Genomic and phenotypic characterisation of Listeria monocytogenes strains isolated from pig feces on farm and from pork meat at retail in France

Evelyne Boscher; Benjamin Félix; Alain Rincé; Patricia Legrandois; Lucas Sajan; Christophe Soumet; Martine Denis

doi:10.1371/journal.pone.0341663

Abstract

Listeria monocytogenes is an ubiquitous foodborne pathogen transmissible to humans through the consumption of contaminated products, including pork. In this study, we characterised the Listeria monocytogenes population circulating from the primary reservoir, pigs, to consumers in France. In 2008 and 2010, 147 strains from on-farm pig feces and 154 strains from retail pork meat were respectively isolated. Serogroups and clonal complexes (CC) were determined (n = 301), and 123 representative strains were further assessed for virulence, biofilm-forming ability, and resistance to Benzalkonium chloride (BC). Serogroup IIb, and CCs such as CC11, CC21, CC20, CC26, CC31, CC59, CC37, CC1 and CC77 strains predominated on farms, whereas serogroup IIc and IIa (respectively CC9 and CC121) were mainly recovered from retail meat. All 123 strains harbored the virulence-associated genes inlA, inlC, inlJ, plcA, prfA, actA, hlyA and iap. Overall virulence did not differ significantly between farm- and retail-derived strains; however, serogroup IVb strains were significantly more virulent, while serogroup IIc strains exhibited lower virulence. In our study, CC1 (mainly present in feces) and CC121 (present only on meat) showed the highest virulence level. Biofilm-forming ability was significantly higher in strains isolated from farm feces than in those from retail meat. Most meat strains, including CC121, CC9, CC14 and CC7, were more resistant to benzalkonium chloride than fecal strains. Our findings highlight the genetic and phenotypic evolution of the Listeria monocytogenes population along the pig-to-pork continuum. They suggest that strains persisting to the retail stage are more strongly associated with resistance to biocides than with robust biofilm formation in the food-processing environment. Contrary to expectations, our CC121 strains exhibited the highest level of virulence, even though this CC is reported hypovirulent.

Citation: Boscher E, Félix B, Rincé A, Legrandois P, Sajan L, Soumet C, et al. (2026) Genomic and phenotypic characterisation of Listeria monocytogenes strains isolated from pig feces on farm and from pork meat at retail in France. PLoS One 21(3): e0341663. https://doi.org/10.1371/journal.pone.0341663

Editor: Shengqian Sun, Yantai Institute of Technology, CHINA

Received: September 17, 2025; Accepted: January 8, 2026; Published: March 2, 2026

Copyright: © 2026 Boscher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data have been added as supplementary table S1.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Listeria monocytogenes is an important foodborne pathogen responsible for listeriosis, a severe disease associated with high mortality rates, particularly among immunocompromised individuals, pregnant women, newborns, and the elderly [1]. All L. monocytogenes isolates have a core set of virulence determinants responsible for the key stages of the Listeria intracellular infection cycle: (i) host cell invasion, (ii) escape from the phagocytic vacuole, (iii) rapid intracellular proliferation, and (iv) actin-based motility and cell-to-cell spread [2; 3].

Pigs and pork products are recognized reservoirs for L. monocytogenes [4]. The presence of the pathogen in these systems represents a dual concern, as it acts both as a vector for human listeriosis and as potential threat to animal health [5]. Several outbreaks of L. monocytogenes linked to ready-to-eat pork meat, jellied pork, pork pies, and pork pâté have been reported highlighting the public health relevance of this contamination route [4].

A major factor contributing to the persistence of L. monocytogenes in food production environments is its remarkable ability to adhere to surfaces and form biofilms. These biofilms, which frequently develop on equipment and surfaces within processing plants, enhance the bacterium’s resistance to environmental stressors and facilitate food contamination [6,7] and thereby increasing public health risks [8]. In addition to biofilm formation, the bacterium’s persistence is further reinforced by its capacity to develop resistance to biocides—chemical disinfectants widely used in the food industry [9,10].

Among the diverse genetic lineages of Listeria monocytogenes, specific clonal complexes (CCs) have been identified as particularly virulent [11] and/or strongly associated with biofilm formation [4]. These clonal complexes exhibit distinct ecological and host-associated adaptations that influence their distribution and prevalence throughout swine production systems. Several in vitro and in vivo model are available to assess the level of virulence of the Listeria monocytogenes strains. Among them, the Galleria mellonella insect larvae model has proven to be a reliable and efficient system for evaluating pathogenicity of Listeria monocytogenes [3].

Resistance to biocide in Listeria monocytogenes can emerge from prolonged exposure to sublethal concentrations of biocides [12], particularly quaternary ammonium compounds (QACs). Such exposure may induce adaptive responses, including efflux pump activation and genetic mutations [13]. These mechanisms not only enhance bacterial survival but also reduce the efficacy of disinfection protocols, especially in the presence of mature biofilms. Within these biofilms, bacteria may display increased tolerance to commonly usedbiocides like benzalkonium chloride and peracetic acid [14].

The aims of this study were:

to identify L. monocytogenes clonal complexes circulating from pig feces on farms to pork meat at retail,
and, to characterize their ability to pass through the food chain and infect human.

We obtained data on their biofilm-forming capacity, their biocide resistance and their virulence level. All these data on these strains are essential for mitigating contamination risks and enhancing food safety.

Materials and methods

Strains

The 301 strains of Listeria monocytogenes analysed in this study were previously isolated during two one-year surveys conducted in mainland France. The first survey, carried out in 2008 at the pig farm level [15] involved pooled fecal samples (sows and fattening pigs) collected from 73 pig farms. From the 34 positive farms, a total of 147 L. monocytogenes strains were recovered. The second survey conducted in 2010 at the retail level [16] involved 320 raw pork meats from supermarkets, yielding 154 strains from the 41 positive meat samples. All fecal and meat samples were analysed for Listeria monocytogenes in our laboratory using the same methodology based on a modified ISO 11290−1 protocol published in 2005 [15]. Characteristic colonies were streaked on Tryptone Soy Agar Yeast Extract (TSAYe) plates (Oxoïd, England) and stored at −80°C in glycerol broth after 24 h at 37°C.

Listeria monocytogenes strains were serogrouped by a multiplex PCR assay following the protocol previously described [17,18]. This multiplex PCR amplifies the genes, lmo0737, lmo1118, ORF2819, ORD2110, and prs (gene for Listeria monocytogenes). The combination of certain amplicons with the prs amplicon allows strains to be classified into serogroup IIa (including serotypes 1/2a, 3a), serogroup IIb (including serotypes 1/2b, 3b, 7), IIc (including serotypes 1/2c, 3c) and serogroup IVb (including serotypes 4b, 4d, 4e). DNA was extracted from fresh bacterial cultures grown on TSAYE plates using the InstaGene® Matrix (BioRad Laboratories, Marnes-la-Coquette France) according to the manufacturer’s instructions.

Pulsed-field gel electrophoresis and clonal complex mapping (n=301 strains)

The 301 strains of Listeria monocytogenes were typed by pulsed-field gel electrophoresis (PFGE). DNA plugs were prepared from fresh bacterial cultures grown on TSAYe plates, and PFGE was performed in accordance with the CDC PulseNet standardized protocol for Listeria monocytogenes [19]. Genomic DNA was digested with two macrorestriction enzymes, ApaI and AscI (Biolabs, Beverly, MA). The ApaI digestion was carried out at 30°C for 6 h, and the AscI digestion at 37°C for 3 h.

Restriction fragments were separated on a 1% SeaKem Gold agarose gel (Cambrex Bio Science, Verviers, Belgium) using the CHEF method in a CHEF-DR III system (Bio-Rad SA). Electrophoresis was performed with a linear ramping factor, applying pulse times from 4.0 to 40.0 seconds at 14°C for 21 h, at a voltage of 6 V/cm. For normalization and reference, Salmonella enterica serotype Branderup H9812 DNA digested with XbaI (New England Biolabs, 6 h at 37°C) was included on every gel [20]. Electrophoretic patterns were analyzed with BioNumerics software version 5.0 (Applied Maths, Sint-Martens-Latem, Belgium), and clonal complexes (CCs) were assigned using a mapping protocol [21,22].

Selection of the strains for virulence genes and phenotypic tests

To investigate the biofilm-forming ability, biocide resistance and virulence (both genes carriage and in vivo assay), we selected a representative panel of 123 Listeria monocytogenes strains based on their CC distribution across farm and retail stages. If two or more strains from the same farm or the same meat sample had the same CC, only one strain was retained. If several CCs were in a farm or on a meat, we retained one strain per CC. In addition, two strains with the same CC were retained, only if one was isolated from feces and the other one from meat. The panel comprised: 22 strains belonging to CCs found exclusively on farms, 10 strains belonging to CCs found exclusively at retail, and 91 strains from CCs present in both compartments (64 from farm and 27 from retail). In total, 86 strains originated from on-farm pig feces and 37 from retail pork meat. Two reference strains from the Pasteur Institute collection, ScottA (CIP 103575) and EGDe (CIP 107776), were included in all assays as positive controls.

Detection of virulence genes (n=123 strains)

The presence of internalin genes (inlA, inlC, and inlJ) and virulence-associated genes (plcA, prfA, actA, hlyA, and iap) was assessed individually by Real-Time PCR developed for this study, using primers previously cited [23] and published by various authors (Table 1). Some primers were slightly modified (one or two bases added or removed) to harmonize melting temperature (indicated by * in the Table 1). Additional primers were designed by our laboratory to generate amplicons ≤ to 800 bp for optimal amplification efficiency (indicated by ^b in the Table 1).

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Table 1. Primer sequences and size of the amplicons.

https://doi.org/10.1371/journal.pone.0341663.t001

DNA was extracted from fresh bacterial cultures on TSAYE plates using InstaGene® Matrix (BioRad Laboratories, Marnes-la-Coquette France) according to the manufacturer’s instructions. Each PCR reaction was performed using 2.5µl DNA extract (adjusted to 10ng/µl) in a total reaction volume of 25µl containing SYBR^® Green JumpStart^TM Taq ReadyMix^TM from Sigma-Aldrich, and 1µl of each primer (10µM). Amplifications of these eight genes were carried out under identical cycling PCR conditions; an initial denaturation at 95°C for 2 min followed by 30 cycles of 95°C for 2 min, 56°C for 1 minute and 72°C for 2 min. The PCRs ended by a step from 50°C to 95°C with an increment of 0.5°C every 5 seconds, to obtain the fusion curve.

Virulence of the strains using Galleria mellonella model (n=123 strains)

We assessed the virulence of Listeria monocytogenes strains using the Galleria mellonella larvae model, by measuring larval survival after inoculation of each strain. Each Listeria monocytogenes strain was streaked on TSAYE plates, and incubated at 37°C for 24 h. A single colony was then inoculated into 20 mL of Brain Heart Infusion (BHI) broth (Biokar), and incubated at 37°C for 24 h. On the day of larvae infection, 5 ml of this culture was transferred into a 10 mL falcon tube and centrifuged at 4,000 rpm for 5 min. The bacteria pellet was washed with one ml of physiological water (distilled water with 0.9% sodium chloride) followed by centrifugation for one minute; this washing step was repeated twice.

The optical density (OD) of the 1 ml bacterial solution was measured at 600nm and adjusted to an OD of 0.2 corresponding to approximately 10⁸ colony-forming unit (CFU)/mL. For each Listeria monocytogenes strain, 10 µL of the bacterial suspension were injected into the hindmost left proleg of 10 larvae of Galleria mellonella (20 mm size) using a 1 mL syringe connected to a syringe pump inoculator (KD Scientific, USA). In each trial, 10 larvae received an injection of 10µl of physiological water in order to monitor any mortality in the absence of Listeria. Inoculated larvae were placed in plastic Petri dishes and placed at 37°C for 7 days. Larval survival was monitored daily, and at the end of the incubation period, the number of surviving larvae was recorded to calculate a survival percentage for each strain. Each assay was performed in duplicate for all strains. The higher the percentage of surviving larvae, the less virulent the L. monocytogenes strain was considered to be, and inversely.

Biofilm-forming ability of Listeria monocytogenes strains (n=123 strains)

We tested the ability of the strains to form biofilm on 96-well plastic microplates using a method previously described [29] with slight modifications. Each Listeria monocytogenes strain was streaked on TSAYe plates and incubated at 37°C for 24 h. A single colony was then transferred into 10 ml of Tryptone Soy Broth Yeast Extract (TSBYe) (AES, Combourg, France) and incubated at 37°C for 24 h. The bacterial suspension was adjusted in TSBYe to an optical density at 600nm of 0.07 corresponding to 10⁸ UFC/ mL, and then diluted 20-fold prior to inoculation into the microplates.

For each strain, 100 µL of the 1:20 bacterial suspension were deposited in four wells of a flat bottom lidded 96-well microplate (Nunclon™ ΔSurface, Danemark). Four wells per microplate filled with 100 µL of TSBYe served as negative control (to account for assay background noise). The microplates were incubated at 37°C for 48h. Bacterial growth was assessed by measuring the optical density (OD) at 600nm. Suspended bacteria were removed by inverting the microplate, followed by three successive washes with 200 µl sterile water per well. The bacterial biofilm was fixed by placing the microplate in an oven at 55°C for 20 min. Next, 125 µL of 1% crystal violet (Sigma-Aldrich, USA) were added to each well and left in contact with the biofilm for 15 min at room temperature. Unbound crystal violet was removed by inverting the microplate, followed by three additional washes with 200 µL of sterile water per well. Microplates were then dried for 15 min at 55°C.

The stained biofilm was solubilized with 200 μL of 95% ethanol for 15 min at room temperature. Then, 125 µL from each well were transferred to another 96-well microplate with flat bottom (Costar 3590, USA). Optical densities (OD) were measured at 600 nm. The final OD values for each strain were determined by subtracting to OD value obtained from the negative controls (wells containing TSBYe without strain). The assay was repeated three times yielding a total of 12 OD values per strain.

Susceptibility to Benzalkonium chloride (n=123 strains)

We tested the susceptibility of the strains to Benzalkonium chloride, as this quaternary ammonium compounds is among the most widely used disinfectants to control the growth and spread of Listeria monocytogenes in food processing facilities. The biocide susceptibility profiles of the 123 Listeria monocytogenes strains were determined by measuring the Minimal Inhibitory Concentrations (MIC) using a broth microdilution method adapted from NF EN 1040. Listeria monocytogenes ScottA (CIP 103575) was included as a reference in each experiments.

Benzalkonium chloride (BC 50%, Stepan Europe, N-CAS: 68391-01-5) was used in this study at final concentrations ranging from 1 to 6 µg/mL. All strains were streaked on TSAYe plates and incubated for 24 h at 37°C. Two to three colonies were inoculated into 4 ml of TSBYe and were incubated for another 24 h at 37°C. From these cultures, 300 µl of were transferred in 10 ml of TSBYe, and incubated at 37°C for only 3–4 h. The optical density (OD) of these bacterial cultures was measured at 600nm and adjusted with sterile tap water to an OD of 0.2 (+/- 0.02) corresponding to approximately 1.5 x 10⁸ to 3 x 10⁸ colony-forming unit (CFU)/mL. These resulting bacterial suspensions were adjusted with sterile tap water to an optical density (OD) of 0.2 (+/- 0.02) at 620 nm, corresponding to approximately 1.5 x 10⁸ à 3 x 10⁸ CFU/mL.

Each well of 96 well-microplates (Dutscher, PS, flat bottom, clear, Greiner bio-one) was filled in duplicates with 20 µL of each 10x concentrated biocide solution (10, 12.5, 15, 30, 40 and 60 µg/mL), and 180 µl of bacterial suspension at OD 0.2, diluted to 1:100, resulting in final concentration of 1.35 x 10⁶ to 2.7 x 10⁶ CFU/mL. Three wells containing only sterile tap water served as negative control, while wells containing bacteria cultures without biocide as positive controls. Minimal Inhibitory Concentrations (MICs) were determined after incubating the microplate for 24 h at 37°C. The experiment was performed three times on different days, and the median of the six MIC measurements was used for MIC distribution analysis.

Statistical analysis

All comparisons of means were performed in R software (version 3.2.4). Holm’s correction was applied to compare the modalities two by two. The strains were subsequently clustered by Ascending Hierarchical Classification (AHC) using the “hclust ward D2” method in R resulting in three classes based of their ability to form biofilm (low L_BF, intermediate I_BF, and high H_BF), and in three classes based on their level of virulence (low L_V, intermediate I_V and high H_V). A Principal Component Analysis (PCA) was conducted in R on normalized data to explore the correlation among the three variables “ability to form biofilms (OD)”, “biocide resistance” (MIC in µg/ml) and “virulence” (% of survival) and to visualize relationships in high-dimensional dataset. The data on the 123 strains are available in the supplementary S1 Table.

Results

Distribution of serogroups and clonal complexes in farm and retail (n=301 strains)

Listeria monocytogenes strain were distributed across four serogroups. The importance of these serogroups were significantly different between farm and retail (χ² test; p-value <to 0.05). Only 2% of the strains isolated from feces belonged to serogroup IIc, whereas this serogroup accounted for nearly half of the strains isolated from meat (48.7%) (Table 2). In contrast, 37.4% of the fecal strains were identified as serogroup IIb, while only 7.1% of meat strains belonged to this serogroup.

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Table 2. Distribution of the 301 strains according their serogroup and origin (number and percentage).

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The strains were distributed across 20 clonal complexes; 18 identified at farm level and 14 at retail level. Of these, 12 CCs were identified at both stages (Table 3). Greater genetic diversity was observed among the fecal strain population (Simpson’s index = 0.90 _IC95% [0.88–0.92]) compared to the meat strain population (Simpson’s index = 0.73 _IC95% [0.66–0.80]).

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Table 3. Distribution in percentage of the 301 strains according their origin, serogroup and clonal complex.

https://doi.org/10.1371/journal.pone.0341663.t003

We identified six clonal complexes (CC11, CC20, CC21, CC26, CC31 and CC59) that were exclusively isolated on farms, while CC121 (13.6% of the meat strains) was found only at retail. All strains belonging to serogroup IIc were classified as clonal complex CC9; which was also the most prevalent CC overall (n = 78) and was predominantly represented at retail (48.9% of the strains isolated at retail) (Table 3). CC37, from serogroup IIa, were mainly present in the feces (21.1% of the strains from pig farm), along with CC77 (10.9%) and CC1 (10.9%). The distribution of these three CCs between farm and retail differed significantly (p-value < 0.05).

Virulence of Listeria monocytogenes strains (n=123 strains)

PCR amplification of the internalin genes (inlA, inlC and inlJ) and virulence-associated genes (plcA, prfA, actA, hlyA and iap) were confirmed for all the 123 strains.

All the larvae with injected water (control) survived during the assays. Analysis of the percentage of survival showed not significant different between strains isolated from feces and those isolated from meat (χ² Kruskal-Wallis, p-value = 0.977). However, significant difference was observed between serogroups (χ² Kruskal-Wallis, p-value <to 0.05) with strains belonging to serogroup IVb exhibiting higher virulence (61% of the IVb strains) and strains from serogroup IIc showing lower virulence.

By hierarchical clustering, the 123 strains were distributed in three classes according their level of virulence, with 17, 59 and 47 strains in the low (L_V), intermediate (I_V) and high (H_V) virulence groups, respectively (Table 4). Fecal strains and meat strains were evenly distributed across these classes. Among the serogroup IVb strains, 60.8% were classified as highly H_V strains (Table 4), while 46.3% of the IIa strains and 54.3% of IIb strains were classified into the intermediate virulence category (I_V).

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Table 4. Distribution of the strains according their origin, serogroup, clonal complex, biofilm classification and virulence classification.

https://doi.org/10.1371/journal.pone.0341663.t004

Regarding clonal complexes, CC121 (found only on meat) and CC1, CC4 and CC6 (from serogroup IVb and predominantly from feces) were mostly associated to highly virulent class (Table 4). Strains belonging to CC37 (n = 16), the most prevalent CC, mainly isolated from feces, were mostly identified as intermediate virulent (56.2%).

Ability of Listeria monocytogenes strains to form biofilm (n=123 strains)

Analysis the OD values revealed that the biofilm-forming capacity of Listeria monocytogenes strains isolated from feces was significantly higher than that of strains isolated from meat (χ² Kruskal-Wallis, p-value <to 0.05). In addition, strains belonging to serogroup IVb exhibited significantly lower biofilm formation compared to other serogroups (χ² Kruskal-Wallis, p-value <to 0.05).

After clustering by Ascending Hierarchical Classification (AHC), the 123 strains were distributed into three classes based on their biofilm-forming capacity: 36, 46, and 41 strains in the low (L_BF), intermediate (I_BF), and high (H_BF) biofilm-forming groups, respectively (Table 4). High biofilm-forming (H_BF) strains represented 40.7% of the strains isolated from feces and 16.2% of those isolated from meat. Conversely, low biofilm-forming (L_BF) strains accounted for 65.2% of the strains belonging to serogroup IVb (Table 4).

Among clonal complexes, CC8, CC59, and CC77 were predominantly high biofilm-formers. The most prevalent CC, CC37, mainly found in feces, was mostly classified as intermediate biofilm-forming (I_BF). CC1 and CC6 from serogroup IVb, which are involved in human infections in France, exhibited mainly low or intermediate biofilm-forming ability.

Susceptibility to Benzalkonium chloride

Most strains isolated from feces showed greater susceptibility to benzalkonium chloride (BC), with mean of MIC values equal to 1.4 µg/ml (Fig 1). Most strains isolated from feces showed greater susceptibility to benzalkonium chloride (BC), with mean of MIC values equal to 1.4 µg/ml ± 0.2 (χ² Kruskal-Wallis, p-value < 0.05). In contrast, strains isolated from retail meat exhibited higher MIC values, ranging from 1.5 to 4 µg/ml, with an mean of MIC values equal to 3.0 µg/ml ± 1.9. This indicates that meat-derived strains are generally more resistant to this biocide than those from fecal samples.

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Fig 1. Principal component Analyses including the tree quantitative variables: Optical density of the biofilm, percentage of survival and Minimal Inhibitory Concentration against Benzalkonium chloride. a. Principal Component Analysis correlation circle with the three considered variables. b. Principal Component Analysis graph of individuals (strains).

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Strains belonging to serogroup IIc also demonstrated increased resistance to benzalkonium chloride compared to other serogroup (χ² Kruskal-Wallis, p-value < 0.05). Additionally, the 16 strains with MIC values ≥ to 3 µg/ml belonged to clonal complexes CC121 (n = 9), CC9 (n = 5), CC14 (n = 1) and CC7 (n = 1).

Interaction between phenotypic traits

A weak negative correlation (−0.238) was observed between the biofilm-forming (OD_Bf) and biocide resistance (p-value = 00081), while no correlation was found with virulence (% of survival). From the correlation circle (Fig 1A) and the map of individuals (Fig 1b) generated by the PCA (Table 5), we observed that strains isolated from feces (in red) would tend to exhibit a higher biofilm-forming capacity, whereas strains isolated from meat (in blue) would tend to exhibit an higher resistance to the biocide.

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Table 5. Complementary data on ACP analysis.

https://doi.org/10.1371/journal.pone.0341663.t005

Discussion

Listeria monocytogenes is the causative agent of listeriosis, a zoonotic disease primarily transmitted to humans through the consumption of contaminated food [30]. Among the 13 known serotypes of L. monocytogenes, serotypes 1/2a, 1/2b, and 4b classified as serogroup IIa, IIb and IVb respectively, are most frequently associated with human listeriosis cases, with serotype 4b often linked to major outbreaks [31]. While serotype 4b strains are responsible for most listeriosis cases and outbreaks, the majority of strains recovered from food products and food-processing environments belong to serotype 1/2a [32].

In our study, serogroup IVb was equally present on farms and at retail, whereas serogroup IIb was more prevalent on farm and serogroup IIc predominated at retail. These results are consistent with previous observations showing that serogroup or serotype distribution varies along the pig and pork production chain [4].

All the 123 strains tested for virulence genes in our study carried internalin genes (inlA, inlC and inlJ) as well as virulence-associated genes (plcA, prfA, actA, hlyA and iap). These genes are commonly detected in Listeria monocytogenes strains [33–36]. They play essential roles in adhesion, cellular invasion, intracellular replication, and dissemination of the pathogen, thereby contributing to its pathogenicity in humans. However, the presence of all these genes for our strains does not provide information on their level of expression, which can vary between strains.

In addition, to further assess virulence, we used the Galleria mellonella insect larvae model- an established system for evaluating Listeria monocytogenes pathogenicity [37,38]. In our study, serogroup IV strains were the most virulent, whereas serogroups IIa and IIb displayed intermediate virulence. This observation aligns with previous findings reporting that serotype 4b strains cause higher G. mellonella lethality than other serotypes [39].

Consistently, CC1 and CC6 strains in our study were mainly classified as highly or intermediately virulent, corroborating earlier findings [11]. Indeed, hypervirulent CCs such as CC1, CC2, CC4 and CC6 are known to cause most severe listeriosis cases — particularly those affecting the central nervous system or maternal–neonatal infections —and are responsible for the majority of outbreaks and sporadic cases worldwide [11,40].

Interestingly, we also found that CC9 and CC121 strains — predominantly isolated from meat — were mostly classified as highly or intermediately virulent. This is unexpected, as these two CCs are generally regarded as hypovirulent, typically associated with infections in highly immunocompromised patients and showing limited virulence in humanized mouse models [11,41]. Previous studies [42,43] highlighted that CC2 strains carry a full-length inlA gene, whereas CC9 and CC121 strains often present a premature stop codon mutation (PMSC) in inlA correlated with reduced virulence. Liu et al., [44], reported that 99.2% of CC9 and 65% of CC121 strains exhibited the highest proportions of inlA with PMSC, though a minority retained the full-length inlA gene which may explain our observations. Alternatively, environmental or selective pressures within the pig and pork production chain could have favoured CC9 and CC121 strains with higher virulence than generally reported. Another possibility is that the Galleria mellonella insect model may not fully capture virulence variability among all L. monocytogenes CCs. However, no study to our knowledge has yet established a direct link between virulence in this model and strain CC.

First, in the initial population (n = 301 strains), we observed greater genetic diversity of Listeria monocytogenes on farms than at retail, and that six clonal complexes identified on farms were not detected in meat. Moreover, four other CCs differed significantly in distribution between farm and retail. These findings are consistent with Lagarde’s review [4]. Such pattern may reflect the inability or ability of certain CCs to persist through the food chain and reach meat products. Their low prevalence on farms can lead to their gradual disappearance along the production chain, or their better adaptation to stressful conditions encountered in food processing environments and during cold storage can lead to their survival [45].

Indeed, Listeria monocytogenes strains circulating from pigs, the primary reservoir, to consumers can survive and multiply under conditions that are stressful for many other bacteria. In particular, their ability to adhere to surfaces and form biofilms allows long-term persistance for years on equipment and in food-processing facilities, as well as resistance to biocides commonly used in slaughterhouses and cutting plants [1,6,7,9,10]. Consequently, contamination of meat products by Listeria monocytogenes frequently occurs during food processing, mainly through cross-contamination from contaminated contact surfaces.

In our study, we observed that strains belonging to serogroup IVb exhibited significantly lower biofilm formation than other serogroups. This finding is consistent with a previous study using similar microplate-based biofilm quantification method [46] which showed that serogroup IVb strains produced significantly less biofilm than serogroup IIa and/or IIb strains.

We also observed that strains isolated from farms had a significantly greater ability to form biofilms than those isolated from retail. Our findings align with several studies reporting that Listeria monocytogenes strains recovered from slaughterhouse environments — such as conveyor belts [34] and the surfaces of equipment and utensils [47,48] — often show poor adhesion to surfaces and limited biofilm formation.

Among our strains, CC121, which was primarily isolated from meat, exhibited low biofilm-forming capacity. This contrasts with other studies reporting that CC121 strains are strong biofilm formers with increased tolerance to quaternary ammonium compounds [6]. Such intra-CC121 variability suggests that biofilm formation is strain-specific rather than strictly CC-dependent. Environmental and selective pressures along the pig-to-meat continuum — such as cleaning procedures, biocide exposure, and temperature conditions — may also modulate the expression of biofilm-associated genes, leading to phenotypic differences observed in our study compared to other [6].

Finally, we observed that strains isolated from meat were significantly more resistant to Benzalkonium chloride than those from feces, with four CCs (16 strains), including CC121, exhibiting high MIC values (≥ 3 µg/ml). Benzalkonium chloride is one of the most widely used disinfectants in the food industry. Similar findings, showing higher MIC values food-derived strains compared to animal isolates, have been reported previously [49]. This increased resistance may result from repeated exposure to sublethal concentrations of biocides, particularly in mature biofilms [12]. Prolonged contact with such concentrations can gradually increased tolerance to cleaning and disinfection over time when present in single-species biofilms, for both peracetic acid and quaternary ammonium disinfectants [50]. Moreover, persistence of Listeria monocytogenes in a pig slaughterhouse has been linked to the presence of Benzalkonium chloride resistance genes [10].

Conclusion

This study provide a genetic and phenotypic characterisation of the Listeria mocytogenes population along the pig-to-retail meat continuum in France. Conducted on the basis of two surveys carried out two years apart (2008 and 2010), the results should be interpreted with caution. Our findings demonstrate that the Listeria monocytogenes population would evolve between the reservoir (pigs) and consumer stage, suggesting that some strains may either be able—or unable—to persist throughout the food chain. This study is original in that it reports highly virulent CC121 strains, a feature rarely described in the literature. Furthermore, our results indicate that strains isolated from retail meat would be more strongly associated with resistance to biocides than with biofilm-forming ability within the food-processing environment. These observations highlight the importance of alternating the types of biocides used in the food industry to limit the emergence and spread of resistant L. monocytogenes strains.

Acknowledgments

The strains isolated from meat were supplied by Annaelle Kérouanton and Valérie Rose. Special thanks to the students Lionel Zapha (university of Caen) and Nathan Stockmann (Anses), for their contribution to the study.

References

1. Osek J, Wieczorek K. Listeria monocytogenes-how this pathogen uses its virulence mechanisms to infect the Hosts. Pathogens. 2022;11(12):1491. pmid:36558825
- View Article
- PubMed/NCBI
- Google Scholar
2. Koopmans MM, Brouwer MC, Vázquez-Boland JA, van de Beek D. Human listeriosis. Clin Microbiol Rev. 2023;36(1):e0006019. pmid:36475874
- View Article
- PubMed/NCBI
- Google Scholar
3. Sousa M, Magalhães R, Ferreira V, Teixeira P. Current methodologies available to evaluate the virulence potential among Listeria monocytogenes clonal complexes. Front Microbiol. 2024;15:1425437. pmid:39493856
- View Article
- PubMed/NCBI
- Google Scholar
4. Lagarde J, Feurer C, Denis M, Douarre P-E, Piveteau P, Roussel S. Listeria monocytogenes prevalence and genomic diversity along the pig and pork production chain. Food Microbiol. 2024;119:104430. pmid:38225039
- View Article
- PubMed/NCBI
- Google Scholar
5. Stein H, Stessl B, Brunthaler R, Loncaric I, Weissenböck H, Ruczizka U, et al. Listeriosis in fattening pigs caused by poor quality silage - a case report. BMC Vet Res. 2018;14(1):362. pmid:30463612
- View Article
- PubMed/NCBI
- Google Scholar
6. Pérez-Baltar A, Pérez-Boto D, Medina M, Montiel R. Genomic diversity and characterization of Listeria monocytogenes from dry-cured ham processing plants. Food Microbiol. 2021;99:103779. pmid:34119091
- View Article
- PubMed/NCBI
- Google Scholar
7. Shedleur-Bourguignon F, Thériault WP, Longpré J, Thibodeau A, Fravalo P. Use of an Ecosystem-Based Approach to Shed Light on the Heterogeneity of the Contamination Pattern of Listeria monocytogenes on Conveyor Belt Surfaces in a Swine Slaughterhouse in the Province of Quebec, Canada. Pathogens. 2021;10(11):1368. pmid:34832524
- View Article
- PubMed/NCBI
- Google Scholar
8. European Food Safety Authority (EFSA), European Centre for Disease Prevention and Control (ECDC). The European union one health 2023 zoonoses report. EFSA J. 2024;22(12):e9106. pmid:39659847
- View Article
- PubMed/NCBI
- Google Scholar
9. Conficoni D, Losasso C, Cortini E, Di Cesare A, Cibin V, Giaccone V, et al. Resistance to Biocides in Listeria monocytogenes collected in meat-processing environments. Front Microbiol. 2016;7:1627. pmid:27807430
- View Article
- PubMed/NCBI
- Google Scholar
10. Cherifi T, Carrillo C, Lambert D, Miniaï I, Quessy S, Larivière-Gauthier G, et al. Genomic characterization of Listeria monocytogenes isolates reveals that their persistence in a pig slaughterhouse is linked to the presence of benzalkonium chloride resistance genes. BMC Microbiol. 2018;18(1):220. pmid:30572836
- View Article
- PubMed/NCBI
- Google Scholar
11. Maury MM, Tsai Y-H, Charlier C, Touchon M, Chenal-Francisque V, Leclercq A, et al. Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. Nat Genet. 2016;48(3):308–13. pmid:26829754
- View Article
- PubMed/NCBI
- Google Scholar
12. Rodríguez-Melcón C, Capita R, Alonso-Calleja C. Exposure to Low Doses of Biocides Increases Resistance to Other Biocides and to Antibiotics in Strains of Listeria monocytogenes. Biology (Basel). 2025;14(5):495. pmid:40427684
- View Article
- PubMed/NCBI
- Google Scholar
13. Palma F, Radomski N, Guérin A, Sévellec Y, Félix B, Bridier A, et al. Genomic elements located in the accessory repertoire drive the adaptation to biocides in Listeria monocytogenes strains from different ecological niches. Food Microbiol. 2022;106:103757. pmid:35690455
- View Article
- PubMed/NCBI
- Google Scholar
14. Saá Ibusquiza P, Herrera JJR, Cabo ML. Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes. Food Microbiol. 2011;28(3):418–25. pmid:21356446
- View Article
- PubMed/NCBI
- Google Scholar
15. Boscher E, Houard E, Denis M. Prevalence and distribution of Listeria monocytogenes serotypes and pulsotypes in sows and fattening pigs in farrow-to-finish farms (France, 2008). J Food Prot. 2012;75(5):889–95. pmid:22564938
- View Article
- PubMed/NCBI
- Google Scholar
16. Kérouanton A, Rose V, Quesne S, Courtillon C, Rouxel S, Denis M. Prevalence and characterization of Listeria monocytogenes in french raw pork meat at the distribution level. Safepork Maastricht. 2011.
- View Article
- Google Scholar
17. Doumith M, Buchrieser C, Glaser P, Jacquet C, Martin P. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J Clin Microbiol. 2004;42(8):3819–22. pmid:15297538
- View Article
- PubMed/NCBI
- Google Scholar
18. Kérouanton A, Marault M, Petit L, Grout J, Dao TT, Brisabois A. Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping. J Microbiol Methods. 2010;80(2):134–7. pmid:19958798
- View Article
- PubMed/NCBI
- Google Scholar
19. Graves LM, Swaminathan B. PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. Int J Food Microbiol. 2001;65(1–2):55–62. pmid:11322701
- View Article
- PubMed/NCBI
- Google Scholar
20. Hunter SB, Vauterin P, Lambert-Fair MA, Van Duyne MS, Kubota K, Graves L, et al. Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J Clin Microbiol. 2005;43(3):1045–50. pmid:15750058
- View Article
- PubMed/NCBI
- Google Scholar
21. Henri C, Félix B, Guillier L, Leekitcharoenphon P, Michelon D, Mariet J-F, et al. Population Genetic structure of Listeria monocytogenes strains as determined by pulsed-field gel electrophoresis and multilocus sequence typing. Appl Environ Microbiol. 2016;82(18):5720–8. pmid:27235443
- View Article
- PubMed/NCBI
- Google Scholar
22. Félix B, Feurer C, Maillet A, Guillier L, Boscher E, Kerouanton A, et al. Population genetic structure of listeria monocytogenes strains isolated from the pig and pork production chain in France. Front Microbiol. 2018;9:684. pmid:29681897
- View Article
- PubMed/NCBI
- Google Scholar
23. Soni DK, Singh M, Singh DV, Dubey SK. Virulence and genotypic characterization of Listeria monocytogenes isolated from vegetable and soil samples. BMC Microbiol. 2014;14:241. pmid:25195727
- View Article
- PubMed/NCBI
- Google Scholar
24. Liu D, Lawrence ML, Austin FW, Ainsworth AJ. A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenes. J Microbiol Methods. 2007;71(2):133–40. pmid:17884210
- View Article
- PubMed/NCBI
- Google Scholar
25. Notermans SH, Dufrenne J, Leimeister-Wächter M, Domann E, Chakraborty T. Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species. Appl Environ Microbiol. 1991;57(9):2666–70. pmid:1662937
- View Article
- PubMed/NCBI
- Google Scholar
26. Suárez M, González-Zorn B, Vega Y, Chico-Calero I, Vázquez-Boland JA. A role for ActA in epithelial cell invasion by Listeria monocytogenes. Cell Microbiol. 2001;3(12):853–64. pmid:11736996
- View Article
- PubMed/NCBI
- Google Scholar
27. Paziak-Domańska B, Bogusławska E, Wieckowska-Szakiel M, Kotłowski R, Rózalska B, Chmiela M, et al. Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods. FEMS Microbiol Lett. 1999;171(2):209–14. pmid:10077846
- View Article
- PubMed/NCBI
- Google Scholar
28. Furrer B, Candrian U, Hoefelein C, Luethy J. Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments. J Appl Bacteriol. 1991;70(5):372–9. pmid:1908450
- View Article
- PubMed/NCBI
- Google Scholar
29. Djordjevic D, Wiedmann M, McLandsborough LA. Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl Environ Microbiol. 2002;68(6):2950–8. pmid:12039754
- View Article
- PubMed/NCBI
- Google Scholar
30. Grigore-Gurgu L, Bucur FI, Mihalache OA, Nicolau AI. Comprehensive review on the biocontrol of Listeria monocytogenes in food products. Foods. 2024;13(5):734. pmid:38472848
- View Article
- PubMed/NCBI
- Google Scholar
31. Orsi RH, den Bakker HC, Wiedmann M. Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics. Int J Med Microbiol. 2011;301(2):79–96. pmid:20708964
- View Article
- PubMed/NCBI
- Google Scholar
32. Mazaheri T, Ripolles-Avila C, Hascoët AS, Rodríguez-Jerez JJ. Effect of an enzymatic treatment on the removal of mature Listeria monocytogenes biofilms: A quantitative and qualitative study. Food Control. 2020;114:107266.
- View Article
- Google Scholar
33. Wu S, Wu Q, Zhang J, Chen M, Yan ZA, Hu H. Listeria monocytogenes prevalence and characteristics in retail raw foods in China. PLoS One. 2015;10(8):e0136682. pmid:26317852
- View Article
- PubMed/NCBI
- Google Scholar
34. Sereno MJ, Viana C, Pegoraro K, da Silva DAL, Yamatogi RS, Nero LA, et al. Distribution, adhesion, virulence and antibiotic resistance of persistent Listeria monocytogenes in a pig slaughterhouse in Brazil. Food Microbiol. 2019;84:103234. pmid:31421784
- View Article
- PubMed/NCBI
- Google Scholar
35. Kawacka I, Olejnik-Schmidt A. High prevalence of virulence-associated genes and length polymorphism in actA and inlB genes identified in Listeria monocytogenes Isolates from Meat Products and Meat-Processing Environments in Poland. Pathogens. 2024;13(6):444. pmid:38921742
- View Article
- PubMed/NCBI
- Google Scholar
36. Żurawik A, Kasperski T, Olechowska-Jarząb A, Szczesiul-Paszkiewicz P, Żak I, Wójcicki M, et al. Genetic diversity, virulence factors and antibiotic resistance of Listeria monocytogenes from food and clinical samples in Southern Poland. Pathogens. 2024;13(9):725. pmid:39338917
- View Article
- PubMed/NCBI
- Google Scholar
37. Rakic Martinez M, Wiedmann M, Ferguson M, Datta AR. Assessment of Listeria monocytogenes virulence in the Galleria mellonella insect larvae model. PLoS One. 2017;12(9):e0184557. pmid:28898264
- View Article
- PubMed/NCBI
- Google Scholar
38. Bah U, de Llanos Frutos R, Donnellan S, Smith A, Flockhart A, Singleton I, et al. The potential virulence of Listeria monocytogenes strains isolated from fresh produce processing facilities as determined by an invertebrate Galleria mellonella model. PLoS One. 2024;19(12):e0311839. pmid:39666623
- View Article
- PubMed/NCBI
- Google Scholar
39. Pan X, Shen J, Hong Y, Wu Y, Guo D, Zhao L, et al. Comparative analysis of growth, survival, and virulence characteristics of Listeria monocytogenes isolated from imported meat. Microorganisms. 2024;12(2):345. pmid:38399749
- View Article
- PubMed/NCBI
- Google Scholar
40. Painset A, Björkman JT, Kiil K, Guillier L, Mariet J-F, Félix B, et al. LiSEQ - whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe. Microb Genom. 2019;5(2):e000257. pmid:30775964
- View Article
- PubMed/NCBI
- Google Scholar
41. Pouillot R, Kiermeier A, Guillier L, Cadavez V, Sanaa M. Updated Parameters for Listeria monocytogenes Dose-response model considering pathogen virulence and age and sex of consumer. Foods. 2024;13(5):751. pmid:38472864
- View Article
- PubMed/NCBI
- Google Scholar
42. Guidi F, Orsini M, Chiaverini A, Torresi M, Centorame P, Acciari VA, et al. Hypo- and hyper-virulent Listeria monocytogenes clones persisting in two different food processing plants of central Italy. Microorganisms. 2021;9(2):376. pmid:33668440
- View Article
- PubMed/NCBI
- Google Scholar
43. Tadielo LE, Dos Santos EAR, Possebon FS, Schmiedt JA, Juliano LCB, Cerqueira-Cézar CK, et al. Characterization of microbial ecology, Listeria monocytogenes, and Salmonella sp. on equipment and utensil surfaces in Brazilian poultry, pork, and dairy industries. Food Res Int. 2023;173(Pt 2):113422. pmid:37803760
- View Article
- PubMed/NCBI
- Google Scholar
44. Liu X, Gao B, Li Z, Liang Y, Shi T, Dong Q, et al. Research on the Genetic Polymorphism and Function of inlA with Premature Stop Codons in Listeria monocytogenes. Foods. 2025;14(17):2955. pmid:40941071
- View Article
- PubMed/NCBI
- Google Scholar
45. Osek J, Wieczorek K. Why does Listeria monocytogenes survive in food and food-production environments?. J Vet Res. 2023;67(4):537–44. pmid:38130454
- View Article
- PubMed/NCBI
- Google Scholar
46. Lee B-H, Cole S, Badel-Berchoux S, Guillier L, Felix B, Krezdorn N, et al. Biofilm formation of Listeria monocytogenes strains under food processing environments and pan-genome-wide association study. Front Microbiol. 2019;10:2698. pmid:31824466
- View Article
- PubMed/NCBI
- Google Scholar
47. Agostinho Davanzo EF, Dos Santos RL, Castro VH de L, Palma JM, Pribul BR, Dallago BSL, et al. Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil. PLoS One. 2021;16(11):e0259687. pmid:34767604
- View Article
- PubMed/NCBI
- Google Scholar
48. Swaminathan B, Gerner-Smidt P. The epidemiology of human listeriosis. Microbes Infect. 2007;9(10):1236–43. pmid:17720602
- View Article
- PubMed/NCBI
- Google Scholar
49. Guérin A, Bridier A, Le Grandois P, Sévellec Y, Palma F, Félix B, et al. Exposure to Quaternary Ammonium Compounds Selects Resistance to Ciprofloxacin in Listeria monocytogenes. Pathogens. 2021;10(2):220. pmid:33670643
- View Article
- PubMed/NCBI
- Google Scholar
50. Fagerlund A, Møretrø T, Heir E, Briandet R, Langsrud S. Cleaning and disinfection of biofilms composed of Listeria monocytogenes and Background Microbiota from Meat Processing Surfaces. Appl Environ Microbiol. 2017;83(17):e01046-17. pmid:28667108
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Osek J, Wieczorek K. Listeria monocytogenes-how this pathogen uses its virulence mechanisms to infect the Hosts. Pathogens. 2022;11(12):1491. pmid:36558825
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Koopmans MM, Brouwer MC, Vázquez-Boland JA, van de Beek D. Human listeriosis. Clin Microbiol Rev. 2023;36(1):e0006019. pmid:36475874
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Sousa M, Magalhães R, Ferreira V, Teixeira P. Current methodologies available to evaluate the virulence potential among Listeria monocytogenes clonal complexes. Front Microbiol. 2024;15:1425437. pmid:39493856
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Lagarde J, Feurer C, Denis M, Douarre P-E, Piveteau P, Roussel S. Listeria monocytogenes prevalence and genomic diversity along the pig and pork production chain. Food Microbiol. 2024;119:104430. pmid:38225039
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Stein H, Stessl B, Brunthaler R, Loncaric I, Weissenböck H, Ruczizka U, et al. Listeriosis in fattening pigs caused by poor quality silage - a case report. BMC Vet Res. 2018;14(1):362. pmid:30463612
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Pérez-Baltar A, Pérez-Boto D, Medina M, Montiel R. Genomic diversity and characterization of Listeria monocytogenes from dry-cured ham processing plants. Food Microbiol. 2021;99:103779. pmid:34119091
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Shedleur-Bourguignon F, Thériault WP, Longpré J, Thibodeau A, Fravalo P. Use of an Ecosystem-Based Approach to Shed Light on the Heterogeneity of the Contamination Pattern of Listeria monocytogenes on Conveyor Belt Surfaces in a Swine Slaughterhouse in the Province of Quebec, Canada. Pathogens. 2021;10(11):1368. pmid:34832524
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. European Food Safety Authority (EFSA), European Centre for Disease Prevention and Control (ECDC). The European union one health 2023 zoonoses report. EFSA J. 2024;22(12):e9106. pmid:39659847
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Conficoni D, Losasso C, Cortini E, Di Cesare A, Cibin V, Giaccone V, et al. Resistance to Biocides in Listeria monocytogenes collected in meat-processing environments. Front Microbiol. 2016;7:1627. pmid:27807430
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Cherifi T, Carrillo C, Lambert D, Miniaï I, Quessy S, Larivière-Gauthier G, et al. Genomic characterization of Listeria monocytogenes isolates reveals that their persistence in a pig slaughterhouse is linked to the presence of benzalkonium chloride resistance genes. BMC Microbiol. 2018;18(1):220. pmid:30572836
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Maury MM, Tsai Y-H, Charlier C, Touchon M, Chenal-Francisque V, Leclercq A, et al. Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. Nat Genet. 2016;48(3):308–13. pmid:26829754
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Rodríguez-Melcón C, Capita R, Alonso-Calleja C. Exposure to Low Doses of Biocides Increases Resistance to Other Biocides and to Antibiotics in Strains of Listeria monocytogenes. Biology (Basel). 2025;14(5):495. pmid:40427684
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Palma F, Radomski N, Guérin A, Sévellec Y, Félix B, Bridier A, et al. Genomic elements located in the accessory repertoire drive the adaptation to biocides in Listeria monocytogenes strains from different ecological niches. Food Microbiol. 2022;106:103757. pmid:35690455
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Saá Ibusquiza P, Herrera JJR, Cabo ML. Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes. Food Microbiol. 2011;28(3):418–25. pmid:21356446
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Boscher E, Houard E, Denis M. Prevalence and distribution of Listeria monocytogenes serotypes and pulsotypes in sows and fattening pigs in farrow-to-finish farms (France, 2008). J Food Prot. 2012;75(5):889–95. pmid:22564938
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Kérouanton A, Rose V, Quesne S, Courtillon C, Rouxel S, Denis M. Prevalence and characterization of Listeria monocytogenes in french raw pork meat at the distribution level. Safepork Maastricht. 2011.
View Article
Google Scholar

[62] View Article

[63] Google Scholar

[ref17] 17. Doumith M, Buchrieser C, Glaser P, Jacquet C, Martin P. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J Clin Microbiol. 2004;42(8):3819–22. pmid:15297538
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref18] 18. Kérouanton A, Marault M, Petit L, Grout J, Dao TT, Brisabois A. Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping. J Microbiol Methods. 2010;80(2):134–7. pmid:19958798
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref19] 19. Graves LM, Swaminathan B. PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. Int J Food Microbiol. 2001;65(1–2):55–62. pmid:11322701
View Article
PubMed/NCBI
Google Scholar

[73] View Article

[74] PubMed/NCBI

[75] Google Scholar

[ref20] 20. Hunter SB, Vauterin P, Lambert-Fair MA, Van Duyne MS, Kubota K, Graves L, et al. Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J Clin Microbiol. 2005;43(3):1045–50. pmid:15750058
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref21] 21. Henri C, Félix B, Guillier L, Leekitcharoenphon P, Michelon D, Mariet J-F, et al. Population Genetic structure of Listeria monocytogenes strains as determined by pulsed-field gel electrophoresis and multilocus sequence typing. Appl Environ Microbiol. 2016;82(18):5720–8. pmid:27235443
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref22] 22. Félix B, Feurer C, Maillet A, Guillier L, Boscher E, Kerouanton A, et al. Population genetic structure of listeria monocytogenes strains isolated from the pig and pork production chain in France. Front Microbiol. 2018;9:684. pmid:29681897
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref23] 23. Soni DK, Singh M, Singh DV, Dubey SK. Virulence and genotypic characterization of Listeria monocytogenes isolated from vegetable and soil samples. BMC Microbiol. 2014;14:241. pmid:25195727
View Article
PubMed/NCBI
Google Scholar

[89] View Article

[90] PubMed/NCBI

[91] Google Scholar

[ref24] 24. Liu D, Lawrence ML, Austin FW, Ainsworth AJ. A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenes. J Microbiol Methods. 2007;71(2):133–40. pmid:17884210
View Article
PubMed/NCBI
Google Scholar

[93] View Article

[94] PubMed/NCBI

[95] Google Scholar

[ref25] 25. Notermans SH, Dufrenne J, Leimeister-Wächter M, Domann E, Chakraborty T. Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species. Appl Environ Microbiol. 1991;57(9):2666–70. pmid:1662937
View Article
PubMed/NCBI
Google Scholar

[97] View Article

[98] PubMed/NCBI

[99] Google Scholar

[ref26] 26. Suárez M, González-Zorn B, Vega Y, Chico-Calero I, Vázquez-Boland JA. A role for ActA in epithelial cell invasion by Listeria monocytogenes. Cell Microbiol. 2001;3(12):853–64. pmid:11736996
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref27] 27. Paziak-Domańska B, Bogusławska E, Wieckowska-Szakiel M, Kotłowski R, Rózalska B, Chmiela M, et al. Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods. FEMS Microbiol Lett. 1999;171(2):209–14. pmid:10077846
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref28] 28. Furrer B, Candrian U, Hoefelein C, Luethy J. Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments. J Appl Bacteriol. 1991;70(5):372–9. pmid:1908450
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref29] 29. Djordjevic D, Wiedmann M, McLandsborough LA. Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl Environ Microbiol. 2002;68(6):2950–8. pmid:12039754
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref30] 30. Grigore-Gurgu L, Bucur FI, Mihalache OA, Nicolau AI. Comprehensive review on the biocontrol of Listeria monocytogenes in food products. Foods. 2024;13(5):734. pmid:38472848
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref31] 31. Orsi RH, den Bakker HC, Wiedmann M. Listeria monocytogenes lineages: Genomics, evolution, ecology, and phenotypic characteristics. Int J Med Microbiol. 2011;301(2):79–96. pmid:20708964
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref32] 32. Mazaheri T, Ripolles-Avila C, Hascoët AS, Rodríguez-Jerez JJ. Effect of an enzymatic treatment on the removal of mature Listeria monocytogenes biofilms: A quantitative and qualitative study. Food Control. 2020;114:107266.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref33] 33. Wu S, Wu Q, Zhang J, Chen M, Yan ZA, Hu H. Listeria monocytogenes prevalence and characteristics in retail raw foods in China. PLoS One. 2015;10(8):e0136682. pmid:26317852
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref34] 34. Sereno MJ, Viana C, Pegoraro K, da Silva DAL, Yamatogi RS, Nero LA, et al. Distribution, adhesion, virulence and antibiotic resistance of persistent Listeria monocytogenes in a pig slaughterhouse in Brazil. Food Microbiol. 2019;84:103234. pmid:31421784
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref35] 35. Kawacka I, Olejnik-Schmidt A. High prevalence of virulence-associated genes and length polymorphism in actA and inlB genes identified in Listeria monocytogenes Isolates from Meat Products and Meat-Processing Environments in Poland. Pathogens. 2024;13(6):444. pmid:38921742
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref36] 36. Żurawik A, Kasperski T, Olechowska-Jarząb A, Szczesiul-Paszkiewicz P, Żak I, Wójcicki M, et al. Genetic diversity, virulence factors and antibiotic resistance of Listeria monocytogenes from food and clinical samples in Southern Poland. Pathogens. 2024;13(9):725. pmid:39338917
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref37] 37. Rakic Martinez M, Wiedmann M, Ferguson M, Datta AR. Assessment of Listeria monocytogenes virulence in the Galleria mellonella insect larvae model. PLoS One. 2017;12(9):e0184557. pmid:28898264
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref38] 38. Bah U, de Llanos Frutos R, Donnellan S, Smith A, Flockhart A, Singleton I, et al. The potential virulence of Listeria monocytogenes strains isolated from fresh produce processing facilities as determined by an invertebrate Galleria mellonella model. PLoS One. 2024;19(12):e0311839. pmid:39666623
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref39] 39. Pan X, Shen J, Hong Y, Wu Y, Guo D, Zhao L, et al. Comparative analysis of growth, survival, and virulence characteristics of Listeria monocytogenes isolated from imported meat. Microorganisms. 2024;12(2):345. pmid:38399749
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref40] 40. Painset A, Björkman JT, Kiil K, Guillier L, Mariet J-F, Félix B, et al. LiSEQ - whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe. Microb Genom. 2019;5(2):e000257. pmid:30775964
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref41] 41. Pouillot R, Kiermeier A, Guillier L, Cadavez V, Sanaa M. Updated Parameters for Listeria monocytogenes Dose-response model considering pathogen virulence and age and sex of consumer. Foods. 2024;13(5):751. pmid:38472864
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref42] 42. Guidi F, Orsini M, Chiaverini A, Torresi M, Centorame P, Acciari VA, et al. Hypo- and hyper-virulent Listeria monocytogenes clones persisting in two different food processing plants of central Italy. Microorganisms. 2021;9(2):376. pmid:33668440
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

[ref43] 43. Tadielo LE, Dos Santos EAR, Possebon FS, Schmiedt JA, Juliano LCB, Cerqueira-Cézar CK, et al. Characterization of microbial ecology, Listeria monocytogenes, and Salmonella sp. on equipment and utensil surfaces in Brazilian poultry, pork, and dairy industries. Food Res Int. 2023;173(Pt 2):113422. pmid:37803760
View Article
PubMed/NCBI
Google Scholar

[168] View Article

[169] PubMed/NCBI

[170] Google Scholar

[ref44] 44. Liu X, Gao B, Li Z, Liang Y, Shi T, Dong Q, et al. Research on the Genetic Polymorphism and Function of inlA with Premature Stop Codons in Listeria monocytogenes. Foods. 2025;14(17):2955. pmid:40941071
View Article
PubMed/NCBI
Google Scholar

[172] View Article

[173] PubMed/NCBI

[174] Google Scholar

[ref45] 45. Osek J, Wieczorek K. Why does Listeria monocytogenes survive in food and food-production environments?. J Vet Res. 2023;67(4):537–44. pmid:38130454
View Article
PubMed/NCBI
Google Scholar

[176] View Article

[177] PubMed/NCBI

[178] Google Scholar

[ref46] 46. Lee B-H, Cole S, Badel-Berchoux S, Guillier L, Felix B, Krezdorn N, et al. Biofilm formation of Listeria monocytogenes strains under food processing environments and pan-genome-wide association study. Front Microbiol. 2019;10:2698. pmid:31824466
View Article
PubMed/NCBI
Google Scholar

[180] View Article

[181] PubMed/NCBI

[182] Google Scholar

[ref47] 47. Agostinho Davanzo EF, Dos Santos RL, Castro VH de L, Palma JM, Pribul BR, Dallago BSL, et al. Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil. PLoS One. 2021;16(11):e0259687. pmid:34767604
View Article
PubMed/NCBI
Google Scholar

[184] View Article

[185] PubMed/NCBI

[186] Google Scholar

[ref48] 48. Swaminathan B, Gerner-Smidt P. The epidemiology of human listeriosis. Microbes Infect. 2007;9(10):1236–43. pmid:17720602
View Article
PubMed/NCBI
Google Scholar

[188] View Article

[189] PubMed/NCBI

[190] Google Scholar

[ref49] 49. Guérin A, Bridier A, Le Grandois P, Sévellec Y, Palma F, Félix B, et al. Exposure to Quaternary Ammonium Compounds Selects Resistance to Ciprofloxacin in Listeria monocytogenes. Pathogens. 2021;10(2):220. pmid:33670643
View Article
PubMed/NCBI
Google Scholar

[192] View Article

[193] PubMed/NCBI

[194] Google Scholar

[ref50] 50. Fagerlund A, Møretrø T, Heir E, Briandet R, Langsrud S. Cleaning and disinfection of biofilms composed of Listeria monocytogenes and Background Microbiota from Meat Processing Surfaces. Appl Environ Microbiol. 2017;83(17):e01046-17. pmid:28667108
View Article
PubMed/NCBI
Google Scholar

[196] View Article

[197] PubMed/NCBI

[198] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Strains

Pulsed-field gel electrophoresis and clonal complex mapping (n=301 strains)

Selection of the strains for virulence genes and phenotypic tests

Detection of virulence genes (n=123 strains)

Virulence of the strains using Galleria mellonella model (n=123 strains)

Biofilm-forming ability of Listeria monocytogenes strains (n=123 strains)

Susceptibility to Benzalkonium chloride (n=123 strains)

Statistical analysis

Results

Distribution of serogroups and clonal complexes in farm and retail (n=301 strains)

Virulence of Listeria monocytogenes strains (n=123 strains)

Ability of Listeria monocytogenes strains to form biofilm (n=123 strains)

Susceptibility to Benzalkonium chloride

Interaction between phenotypic traits

Discussion

Conclusion

Acknowledgments

References