Abstract
Listeria monocytogenes is an important foodborne zoonotic bacterium. It is a heterogeneous species that can be classified into lineages, serogroups, clonal complexes, and sequence types. Only scarce information exists on the properties of L. monocytogenes from game and game meat. We characterised 75 L. monocytogenes isolates from various game sources found in Finland between 2012 and 2020. The genetic diversity, presence of virulence and antimicrobial genes were studied with whole genome sequencing. Most (89%) of the isolates belonged to phylogenetic lineage (Lin) II and serogroup (SG) IIa. SGs IVb (8%) and IIb (3%) of Lin I were sporadically identified. In total, 18 clonal complexes and 21 sequence types (STs) were obtained. The most frequent STs were ST451 (21%), ST585 (12%) and ST37 (11%) found in different sample types between 2012 and 2020. We observed 10 clusters, formed by closely related isolates with 0–10 allelic differences. Most (79%) of the virulence genes were found in all of the L. monocytogenes isolates. Only fosX and lin were found out of 46 antimicrobial resistance genes. Our results demonstrate that potentially virulent and antimicrobial-sensitive L. monocytogenes isolates associated with human listeriosis are commonly found in hunted game and game meat in Finland.
Keywords: Listeria monocytogenes, game, whole genome sequencing, sequence type, virulence, antimicrobial resistance
1. Introduction
Listeria monocytogenes has emerged over recent decades as an important foodborne pathogen responsible for numerous outbreaks [1]. L. monocytogenes is responsible for listeriosis, a disease affecting both humans and animals. Foodborne listeriosis typically causes a self-limited gastroenteritis among healthy people [2]. However, invasive infection leading to hospitalisation and even death may occur, especially among immunocompromised people [1]. Invasive listeriosis may also lead to abortion in pregnant women. The severity of listeriosis depends, inter alia, on the virulence of the bacterial strain [2]. Invasive listeriosis, in particular, requires antimicrobial treatment. Listeriosis had the highest proportion of hospitalised cases of all zoonoses in 2020 in the EU [3].
L. monocytogenes is a ubiquitous bacterium that can survive in a variety of environments and grow at low temperatures, e.g., in cold-stored foods [4]. Soil and decaying organic material are important sources of L. monocytogenes, and mammals and birds can spread this pathogen through faecal shedding [5]. L. monocytogenes-contaminated food is an important source attributed to human infections [6]. The consumption of contaminated food has been linked to both epidemic and sporadic listeriosis. Poor hygiene practices and inadequate sanitation procedures in the food processing industry can lead to listeriosis outbreaks [4,7].
L. monocytogenes has sporadically been found in game animals and on game carcasses [8,9,10,11]. Detection rates of L. monocytogenes in deer and wild boar faeces have been low, varying between 0 and 6% [12]. However, this pathogen is more frequently present in the tonsils than in faeces [12,13,14,15]. In Spain, L. monocytogenes was detected in 44% and 41% of deer and wild boar tonsils, respectively [12]. Recently, L. monocytogenes was detected in 5% of deer carcasses in Austria using an antigen test [16] and in 12% of deer carcasses in Finland using a polymerase chain reaction (PCR) [17]. This relatively high prevalence of L. monocytogenes on deer carcasses shows the importance of observing hygiene practices during hunting and slaughtering.
L. monocytogenes is a very heterogeneous species, which can be divided into at least 14 serotypes based on variation in the somatic (O) and flagellar (H) antigens [18,19]. Over 95% of the human and food strains are linked to four (1/2a, 1/2b, 1/2c, and 4b) serotypes. Genetically, L. monocytogenes can be divided into four phylogenetic lineages (Lin), six serogroups (SGs), multiple clonal complexes (CCs), and sequence types (STs) [2,5]. Most clinical strains found in humans belong to Lin I (SGs IIb and IVb) and II (SG IIa) [20], whereas food strains more frequently belong to SG IIa [21,22].
Whole genome sequencing (WGS)—an accurate method with a high resolution—is currently becoming the method of choice for characterising L. monocytogenes isolates [20]. It has emerged as a powerful tool for outbreak investigations and is increasingly also used for the surveillance and monitoring of listeriosis [23]. Investigating the diversity of L. monocytogenes isolates from game and game meat will provide valuable information on the significance of game in the meat production chain and in human infections.
Studies on the genetic relationships between L. monocytogenes isolates from game sources remain scarce. Game and game meat may play an important role in the L. monocytogenes infection cycle. The aim of our study was to use WGS to investigate the diversity and genetic relationships between L. monocytogenes isolates from hunted game and game meat in Finland. Furthermore, we studied the presence of important virulence and resistance genes obtained from the WGS data.
2. Materials and Methods
2.1. Listeria Monocytogenes Isolates
L. monocytogenes has been detected in hunted game and game meat in Finland between 2012 and 2020, especially in deer and mallard meat (Table 1). We characterised a total of 75 L. monocytogenes isolates from various game sources in this study (Table 1). One isolate per positive sample from the earlier studies was characterised. The sampling plan and time frame differed between the earlier studies (Table 1). Moose, deer, and wild boar samples were collected from wild hunted animals and game bird samples from game birds that were farmed for hunting. Deer and mallard meat samples were collected from a small meat processing plant. L. monocytogenes isolates were found after PCR screening in our microbiological laboratory at the Department of Food Hygiene and Environmental Health of the Faculty of Veterinary Medicine, University of Helsinki, in Helsinki, Finland. PCR screening and isolation of the isolates have been described in earlier studies [17,24].
Table 1.
Isolation rates of Listeria monocytogenes from hunted game and game meat in Finland between 2012 and 2020.
| Scheme 2012 | Sampling Year | Number of Samples | Positive Samples | Reference | |
|---|---|---|---|---|---|
| Moose carcass | 2012–2014 | 100 | 5 | (5%) | [17] |
| Deer carcass | 2013–2015 | 100 | 5 | (5%) | [17] |
| Deer meat | 2019–2020 | 50 | 9 | (18%) | Not published |
| Wild boar organ | 2016 | 130 | 40 | (31%) | [25] |
| Pheasant faeces | 2013–2014 | 101 | 9 | (9%) | [24] |
| Teal faeces | 2013 | 30 | 1 | (3%) | [24] |
| Mallard faeces | 2013–2014 | 110 | 15 | (14%) | [24] |
| Mallard meat | 2016 | 100 | 13 | (13%) | [24] |
2.2. Whole Genome Sequencing (WGS)
DNA of L. monocytogenes isolates was purified from overnight enrichment at 37 °C in tryptic soya broth using PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbaden, CA, USA) according to the manufacturer’s protocol. DNA quality was measured with a NanoDrop™ spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and DNA quantity with a Qubit fluorometer (ThermoFisher Scientific). WGS was performed on the Illumina platform by CeGaT (Center for Genomics and Transcriptomics, Tübingen, Germany). Illumina DNA Prep library preparation kit and NovaSeq6000 were used to generate 100 bp paired end reads. The short raw reads were assembled de novo using a Unicycler v0.4.8 assembler available on the PATRIC 3.6.12 platform (https://www.patricbrc.org/app/Assembly, accessed on 11 November 2022).
2.3. Characterisation of Listeria Monocytogenes Isolates
Species identification was confirmed in silico from the assemblies with KmerFinder v3.2 and SpeciesFinder v2.0 [26] available on the CGE (Center for Genomic Epidemiology) platform (http://www.genomicepidemiology.org/services/, accessed on 11 November 2022). In silico typing using 7-gene multi-locus sequence typing (MLST) [27] was performed on the CGE and BIGSdb-Lm (https://bigsdb.pasteur.fr/listeria/, accessed on 11 November 2022) platforms. STs obtained through the 7-gene MLST were grouped into CCs and phylogenetic Lin [27].
Assembled sequence data of 55 L. monocytogenes isolates were genotyped with core genome MLST (cgMLST) based on 1748 genes [28] using the open-source tool available on the BIGSdb-Lm platform. The nearest cgMLST profile (CT) from the database was recorded. Additionally, cgMLST targeting 1701 genes was performed using Ridom SeqSphere+ software v7.7.5 (Ridom GmbH, Muenster, Germany) [29] and the results were visualised with a minimum spanning tree (MST). Isolates forming a cluster (CL) displayed a maximum of 10 allelic differences from each other. The CLs were shaded in grey, and the number of allelic differences between the isolates was indicated on the connecting lines. Using the default parameters in the Ridom software, (1) STs, (2) PCR serogroups (SGs), (3) virulence genes and (4) antimicrobial resistance genes were also determined. Presence of the virulence genes was additionally studied with the VirulenceFinder 2.0 available on the CGE platform and on the Virulence Factor Database (VFDB) [30] (http://www.mgc.ac.cn/VFs/, accessed on 11 November 2022). In total, the presence of 33 virulence genes and 46 AMR genes among the 55 L. monocytogenes isolates was recorded.
3. Results
In total, 75 L. monocytogenes isolates from 75 hunted game and game meat samples—isolated in Finland between 2012 and 2020—were serotyped and characterised by seven-gene MLST (Table 2). Most (89%) of the isolates belonged to serotype 1/2a and were found in all sample types. Serotypes 4b and 2b were identified among 8% and 3% of the isolates, respectively. L. monocytogenes 4b was found on deer carcasses (n = 3), wild boar organs (n = 2) and in pheasant faeces (n = 1) (Table 2). L. monocytogenes 2b was only found in mallard faeces.
Table 2.
Serotypes and sequence type (STs) using 7-gene multi-locus sequence typing (MLST) of 75 Listeria monocytogenes isolates obtained from hunted game and game meat in Finland between 2012 and 2022.
| Source | Isolation Year | Number of Isolates | Serotype | MLST |
|---|---|---|---|---|
| Moose carcass | 2012–2013 | 5 | 1/2a | ST7, 29, 37, 451 |
| Deer carcass | 2013–2014 | 5 | 1/2a (2), 4b (3) | ST4, 18, 315, 412 |
| Deer meat | 2019 | 9 | 1/2a | ST8, 11, 155 |
| Mallard faeces | 2013–2014 | 15 | 1/2a (13), 1/2b (2) | ST11, 37, 224, 391, 585 |
| Mallard meat | 2016 | 13 | 1/2a | ST8, 18, 412, 451, 585 |
| Teal faeces | 2013 | 1 | 1/2a | ST37 |
| Pheasant faeces | 2013–2014 | 9 | 1/2a (8), 4b (1) | ST1, 7, 20, 37, 585 |
| Wild boar faeces | 2020 | 1 | 1/2a | ST451 |
| Wild boar organ | 2016 | 17 | 1/2a (15), 4b (2) | ST1, 7, 8, 18, 20, 21, 37, 91, 399, 451, 573 |
Based on the seven-gene MLST, 75 L. monocytogenes isolates from hunted game and game meat samples (n = 75) were classified into Lin I and II, 18 CCs and 21 STs (Table 3). Most of the isolates (89%) belonged to Lin II, including all serotype 1/2a isolates. Lin I included isolates of serotypes 1/2b and 4b. ST451 (16/75) was the most frequent ST followed by ST585 (9/75) and ST37 (8/75). These STs were found from different sample types between 2012 and 2020 (Table 3). ST451 and ST37 have frequently been identified in human listeriosis during recent years in Finland (Table 3). In total, 8 out of 21 STs found in game have been identified in cases of human listeriosis in Finland between 2016 and 2021. Most (17/75) of the isolates from wild boars hunted in 2016 belonged to several (11/21) STs (Figure 1). L. monocytogenes isolates were also frequently found from mallard faeces (15/75) and mallard meat (13/75) (Table 2). These samples were contaminated with less (5/75) STs compared with wild boar organs.
Table 3.
Characteristics of 75 Listeria monocytogenes isolates from hunted game and game meat from Finland between 2012 and 2020.
| MLST | Finland b | Clonal Complex | Lineage | Serotype | Number of Isolates | Source (Isolation Year) |
|---|---|---|---|---|---|---|
| ST1 a | 2016, 2017 | CC1 | I | 4b | 3 | Pheasant faeces (2013), Wild boar organ (2016) |
| ST4 | CC4 | I | 4b | 1 | Deer carcass (2014) | |
| ST7 | 2018–2021 | CC7 | II | 1/2a | 4 | Moose carcass (2012), Pheasant faeces (2013), Wild boar organ (2016) |
| ST8 | 2017–2018, 2020–2021 | CC8 | II | 1/2a | 3 | Mallard meat (2016), Wild boar organ (2016), Deer meat (2019) |
| ST11 | CC11 | II | 1/2a | 1 | Deer meat (2019) | |
| ST18 | 2016 | CC18 | II | 1/2a | 5 | Deer carcass (2013), Wild boar organ (2016), Mallard meat (2019) |
| ST20 | CC20 | II | 1/2a | 3 | Pheasant faeces (2013), Wild boar organ (2016) | |
| ST21 | CC21 | II | 1/2a | 2 | Wild boar organ (2016) | |
| ST29 | CC29 | II | 1/2a | 1 | Moose carcass (2013) | |
| ST37 | 2016, 2018–2020 | CC37 | II | 1/2a | 8 | Moose carcass (2013), Teal faeces (2013), Mallard faeces (2013–2014), Pheasant faeces (2013–2014) Wild boar organ (2016) |
| ST91 | 2021 | CC14 | II | 1/2a | 1 | Wild boar organ (2019) |
| ST155 | 2020 | CC155 | II | 1/2a | 3 | Deer meat (2019) |
| ST224 | CC224 | I | 1/2b | 2 | Mallard faeces (2013) | |
| ST249 | CC315 | I | 4b | 2 | Deer carcass (2013) | |
| ST391 | CC89 | II | 1/2a | 2 | Mallard faeces (2013) | |
| ST399 | CC14 | II | 1/2a | 1 | Wild boar organ (2016) | |
| ST400 | CC11 | II | 1/2a | 2 | Mallard faeces (2013) | |
| ST412 | CC412 | II | 1/2a | 5 | Deer carcass (2013), Mallard meat (2019) | |
| ST451 | 2017–2021 | CC11 | II | 1/2a | 16 | Moose carcass (2012), Mallard faeces (2013–2014), Mallard meat (2016), Wild boar organ (2016), Deer meat (2019), Wild boar faeces (2020) |
| ST573 | CC573 | II | 1/2a | 1 | Wild boar organ (2016) | |
| ST585 | ST585 | II | 1/2a | 9 | Pheasant faeces (2013–2014), Mallard faeces (2013–2014), Mallard meat (2016) |
Figure 1.
Sequence types (ST), which include at least three Listeria monocytogenes isolates, found in hunted game and game meat in Finland between 2012 and 2020.
A subset of 55 out of 75 L. monocytogenes isolates were characterised with cgMLST based on 1748 genes. In total, 35 CTs among 21 STs were obtained using the BIGSdb-Lm platform (Table 4). Overall, 10 CTs (CT5208, 11797, 20896, 20939, 25365, 26674, 26763, 28125, 28250, and 28251) included more than one L. monocytogenes isolate. In total, 10 CLs (CL1, 8, 18, 155, 412, 451a, 451b, 451c, 451d, and 585) formed by closely related genotypes were obtained with the cgMLST based on 1701 genes using the Ridom software (Table 4). Isolates belonging to the same CL showed an allelic difference between 0 and 10 (Figure 2). All CLs obtained by Ridom software had their own CT obtained from the BIGSSdb-Lm platform (Table 4). Most (9/10) of the CLs included isolates of SG IIa. CL1 included two undistinguishable isolates of SG IVb, both found from wild boar organs. Five CLs (CL1, 18, 155, 412 and 451c) included very closely related isolates, each with 0 to 5 allelic differences (Figure 2). Each of these CLs included L. monocytogenes isolates found from one source during the same year (Table 4). CL18 and CL412 were formed by isolates from mallard meat and CL155 and CL451c from deer meat. The other CLs (CL8, 451a, 451b, 451d and 585) were formed by closely related isolates (0 to 10 allele differences) found from different sources between 2012 and 2019 (Table 4).
Table 4.
Distribution of different serogroups (SGs), MLST (STs) and cgMLST (CTs) profiles, clusters (CLs) and virulence profiles (VPs) among 55 Listeria monocytogenes isolates from hunted game and game meat between 2012 and 2020 in Finland.
| ST a | SG | CT b | CL c | VP d | No. | Source | Year | |
|---|---|---|---|---|---|---|---|---|
| 1 | IVb | 1430 | 2c | 1 | Pheasant | Faeces | 2013 | |
| 1 | IVb | 25365 | 1 | 2c | 2 | Wild boar | Organ | 2016 |
| 4 | IVb | 27292 | 1d | 1 | Deer | Carcass | 2014 | |
| 7 | IIa | 21218 | 2a | 1 | Wild boar | Organ | 2016 | |
| 7 | IIa | 22874 | 1e | 1 | Moose | Carcass | 2012 | |
| 8 | IIa | 19030 | 2a | 1 | Wild boar | Organ | 2016 | |
| 8 | IIa | 28125 | 8 | 1e | 2 | Mallard, Deer | Meat | 2016, 2019 |
| 11 | IIa | 30149 | 1b | 1 | Deer | Meat | 2019 | |
| 18 | IIa | 26674 | 18 | 1a | 3 | Mallard | Meat | 2016 |
| 18 | IIa | 28197 | 1a | 1 | Wild boar | Organ | 2016 | |
| 20 | IIa | 21358 | 1a | 1 | Wild boar | Organ | 2016 | |
| 20 | IIa | 26681 | 0 | 1 | Pheasant | Faeces | 2013 | |
| 21 | IIa | 9841 | 2a | 1 | Wild boar | Organ | 2016 | |
| 21 | IIa | 25363 | 2a | 1 | Wild boar | Organ | 2016 | |
| 29 | IIa | 23920 | 1e | 1 | Moose | Carcass | 2013 | |
| 37 | IIa | 22893 | 1e | 1 | Teal | Faeces | 2013 | |
| 37 | IIa | 28230 | 1e | 1 | Pheasant | Faeces | 2014 | |
| 37 | IIa | 30787 | 2a | 1 | Wild boar | Organ | 2016 | |
| 91 | IIa | 20232 | 1a | 1 | Wild boar | Organ | 2016 | |
| 155 | IIa | 26763 | 155 | 0 | 3 | Deer | Meat | 2019 |
| 224 | IIb | 8887 | 1c | 1 | Mallard | Faeces | 2013 | |
| 249 | IVb | 20958 | 1d | 1 | Deer | Carcass | 2013 | |
| 391 | IIa | 29935 | 0 | 1 | Mallard | Meat | 2013 | |
| 399 | IIa | 22031 | 2b | 1 | Wild boar | Organ | 2016 | |
| 400 | IIa | 28173 | 1b | 1 | Mallard | Meat | 2013 | |
| 412 | IIa | 8287 | 3b | 1 | Deer | Carcass | 2013 | |
| 412 | IIa | 20896 | 412 | 2d | 4 | Mallard | Meat | 2016 |
| 451 | IIa | 24184 | 0 | 1 | Deer | Meat | 2019 | |
| 451 | IIa | 11793 | 0 | 1 | Wild boar | Faeces | 2020 | |
| 451 | IIa | 5208 | 451a | 1a | 3 | Mallard, wild boar | Meat, organ | 2016 |
| 451 | IIa | 11797 | 451b | 0,1a | 2 | Moose, wild boar | Carcass, organ | 2012, 2016 |
| 451 | IIa | 20939 | 451c | 0 | 3 | Deer | Meat | 2019 |
| 451 | IIa | 28250 | 451d | 0,1a | 4 | Mallard, wild boar | Faeces, organ | 2014, 2016 |
| 573 | IIa | 1569 | 2a | 1 | Wild boar | Faeces | 2016 | |
| 585 | IIa | 28251 | 585 | 1e,2a,3a | 4 | Mallard | Faeces, meat | 2013, 2016 |
a ST based on 7-gene MLST using the BIGSdb-Lm platform and Ridom software. b Nearest CT based on cgMLST (1748 target genes) using the BIGSdb-Lm platform. c CL based cgMLST (1708 target genes) using the Ridom software. d VPs using the Ridom software and CGE platform.
Figure 2.
Minimum spanning tree of 55 Listeria monocytogenes isolates from hunted game and game meat in Finland during 2012–2020. The tree was calculated in Ridom SeqSphere+ with 1708 core genome multi-locus sequence typing (MLST) targets and 7-gene MLST targets (pairwise ignoring missing values, logarithmic scale). Nodes are coloured according to sequence type. Number of allelic differences between the isolates are indicated on the connecting lines. Clusters are shaded in grey and a cluster distance threshold of maximum10 was used according to Ruppitsch et al. [29].
We studied the presence of 33 virulence genes available in the Ridom software [34] among the 55 L. monocytogenes isolates. Most (26/33) of the genes were detected in all isolates. Seven virulence genes (act, ami, aut, inlF, inlJ, lapB and vip) were not present in all isolates. We designed 12 virulence profiles (VPs) based on these genes (Table 5). All virulence genes (VP0) were detected in 12 (22%) L. monocytogenes isolates, all belonging to SG IIa. The most frequently missing genes were ami and vip, which were missing in 42% and 38% of L. monocytogenes isolates, respectively. The VP did not correlate with ST, but isolates belonging to the same cluster mostly (66%) had the same VP (Table 4).
Table 5.
Virulence profiles detected among 55 Listeria monocytogenes isolates.
| Virulence Profile |
Number of Isolates | Sequence Types | Missing Virulence Genes (=1) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| act | ami | aut | inlF | inlJ | lapB | vip | |||
| VP0 | 12 | ST20,155,391,451 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| VP1a | 13 | ST18,20,91,451 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
| VP1b | 2 | ST11,400 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
| VP1c | 1 | ST224 | 0 | 0 | 0 | 0 | 1 | 0 | 0 |
| VP1d | 2 | ST4,249 a | 0 | 0 | 1 | 0 | 0 | 0 | 0 |
| VP1e | 7 | ST7,8,29,37, 585 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| VP2a | 7 | ST7,8,21,37,573,585 | 0 | 1 | 0 | 0 | 0 | 0 | 1 |
| VP2b | 1 | ST399 | 0 | 1 | 0 | 1 | 0 | 0 | 0 |
| VP2c | 3 | ST1 | 0 | 0 | 1 | 0 | 1 | 0 | 0 |
| VP2d | 4 | ST412 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
| VP3a | 2 | ST585 | 0 | 1 | 0 | 0 | 1 | 0 | 1 |
| VP3b | 1 | ST412 | 1 | 0 | 0 | 0 | 0 | 1 | 1 |
a Isolates with sequence types in bold belong to serogroup IVb.
We studied the presence of 46 AMR genes available in the Ridom software. Only the fosX and lin genes were detected in all 55 L. monocytogenes isolates.
4. Discussion
L. monocytogenes is a common finding in hunted game and game meat in Finland. Most of the L. monocytogenes isolates originating from game belonged to serotype 1/2a (SG IIa, Lin II) but serotype 4b (SG IVb, Lin I) was also found. L. monocytogenes strains belonging to SG IIa/Lin II and SG IVb/Lin I are responsible for the largest share of listeriosis [20,35,36]. However, SG IVb is more frequently associated with human diseases and outbreaks than SG IIa, which is more often identified among isolates found in animal, environmental and food samples [6,18,21]. Recently, L. monocytogenes IIa and IVb were found in deer and wild boar tonsils in Spain [12]. Serotyping and serogrouping provide useful information about L. monocytogenes isolates found in epidemiological studies, surveys and during monitoring.
Very little is known about the genetic diversity of L. monocytogenes isolates from game origin [12]. We found several CCs and STs in hunted game and game meat from Finland showing a large genetic diversity among the L. monocytogenes isolates studied. This was expected because L. monocytogenes isolates were found from various sources and locations during a ten-year period [17,24,25]. All CCs identified among our hunted game and game meat isolates from Finland have recently been identified among various environmental and animal sources in Europe [5]. In our data, the most common CC was CC11 (25%), which included three STs: ST11, ST400 and ST451. CC11 is also a prevalent clonal type found in Europe [23,31]. Most (67%) of the STs found in our study have also been found in Europe from various sources [23,31,32]. Several (7/21) STs found in game in our study have been associated with human listeriosis in Finland (https://thl.fi/en/web/infectious-diseases-and-vaccinations accessed on 11 November 2022). ST451 (21%) was the most frequently found ST in our study, as it was found in different sample types between 2012 and 2020. This type has also been reported in human listeriosis in Finland yearly between 2017 and 2021. ST451 is a common universal ST found in humans, animals, foods, and the environment in Europe [23,31,32,37]. To obtain more accurate information about the link between human and game isolates, STs based on the core or whole genome (cgMLST or wgMLST) should be used instead of seven-gene MLST.
Wild boar organs were contaminated with several L. monocytogenes isolates of different STs. This is very understandable because the isolates originated from wild boars hunted in various geographical locations in Finland [25]. Fewer STs were found in isolates from deer and mallard meat than from wild boar. Deer and mallard meat were processed in one meat processing plant each, which may explain the limited genetic diversity among the meat isolates. Interestingly, only 4 STs were identified among 13 L. monocytogenes SG IIa isolates from mallard faeces. The hunted mallards were reared and fed in a natural pond before being hunted, which could be a common contamination source for the mallards [24]. L. monocytogenes is relatively commonly found in various environments, and L. monocytogenes-contaminated soil and water are therefore important L. monocytogenes sources [5,38]. ST18, ST20, ST37, ST91 and ST451, identified among our game isolates, are reportedly common STs among isolates from environmental samples in Finland [37] and Latvia [31].
We identified some CLs of L. monocytogenes isolates with 0 to 10 allelic differences among the hunted game and game meat isolates using cgMLST, which is the method capable of differentiating related strains from unrelated ones [39]. Very closely related isolates, with a maximum of five allelic differences, were found in five CLs, and they originated from the same source and year, which could explain the high genetic similarity and may indicate a common source of contamination. Three very closely related L. monocytogenes isolates—forming CL18—were isolated from mallard meat originated from various mallards sampled on the same day in the same game meat processing plant, indicating a cross-contamination during processing. In CL412, four very closely related isolates from mallards were sampled on two different days in the same plant, indicating a plant contamination possibly due to inadequate cleaning. Deer meat isolates also formed two clusters—CL155 and CL451c—both with three very closely related isolates. The isolates in CL155 and CL451c were from deer meat samples cut on different days in the same plant. Cross-contamination during meat cutting occurs easily if working hygiene is poor. L. monocytogenes can easily persist in the plant, and thorough cleaning of the meat processing plant after each working day is therefore very important.
L. monocytogenes has shown heterogeneity in its virulence [35,40]. Virulence factors are essential for adapting L. monocytogenes to spread optimally within the environment [35]. The virulence of L. monocytogenes is encoded by a wide range of virulence genes [2]. In our study, most (79%) of the 33 studied virulence genes were present in all 55 L. monocytogenes isolates of game origin in Finland. The actA gene located on the Listeria pathogenicity island (LIPI-1) was missing in only one isolate (a deer carcass isolate). LIP-I is composed of important virulence genes (including actA, hly, mpl, plcA, plcB, prfA and orfX) and is necessary for intracellular survival and spread from cell to cell [35]. LPI-1 is typically present in all L. monocytogenes strains [2,41]. This actA-negative deer carcass isolate (with VP3b, SG IIa and ST412) also missed the lapB (coding for an adhesion protein) and vip (coding for an invasion protein) genes, indicating a reduced virulence in this isolate. The most frequently missing virulence gene ami, which is coding an autolysin protein for adherence, was not found in 42% of the isolates. However, the meaning of ami in the virulence remains unclear. The invasion gene aut was missing only in the isolates belonging to SG IVb. All SG IVb isolates (with ST1, ST4 and ST249) were aut-negative. The three SG IVb isolates belonging to ST1 were also inlJ-negative. The aut gene codes for an autolysin protein needed for invasion and the inlJ for an internalin protein needed for adherence [35]. How the absence of these two genes affects the virulence of L. monocytogenes IVb isolates needs to be further studied. Typically, ST1 (CC1) and ST4 (CC4) have been associated with clinical cases more often than other STs [35,41].
AMR is a serious public health issue due to increasing resistance. There is also a trend of increasing AMR among L. monocytogenes strains of animal and food origin. Resistance, e.g., to penicillin, ampicillin, gentamycin, streptomycin, tetracycline, and trimethoprim-sulfamethoxazole has been reported [42,43]. However, in our study, only the fosX and lin genes were detected. These two genes were present in all 55 L. monocytogenes isolates. Earlier studies have shown fosX and lin to be present in nearly all L. monocytogenes isolates [42]. This can be explained by native resistance to fosfomycin and lincosamides reported in L. monocytogenes strains [34,43]. Our results indicate that L. monocytogenes of game and game meat origin found in Finland are so far sensitive to antimicrobials. One explanation may be that hunted game in Finland have no access to feed contaminated with resistant L. monocytogenes strains.
5. Conclusions
In this study, we analysed the sequence data of L. monocytogenes isolates of game origin using tools available on open-source platforms and Ridom software. Our study demonstrates that game meat is contaminated with various STs associated with human listeriosis. All L. monocytogenes isolates were potentially pathogenic, carrying most important virulence genes. No acquired AMR genes were found, indicating that all isolates were sensitive to most of the important antimicrobials used to treat listeriosis. Some of the isolates from mallard and deer meat belonged to CLs that were formed by very closely related isolates, indicating common contamination sources. Contaminated game meat may pose a public health problem, and game meat should therefore be handled and stored correctly.
Acknowledgments
Open access funding provided by University of Helsinki. Maria Stark is gratefully acknowledged for her technical assistance.
Author Contributions
Conceptualisation: all authors; Investigation: M.S., P.K. and V.H.; Methodology: M.S. and A.H.; Software: P.K. and V.H.; Visualisation: P.K.; Validation: all authors; Analysis: M.F.-A.; Writing—original draft preparation: M.F.-A.; Writing—review and editing: P.P.; Resources: M.F.-A., A.H. and P.P.; Supervision, project administration and funding acquisition: M.F.-A. All authors have read and agreed to the published version of the manuscript.
Data Availability Statement
Data are contained within the article.
Conflicts of Interest
The authors declare no conflict of interest.
Funding Statement
This study was partly funded by the “Kyllikki ja Uolevi Lehikoinen” (IQUJA:10019) and “Walter Ehrström” Foundations. Proofreading and APC were funded by the University of Helsinki, Finland.
Footnotes
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Lecuit M. Listeria Monocytogenes, a Model in Infection Biology. Cell. Microbiol. 2020;22:e13186. doi: 10.1111/cmi.13186. [DOI] [PubMed] [Google Scholar]
- 2.Quereda J.J., Morón-García A., Palacios-Gorba C., Dessaux C., García-del Portillo F., Pucciarelli M.G., Ortega A.D. Pathogenicity and Virulence of Listeria Monocytogenes: A Trip from Environmental to Medical Microbiology. Virulence. 2021;12:2509–2545. doi: 10.1080/21505594.2021.1975526. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.EFSA. ECDC The European Union One Health 2020 Zoonoses Report. EFSA J. 2021;19:e06971. doi: 10.2903/j.efsa.2021.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Matereke L.T., Okoh A.I. Listeria Monocytogenes Virulence, Antimicrobial Resistance and Environmental Persistence: A Review. Pathogens. 2020;9:528. doi: 10.3390/pathogens9070528. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Félix B., Sevellec Y., Palma F., Douarre P.E., Felten A., Radomski N., Mallet L., Blanchard Y., Leroux A., Soumet C. A European-Wide Dataset to Uncover Adaptive Traits of Listeria Monocytogenes to Diverse Ecological Niches. Sci. Data. 2022;9:1–12. doi: 10.1038/s41597-022-01278-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Acciari V.A., Ruolo A., Torresi M., Ricci L., Pompei A., Marfoglia C., Valente F.M., Centorotola G., Conte A., Salini R. Genetic Diversity of Listeria Monocytogenes Strains Contaminating Food and Food Producing Environment as Single Based Sample in Italy (Retrospective Study) Int. J. Food Microbiol. 2022;366:109562. doi: 10.1016/j.ijfoodmicro.2022.109562. [DOI] [PubMed] [Google Scholar]
- 7.Osek J., Lachtara B., Wieczorek K. Listeria Monocytogenes in Foods—From Culture Identification to Whole-genome Characteristics. Food Sci. Nutr. 2022;10:2825–2854. doi: 10.1002/fsn3.2910. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Gomes-Neves E., Abrantes A.C., Vieira-Pinto M., Müller A. Wild Game Meat—A Microbiological Safety and Hygiene Challenge? Curr. Clin. Microbiol. Rep. 2021;8:31–39. doi: 10.1007/s40588-021-00158-8. [DOI] [Google Scholar]
- 9.Obwegeser T., Stephan R., Hofer E., Zweifel C. Shedding of Foodborne Pathogens and Microbial Carcass Contamination of Hunted Wild Ruminants. Vet. Microbiol. 2012;159:149–154. doi: 10.1016/j.vetmic.2012.03.031. [DOI] [PubMed] [Google Scholar]
- 10.Paulsen P., Winkelmayer R. Seasonal Variation in the Microbial Contamination of Game Carcasses in an Austrian Hunting Area. Eur. J. Wildl. Res. 2004;50:157–159. doi: 10.1007/s10344-004-0054-z. [DOI] [Google Scholar]
- 11.Sasaki Y., Goshima T., Mori T., Murakami M., Haruna M., Ito K., Yamada Y. Prevalence and Antimicrobial Susceptibility of Foodborne Bacteria in Wild Boars (Sus Scrofa) and Wild Deer (Cervus Nippon) in Japan. Foodborne Pathog. Dis. 2013;10:985–991. doi: 10.1089/fpd.2013.1548. [DOI] [PubMed] [Google Scholar]
- 12.Palacios-Gorba C., Moura A., Leclercq A., Gómez-Martín Á., Gomis J., Jiménez-Trigos E., Mocé M.L., Lecuit M., Quereda J.J. Listeria spp. Isolated from Tonsils of Wild Deer and Boars: Genomic Characterization. Appl. Environ. Microbiol. 2021;87:e02651-20. doi: 10.1128/AEM.02651-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Stella S., Tirloni E., Castelli E., Colombo F., Bernardi C. Microbiological Evaluation of Carcasses of Wild Boar Hunted in a Hill Area of Northern Italy. J. Food Prot. 2018;81:1519–1525. doi: 10.4315/0362-028X.JFP-18-077. [DOI] [PubMed] [Google Scholar]
- 14.Wacheck S., Fredriksson-Ahomaa M., König M., Stolle A., Stephan R. Wild Boars as an Important Reservoir for Foodborne Pathogens. Foodborne Pathog. Dis. 2010;7:307–312. doi: 10.1089/fpd.2009.0367. [DOI] [PubMed] [Google Scholar]
- 15.Weindl L., Frank E., Ullrich U., Heurich M., Kleta S., Ellerbroek L., Gareis M. Listeria Monocytogenes in Different Specimens from Healthy Red Deer and Wild Boars. Foodborne Pathog. Dis. 2016;13:391–397. doi: 10.1089/fpd.2015.2061. [DOI] [PubMed] [Google Scholar]
- 16.Paulsen P., Smulders F., Kukleci E., Tichy A. Bacterial Surface Counts and Visually Assessed Cleanliness of Carcasses from Hunted Roe Deer (Capreolus Capreolus) Wien. Tierarztl. Mon. 2022;109:doc3. [Google Scholar]
- 17.Sauvala M., Laaksonen S., Laukkanen-Ninios R., Jalava K., Stephan R., Fredriksson-Ahomaa M. Microbial Contamination of Moose (Alces Alces) and White-Tailed Deer (Odocoileus Virginianus) Carcasses Harvested by Hunters. Food Microbiol. 2019;78:82–88. doi: 10.1016/j.fm.2018.09.011. [DOI] [PubMed] [Google Scholar]
- 18.Alía A., Andrade M.J., Córdoba J.J., Martín I., Rodríguez A. Development of a Multiplex Real-Time PCR to Differentiate the Four Major Listeria Monocytogenes Serotypes in Isolates from Meat Processing Plants. Food Microbiol. 2020;87:103367. doi: 10.1016/j.fm.2019.103367. [DOI] [PubMed] [Google Scholar]
- 19.Li F., Ye Q., Chen M., Zhou B., Xiang X., Wang C., Shang Y., Zhang J., Pang R., Wang J. Mining of Novel Target Genes through Pan-Genome Analysis for Multiplex PCR Differentiation of the Major Listeria Monocytogenes Serotypes. Int. J. Food Microbiol. 2021;339:109026. doi: 10.1016/j.ijfoodmicro.2020.109026. [DOI] [PubMed] [Google Scholar]
- 20.Hyden P., Pietzka A., Lennkh A., Murer A., Springer B., Blaschitz M., Indra A., Huhulescu S., Allerberger F., Ruppitsch W. Whole Genome Sequence-Based Serogrouping of Listeria Monocytogenes Isolates. J. Biotechnol. 2016;235:181–186. doi: 10.1016/j.jbiotec.2016.06.005. [DOI] [PubMed] [Google Scholar]
- 21.Lachtara B., Wieczorek K., Osek J. Genetic Diversity and Relationships of Listeria Monocytogenes Serogroup IIa Isolated in Poland. Microorganisms. 2022;10:532. doi: 10.3390/microorganisms10030532. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Zhang H., Chen W., Wang J., Xu B., Liu H., Dong Q., Zhang X. 10-Year Molecular Surveillance of Listeria Monocytogenes Using Whole-Genome Sequencing in Shanghai, China, 2009–2019. Front. Microbiol. 2020;11:551020. doi: 10.3389/fmicb.2020.551020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Fagerlund A., Idland L., Heir E., Møretrø T., Aspholm M., Lindbäck T., Langsrud S. Whole-Genome Sequencing Analysis of Listeria Monocytogenes from Rural, Urban, and Farm Environments in Norway: Genetic Diversity, Persistence, and Relation to Clinical and Food Isolates. Appl. Environ. Microbiol. 2022;88:e02136-e21. doi: 10.1128/aem.02136-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Sauvala M., Woivalin E., Kivistö R., Laukkanen-Ninios R., Laaksonen S., Stephan R., Fredriksson-Ahomaa M. Hunted Game Birds–Carriers of Foodborne Pathogens. Food Microbiol. 2021;98:103768. doi: 10.1016/j.fm.2021.103768. [DOI] [PubMed] [Google Scholar]
- 25.Fredriksson-Ahomaa M., London L., Skrzypczak T., Kantala T., Laamanen I., Biström M., Maunula L., Gadd T. Foodborne Zoonoses Common in Hunted Wild Boars. EcoHealth. 2020;17:512–522. doi: 10.1007/s10393-020-01509-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Larsen M.V., Cosentino S., Lukjancenko O., Saputra D., Rasmussen S., Hasman H., Sicheritz-Pontén T., Aarestrup F.M., Ussery D.W., Lund O. Benchmarking of Methods for Genomic Taxonomy. J. Clin. Microbiol. 2014;52:1529–1539. doi: 10.1128/JCM.02981-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ragon M., Wirth T., Hollandt F., Lavenir R., Lecuit M., Le Monnier A., Brisse S. A New Perspective on Listeria Monocytogenes Evolution. PLoS Pathog. 2008;4:e1000146. doi: 10.1371/journal.ppat.1000146. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Moura A., Criscuolo A., Pouseele H., Maury M.M., Leclercq A., Tarr C., Björkman J.T., Dallman T., Reimer A., Enouf V. Whole Genome-Based Population Biology and Epidemiological Surveillance of Listeria Monocytogenes. Nat. Microbiol. 2016;2:16185. doi: 10.1038/nmicrobiol.2016.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Ruppitsch W., Pietzka A., Prior K., Bletz S., Fernandez H.L., Allerberger F., Harmsen D., Mellmann A. Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Listeria Monocytogenes. J. Clin. Microbiol. 2015;53:2869–2876. doi: 10.1128/JCM.01193-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Liu B., Zheng D., Zhou S., Chen L., Yang J. VFDB 2022: A General Classification Scheme for Bacterial Virulence Factors. Nucleic Acids Res. 2022;50:D912–D917. doi: 10.1093/nar/gkab1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Terentjeva M., Šteingolde Ž., Meistere I., Elferts D., Avsejenko J., Streikiša M., Gradovska S., Alksne L., Ķibilds J., Bērziņš A. Prevalence, Genetic Diversity and Factors Associated with Distribution of Listeria Monocytogenes and Other Listeria spp. in Cattle Farms in Latvia. Pathogens. 2021;10:851. doi: 10.3390/pathogens10070851. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Filipello V., Mughini-Gras L., Gallina S., Vitale N., Mannelli A., Pontello M., Decastelli L., Allard M.W., Brown E.W., Lomonaco S. Attribution of Listeria Monocytogenes Human Infections to Food and Animal Sources in Northern Italy. Food Microbiol. 2020;89:103433. doi: 10.1016/j.fm.2020.103433. [DOI] [PubMed] [Google Scholar]
- 33.Fagerlund A., Langsrud S., Møretrø T. Microbial Diversity and Ecology of Biofilms in Food Industry Environments Associated with Listeria Monocytogenes Persistence. Curr. Opin. Food Sci. 2021;37:171–178. doi: 10.1016/j.cofs.2020.10.015. [DOI] [Google Scholar]
- 34.Parra-Flores J., Holý O., Bustamante F., Lepuschitz S., Pietzka A., Contreras-Fernández A., Castillo C., Ovalle C., Alarcón-Lavín M.P., Cruz-Córdova A. Virulence and Antibiotic Resistance Genes in Listeria Monocytogenes Strains Isolated from Ready-to-Eat Foods in Chile. Front. Microbiol. 2021;12:796040. doi: 10.3389/fmicb.2021.796040. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Disson O., Moura A., Lecuit M. Making Sense of the Biodiversity and Virulence of Listeria Monocytogenes. Trends Microbiol. 2021;29:811–822. doi: 10.1016/j.tim.2021.01.008. [DOI] [PubMed] [Google Scholar]
- 36.Gorski L., Cooley M.B., Oryang D., Carychao D., Nguyen K., Luo Y., Weinstein L., Brown E., Allard M., Mandrell R.E. Prevalence and Clonal Diversity of over 1,200 Listeria Monocytogenes Isolates Collected from Public Access Waters near Produce Production Areas on the Central California Coast during 2011 to 2016. Appl. Environ. Microbiol. 2022;88:e00357-22. doi: 10.1128/aem.00357-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Castro H., Douillard F.P., Korkeala H., Lindström M. Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria Monocytogenes Strains Persisting on Dairy Farms. Msphere. 2021;6:e00383-21. doi: 10.1128/mSphere.00383-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Farber J.M., Zwietering M., Wiedmann M., Schaffner D., Hedberg C.W., Harrison M.A., Hartnett E., Chapman B., Donnelly C.W., Goodburn K.E. Alternative Approaches to the Risk Management of Listeria Monocytogenes in Low Risk Foods. Food Control. 2021;123:107601. doi: 10.1016/j.foodcont.2020.107601. [DOI] [Google Scholar]
- 39.Chen Y., Gonzalez-Escalona N., Hammack T.S., Allard M.W., Strain E.A., Brown E.W. Core Genome Multilocus Sequence Typing for Identification of Globally Distributed Clonal Groups and Differentiation of Outbreak Strains of Listeria Monocytogenes. Appl. Environ. Microbiol. 2016;82:6258–6272. doi: 10.1128/AEM.01532-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Vázquez-Boland J.A., Kuhn M., Berche P., Chakraborty T., Domínguez-Bernal G., Goebel W., González-Zorn B., Wehland J., Kreft J. Listeria Pathogenesis and Molecular Virulence Determinants. Clin. Microbiol. Rev. 2001;14:584–640. doi: 10.1128/CMR.14.3.584-640.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Schiavano G.F., Ateba C.N., Petruzzelli A., Mele V., Amagliani G., Guidi F., De Santi M., Pomilio F., Blasi G., Gattuso A. Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria Monocytogenes Food and Human Isolates and in Vitro Adhesion/Invasion Assessment. Microorganisms. 2021;10:62. doi: 10.3390/microorganisms10010062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Hanes R.M., Huang Z. Investigation of Antimicrobial Resistance Genes in Listeria Monocytogenes from 2010 through to 2021. Int. J. Environ. Res. Public Health. 2022;19:5506. doi: 10.3390/ijerph19095506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Olaimat A.N., Al-Holy M.A., Shahbaz H.M., Al-Nabulsi A.A., Abu Ghoush M.H., Osaili T.M., Ayyash M.M., Holley R.A. Emergence of Antibiotic Resistance in Listeria Monocytogenes Isolated from Food Products: A Comprehensive Review. Compr. Rev. Food Sci. Food Saf. 2018;17:1277–1292. doi: 10.1111/1541-4337.12387. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data are contained within the article.