Multi-species biofilms of environmental microbiota isolated from fruit packing facilities promoted tolerance of Listeria monocytogenes to benzalkonium chloride

Abstract

Listeria monocytogenes may survive and persist in food processing environments due to formation of complex multi-species biofilms of environmental microbiota that co-exists in these environments. This study aimed to determine the effect of selected environmental microbiota on biofilm formation and tolerance of L. monocytogenes to benzalkonium chloride in formed biofilms. The studied microbiota included bacterial families previously shown to co-occur with L. monocytogenes in tree fruit packing facilities, including Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae. Biofilm formation ability and the effect of formed biofilms on the tolerance of L. monocytogenes to benzalkonium chloride was measured in single- and multi-family assemblages. Biofilms were grown statically on polystyrene pegs submerged in a R2A broth. Biofilm formation was quantified using a crystal violet assay, spread-plating, confocal laser scanning microscopy, and its composition was assessed using amplicon sequencing. The concentration of L. monocytogenes in biofilms was determined using the most probable number method. Biofilms were exposed to the sanitizer benzalkonium chloride, and the death kinetics of L. monocytogenes were quantified using a most probable number method. A total of 8, 8, 6, and 3 strains of Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae, respectively, were isolated from the environmental microbiota of tree fruit packing facilities and were used in this study. Biofilms formed by Pseudomonadaceae, Xanthomonadaceae, and all multi-family assemblages had significantly higher concentration of bacteria, as well as L. monocytogenes, compared to biofilms formed by L. monocytogenes alone. Furthermore, multi-family assemblage biofilms increased the tolerance of L. monocytogenes to benzalkonium chloride compared to L. monocytogenes mono-species biofilms and planktonic multi-family assemblages. These findings suggest that L. monocytogenes control strategies should focus not only on assessing the efficacy of sanitizers against L. monocytogenes, but also against biofilm-forming microorganisms that reside in the food processing built environment, such as Pseudomonadaceae or Xanthomonadaceae.

Keywords: Antimicrobial tolerance; Benzalkonium chloride; Biofilms; Environmental microbiomes; Flavobacteriaceae; Listeria monocytogenes; Microbacteriaceae; Microbiota; Pseudomonadaceae; Sanitizers; Xanthomonadaceae.

Fig. 1

Phenotypic and genotypic resistance to benzalkonium chloride of L. monocytogenes and environmental isolates from tree fruit packing facilities. (a) Bar plots show the minimal inhibitory concentration (MIC) to benzalkonium chloride (BAC), determined using the broth microdilution assay at 30°C. Each panel and distinct colors represent different taxonomic family of bacterial isolates. (b) Heatmap shows the presence (dark green) and absence (light green) of known genes associated with resistance to BAC. Isolates are grouped by taxonomic family: F, Flavobacteriaceae; L, L. monocytogenes; M, Microbacteriaceae; P, Pseudomonadaceae; X, Xanthomonadaceae. Genes are grouped by function. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Biofilm formation by single- and multi-family assemblages of environmental isolates and L. monocytogenes. (a) Biofilm formation after incubation of single- and multi-family assemblages in Minimal Biofilm Eradication Concentration (MBEC) devices in Reasoner's 2 A (R2A) broth for 3 days at 15 °C with daily replacement of media, as determined by the crystal violet assay. Biofilms with an absorbance above 1 were diluted and the final absorbance was calculated using the dilution factor. (b) Total concentration of bacteria in biofilms, as determined after detachment and spread plating onto R2A. (c) Change of L.monocytogenes concentration in single- and multi-family assemblage biofilms compared to the initial concentration of L. monocytogenes. (d) Relative abundance of bacteria in biofilms determined by viability amplicon sequencing. Bars are color coded by taxonomic genus of each Amplicon Sequence Variant (ASV) and, when possible, the ASV number was added to the bar. (e) Representative CLSM projections of single- and multi-family assemblage biofilms grown in R2A broth at 15 °C for 3 days in black microtiter plates with an optical grade base are shown. Biofilms were stained with LIVE/DEAD fluorescent stain; live cells shown in green (SYTO 9) and dead cells shown in magenta (propidium iodide). For each image, nine z-stacks were taken with 1 μm between each image and composed 3D projections of the biofilm structure were produced using ImageJ software. Each image represents an 84 × 84 μm square. In panels a, b, and c, error bars represent the mean ± standard error of the mean. Different lowercase letters show significant differences (α = 0.05) between microbial assemblages, as determined using one-way ANOVA followed by Tukey's Honest Difference post hoc test for panels a, c, and d; or the Kruskal-Wallis tests followed by Dunn's test for panel b. Labels for microbial assemblage treatments were as follows: F, Flavobacteriaceae; L, L. monocytogenes; M, Microbacteriaceae; P, Pseudomonadaceae; X, Xanthomonadaceae. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Changes in microbial composition of multi-family assemblages during exposure to benzalkonium chloride. Reduction in total aerobic mesophilic bacteria (a) and L. monocytogenes (b) concentration in biofilms (circles with full lines) and planktonic cultures (triangles with dashed lines) exposed to 12.5 ppm of benzalkonium chloride (BAC). Data points represent the mean ± standard error of three independent biological replicates. Each microbial assemblage is color coded and presented in a separate panel. In each panel, different uppercase and lowercase letters indicate statistical differences (α = 0.05) in the bacterial concentration of planktonic and biofilm assemblages, respectively, as determined using one-way ANOVA followed by post-hoc Tukey Honest Significant Different test. Dashed grey line represents the limit of quantification of the aerobic plate count method (0.6 log₁₀ CFU/peg or ml) and of the most probable number method (0.9 log₁₀ MPN/peg or ml). Labels of microbial assemblage treatments: F, Flavobacteriaceae; L, L. monocytogenes; M, Microbacteriaceae; P, Pseudomonadaceae; X, Xanthomonadaceae. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)