Mining Of Molecular Targets And Development Of Multiplex Pcr Methods For Serogouping And Serotyping Salmonella Spp.

Salmonella is one of the most common foodborne pathogens, which often causes food poisoning around the world. Salmonella strains are divided into over 50 serogroups according to the somatic (O-) antigens, which are further divided into more than 2500 serovars based upon the ?agellar (H-) antigens. Certain food products are associated with specific Salmonella serovars, particularly belonging to serogroups A through D, constituting about 70% of Salmonella infections in humans and animals. However, the traditional serotyping method is labor-intensive, expensive, complicated, and time-consuming. Another limitation of this method is that there is a lack of standardization of the antisera used for serotyping. Molecular typing methods offer promising alternatives to overcome the limitations associated with traditional serotyping methods.Using a bioinformatics platform for mining targets by comparative genomics, and specific targets for PCR detection were identified for serogroups A-D and 8 serotypes. Targeting these specific sequences, two multiplex PCR assays were developed: one assay for serogrouping and another assay for serotyping Salmonella stains.More than 20 geomic sequences of Salmonella were downloaded from the databanks of the NCBI, the Sanger Institute, the University of Illinois and the Genome Sequencing Center at University of Washington, and then divided into 6 local databanks of Salmonella serogoups: A-, B-, C1-, C2-, D- and Others-databanks. Except for the Others-databank, the gemomic sequence of a refrence strain from each serogoup's databank was splited in thousands of 986-bp fragments in silico. These fragments from one strain were aligned with the gemomic sequences of orther strains using the BLASTN program. Serogroup-specific fragmemts, which were only presented in gemomes of strains in the same serogroups but were not present in the gemomes of the orther serogroups, were selected to serve as target candidates for PCR. As a result, 2, 6, 7, 10, and 7 specific fragments were found for serogroups A, B, C1, C2 and D, respectively. Specific primer sets targeting these DNA fragments were designed for multiplex PCR assays identifying 21 Salmonella standard strains and 86 food isolates. The PCR results demonstrated good agreement with those from traditional Salmonella serogrouping. This means that the PCR assay may be able to identify 5 (A, B, C1, C2 and D) Salmonella serogroups and elucidate differences among them. The sensitivity of this PCR assay for serogrouping Salmonella was 1.65 pg gemomic DNA per PCR.Similarly, 8 genomic sequence databanks of Salmonella serotypes were constructed: Typhi/Paratyphi C, Typhimurium, Paratyphi B, Choleraesuis, Infantis, Enteritidis, Gallinarum and Others. Comparative genomics revealed 1, 11, 18, 1, 22, 4 and 3 serotype-specific fragments for the 8 serotypes, respectively. Based on 7 highly specific and sensitive primer sets targeting these DNA fragments, a novel multiplex PCR was developed for serotyping the 8 Salmonella serovars. PCR results from a collection of 153 Salmonella strains demonstrated that this method could identify 8 serotypes, which agreed with from Salmonella serotyping. Using these two PCR methods to detect Salmonella in artificially samples, as few as 5 cfu/10 mL of milk sample was detectable after a brief (12 h) non-selective culture enrichment.According to the gene annotations, the 32 serogroup-specific fragments were divided into 3 categories (membrane protein genes, rfb gene clusters, and fimbrial genes). Each gene from these three groups was conserved within the serogroup and was closely correlated with phenotypic characterization. This finding implies that these genes, which are associated with sugar synthesis and metabolism or glycosyl and O-actetyl transfer, impart the differences among Salmonella serogroups. The serotype-specific fragments consist of phage genes, pathogenic factor genes, transposon genes, and restriction enzyme genes and orhers.These genes are closely related with mobile elements and virulence genes, which help the pathogens to adapt to the host environment, suggesting that the pathogens generate different serotypes to better adapt to different hosts and the environments.The two PCR assays were used to characterize 437 isolates from the food, poultry farms and patients and were able to identify 5 Salmonella serogroups (A, B, C1, C2 and D) and 10 Salmonella serotypes (Typhi, Paratyphi A~C, Typhimurium, Choleraesuis, Infantis, Enteritidis, Gallinarum and Agona). The Kappa value was greater than 80%, demonstrating the agreement between the developed PCR assays and serotyping methods. Of these strains, 40 to 50% were Salmonella Enteritidis or Typhimurium. These strains were subtyped by MLST and tested antimicrobial resistance. Almost all of Salmonella Enteritidis strains were of the same sequence type (ST11), which is a common subtype worldwide. There are five sequence types among the Salmonella Typhimurium isolates, including ST19 (mainly clinical and farm isolates), ST34 (mainly food strains) and three new subtypes, which were only found in East and Southeast Asia. The sequence types of these two serotypes were correlated with their antibiotics resistance according to the results of antibiotic testing. The ST34 strains usually had six to eight antibiotics resistances. The close evolutionary relationship between food and clinical isolates, as revealed by MLST, suggests that the patients may have been infected by food.