A genoserotyping system for a fast and objective identification of Salmonella serotypes commonly isolated from poultry and pork food sectors in Belgium

Humans are mostly contaminated by Salmonella through the consumption of pork- and poultry-derived food products. Therefore, a strict monitoring of Salmonella serotypes in food-producing animals is needed to limit the transmission of the pathogen to humans. Additionally, Salmonella can lead to economic loss in the food sector. Previously, a genoserotyping method using the MOL-PCR and Luminex technology was developed for the identification of the 6 Salmonella serotypes, and their variants, subjected to an official control in the Belgian food sector. In this study, 3 additional assays using the same technology were developed for the rapid and cost-effective detection of 13 dangerous highly invasive serotypes or other serotypes frequently isolated from the Belgian poultry and pork sector, i.e. Agona, Anatum, Brandenburg, Choleraesuis, Derby, Enteritidis vaccine strains, Gallinarum var. Gallinarum/Pullorum, Livingstone, Mbandaka, Minnesota, Ohio, Rissen and Senftenberg. Moreover, the previously developed first MOL-PCR assay was improved for S. Paratyphi B and serogroup O:3 detection. Finally, a Decision Support System hosted by a web application was created for an automatic and objective interpretation of the Luminex raw data. The 3 new assays and the modifications of the first assay were validated with a 100% accuracy, using 553 Salmonella and non-Salmonella strains in total

Development of a genoserotyping system for the identification of Salmonella serotypes

Salmonella is a major pathogen, widely spread and responsible of salmonellosis. It can cause different symptoms, from simple gastroenteritis to the more dangerous typhoid fever. Salmonella can infect humans, but also animals which are its main reservoir. Humans are mostly infected through the consumption of animal-derived food products. Besides its impact on public health, another major concern of Salmonella is economic loss for the professional of the food sectors due to contaminated animals or food products, and economic inactivity due to sickness leave. The genus Salmonella is divided by a complex classification system into 2 species, 6 subspecies and more than 2500 serotypes. The severity of salmonellosis is highly conditioned by, amongst other factors, the infected host species and the serotype of the infecting Salmonella. Therefore, the determination of the serotype is a first key diagnostic for Salmonella control. Moreover, 6 serotypes and their variants, i.e. S.&nbsp;Enteritidis, S. Hadar, S. Infantis, S.&nbsp;Paratyphi B var. Java, S. Typhimurium including its monophasic variant 1,4,[5],12:i:- and S. Virchow are particularly targeted by the legislation as to be excluded from the food chain (EU regulation N°2160/2003, Belgian royal decree 27/04/2007 and Belgian FASFC note BP-MN-FDS/LABO/1557457 v8), with the aim to limit their transmission to humans. Unfortunately, the classical methods for Salmonella serotyping, i.e. slide-agglutination and biochemical tests, are expensive, time-consuming and subjective. Therefore, highly trained and experienced technicians are required to perform these techniques which are usually only fully mastered at National Reference Centers (NRCs). Despite the fact that these techniques are implemented worldwide since more than 80 years, they are not fully adapted to the need of the field, especially for the professionals of the food sector who need to rapidly, accurately and cost-efficiently detect the serotypes targeted by the legislation. Fortunately, during these last years, molecular techniques have shown their potential as replacement method for Salmonella serotype identification. A wide range of different molecular technologies, based on the detection of molecular markers or on the analysis of sequencing data, are described in the scientific literature. Based on a critical review of some of these techniques, the Multiplex Oligonucleotide Reaction-PCR (MOL-PCR) &amp; Luminex method was selected as the principle in this PhD study to develop a fast, cost-effective and accurate Salmonella genoserotyping&nbsp;system. The first step of the new method development was to choose the serotypes to be targeted. Eighteen serotypes and their variants were selected based on their occurrence in the legislation, their clinical relevance (invasive serotypes) and their prevalence in the poultry and pork sectors in Belgium, i.e. S.&nbsp;Agona, S. Anatum, S. Brandenburg, S. Choleraesuis, S. Derby, S. Enteritidis including its vaccine variants AviPro SALMONELLA VAC E and Salmovac SE, S. Gallinarum including its variants Gallinarum and Pullorum, S. Hadar, S. Infantis, S. Livingstone, S. Mbandaka, S. Minnesota, S. Ohio, S.&nbsp;Paratyphi B var.&nbsp;Java, S. Rissen, S. Senftenberg, S. Typhimurium including its monophasic variant 1,4,[5],12:i:- and S. Virchow. Secondly, molecular markers specific to the targeted serotypes were selected from Salmonella EnteroBase (a database with the MLST sequences of more than 230 000 Salmonella isolates), from the scientific literature and from genomic studies using publicly available and in-house produced Whole Genome Sequencing (WGS) data (achieving a number of 100 genomes used for comparisons) and bioinformatics tools such as Gegenees and BioNumerics. This marker selection was particularly complex for the detection of the heterogeneous population of S. Paratyphi B var. Java where only one suitable marker could be retrieved among more than 3 million Single Nucleotide Polymorphism (SNP) positions obtained from a genomic comparison. This valuable SNP marker was used to develop in addition a real-time PCR as an alternative method for the rapid identification of S. Paratyphi B and the determination of its variant Java, replacing a complex and subjective biochemical test. From this molecular markers’ selection, 4 MOL-PCR assays were developed, i.e. the molecular markers were recognized by probes through a ligation-amplification reaction (MOL-PCR), followed by a capturing of the created amplicons by specific oligonucleotides coated on color-coded microspheres, which are themselves detected by a device through a fluorescence reaction (Luminex technology). Additionally, a Decision Support System (DSS), hosted by a web-application, was created for an automatic interpretation of the Luminex results with recommendations provided to the users, and for a centralization of the results in a database to improve the Salmonella surveillance in Belgium. The 4 modules and the DSS were validated by comparison with the classical method, including more than 1300 strains and reaching an accuracy above 99%. Finally, the complete genoserotyping system was evaluated for its ability to completely identify auto-agglutinable isolates which cannot be typed by the slide-agglutination&nbsp;technique. This PhD work showed that a targeted molecular method such as the MOL-PCR &amp; Luminex technology, even though not the most complete technique as compared to WGS, has the potential to improve the accuracy, cost- and time-effectiveness of Salmonella serotype identification in a routine setting. The 4 MOL-PCR assays developed here are up to 7.5 less expensive than the classical methods and they are able to completely identify, in 1 to 2 days, more than 75% of the serotypes usually encountered in Belgium. The developed genoserotyping system is complementary to WGS and an ideal workflow including both techniques was proposed for global Salmonella surveillance and control at a national&nbsp;level.</p

Modélisation du développement de bactéries potentiellement altérantes ou biopréservatrices sur des pâtes pré-cuites conservées à différentes conditions de températures et analysées par microbiologie classique et métagénomique (ADNr 16S)

An important way to prevent the spoilage of food is the respect of the cold chain during the storage. While the temperature instructions are generally respected during process and distribution of food products, it is not always the case with the consumers. Indeed, a few persons reach the right temperature level required for a safe storage of foodstuffs in their refrigerator. Besides, the food can sometimes spend a few hours in ambient temperature between the buying in the supermarket and the storage in cold temperature. In this study, we propose to model the growth of microorganisms on precooked pasta, stored in different conditions of temperature that reflect the situations described above (constant 4°C, constant 8°C, constant 12°C, 1/3 4°C – 2/3 8°C, 1/3 4°C – breach during 4h at 20°C – 2/3 8°C and 1/3 8°C – breach during 4h at 20°C – 2/3 8°C. The product was surface inoculated with potential spoilage and biopreservative strains (Lactococcus piscium, Leuconostoc mesenteroides and Leuconostoc citreum). Analyses by classical microbiology and V1-V3 16S rDNA metagenomics were done each day until the out of date of the food matrix. The transition from 4 to 8°C and the breach at 20°C during 4h have clearly boosted the growth of the microorganisms. The metagenomic analysis was a powerful tool to follow separately each population in each condition of storage. The results of this communication show the importance of keeping the foodstuffs in 4°C or lower in the refrigerator with the goal to avoid the spoilage or the development of pathogens and the potential of metagenomics for selection of biopreservative strains

The use of 16S rRNA gene metagenetic monitoring of refrigerated food products for understanding the kinetics of microbial subpopulations at different storage temperatures: the example of white pudding

In order to control food losses and wastage,monitoring the microbial diversity of food products, during processing and storage is important, as studies have highlighted the metabolic activities of somemicroorganismswhich can lead to spoilage. Knowledge of this diversity can be greatly improved by using a metagenetic approach based on high throughput 16S rRNA gene sequencing, which enables a much higher resolution than culture-based methods. Moreover, the Jameson effect, a phenomenon described by Jameson in 1962, is often used to classify bacterial strains within an ecosystem. According to this, we have studied the bacterial microbiota of Belgian white pudding during storage at different temperatures using culture-dependent and independent methods. The product was inoculated with a mix of dominant strains previously isolated from this foodstuff at the end of its shelf life (Carnobacterium maltaromaticum, Lactobacillus fuchuensis, Lactobacillus graminis, Lactobacillus oligofermentans, Lactococcus lactis, Leuconostoc mesenteroides, Raoultella terrigena and Serratia sp.). Daily during 16 days, the absolute abundance of inoculated strain was monitored by combining total count on plate agar and metagenetic analysis. The resultswere confirmed by qPCR analysis. The growth of each specieswasmodelled for each temperature conditions, representative of good or bad storage practices. These data allowed the bacterial strains subdivision into three classes based on criteria of growth parameters for the studied temperature: the “dominant”, the “subdominant” and the “inhibited” bacterial species, according to their maximal concentration (Nmax, log CFU/g), growth rate (μmax, 1/h) and time to reach the stationary phase (TRSP, days). Thereby, depending on the storage conditions, these data have permitted to follow intrinsically the evolution of each strain on the bacterial ecosystemof Belgianwhite pudding. Interestingly, it has shown that the reliability of the Jameson effect can be discussed. For example, at 4 °C when Lactococcus lactis and Serratia sp. stopped growth at day 12, at the same time Carnobacterium maltaromaticum reached its maximal concentration and entered its stationary phase. In opposition to this, it can be noticed that in the same condition, the “sub-dominant” organisms continued their growth independently of the “dominant” species behaviour. In this case, the Jameson effect was not illustrated. This pattern is described for all storage conditions with the same strain classifications. These results highlighted the importance of combining metagenetic analysis and classical methods, with modelling, to offer a new tool for studying the evolution ofmicroorganisms present in perishable foodwithin different environmental conditions.METAMODE

Modélisation du développement de bactéries potentiellement altérantes ou biopréservatrices sur du boudin blanc conservé à différentes conditions de températures et analysées par microbiologie classique et métagénomique (ADNr 16S)

An important way to prevent the spoilage of food is the respect of the cold chain during the storage. While the temperature instructions are generally respected during process and distribution of food products, it is not always the case with the consumers. Indeed, a few persons reach the right temperature level required for a safe storage of foodstuffs in their refrigerator. Besides, the food can sometimes spend a few hours in ambient temperature between the buying in the supermarket and the storage in cold temperature. In this study, we propose to model the growth of microorganisms on white pudding, stored in different conditions of temperature that reflect the situations described above (constant 4°C, constant 8°C, constant 12°C, 1/3 4°C – 2/3 8°C, 1/3 4°C – breach during 4h at 20°C – 2/3 4°C and 1/3 4°C – breach during 4h at 20°C – 2/3 8°C. The product was surface inoculated with potential spoilage and biopreservative strains (Raoultella terrigena, Serratia quinivorans, Carnobacterium maltaromaticum, Lactobacillus oligofermentans, Lactobacillus nenjiangensis, Lactobacillus fuchuensis, Leuconostoc mesenteroides, lactococcus lactis and Lactobacillus graminis). Analyses by classical microbiology and V1-V3 16S rDNA metagenomics were done each day until the out of date of the food matrix. The transition from 4 to 8°C and the breach at 20°C during 4h have clearly boosted the growth of the microorganisms. The metagenomic analysis was a powerful tool to follow separately each population in each condition of storage. The results of this communication show the importance of keeping the foodstuffs in 4°C or lower in the refrigerator with the goal to avoid the spoilage or the development of pathogens and the potential of metagenomics for selection of biopreservative strains