Biofilm Formation by Listeria monocytogenes 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups

Abstract

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants.IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.

Keywords: Listeria monocytogenes; biofilms; food safety; mutagenesis.

FIG 1

Comparison of biofilm formation by L. monocytogenes 15G01 (wt), the uvrB and mltD mutants (33E11 and 39G5), and mutants containing a wild-type copy of the corresponding gene (complemented strains [-C]) or the empty vector pIMK (-EV). Error bars represent the SD for three independent experiments (n = 6). Biofilm formation was determined by measuring the OD₅₉₅ as part of the CV assay.

FIG 2

Images of the biofilms produced by L. monocytogenes 15G01 (wild type) (a) and selected transposon mutants with altered biofilm formation (34F11 [b], 33E11 [c], 39G5 [d], and 44D3 [e]) grown on polystyrene surfaces in MWB for 48 h at 30°C. The biofilms were stained with a Live/Dead BacLight bacterial viability kit according to the manufacturer’s instructions (Life Technologies, Thermo Fisher, New Zealand). Living cells were labeled with SYTO9 (green) and dead cells with propidium iodide (red). Scale bars, 20 μm. Mutants: 34F11, clsA mutant; 33E11, uvrB mutant; 39G5, mltD mutant; 44D3, mgtB mutant.

FIG 3

Images of the coupon coated with mussel juice (k and l) and the biofilms produced by L. monocytogenes 15G01 (wild type) (a and b) and selected transposon mutants with altered biofilm formation (34F11, clsA mutant [c and d]; 33E11, uvrB mutant [e and f]; 39G5, mltD mutant [g and h]; 44D3, mgtB mutant [i and j]) grown on stainless steel coupons coated with mussel juice for 7 days at 30°C. The images were obtained with a scanning electron microscope at 5,000× and 10,000× magnifications. The cracks are features of the stainless-steel surface. The white arrows point at coccoid-shaped bacteria (f) and two different types of biofilm (i).

FIG 4

Biofilm formation by the wild type and selected mutants in MWB (light gray bars) and MWB with a Mg²⁺ concentration of 5 mM (dark gray bars) at 30°C (a) and 37°C (b) after 48 h of incubation measured with a CV assay. Error bars represent the SD of three independent experiments (n = 6). Letters in common indicate no significant difference. (c to h) Isosurface images of biofilms of wild-type (c), 39G5 (e), and 44D3 (g) strains formed on glass after 7 days of incubation in MWB (1.67 mM Mg²⁺) and in MWB with a final Mg²⁺ concentration of 5 mM (wild type [d], 39G5 [f], and 44D3 [h]). Biofilms were grown at 30°C (wild type, 44D3) or 37°C (39G5) and stained with SYTO9. Orthogonal view of biofilms formed on a glass surface after 7 days at 30°C by the wild type in MWB (i), by the mgtB mutant (44D3) in MWB (j), and by the mgtB mutant (44D3) in the presence of 5 mM Mg²⁺ (k). Images were taken after removal of media and staining with a Live/Dead BacLight kit with a confocal laser scanning microscope.

FIG 5

(a) Triton X-100-induced autolysis of L. monocytogenes 15G01 (wt) and the mltD mutant (39G5) determined by optical density measurement at 595 nm and (b) motility of L. monocytogenes 15G01 (top left), the mltD mutant (39G5 [top right]), and the mltD mutant containing a wild-type copy of the corresponding gene (39G5-C [bottom left]) or the empty vector pIMK (39G5-EV [bottom right]) after 24 h at 30°C.