Small-Molecule Modulators of Listeria monocytogenes Biofilm Development

Abstract

Listeria monocytogenes is an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay for L. monocytogenes and, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains of Listeria at micromolar concentrations, as well as molecules that decreased (<50% of vehicle control) or increased (>200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules-including the protein kinase C antagonist sphingosine-had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-D,L-carnitine showed that while Listeria biofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ؎ 0.24 M, D,L-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C 9:0 and C 14:0 were Listeria biofilm inhibitors, whereas fatty acids of C 16:0 or longer were stimulators, showing chain length specificity. De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes of L. monocytogenes biology and promising leads for the further development of antibiofilm strategies.

Carnitine in bacterial physiology and metabolism

“…1), which can serve as sources of carnitine, but also can alter bacterial physiology directly (Nguyen et al, 2012). Pseudomonas aeruginosa can utilize acylcarnitines with 2-16 fatty acid chain lengths as sole carbon, nitrogen and energy sources, with the exception of octanoylcarnitine, although the reason for this utilization gap is unknown (Meadows & Wargo, 2013).…”

Role of PBPD1 in Stimulation of Listeria monocytogenes Biofilm Formation by Subminimal Inhibitory β-Lactam Concentrations

“…Determination of the MICs for L. monocytogenes strains was performed as previously described (29). L. monocytogenes does not grow in Mueller-Hinton Broth (MHB), the standard medium for MIC determination, according to Clinical and Laboratory Standards Institute guidelines; it is considered a fastidious bacterium requiring blood or blood products and modified atmospheric conditions such as 5% CO 2 (CLSI guideline M45-A2) (34).…”

“…monocytogenes biofilm formation and dispersal assays. Biofilm assays were performed as previously described (29). Briefly, in the biofilm formation assay, overnight cultures of L. monocytogenes were diluted to an OD 600 of ϳ0.03 in TSB.…”

“…Following the wash step, the biofilms on the pegs were transferred to a 96-well plate that contained 148.5 l TSB plus 1.5 l ␤-lactams in DMSO (at the indicated concentrations) or DMSO alone (vehicle control) in each well and then incubated at 37°C for 24 h, at 200 rpm. The amount of biofilm that formed (or remained) on the pegs was quantified using 0.1% (wt/vol) crystal violet (CV), and the absorbance of the eluted CV was read at 600 nm using a plate reader as previously described (29). The planktonic cell optical density and CV absorbance graphs were generated using GraphPad Prism 5 (GraphPad Software, Inc.).…”

“…Here we used a previously developed L. monocytogenes biofilm assay (29) to screen a library of 1,120 previously FDA-approved, off-patent drugs, in order to identify molecules that modulate L. monocytogenes biofilm formation, prevent biofilm development, and/or disperse established biofilms. Since many of these drugs have known modes of action, they represent useful probes for identifying targets that affect biofilm formation.…”