Biomimetic Molecularly Imprinted Polymers: A New Quorum Sensing Capturing Agent to Prevent Bacterial Biofilm Formation

Introduction: Biofilms are prevalently formed by monospecies and multispecies bacteria in food processing environments. Compared to planktonic cells, biofilms are up to 10-500 times more resistant to antimicrobial agents and disinfectants, thus constituting a threat to public health and food industry. The formation of biofilms is mediated by quorum sensing (QS). QS is a mechanism by which bacteria can assess their population density through the secretion and sensing of autoinducers (AIs) and regulate the expression of certain genes in a cell-density dependent manner. Attenuating QS via sequestration of AIs represents a promising strategy to control biofilm development. However, conventional sequestration agents (i.e., antibodies) are unstable and their large-scale production is difficult to achieve. To address these disadvantages, molecularly imprinted polymers (MIPs), known as “artificial antibodies,” that were developed to specifically capture AIs. 

Purpose: The overall objective of this study was to develop a MIP and evaluate its inhibitory effect against bacterial biofilm formation. 

Methods: Pseudomonas aeruginosa was selected as it is a model organism for the study of both biofilms and QS. As one of the major AIs produced by P. aeruginosa, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C₁₂-AHL) was served as the template molecule for the synthesis of MIPs. Binding capacity and selectivity of MIPs towards 3-oxo-C₁₂-AHL was evaluated in equilibrium binding tests in aqueous condition (i.e., 20% acetonitrile). To evaluate the inhibitory effect of MIPs against biofilm formation, biofilms of P. aeruginosa and/or Salmonella enterica and Campylobacter jejuni were incubated with or without the presence of MIPs. Confocal laser scanning microscopy staining assay and crystal violet staining assay were conducted to quantify biofilm formation levels. Statistical significance was determined using Student’s t-test.

Results: The binding capacity of MIPs was 3.81 mg/g, which was 1.22 times higher than the control group.  It indicated highly specificity of MIPs towards 3-oxo-C₁₂-AHL.The results showed that MIPs could significantly (P<0.05) prevent biofilm formation at the early and middle stage (i.e., 4 to 12 h).

Significance: This study developed and evaluated a novel strategy to control both monospecies and multispecies bacterial biofilm formation and can be potentially applied to inhibit other QS-regulated bacterial behaviors.