Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for a serious food-borne zoonosis called listeriosis. This pathogen is transmissible to humans through the consumption of contaminated food. It can lead to mild gastroenteritis and also to serious infections of the bloodstream or the central nervous system as well as abortions. The organism is able to pass from cell to cell, allowing it to cross blood–brain and placental barriers (Janakiraman, 2008). Immuno-compromised persons, pregnant women, infants, and the elderly are the most vulnerable to listeriosis (Vazquez-Boland et al., 2001). In 2015–2019, the listeriosis trend in Europe remained stable after a long period of increase but had the highest case-fatality rate (8.9%) among the outbreak-related illness (EFSA and ECDC, 2021). In Slovakia, 18 human listeriosis cases and four deaths were reported in the year 2019, which is 6% above the 5-year average (Slovak Focal Point for the Scientific, and Technical Matters of the Efsa., 2020).

Some L. monocytogenes strains can persist for a long period of time in various types of food-processing environment (FPE), including chilled processing plants (Lundén et al., 2004; Schmitz-Esser et al., 2015). Listeria monocytogenes contamination may occur as a result of poor hygiene during food processing or packaging (Carpentier and Cerf, 2011).

Animal listeriosis is generally subclinical, but more serious forms have also been observed (OIE, 2018), with symptoms including septicaemia, encephalitis, meningitis, meningoencephalitis, rhombencephalitis, abortion, stillbirth, perinatal infections, and/or gastroenteritis (Okada et al., 2011; OIE, 2018).

Listeria monocytogenes is a genetically heterogeneous species divided into four phylogenetic lineages, of which lineages I and II are the most frequently encountered. The strains from lineage I (serotypes 1/2b, 4b) are in general found in higher frequency in human outbreaks than lineage II strains (serotypes 1/2a, 1/2c), which are commonly isolated from food, natural and farm environments, and animal and sporadic human listeriosis cases (Orsi et al., 2011).

Several methods have been developed to investigate the genetic diversity of L. monocytogenes strains. Agglutination serotyping is considered to be the first level of discrimination between isolates and can differentiate 13 serotypes. For many years, pulsed-field gel electrophoresis (PFGE) had been the gold standard for L. monocytogenes subtyping (Graves and Swaminathan, 2001). Another key typing approach has been multi-locus sequence typing (MLST) based on the standardized nomenclature derived from the sequences of seven housekeeping genes (Ragon et al., 2008). Unique combinations of alleles from MLST analysis determine strain sequence types (STs) and clonal complexes (CCs), which are now systematically used to describe the population structure of L. monocytogenes (Ragon et al., 2008; Cantinelli et al., 2013; Maury et al., 2016). Certain CCs account for the majority of outbreaks and sporadic cases in humans (Maury et al., 2016) and animals (Dreyer et al., 2016). More recently, typing based on whole-genome sequencing (WGS) of L. monocytogenes has become a very powerful tool and more and more studies are employing this method for national surveillance, outbreak detection, or tracking of the sources of listeriosis (Jackson et al., 2016; Moura et al., 2017; Pietzka et al., 2019).

The State Veterinary and Food Institute (SVFI) has been the designated Slovak National Reference Laboratory (NRL) for L. monocytogenes since 2007. This national-level laboratory is engaged in the surveillance and typing of L. monocytogenes isolates from food items and animals. It participates in the European surveillance network for L. monocytogenes, along with 34 other NRLs coordinated by the European Reference Laboratory (EURL).¹ The PFGE method is routinely performed in the Slovak NRL. The isolates are assigned to an MLST CC using a mapping method detailed in Félix et al. (2018). Whole-genome sequencing of the isolates is currently being introduced.

Only a few studies are available on the genomic or genetic diversity of L. monocytogenes in Slovakia, and they focus only on selected food manufacturers, either in the dairy sector (Véghová et al., 2015) or in the meat sector (Véghová et al., 2016). To date, no data are available on a large and diverse panel of food isolates for Slovakia. The purpose of this study was thus to provide an overview of the population structure of the L. monocytogenes isolates over the past 11 years in Slovakia. This survey should lead to better management and understanding of food-related health risks.

This study had two objectives. The first was to analyse the genetic diversity of all L. monocytogenes isolates from food, FPEs, and animals available in the NRL collection. The second was to compare this diversity to that of isolates from human outbreaks during the same time period. The genetic diversity of 988 isolates was assessed using conventional agglutination serotyping. In a subset of 176 isolates, CCs were deduced either from PFGE profiles (127 isolates) or from WGS analysis (49 isolates).

Listeriosis is a serious public health and food safety problem in European countries, including Slovakia. Little data are available on the population genetic structure of food isolates in this country. Considering that food products are the main sources of human infections, it is very important to study the overall distribution of isolates throughout the country to improve understanding of the circulation of this disease.

The 988 L. monocytogenes isolates analyzed in this study by Denka Seiken kit which is often considered the “gold standard” for its serotyping (Burall et al., 2011) were isolated throughout the 2010–2020 period, providing a representative coverage of isolates circulating in Slovakia. Out of the 988 isolates tested using conventional serotyping, more than half (61.1%) were serotype 1/2a. This prevalence is consistent with many previous studies performed on isolates from various types of foods, mostly milk and dairy and meat products (Kramarenko et al., 2013; Martín et al., 2014; Ebner et al., 2015). The distribution of serotypes throughout the years suggested an even distribution of serotypes 1/2a, 1/2c, and 4b. The descending trend in distribution of 1/2b was observed in the 2010–2020 period.

In this study, CC1, CC2, CC4, and CC6 made up 66.7% of lineage I isolates and were isolated from food, FPEs, and animals, as well as from human cases. This result is similar to that reported in Lachtara et al. (2021) on the prevalence of CC1, CC2, and CC6 in food and FPE isolates in Poland. These CCs have previously been described as hypervirulent (Maury et al., 2016). CC1 and CC2 were concluded as being predominant in all world regions with an exception of northern Africa for CC1. Interestingly, CC6 had been observed mainly in Europe in a study of Chenal-Francisque et al. (2011).

Regarding the food isolates, serotypes 1/2b and 4b were detected mainly among the isolates from milk and dairy products and other food products, this latter category being predominated by ready-to-eat delicatessen products (mainly ready-to-eat salads). These isolates were collected mainly from production plants. Considering that the typing data in this category were only available for a limited number of isolates mainly from 2014, more extensive evaluation of the CC’s diversity should be performed in future to clarify its importance in public health protection. The decreasing trend of the 1/2b serotype in the period of 2010–2020 might be explained by the reduced popularity of rather unhealthy delicatessen products and increased healthier eating habits of Slovak population (Holotová et al., 2021). Prevalence of serotypes 1/2b and 4b has been reported in several studies on milk, meat, and ready-to eat products (Beak et al., 2000; Braga et al., 2017).

Serotype 1/2c was prevalent in isolates from meat, corroborating results from other studies conducted on the meat sector in Europe (Gianfranceschi et al., 2009; Ortiz et al., 2010; Prencipe et al., 2012; Meloni et al., 2014; Ebner et al., 2015; Morganti et al., 2015; Félix et al., 2018).

An indication of potential persistence of a CC9 L. monocytogenes isolate was observed at one meat production site between 2014 and 2018. This result substantiates previous studies demonstrating persistence of CC9 over many years in meat-processing facilities in Switzerland (Stoller et al., 2019) and in meat products and in pig slaughterhouse environments worldwide (Martín et al., 2014; Wang et al., 2015; Véghová et al., 2016; Félix et al., 2018; Maury et al., 2019; Demaître et al., 2020; Fagerlund et al., 2020).

In this study, two CCs, CC1 and CC37, were frequently observed among the isolates sourced from milk and dairy products and from sheep. This result is in accordance with a 2019 study (Papić et al., 2019) in Slovenia that showed that CC1 and CC37 were responsible for the majority of the clinical cases in animals, mainly small ruminants and cattle. CC37 is also the most prevalent isolate found on pig and ruminant farms in France (Félix et al., 2018) and frequently reported from ruminant farms in Switzerland (Dreyer et al., 2016), in the natural environment throughout Europe (Haase et al., 2014) and in the natural environment in Austria (Linke et al., 2014). Similar to our findings, CC7 and CC37 were the most abundant in the study focusing on milk and milk-related environment in the United States (Kim et al., 2018).

Interestingly, CC121 isolates, which are overrepresented in all food sectors in Europe and worldwide (Chenal-Francisque et al., 2011; Henri et al., 2016; Félix et al., 2018) and which can persist in FPEs (Holch et al., 2013; Rychli et al., 2017; Stoller et al., 2019), were of minor occurrence in our study, with only a few CC121 isolates from food sectors being observed. Also, CC121 belongs to the most prevalent clones worldwide (Chenal-Francisque et al., 2011). No CC121 isolates were isolated from animals, similar to previous reports (Linke et al., 2014; Dreyer et al., 2016; Félix et al., 2018).

In this study, CC11-ST451 was prevalent in the milk and dairy products sector. This result is concordant with an Austrian study that revealed that (i) dairy products are three times more likely to be contaminated with CC11-ST451 than the products from other food categories and (ii) a large part of CC11-ST451 food isolates could be traced back to a specific cheese-producing facility (Cabal et al., 2019). However, of the three ST451 human cases reported in Austria in 2017, none were related to the cheese-producing facility and all three were distinguishable, thus most likely arising from different contamination sources. CC11-ST451 isolates have been also reported in dairy products in the Czech Republic (Tomaštíková et al., 2019).

These findings, together with the frequent detection of CC7 in dairy products in Austria (Cabal et al., 2019) and the location of Austria and Czech Republic in Central Europe, are in accordance with our observation of CC11-ST451 and CC7 in the isolates from the dairy sector and the hypothesis of a multiple sources of introduction of these clones that likely circulate intensively.

CC11-ST451 isolates were also detected in the meat production sector, particularly in one rabbit meat-processing plant in Czech Republic. Persistence of these isolates in FPEs was first suggested by Gelbíčová et al. (2019). Interestingly, only one CC11-ST451 isolate was detected among the FPE isolates in our study.

Human cases of listeriosis caused by CC11-ST451 have been reported in Germany (Halbedel et al., 2018), France (Moura et al., 2017), Austria (Cabal et al., 2019), and Poland (Kuch et al., 2018). A nosocomial outbreak linked to ST451 was described lately in Italy (Russini et al., 2021). Furthermore, this clone is considered hypervirulent based on its prevalence in human cases and in food contamination in France (Fritsch et al., 2018).

Conversely, a very low occurrence of ST451 (fewer than 10 isolates) was reported in a wide-scale study (Painset et al., 2019) on 1,142 European isolates (isolated in 22 EU Member States and 1 Non-Member State). The frequent occurrence of CC11-ST451 in Slovakia and in neighboring countries suggests that this ST is specific to Central Europe.

The isolate distribution in Central Europe over a period of 11 years highlights a possible distribution of the CC11-ST451 clone in the milk and meat sectors in Slovakia. The seven genomes of Slovak CC11-ST451 isolates were dispersed among the available known genomes of ST451 isolates collected worldwide. The source of the CC11-ST451 isolates in Slovakia can potentially be determined with the systematic sequencing of the ST451 isolate genomes, now being collected by the Slovak NRL.

The importance of subtyping of L. monocytogenes isolates in Slovakia and in Europe in general is an important tool for food safety management. With the increasing production of food, globalization of the market, and consumers’ focus on traditional farm products with the characteristic small production and less trained staff in the field of the food safety, there is a higher risk of contamination of FPE and final food products. Isolate typing allows the identification of such threats and control measures implementation.

This study is the first comprehensive insight into L. monocytogenes isolate diversity in Slovakia, covering isolates from the different food sectors available in the NRL collection, as well as isolates from clinical animal cases and several human outbreaks. The distribution of 988 serotypes was similar to that generally observed in Europe. The CC11-ST451 type, frequently reported in Central Europe, predominated in the Slovakia dataset, being detected mainly in isolates from dairy products.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: http://www.ebi.ac.uk/ena/, PRJEB38828, PRJNA475189, and PRJEB45859.

LC was in charge of serotyping. ZK was in charge of PFGE data production, analysis, and interpretation. ZK and BF performed mapping of PFGE profiles to MLST CCs and WGS analysis. ZK, SR, and BF wrote the manuscript. All authors read, commented, and approved the final version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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