Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004–2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.


Introduction
Listeria monocytogenes is a gram-positive bacterium and the causative agent of listeriosis, a zoonotic disease that affects mammal species, including humans [1,2]. L. monocytogenes is amongst the major food-borne pathogens in the world and has attracted much research attention from public health, veterinary medicine, and food authorities over the recent decades [3]. L. monocytogenes has been linked to sporadic episodes, as well as large outbreaks, of human listeriosis worldwide [4,5]. Although relatively rare (the annual incidence rate ranges from 0.2 to 0.5 cases per 100,000 population), listeriosis is responsible for high hospitalization rates of over 95% and mortality rates (average fatality rates are estimated to be 20-30% of hospitalized patients and can reach 40% in outbreaks related to (III and IV) have subsequently been identified, are rarely isolated, and mostly occur in ruminants [19,20].
The majority of STs can be assigned to one of the seven major clones or clonal complexes (CC). The predominant CCs were CC1, CC2, and CC3 (lineage I). Lineage II was more heterogeneous and comprised different CCs [16,21].
Currently, the distribution of L. monocytogenes STs in different world regions is being intensively studied. However, for some countries only limited information on the diversity of L. monocytogenes genotypes is available. In addition, there is relatively little information on the sequences of surface and secreted proteins by L. monocytogenes from the internalin family, which are the main essential virulence factors allowing L. monocytogenes to cross the intestinal barrier in order to invade the epithelial and endothelial cells of the host [1].
Recently, the initial L. monocytogenes MLST scheme was successfully supplemented with an analysis of the genes expressing the proteins of a protein family known as the internalins, which contribute to the virulence of this microorganism-multi-virulent-locus sequence typing (MvLST) [22]. MLST and MvLST are considered great tools for studying L. monocytogenes genotype distribution among animal and human clinical cases, foods, and other sources, as well as for unraveling food-borne outbreaks [2,15].
The Republic of Serbia is a country in central and southeastern Europe, in the southern Pannonian Plain and central Balkans. It borders Hungary in the north, Romania in the northeast, Bulgaria in the southeast, North Macedonia in the south, Croatia and Bosnia and Herzegovina in the west, and Montenegro in the southwest ( Figure 1). According to the report on infectious diseases by the Institute of Public Health of Serbia (Belgrade), a small number of cases of human listeriosis are registered in the Republic of Serbia, reflecting the sporadic nature of invasive listeriosis in this country. From 2014 to 2018, the annual incidence rate ranged from 0.04 to 0.19 cases per 100,000 population, but the mortality rate in 2018 was 12.5% [23]. In the Republic of Serbia to this day, L. monocytogenes have only been studied in food, the food industry environment, and humans, and, unlike some neighboring countries, there is limited knowledge about their genetic characteristics, distribution, and effects, especially on ruminant populations, and information about the veterinary significance of L. monocytogenes is still insufficient. In fact, no data have been reported on the distribution of L. monocytogenes STs there, although the Republic of Serbia is one of the European countries with the largest population of small ruminants.
The aims of this study were: (a) to reveal genetic relationships of STs among the L. monocytogenes strains in the Republic of Serbia and worldwide by MLST technique; and (b) to determine the partial sequences of L. monocytogenes virulence genes which encode the proteins of the internalin family (namely, inlA, inlB, inlC and inlE), making them adhere and invade host cells [22,[24][25][26], by MvLST analysis. Figure 1. Location of the study area. The territory of the Republic of Serbia is illustrated in a separate square. The L. monocytogenes strains of different STs are highlighted in the colored circles: red-ST1, green-ST3, blue-ST9, yellow-ST32, plum-ST734, lime-ST1232, brown-ST1233, teal-ST1234, maroon-ST1235, white-ST1236, azure-ST1237, hunter green-ST1238, and hot pink-ST1242.

L. Monocytogenes Strains and Culture Cultivation
A total of five L. monocytogenes strains were obtained from the Veterinary Specialized Institute Kraljevo (Kraljevo, the Republic of Serbia) for further analysis. The isolates were collected throughout 2019 from animals which had died of listerial neuroinfection (postmortem sectional material), fetus of small ruminants from farms from the Republic of Serbia, and from food and food production environment (Table S1, Figure 1, Supplementary Materials), and designated as Serbia-2019 isolates. The strains were kept frozen at −70 °C. The bacterial cultures were plated on brain-heart infusion (BHI) (BD Bioscience, EU) agar and grown overnight before an experiment.

L. Monocytogenes Strains and Culture Cultivation
A total of five L. monocytogenes strains were obtained from the Veterinary Specialized Institute Kraljevo (Kraljevo, the Republic of Serbia) for further analysis. The isolates were collected throughout 2019 from animals which had died of listerial neuroinfection (postmortem sectional material), fetus of small ruminants from farms from the Republic of Serbia, and from food and food production environment (Table S1, Figure 1 (Table S1, Supplemental Data 1).

DNA Extraction
Lysates of overnight L. monocytogenes cultures of Serbia-2019 were obtained as previously described, with modifications [27]. Then, 100 µL of a 24-h culture grown in heart-brain broth (Difco, Detroit, MI, USA) at a temperature of 37 • C was resuspended in a pre-lysis buffer of the following composition: 20 mM Tris (hydroxymethyl) aminomethane (Tris)/hydrochloric acid (HCl); 2 mM EDTA ethylenediaminetetraacetic acid (EDTA); 1% Triton X-100; pH 8; all components (Sigma-Aldrich, St. Louis, MO, USA) containing 20 mg/mL were added directly before the use of lysozyme (50 mg/mL) (Serva, Heidelberg, Germany). After the incubation of the mixture for 1 h at a temperature of 37 • C, 1 µL proteinase K (10 mg/mL, Sigma) was added and incubated for 1 h at a temperature of 56 • C. The lysates were boiled for 10 min to inactivate proteinase K and stored at a temperature of 4 • C.

PCR
The PCR was run with 1 µL of lysate in the CFX96 amplifier (Bio Rad, Hercules, CA, USA). An Encyclo Plus PCR kit (Evrogen, Moscow, Russia) and primers (Syntol, Moscow, Russia) were used for DNA amplification (Supplementary Materials).
For the MvLST, the PCR amplification of the internal fragments of four internalin genes (inlA, inlB, inlC, and inlE) (Supplemental Data 1) for virulence gene analysis was performed as previously described [1,28], with modifications for inlB, in which the following parameter was changed: Tm = 48 • C. PCR products were evaluated by electrophoresis in 1% agarose gel. Amplified DNA was eluted from the reaction mixture using Cleanup Standard (Evrogen, Moscow, Russia) according to the manufacturer's instructions. Verification on 1.5% agarose gel followed, after which it was sent for sequencing to the "Genome" center (http://www.genomecenter.ru, accessed on 4 August 2020).

PCR Product Sequencing
Individual PCR product sequencing was performed using a set of reagents ABI PRISM ® BigDye ™ Terminator v. 3.1, followed by the analysis of the reaction products on an automatic sequencer, Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Electrophoretic DNA separation was performed in 50-cm capillaries with POP7 polymer.
The identification of obtained sequences was executed with the help of the BIGSdb-Lm database (https://bigsdb.pasteur.fr/listeria/primers_used.html, accessed on 22 March 2021) and included a search for allele numbers, relevant sequence type (ST), clonal complex (CC), and phylogenetic lineage for each L. monocytogenes isolate.
The determination of allelic profiles for each consensus gene sequence of internalins derived from Serbia-2019 strains was carried out by means of comparing the relevant sequences against loci lmo0433 (inlA), lmo0434 (inlB), lmo1786 (inlC), and lmo0264 (inlE) of the reference nucleotide sequences of L. monocytogenes strains that were available in the BIGSdb-Pasteur MLST database (https://bigsdb.pasteur.fr/listeria/listeria.html, accessed on 4 April 2021).

Phylogenetic and Data Analysis
The sequences were proofread and assembled in Chromas Lite MFC Application A minimum-spanning tree from allelic profiles based on MLST loci scheme was generated and visualized using GrapeTree [29], in order to demonstrate the phylogenetic relationships between the Serbia-2019 isolates and L. monocytogenes strains ( (Table S1, Supplemental Data 1).
The phylogenetic trees were constructed using the Maximum Likelihood algorithm with 1000 bootstrap replicates. The concatenated sequences of 27 reference representative L. monocytogenes isolates from the BIGSdb-Pasteur MLST database (https://bigsdb.pasteur. fr/listeria/listeria.html, accessed on 19 May 2021) (Supplemental Data 1) were aligned and analyzed in MEGA 7 [30].
The results of MLST typing are now available in the BIGSdb-Lm MLST database (http://www.pasteur.fr/mlst, accessed on 11 June 2021) with IDs, as indicated in Table 1.

Cartographic and Phylogeographic Analysis
The distribution of L. monocytogenes ST1, ST3, ST9, ST32, ST734, and ST1232-ST1238, ST1242, which are available in the BIGSdb-Pasteur MLST database (https://bigsdb.pasteur. fr/listeria/listeria.html, accessed on 11 June 2021) for spatial data processing and visualization in the Republic of Serbia and worldwide was performed using the ESRI geographic information system ArcGIS 10.6.1 (Esri, Redlands, CA, USA) and the DSW electronic map of the world (the Russian Federation). To display the multiple STs on the map, a color pie chart tool (http://www.esri.com/software/arcgis/arcview/index.html, accessed on 21 May 2021) was used.

Results and Discussion
In this study, the polymorphism in the housekeeping genes of all 13 L. monocytogenes strains that were isolated during the routine monitoring to control of the spread of listeriosis in the Republic of Serbia in 2004-2019 was carefully examined. Initially, five L. monocytogenes strains isolated recently, in 2019, from the Republic of Serbia were carefully investigated using MLST and MvLST techniques [14,17,18]. Among them three strains were derived from food-associated samples and two isolates had an animal origin (Table S1, Supplemental Data 1). By MLST, five different STs, namely ST1, ST3, ST9, ST32, and ST734, were revealed. Only two STs, ST1 and ST734, belonged to the identical CC1 while the other STs corresponded to three different clonal complex CCs: (i) ST3-CC3, (ii) ST9-CC9, and (iii) ST32-ST32, respectively. Overall, all the STs were assigned to two phylogenetic lineages, I and II. In fact, the genetic lineage I was prevalent, as the majority of STs (4/5, 80%) were represented by the relevant ST1, ST3, ST32, and ST734. Only a single ST9 (1/5, 20%) could be allocated to the genetic lineage II.
Molecular phylogenetic analysis based on the MLST data obtained, showed that each of the Serbia-2019 strains was clearly clustered with the reference L. monocytogenes organisms, possessing the same STs and isolated earlier in either the Republic of Serbia or worldwide ( Figure 2). In addition, we demonstrated that two STs, ST734 and ST32, and the relevant clonal complexes, CC1 and ST32, detected among Serbia-2019 strains were found in Europe for the first time ( Figure 2).
Microorganisms 2021, 9,1289 8 of 17 ST1242) except the only ST1234 (CC5), which was detected in a food specimen (Table S1, Supplemental Data 1). Interestingly, previous epidemiologic studies demonstrated that lineage I is generally overrepresented among human clinical listeriosis cases, while lineage II strains were found to be major contaminants of food products [5,9,19]. However, in fact, among the Serbia-2019 strains, non-human clinical isolates of animal and food origin belonging to lineage I were first detected (Table 1)  Importantly, only a single strain (#76380/DP 1093/2019) from Serbia-2019 belonged to CC3 that had also previously been revealed in the Republic of Serbia in humans with L. monocytogenes-associated infection (in 2010, #16,160/2184/2010), while the rest, CC1, CC9, and ST32, were recognized here for the first time. However, both CC3 strains had different STs, namely, ST3 for the organism detected in 2019, and ST1236 for the strain isolated in this country in 2010. These strains demonstrated identical alleles, with six out of the seven loci found in ST3 and ST1236, respectively, and they differed by a single allele ldh (Table S1, Supplemental Data 1). Although these strains originated from different sources, food and human specimens, they formed a single cluster, indicating that both isolates could have a common ancestor (Figure 2).
Interestingly, previous epidemiologic studies demonstrated that lineage I is generally overrepresented among human clinical listeriosis cases, while lineage II strains were found to be major contaminants of food products [5,9,19]. However, in fact, among the Serbia-2019 strains, non-human clinical isolates of animal and food origin belonging to lineage I were first detected (Table 1) (Figure 4a,b). These findings could be correlated with the observations of previous studies suggesting that ruminants and food could represent a possible source of human listeriosis [8][9][10].      Indeed, at least eight novel STs (ST1232-ST1238 and ST1242) were only detected in the Republic of Serbia, in 2004-2012, and have not yet been found worldwide (Table S1, Figure 4, Supplemental Data 1). This indicates the possible presence of some undetectable sources and hidden reservoirs of L. monocytogenes in this territory.
Source category analysis showed a certain association between the sample origin and the ST and CC identified. Thus, Serbia-2019 strains belonging to CC1 (ST1 and ST734) were isolated from animal specimens, while CC3 and CC9 were derived from food sources, similarly to CC1 (ST1), CC3 (ST3), and CC9 (ST9) that had been detected earlier The majority of the L. monocytogenes strains belonging to ST1, ST3, and ST9 that were similar to the Serbia-2019 isolates of the identical STs (ST1, # 76379/DP 9228/2019, ST3, #76380/DP 1093/2019 and ST9, #76382/DP 7675/2019, respectively) were regularly detected in Australia (n = 230), France (n = 100), Chile (n = 58), Germany (n = 45), the USA (n = 38) etc. (Figure 4a). These STs were more often found in Europe, Oceania, and South America, rarely in North America and Asia, and occasionally in the Middle East, Africa, Central America, etc. (Figure 4b). Both ST1 and ST9 had been occasionally isolated in some neighboring countries such as: Bulgaria (1965) and Greece (1959) from animal specimens, and Greece (2018) from human samples (Supplemental Data 1) as well. No ST3 (CC3) was detected in the countries neighboring the Republic of Serbia, whereas since 1955-1957 the ST3 strains have been widely isolated in other European countries, namely France, Spain, Poland, Germany, Finland, Russia, Italy, Austria, Switzerland, Denmark, Sweden, Greece, Ireland, Portugal (Figure 4a, Supplemental Data 1). Interestingly, CC3 is the fourth most common clonal complex in Europe [16], and is predominant in Australia and has been detected in South and North America, Japan, and Oceania. The data obtained correlated with the observations of Parisi et al. [21] on the worldwide distribution of large clonal complexes CC1, CC3, and CC9 and the phylogenetic lineages I and II.
Surprisingly, two STs, ST734 and ST32, belonging to the clonal complexes, CC1 and ST32, and detected among Serbia-2019 strains were found in Europe for the first time. Thus, each of these STs and CCs were revealed only once, ST32 in Mexico, North America in 1999 (#4258/LM88502, clonal complex ST32) and ST734 in Chile, South America in 2009 (#5776/L-309-09, clonal complex CC1) (Figure 4a-d, Supplemental Data 1). Seemingly, L. monocytogenes strains circulating in the Republic of Serbia may possess more genetic diversity than those in some other European countries.
Indeed, at least eight novel STs (ST1232-ST1238 and ST1242) were only detected in the Republic of Serbia, in 2004-2012, and have not yet been found worldwide (Table S1, Figure 4, Supplemental Data 1). This indicates the possible presence of some undetectable sources and hidden reservoirs of L. monocytogenes in this territory.
Source category analysis showed a certain association between the sample origin and the ST and CC identified. Thus, Serbia-2019 strains belonging to CC1 (ST1 and ST734) were isolated from animal specimens, while CC3 and CC9 were derived from food sources, similarly to CC1 (ST1), CC3 (ST3), and CC9 (ST9) that had been detected earlier worldwide and principally had the same origin, from animal and food-associated isolates ( Figure 5). In fact, the clonal complexes CC1 and CC3 were represented by the largest number of strains, being recognized as the most common globally prevalent isolates [29].
Microorganisms 2021, 9,1289 12 of 17 worldwide and principally had the same origin, from animal and food-associated isolates ( Figure 5). In fact, the clonal complexes CC1 and CC3 were represented by the largest number of strains, being recognized as the most common globally prevalent isolates [29].
Another significant finding of our research was the detection of ST1 belonging to CC1 and the phylogenetic line I (DP 9228/2019, Table S1, Supplemental Data 1) in the brain of dead sheep with listerial neuroinfection. This is consistent with the data of European researchers, who reported neurotropism and a strong association of ST1 with rhombencephalitis in ruminants which died during listeriosis outbreaks [2,32,33]. The majority of STs and CCs of L. monocytogenes strains isolated in the Republic of Serbia earlier, in 2004-2012, originated from human specimens (ST1236, ST1232, ST1235, ST1238, ST1242, ST1233 and ST1237, and CC3, CC19, CC21, CC37, CC155 and CC315, respectively) while only one of them (CC5, ST1234) was from a food source (Table S1, Supplemental Data 1).
Another significant finding of our research was the detection of ST1 belonging to CC1 and the phylogenetic line I (DP 9228/2019, Table S1, Supplemental Data 1) in the brain of dead sheep with listerial neuroinfection. This is consistent with the data of European researchers, who reported neurotropism and a strong association of ST1 with rhombencephalitis in ruminants which died during listeriosis outbreaks [2,32,33].
It is known that L. monocytogenes CC3 and CC9 are more often isolated in highly immuno-compromised patients [2,26] (Figure 5) and who were predominantly associated with food production sectors [26,34]. In our research, ST3 and CC3 and ST9 and CC9 was successfully isolated from cheese with prosciutto and smoked pork ribs (DP 1093/2019, DP 7675/2019, Table S1, Supplemental Data 1), respectively, which are related to meatcontaining products. Our result correlates with many studies that have shown a high adaptability of ST9 isolates to meat production conditions. An overview of the diversity of strains circulating at all levels of the pork production chain in France showed that CC9 was directly associated with finished meat products [35]. This type of sequence was often found to be dominant in raw pork meat and usually preserved in slaughterhouses, meat processing plants, and meat products [36]. Thus, L. monocytogenes persistence in food processing environments (FPEs) increases the risk of food contamination and represents a major concern for the food industry and food safety [3,36,37].
Interestingly, the rare ST32 clonal complex ST32 showed an identical source (Production environment) when it was found in both cases, namely in the Republic of Serbia in 2019 and in Mexico in 1999. In contrast, the second rare CC1 ST734 was isolated in the Republic of Serbia from animal specimens, whereas the first isolate from Chile, 2009, was derived from a human sample (Supplemental Data 1). Notably, ST734 from Serbia-2019 was isolated from an aborted goat fetus and belonged to the hypervirulent clonal complex CC1 that was earlier detected among the L. monocytogenes-associated cases of abortion in ruminants in European countries (Slovenia, France, Great Britain, and Switzerland) [32]. It is quite possible, that this could be explained by the presence of internalins in the L. monocytogenes strains, especially the InlA protein that is required to cross the human maternofetal barrier and to cause fetal infection [38]. Importantly, ST32 detected among Serbia-2019 strains was obtained from the specimen derived from a fruit sorting machine (Table S1, Supplemental Data 1). Fruit processing facilities are reported to be potential sources of persisting L. monocytogenes contamination [39]. The 2014-2015 U.S. nationwide outbreak of listeriosis linked to apples was the first implication of fruit processing in the outbreaks of L. monocytogenes-associated food-borne illnesses [40]. In fact, 35 outbreaks of this listerial infection were identified in 12 US states from October 2014 to February 2015; 34 (97%) listeriosis patients were hospitalized and seven (20%) died. According to MLST analysis, environmental samples from the grower's packing facility and distribution-chain apples yielded outbreak-related L. monocytogenes isolates. These observations highlighted the importance of minimizing fruit processing facility contamination with L. monocytogenes [41]. In addition, the MvLST method showed the presence of alleles of the internalin gene (inlA1), which is one of the main characteristics of the most virulent strains from the CC1 and CC2 clones [1,32] in the relatively rare sequence type ST32, which did not belong to any of the clonal complexes associated with outbreaks of listeriosis in both farm animals and humans.
Finally, internal fragments of the internalin genes, such as inlA, inlB, inlC, and inlE, were carefully studied to supplement the MLST data for the Serbia-2019 strains with virulence gene analysis, according to recommendations [1,28,42]. Overall, five inlA/inlE, four inlB, and three inlC allelic variants were detected that correlated with a number of reports on the evident polymorphisms of the internalin proteins [22,24]. There was a certain correlation between the inlB/inlC/inlE allele profiles (841/82/177, respectively) and CC1. In fact, this clonal complex was represented by two different STs, namely ST1 and ST734, that were both isolated from an animal source. Additionally, the inlC allele profiles for CC3 (ST3) and ST32 were identical, as the same allele 89 was identified in both L. monocytogenes, although these strains were revealed to be from different sources, i.e., food and production environment (Supplemental Data 1).
The analysis of nucleotide sequences of the internalins inlA, inlB, inlC, and inlE genes of the L. monocytogenes strains isolated in the Republic of Serbia in 2019 from small ruminants and food products of animal origin did not reveal the presence of single nucleotide polymorphisms (SNPs) related to the reference sequences (Figure 6a-d, Supplemental Data 1). There was a certain identity between the investigated sequences of Serbia-2019 strains and the relevant gene of the reference sequences available in the BIGSdb-Pasteur MLST database https://bigsdb.pasteur.fr/listeria/listeria.html, accessed on 11 June 2021).

Conclusions
To the best of our knowledge, this study is the first detailed investigation of the polymorphism in the housekeeping genes of the L. monocytogenes strains that were isolated in the European country, the Republic of Serbia in 2004-2019. These strains belonged to the phylogenetic lineages I and II and were represented by the ST1, ST3, and ST9 of the clonal complexes CC1, CC3, and CC9, which were common in the majority of the European countries and worldwide, as well as by the novel STs (ST1232, ST1233, ST1234, ST1235, ST1238, ST1236, ST1237, and ST1242) of CC19, CC155, CC5, CC21, CC3, CC315 and CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1) reported in the Republic of Serbia for the first time. At least four genes of the internalins (inlA, inlB, inlC, and Thus, both the MLST and MvLST analysis allowed characterizing the strains isolated from the Republic of Serbia and performing a comparison with modern data. Until now, MLST-based population studies in this territory have mainly been conducted on isolates from human clinical cases in the Republic of Serbia. Our study adds further information on the diversity of L. monocytogenes genotypes in small ruminant and food products, as until now strain distribution in animal reservoirs in the Republic of Serbia has not been evaluated. The extension of the present analysis to a higher number of isolates could contribute to a better knowledge of the structure of the L. monocytogenes population in this country, in the EU, and worldwide.

Conclusions
To the best of our knowledge, this study is the first detailed investigation of the polymorphism in the housekeeping genes of the L. monocytogenes strains that were isolated in the European country, the Republic of Serbia in 2004-2019. These strains belonged to the phylogenetic lineages I and II and were represented by the ST1, ST3, and ST9 of the clonal complexes CC1, CC3, and CC9, which were common in the majority of the European countries and worldwide, as well as by the novel STs (ST1232, ST1233, ST1234, ST1235, ST1238, ST1236, ST1237, and ST1242) of CC19, CC155, CC5, CC21, CC3, CC315 and CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1) reported in the Republic of Serbia for the first time. At least four genes of the internalins (inlA, inlB, inlC, and inlE) that were earlier found in the more virulent CC1 and CC2 L. monocytogenes isolates were successfully detected in Serbia-2019 strains. Importantly, these genes of the internalins were present in the relatively rare sequence type ST32, which does not belong to any of the clonal complexes associated with outbreaks of listeriosis among farm animals and humans. Our study could confirm the association of CC1 with cases of neuroinfection and abortions among small ruminants, and CC3 and CC9 with food products of animal origin, respectively. Given the small number of L. monocytogene isolates studied by molecular methods in the Republic of Serbia, large-scale studies are needed to better understand the origin and distribution of virulent strains of this pathogen from different sources, in this country and worldwide.
Therefore, a future strategy should focus on extended multi-laboratory studies applying an appropriate molecular typing technique that allows exchanging data to better understand the origin and distribution of virulent strains of this pathogen in all ecological niches.  Acknowledgments: The authors would like to thank Ermolaeva, S.A. and Psareva, E.K. for her excellent and valuable comments and suggestions and to Lunina, D.A. for her great assistance in the map creation.

Conflicts of Interest:
The authors declare no conflict of interest.