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Article

Pathogenic Yersinia enterocolitica’s Contamination of Cheeks, Tongues, and Other Pork Meats at Retail in France, 2023

by
Martine Denis
1,*,
Arnaud Felten
2,
Linda Ducret
1,
Emmanuelle Houard
1,
Manon Tasset
2,
Delphine Novi
3 and
Marianne Chemaly
1
1
Ploufragan-Plouzané-Niort Laboratory, Hygiene and Quality of Poultry and Pork Products Unit, ANSES (French Agency for Food, Environmental and Occupational Health and Safety), 22440 Ploufragan, France
2
Ploufragan-Plouzané-Niort Laboratory, Viral Genetics and Biosafety Unit, ANSES (French Agency for Food, Environmental and Occupational Health and Safety), 22440 Ploufragan, France
3
DGAL (French General Directorate for Food), Integrated Risk Management Office, Sub-Directorate of Europe, of International and of Integrated Risk Management, 75015 Paris, France
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2025, 5(1), 15; https://doi.org/10.3390/applmicrobiol5010015
Submission received: 26 December 2024 / Revised: 22 January 2025 / Accepted: 29 January 2025 / Published: 1 February 2025
(This article belongs to the Special Issue Applied Microbiology of Foods, 2nd Edition)
Browse Figures
Figure 1
<p>The distribution of the samples in the 13 regions of mainland France.</p> "> Figure 2
<p>The strain Y23PE0281 (BT4, isolated from tongue) and strain Y23PE0341 (BT4, isolated from tongue) on CR-MOX after 28 h at 37 °C. In the absence of the plasmid, the colonies were colorless (strain Y23PE0281). The strain Y23PE0341 showed two types of colonies, colorless colonies and colonies that were orange–red pinheads, indicating the presence of the plasmid.</p> "> Figure 3
<p>The cgMLST tree from Enterobase of the 57 BT4 strains. The number on the branch indicates the allelic distance. Clusters were created when the allelic distance was ≤5. A total of 56 cgMLST genotypes were identified and could not all be presented on the tree.</p> "> Figure 4
<p>The cgSNP tree for the BT4 strains (<span class="html-italic">n</span> = 57). Strains were considered closely related when the allelic distance was ≤4 (with wgMLST, tree not shown) and the number of different SNPs was ≤10.</p> ">
Versions Notes

Abstract

:
Pathogenic Y. enterocolitica’s contamination of cheeks, tongues, and other pork meats at retail was assessed in 2023, over 9 months. A total of 111 samples of cheeks, 104 of tongues, and 160 of fresh meat were taken at retail from the 13 regions of mainland France. The level of contamination was 16.0%, with a higher contamination in tongues (39.4%), followed by cheeks (16.4%). Only one meat sample was contaminated. Of the 128 isolated strains, 97.6% were of the BT4 biotype. Depending on the method used to check the presence of the plasmid—yadA-PCR, CR-MOX testing, or sequencing—the results were not consistent for some strains, but most of the strains (≥ to 65%) had the virulent plasmid pYV. All the BT4 strains (except two strains) carried the sequence ST18; they were distributed in 54 cgMLST genotypes. The genetic diversity of the strains was very high, whatever the typing method used, including cgMLST, wgMLST, and cgSNP. There was higher contamination in tongues and cheeks, and lower contamination in meat, suggesting that the head deboning step is riskier than the evisceration step for contamination by pathogenic Y. enterocolitica. This pathogen remains a zoonotic agent of public health importance to be monitored in pigs.

1. Introduction

For many years, Yersinia enterocolitica has been identified as the third most common foodborne bacterial agent in Europe responsible for gastroenteritis, after Campylobacter and Salmonella [1]. In France, the average number of cases per year was estimated as 23,674 over the period from 2008 to 2013 [2].
Y. enterocolitica is distributed in six biotypes. Among them, biotype 1A strains, frequently isolated from the environment, are considered avirulent, while some strains are able to cause gastrointestinal symptoms [3]. The other biotypes are considered pathogenic Y. enterocolitica strains that carry several chromosomal markers of pathogenicity and the virulence plasmid (pYV). The presence of the plasmid pYV is decisive for the pathogenicity of the strains. The yadA gene, located on the virulence plasmid of all pathogenic biotypes, encodes the adhesion protein, YadA, a polymeric protein associated with the outer membrane, which is a decisive virulent factor in the pathogenesis of Y. enterocolitica [4].
The analysis of the biotypes and serotypes in the collection of Y. enterocolitica strains at the National Reference Centre (CNR) for plague and other yersinioses (Pasteur Institute, Paris, France) confirms the significant contribution of the pig reservoir to human cases of yersiniosis [5]. Indeed, it is the BT4 biotype, with its serotype O:3, that is found in the majority of human infections (66.8% of strains), and this biotype is also very prevalent in pigs in France (91.9% of strains) [6]. Pork and pork products are often described as being primarily responsible for sporadic Y. enterocolitica infections.
In pigs, Y. enterocolitica has tonsillar and intestinal tropism, with higher levels in the tonsils than in intestinal contents and in feces [7,8]. This tropism in tonsils, and on the tongue, was demonstrated in an experimental trial on pigs inoculated with a BT4 Y. enterocolitica strain of porcine origin [9]. As a result, Y. enterocolitica is regularly isolated from the tonsils and tongues of pigs. A survey conducted by our laboratory in 2010–2011 (16 abattoirs, 3120 pigs, 96 batches of pigs) using tonsil swabs showed that 74% of batches of pigs at the abattoir were contaminated with pathogenic Y. enterocolitica at the tonsil level and that the individual prevalence was 14% [6]. The presence of this bacterium in the oral cavity of pigs can lead to the contamination of the muscles of a pig’s head, tongue, and cheeks when the head is boned. However, no data on these matrices were available until this survey at retail due to the lack of regulatory criteria and monitoring for pathogenic Y. enterocolitica in pork products.
Directive 2003/99/EC requires member states to set up a system for monitoring zoonoses and zoonotic agents. Yersinia enterocolitica is on list B of this directive, ‘Zoonoses and zoonotic agents to be monitored according to the epidemiological situation’. Also, the objectives of this exploratory survey, planned by the DGAL (French General Directorate for Food), were to supplement prevalence data by focusing on the retail stage and on three matrices: cheeks, tongues, and other fresh pork meats. The objectives of this survey was to assess (1) the risk of contamination by Yersinia enterocolitica during the slaughtering process regarding the prevalence observed for these different matrices and (2) the pathogenicity of strains through their biotype and the presence of the virulence plasmid pYV. In addition, we tested several typing methods to evaluate the genetic diversity of the isolated strains.

2. Materials and Methods

2.1. Sampling

This exploratory survey was conducted from 15 April to 15 December in 2023. A total of 375 samples (111 cheek samples, 104 tongue samples, and 160 fresh meat samples) were taken at retail and sent under cold conditions to our laboratory, ANSES. The samples were distributed across the 13 regions of mainland France (Figure 1).
The number of samples to be taken per region was established by the DGAL in proportion to human consumption with an identical distribution between the three matrices: cheeks, tongues, and fresh pork meat.

2.2. Detection of Pathogenic Y. enterocolitica

Ten grams of samples were placed in a stomacher filter bag and diluted to 1:10 in peptone sorbitol bile (PSB) broth (prepared in the laboratory, as described in the ISO 10273:2017 method [10]). Then, 1 mL of suspension was added to 9 mL of irgasan–ticarcillin–potassium chlorate (ITC) broth (Bio-Rad, Marnes La Coquette, France). The PSB broth and ITC broth were incubated for 48 h at 25 °C. Before streaking, 0.5 mL of each broth was treated by KOH, as described in the ISO 10273:2017 method [10] and 10 μL was then streaked on cefsulodin–irgasan–novobiocin (CIN) agar plates (Yersinia Selective Agar Base and Yersinia Selective Supplement, Oxoid, Basingstoke, UK). All the plates were incubated at 30 °C for 24 h.
A maximum of four typical colonies per CIN were streaked on plates containing Y. enterocolitica chromogenic medium (YeCM) prepared in our laboratory [11] for the selection of presumptive pathogenic Y. enterocolitica isolates (red “bull’s-eye” colonies). All the YeCM plates were incubated at 30 °C for 24 h. Then, only the “bull’s-eye” cultures on the YeCM plates were kept, and one isolated colony from these plates was then subcultured two times successively on Plate Count Agar (PCA) plates (AES, Bruz, France) with incubation at 30 °C for 24 h each time. The culture from the second PCA was used for PCR, biochemical assays, and sequencing. Strains were stored in peptone glycerol broth at −80 °C.

2.3. Assays for Testing the Pathogenicity of the Strains

Strains sub-cultured on PCA were used for confirming their pathogenicity through three assays.

2.3.1. Biotyping

Pathogenic isolates were biotyped, as described in the ISO 10273:2017 method, with the following tests: esculin hydrolysis, indole production, and the fermentation of xylose and trehalose. Strains of the biotype 1A (IP124), biotype 4/0:3 (IP134), biotype 3/0:5.27 (IP29228), and biotype 2/0:9 (IP383), purchased from the Pasteur Institute (Paris, France), were used as controls.

2.3.2. CR-MOX Test

The strains were tested on CR-MOX (Congo-red magnesium oxalate) plates, prepared in the laboratory as described in the ISO 10273:2017 method [10]. Using an inoculating loop, three colonies of the pure culture were picked up and streaked onto CR-MOX agar to obtain separate colonies, and the plates were incubated at 37 °C for 24 h–48 h. If the colonies were orange–red pinheads, this indicated the presence of the plasmid pYV for this strain of Y. enterocolitica. In the absence of the plasmid, the colonies were colorless. Strains of the biotype 1A (IP124) and biotype 4/0:3 (IP134) were used as controls.

2.3.3. DNA Extraction and Real-Time PCR for Detection of Virulence Genes

Real-time PCR was used to evaluate the presence of virulence gene ail, carried by the genome, and the gene yadA, carried by the pYV plasmid, in order to confirm the pathogenicity of the strains. The strains were sub-cultured on PCA at 30 °C for 24 h. DNA was extracted from some colonies with an InstaGene™ Matrix kit (Biorad, Marnes-la-Coquette, France) following the manufacturer’s instructions. Two PCRs were performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), with a final volume of 25 µL, with the Sybr® Green JumpstartTM Taq ReadyMix TM (Sigma-Aldrich, Saint Louis, MI, USA) at 1X. Gene ail and gene yadA were detected with specific primers [12]. The final concentration of primers in the PCR reaction was 0.3 µM for ail and 0.2 µM for yadA. The primer sequence and amplification conditions for each gene are detailed in Table 1 [13].

2.4. Sequencing and Genome Analysis

The selection of sixty strains (one per positive sample) was submitted for sequencing with Illumina technology after DNA extraction with the QIAamp Fast DNA Tissue kit (Quiagen, Les Ulis, France) following the manufacturer’s instructions. Libraries were prepared using the Illumina DNA Prep Extraction Kit and sequenced in a 2 × 150 bp paired-end mode using an Illumina NextSeq 2000. The data on the assembled genomes of the selected strains are indicated in Table S1.
Short paired-end reads were trimmed using fastp [14] v0.20.1 (i.e., adapter sequences with mean quality phred score of 25 and correction in overlapped region). The trimmed reads were used to check the Yersinia enterocolitica species using speciesFinder (https://bitbucket.org/genomicepidemiology/speciesfinder/src/master/, accessed on 1 July 2024). The trimmed reads were mapped against the closely related reference genome identified by estimating the Jaccard index with Mash v2.0 [15] among a collection of high-quality fully closed assemblies from Refseq, representative of the species. The trimmed reads were assembled using Shovill v1.1.0 (https://github.com/tseemann/shovill, accessed on 1 July 2024). Contigs smaller than 200 bp or contigs with a coverage less than 2xwere removed from the assembly. Then, the finalized assembly was annotated with Bakta v1.8.1 [16].
Contigs from the assembly were classified as either plasmid or chromosomal using Plascope [17] v1.3.1. For this analysis, a database was constructed comprising complete Yersinia chromosomal sequences from RefSeq and plasmid sequences specific to Yersinia obtained from the PLSdb [18] database (version 2020_06_23).
Raw reads were uploaded to enterobase [19] for multi-locus sequence typing (MLST) and core genome MLST (cgMLST). The MLST was performed according to the McNally scheme [20]. The cgMLST profiles were grouped and visualized by the MSTree V2 algorithm in GrapeTree [21]. Strains were considered closely related when the allelic distance was ≤5 for strains of BT4, as defined by Le Guern et al. [22] for their cgMLST.
The wgMLST (6344 genes) analysis was performed on the assemblies using Chewbbaca [23] v3.2.0 with the INNUENDO schema provided by Chewie-NS [24] (31 May 2021). The resulting allele profiles were converted into a distance matrix based on Hamming distances. These calculated distances were subsequently used to construct a phylogenetic tree employing the Neighbor-Joining (NJ) clustering method.
The core-genome SNP analysis was conducted using Snippy, with the trimmed reads mapped to the reference genome NZ_CP030980.1. The resulting core.full.aln alignment file was analyzed with IQ-TREE [25] v2.2.0.3, which generated a phylogenetic tree by selecting the optimal model based on statistical criteria. The best-fitting model, K3Pu + F + I, was chosen according to the Bayesian Information Criterion (BIC). The log-likelihood score of the consensus tree was −6,341,037.854 with 1000 bootstraps. The minimum spanning tree, illustrating the clustering of strains based on the number of SNPs separating them, was generated using Grapetree [21] v1.5.0.
The wgMLST and cgSNP trees were annotated with iTol [26]. The strains were considered closely related when the allelic distance was ≤4 and the number of different SNPs was ≤10.

3. Results

3.1. Prevalence of Pathogenic Y. enterocolitica

Of the 375 samples, 60 were found to be contaminated with pathogenic Y. enterocoli- tica, giving an overall contamination rate of 16.0% IC95% [12.6–20.1]. There was a significant difference between the three types of matrices, with a much higher rate of pathogenic Y. enterocolitica contamination for tongues (39.4%), followed by cheeks (16.4%) (Table 2). Only one meat sample out of 160 was contaminated (0.6%). Positive samples were found in all the 13 French regions covered by this exploratory survey.
Depending on the enrichment broth, the detection of positive samples in pathogenic Y. enterocolitica was favored by PSB compared to ITC. Indeed, of the 60 positive samples, 56 were detected as positive by the PSB-CIN pathway and 21 by the ITC-CIN pathway, of which 17 were positive with both broths. Four samples were positive with ITC and negative with PSB, and 39 were positive with PSB and negative with ITC.
These matrices were packed under film (48.0%), modified atmosphere (38.6%), or vacuum (12.3%). A matrix was not associated with a particular type of packaging. The conditioning had no impact on the prevalence of pathogenic Y. enterocolitica (Table 2).
The seasonal effect was considered, taking into account the absence of sampling during the winter period. Contamination rates ranged from 14.9% to 18.2%, with no significant difference between the three seasons (Table 2).

3.2. Biotype and Plasmid pYV

For these tests, we prioritized per positive sample at least two strains with a pathogenic profile on YeCM. A total of 128 strains were thus tested for the presence of the ail gene and the yadA gene by PCR and biotyped. The strains were distributed as follows according to the biotype: 125 BT4 strains (97.6% of strains), 2 BT3 strains isolated from two cheeks (1.6%), and 1 BT2 strain isolated from one cheek (0.8%).
All the 128 strains had a positive ail-PCR confirming pathogenic Y. enterocolitica, and 71.1% of them had a positive yadA-PCR, suggesting that the other strains (n = 37) had lost their plasmid (35 of the BT4 strains, 1 of the BT3 strains, and the BT2 strain). Among the 60 samples positive for Y. enterocolitica, 15 samples had strains with a positive yadA-PCR and strains with a negative yadA-PCR, 33 samples had only positive yadA-PCR strains, and 12 samples had only negative yadA-PCR strains. Therefore, strains with and strains without plasmids could coexist in the same sample. There was no statistical relationship between the presence or absence of the yadA gene and the type of matrix (tongues, cheeks, meat; Pearson’s chi-square test: p = 0.740), the enrichment broth (PSB, ITC; Pearson’s chi-square test: p = 0.410), and the type of sample conditioning (modified atmosphere, film, vacuum; Pearson’s chi-square test: p = 0.287).
The 128 strains were tested on CR-MOX plates; 84 strains (65.6%) showed a mix of colorless colonies and colonies that were orange–red pinheads, with the latter indicating the presence of the plasmid for these strains of Y. enterocolitica (Figure 2). These 84 strains also had a positive yadA-PCR. The other strains (n = 44) had only colorless colonies, which suggested the absence of the plasmid. However, among the latter, seven strains had a positive yadA-PCR.
Within the 60 strains sequenced (1 per positive sample), the presence of the plasmid was confirmed for 40 strains after sequencing. They all had a positive yadA-PCR, except one strain, suggesting that this strain had a plasmid pYV without a yadA gene.

3.3. Genetic Diversity of BT4 Strains (n = 57)

The MLST analyses identified 55 BT4 strains with the sequence type ST18; the others were of the ST203 type. The cgMLST analyses revealed 56 cgMLST genotypes, and the ST18 type was represented by 54 of them. Among them, 12 strains could be grouped into five cgMLST clusters (Figure 3). With a Simpson diversity index of 0.994 IC95% [0.99–1.00], this population was highly genetically diverse.
The wgMLST and SNP analysis also showed high genetic diversity in this Y. enterocolitica population, with a Simpson diversity index of 0.998 IC95% [0.99–1.00]. Only six strains among the BT4 strains were distributed in three clusters and could be considered very genetically close, with or without the plasmid (Figure 4): Y23PE0610 (cheek, 9 November 9, with plasmid) and Y23PE0147 (tongue, 5 May, with plasmid) with nine SNPs and four alleles of difference; Y23PE0673 (tongue, 11 November, with plasmid) and Y23PE0465 (cheek, 29 September, without plasmid) with seven SNPs and three alleles of difference; Y23PE0731 (cheek, 30 November, with plasmid) and Y23PE 0569 (tongue, 26 October, without plasmid) with one SNP and two alleles of difference. None of these strains were isolated on the same date or in the same region in France.

4. Discussion

This exploratory survey provides data on Y. enterocolitica contamination at retail and in matrices (cheeks, tongues, and fresh pork meat) that have not been explored until now in all regions of mainland France. The contamination rate for pathogenic Y. enterocolitica was 16.0% in the matrices covered by this survey. This is of the same order (14.0%) as that observed in a survey carried out previously in France [6] whose samples consisted of tonsil swabs before the separation of the whole head from the pig carcass.
Positive samples were found in the 13 French regions covered by this exploratory survey. In France, 68% of pig production is located in the west of the country in two regions (56.3% in Brittany and 11.7% in Pays de Loire) [27] and carcasses or meat from these pigs is distributed throughout the country. According to Fondrevez et al., [6] who showed that 74.3% of pig batches tested (n = 96) contained at least one pig positive for pathogenic Y. enterocolitica, the bacterium can be expected to spread throughout the country via pork products distributed at retail.
We detected a higher number of positive samples by the PSB-CIN pathway than by the ITC-CIN pathway. The highest recovery rate of the pathogen from the tonsils was found when alkali-treated PSB and CIN agar were combined [28]. In a previous study, we demonstrated that the ITC-CIN pathway was more effective at detecting pathogenic Y. enterocolitica from tonsil swabs [29]. This difference may have been due to the KOH treatment that was applied prior to isolation on CIN in this survey at retail, a treatment that we did not apply to the tonsil swabs sampled at slaughterhouses using our ITC-CIN method [6]. The saliva of pigs is rich in bacterial flora [30]; the absence of KOH treatment after using PSB, which is a weakly selective enrichment medium, could result in abundant background flora on CIN after the enrichment of the tongues, making it difficult to observe typical colonies of Y. enterocolitica. To limit competition with background flora during enrichment, better results could be obtained using pork products with only a 1-day enrichment in PSB, without carrying out KOH treatment [31]. With this survey, we believed that 2-day enrichment in PSB and in ITC followed by KOH treatment, as recommended by the standard ISO 10273-2017, was optimal for detecting and isolating pathogenic Y. enterocolitica and, in particular, for samples expected to have a high level of associated flora.
The results showed that 39.4% of the tongues and 16.2% of the cheeks were contaminated with pathogenic Y. enterocolitica, while only one sample of meat was contaminated (<1%). If only the head matrices were considered (tongues and cheeks), the contamination rate rose to 27.4% for these matrices taken at retail. This presence of pathogenic Y. enterocolitica on tongues was consistent with what was observed after the inoculation of pigs with a BT4 strain [9] or in naturally contaminated pigs at slaughter [32] or at retail [33,34].
Head muscles and tongues appear to be food matrices at risk from pathogenic Y. enterocolitica but also from other pathogens such as Salmonella and Listeria, two important bacterial pathogens in human health. One study reported that the prevalence of Salmonella on the tongues (9.3%) and tonsils (19.6%) of pigs could also be high and higher than that observed on the carcasses of the same pigs (1.4%) [35]. Another study revealed a prevalence of 14% on tongues and 12% on tonsils for Listeria monocytogenes, similar to that observed on carcasses (12%) [36].
This contamination on these two matrices, which is higher than that obtained on whole-head tonsils (14%) [6], suggests that cross-contamination may occur during the boning of pig heads. Indeed, at the end of the slaughter line, the whole head is separated from the carcass and sent to the pig head boning line in almost all French pig slaughterhouses. This step, which is generally manual, consists of separating the various parts of the head (ear, snout, rind without meat, rind with meat, tongue, cheek meat, temple meat, etc.) in order to recover at least 50% of the pig’s head. Some of these parts are intended for animal feed and others are intended for human consumption in the form of processed or unprocessed products, such as cheeks and tongues available at retail.
The low level of contamination of pathogenic Y. enterocolitica on the meat (0.6%) was consistent with the results of a study conducted by the French Pig and Pork Institute (IFIP) showing no contamination of pig carcasses swabbed at slaughterhouses [37]. A lower prevalence on carcasses compared to tonsils was also observed in another study [38]. Meanwhile, other studies have shown an equally lower prevalence of pathogenic Y. enterocolitica on meat compared to tongues at retail [33,34]. These results suggest that, for Y. enterocolitica, the evisceration stage at the abattoir is a less contaminating step than the boning step and that splitting the carcass in two at the slaughterhouse without including the head is less contaminating than splitting the carcass in two with the head, as is performed in some slaughterhouses.
With a contamination of 16% and a BT4 biotype predominantly found in these matrices, this zoonotic agent remains an agent to be monitored in pigs, especially because, as we recently demonstrated, BT4 strains of porcine origin can survive and multiply at 4 °C on ham for 10 days, thus confirming the psychrotrophic nature of this pathogen [39].
The packaging of the samples had no impact on the rate of contamination by pathogenic Y. enterocolitica in the matrices, nor did the season of slaughter at retail. A seasonal effect was, however, observed in our previous study [6], where prevalence on tonsils was higher in the warm period (15.3%) compared to the cold period (5.9%). But, in this survey at retail, no samples were sampled in winter, which could explain this difference.
During this exploratory survey, it was mainly strains of the BT4 pathogenic biotype that were isolated (97.6% of strains). The BT4 pathogenic biotype is mainly isolated from pigs. This is in line with what we previously obtained from pig tonsils [6] and feces during a French survey conducted by the IFIP in 2010 [37]. The French NRC of Plague and other Yersinioses concluded that there was a strong association between the BT4 biotype found in human infections and the BT4 biotype isolated from pigs [5].
The Yersinia virulence plasmid pYV confers on strains of Y. enterocolitica an adhesive potential superior to the one encoded by the chromosome alone. Moreover, the YadA protein is crucial for adhesion to intestinal tissues, extracellular matrix proteins, and other surfaces. Without YadA, Y. enterocolitica shows reduced adhesion capabilities, which affects its ability to colonize hosts effectively [40]. The absence of the yadA gene for some of our strains was confirmed by the absence of the plasmid after sequencing and suggested that these strains had lost their plasmid. From tonsils from pig carcasses from French slaughterhouses, we identified that 12.0% of pathogenic Y. enterocolitica strains did not have the yadA gene [13]. The arrival of plasmid-free strains at slaughterhouses reflects the situation in farms. Through the oral inoculation of pigs with a BT4 yadA+ strain, it has been demonstrated that this plasmid could be partially lost over the duration of an experimental trial [9]. In our study, realized at retail using pork products in France, we observed a higher number of strains without the yadA gene (28.9%) compared to that which we observed on pig tonsils (12.0%) [13]. This situation suggests that the loss of the plasmid may be linked in part to stress conditions generated by the duration and conditions of the slaughter process, of the distribution of pork products, and of the conservation of these products at retail. Nevertheless, we had samples in our survey in which some of the isolated strains had the plasmid and others did not after yadA-PCR.
Among the sequenced strains, one strain did not have the yadA gene while the plasmid was present. The presence of the virulence plasmid without the yadA gene leads to a marked decrease in the virulence of Y. enterocolitica. The culture of pathogenic Y. enterocolitica strains on the CR-MOX (Congo-red magnesium oxalate) plates allowed us to observe that some colonies could have a positive plasmid reaction (pinhead orange–red colonies) and others on the same plate could have a negative plasmid reaction (colorless colonies) for the same strain. This suggest that a strain could be a mix of cells with and without the plasmid and that a sub-culture can be made from a bacterial cell without the plasmid giving a negative PCR result, as observed in our study. It is usually possible to identify a pathogenic biotype with a percentage of its population (colonies) still containing the plasmid and the other not on CR-MOX plates [41]. The virulence plasmid can also easily be lost when the strains are subcultured at temperatures higher than 30 °C, if they are repeatedly subcultured, or if they are stored over time [41,42]. To prevent this scenario, after the isolation of the strains, we carried out only two successive streakings of the strains on PCA with incubation at 30 °C to simultaneously carry out the CR-MOX test, DNA extraction for detecting the ail and yadA gene by PCR, and DNA extraction for genome sequencing. The aim was to characterize as accurately as possible the strains found on the meat. We observed that seven strains had a positive yadA-PCR whereas they were negative on the CR-Mox; this agar test was less effective than PCR or sequencing in determining the presence of the plasmid pyV.
The comparison of the genomes of our BT4 strains revealed a high genetic diversity in the strains isolated from pork matrices at retail. This high diversity of BT4 swine strains in France was previously demonstrated by MLVA typing [43] from strains isolated from tonsils at slaughterhouses. The most prevalent sequence type, ST18 (96.5% of our BT4 strains), was distributed in 54 cgMLST genotypes. Similar results were observed in a study carried out in Latvia [38] using pig tonsils and carcasses at slaughterhouses. Their BT4 strains were only represented by the sequence type ST18, and among them, 43 cgMLST genotypes were identified. This high genetic diversity in our Y. enterocolitica populations was confirmed by the wgMLST and SNP analyses that we carried out, from which the three clusters were similar to three of the five clusters obtained in the cgMLST.

5. Conclusions

Our study is the first to provide data on the prevalence of pathogenic Yersinia enterocolitica at retail all over France. While the biotype is a good marker for assessing the pathogenicity of Y. enterocolitica strains, it is important to ensure the presence of the pyV plasmid, which plays an important role in infection. The higher contamination on tongues and cheeks, compared to pork meat, suggests that the pig head deboning step at slaughterhouses is more at risk than the evisceration step of contamination by pathogenic Yersinia enterocolitica. This also suggests that slaughter processes that split the head at the same time as the carcass could lead to a higher prevalence on carcasses and consequently on meat, due to the contamination of the cutting tools by Y. enterocolitica. The same conclusion can also be made concerning other pathogens such as Salmonella and Listeria monocytogenes, which can be found in the oral cavity of pigs.
In conclusion, this zoonotic agent remains an agent to be monitored in pigs. Because of its psychrotrophic nature, this pathogen can represent a real health problem at the slaughterhouse, cutting plant, and consumer stages of a product’s life, where refrigeration is used as a means of controlling pathogens.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/applmicrobiol5010015/s1. Table S1: Sequencing data on the assembled genomes.

Author Contributions

M.D. was the co-ordinator of this survey in the laboratory, analyzed the data, carried out the MLST and cgMLST analyses using Enterobase, and wrote the manuscript. D.N. was the co-ordinator of this survey for the technical instruction and the sampling planning with the French DDPP (Departmental directorate for population protection). L.D. and E.H. analyzed the samples, isolated the strains, and realized the assays and DNA extraction. M.T. realized the libraries and sequencing. A.F. carried out the wgMLST and cgSNPs analyses and deposited the reads in SRA. M.C. supervised the survey. All authors have read and agreed to the published version of the manuscript.

Funding

The DGAL financed the sampling and the analyses carried out as part of this survey; financial agreement number DGAL/SDEIGIR/2023-173.

Data Availability Statement

The reads are accessible in SRA in BioProject PRJNA1211310.

Acknowledgments

We would like to thank all the staff of the HQPAP Unit, Anses de Ploufragan, who contributed to the completion of this survey, in particular J. Gateau and V. Rose for their technical assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. EFSA; ECDC. The European Union one health 2023 zoonoses report. EFSA J. 2024, 22, e9106. [Google Scholar]
  2. Van Cauteren, D.; Le Strat, Y.; Sommen, C.; Bruyand, M.; Tourdjman, M.; Jourdan-Da Silva, N.; Couturier, E.; Fournet, N.; De Valk, H.; Desenclos, J.-C. Estimation de la morbidité et de la mortalité liées aux infections d’origine alimentaire en France métropolitaine, 2008-2013. Bull. Epidémiol. Hebd. 2018, 2–10. [Google Scholar]
  3. Tennant, S.M.; Grant, T.H.; Robins-Browne, R.M. Pathogenicity of Yersinia enterocolitica biotype 1A. FEMS Immunol. Med. Microbiol. 2003, 38, 127–137. [Google Scholar] [CrossRef] [PubMed]
  4. Mühlenkamp, M.; Oberhettinger, P.; Leo, J.C.; Linke, D.; Schütz, M.S. Yersinia adhesin A (YadA)--beauty & beast. Int. J. Med. Microbiol. 2015, 305, 252–258. [Google Scholar] [CrossRef] [PubMed]
  5. Le Guern, A.S.; Martin, L.; Savin, C.; Carniel, E. Yersiniosis in France: Overview and potential sources of infection. Int. J. Infect. Dis. 2016, 46, 1–7. [Google Scholar] [CrossRef] [PubMed]
  6. Fondrevez, M.; Minvielle, B.; Labbé, A.; Houdayer, C.; Rose, N.; Esnault, E.; Denis, M. Prevalence of pathogenic Yersinia enterocolitica in slaughter-aged pigs during a one-year survey, 2010-2011, France. Int. J. Food Microbiol. 2014, 174, 56–62. [Google Scholar] [CrossRef]
  7. Nesbakken, T.; Eckner, K.; Høidal, H.K.; Røtterud, O.J. Occurrence of Yersinia enterocolitica and Campylobacter spp. in slaughter pigs and consequences for meat inspection, slaughtering, and dressing procedures. Int. J. Food Microbiol. 2003, 80, 231–240. [Google Scholar] [CrossRef] [PubMed]
  8. Liang, J.; Wang, X.; Xiao, Y.; Cui, Z.; Xia, S.; Hao, Q.; Yang, J.; Luo, L.; Wang, S.; Li, K.; et al. Prevalence of Yersinia enterocolitica in pigs slaughtered in Chinese abattoirs. Appl. Environ. Microbiol. 2012, 78, 2949–2956. [Google Scholar] [CrossRef] [PubMed]
  9. Esnault, E.; Rouaud, A.; Labbé, A.; Houdayer, C.; Bailly, Y.; Houard, E.; Bougeard, S.; Paboeuf, F.; Eterradossi, N.; Chemaly, M.; et al. Controlled Experimental Infection in Pigs with a Strain of Yersinia enterocolitica Harboring Genetic Markers for Human Pathogenicity: Colonization and Stability. Infect. Immun. 2023, 91, e0015723. [Google Scholar] [CrossRef] [PubMed]
  10. ISO 10273:2017; Microbiology of food chain—Horizontal method for the detection of pathogenic Yersinia enterocolitica. ISO: Geneva, Switzerland, 2017.
  11. Weagant, S.D. A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica. J. Microbiol. Methods 2008, 72, 185–190. [Google Scholar] [CrossRef]
  12. Kot, B.; Piechota, M.; Jakubczak, A. Analysis of occurrence of virulence genes among Yersinia enterocolitica isolates belonging to different biotypes and serotypes. Pol. J. Vet. Sci. 2010, 13, 13–19. [Google Scholar] [PubMed]
  13. Fondrevez, M.; Labbé, A.; Houdayer, C.; Denis, M. Genetic characterization of Yersinia enterocolitica collected from tonsils of slaughtered pigs. In Proceedings of the Fifth International Symposium on the Epidemiology and Control of Foodborn Pathogens in Pork, Maastricht, The Netherlands, 19–22 June 2011. [Google Scholar]
  14. Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018, 34, i884–i890. [Google Scholar] [CrossRef]
  15. Ondov, B.D.; Treangen, T.J.; Melsted, P.; Mallonee, A.B.; Bergman, N.H.; Koren, S.; Phillippy, A.M. Mash: Fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016, 17, 132. [Google Scholar] [CrossRef] [PubMed]
  16. Schwengers, O.; Jelonek, L.; Dieckmann, M.A.; Beyvers, S.; Blom, J.; Goesmann, A. Bakta: Rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb. Genom. 2021, 7, 000685. [Google Scholar] [CrossRef] [PubMed]
  17. Royer, G.; Decousser, J.W.; Branger, C.; Dubois, M.; Médigue, C.; Denamur, E.; Vallenet, D. PlaScope: A targeted approach to assess the plasmidome from genome assemblies at the species level. Microb. Genom. 2018, 4, e000211. [Google Scholar] [CrossRef] [PubMed]
  18. Galata, V.; Fehlmann, T.; Backes, C.; Keller, A. PLSDB: A resource of complete bacterial plasmids. Nucleic Acids Res. 2019, 47, D195–D202. [Google Scholar] [CrossRef] [PubMed]
  19. Zhou, Z.; Alikhan, N.F.; Mohamed, K.; The Agama Study Group; Achtman, M. The EnteroBase user’s guide, with case studies on Salmonella transmissions, Yersinia pestis phylogeny and Escherichia core genomic diversity. Genome Res. 2020, 30, 138–152. [Google Scholar] [CrossRef]
  20. Hall, M.; Chattaway, M.A.; Reuter, S.; Savin, C.; Strauch, E.; Carniel, E.; Connor, T.; Van Damme, I.; Rajakaruna, L.; Rajendram, D.; et al. Use of whole-genus genome sequence data to develop a multilocus sequence typing tool that accurately identifies Yersinia isolates to the species and subspecies levels. J. Clin. Microbiol. 2015, 53, 35–42. [Google Scholar] [CrossRef] [PubMed]
  21. Zhou, Z.; Alikhan, N.F.; Sergeant, M.J.; Luhmann, N.; Vaz, C.; Francisco, A.P.; Carrico, J.A.; Achtman, M. GrapeTree: Visualization of core genomic relationships among 100,000 bacterial pathogens. Genome Res. 2018, 28, 1395–1404. [Google Scholar] [CrossRef]
  22. Le Guern, A.-S.; Savin, C.; Chereau, F.; Tessier, S.; Guglielmini, J.; Brémont, S.; Pizarro-Cerdá, J. A novel cgMLST for genomic surveillance of Yersinia enterocolitica infections in France allowed the detection and investigation of outbreaks in 2017−2021. Microbiol. Spectr. 2024, 12, e0050424. [Google Scholar] [CrossRef] [PubMed]
  23. Silva, M.; Machado, M.P.; Silva, D.N.; Rossi, M.; Moran-Gilad, J.; Santos, S.; Ramirez, M.; Carriço, J.A. chewBBACA: A complete suite for gene-by-gene schema creation and strain identification. Microb. Genom. 2018, 4, 000166. [Google Scholar] [CrossRef]
  24. Mamede, R.; Vila-Cerqueira, P.; Silva, M.; Carriço, J.A.; Ramirez, M. Chewie Nomenclature Server (chewie-NS): A deployable nomenclature server for easy sharing of core and whole genome MLST schemas. Nucleic Acids Res. 2021, 49, D660–D666. [Google Scholar] [CrossRef] [PubMed]
  25. Nguyen, L.T.; Schmidt, H.A.; von Haeseler, A.; Minh, B.Q. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [Google Scholar] [CrossRef]
  26. Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021, 49, W293–W296. [Google Scholar] [CrossRef] [PubMed]
  27. Roguet, C. Pig and pork production in France: Characteristics, changes and challenges. In Proceedings of the EPP (European Pig Production) Congress, Nantes, France, 29 May 2024. [Google Scholar]
  28. Zdolec, N.; Kiš, M.; Jankuloski, D.; Blagoevska, K.; Kazazić, S.; Pavlak, M.; Blagojević, B.; Antić, D.; Fredriksson-Ahomaa, M.; Pažin, V. Prevalence and Persistence of Multidrug-Resistant Yersinia enterocolitica 4/O:3 in Tonsils of Slaughter Pigs from Different Housing Systems in Croatia. Foods 2022, 11, 1459. [Google Scholar] [CrossRef]
  29. Fondrevez, M.; Labbé, A.; Houard, E.; Fravalo, P.; Madec, F.; Denis, M. A simplified method for detecting pathogenic Yersinia enterocolitica in slaughtered pig tonsils. J. Microbiol. Methods 2010, 83, 244–249. [Google Scholar] [CrossRef] [PubMed]
  30. Murase, K.; Watanabe, T.; Arai, S.; Kim, H.; Tohya, M.; Ishida-Kuroki, K.; Võ, T.H.; Nguyễn, T.P.B.; Nakagawa, I.; Osawa, R.; et al. Characterization of pig saliva as the major natural habitat of Streptococcus suis by analyzing oral, fecal, vaginal, and environmental microbiota. PLoS ONE 2019, 14, e0215983. [Google Scholar] [CrossRef] [PubMed]
  31. Peruzy, M.F.; Aponte, M.; Proroga, Y.T.R.; Capuano, F.; Cristiano, D.; Delibato, E.; Houf, K.; Murru, N. Yersinia enterocolitica detection in pork products: Evaluation of isolation protocols. Food Microbiol. 2020, 92, 103593. [Google Scholar] [CrossRef] [PubMed]
  32. Doyle, M.P.; Hugdahl, M.B.; Taylor, S.L. Isolation of virulent Yersinia enterocolitica from porcine tongues. Appl. Environ. Microbiol. 1981, 42, 661–666. [Google Scholar] [CrossRef] [PubMed]
  33. Fredriksson-Ahomaa, M.; Hielm, S.; Korkeala, H. High prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland. J. Food Prot. 1999, 62, 123–127. [Google Scholar] [CrossRef]
  34. Lorencova, A.; Slany, M. Prevalence of pathogenic Yersinia enterocolitica in minced meat, pig tongues and hearts at the retail level in the Czech Republic detected by real time PCR. Potravinarstvo Slovak. J. of Food Sci. 2016, 10, 282–286. [Google Scholar] [CrossRef] [PubMed]
  35. Swanenburg, M.; Urlings, H.A.; Snijders, J.M.; Keuzenkamp, D.A.; van Knapen, F. Salmonella in slaughter pigs: Prevalence, serotypes and critical control points during slaughter in two slaughterhouses. Int. J. Food Microbiol. 2001, 70, 243–254. [Google Scholar] [CrossRef]
  36. Autio, T.; Säteri, T.; Fredriksson-Ahomaa, M.; Rahkio, M.; Lundén, J.; Korkeala, H. Listeria monocytogenes contamination pattern in pig slaughterhouses. J. Food Prot. 2000, 63, 1438–1442. [Google Scholar] [CrossRef]
  37. Feurer, C.; Piaudel, G.; Le Roux, A.; Minvielle, B. Yersinia enterocolitica contamination of pig tonsils, carcasses and feces in one French slaughterhouse. In Proceedings of the 14èmes Journées Sciences du Muscle et Technologies des Viandes, Caen, France, 13–14 November 2012; pp. 65–66. [Google Scholar]
  38. Terentjeva, M.; Ķibilds, J.; Gradovska, S.; Alksne, L.; Streikiša, M.; Meistere, I.; Valciņa, O. Prevalence, virulence determinants, and genetic diversity in Yersinia enterocolitica isolated from slaughtered pigs and pig carcasses. Int. J. Food Microbiol. 2022, 376, 109756. [Google Scholar] [CrossRef] [PubMed]
  39. Denis, M.; Houard, E.; Ouedraogo, A.; Le Berre, L.; Feurer, C.; Pizarro-cerda, J.; Savin, C.; Le Guern, A.-S. Cold survival and pathogenicity of Yersinia enterocolitica BT4 strains isolated from pigs and genetically close or not to strains isolated from human cases. Anim.—Sci. Proc. 2024, 15, 265. [Google Scholar] [CrossRef]
  40. Paerregaard, A.; Espersen, F.; Skurnik, M. Role of the Yersinia outer membrane protein YadA in adhesion to rabbit intestinal tissue and rabbit intestinal brush border membrane vesicles. APMIS 1991, 99, 226–232. [Google Scholar] [CrossRef] [PubMed]
  41. Farmer, J.J., 3rd; Carter, G.P.; Miller, V.L.; Falkow, S.; Wachsmuth, I.K. Pyrazinamidase, CR-MOX agar, salicin fermentation-esculin hydrolysis, and D-xylose fermentation for identifying pathogenic serotypes of Yersinia enterocolitica. J. Clin. Microbiol. 1992, 30, 2589–2594. [Google Scholar] [CrossRef] [PubMed]
  42. Thoerner, P.; Bin Kingombe, C.I.; Bögli-Stuber, K.; Bissig-Choisat, B.; Wassenaar, T.M.; Frey, J.; Jemmi, T. PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution. Appl. Environ. Microbiol. 2003, 69, 1810–1816. [Google Scholar] [CrossRef]
  43. Raymond, P.; Houard, E.; Denis, M.; Esnault, E. Diversity of Yersinia enterocolitica isolated from pigs in a French slaughterhouse over 2 years. MicrobiologyOpen 2019, 8, e00751. [Google Scholar] [CrossRef] [PubMed]
Applmicrobiol 05 00015 g001
Figure 1. The distribution of the samples in the 13 regions of mainland France.
Figure 1. The distribution of the samples in the 13 regions of mainland France.
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Figure 2. The strain Y23PE0281 (BT4, isolated from tongue) and strain Y23PE0341 (BT4, isolated from tongue) on CR-MOX after 28 h at 37 °C. In the absence of the plasmid, the colonies were colorless (strain Y23PE0281). The strain Y23PE0341 showed two types of colonies, colorless colonies and colonies that were orange–red pinheads, indicating the presence of the plasmid.
Figure 2. The strain Y23PE0281 (BT4, isolated from tongue) and strain Y23PE0341 (BT4, isolated from tongue) on CR-MOX after 28 h at 37 °C. In the absence of the plasmid, the colonies were colorless (strain Y23PE0281). The strain Y23PE0341 showed two types of colonies, colorless colonies and colonies that were orange–red pinheads, indicating the presence of the plasmid.
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Figure 3. The cgMLST tree from Enterobase of the 57 BT4 strains. The number on the branch indicates the allelic distance. Clusters were created when the allelic distance was ≤5. A total of 56 cgMLST genotypes were identified and could not all be presented on the tree.
Figure 3. The cgMLST tree from Enterobase of the 57 BT4 strains. The number on the branch indicates the allelic distance. Clusters were created when the allelic distance was ≤5. A total of 56 cgMLST genotypes were identified and could not all be presented on the tree.
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Figure 4. The cgSNP tree for the BT4 strains (n = 57). Strains were considered closely related when the allelic distance was ≤4 (with wgMLST, tree not shown) and the number of different SNPs was ≤10.
Figure 4. The cgSNP tree for the BT4 strains (n = 57). Strains were considered closely related when the allelic distance was ≤4 (with wgMLST, tree not shown) and the number of different SNPs was ≤10.
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Table 1. Primer sequences and real-time PCR conditions for detection of ail and yadA genes.
Table 1. Primer sequences and real-time PCR conditions for detection of ail and yadA genes.
GenesPrimer SequenceFirst StepCycle of AmplificationMelt CurveSize in bpExpected Tm
ailF—TGG TTA TGC GCA AAG CCA TGT94 °C,
2 min		35 cycles of following:55 °C -> 89 °C
R—TGG AAG TGG GTT GAA TTG CA Denaturation, 94 °C, 30 s;
Annealing, 57 °C, 30 s;
Extension, 72 °C, 60 s.		increment of 0.5 °C every 5 s35682.5 °C
yadAF—CAG ATA CAC CTG CCT TCC ATC T94 °C,
2 min		35 cycles of following:55 °C -> 95 °C
R—CTC GAC ATA TTC CTC AAC ACG C Denaturation, 94 °C, 60 s;
Annealing, 58 °C, 60 s;
Extension, 72 °C, 60 s.		increment of 0.5 °C every 5 s74786 °C
Table 2. The level of contamination by pathogenic Y. enterocolitica according to the matrices, the packaging, and the seasons. The p-value was obtained from Pearson’s chi-square test.
Table 2. The level of contamination by pathogenic Y. enterocolitica according to the matrices, the packaging, and the seasons. The p-value was obtained from Pearson’s chi-square test.
NegativePositiveTotal% of PositiveIC95%p-Value
MatrixCheek931811116.210.4–21.9
Tongue634110439.431.5–47.3p = 4.6 × 10−16
Meat15911600.60.02–3.4
Total3156037516.012.8–19.1
PackagingModified atmosphere1172814519.313.9–24.7
Under film1542618014.410.1–18.7p = 0.395
Under vacuum4164712.74.8–20.7
nd **3030-
Total3156037516.012.8–19.1
SeasonSpring81189918.211.8–24.5
Summer62127416.29.2–23.2p = 0.759
Autumn1723020214.910.7–18.9
Total3156037516.012.8–19.1
p value from Pearson’s chi-square test; nd: not determined; **: data not considered in chi-square test.
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Denis, M.; Felten, A.; Ducret, L.; Houard, E.; Tasset, M.; Novi, D.; Chemaly, M. Pathogenic Yersinia enterocolitica’s Contamination of Cheeks, Tongues, and Other Pork Meats at Retail in France, 2023. Appl. Microbiol. 2025, 5, 15. https://doi.org/10.3390/applmicrobiol5010015

AMA Style

Denis M, Felten A, Ducret L, Houard E, Tasset M, Novi D, Chemaly M. Pathogenic Yersinia enterocolitica’s Contamination of Cheeks, Tongues, and Other Pork Meats at Retail in France, 2023. Applied Microbiology. 2025; 5(1):15. https://doi.org/10.3390/applmicrobiol5010015

Chicago/Turabian Style

Denis, Martine, Arnaud Felten, Linda Ducret, Emmanuelle Houard, Manon Tasset, Delphine Novi, and Marianne Chemaly. 2025. "Pathogenic Yersinia enterocolitica’s Contamination of Cheeks, Tongues, and Other Pork Meats at Retail in France, 2023" Applied Microbiology 5, no. 1: 15. https://doi.org/10.3390/applmicrobiol5010015

APA Style

Denis, M., Felten, A., Ducret, L., Houard, E., Tasset, M., Novi, D., & Chemaly, M. (2025). Pathogenic Yersinia enterocolitica’s Contamination of Cheeks, Tongues, and Other Pork Meats at Retail in France, 2023. Applied Microbiology, 5(1), 15. https://doi.org/10.3390/applmicrobiol5010015

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