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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1999 Jul;37(7):2255–2261. doi: 10.1128/jcm.37.7.2255-2261.1999

Development of Amplified 16S Ribosomal DNA Restriction Analysis for Identification of Actinomyces Species and Comparison with Pyrolysis-Mass Spectrometry and Conventional Biochemical Tests

Val Hall 1,*, G L O’Neill 1,, J T Magee 1, B I Duerden 1
PMCID: PMC85130  PMID: 10364594

Abstract

Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinical isolates (n = 36) of Actinomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination of HaeIII and HpaII endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification of Actinomyces spp. of clinical origin.


The genus Actinomyces comprises a heterogeneous group of anaerobic and facultatively anaerobic, asporogenous, nonmotile, non-acid-fast, gram-positive rods with a G+C content of 55 to 71 mol% (2). Many Actinomyces spp. are known to be indigenous to mucous membranes, particularly those in the oral cavity, in humans and other animals. Members of the genus are known to cause actinomycosis and may be found in polymicrobial infections arising from tissue invasion by oral anaerobes (16). Some species are significant in periodontal disease (16).

Detection of the presence of Actinomyces spp. in clinical specimens may affect the prognosis and patient management, but identification by conventional phenotypic methods is notoriously difficult and unreliable (6). Problems arise from the slow and granular growth of some isolates (particularly Actinomyces israelii), the poor reproducibilities of biochemical tests, and the lack of discriminatory power of biochemical tests. The lack of discriminatory power may indicate heterogeneity within species. Indeed, for some species, subdivisions based on biochemical or serological differences have been described (17, 18), and combination in a single genospecies of some serotypes of Actinomyces naeslundii and Actinomyces viscosus has been proposed (8).

Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. These include Actinomyces europaeus (4), Actinomyces graevenitzii (12), Actinomyces neuii (5), Actinomyces radingae (22), and Actinomyces turicensis (22). Other new species have been assigned to other, similar genera: Arcanobacterium bernardiae (13), Arcanobacterium phocae (13), Arcanobacterium pyogenes (13), Actinobaculum schaalii (9), and Actinobaculum suis (9). Currently, little is known of the natural habitats, clinical prevalence, and pathogenic potential of these species. A discriminatory, reproducible, and practical method for characterization of clinical isolates may help elucidate their occurrence and significance.

Pyrolysis mass-spectrometry (PMS) has been shown to be a useful tool in differentiating groups of organisms to the species or subspecies level (10) and represents a whole-cell analysis approach independent of conventional biochemical tests. However, PMS is a fingerprinting method and is best suited to examination of large batches of organisms. Barsotti et al. (1) demonstrated the potential of rRNA gene (rDNA) restriction patterns as taxonomic tools for Actinomyces. This approach may be enhanced by initial amplification of rDNA by PCR. Amplified rDNA restriction analysis (ARDRA) has been used to identify various fungi (21) and bacteria (19, 20).

In this study, ARDRA was applied to reference strains and clinical isolates of Actinomyces spp. and some other gram-positive rods. Clinical isolates were identified initially to the species level by conventional biochemical tests and comparison of the results with those for reference strains. However, given the low degree of confidence in this method, the findings were also compared with those obtained by PMS.

MATERIALS AND METHODS

Bacterial strains.

A total of 63 strains were examined (Table 1). Reference strains (n = 27) represented 17 species of Actinomyces and 5 species of other, similar genera. Clinical strains comprised 35 human isolates and 1 animal isolate from the collection held by the Anaerobe Reference Unit, Public Health Laboratory Service. Strains were selected to represent the range of species and sites of isolation of strains referred for identification from laboratories throughout England and Wales. Strains were stored at −80°C on Microbank beads (Pro-lab Diagnostics, Wirral, United Kingdom) and were recovered on Fastidious Anaerobe Agar (FAA; LabM, Bury, United Kingdom) incubated anaerobically at 37°C for 48 h.

TABLE 1.

Sources of bacterial strainsa

Clinical isolates
Reference strainsb
Strain Organism Source Strain Organism Reference
VH20 A. denticolens Abdominal abscess VH45 A. israelii ATCC 12102
VH29 A. georgiae Cerebral abscess VH34 A. israelii NCTC 10236
VH24 A. georgiae IUCD VH28 A. gerencseriae ATCC 23860
VH48 A. gerencseriae Facial abscess VH14 A. naeslundii serotype I NCTC 10301
VH16 A. gerencseriae Eyelid puncta VH55 A. naeslundii serotype II ATCC 44339
VH4 A. gerencseriae Jaw abscess VH56 A. naeslundii serotype III ATCC 44340
VH32 A. gerencseriae Vertebral abscess VH36 A. viscosus serotype I NCTC 10951
VH15 A. israelii Pleural pus VH54 A. viscosus serotype II ATCC 27044
VH5 A. israelii IUCD VH6 A. odontolyticus NCTC 09935
VH1 A. israelii Mandibular sinus VH10 A. meyeri ATCC 35568
VH38 Most like A. israelii Perinephric abscess VH11 A. georgiae ATCC 49285
VH21 A. israelii IUCD VH47 A. denticolens NCTC 11490
VH44 A. meyeri IUCD VH43 A. slackii NCTC 11923
VH35 A. meyeri IUCD VH25 A. bovis NCTC 11535
VH22 A. meyeri Groin abscess VH7 Actinomyces hordeovulneris ATCC 35275
VH41 A. meyeri Peritoneal pus VH30 A. howellii NCTC 11636
VH12 A. meyeri Pleural effusion VH51 A. graevenitzii CCUG 27294
VH8 A. naeslundii Gingival swab VH52 A. europaeus CCUG 32789A
VH26 Most like A. naeslundii Blood culture VH57 A. neuii subsp. neuii DSM 8576
VH42 Most like A. naeslundii Parotid duct VH58 A. neuii subsp. anitratus DSM 8577
VH2 A. naeslundii IUCD VH60 A. turicensis DSM 9168
VH39 A. naeslundii IUCD VH61 A. radingae DSM 9169
VH27 Most like A. odontolyticus Jaw pus VH53 A. schaalii CCUG 27420
VH50 Most like A. odontolyticus Liver abscess VH63 A. suis DSM 20639
VH13 A. odontolyticus Oral bone plate VH19 A. pyogenes NCTC 05224
VH40 A. odontolyticus Blood culture VH59 A. bernardiae DSM 9152
VH33 Most like A. odontolyticus IUCD VH62 A. phocae DSM 10002
VH37 Most like A. pyogenes Cow jaw
VH18 A. viscosus Submandibular abscess
VH3 Most like A. viscosus Groin
VH49 Most like A. viscosus IUCD
VH46 Most like A. viscosus Fractured mandible
VH23 Most like A. viscosus IUCD
VH17 Most like A. viscosus Lacrimal fluid
VH9 Actinomyces species Dental abscess
VH31 Actinomyces species Osteomyelitis
a

Abbreviations: IUCD, intrauterine contraceptive device; ATCC, American Type Culture Collection, Rockville, Md.; NCTC, National Collection of Type Cultures, London, United Kingdom; CCUG, Culture Collection, University of Goteborg, Goteborg, Sweden; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany. 

b

Reference strains other than A. israelii NCTC 10236 represent type strains for species or subspecies. 

Conventional tests.

Strains were determined to be members of the genus Actinomyces on the basis of volatile and nonvolatile fatty acid end products of glucose metabolism, as detected by gas-liquid chromatography as described elsewhere (7).

Cell and colonial morphologies, pigment production, fluorescence under long-wave UV illumination, ability to grow in air and in air plus 5% CO2, and production of catalase and indole were recorded. Hydrolysis of esculin and starch and production of acid from amygdalin, arabinose, cellobiose, glucose, mannitol, raffinose, ribose, salicin, sucrose, trehalose, and xylose were tested by the method of Phillips (14). Production of nitrate reductase, urease, pyrazinamidase, β-galactosidase, α-glucosidase, and β-N-acetyl-glucosaminidase was detected after incubation for 18 to 24 h with Rosco diagnostic tablets (BioConnections, Leeds, United Kingdom). Identifications were made initially by reference to the scheme of Brazier and Hall (3) and subsequently by reference to the publications of Funke et al. (5), Pascual Ramos et al. (12), Funke et al. (4) and Lawson et al. (9).

PMS.

Pyrolysis was performed with colonies from 48-h anaerobic FAA cultures, and the mass spectra were analyzed as described elsewhere (11).

ARDRA.

All strains were tested in triplicate. Strains were cultured anaerobically on FAA for 48 h.

For DNA extraction, a 1-μl loopful of bacteria was suspended in 100 μl of Chelex resin (5%; Bio-Rad Laboratories, Richmond, Calif.) in sterile distilled water, boiled for 8 min, and centrifuged at 17,000 × g for 10 min. The supernatant was decanted.

For PCR, DNA extract (5 μl) was added to 45 μl of a reaction mixture containing 1 U of Taq polymerase (Pharmacia Biotech, St. Albans, United Kingdom), each deoxynucleoside triphosphate at a concentration of 200 mM, 20 pmol of each primer (pA and pH′), 2.25 mM magnesium chloride, 10 mM Tris HCl (pH 9), 50 mM KCl, and 0.1% Triton X-100. The primer sequences were AGAGTTTGATCCTGGCTCAG (pA) and AAGGAGGTGATCCAGCCGCA (pH′). Amplification was accomplished by 31 cycles of denaturation at 92°C for 2 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min, with a final extension period of 5 min. The specific PCR product (approximately 1,600 bp) was detected by electrophoresis of a 5-μl sample for 40 min at 5 V/cm in 1% agarose in TAE (Tris-acetate-EDTA) buffer with ethidium bromide (0.5 μg/ml) and was visualized under UV illumination.

Restriction endonuclease digestion was performed with HaeIII and HpaII endonucleases in separate reaction mixtures containing 1 μl of endonuclease (10 U/μl), 1.5 μl of matched incubation buffer, and 12.5 μl of PCR product; the mixture was incubated for 1.5 h at 37°C. For electrophoresis, 3 μl of 6× loading buffer (Advanced Biotechnologies, Epsom, United Kingdom) was added to each sample, and 15 μl was electrophoresed for 1.75 h at 5 V/cm in Metaphor agarose (3.5%; FMC, Rockland, Maine) in TAE buffer with ethidium bromide (0.5 μg/ml). Molecular size markers (2 kb; Sigma, Poole, United Kingdom) were run in duplicate or triplicate alongside the samples. The gels were destained in deionized water for 10 min and photographed under UV illumination, and the images were stored on a floppy disk.

Gel data were analyzed with GelCompar software (Applied Maths, Kortrijk, Belgium). Distinct banding patterns were assigned three-digit codes, arbitrarily numbered in the order encountered, and were stored as types in HAE and HPA libraries. The combined results for each strain were recorded as, for example, 001/003, which represent the HaeIII and HpaII profiles, respectively.

RESULTS

Conventional biochemical tests.

For all strains, minor amounts of acetic acid and major amounts of lactic and succinic acids were detected as products of glucose metabolism.

(i) Reference strains.

The identities of 18 of the 27 strains were confirmed by conventional tests. Strains that gave anomalous results are listed in Table 2.

TABLE 2.

Reference strains demonstrating results anomalous to those published elsewherea

Strain Species and strain designation Conventional identification Anomaly
VH28 A. gerencseriae ATCC 23860 A. georgiae Raffinose negative
VH36 A. viscosus ser. I NCTC 10951 A. naeslundii Catalase negative
VH47 A. denticolens NCTC 11490 No identification Trehalose positive
VH52 A. europaeus CCUG 32789A No identification Nitrate positive
VH59 A. bernardiae DSM 9152 No identification β-N-Acetyl-glucosaminidase positive
VH60 A. turicensis DSM 9168 Poor differentiation Pyrazinamidase positive
VH61 A. radingae DSM 9169 No identification Esculin negativeb
VH62 A. phocae DSM 10002 A. georgiae Xylose positive
VH63 A. suis DSM 20639 A. meyeri or A. naeslundii β-Galactosidase negative
a

The previous publications are references 4, 5, 9, and 12

b

Reaction listed as positive in the previous studies but negative or slowly positive in the original description of Wust et al. (22). 

(ii) Clinical strains.

Identifications obtained by conventional tests are listed in Table 1. Interpretation of results was sometimes difficult due to insufficient growth or the poor reproducibility of reactions. When results were inconsistent with those described for recognized species, strains are designated “most like” followed by the species name.

PMS.

By PMS, strains VH20, VH21, and VH28 were contaminated and thus were excluded from analysis. The remaining strains formed four superclusters (superclusters A, B, C, and E) plus cluster with a single strain (cluster D) (Fig. 1; Table 3). The majority of strains clustered with members of the same species. However, five clinical strains identified by conventional tests as Actinomyces meyeri clustered closely with the type strain of A. turicensis and remotely from the type strain of A. meyeri. Also, two strains conventionally identified as Actinomyces georgiae clustered with four Actinomyces gerencseriae strains; one strain (strain VH29) was subsequently reidentified as A. gerencseriae; the identity of the other strain (strain VH24) remains uncertain. A reference strain of A. israelii (strain VH34) inexplicably clustered with this A. gerencseriae group. One strain (strain VH23), conventionally identified as most like A. viscosus, clustered with the type strain of A. neuii subsp. neuii and was subsequently reidentified as A. neuii subsp. neuii by virtue of its abilities to produce catalase, ferment mannitol, and reduce nitrate. Strains VH7, VH9, VH19, VH27, VH31, VH43, VH47, VH52, VH58, VH59, and VH61 each formed single-strain clusters.

FIG. 1.

FIG. 1

Dendrogram of similarities between strains on the basis of PMS, which reflects whole-cell composition. Strain membership of the clusters is outlined in Table 3. dof, degrees of freedom.

TABLE 3.

Results by ARDRA, PMS, and conventional biochemical tests, arranged by HpaII profile

ARDRA profile PMS cluster Strain Conventional identification True identification
001/001 B2 VH6 A. odontolyticus A. odontolyticus NCTC 09935
001/001 B2 VH13 A. odontolyticus A. odontolyticus
001/001 B2 VH33 Most like A. odontolyticus A. odontolyticus
001/001 B2 VH50 Most like A. odontolyticus A. odontolyticus
025/001 B2 VH40 A. odontolyticus A. odontolyticus
002/002 C9 VH7 A. hordeovulneris A. hordeovulneris ATCC 35275
001/003 B3 VH10 A. meyeri A. meyeri ATCC 35568
001/004 B3 VH11 A. georgiae A. georgiae ATCC 49285
003/005 B1 VH2 A. naeslundii A. naeslundii
003/005 C1 VH14 A. naeslundii A. naeslundii serotype I NCTC 10301
003/005 C1 VH26 Most like A. naeslundii A. naeslundii
007/005 B4 VH30 A. howellii A. howellii NCTC 11636
009/005 C1 VH39 A. naeslundii A. naeslundii
014/005 C1 VH8 A. naeslundii A. naeslundii
014/005 B4 VH56 A. naeslundii A. naeslundii serotype III ATCC 44340
014/005 B4 VH42 Most like A. naeslundii A. naeslundii
017/005 C2 VH55 A. naeslundii A. naeslundii serotype II ATCC 44339
017/005 C2 VH18 A. viscosus A. viscosus
021/005 C2 VH54 A. viscosus A. viscosus serotype II ATCC 27044
021/005 C2 VH17 Most like A. viscosus A. viscosus
021/005 C2 VH46 Most like A. viscosus A. viscosus
021/005 C2 VH49 Most like A. viscosus A. viscosus
021/005 C1 VH3 Most like A. viscosus A. viscosus
004/006 D VH19 A. pyogenes A. pyogenes NCTC 05224
005/007 B4 VH25 A. bovis A. bovis NCTC 11535
006/008 A2 VH4 A. gerencseriae A. gerencseriae
006/008 A2 VH16 A. gerencseriae A. gerencseriae
006/008 A2 VH29 A. georgiae A. gerencseriae
006/008 A2 VH32 A. gerencseriae A. gerencseriae
006/008 A2 VH48 A. gerencseriae A. gerencseriae
006/008 NDa VH28 A. georgiae A. gerencseriae ATCC 23860
008/009 A1 VH45 A. israelii A. israelii ATCC 12102
008/009 A2 VH34 A. israelii A. israelii NCTC 10236
008/009 ND VH21 A. israelii A. israelii
018/009 A1 VH5 A. israelii A. israelii
018/009 ND VH20 A. denticolens A. israelii
020/009 A1 VH1 A. israelii A. israelii
020/009 A1 VH15 A. israelii A. israelii
020/009 A1 VH38 Most like A. israelii A. israelii
009/010 B1 VH36 A. naeslundii A. viscosus serotype I NCTC 10951
010/011 C5 VH43 A. slackii A. slackii NCTC 11923
024/011 B7 VH31 Actinomyces species Uncertain
011/012 B5 VH47 Uncertain A. denticolens NCTC 11490
013/013 B1 VH51 A. graevenitzii A. graevenitzii CCUG 27294
012/014 C6 VH52 Uncertain A. europaeus CCUG 35789A
004/015 B8 VH53 A. schaalii A. schaalii CCUG 27420
001/016 E2 VH27 Most like A. odontolyticus Uncertain
015/016 E1 VH60 A. turicensis or A. meyeri A. turicensis DSM 9168
015/016 E1 VH12 A. meyeri A. turicensis
015/016 E1 VH22 A. meyeri A. turicensis
015/016 E1 VH35 A. meyeri A. turicensis
015/016 E1 VH41 A. meyeri A. turicensis
015/016 E1 VH44 A. meyeri A. turicensis
019/017 A3 VH9 Actinomyces species Uncertain
022/019 C3 VH57 A. neuii subsp. neuii A. neuii subsp. neuii DSM 8576
022/019 C3 VH23 A. viscosus A. neuii subsp. neuii
022/019 C4 VH58 A. neuii subsp. anitratus A. neuii subsp. anitratus DSM 8577
023/020 A2 VH24 Most like A. georgiae Uncertain
026/021 C8 VH59 Uncertain A. bernardiae DSM 9152
027/022 B6 VH37 Most like A. pyogenes Uncertain
028/023 C7 VH61 Uncertain A. radingae DSM 9169
029/024 B6 VH62 A. georgiae A. phocae DSM 10002
030/025 B8 VH63 A. meyeri or A. naeslundii A. suis DSM 20639
a

ND, no data available. 

ARDRA.

All strains were cleaved by both endonucleases, yielding 6 to 10 bands by HaeIII and 5 to 12 bands by HpaII typing (Fig. 2 and 3, respectively). The resulting HaeIII and HpaII profiles were found to be highly reproducible, allowing the assignation of permanent types to each strain (Table 3). Clinical strains yielding HaeIII and HpaII profiles indistinguishable from those of a reference strain were assigned to that species. The types obtained for A. naeslundii reference serovars indicated subspecies variation in HaeIII profiles and the possible species specificity of the HpaII profiles. Therefore, clinical strains were assigned to species for which the HpaII profile was indistinguishable from that for a reference strain and identification by conventional biochemical tests concurred. When the latter did not concur or when strains yielded distinct profiles with both enzymes, the identity was considered to be uncertain. This approach was supported by the findings obtained by conventional biochemical tests and PMS.

FIG. 2.

FIG. 2

HaeIII restriction profiles for some reference strains. Lanes 1, 8, and 15, 2-kb marker; lane 2, A. israelii ATCC 12102; lane 3, A. gerencseriae ATCC 23860; lane 4, A. naeslundii serotype I NCTC 10301; lane 5, A. viscosus serotype II ATCC 27044; lane 6, A. neuii subsp. neuii DSM 8576; lane 7, A. graevenitzii CCUG 27294; lane 9, A. odontolyticus NCTC 09935; lane 10, A. georgiae ATCC 49285; lane 11, A. meyeri ATCC 35568; lane 12, A. turicensis DSM 9168; lane 13, A. radingae DSM 9169; lane 14, A. europaeus CCUG 32789A.

FIG. 3.

FIG. 3

HpaII restriction profiles for some reference strains. Lanes 1, 8, and 15, 2-kb marker; lane 2, A. israelii ATCC 12102; lane 3, A. gerencseriae ATCC 23860; lane 4, A. naeslundii serotype I NCTC 10301; lane 5, A. viscosus serotype II ATCC 27044; lane 6, A. neuii subsp. neuii DSM 8576; lane 7, A. graevenitzii CCUG 27294; lane 9, A. odontolyticus NCTC 09935; lane 10, A. georgiae ATCC 49285; lane 11, A. meyeri ATCC 35568; lane 12, A. turicensis DSM 9168; lane 13, A. radingae DSM 9169; lane 14, A. europaeus CCUG 32789A.

(i) Reference strains.

By HaeIII typing, 17 of the 22 species represented and the three serotypes of A. naeslundii and two of A. viscosus were clearly differentiated. Actinomyces odontolyticus, A. meyeri, and A. georgiae strains were indistinguishable (type 001), as were A. pyogenes and A. schaalii (type 004). Both A. israelii strains were type 008. The two A. neuii subspecies were type 022.

By HpaII typing, 20 species were clearly differentiated; but A. naeslundii serotypes I, II, and III, A. viscosus serotype II, and A. howellii were indistinguishable (type 005). Both A. israelii strains were type 009. The two A. neuii subspecies were type 019. The combination of HaeIII and HpaII types allowed differentiation of all 22 species represented by reference strains.

(ii) Clinical strains.

In reactions with both endonucleases, 16 of the 36 strains were indistinguishable from reference strains of the same species, as determined by conventional tests, and were assigned to species with confidence. Eight strains were indistinguishable by ARDRA from the reference strains of a species other than that identified by conventional tests. These comprised the five strains conventionally identified as A. meyeri, which were indistinguishable from A. turicensis; VH23, which was originally most like A. viscosus but which was redesignated A. neuii subsp. neuii and which gave the same profile as the A. neuii strains; VH29, which was originally considered to be A. georgiae but which was indistinguishable from A. gerencseriae; and VH18 (A. viscosus), which gave the same profile as A. naeslundii serotype II. These were assigned to the species determined by ARDRA, with strain VH18 being deemed a member of A. naeslundii genospecies 2, which includes A. viscosus serotype II.

Seven strains had distinct HaeIII profiles but gave HpaII profiles indistinguishable from those of reference strains of the species determined by conventional tests and were assigned to that species. One strain (strain VH31), which had a distinct HaeIII profile and which was indistinguishable from the reference strain of Actinomyces slackii by HpaII typing, was not clearly identified by conventional tests and was deemed to be of uncertain identity.

One strain (strain VH27) that was identified by conventional tests as most like A. odontolyticus gave profiles indistinguishable from those of reference strains of A. odontolyticus by HaeIII typing and A. turicensis by HpaII typing. The remaining three strains (strains VH9, VH24, and VH37) gave unique profiles in both reactions. The true identities of these four strains remain uncertain.

DISCUSSION

The application of chemotaxonomic and molecular methods has clarified the taxonomy of the genus Actinomyces, but the description of novel species has rendered previously published identification schemes obsolete. When few strains of a species have been studied, the reliability of identification by conventional tests is questionable. Furthermore, given the diversity within the genus, further modification of the taxonomic status of strains may be warranted.

Currently, the Public Health Laboratory Service Anaerobe Reference Unit receives over 100 referrals each year from clinical laboratories throughout England and Wales for confirmation of isolates presumptively identified as Actinomyces spp. There is a need for a practical and discriminatory method for the identification of clinical isolates.

The identification of Actinomyces to the species level by conventional biochemical tests is beset with problems. These include technical difficulties, poorly discriminatory tests, and heterogeneity within described species. In this study, nine reference strains demonstrated reactions anomalous from those published elsewhere, and this resulted in mis- or nonidentification (Table 2). Notably, A. europaeus was found to reduce nitrate to nitrite, and A. bernardiae produced β-N-acetyl-glucosaminidase. Anomalies, particularly in enzyme reactions, may be due to differences in test methodologies.

PMS has been shown to be highly discriminatory for a wide range of organisms but is best suited to the testing of large batches of isolates. Direct comparisons between batches are not readily made. Thus, PMS would be impractical for examination of occasional clinical isolates. However, given the low degree of confidence in results obtained by conventional tests for the identification of Actinomyces, evaluation of the efficacy of novel methods is difficult. In this study, PMS provided a valuable independent approach with which to compare findings. It is noteworthy and reassuring that the dendrogram derived from PMS analysis reflects the taxonomic relationships generated by 16S rDNA sequencing (4, 9, 13). A. israelii and A. gerencseriae strains formed a supercluster (supercluster A) remote from other species. Superclusters B and C were linked and contained strains identified as A. odontolyticus, A. naeslundii, A. viscosus, and several other species, largely represented by single reference strains. Within this group, the type strains of A. meyeri and A. georgiae formed a cluster (cluster B3) closely linked to A. odontolyticus strains (cluster B2). The remoteness of cluster E (A. turicensis strains and strain VH27) from other species raises doubts as to their place in the genus Actinomyces.

ARDRA has proven to be useful for discrimination of various bacterial species. In this study, the types generated by the combination of HaeIII and HpaII endonuclease digestion profiles correlated well with the findings obtained by PMS and conventional tests and enabled the identification of 31 of 36 clinical strains to the species level. The remaining five strains were not clearly identified by conventional tests, and three strains formed distinct clusters by PMS; the latter strains may well represent novel species or other genera.

Seven strains were identified by ARDRA and PMS as species other than those initially assigned by conventional tests. Five of these seven strains initially identified as A. meyeri clustered tightly with the A. turicensis type strain by PMS and were indistinguishable from A. turicensis by ARDRA. Review of conventional test results showed that the two species are poorly differentiated by the range of biochemical tests performed. The identity of one of the five strains (strain VH12) was confirmed as A. turicensis by 16S rDNA sequence analysis (data not shown). The strain that was apparently misidentified by PMS and ARDRA as A. gerencseriae had been designated A. georgiae by conventional tests by virtue of its inability to ferment raffinose; the morphology and other reactions were consistent with A. gerencseriae. This property was also demonstrated by the A. gerencseriae type strain; thus, raffinose fermentation appears to be an unreliable determinant in the identification of this species. The strain originally designated most like A. viscosus and identified as A. neuii by PMS and ARDRA was subsequently redesignated A. neuii subsp. neuii by virtue of catalase production, nitrate reduction, and fermentation of mannitol.

A. gerencseriae and A. turicensis showed homogeneous, distinct ARDRA profiles. The subdivisions observed within A. israelii, A. odontolyticus, A. naeslundii, and A. viscosus may correspond to those previously recognized by other workers (8, 17, 18). One strain of A. viscosus was indistinguishable from A. naeslundii serotype II by PMS and ARDRA, adding to the body of evidence that A. naeslundii and A. viscosus isolates form a heterogeneous complex that requires further investigation.

When species were represented by single reference strains, all strains gave distinct profiles, and none was misidentified. However, no further conclusions regarding the efficacy of ARDRA for identification of these species can be drawn without data derived from additional strains.

Current consumable costs, based on a batch of 10 isolates, were £2.80 (as of April 1999, £1 is equal to $1.60) per isolate. Each batch required approximately 3.5 h of labor spread over 1.5 days.

In conclusion, ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for identification of Actinomyces spp. of clinical origin. Application of the method to further clinical and veterinary isolates may confirm its usefulness, and an extensive investigation of strains referred to the Anaerobe Reference Unit is in progress. Within the limitations of the current study, identification of a strain as a member of the genus Actinomyces by gas-liquid chromatography is a prerequisite. However, preliminary investigations of strains of Propionibacterium spp., Lactobacillus spp., and Bifidobacterium spp. have demonstrated the clear differentiation of strains by ARDRA (unpublished data), indicating potential for identification to the species level of members of these genera and obviating the need for gas-liquid chromatography.

Identification of clinical isolates of Actinomyces to the species level may be important for patient management. Additionally, a reliable identification system is essential for the discovery of the natural habitats, prevalence, and pathogenicity of recently described species. With increasing knowledge of these aspects, species-level identification of clinical isolates may become more relevant to patient management. Some species, e.g., A. turicensis, have been shown to be identifiable in commercial biochemical systems (15). However, genotypic methods may be advantageous in their ability to detect novel species as well as those listed in commercial databases. A practical, highly discriminatory, and cost-effective method such as ARDRA applied to many strains may greatly aid in the elucidation of the ecology and clinical spectra of Actinomyces species and may further clarify the taxonomy of the genus.

ACKNOWLEDGMENTS

We thank Margaret Heginbotham and Paul Talbot for technical assistance with PMS and conventional tests.

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