Dataset Information

Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

ABSTRACT: Salmonella enterica is a pathogenic bacterium that causes foodborne illness. One of the vehicle foods of S. enterica are chicken eggs. Efficient collection of the bacterium is necessary to detect it specifically. We developed a method to detect S. enterica by PCR on a microfluidic disc device using a fluorescent probe. Salmonella enterica cells were isolated in the microchambers on the device, followed by thermal lysis and PCR targeting with the invA gene, a gene specific to S. enterica, were observed by measurement of the fluorescent signal that resulted from gene amplification. However, the developed method was unable to discriminate viable cells from dead cells. Consequently, in this study, magnetic beads modified with anti-Salmonella antibody were utilized to detect viable Salmonella cells from egg yolk prior to PCR on the device. While using the antibody-modified beads, egg yolk components, which inhibit PCR, were removed. The collected cells were subsequently detected by PCR of the invA gene on a microfluidic disc device. This method enabled the detection of viable cells without the inhibition of PCR by any egg component. S. enterica was detected at 5.0×104 cells mL-1 or at a higher concentration of egg yolk within 6 h including the sampling time.

SUBMITTER: Kubo I

PROVIDER: S-EPMC7070913 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

Kubo Izumi I Kajiya Mitsutoshi M Aramaki Narumi N Furutani Shunsuke S

Sensors (Basel, Switzerland) 20200215 4

Salmonella enterica is a pathogenic bacterium that causes foodborne illness. One of the vehicle foods of S. enterica are chicken eggs. Efficient collection of the bacterium is necessary to detect it specifically. We developed a method to detect S. enterica by PCR on a microfluidic disc device using a fluorescent probe. Salmonella enterica cells were isolated in the microchambers on the device, followed by thermal lysis and PCR targeting with the invA gen ...[more]

PMID: 32075315

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Project description:Contribution of food vehicles to pathogenicity of disease-causing microorganisms is an important but overlooked research field. The current study was initiated to reveal the relationship between virulence of Salmonella enterica serovar Enteritidis and egg yolk as a hosting medium. Mice were orally challenged with Salmonella Enteritidis cultured in egg yolk or tryptic soy broth (TSB). Additionally, mice were challenged with Salmonella Enteritidis cultured in TSB, followed by administration of sterile egg yolk, to discern the difference between pre-growth of the pathogen and its mere presence in egg yolk during infection. The pathogen's Lethal dose 50 (LD50) was the lowest when grown in yolk (2.8×102 CFU), compared to 1.1×103 CFU in TSB, and 4.6×103 CFU in TSB followed by administration of sterile yolk. Additionally, mice that orally received Salmonella Enteritidis grown in egg yolk expressed a high death rate. These findings were supported by transcriptional analysis results. Expression of promoters of virulence-related genes (sopB and sseA) in genetically modified Salmonella Enteritidis reporter strains was significantly higher (p < 0.05) when the bacterium was grown in the yolk, compared to that grown in TSB. Sequencing of RNA (RNA-seq) revealed 204 differentially transcribed genes in Salmonella Enteritidis grown in yolk vs. TSB. Yolk-grown Salmonella Enteritidis exhibited upregulated virulence pathways, including type III secretion systems, epithelial cell invasion, and infection processes; these observations were confirmed by RT-qPCR results. The transcriptomic analysis suggested that upregulation of virulence machinery of Salmonella Enteritidis grown in egg yolk was related to increased iron uptake, biotin utilization, flagellar biosynthesis, and export of virulence proteins encoded on Salmonella pathogenicity island 1, 2, 4, and 5. These biological responses may have acted in concert to increase the virulence of Salmonella infection in mice. In conclusion, growth in egg yolk enhanced Salmonella Enteritidis virulence, indicating the significance of this food vehicle to the risk assessment of salmonellosis.

Project description:The survival mechanism of Salmonella enterica serovar Enteritidis in antibacterial egg white is not fully understood. In our lab, an egg white-resistant strain, S. Enteritidis SJTUF 10978, was identified. Cell envelope damage and osmotic stress response (separation of cell wall and inner membrane as well as cytoplasmic shrinkage) of this strain surviving in egg white were identified through microscopic observation. RNA-Seq analysis of the transcriptome of Salmonella survival in egg white showed that a considerable number of genes involved in DNA damage repair, alkaline pH adaptation, osmotic stress adaptation, envelope damage repair, Salmonella pathogenicity island 2 (SPI-2), iron absorption, and biotin synthesis were significantly upregulated (fold change ? 2) in egg white, indicating that these pathways or genes might be critical for bacterial survival. RNA-Seq results were confirmed by qRT-PCR, and the survival analysis of six gene deletion mutants confirmed their importance in the survival of bacteria in egg white. The importance of alkaline pH adaptation and envelope damage repair for Salmonella to survive in egg white were further confirmed by analysis of nhaA, cpxR, waaH, and eco deletion mutants. According to the RNA-Seq results, we propose that alkaline pH adaptation might be the cause of bacterial osmotic stress phenotype and that the synergistic effect between alkaline pH and other inhibitory factors can enhance the bacteriostatic effect of egg white. Moreover, cpxR and sigE were recognized as the central regulators that coordinate bacterial metabolism to adapt to envelope damage and alkaline pH.IMPORTANCE Salmonella enterica serovar Enteritidis is a major foodborne pathogen that causes salmonellosis mainly through contaminated chicken eggs or egg products and has been a worldwide public health threat since 1980. Frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival ability in the antibacterial egg white. Research on the survival mechanism of S. Enteritidis in egg white will help to further understand the complex and highly effective antibacterial mechanisms of egg white and lay the foundation for the development of safe and effective vaccines to prevent egg contamination by this Salmonella serotype. Key pathways and genes that were previously overlooked under bactericidal conditions were characterized as being induced in egg white, and synergistic effects between different antimicrobial factors appear to exist according to the gene expression changes. Our work provides new insights into the survival mechanism of S. Enteritidis in egg white.

Dataset Information

Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

Publications

Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

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