Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

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

Cellulose degradation and biofilm formation in the developmental life cycle of the cellulolytic actinomycete Thermobifida fusca

Actinomycetes have been used with enormous success in industrial processes; however, little is known about biofilm development by these filamentous microbes, and the presence of community development on insoluble cellulosic substrates such as cellulose. Cellulose is the most abundant biopolymer and renewable energy source on Earth, and its decomposition, which is carried out almost exclusively by microorganisms, is a key step in the cycling of carbon in the biosphere. It has long been known that cellulolytic bacteria may adhere to their insoluble substrate as it is degraded, although surprisingly little is known about microbial growth, colonization and community development on insoluble cellulosic substrates and non-nutritive surfaces. Previous investigations indicated that two Gram-positive cellulolytic soil bacteria, Cellulomonas uda, a facultative aerobe, and Clostridium phytofermentans , an obligate anaerobe, specifically adhered to nutritive surfaces forming a biofilm, but cells did not colonize non-nutritive surfaces. In this study is hypothesized that biofilm formation is a general strategy used by microbes in the degradation of insoluble substrates, and that it may serve as a means for microbes to secure a nutrient and persist in their environments. The objective of this study was to characterize biofilms produced by Thermobifida fusca, a Gram-positive cellulolytic actinomycete isolated from compost that rapidly degrades cellulose by means of a well-characterized extracellular cellulase system, and is a causative agent of Farmers Lung, the most common type of hypersensitivity pneumonitis. T. fusca was cultured with dialysis tubing as a nutritive surface for biofilm formation, and by using non-nutritive surfaces such as glass, plastic, metal and Teflon. Dialysis tubing was colonized by T. fusca aleuriospores but not by mycelial pellets. Surface-attached growth, examined by confocal scanning laser and scanning electron microscopy revealed structures resembling biofilms with cells embedded in fibrous material suggestive of an exopolymeric (EPS) matrix. T. fusca cells possessed higher hydrophobicity than C. uda and C. phytofermentans cells implicating higher capacity to bind to surfaces. DNase1 inhibited biofilm formation when assayed on microtiter plates suggesting a role for extracellular DNA in T. fusca biofilm formation. Concanavalin-A bound to the EPS material of biofilms and mycelial pellets, indicating alpha-linked D-mannosyl and/or alpha-linked D-glucosyl residues. The carbohydrate content of biofilms and mycelial pellets increased during growth. T. fusca biofilm formation is reduced when lack or excess of nutrients such as; iron, nitrogen and salt. Robust biofilms were developed between pHs 7 and 9, whereas minimum biofilms were produced at pH 3 and 11. Cellulose degradation rate and celE (endoglucanase E5) expression was similar for T. fusca biofilms and mycelial pellets. Also, results of this study indicate that in the life cycle of this actinomycete, cellulose is specifically colonized by aleuriospores, which germinate and degrade cellulose, ultimately developing into biofilms encased in a carbohydrate-containing EPS matrix, a hallmark of biofilm production

Hydrophobic nature and effects of culture conditions on biofilm formation by the cellulolytic actinomycete <em>Thermobifida fusca</em>

Thermobifida fusca produces a firmly attached biofilm on nutritive and non-nutritive surfaces, such as cellulose, glass, plastic, metal and Teflon&reg;. The ability to bind to surfaces has been suggested as a competitive advantage for microbes in soil environments. Results of previous investigations indicated that a Gram-positive cellulolytic soil bacteria, Cellulomonas uda, a facultative aerobe, specifically adhered to nutritive surfaces forming biofilms, but cells did not colonize non-nutritive surfaces. Cell surface hydrophobicity has been implicated in the interactions between bacteria and the adhesion to surfaces. It was recently described that the cellulolytic actinomycete T. fusca cells hydrophobicity was measured and compared to the cellulolytic soil bacteria C. uda. Also, T. fusca biofilm formation on non-nutritive surface, such as polyvinyl chloride, was examined by testing various culture ingredients to determine a possible trigger mechanism for biofilm formation. Experimental results showed that partitioning of bacterial cells to various hydrocarbons was higher in T. fusca cells than in C. uda. The results of this study suggest that the attachment to multiple surfaces by T. fusca could depend on nutrient availability, pH, salt concentrations, and the higher hydrophobic nature of bacterial cells. Possibly, these characteristics may confer T. fusca a selective advantage to compete and survive among the many environments it thrives

CSLM analysis of <i>L. monocytogenes</i> biofilm production.

<p>Results presented are the means ±SD from two independent experiments performed in triplicate.</p><p>Student's <i>t-test</i> indicated a statistically significant difference between biofilm thickness formed by <i>L. monocytogenes</i> 10403S compared to mutant bacterial strains (p ≤ 0.05).</p><p>CSLM analysis of <i>L. monocytogenes</i> biofilm production.</p

Identified <i>L. monocytogenes</i> biofilm-formation genes.

<p><i>Putative functions were obtained from <a href="http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html</a></i>.</p><p><i>Based on DNA homologies with the L. monocytogenes 10403S genome database; lmrg refers to genetic loci within strain 10403S</i>.</p><p><i>% Compared to wild-type L. monocytogenes 10403S biofilm formation in two independent experiments</i>.</p><p>Identified <i>L. monocytogenes</i> biofilm-formation genes.</p

Transmission and scanning electron microscopy analysis of <i>L. monocytogenes</i> EPS production.

<p><i>L. monocytogenes</i> 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 <i>L. monocytogenes</i> 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.</p

Biofilm formation by Δ<i>phoPR</i> and Δ<i>dltABCD L. monocytogenes</i>.

<p>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).</p

Scanning electron microscopy of a bean sprout inoculated with <i>L. monocytogenes</i>.

<p>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 <i>L. monocytogenes</i> (bar = 10 µm) (D) magnification of (C) (bar = 10 µm).</p