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This study investigated the prevalence of Actinomyces spp. in shallow, deep and very deep pockets of patients with chronic periodontitis compared to healthy controls and correlated the results with clinical status. Twenty patients with chronic periodontitis and 15 healthy subjects were enrolled in this study. Clinical indices were recorded in a six-point measurement per tooth. From each patient samples of supra and subgingival plaque were taken separately from teeth with shallow, deep and very deep pockets. Samples of supragingival plaque and sulcular microflora were collected from the healthy subjects. All the samples were cultivated on different media at 37̊C in an anaerobic atmosphere for 7 days. All the suspect colonies were identified using a rapid ID 32 A system (bioMèrieux) and MALDI-TOF-MS analysis using an Autoflex II Instrument (Bruker Daltonics) together with in house developed identification software and a reference spectra database. A total of 977 strains were identified as Actinomyces. Actinomyces naeslundii/oris/johnsonii (430 isolates) was the most prevalent species and was found in all patients and in almost all of the healthy subjects. Significant differences (p=0.003) between the groups were found for Actinomyces odontolyticus/meyeri and Actinomyces israelii which were associated with periodontitis patients. Actinomyces dentalis was found in higher percentage (p=0.015) in the periodontitis group. Actinomyces gerencseriae and Actinomyces massiliensis were significantly more often found supragingivally than subgingivally (p=0.004, p=0.022, respectively) in the periodontitis group. Whether some Actinomyces species, definitely important plaque formers, are actively involved in the pathogenicity of chronic periodontitis needs further investigation., (Copyright © 2015 Elsevier GmbH. All rights reserved.)