Purpose: The influence of organic leafy greens wash water reuse on biofilm formation over contact surface by Escherichia coli O157:H7, curli-deficient E. coli O157:H7 and non-pathogenic E. coli K12 was studied.
Methods: Wash waters were prepared by washing, with and without filter sterilization, 1 batch, 6 batches and stomaching organic- baby spinach, bunched mature spinach, iceberg lettuce and romaine lettuce to simulate light, moderate and extensive reuse; and to study the influence of native produce microbiota on biofilm formation. Biofilm was allowed to develop over 48 hours at 30°C in 96-well polystyrene plates. Biofilm was quantified using Crystal Violet assay.
Results: Biofilms formed on unfiltered baby spinach wash water contact surfaces after one wash (light reuse) by E. coli O157:H7, curli-deficient E. coli O157:H7 and E. coli K12 resulted in OD600 of 0.49 ± 0.17, 0.57 ± 0.20 and 0.31 ± 0.08, respectively, while on pummeled (extensive reuse) unfiltered spinach wash water contact surface, OD600 of 1.38 ± 0.26, 2.59 ± 0.95 and 2.08 ± 0.12 resulted. On stomached filtered baby spinach wash water contact surface that was free of native microbiota, E. coli O157:H7, curli-deficient E. coli O157:H7 and E. coli K12 formed biofilms resulting in OD600 of 0.66 ± 0.18, 0.13 ± 0.12, 1.21 ± 0.47 indicating that wash water reuse and the resulting dislodged native microbiota caused synergistic biofilm formation in curli deficient E. coli O157:H7. On iceberg lettuce wash water contact surface containing E. coli O157:H7 and curli-deficient E. coli O157:H7, 6 washes resulted in increased biofilm formation than pummeling.
Significance: The increased biofilm formation by curli deficient E. coli O157:H7 with native microbiota, could indicate a strategy of environmental survival in conditions not conducive to curli expression. Curli expression but not pathogenicity seemed to play an important role in biofilm formation on produce wash water contact surfaces.