N-acetylcysteine effects on extracellular polymeric substances of Xylella fastidiosa: A spatiotemporal investigation with implications for biofilm disruption

Background: The matrix of extracellular polymeric substances (EPS) present in biofilms greatly amplifies the problem of bacterial infections, protecting bacteria against antimicrobial treatments and eventually leading to bacterial resistance. The need for alternative treatments that destroy the EPS matrix becomes evident. N-acetylcysteine (NAC) is one option that presents diverse effects against bacteria; however, the different mechanisms of action of NAC in biofilms have yet to be elucidated. Objectives: In this work, we performed microscopy studies at micro and nano scales to address the effects of NAC at single cell level and early-stage biofilms of the Xylella fastidiosa phytopathogen. Methods: We show the physical effects of NAC on the adhesion surface and the different types of EPS, as well as the mechanical response of individual bacteria to NAC concentrations between 2 and 20 mg/mL. Results: NAC modified the conditioning film on the substrate, broke down the soluble EPS, resulting in the release of adherent bacteria, decreased the volume of loosely bound EPS, and disrupted the biofilm matrix. Tightly bound EPS suffered structural alterations despite no solid evidence of its removal. In addition, bacterial force measurements upon NAC action performed with InP nanowire arrays showed an enhanced momentum transfer to the nanowires due to increased cell mobility resulting from EPS removal. Conclusions: Our results clearly show that conditioning film and soluble EPS play a key role in cell adhesion control and that NAC alters EPS structure, providing solid evidence that NAC actuates mainly on EPS removal, both at single cell and biofilm levels.