Identification of Listeria monocytogenes determinants required for biofilm formation

Abstract

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.

Figure 1. Scanning electron microscopy of a bean sprout inoculated with L. monocytogenes.

Sterile bean sprouts were placed in HTM agar media with 3% glucose and inoculated with 10 µl of a 1:10 dilution of a 24-hour culture of 10403S. Following a 24 hour incubation, bean sprouts were processed for scanning electron microscopy (A) Bean sprout (bar = 1 mm) (B) magnified view of the white square from (A) (bar = 100 µm). (C) Bean sprout vegetative tissue colonized with L. monocytogenes (bar = 10 µm) (D) magnification of (C) (bar = 10 µm).

Figure 2. Transmission and scanning electron microscopy analysis of L. monocytogenes EPS production.

L. monocytogenes 10403S bacteria in biofilms formed on dialysis tubing membranes (regenerated cellulose) (A) (bar = 100 nm) or planktonic bacteria grown in broth culture (B) (bar = 500 nm) were examined by TEM at 72 hours post-inoculation. (C) SEM of a L. monocytogenes biofilm developed on regenerated cellulose at 24 hours post-inoculation (bar = 10 µm). Arrows indicate EPS. For TEM, samples were fixed with 25% glutaraldehyde, rinsed with cacodylate buffer and stained with ruthenium red to visualize EPS material. For SEM, samples were rinsed with multiple dilutions of ethanol prior to visualization.

Figure 3. Biofilm formation by ΔphoPR and ΔdltABCD L. monocytogenes.

Bacterial strains were inoculated into TSBYE media in 96-well plates and grown at 35°C for 24 hours. Cultures were then diluted 1:10 into fresh HTM media with 3% glucose and 0.1 mg/mL each cysteine and methionine in new 96-well PVC microtiter plates. Plates were incubated at 35°C for 96 hours, rinsed with dH₂O using a semi-automated cell washer, stained with crystal violet, rinsed with acetic acid and the OD₅₉₅ ±SD determined using a spectrophotometer. The data presented are representative of three independent experiments. *, p <0.05 (One-way ANOVA test).