Sugar Modification of Wall Teichoic Acids Determines Serotype-Dependent Strong Biofilm Production in Listeria monocytogenes

Abstract

Biofilm production is responsible for persistent food contamination by Listeria monocytogenes, threatening food safety and public health. Human infection and food contamination with L. monocytogenes are caused primarily by serotypes 1/2a, 1/2b, and 4b. However, the association of biofilm production with phylogenic lineage and serotype has not yet been fully understood. In this study, we measured the levels of biofilm production in 98 clinical strains of L. monocytogenes at 37°C, 25°C, and 4°C. The phylogenetic clusters grouped by core genome multilocus sequence typing (cgMLST) exhibited association between biofilm production and phylogenetic lineage and serotype. Whereas clusters 1 and 3 consisting of serotype 4b strains exhibited weak biofilm production, clusters 2 (serotype 1/2b) and 4 (serotype 1/2a) were composed of strong biofilm formers. Particularly, cluster 2 (serotype 1/2b) strains exhibited the highest levels of biofilm production at 37°C, and the levels of biofilm production of cluster 4 (serotype 1/2a) strains were significantly elevated at all tested temperatures. Pan-genome analysis identified 22 genes unique to strong biofilm producers, most of which are related to the synthesis and modification of teichoic acids. Notably, a knockout mutation of the rml genes related to the modification of wall teichoic acids with l-rhamnose, which is specific to serogroup 1/2, significantly reduced the level of biofilm production by preventing biofilm maturation. Here, the results of our study show that biofilm production in L. monocytogenes is related to phylogeny and serotype and that the modification of wall teichoic acids with l-rhamnose is responsible for serotype-specific strong biofilm formation in L. monocytogenes. IMPORTANCE Biofilm formation on the surface of foods or food-processing facilities by L. monocytogenes is a serious food safety concern. Here, our data demonstrate that the level of biofilm production differs among serotypes 1/2a, 1/2b, and 4b depending on the temperature. Furthermore, sugar decoration of bacterial cell walls with l-rhamnose is responsible for strong biofilm production in serotypes 1/2a and 1/2b, commonly isolated from foods and listeriosis cases. The findings in this study improve our understanding of the association of biofilm production with phylogenetic lineage and serotype in L. monocytogenes.

Keywords: Listeria; biofilms; cell wall; rhamnosylation; serotype.

FIG 1

Phylogenetic association of 98 clinical isolates of L. monocytogenes with biofilm productivity. (A) A phylogenetic tree was generated using the cgMLST scheme based on 1,748 loci (27). The relative level of biofilm production was measured by comparing biofilm levels between the clinical isolates and a control strain (L. monocytogenes ATCC 19115). (B to D) The biofilm productivity of the four phylogenetic clusters at 37°C (B), 25°C (C), and 4°C (D). Statistical analysis was conducted by the one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison tests; ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

(A and B) Biofilm production of the CCs of 98 clinical isolates of L. monocytogenes at 37°C (A) and 4°C (B). The four phylogenetic clusters identified by cgMLST (Fig. 1A) are indicated in different colors. A solid black line indicates the mean; UT, untypeable.

FIG 3

Linearized pan-genomic view of 98 L. monocytogenes strains. The assembled genomes of 98 L. monocytogenes strains were annotated with Prokka v1.14.6 and used for a pan-genome analysis using Roary v3.11.2. The resulting presence and absence matrix of orthologous genes was visualized using FriPan. The red box and line indicate the location of the genes unique to strong biofilm formers listed in Table 1. The gene numbers are indicated on top.

FIG 4

Relative biofilm productivity of additional 73 L. monocytogenes strains. (A) Biofilm production of L. monocytogenes in association with the presence and absence of genes identified by the pan-genome analysis (Table 1). The relative level of biofilm production was measured by comparing biofilm levels between the isolates and a control strain (L. monocytogenes ATCC 19115). The filled and open squares represent the presence and absence of a gene, respectively. The numbers of the isolates beneath the figure correspond to those in Table S2 in the supplemental material. (B to D) The biofilm productivity of the four phylogenetic clusters at 37°C (B), 25°C (C), and 4°C (D). A solid black line indicates the mean. Statistical analysis was conducted with the one-way ANOVA with Tukey’s multiple-comparison tests; ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

Effects of l-rhamnosylation on biofilm formation in L. monocytogenes. (A) Defective biofilm production in ΔrmlD and ΔrmlTACBD mutants. Statistical analysis was conducted with the Student’s t test in comparison with wild-type (WT); ****, P < 0.0001; rmlD comp, a rmlD-complemented strain. The results are representative of three independent experiments, which produced similar results. (B) Compromised biofilm maturation in a ΔrmlD mutant. Fluorescence microscopic images show that a ΔrmlD mutant cannot produce mature biofilms compared to WT.