Virulence Genotyping of Salmonella spp. with Multiplex PCR

Abstract

The purpose of this study was to develop a multiplex polymerase chain reaction (PCR) protocol useful in the virulence genotyping of Salmonella spp. with the idea that genotyping could augment current Salmonella characterization and typing methods. Seventeen genes associated with Salmonella invasion, fimbrial production, toxin production, iron transport, and intramacrophage survival were targeted by three PCR reactions. Most of these genes are required for full Salmonella virulence in a murine model, and many are also located onSalmonella pathogenicity islands (PAIs) and are associated with type III secretion systems (TTSSs). Once the success of procedures that used positive and negative control strains was verified, the genotypes of 78 Salmonella isolates incriminated in avian salmonellosis (primarily from sick, commercially reared chickens and turkeys) and 80 Salmonella isolates from apparently healthy chickens or turkeys were compared. Eleven of the 17 genes tested (invA, orgA, prgH, tolC, spaN [invJ], sipB, sitC, pagC, msgA, spiA, and iroN) were found in all of the isolates. Another (sopB) was present in all isolates from sick birds and all but one isolate from healthy birds. The remaining five genes (lpfC, cdtB, sifA, pefA, and spvB) were found in 10%-90% of the isolates from sick birds and 3.75%-90% of the healthy birds. No significant differences in the occurrence of these genes between the two groups of isolates were detected. These results suggest that these virulence genes, and presumably the PAIs and TTSSs with which they are associated, are widely distributed among Salmonella isolates of birds, regardless of whether their hosts of origin have been identified as having salmonellosis. SUMMARY. The purpose of this study was to develop a multiplex polymerase chain reaction (PCR) protocol useful in the virulence genotyping of Salmonella spp. with the idea that genotyping could augment current Salmonella characterization and typing methods. Seventeen genes associated with Salmonella invasion, fimbrial production, toxin production, iron transport, and intramacrophage survival were targeted by three PCR reactions. Most of these genes are required for full Salmonella virulence in a murine model, and many are also located on Salmonella pathogenicity islands (PAIs) and are associated with type III secretion systems (TTSSs). Once the success of procedures that used positive and negative control strains was verified, the genotypes of 78 Salmonella isolates incriminated in avian salmonellosis (primarily from sick, commercially reared chickens and turkeys) and 80 Salmonella isolates from apparently healthy chickens or turkeys were compared. Eleven of the 17 genes tested (invA, orgA, prgH, tolC, spaN [invJ], sipB, sitC, pagC, msgA, spiA, and iroN) were found in all of the isolates. Another (sopB) was present in all isolates from sick birds and all but one isolate from healthy birds. The remaining five genes (lpfC, cdtB, sifA, pefA, and spvB) were found in 10%-90% of the isolates from sick birds and 3.75%-90...

Isolation and characterization of Salmonella Enteritidis and Salmonella Typhimurium from chicken meat in Egypt

“…Targeted genes and their primer sequences used in the amplification studies are summarized in Table 1. The primer sequence and PCR conditions were carried based on Skyberg et al [22]. Three reactions were used to amplify the 17 genes; the first reaction (set 1) was used to amplify spvB, spiA, pagC, cdtB, and msgA; set 2 was used to amplify invA, sipB, prgH, spaN, orgA, and tolC; and set 3 was used to amplify iroN, sitC, lpfC, sifA, sopB, and pefA genes.…”

Salmonella infection in healthy pet reptiles: Bacteriological isolation and study of some pathogenic characters

“…The presence of plasmid virulence genes (spvC, spvB, and spvR) was determined using PCR assays which amplified fragments of 235 bp, 717 bp, and 202 bp, respectively [18,19].…”

Antimicrobial and molecular analysis of Salmonella serovar Livingstone strains isolated from humans in Tunisia and Belgium

“…Virulence genes have been screened using simple [25] or multiplex PCR [26] to determine the distribution of these genes among Salmonella enterica strains [25,27] and to genotype many Salmonella serovars [15,[28][29].…”