Oral Actinomyces Isolates Forming Red Colonies on Brain Heart Blood Agar Can Be Unambiguously Classified as A. odontolyticus by Macroscopic Examination

Institute of Biochemistry, Medical Faculty, University of Leipzig, D-04103 Leipzig,¹ Department of Preventive Dentistry, Medical Faculty, Friedrich-Schiller University Jena, D-99089 Erfurt, Germany²

Received 23 December 2002/ Returned for modification 9 May 2003/ Accepted 2 June 2003

	ABSTRACT

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The accurate classification of oral Actinomyces isolates as one species is difficult. Out of 18 Actinomyces isolates forming red colonies on brain heart blood agar, 12 could be straightforwardly assigned as Actinomyces odontolyticus by biochemical, morphological, and chemotaxonomic characteristics. For the remaining six isolates, the results of the different identification methods were inconsistent. By sequencing a 16S ribosomal DNA fragment by a rapid mass spectrometric method, all isolates could be identified unambiguously as A. odontolyticus. This result proves the importance of red colony pigmentation on brain heart blood agar together with the characteristic cell morphology for unequivocal assignment of oral Actinomyces isolates to the species A. odontolyticus.

	INTRODUCTION

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The identification of Actinomyces isolates on the basis of biochemical traits is difficult and sometimes results in misidentification (5). Alternative techniques (e.g., sequencing of the 16S ribosomal DNA ) are often not available in routine clinical laboratories. For this reason, it would be desirable to have phenotypic features at hand that enable reliable discrimination of Actinomyces spp. The pigmentation of colonies plays an important role in the flowchart for the differentiation of Actinomyces species (10) and is easy to detect. All of the strains of Actinomyces odontolyticus studied by Batty (2) developed red-pigmented colonies on blood agar.

From the experimental group of the Caries Risk Assessment Study of Erfurt (11, 12), the cultivable bacterial flora (9) of interdental plaque and saliva was estimated on brain heart blood agar. Among the isolates identified as members of the genus Actinomyces by biochemical and physiological traits (7) and by membrane fatty acid spectra (W. E. C. Moore, VPI broth grown anaerobe library, 35°C, PYGT-broth, version 3.9 software, Sherlock version 2.11; MIDI, Newark, Del., 1995), the species A. naeslundii and A. odontolyticus frequently were found. A. naeslundii dominated in the plaque flora, and A. odontolyticus dominated in the salivary flora (9). Other Actinomyces species were represented by A. israelii, A. gerencseriae, A. meyeri, and A. georgiae. From the strains preliminarily identified as A. odontolyticus, about one-third produced crème-colored colonies on brain heart blood agar, and about two-thirds produced red-pigmented colonies. Additionally, five red-pigmented A. odontolyticus strains from a study by D. Beighton (GKT Dental Institute, King's College London, United Kingdom) were used.

According to chemotaxonomic characteristics, 6 out of 18 red-pigmented strains could not be unequivocally assigned to the species A. odontolyticus. Remarkably, two of them were even not assigned to the genus Actinomyces. This result seemed doubtful in the light of the results published by Batty (2) and was in clear contradiction to the typical rod-shape appearance microscopically observed after Gram staining.

In this paper, all oral Actinomyces isolates forming red colonies on brain heart blood agar could unambiguously be assigned to the species A. odontolyticus by 16S rDNA sequencing using a recently developed mass spectrometric method (8).

	MATERIALS AND METHODS

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Bacterial strains. Five isolates which formed red colonies on brain heart blood agar were kindly provided by D. Beighton. By membrane fatty acid analysis four of them could be identified as A. odontolyticus according to the average similarity index and Euclidian distance (VPI broth grown anaerobe library, 35°C, PYGT-broth, version 3.9 software, Sherlock version 2.11; MIDI). The fifth was classified as Actinomyces sp. strain DO1.

The isolates from the Caries Risk Assessment Study of Erfurt (11, 12) used in this study were cultivated on brain heart infusion agar (Merck KG, Darmstadt, Germany) with 5% (vol/vol) human blood obtained from a local blood bank according to Edwardsson (4) and Heinrich and Kneist (6). Out of 13 Actinomyces isolates forming red colonies on brain heart blood agar, 8 could be identified as A. odontolyticus by membrane fatty acid analysis (3; VPI broth grown anaerobe library, 35°C, PYGT-broth, version 3.9 software, Sherlock version 2.11; MIDI). Three of the remaining five isolates were assigned to the genus Actinomyces, while the other two were classified as Bifidobacterium spp.

Chemicals and enzymes. Deoxynucleoside triphosphates (dNTPs), dideoxynucleoside triphosphates (ddNTPs), and ThermoSequenase DNA polymerase were obtained from Amersham Pharmacia Biotech (Freiburg, Germany). Ampli-Taq DNA polymerase was purchased from Applied Biosystems (Weiterstadt, Germany). 3-Hydroxypicolinic acid and ammonium citrate were obtained from Sigma-Aldrich (Steinheim, Germany). Primers for PCR and DNA sequencing were custom synthesized by Metabion GmbH (Martinsried, Germany). The primers are named according to Alm et al. (1), and the purity of the oligonucleotides was checked by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

PCR amplification of 16S rDNA fragments. The PCR amplification of the 16S rDNA fragments was carried out in two steps. In the first step, 1,400-bp fragments of the 16S rRNA gene of the Actinomyces isolates were amplified by PCR with primers S-D-Bact-0006-S-18 (5'-GAGAGTTTGATCCTGGCT-3') and S-D-Bact-1392-A-16 (5'-TGACGGGCGGTGTGTA-3'). The reaction mixture consisted of 10⁶ bacteria cells, PCR buffer, 2 mM MgCl₂, 200 µM dNTPs, 25 pmol of each primer, 1.25 U of Ampli-Taq DNA polymerase, and water to a final volume of 50 µl. The cycling conditions were 94°C for 7 min, 35 cycles (denaturation at 94°C for 30 s, primer annealing for 30 s, and primer extension at 72°C for 30 s), and a final extension step (72°C for 10 min). The initial annealing temperature of 63°C was decreased by 0.5°C at every cycle until 53°C was reached. Aliquots of the PCR products (10³-fold diluted) served as template for the second PCR with 25 pmol of primers S-D-Bact-925-S-14 (5'-AAAGGAATTGACGG-3') and S-D-Bact-1045-a-A-13 (5'-CCATGCACCACCT-3'). After an initial denaturation of 94°C for 5 min, 35 cycles of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C were performed followed by a final extension step of 72°C for 10 min.

Sanger cycle sequencing of PCR-amplified 16S rDNA fragments. The PCR products were purified with Genopure_ds (Bruker Daltonics, Bremen, Germany) according to the manufacturer's instructions and used as templates for the sequencing reactions. Depending on the evaluation method applied, the sequencing reactions were performed by two different protocols.

In the case of the intended MALDI-TOF MS analysis of the sequencing products, the second PCR products were used as templates. Twenty microliters of the sequencing reaction mixture contained buffer consisting of 5 mM (NH₄)₂SO₄, 2 mM MgCl₂, 10 mM Tris-HCl (pH 9.5), 20 pmol of S-D-Bact-1045-a-A-13, 200 µM dNTPs, 20 µM ddNTPs, 2 to 3 µl of template, and 2 U of ThermoSequenase. Cycle sequencing was performed by initial denaturation at 94°C for 5 min followed by 40 cycles of 94°C for 30 s, 42°C for 30 s, and 72°C for 1 min.

To get the sequence information from a larger 16S rDNA region, the 1,400-bp PCR products of the isolates were sequenced with the primer S-D-Bact-0006-18 on an ABI 377 sequencer by using the ABI-PRISM Big Dye Terminator sequencing kit (Applied Biosystems, Weiterstadt, Germany).

The sequences obtained were compared to those in the National Center for Biotechnology Information (NCBI) GenBank database.

MALDI-TOF MS. The sample preparations and the MALDI-TOF MS measurements were performed as previously described (8). For each sequencing reaction, at least three spectra consisting of 45 shots were recorded. The spectrum with the best signal/noise ratio was selected for reading the sequence. The identity of an incorporated base was determined by comparing the experimentally obtained mass difference between neighboring peaks with the masses of dAMP (313.2 Da), dGMP (329.2 Da), dCMP (289.2 Da), dTMP (304.2 Da), ddAMP (297.2 Da), ddGMP (313.2 Da), ddCMP (273.2 Da), and ddTMP (288.2 Da).

	RESULTS AND DISCUSSION

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From the 18 oral Actinomyces isolates that formed red colonies on brain heart blood agar, a partial sequence of the 16S rDNA was obtained by nested PCRs using the primer pair S-D-Bact-0006-18/S-D-Bact-1392-A-16 and S-D-Bact-925-S-14/S-D-Bact-1045-a-A-13, respectively. The PCR products served as templates for Sanger DNA sequencing reactions. The primer extension products were analyzed by a recently developed rapid method based on MALDI-TOF MS (8). Figure 1 exemplifies the mass spectrum of the sequence ladder obtained from the 16S rDNA fragment of an isolate from the Caries Risk Assessment Study of Erfurt. The spectra recorded from the sequencing reactions of all investigated isolates yielded identical sequences of 12 to 16 nucleotides in length. The comparison to the NCBI GenBank database allowed confirmation of the genus Actinomyces for all 18 isolates. Furthermore, with the exception of different A. odontolyticus strains and the species A. lingnae, all other Actinomyces species for which 16S rDNA sequences are available in the NCBI database could be unambiguously excluded as nearest neighbors (Table 1). This is especially important for species such as A. gerencseriae (e.g., X80414), A. naeslundii (e.g., AJ234048), A. meyeri (e.g., X82451), A. georgiae (X80413), and A. israelii (e.g., AF479270), which are typical oral actinomycetes like A. odontolyticus. The species A. lingnae was isolated from human tongue but did not form red colonies on brain heart blood agar (M. D. Collins, personal communication). This excludes a classification of the isolates studied in this paper to A. lingnae. Nevertheless, the affiliation of the isolates with the species A. odontolyticus was proved by conventional Sanger DNA sequencing of a PCR-amplified 16S rDNA region, for which the sequences of A. odontolyticus, A. lingnae, Actinomyces sp. oral strain Hal-1083, and Actinomyces sp. oral strain C29KA differ significantly.

fig.ommitted FIG. 1. MALDI-TOF mass spectrum of the Sanger sequencing products of the PCR-amplified 16S rDNA fragment of an Actinomyces isolate from the Caries Risk Assessment Study of Erfurt. The first peak (m/z 3,840.4 Da) represents the unextended primer.

 

fig.ommitted TABLE 1. Nearest neighbors to the sequence determined for the 16S rDNA fragment of the isolates in this studya

 
The combination of phenotypic and genotypic characters of an isolate increases the reliability of its identification and is necessary for the description of a new species (13). However, as this study shows, the typical cell morphology together with a biochemical trait like the pigmentation on brain heart blood agar is sufficient for an unambiguous assignment of oral Actinomyces isolates to the species A. odontolyticus. The pigment may appear in as little as 48 h, but usually requires 5 to 10 days to develop. In these cases, diphtheroidal forms predominate in stained smears. Two-thirds of the strains of A. odontolyticus studied were red pigmented, and in all of these cases, pigmentation together with diphtheroidal rods in Gram-stained smears proved sufficient for identification. A misidentification can be excluded, because all other oral microorganisms that produce red colonies on brain heart blood agar are gram negative. This finding contributes to acceleration of the detection of A. odontolyticus without the use of biochemical characteristics, membrane fatty acid spectra, or highly sophisticated new methods that are not available in routine clinical or oral microbiological laboratories.

　

	ACKNOWLEDGMENTS

 
We thank D. Beighton (GKT Dental Institute, King's College London, United Kingdom) for providing the A. odontolyticus strains.

This work was funded by the Sächsisches Staatsministerium für Umwelt und Landwirtschaft (grant no. 13-8802.3527).

	REFERENCES

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