Dataset Information

Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis.

ABSTRACT: The genotype of Salmonella enterica serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whereas the PT4 genome to which they were compared, as well as a PT4 field isolate, had a DNA sequence with some similarity to the bacteriophage ST64b sequence. Phenotype MicroArray analysis indicated that the two PT13A strains and the PT4 field isolate had similar respiratory activity profiles at 37 degrees C. However, the wild-type S. enterica serovar Enteritidis PT13A strain grew significantly better in 20% more of the 1,920 conditions tested when it was assayed at 25 degrees C than the biofilm-forming PT13A strain grew. Statistical analysis of the respiratory activity suggested that S. enterica serovar Enteritidis PT4 had a temperature-influenced dimorphic metabolism which at 25 degrees C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37 degrees C the metabolism was nearly identical to that of the wild-type PT13A strain. Although it is possible that lysogenic bacteriophage alter the balance of phage types on a farm either by lytic competition or by altering the metabolic processes of the host cell in subtle ways, the different physiologies of the S. enterica serovar Enteritidis strains correlated most closely with minor, rather than major, genomic changes. These results strongly suggest that the pandemic of egg-associated human salmonellosis that came into prominence in the 1980s is primarily an example of bacterial adaptive radiation that affects the safety of the food supply.

SUBMITTER: Morales CA

PROVIDER: S-EPMC1183325 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis.

Morales Cesar A CA Porwollik Steffen S Frye Jonathan G JG Kinde Hailu H McClelland Michael M Guard-Bouldin Jean J

Applied and environmental microbiology 20050801 8

The genotype of Salmonella enterica serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whe ...[more]

PMID: 16085829

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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.

Project description:Purpose: To elucidated key metabolic pathways important for S. Enteritidis survival in egg white. Methods: RNA sample of Mid-log phase bacteria cultures that grown in M9FeS medium were taken as 0h sample, and bacteria incubated with egg white for 6h 12h and 24h were taken as 6,12,24h RNA sample respectively. Three biological replicates were carried out per sample. RNA sample was sequenced with Illumina HiSeq 2000 System. DESeq2 method was used to calculate the differentially expressed genes. qRT–PCR and gene mutation were used to validate RNA-seq result. Results: A total of 12 samples were sequenced. The average output of each sample was 1.33 Gb, and the average comparison rate with the reference genome was 97.51%. M9FeS and egg whites cultivation resulted in 2400 to 2700 expressed genes accounting for 53 to 58% of the whole genome. In this group, 1240 to 1470 genes had significant levels of log2 fold changes ≥1, (Padj <0.05) compared with control. These accounted for 27.3 to 32.2% of the whole genome indicating a large shift in the transcriptional response to egg whites. qRT–PCR result of 21 selected genes highly consistent with the RNAseq results at all three timepoints. 8 out of 12 DEGs mutants showed decreased survival ability in egg white, suggested that the DEGs from the RNAseq results correlated significantly with the egg white resistance phenotype. Conclusions: Using strand-specific RNA sequencing, we found pathways significantly changed in egg white. Genes involved in SOS-dependent and independent DNA damage repair, alkaline pH adaption, osmotic stress adaption, envelope damage repair including maintaining periplasm homeostasis and reinforcing peptidoglycan layer, nitrogen assimilation, Salmonella pathogenicity island 2, iron absorption and biotin synthesis, were significantly up regulated in bacteria surviving in egg whites. Differentially expressed genes involved in energy metabolism, translation, and cell motility, Salmonella pathogenicity island 1 were significantly down-regulated in egg whites.

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:Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.CGH using an oligonucleotide array based upon chromosomal coding sequences of S. enterica serovar Typhimurium strain LT2 and the Salmonella genomic island 1 successfully determined major genetic differences between S. Typhimurium and S. Enteritidis PT13, but no significant strain-to-strain differences were observed between S. Enteritidis PT13 isolates. Individual loci (safA and fliC) that were identified as potentially divergent in the CGH data set were sequenced in a panel of S. Enteritidis strains, and no differences were detected between the PT13 strains. Additional sequence-based typing was performed at the fimA, mdh, manB, cyaA, citT, caiC, dmsA, ratA and STM0660 loci. Similarly, no diversity was observed amongst PT13 strains. Variation in plasmid content between PT13 strains was observed, but macrorestriction with BglII did not identify further differences. Automated rep-PCR patterns were variable between serovars, but S. Enteritidis PT13 strains could not be differentiated.None of the methods identified any significant variation between PT13 strains. Greater than 11,300 base pairs of sequence for each of seven S. Enteritidis PT13 strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of S. Enteritidis was observed. These data suggest that Canadian S. Enteritidis PT13 strains are highly related genetically.

Dataset Information

Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis.

Publications

Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis.

Similar Datasets

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