Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes–Escherichia coli dual-species biofilms

Abstract

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes–Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r² = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Keywords:

• Benzalkonium chloride
• biofilm
• disinfection
• fluorescence microscopy
• Listeria monocytogenes
• Escherichia coli
• pronase

Acknowledgements

All authors acknowledge S. Rodríguez, T. Blanco, V. Nimo and A. Carrera (laboratory technicians, IIM-CSIC) for their help throughout the whole study.