Environmental monitoring and equipment sanitation to mitigate <i>Listeria</i> risks in a soil-based controlled environment agriculture system

Controlled environment agriculture (CEA) offers protection from external contaminants but introduces unique food safety challenges, particularly related to equipment sanitation. This study evaluated the occurrence and persistence of <i>Listeria monocytogenes</i> and related species in a commercial soil-based CEA facility through a 1-year environmental monitoring program (EMP). A total of 169 samples, including baby leaves, soil, water, and swabs from equipment surfaces, were collected across three visits. <i>L. monocytogenes</i> was detected in only one sample (0.59% prevalence), whereas <i>Listeria innocua</i> was isolated from harvesting crates and structural surfaces (3/169). Molecular confirmation and characterization were conducted using PCR (<i>hly, iap, sigB</i>), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and whole genome sequencing (WGS). To assess the potential for cross-contamination, <i>L. innocua</i> isolates were used for inoculation studies on reusable plastic crates. Viable cells survived up to 24 h post-inoculation despite water rinsing under 25°C and 60%-70% relative humidity. These findings suggest a low risk of <i>L. monocytogenes</i> contamination in this soil-based CEA system but underscore the importance of targeted sanitation, particularly for harvesting equipment, to prevent harborage and transfer of <i>Listeria</i> spp.IMPORTANCEControlled environment agriculture (CEA) is a rapidly expanding approach to producing fresh food year-round with greater resource efficiency. However, it presents unique challenges for managing foodborne pathogens. This study demonstrates that in this soil-based indoor system, contamination risks persist, particularly via inadequately cleaned harvesting equipment. The boot cover finding is interpreted as an indicator of environmental contamination, but it does not constitute evidence of footwear-to-crop transfer risk. Although <i>Listeria monocytogenes</i> was rarely detected, related species like <i>Listeria innocua</i> persisted on reusable plastic crates and structural surfaces, highlighting weaknesses in current sanitation protocols. The use of boot covers as environmental monitoring tools proved valuable for detecting contamination sources. These findings underscore the need for tailored sanitation strategies and comprehensive environmental monitoring programs to enhance food safety in CEA systems and prevent pathogen harborage and spread.