Sources and survival of listeria monocytogenes on fresh, leafy produce

Abstract

Listeria monocytogenes is an intracellular bacterial pathogen that has been responsible for many severe disease outbreaks resulting in multiple fatalities. L. monocytogenes enters the body through contaminated food and is known to contaminate fresh leafy produce such as spinach and lettuce. In the UK there are many stakeholders in the fresh leafy produce supply chain (FLPSC) including growers, processors and retailers, making the FLPSC complex and the management of bacterial contamination in the supply chain difficult. L. monocytogenes is sporadically detected in the FLPSC by routine testing, but currently, little is known about the strains of L. monocytogenes present in the UK FLPSC, their potential pathogenicity and phenotypic characteristics. Furthermore, data on L. monocytogenes survival in environments associated with the FLPSC, like horticultural soils and the leaf surface, is scarce and requires further investigation. Due to the potential risk of the bacterium, source tracking, risk assessment and understanding the ability of L. monocytogenes to survive in the FLPSC should be considered key factors in tackling L. monocytogenes contamination of fresh leafy produce and reducing risk to the consumer.
Given the importance of these factors, 15 L. monocytogenes strains isolated from the UK FLPSC were characterised using a range of genotypic (MLST and WGS) and phenotypic (including biofilm formation and rates of swarming motility) methods. WGS revealed a genetically diverse population spanning two lineages but showed some indistinguishable pairs of isolates, suggesting cross contamination may have occurred in the supply chain. Following characterisation, selected isolates (from lineages I and II) were examined for their ability to survive in environments associated with the FLPSC including horticultural soils and spinach leaves. Results showed no differences in soil survival between strains, but strains were detected at a higher level (in sterilised soil) for longer in a clay loam soil compared to other types of soil in the short term (over 70 days). In contrast, L. monocytogenes was detected at higher levels in sandy soil using an illumina based NGS method in the long term. Washing contaminated spinach in chlorine water at operational levels (60ppm) was found to reduce L. monocytogenes number by 1log but electrolysed water (a chlorine alternative) showed lower efficacy at operational free levels of free chlorine, suggesting electrolysed water is not a viable alternative to chlorine wash at these levels. Survival on spinach leaves was influenced by temperature and whether the leaves were subjected to a chlorine wash, or not. L. monocytogenes populations grew to a higher level on contaminated spinach at room temperature vs. when contaminated leaves were refrigerated. This demonstrated the importance of maintaining refrigeration temperatures throughout the supply chain. At both room and refrigeration temperatures, washing spinach leaves in a chlorine wash meant that L. monocytogenes populations grew to a higher level over the shelf life of the spinach. In addition, the endogenous microflora present on ready to eat (washed) spinach was less diverse and abundant than spinach which hadn’t been washed.
Overall, the results of this work suggest that WGS technology should be phased into L. monocytogenes surveillance programmes in the FLPSC for purposes of source tracking and risk assessment so incidences of L. monocytogenes contamination can be controlled or reduced e.g. by cleaning and sanitation of affected areas. Furthermore, implementing this technology may give customers (retail) and consumers added confidence that growers and processors are informed of the L. monocytogenes risk in their supply chain and demonstrates a precautionary, rather than reactionary approach to consumer safety. Results from soil survival experiments suggest that L. monocytogenes found in the FLPSC can survive for extended periods in horticultural soils and thus, it was concluded that soil is a potential source of contamination in the FLPSC. However, the level of L. monocytogenes in horticultural soil declines quickly and transfer of the bacteria to soil on a large scale (in the field for example) is unlikely, suggesting that this risk of contamination from this source is low. Furthermore, sporadic contamination from this source is hard to prevent due to the intimate nature of the growing environment (soil) with fresh produce products. Results from the final chapter of this work show the chlorine wash at operation levels for the UK FLPSC is an effective sanitiser with regards to L. monocytogenes in a pre-wash contamination scenario. On the other hand, it was shown that chlorine wash may enable L. monocytogenes populations to reach a higher level on spinach leaves when the leaves have been contaminated post-wash by reducing the abundance and diversity of endogenous leaf microflora.