Use of plant essential oils to control single and mixed species biofilms formed by foodborne pathogens and spoilage bacteria

Pathogenic bacteria, e.g., Listeria monocytogenes, located in biofilms on processing equipment can become transferred to foods thereby jeopardizing food safety. Antimicrobial essential oils(EOs) have potential uses as natural disinfectants in the food industry. The objective of this study was to determine the antimicrobial and anti-biofilm effects of EOs (thymol, carvacrol, trans-cinnamaldehydeand lemongrass) on single and mixed species biofilms of Listeria monocytogenes, Salmonella entericaTyphimurium, Staphylococcus aureus, Pseudomonas fluorescens and Shewanella baltica. Minimum inhibition concentrations (MIC), minimum biofilm inhibition concentrations (MBIC), and minimum biofilm eradication concentrations (MBEC) were determined for single and mixed species biofilms in microtiterplate assays at 15°C using crystal violet and 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazoliumbromide (MTT) staining to measure biofilm mass and metabolic activity, respectively. Mixed biofilmpopulations were characterized by enumeration on selective agars. Biofilms were also characterized using Scanning Electron Microscopy (SEM).All EOs inhibited biofilm formation and planktonic growth, however, biofilm eradiation depended on the bacteria, EO, maturity of the biofilm and mixed species interactions. Biofilm cells (MBICs) showed greater resistance to the EOs than planktonic cells (MICs). Only carvacrol (1.5-12 mM) and thymol (2-8 mM) eradicated the metabolic activity (MBEC) in all single species preformed (3 days) biofilms. Increasing biofilmmaturity led to increasing MBEC values. Presence of EOs repressed formation of mixed biofilms with L.monocytogenes and P. fluorescens, S. Typhimurium or Sh. baltica. Proportions of bacteria in mixed species biofilm depended on EO treatments. The proportion of L. monocytogenes in mixed species biofilms rangedfrom 30 to 82% prior to application of EOs. SEM images supported the quantitative findings, showing reduced biofilm formation and abnormal cell morphologies following effective EO treatments. In conclusion, the tested EOs, in particular thymol and carvacrol, inhibited biofilms formed by pathogenic and spoilage bacteria and showed promise as disinfectants in the food industry.