Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis

Abstract

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

“…PCR amplification of the sefA gene, encoding SEF14 fimbrial antigen, was used for preliminary confirmation of the identity of Salmonella isolates (Doran et al, 1996). A chromosomally located sdfI gene reported to be unique to the serovar S. Enteritidis was targeted for the serotype-specific identification of S. Enteritidis (Agron et al, 2001;Alvarez et al, 2004;Clavijo et al, 2006;Trafny et al, 2006). A 60 kb virulence-plasmid-associated spvB gene was used as a marker for determination of the presence or absence of a typical virulence plasmid (pSEV) among S. Enteritidis isolates (Cho et al, 2007;Rotger & Casadesú s, 1999;Rychlík et al, 2006Rychlík et al, , 2008.…”

Emergence and clonal dissemination of Salmonella enterica serovar Enteritidis causing salmonellosis in Mauritius

“…The serotypes were assigned according to the Kauffmann-White scheme [10]. At CDC, the serotype was confirmed by PCR amplification of the Sdf I gene specific for Salmonella serovar Enteritidis [11].…”

Gastrointestinal tract distribution of Salmonella enteritidis in orally infected mice with a species-specifi c fl uorescent quantitative polymerase chain reaction

“…We evaluated the specific fragment (SdfⅠ) reported by Agron [6] , which was screened for using the Supression Subtractive Hybridization method. SdfⅠappears to only be found in serovar enteritidis strains, which includes a wide range of clinical and environmental species.…”