Characterisation of Listeria monocytogenes Isolates from Hunted Game and Game Meat from Finland

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.


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].

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).
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.

Results
In total, 75 L. monocytogenes isolates from 75 hunted game and game meat samplesisolated in Finland between 2012 and 2020-were serotyped and characterised by sevengene 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.
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).  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.

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/infectiousdiseases-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. monocytogenescontaminated 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 isolatesforming 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 CL451cboth 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, prf A and orf X) 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 trimethoprimsulfamethoxazole 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.

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.