Abstract

Background/Objectives: Aggregatibacter actinomycetemcomitans is an important periodontal pathogen whose biofilm formation and bacterial aggregation contribute to colonization and persistence within the oral cavity. This study aimed to generate random transposon mutants of A. actinomycetemcomitans and screen for alterations in biofilm- and aggregation-related phenotypes. Methods: A transposon mutagenesis library was created in the serotype d strain SA269 using the EZ-Tn5 transposon system. Transformants were screened in vitro for biofilm formation, autoaggregation, and coaggregation with Fusobacterium nucleatum. Biofilm biomass was quantified using a standardized microtiter plate assay, while aggregation assays were performed by optical density measurements. Results: Among 44 transformants, 12 exhibited a markedly reduced ability to form biofilms compared with the wild-type strain, indicating that disruption of certain loci compromises surface adherence and biofilm biomass accumulation. Five representative mutants were selected for aggregation studies. Three mutants showed reduced autoaggregation, suggesting impaired intraspecies cell–cell interactions, whereas four mutants demonstrated enhanced coaggregation with F. nucleatum, reaching approximately 50% coaggregation after 120 min. These findings indicate that insertional inactivation of genes in A. actinomycetemcomitans can differentially affect biofilm formation, autoaggregation, and interspecies coaggregation. Conclusions: This preliminary study demonstrates the utility of random transposon mutagenesis for identifying mutants with altered adhesion- and biofilm-related phenotypes. Although the disrupted genes were not identified, the mutant library provides a foundation for future molecular characterization of genetic determinants involved in biofilm development and microbial community interactions.

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