Isolation of novel <i>Actinomyces oris</i> and <i>Aggregatibacter actinomycetemcomitans</i> bacteriophages and study of their biological characteristics <i>in vitro</i>

Abstract

SCIENTIFIC RELEVANCE. The incidence of periodontal infections with antibiotic-resistant strains of Actinomyces oris and Aggregatibacter actinomycetemcomitans capable of forming biofilms in the oral cavity is increasing, and the effectiveness of antimicrobials against biofilms is insufficient. Therefore, the isolation of bacteriophages active against A. oris and Ag. actinomycetemcomitans is an urgent task.AIM. This study aimed to isolate bacteriophages active against A. oris and Ag. actinomycetemcomitans, study their biological properties, and select optimum culture conditions providing pure lines and high titres of bacteriophages.MATERIALS AND METHODS. Bacteriophages were isolated from biosamples of saliva, dental plaque, and periodontal pocket contents. The study determined bacteriophage characteristics, including the optimum multiplicity of infection, phage–cell system cultivation time, resistance to various temperatures and pH, and storage stability. Bacteriophage morphology was studied using transmission electron microscopy. Negative colony morphology, lytic activity, host range, and specificity were assessed using spot testing and the Gratia method. Bacterial strains were obtained from the strain collection of the Micromir research and production centre.RESULTS. The authors isolated and studied 3 novel bacteriophages active against A. oris (vB_AorP_1/G-12, vB_AorP_2/Ch-28, and vB_AorP_3/Bl-35) and 1 bacteriophage active against Ag. actinomycetemcomitans (vB_AacS_1/Dc-1). The A. oris bacteriophages were classified as podoviruses, and the vB_AacS_1/Dc-1 bacteriophage was classified as a siphovirus. The phages formed completely transparent round plaques without a halo, with a diameter ranging from 0.8±0.1 to 4.0±0.2 mm. The optimum parameters established to obtain pure phages with maximum titres included a multiplicity of infection of 0,1–10 and phage–cell system cultivation time of 8–12 hours. The study demonstrated the ability of A. oris bacteriophages to lyse Actinomyces naeslundii strains. Of the 15 A. oris bacteriophage strains studied, vB_AorP_1/G-12, vB_AorP_2/Ch-28, and vB_AorP_3/Bl-35 lysed 10, 8, and 12 bacterial strains, respectively. The vB_AacS_1/Dc-1 phage isolate exhibited lytic activity against both tested strains of Ag. actinomycetemcomitans. The studied phages demonstrated stability under abiotic stress and long-term storage conditions.CONCLUSIONS. The authors isolated 3 novel bacteriophages active against A. oris and 1 bacteriophage active against Ag. actinomycetemcomitans and studied their biological properties. The isolated bacteriophages are promising as candidates for further research using clinical strains and whole-genome sequencing.