The impact of DNA extraction methods on species quantification and apparent community composition of in vitro oral biofilms.

In vitro multispecies biofilm models are widely used to study oral diseases and to evaluate treatment strategies for conditions such as peri-implantitis, often relying on accurate species quantification to assess treatment efficiency. However, the influence of DNA extraction methodology on downstream quantitative analysis has not yet been addressed for such model systems. Here, we evaluated three mechanistically distinct protocols, a custom phenol-chloroform approach, and two commercial kits employing different lysis strategies. These were applied to planktonic cultures of six peri-implantitis-associated species individually, as well as to defined multispecies biofilms grown on implant surfaces. Pure culture DNA yields differed substantially between methods, revealing pronounced species-dependent variation. Species-specific quantification of biofilm replicates by quantitative PCR resulted in community profiles that appeared dominated by either commensal early- or pathogenic late-colonizers, depending solely on the extraction approach employed. These findings demonstrate that DNA extraction is a critical yet often overlooked variable, capable of fundamentally altering the apparent community composition of in vitro biofilm models. Our work is intended to serve as a warning, emphasizing the need for method validation and standardization when applying DNA-based community profiling to biofilm models before drawing conclusions on relative species abundances.