Identification of pathways required for
Salmonella
to colonize alfalfa using TraDIS-
Xpress
Authors: Holden Emma R. , Abi Assaf Justin , Al-Khanaq Haider , Vimont Noemie , Webber Mark A. , Trampari Eleftheria 
Published: June 2024

Enteropathogenic bacteria, such as
Salmonella
, have been linked to numerous fresh produce outbreaks, posing a significant public health threat. The ability of
Salmonella
to persist on fresh produce for extended periods is partly attributed to its capacity to form biofilms, which pose a challenge to food decontamination and can increase pathogenic bacterial load in the food chain. Preventing
Salmonella
colonization of food products and food processing environments is crucial for reducing the incidence of foodborne outbreaks. Understanding the mechanisms of establishment on fresh produce will inform the development of decontamination approaches. We used Transposon-Directed Insertion site Sequencing (TraDIS-
Xpress
) to investigate the mechanisms used by
Salmonella enterica
serovar Typhimurium to colonize and establish on fresh produce over time. We established an alfalfa colonization model and compared the findings to those obtained from glass surfaces. Our research identified distinct mechanisms required for
Salmonella
establishment on alfalfa compared with glass surfaces over time. These include the type III secretion system (
sirC
), Fe-S cluster assembly (
iscA
), curcumin degradation (
curA
), and copper tolerance (
cueR
). Shared pathways across surfaces included NADH hydrogenase synthesis (
nuoA
and
nuoB),
fimbrial regulation (
fimA
and
fimZ
), stress response (
rpoS),
LPS O-antigen synthesis (
rfbJ),
iron acquisition (
ybaN
), and ethanolamine utilization (
eutT
and
eutQ
). Notably, flagellum biosynthesis differentially impacted the colonization of biotic and abiotic environments over time. Understanding the genetic underpinnings of
Salmonella
establishment on both biotic and abiotic surfaces over time offers valuable insights that can inform the development of targeted antibacterial therapeutics, ultimately enhancing food safety throughout the food processing chain.

IMPORTANCE

Salmonella
is the second most costly foodborne illness in the United Kingdom, accounting for £0.2 billion annually, with numerous outbreaks linked to fresh produce, such as leafy greens, cucumbers, tomatoes, and alfalfa sprouts. The ability of
Salmonella
to colonize and establish itself in fresh produce poses a significant challenge, hindering decontamination efforts and increasing the risk of illness. Understanding the key mechanisms of
Salmonella
to colonize plants over time is key to finding new ways to prevent and control contamination of fresh produce. This study identified genes and pathways important for
Salmonella
colonization of alfalfa and compared those with colonization of glass using a genome-wide screen. Genes with roles in flagellum biosynthesis, lipopolysaccharide production, and stringent response regulation varied in their significance between plants and glass. This work deepens our understanding of the requirements for plant colonization by
Salmonella
, revealing how gene essentiality changes over time and in different environments. This knowledge is key to developing effective strategies to reduce the risk of foodborne disease.