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. Enteritidis, S. Hadar, S. Infantis, S. 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) & Luminex method was selected as the principle in this PhD study to develop a fast, cost-effective and accurate Salmonella genoserotyping 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. 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. Paratyphi B var. 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 technique.
This PhD work showed that a targeted molecular method such as the MOL-PCR & 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 level.