Listeria monocytogenes prevalence and genomic diversity along the pig and pork production chain.

The facultative intracellular bacterium Listeria monocytogenes (L. monocytogenes) is the causative agent of listeriosis, a severe invasive illness. This ubiquitous species is widely distributed in the environment, but infection occurs almost exclusively through ingestion of contaminated food. The pork production sector has been heavily affected by a series of L. monocytogenes-related foodborne outbreaks in the past around the world. Ready-to-eat (RTE) pork products represent one of the main food sources for strong-evidence listeriosis outbreaks. This pathogen is known to be present throughout the entire pig and pork production chain. Some studies hypothesized that the main source of contamination in final pork products was either living pigs or the food-processing environment. A detailed genomic picture of L. monocytogenes can provide a renewed understanding of the routes of contamination from pig farms to the final products. This review provides an overview of the prevalence, the genomic diversity and the genetic background linked to virulence of L. monocytogenes along the entire pig and pork production chain, from farm to fork.

Holistic integration of omics data reveals the drivers that shape the ecology of microbial meat spoilage scenarios.

Background: The use of omics data for monitoring the microbial flow of fresh meat products along a production line and the development of spoilage prediction tools from these data is a promising but challenging task. In this context, we produced a large multivariate dataset (over 600 samples) obtained on the production lines of two similar types of fresh meat products (poultry and raw pork sausages). We describe a full analysis of this dataset in order to decipher how the spoilage microbial ecology of these two similar products may be shaped differently depending on production parameter characteristics. Methods: Our strategy involved a holistic approach to integrate unsupervised and supervised statistical methods on multivariate data (OTU-based microbial diversity; metabolomic data of volatile organic compounds; sensory measurements; growth parameters), and a specific selection of potential uncontrolled (initial microbiota composition) or controlled (packaging type; lactate concentration) drivers. Results: Our results demonstrate that the initial microbiota, which is shown to be very different between poultry and pork sausages, has a major impact on the spoilage scenarios and on the effect that a downstream parameter such as packaging type has on the overall evolution of the microbial community. Depending on the process, we also show that specific actions on the pork meat (such as deboning and defatting) elicit specific food spoilers such as Dellaglioa algida, which becomes dominant during storage. Finally, ecological network reconstruction allowed us to map six different metabolic pathways involved in the production of volatile organic compounds involved in spoilage. We were able connect them to the different bacterial actors and to the influence of packaging type in an overall view. For instance, our results demonstrate a new role of Vibrionaceae in isopropanol production, and of Latilactobacillus fuchuensis and Lactococcus piscium in methanethiol/disylphide production. We also highlight a possible commensal behavior between Leuconostoc carnosum and Latilactobacillus curvatus around 2,3-butanediol metabolism. Conclusion: We conclude that our holistic approach combined with large-scale multi-omic data was a powerful strategy to prioritize the role of production parameters, already known in the literature, that shape the evolution and/or the implementation of different meat spoilage scenarios.

Identification by High-Throughput Real-Time PCR of 30 Major Circulating Listeria monocytogenes Clonal Complexes in Europe.

Listeria monocytogenes is a ubiquitous bacterium that causes a foodborne illness, listeriosis. Most strains can be classified into major clonal complexes (CCs) that account for the majority of outbreaks and sporadic cases in Europe. In addition to the 20 CCs known to account for the majority of human and animal clinical cases, 10 CCs are frequently reported in food production, thereby posing a serious challenge for the agrifood industry. Therefore, there is a need for a rapid and reliable method to identify these 30 major CCs. The high-throughput real-time PCR assay presented here provides accurate identification of these 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations, along with the molecular serogroup of a strain. Based on the BioMark high-throughput real-time PCR system, our assay analyzes 46 strains against 40 real-time PCR arrays in a single experiment. This European study (i) designed the assay from a broad panel of 3,342 L. monocytogenes genomes, (ii) tested its sensitivity and specificity on 597 sequenced strains collected from 24 European countries, and (iii) evaluated its performance in the typing of 526 strains collected during surveillance activities. The assay was then optimized for conventional multiplex real-time PCR for easy implementation in food laboratories. It has already been used for outbreak investigations. It represents a key tool for assisting food laboratories to establish strain relatedness with human clinical strains during outbreak investigations and for helping food business operators by improving their microbiological management plans. IMPORTANCE Multilocus sequence typing (MLST) is the reference method for Listeria monocytogenes typing but is expensive and takes time to perform, from 3 to 5 days for laboratories that outsource sequencing. Thirty major MLST clonal complexes (CCs) are circulating in the food chain and are currently identifiable only by sequencing. Therefore, there is a need for a rapid and reliable method to identify these CCs. The method presented here enables the rapid identification, by real-time PCR, of 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations. The assay was then optimized on different conventional multiplex real-time PCR systems for easy implementation in food laboratories. The two assays will be used for frontline identification of L. monocytogenes isolates prior to whole-genome sequencing. Such assays are of great interest for all food industry stakeholders and public agencies for tracking L. monocytogenes food contamination.

Tell me if you prefer bovine or poultry sectors and I'll tell you who you are: Characterization of Salmonella enterica subsp. enterica serovar Mbandaka in France.

Introduction: In north-western France, Salmonella enterica susp. enterica serovar Mbandaka (S. Mbandaka) is most frequently isolated from bovine and dairy samples. While this serovar most often results in asymptomatic carriage, for a number of years it has caused episodes of abortions, which have serious economic consequences for the sector. Interestingly, this serovar is also isolated from Gallus gallus in the same geographic zone. Despite its prevalence in bovines in north-western France, S. Mbandaka has not been broadly studied at the genomic level, and its prevalence and host adaptation are still not fully understood. Methods: In this study, we analyzed the genomic diversity of 304 strains of S. Mbandaka isolated from the bovine and poultry sectors in this area over a period of 5 years. A phylogenetic analysis was carried out and two approaches were followed to identify conserved genes and mutations related to host associations. The first approach targeted the genes compiled in the MEGARESv2, Resfinder, VFDB and SPI databases. Plasmid and phage contents were also investigated. The second approach refers to an in-house algorithm developed for this study that computes sensitivity, specificity, and accuracy of accessory genes and core variants according to predefined genomes groups. Results and discussion: All the analyzed strains belong to the multi-locus sequence type profile ST413, and the phylogenomic analysis revealed main clustering by host (bovine and poultry), emphasizing the circulation of 12 different major clones, of which seven circulate in poultry and five in the bovine sector in France and a likely food production chain adaptation of these clones. All strains present resistance determinants including heavy metals and biocides that could explain the ability of this serovar to survive and persist in the environment, within herds, and in food processing plants. To explore the wild animal contribution to the spread of this serovar in north-western France, we retrieved S. Mbandaka genomes isolated from wild birds from EnteroBase and included them in the phylogenomic analysis together with our collection. Lastly, screening of accessory genes and major variants allowed us to identify conserved specific mutations characteristic of each major cluster. These mutations could be used to design useful probes for food safety surveillance.

Contribution of omics to biopreservation: Toward food microbiome engineering.

Biopreservation is a sustainable approach to improve food safety and maintain or extend food shelf life by using beneficial microorganisms or their metabolites. Over the past 20 years, omics techniques have revolutionised food microbiology including biopreservation. A range of methods including genomics, transcriptomics, proteomics, metabolomics and meta-omics derivatives have highlighted the potential of biopreservation to improve the microbial safety of various foods. This review shows how these approaches have contributed to the selection of biopreservation agents, to a better understanding of the mechanisms of action and of their efficiency and impact within the food ecosystem. It also presents the potential of combining omics with complementary approaches to take into account better the complexity of food microbiomes at multiple scales, from the cell to the community levels, and their spatial, physicochemical and microbiological heterogeneity. The latest advances in biopreservation through omics have emphasised the importance of considering food as a complex and dynamic microbiome that requires integrated engineering strategies to increase the rate of innovation production in order to meet the safety, environmental and economic challenges of the agri-food sector.

Spoilage of fresh turkey and pork sausages: Influence of potassium lactate and modified atmosphere packaging.

Fresh poultry and pork meat products represent highly perishable products which are susceptible to spoil within a few days after production. Lactate addition and modified atmosphere packaging are common preservation strategies used to overcome spoilage. This study aimed to identify the effects of these strategies and their possible interactions on spoilage indicators simultaneously on fresh pork and turkey sausages. Ten batches of raw meat (turkey or pork) sausages were industrially produced with different lactate concentrations (0, 1 or 2% w/w in turkey and 0, 0.57 and 1.13% w/w in pork), packed under different gas mixtures (air, MAP1: 70% O2 - 30% CO2 and MAP2: 50% CO2 - 50% N2) and chill stored during 22 days. Spoilage responses including enumeration of total aerobic mesophilic and lactic acid bacteria, measurement of pH and colour, evaluation of visual defects and off-odour, were monitored. Effects of lactate and modified atmosphere packaging (MAP) as well as random effect of the batch variability were studied using a mixed effect model. Despite initial batch variability, significant effects of lactate and gas packaging were observed but in a different way in turkey and pork. Our results suggest that for fresh turkey sausages, the gas mixture enriched in oxygen enhanced off-odour perception and sausage discolouration from red to dark grey / brown colour. Unlike turkey sausages, in pork sausages, lactate did not significantly influence the monitored spoilage responses, whereas MAP (70% O2-30% CO2) reduced the off-odour perception. The developed model could be useful to estimate the effect of preservation strategies on spoilage occurrence while considering industrial batch variability.

Large-scale multivariate dataset on the characterization of microbiota diversity, microbial growth dynamics, metabolic spoilage volatilome and sensorial profiles of two industrially produced meat products subjected to changes in lactate concentration and packaging atmosphere.

Data in this article provide detailed information on the diversity of bacterial communities present on 576 samples of raw pork or poultry sausages produced industrially in 2017. Bacterial growth dynamics and diversity were monitored throughout the refrigerated storage period to estimate the impact of packaging atmosphere and the use of potassium lactate as chemical preservative. The data include several types of analysis aiming at providing a comprehensive microbial ecology of spoilage during storage and how the process parameters do influence this phenomenon. The analysis includes: the gas content in packaging, pH, chromametric measurements, plate counts (total mesophilic aerobic flora and lactic acid bacteria), sensorial properties of the products, meta-metabolomic quantification of volatile organic compounds and bacterial community metagenetic analysis. Bacterial diversity was monitored using two types of amplicon sequencing (16S rRNA and GyrB encoding genes) at different time points for the different conditions (576 samples for gyrB and 436 samples for 16S rDNA). Sequencing data were generated by using Illumina MiSeq. The sequencing data have been deposited in the bioproject PRJNA522361. Samples accession numbers vary from SAMN10964863 to SAMN10965438 for gyrB amplicon and from SAMN10970131 to SAMN10970566 for 16S.

Impact of cleaning and disinfection procedures on microbial ecology and Salmonella antimicrobial resistance in a pig slaughterhouse.

To guarantee food safety, a better deciphering of ecology and adaptation strategies of bacterial pathogens such as Salmonella in food environments is crucial. The role of food processing conditions such as cleaning and disinfection procedures on antimicrobial resistance emergence should especially be investigated. In this work, the prevalence and antimicrobial resistance of Salmonella and the microbial ecology of associated surfaces communities were investigated in a pig slaughterhouse before and after cleaning and disinfection procedures. Salmonella were detected in 67% of samples and isolates characterization revealed the presence of 15 PFGE-patterns belonging to five serotypes: S.4,5,12:i:-, Rissen, Typhimurium, Infantis and Derby. Resistance to ampicillin, sulfamethoxazole, tetracycline and/or chloramphenicol was detected depending on serotypes. 16S rRNA-based bacterial diversity analyses showed that Salmonella surface associated communities were highly dominated by the Moraxellaceae family with a clear site-specific composition suggesting a persistent colonization of the pig slaughterhouse. Cleaning and disinfection procedures did not lead to a modification of Salmonella susceptibility to antimicrobials in this short-term study but they tended to significantly reduce bacterial diversity and favored some genera such as Rothia and Psychrobacter. Such data participate to the construction of a comprehensive view of Salmonella ecology and antimicrobial resistance emergence in food environments in relation with cleaning and disinfection procedures.

Correction for Sévellec et al., "Complete Genome Sequence of Salmonella enterica subsp. enterica Serotype Derby, Associated with the Pork Sector in France".

[This corrects the article DOI: 10.1128/MRA.01027-18.].

Complete Genome Sequence of Salmonella enterica subsp. enterica Serotype Derby, Associated with the Pork Sector in France.

In the European Union, Salmonella enterica subsp. enterica serovar Derby is the most abundant serotype isolated from pork. Recent studies have shown that this serotype is polyphyletic. However, one main genomic lineage, characterized by sequence type 40 (ST40), the presence of the Salmonella pathogenicity island 23, and showing resistance to streptomycin, sulphonamides, and tetracycline (STR-SSS-TET), is pork associated. Here, we describe the complete genome sequence of a strain from this lineage isolated in France.

Polyphyletic Nature of Salmonella enterica Serotype Derby and Lineage-Specific Host-Association Revealed by Genome-Wide Analysis.

In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify genetic factors associated with host adaptation and markers for the monitoring of these different lineages within the corresponding animal sectors. The recognition of these four lineages is of primary importance for epidemiological surveillance throughout the food production chains and constitutes the first step toward refining monitoring and preventing dispersal of this pathogen.

Population Genetic Structure of Listeria monocytogenes Strains Isolated From the Pig and Pork Production Chain in France.

Listeria monocytogenes is an ubiquitous pathogenic bacterium, transmissible to humans through the consumption of contaminated food. The pork production sector has been hit hard by a series of L. monocytogenes-related food poisoning outbreaks in France. An overview of the diversity of strains circulating at all levels of the pork production chain, from pig farming (PF) to finished food products (FFP), is needed to identify the contamination routes and improve food safety. Until now, no typing data has been available on strains isolated across the entire pig and pork production chain. Here, we analyzed the population genetic structure of 687 L. monocytogenes strains isolated over the last 20 years in virtually all the French départements from three compartments of this production sector: PF, the food processing environment (FPE), and FFP. The genetic structure was described based on Multilocus sequence typing (MLST) clonal complexes (CCs). The CCs were obtained by mapping the PFGE profiles of the strains. The distribution of CCs was compared firstly between the three compartments and then with CCs obtained from 1106 strains isolated from other food production sectors in France. The predominant CCs of pig and pork strains were not equally distributed among the three compartments: the CC37, CC59, and CC77 strains, rarely found in FPE and FFP, were prevalent in PF. The two most prevalent CCs in the FPE and FFP compartments, CC9 and CC121, were rarely or never detected in PF. No CC was exclusively associated with the pork sector. Three CCs (CC5, CC6, and CC2) were considered ubiquitous, because they were observed in comparable proportions in all food production sectors. The two most prevalent CCs in all sectors were CC9 and CC121, but their distribution was disparate. CC9 was associated with meat products and food products combining several food categories, whereas CC121 was not associated with any given sector. Based on these results, CC121 is likely able to colonize a larger diversity of food products than CC9. Both CCs being associated with the food production suggests, that certain processing steps, such as slaughtering or stabilization treatments, favor their settlement and the recontamination of the food produced.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Pulsed-field gel electrophoresis subtyping database for foodborne Salmonella enterica serotype discrimination.

Nontyphoid Salmonella is one of the main causes of bacterial gastroenteritis worldwide and is responsible for 65% of reported outbreaks of foodborne diseases in France. Serotyping is widely used for isolate preliminary identification, but it poorly discriminates strains. Rapid, efficient molecular subtyping tools have therefore been developed for the investigation of outbreaks. We evaluated the performance of the pulsed-field gel electrophoresis (PFGE) method for discrimination of 31 Salmonella serotypes frequently isolated in France. We set up a genomic database of Salmonella strains isolated from food, animals, the environment, and humans to improve the management of contamination and reactions to foodborne disease outbreaks. We studied 1128 isolates by PFGE, according to the standardized PulseNet protocol. We identified 452 PFGE patterns, 67.5% of which corresponded to a single isolate. The ability of this method to distinguish between isolates was estimated by calculating the Simpson index and the 95% confidence interval. Values obtained ranged between 0.33 (0.11-0.54) to 0.99 (0.96-1.00), depending on serotype. Epidemiological information about isolates was used for analyses of intra- and interserotype diversity results and for determining whether PFGE patterns were linked to the source of the isolate. Clustering analysis of the PFGE patterns obtained confirmed that serotype and PFGE genotype were closely linked. Some PFGE patterns were identified as major patterns, each of these patterns being found in at least 10 isolates. The database generated has already proved its effectiveness in epidemiological investigations in livestock production and foodborne outbreaks.

Taxonomic characterization of nine strains isolated from clinical and environmental specimens, and proposal of Corynebacterium tuberculostearicum sp. nov.

Nine unidentified Gram-positive, lipophilic corynebacteria were isolated from clinical and food samples and subjected to a polyphasic taxonomic analysis. The bacteria were distinguished from Corynebacterium species with validly published names by biochemical tests, fatty acid content and whole-cell protein analysis. Comparative 16S rRNA gene sequence analysis demonstrated unambiguously that the nine strains were related phylogenetically to the species 'Corynebacterium tuberculostearicum' and represented a distinct subline within the genus Corynebacterium. On the basis of both phenotypic and phylogenetic evidence, the formal description of Corynebacterium tuberculostearicum sp. nov. is proposed. The type strain of C. tuberculostearicum is Medalle XT (=LDC-20T=CIP 107291T=CCUG 45418T=ATCC 35529T).