 Bm10 VIII C. glutamicum [6]

DSM 20412 L-glutamic acidproducer

M3 T4 N4 S3 B11 R11 E12 Bm10 VIII C. glutamicum [6]

C. glutamicum ATCC 13032T L-glutamic acidproducer

M3 T4 N4 S3 B12 R12 E13 Bm11 VIII

ATCC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen; BCCM/LMG, Belgian CoordinatedCollections of Microorganisms^Laboratorium voor Microbiologie Universiteit Gent; CCUG, Culture Collection University of Go«teborg.

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discriminated from one another by pigment production,since B. iodinum produces iodinin and B. linens producesan orange pigment.

3.2. Analysis of BOX- and REP-PCR genomic ¢ngerprints

Both approaches yielded complex genomic ¢ngerprintsbut the ampli¢cation reactions performed with primerBOXA1R resulted in a greater number of bands thanthe reactions made with the primer set REP1R andREP2I as can be seen in Fig. 2. The BOXA1R ¢ngerprintsare clear, with intense bands in the size range between 500bp and 4000 bp allowing easy visual comparisons betweenstrains. Two independent PCR reactions performed bydi¡erent persons and on di¡erent DNA samples of thesame strains yielded identical results, thus revealing thehigh reproducibility of the rep-PCR genomic ¢ngerprint-ing protocol. The individual and combined BOX- andREP-PCR patterns revealed considerable genetic diversity.Two groups of B. linens strains clearly emerge, corre-sponding to the two genotypes detected by ARDRA.The separation between B. casei and B. linens is also ob-

vious. Brevibacterium linens strains CCUG 12168, CCUG23896, and LMG 3915 always exhibited the same patternin both cases. The same was observed with B. casei strainsDSM 20657 and DSM 20658, and also with B. iodinumstrains DSM 20626 and LMG 2201.As seen in the case of ARDRA, the strains of ‘B. lacto-

fermentum’ and C. glutamicum form a consistent group,sharing a large number of common bands. The strainsof B. epidermidis, ‘B. vitarumen’, and B. liquefaciensyielded unique patterns quite di¡erent from those obtainedfor the other species analyzed. It is not known if thesesequences exist in the genome of brevibacteria, but rep-PCR appears as a good DNA ¢ngerprinting techniquethat can be used for the molecular typing of Brevibacte-rium spp. and other coryneform bacteria. These resultsreinforce the consistency of the ones obtained with AR-DRA, allowing the di¡erentiation between the species B.linens and B. casei, and also B. iodinum and B. epidermidis.

3.3. Ribotyping ¢ngerprints

For ribotyping analysis two low-cost restriction endo-

Fig. 1. ARDRA patterns obtained by 2% agarose gel electrophoresis of digests of the ampli¢ed 16S rDNA after restriction with MboI, TaqI, NciI andSmaI. Lanes: M, GeneRuler 1-kb DNA Ladder; MP, GeneRuler 100-bp DNA Ladder Plus; 1^16: B. linens (ATCC 9172, ATCC 19391, CCUG 12168,CCUG 23846, CCUG 23896, LMG 3915), B. casei (DSM 20657, DSM 20658), B. iodinum (LMG 2201, DSM 20626), ‘B. vitarumen’ DSM 20294, B. epi-dermidis DSM 20660, ‘B. liquefaciens’ LMG 16159, C. glutamicum ATCC 13032, and ‘B. lactofermentum’ (ATCC 13869, DSM 20412).

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nucleases (EcoRI and BamHI) were chosen. After check-ing 16S nucleotide sequences of bacteria belonging to thegenus Brevibacterium deposited in the database GenBank,it was noted that EcoRI had one restriction site inside the16S rDNA gene, while BamHI did not have any restrictionsites. RFLPs of 16S rRNA genes were examined using asprobe the PCR-ampli¢ed 16S rDNA gene from strainATCC 9172. With this protocol, di¡erences at restrictionsites surrounding the 16S rRNA genes can be assigned. Ascan be seen in Fig. 3, the hybridization of the probe toSouthern blots of EcoRI digestions gave more complexpatterns than those obtained with BamHI digestions.As in the procedures described above two genotypes can

be distinguished among B. linens strains. The strainsCCUG 12168, CCUG 23896 and LMG 3915 again exhib-ited similar patterns with the exception of a band withapproximately 8.5 kb present in the EcoRI pattern ofstrain CCUG 12168. In a previous work [17] strainsCCUG 12168 and CCUG 23896 were reported as identicalusing ribotyping and pulsed ¢eld gel electrophoresis anal-ysis. The two strains of B. casei and B. iodinum also hadequivalent patterns in both cases. The ‘B. lactofermentum’and ‘C. glutamicum’ strains revealed identical patterns,thus reinforcing the consistency of the group. Finally B.epidermidis, ‘B. vitarumen’, and B. liquefaciens yielded pat-terns quite di¡erent from the other strains.From the results presented in Fig. 3 it is possible to

estimate the number of copies of operons encoding ribo-

somal RNA present in the genome of these bacteria. Threeto four copies are present in the genomes of the true Bre-vibacterium species, a result that is in accordance to thosepreviously published [17] and the same number is foundfor ‘B. vitarumen’. Six copies are detected for ‘B. liquefa-ciens’ and ¢ve to six copies are found in ‘B. lactofermen-tum’ and C. glutamicum. In another publication [18], ¢verRNA operons were described for C. glutamicum. In thisstudy, ribotyping gave good results and revealed high dis-criminatory power at species and strain level.

3.4. Analysis of combined ARDRA, rep-PCR andribotyping ¢ngerprints

A cluster analysis was performed on the combination ofARDRA, rep-PCR, and ribotyping genomic ¢ngerprints.The dendrogram obtained (Fig. 4) re£ects the overall clas-si¢cation of this group of bacteria. As can be seen, ‘B.lactofermentum’ and C. glutamicum formed a consistentgroup reinforcing the reclassi¢cation of ‘B. lactofermen-tum’ as C. glutamicum [6]. Also, ‘B. vitarumen’, whichclustered close to the species of Corynebacterium, has al-ready been reclassi¢ed in this genus as C. vitaruminis [5].The same authors proposed the transfer of ‘B. liquefaciens’to the genus Corynebacterium but that has been proven tobe erroneous [4]. The species ‘B. liquefaciens’ in Bergey’sManual is suggested to be close to the genus Arthrobacter,but that needs further investigation.

Fig. 2. Rep-PCR ¢ngerprints generated with the primer set REP1R/REP2I (REP-PCR) and BOXA1R (BOX-PCR). Lanes: M, 1-kb Plus DNA Ladder;1^16: B. linens (ATCC 9172, ATCC 19391, CCUG 12168, CCUG 23846, CCUG 23896, LMG 3915), B. casei (DSM 20657, DSM 20658), B. iodinum(LMG 2201, DSM 20626), ‘B. vitarumen’ DSM 20294, B. epidermidis DSM 20660, ‘B. liquefaciens’ LMG 16159, C. glutamicum ATCC 13032, and‘B. lactofermentum’ (ATCC 13869, DSM 20412).

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A cluster comprising the species B. linens, B. casei, and‘B. liquefaciens’ was resolved consistently into three groupseach one representing a distinct species, with the group ofB. linens being subdivided in two subgroups correspondingto genotypes A and B respectively. A second cluster com-prised the species B. iodinum and B. epidermidis clusteringin the same branch, with the representatives of ‘B. vitaru-men’, ‘B. lactofermentum’, and C. glutamicum in anotherbranch.The results presented show that the strategy of combin-

ing genomic ¢ngerprints obtained with di¡erent ap-proaches yields taxonomically sound groupings for this

group of bacteria. All methods revealed a high degree ofheterogeneity amongst B. linens strains as revealed by theclear distinction of two genotypes, A and B. This fact isconsistent with the existence of two distinct DNA^DNAhomology groups among B. linens strains [2], with thegenomic divergence being an argument for the establish-ment of two species. The non-pigmented species, B. caseiand B. epidermidis, which are impossible to discriminateby simple biochemical tests, can be distinguished on thebasis of ARDRA patterns, as reported by other authors[7].

4. Conclusions

The techniques used revealed a high degree of geneticvariation between the group B. linens/B. casei and thegroup B. iodinum/B. epidermidis. The latter two speciesseem to be more closely related to species of the genusCorynebacterium. These results, together with the highgenotypic diversity found, emphasize the fact that thetaxonomy of the genus Brevibacterium needs to be furtherdeveloped. Future studies including more strains and somerecently described species should be performed.Together, our ARDRA, rep-PCR, and ribotyping data

indicate a large genotypic diversity from genus to strainlevels, and provide a good analysis of the phylogeneticposition and genotypic diversity of these bacteria. The

Fig. 3. Ribotyping patterns of the strains, obtained with the restriction endonucleases EcoRI and BamHI. Lanes: M, V DNA digested with HindIII;1^16: B. linens (ATCC 9172, ATCC 19391, CCUG 12168, CCUG 23846, CCUG 23896, LMG 3915), B. casei (DSM 20657, DSM 20658), B. iodinum(LMG 2201, DSM 20626), ‘B. vitarumen’ DSM 20294, B. epidermidis DSM 20660, ‘B. liquefaciens’ LMG 16159, C. glutamicum ATCC 13032, and‘B. lactofermentum’ (ATCC 13869, DSM 20412).

Fig. 4. Dice/UPGMA cluster analysis of combined ARDRA, rep-PCRand ribotyping genomic ¢ngerprints. Similarity is indicated as a percent-age. r=0.98.

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two di¡erent genotypes detected for B. linens suggest thatdelimitation of species within the genus Brevibacteriumstill deserves extensive research.In conclusion, all ¢ngerprinting methods applied were

found to be useful for molecular typing and identi¢cationof Brevibacterium spp. and other coryneform bacteria atthe species and/or strain level. Although ribotyping hasproven useful for taxonomic studies [10], this approachhas a distinct disadvantage over PCR-based techniques,that is the need for performing a Southern transfer andsubsequent hybridization with a labeled probe, making itmore expensive, time-consuming, and more di⁄cult toperform. Major advantages of PCR-based ¢ngerprintingmethods are technical simplicity, low cost, and rapidness[20]. These DNA ¢ngerprinting techniques present them-selves as good tools for environmental, epidemiologicaland clinical follow-up studies of Brevibacterium isolatesas well as other coryneform bacteria.

Acknowledgements

This work was supported by the Instituto de Investi-gacOa‹o da Universidade de Aveiro and by FundacOa‹o paraa Cie“ncia e a Tecnologia in the form of an MsC grant(SFRH/BM/419/2000) to A.A. The authors wish to thankDr. Alan Phillips for critical reading of the manuscript.

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