2.1 Lineage groups and serotypes

Serotyping is a first-level subtyping method used for epidemiological purposes and was the first L. monocytogenes subtyping scheme. It is a typing method based on the presence of somatic (O) and flagellar (H) antigens (Allerberger, 2003; Capita et al., 2019; Hyden et al., 2016). Fifteen possible O antigens and four possible H antigens occur, and the serogroups are assigned based on the unique combination. L. monocytogenes was until recently classified into 13 serotypes (Allerberger, 2003; Jami et al., 2014; Jamshidi & Zeinali, 2019). Recent studies employed methods to rapidly characterize a novel serotype 4h (hybrid sublineage II) displaying hypervirulent properties (Feng et al., 2020). Different serotypes are associated with different environments or ability to cause disease (Feng et al., 2020; Jamshidi & Zeinali, 2019).

L. monocytogenes is further categorized into four genetic lineages (I–IV), with each lineage comprised of distinct serotypes (Abdollahzadeh, Ojagh, Hosseini, Irajian, et al., 2016; Matle et al., 2020). The majority of L. monocytogenes isolates fall into lineages I and II (Orsi et al., 2011). Lineage I show a preference for human and animal tissue and cell types, while lineage II is more adaptive to environmental conditions (Kaszoni-Rückerl et al., 2020). Human listeriosis cases are mostly linked to serotypes 4b, 1/2b and 1/2a (Alvarez-Molina et al., 2021; Batt, 2014; Hyden et al., 2016; Jami et al., 2014). Additionally, serotype 1/2a is mostly isolated from food (Basha et al., 2019; Matle et al., 2020; Orsi et al., 2011). Serotype 4b is more frequently isolated from clinical cases than serotypes 1/2a and 1/2c relative to its frequency in food and the food processing environment (Maury et al., 2016). Food items are typically linked with L. monocytogenes characterized by reduced pathogenicity and virulence (Maury et al., 2016). A reason that some serotypes are more associated with clinical cases is their virulence and ability to adapt to the human host (Matle et al., 2020). The antigenic group 1/2 (1/2a, 1/2b and 1/2c) has been overrepresented in food isolates and is also increasing in human clinical cases (Wagner et al., 2022). Table 1 illustrates the distribution of L. monocytogenes lineages and serotypes in seafood and its food processing environment, reported globally.

In a South African study, lineage II was the most prevalent (p < 0.01) among isolates from seafood and its processing environment (n = 38) (Lambrechts et al., 2024). A similar trend of lineage II in seafood and its processing environment has been reported by researchers globally (Iran, India, Poland, France, and Norway) (Abdollahzadeh, Ojagh, Hosseini, Ghaemi, et al., 2016; Fagerlund, Idland et al., 2022; Maury et al., 2019; Wieczorek & Osek, 2017; Wieczorek et al., 2020). The opposite was also reported with a majority of lineage I isolates found in seafood and its processing environment from Finland, Korea and South Africa (Table 1) (Keet & Rip, 2021; Lee et al., 2017; Miettinen & Wirtanen, 2005). The study conducted in the Western Cape, South Africa, on locally sourced fish (raw hake) categorized isolates only into lineage groups (Keet & Rip, 2021). It found that lineage I comprised a significantly higher proportion of the raw hake samples (95%) compared to lineage II. However, these results were derived from multiple sampling occasions at a single factory.

From the literature, the predominant serotype isolated in seafood and its processing environment is serotype 1/2a followed by serotypes 4b, 1/2b and 1/2c (Table 1). Seafood contaminated with L. monocytogenes lineage I, serotype 4b, originated from various types of fish, with many associated with RTE fish products in countries such as China, Korea, Spain, Finland, Ireland, Iran and South Africa.

2.2 Sequence types and clonal complexes

Ninety-five per cent of invasive listeriosis infections are caused by three serotypes (4b, 1/2b, and 1/2a), stressing the importance of greater strain determination. The four genetic lineages and subsequent serotypes of L. monocytogenes can be further subdivided into sequence types (STs) and clonal complexes (CCs). STs are categorized based on seven housekeeping genes and depending on the variation of the nucleotide sequences of these genes, an ST is assigned (Enright et al., 2000, 2002; Seidl et al., 2015). MLST describes population structures and the phylogeny of many bacterial pathogens (Painset et al., 2019). STs can be grouped into CCs, with every ST in that group sharing at least five of the seven identical alleles with one other ST in that group (Enright et al., 2000, 2002; Seidl et al., 2015).

Some clones and STs are more associated with the food processing environment and others more with clinical cases. These different host tropisms are not fully understood yet and can be linked to virulence potential or the ability to adapt to harsh environmental conditions, allowing for multiplication, persistence and survival (Fagerlund, Wagner, et al., 2022). Although research shows that some STs are more pathogenic than others, the food safety and regulatory authorities consider all isolates to display the same risk from a food safety point of view (Wagner et al., 2022).

Table 2 shows the CCs of L. monocytogenes in seafood and its food processing environment that other researchers reported globally. In a study done in Poland by Wieczorek et al. (2020), CC121 (ST121) was isolated from most fish (cold-smoked salmon and raw salmon) and its processing environment, followed by CC8 (ST8). The authors also found that 96% of these isolates were from lineage II (Table 2).

Painset et al. (2019) found that the most prevalent CC in fish products and their food processing environment in various countries in the EU was CC121, followed by CC8, CC9 and CC155. Maury et al. (2019) found similar results in France, with CC121 being the most prevalent in seafood, followed by CC9. Several researchers also found CC321 to be prevalent in seafood in the United States and Canada, among other countries (Cooper et al., 2021; Daeschel et al., 2022; Harrand et al., 2020). Fagerlund, Idland, et al. (2022) examined Norwegian clinical isolates, and ST121 (CC121) was the second most prevalent (13%) ST found within the clinical isolates examined (Table 2). The authors also found that 80% of the clinical isolates examined belonged to lineage II. ST121 is considered hypovirulent; however, the relatively high prevalence of clinical cases attributed to these strains in Norway shows their ability to cause disease (Fagerlund, Idland, et al., 2022). A study characterized L. monocytogenes from fish and fish-processing environments in Poland. Authors reported CC6 and CC121 to be the most prevalent (Zakrzewski et al., 2023). Thus far, one study in South Africa has categorized L. monocytogenes isolates from locally caught seafood and its food processing environment into serotype, ST or CC using WGS (Lambrechts et al., 2024). The authors reported ST3, ST5, ST54, ST87, ST121, ST155, ST204 and ST515 over their subset of isolates.

4.1 Contamination of raw incoming fish

Various studies have determined the route of contamination of L. monocytogenes in a seafood processing facility and have shown that contamination of the final product does not always originate from the raw incoming fish. The prevalence of L. monocytogenes in raw fish varies from 0% to 10% but is generally quite low, with a higher probability of contamination in fish sourced from water receiving a heavy runoff from land (Chen et al., 2016; Fonnesbech Vogel et al., 2001; Miettinen & Wirtanen, 2005).

A systematic review and meta-analysis detailing the prevalence of Listeria and L. monocytogenes prevalence in fish, fish products and the fish processing environment, reported a prevalence of 5.8% L. monocytogenes from raw fish versus a 14.5% prevalence in ready-to-eat products (Zakrzewski et al., 2024). The study also found a significantly higher prevalence of L. monocytogenes in raw fish in high-income countries (15%) compared to 3.0% in lower to middle-income countries. This study further categorized L. monocytogenes from raw fish by family, with pooled results showing the highest contamination in Pleuronectidae and Salmonidae, at 21.4% and 28.5%, respectively (Zakrzewski et al., 2024). Pleuronectidae includes commercially important flatfish like halibut, and sole, typically found in cold and temperate waters, while Salmonidae, found in cold-water environments mainly in the Northern Hemisphere, includes species such as salmon and trout. Additionally, species characterization in this study showed a 50% prevalence of L. monocytogenes in Mugil cephalus, the flathead gray mullet commonly found in coastal and surface waters of lakes, where Listeria is also commonly found (Zakrzewski et al., 2024). Consequently, contamination of processing facilities with this species may be linked to imported raw fish; the longer time between catching and sale enhances the detection rate (Zakrzewski et al., 2024). Further, species Oncorhynchus mykiss and Salmo salar were associated with prevalence rates of 36.9% and 30.3%, respectively (Zakrzewski et al., 2024), known as rainbow trout/steelhead and Atlantic salmon, respectively.

A study by Miettinen and Wirtanen (2005) on farmed rainbow trout found that most of the L. monocytogenes contamination was in the gills of the fish and only occasionally in the skin and viscera. Gills serve as an ideal environment for bacterial growth and are the first part of the fish to be contaminated whenever volumes of contaminated water filter through (Miettinen & Wirtanen, 2005). Knowledge of the location of L. monocytogenes on the fish is important for the development and choice of transportation, handling and processing methods to avoid L. monocytogenes contamination to the rest of the fish, such as the pure fish flesh (Miettinen & Wirtanen, 2005). Additionally, minimizing the contamination of raw fish is vital to prevent the contamination of processing equipment and surfaces (Miettinen & Wirtanen, 2005). Furthermore, Miettinen and Wirtanen (2006) established that the seasons and weather had a strong influence on the presence of Listeria spp. in fish farms. Fagerlund, Idland, et al. (2022) also verified that the presence of L. monocytogenes was positively correlated with rainfall.

Chen et al. (2016) found no contamination of transportation tanks, the aquaculture environment or raw fish samples before processing in an RTE tilapia sashimi processing plant. However, final products, workers' hands and facility surfaces were contaminated with L. monocytogenes. This suggested that contamination was most likely due to inadequate cleaning and sanitation processes rather than the aquaculture or transportation tanks.

4.2 Contamination of products from the food processing environment

Although raw incoming fish is an important and valid source of contamination, contamination from the food processing environment and equipment during processing of the fish and after processing of the final product is the main source of contamination (Chen et al., 2010, 2016; Miettinen & Wirtanen, 2006; Pagadala et al., 2012; Rørvik et al., 2000). Contamination from the food processing environment includes food contact surfaces such as trimming boards, slicing machines, conveyor belts, weighing tables, skinning units, salting units and smokehouses (Chen et al., 2010; Johansson et al., 1999; Rørvik et al., 2000). Fagerlund, Wagner, et al. (2022) found that drains, floors, conveyor belts and gutting machines were most commonly associated with L. monocytogenes in salmon processing facilities.

Lin et al. (2006) studied the survival and adherence of L. monocytogenes on surfaces of commercial slicers and found that L. monocytogenes could be transferred from the slicer onto deli meats. Cross-contamination and recontamination of RTE food within the food processing environment are a common source of L. monocytogenes contamination (Pagadala et al., 2012). Chen et al. (2010) found a prevalence of L. monocytogenes in chiller water and on trimming boards, suggesting that these could be a source of cross-contamination to fish fillets.

Preventing the introduction of L. monocytogenes into the food processing environment is a very important step in controlling L. monocytogenes in seafood production because contamination of the facility can lead to contamination of the food itself (Leong et al., 2015; Rørvik et al., 2000). Rørvik et al. (2000) found L. monocytogenes on pallets and floors of the loading vehicles at the transport terminal, concluding that this might be an important factor in contaminating the processing plants with L. monocytogenes.

The specific source of environmental contamination (food contact surfaces and nonfood contact surfaces) is difficult to identify within a processing plant, and contamination can come from many sources including raw material, personnel and packaging material (Pagadala et al., 2012).

4.3 Contamination during and after distribution from the food processing environment

A study done by Miya et al. (2010) showed that the cell count of L. monocytogenes in final seafood products increased considerably with temperature abuse over 2 days at 10°C. Inappropriate storage or temperature abuse can happen during distribution or before consumption.

In an EU-wide baseline study between 2010 and 2012, 3053 smoked or gravad fish samples were tested for L. monocytogenes prevalence and cell counts at the retail level. At the time of sampling the fish products, 10.4% of products were positive for L. monocytogenes and at the end of shelf life, 10.3% of samples were positive for L. monocytogenes. Furthermore, 1.7% of these samples had a cell count higher than 100 CFU/g at the end of the shelf life (European Food Safety Authority, 2013).

A study done by González et al. (2013) in Spain found that all the RTE smoked salmon products bought in the retail market were at temperatures higher than the recommended 4°C. Furthermore, the temperature of the smoked salmon was on average 8.0°C (5–14°C) (González et al., 2013). The authors also observed that when transported using regular/plastic shopping bags, the product's temperature could increase by an average of 6.2°C on the way home (González et al., 2013). Garrido et al. (2010) found that most consumers were unaware of the temperature of their refrigerators and that many consumers stored their RTE products at temperatures above 6°C. Based on this information, the consumer has a responsibility for the proper handling and storage of RTE food. Education and communication with the consumer about appropriate practices for buying, transporting and storing these high-risk foods are important (González et al., 2013).

It is difficult to produce fish products, in particular cold-smoked salmon, completely L. monocytogenes free. Therefore, it is important to introduce food safety programs at the preprocessing and processing steps. Additionally, new disinfectants must be incorporated to control persistent L. monocytogenes strains that could have adapted to the routine disinfectants used (Mahgoub et al., 2022). According to the guidelines of the European Salmon Smokers Association, the standard for L. monocytogenes is that it must be absent in 25 g of food before it has left the immediate control of the food processing environment. If L. monocytogenes is present in the final product, the food business operator may set intermediate limits if they can prove that the product will not exceed the limit of 100 CFU/g over the shelf life of the product (European Salmon Smokers Association, 2018).

Many studies reported higher frequencies of Listeria spp. over L. monocytogenes (Pagadala et al., 2012). The detection of any Listeria spp., however, is an indication of poor sanitation and cleaning practices and indicates the high probability that L. monocytogenes will be present sooner or later (Cocolin et al., 2002; Cossu et al., 2016).

4.4 Persistence

Once L. monocytogenes enters a food processing facility, it is very difficult to eradicate if no proper monitoring programs are in place. The survival of L. monocytogenes is influenced by several complex factors, and it can grow under stress conditions, as noted earlier. Persistent strains can be defined as a specific strain of L. monocytogenes isolated repeatedly from the same environment over an extended time (6 months and longer) (Leong et al., 2015). The presence of persistent strains increases the possibility of cross-contamination of final food products as these strains are not eliminated from the food processing environment (Palaiodimou et al., 2021). Several cases of persistent strains have been documented in seafood processing environments (Chen et al., 2016; Fonnesbech Vogel et al., 2001; Palma et al., 2020).

Several factors can result in the persistence of L. monocytogenes in food processing environments. Harborage sites such as cracks, the inside of equipment and areas protected from cleaning and sanitation are areas where the sanitizer cannot reach properly and as a result favor the proliferation and growth of L. monocytogenes (Møretrø et al., 2017; Palaiodimou et al., 2021). This persistence is further complicated by various studies showing that L. monocytogenes displays tolerance to sanitizers commonly used in the food processing environment (Matle et al., 2019). The formation of biofilms in food processing environments contributes to L. monocytogenes' sanitizer tolerance (Møretrø & Langsrud, 2004; Rip & Gouws, 2020).

Persistent strains of L. monocytogenes can also be present due to the introduction or adaptation of specific strains to the selection pressures within the food processing environment. Suboptimal conditions (temperatures and disinfection concentrations) within the food processing environment can promote the survival and multiplication of these strains (Larsen et al., 2014; Palma et al., 2020). These adaptations (i.e. sanitizer tolerance genes or stress survival genes) may protect the isolates from the sanitizers or stresses encountered in the food processing environment (Palma et al., 2020). Persistence can indicate inadequate cleaning and sanitation processes or inadequate implementation thereof. Staff should be properly trained to prevent cross-contamination of L. monocytogenes from the environment to products, ensuring a safe product (Chen et al., 2016; Palaiodimou et al., 2021; Palma et al., 2020). ST121 has been shown in many studies to be able to persist in the food processing environment for months or even years (Fagerlund, Wagner, et al., 2022; Harter et al., 2017; Holch et al., 2013; Maury et al., 2016; Myintzaw et al., 2023; Painset et al., 2019; Pasquali et al., 2018). CC121 has been the most common CC identified in many seafood factories (Maury et al., 2019; Painset et al., 2019; Wagner et al., 2022; Wieczorek et al., 2020), and persisting in factories in many European countries (Holch et al., 2013; Maury et al., 2016; Myintzaw et al., 2023; Painset et al., 2019). CC1 has been reported as one of the most common CCs in a salmon processing facility in Norway and has also been found to be persistent in these factories (Fagerlund, Wagner, et al., 2022; Wagner et al., 2022). Furthermore, ST3 (CC3) has also been found to persist in two king oyster mushroom production plants (Xu et al., 2023).