# The microbiota of wooden cheese-ripening boards is a rich source of antimicrobial-producing bacteria against Listeria monocytogenes

## TL;DR

Wooden cheese boards host bacteria that produce antimicrobials, which can inhibit Listeria monocytogenes, a dangerous foodborne pathogen.

## Contribution

Identification of Bacillus safensis and other antimicrobial-producing bacteria from wooden cheese boards as potential biocontrol agents.

## Key findings

- L. monocytogenes declines on clean wooden board surfaces but can grow on cheese-covered areas.
- Seven bacterial species, including Bacillus safensis, inhibit L. monocytogenes through antimicrobial factors.
- B. safensis supernatant downregulates prophage elements and upregulates stress responses in L. monocytogenes.

## Abstract

Wooden boards are essential tools in cheese ripening, and there is accumulating evidence that they have antimicrobial effects against foodborne bacterial pathogens, such as Listeria monocytogenes. However, poor bacterial recovery of bacteria from wood can confound the quantification of pathogen burden. To assess L. monocytogenes survival on wooden cheese boards, we applied a disruptive grinding method and tracked native board-associated bacterial counts as controls. Our data revealed that L. monocytogenes declines on clean zones of wooden boards but can replicate on areas where there is suitable cheese. Our microbiota analysis revealed diverse bacterial communities on wooden board surfaces, with a prominent presence of bacteria in the genera Brevibacterium, Brachybacterium, and Staphylococcus. We further identified seven bacterial species that inhibit L. monocytogenes, belonging to Bacillus, Staphylococcus, and Serratia phyla, in addition to Lactococcus lactis. One isolate, Bacillus safensis, was found to be a potential biocontrol agent, potently inhibiting L. monocytogenes via secreted antimicrobial factors. Our genomic, bioinformatic, and biochemical analyses indicate that those factors are likely antimicrobial peptides encoded by multiple biosynthetic gene clusters, many of which have not been characterized. A sub-inhibitory concentration of B. safensis supernatant induces a significant downregulation of prophage elements and an upregulation of antimicrobial stress responses in L. monocytogenes. Taken together, our findings indicate that the wooden board microbiota is a rich source of antimicrobial-producing bacteria with potential applications in foodborne pathogen control strategies.

Despite stringent food safety measures, L. monocytogenes foodborne outbreaks remain frequent with high hospitalization and mortality rates. Removal of L. monocytogenes from food processing environments is extremely challenging because this pathogen is ubiquitous and encodes a wide array of stress response mechanisms that enable it to thrive under harsh conditions. Our study found that clean wooden boards used in cheese ripening inhibit L. monocytogenes, causing a noticeable decline in pathogen population following surface inoculation. Bacterial communities on wooden cheese boards are rich and diverse and harbor many species that produce antimicrobial compounds against L. monocytogenes, with the example of a new B. safensis isolate. Therefore, the wooden cheese-ripening board microbiota is a promising source for future antimicrobial discovery efforts.