In early childhood, the human mouth is already colonized by actinomycetes. Due to recent taxonomic changes within the genus Actinomyces, up-to-date data are warranted on the time and succession of different Actinomyces species in the oral cavity. By using a longitudinal study design and culture techniques, we examined the age-related occurrence of Actinomyces species in saliva from 39 healthy infants at 2, 6, 12, 18, and 24 months of age. Altogether 428 Actinomyces isolates were available for this study. Identification was based on biochemical tests and gas chromatographic demonstration of metabolic end-products, and when needed, cellular fatty acid profiles were determined. The frequency of the total actinomycetal flora increased from 31% to 97% within 2 years. A. odontolyticus was the most prominent Actinomyces colonizer at all five sampling occasions. A. naeslundii was the second most common Actinomyces sp. but was not detected before the age of 1 year. As a novel observation, we found A. graevenitzii in the oral cavity. The number of A. graevenitzii isolates indicates that this species is not just occasionally present in infants' mouths. We also found A. viscosus, A. gerencseriae, A. israelii, and A. georgiae. Based on the present results, we suggest that A. odontolyticus is the main primary Actinomyces species on oral mucosal surfaces in infants up to 2 years of age.

Recent advancements in chemotaxonomic and molecular biology-based identification methods have clarified the taxonomy of the genus Actinomyces and have led to the recognition of several new Actinomyces and related species. Actinomyces-like gram-positive rods have increasingly been isolated from various clinical specimens. Thus, an easily accessible scheme for reliable differentiation at the species level is needed in clinical and oral microbiology laboratories, where bacterial identification is mainly based on conventional biochemical methods. In the present study we designed a two-step protocol that consists of a flowchart that describes rapid, cost-efficient tests for preliminary identification of Actinomyces and closely related species and an updated more comprehensive scheme that also uses fermentation reactions for accurate differentiation of Actinomyces and closely related species.

We evaluated four commercially available kits for rapid identification of Actinomyces and related species. The kits identified correctly 26 to 65% of "classical" Actinomyces strains to the species level and 13 to 49% of newly described Actinomyces strains to the genus level, thus indicating relatively poor applicability and a need to update these kits.The genus Actinomyces embraces a heterogeneous group of facultatively anaerobic, gram-positive, mainly branching rods, which occur as frequent inhabitants on mucosal surfaces but also as common opportunistic pathogens in various infectious processes (3,5,7,10,15,17). Improvements in identification methods have resulted in expanded recognition of new Actinomyces species from clinical specimens (3,4,6,8,13,18). On the other hand, some former Actinomyces species have been moved to other genera, such as Arcanobacterium and Actinobaculum (11,14). Over 80% of the problematic isolates sent to our reference laboratory turn out to belong to the genus Actinomyces, indicating that clinical microbiology laboratories need updated information on these slowly growing, asporogenous rods. Recently, Sarkonen et al. published an extensive identification scheme for Actinomyces species for the purpose of clarifying their accurate identification on the basis of phenotypic testing alone (16); however, many laboratories rely solely on commercial kits because of lack of manpower and facilities. In the present study, we demonstrated the performance of four commercial test kits in the identification of "classical" (i.e., described before 1994) Actinomyces species and tested the reactivity of some newly described species not yet included in the databases.The RapID ANA II system (Remel, Lenexa, Kans.) consists of 10 wells (including dehydrated reactants for 18 biochemical reactions) and the Rapid ID 32 A system (bioMérieux, Marcyl'Etoile, France) consists of 32 cupules (including 29 containing dehydrated test substrates) for the testing of anaerobic bacteria. The RapID CB Plus system (Remel) consists of 18 wells (including 4 for carbohydrate utilization and 14 for single-substrate enzyme tests) for the testing of coryneform and related bacteria. The BBL Crystal ANR ID system (Becton Dickinson Microbiology System, Cockeysville, Md.) consists of 30 wells (including 17 wells with fluorescent substrates, 12 wells with chromogenic substrates, and a fluorescence control well); the assay includes tests for fermentation, oxidation, degradation, and hydrolysis of various substrates.A total of 21 reference strains from international culture collections and 86 clinical Actinomyces strains (65 of infectious origin and 21 from human resident floras) were included in this study. All strains were previously identified by conventional methods (16) or by the ARDRA (PCR-restriction fragment length polymorphism) method (8) or by both. The organisms were revived from frozen (Ϫ70°C) stocks, grown twice in subcultures on brucella blood agar, and incubated anaerobically at 35°C for 3 to 4 days before testing was con...

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