Evolution of Listeria monocytogenes in a Food Processing Plant Involves Limited Single-Nucleotide Substitutions but Considerable Diversification by Gain and Loss of Prophages

Abstract

Whole-genome sequencing (WGS) is becoming the standard method for subtyping Listeria monocytogenes. Interpretation of WGS data for isolates from foods and associated environments is, however, challenging due to a lack of detailed data on Listeria evolution in processing facilities. Here, we used previously collected WGS data for 40 L. monocytogenes isolates obtained from a cold-smoked salmon processing facility between 1998 and 2015 to probe the L. monocytogenes molecular evolution in this facility, combined with phenotypic assessment of selected isolates. Isolates represented three clusters (1, 2, and 3); cluster 3 isolates (n = 32) were obtained over 18 years. The average mutation rate for cluster 3 was estimated as 1.15 × 10−7 changes per nucleotide per year (∼0.35 changes per genome per year); the most recent common ancestors (MRCAs) of subclusters 3a and 3b were estimated to have occurred around 1958 and 1974, respectively, within the age of the facility, suggesting long-term persistence in this facility. Extensive prophage diversity was observed within subclusters 3a and 3b, which have one shared and six unique prophage profiles for each subcluster (with 16 prophage profiles found among all 40 isolates). The plasmid-borne sanitizer tolerance operon bcrABC was found in all cluster 2 and 3 isolates, while the transposon-borne sanitizer tolerance gene qacH was found in one cluster 1 isolate; presence of these genes was correlated with the ability to survive increased concentrations of sanitizers. Selected isolates showed significant variation in the ability to attach to surfaces, with persistent isolates attaching better than transient isolates at 21°C. IMPORTANCE Knowledge about the genetic evolution of L. monocytogenes in food processing facilities over multiple years is generally lacking. This information is critical to interpret WGS findings involving food or food-associated isolates. This study suggests that L. monocytogenes that persists in processing facilities may evolve with a low single-nucleotide mutation rate mostly driven by negative (i.e., purifying) selection but with rapid diversification of prophages. Hence, isolation of L. monocytogenes with few single-nucleotide polymorphism (SNP) differences in different locations (e.g., supplier plants and receiving plants) is possible, highlighting the importance of epidemiological and detailed isolate metadata for interpreting WGS data in traceback investigation. Our study also shows how advanced WGS data analyses can be used to support root cause analysis efforts and may, for example, pinpoint the time when a persistence event started (which then potentially could be linked to facility changes, introduction of new equipment, etc.).

Dynamics of mobile genetic elements of Listeria monocytogenes persisting in ready-to-eat seafood processing plants in France

“…Our results show that CCs recurring in plants A, B and C represented persisting and even "truly persisting" plant-specific clones including long-term persistent strains with less than 25 pairwise SNPs differences. As already observed in previous studies [18,78,79], the repeated detection of the genetically close or even indistinguishable strains from different foods and FPEs during the whole sampling period (2-6 years) bolstered the intra-plant clonality of L. monocytogenes strains included in the present study. The only exceptions were single strains for CC155 (DSS836-CS1-LmUB3PA), CC101 (C1530-O-LmUB3PA) and CC121 (CS461-S1-LmUB3PA), considered as transient and likely reintroduced in the FPEs due to the high genetic differences from the persisting clones.…”

“…Plasmids encoding genes conferring increased tolerance under multiple stress conditions related to food and FPEs were identified in the analysed L. monocytogenes from CC121, CC7, CC155 and CC204. These plasmids showed high similarity with globally spread publicly available plasmids harboured by persistent L. monocytogenes strains from ST121, ST5 and ST204 isolated in food products and related FPEs [17,21,22,79]. Experimental evidence that such plasmids from L. monocytogenes ST121 and ST5 contribute to cells survival under stress condition met in FPEs have been recently proved [28].…”

“…We tested this hypothesis focusing on the core and accessory genomes of L. monocytogenes subtypes repeatedly collected over 2-6 years from food and food-related environments of three closely located plants producing RTE seafood in France. Persistent strains and newly introduced strains in the FPEs by raw material have been described in relation to specific STs or CCs [18,20,78,79]. With a similar objective, some authors [18,78,80] proposed arbitrary thresholds ranging from 4 to less than 10 or 20 until 25 SNPs to identify persisting and "truly persisting" clones of L. monocytogenes.…”

“…Adaptation to specific environmental niches in FPEs and short-term evolution of both distantly and closely related L. monocytogenes strains have been linked to the diversification of prophages [50,79,81,82]. ST-specific prophages showing a high degree of conservation in FPEs have been described for strains from ST121 and ST8, while prophages from strains belonging to ST204 presented a lower degree of conservation [16,17,22].…”

Characterization of Mobile Genetic Elements Using Long-Read Sequencing for Tracking Listeria monocytogenes from Food Processing Environments

“…For example, the average substitution rate per year for 1696 Lm strains from both Lineages I and II is known to be about 2.6 × 10 −7 (95% HPD, 1.9×10 –7 to 3.4×10 –7 ) substitutions/site/year [ 15 ], which was in the same range as our analysis. The mutation rate on 33 CC321 strains from lineage II was 1.15 × 10 −7 (95% HPD, 0.79 × 10 −7 to 1.52 × 10 −7 ) substitutions/site/year [ 34 ]. In another study that calculated substitution rates among isolates associated with specific outbreaks, the average substitution rates were slightly higher, 5.5 × 10 −7 to 5.8 × 10 −7 substitutions/site/year for CC6 isolates associated with two different outbreaks linked to contaminated cheese products [ 9 ].…”