Computational protocol: Whole Genome Sequence Analysis of Salmonella Enteritidis PT4 Outbreaks from a National Reference Laboratory’s Viewpoint

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Protocol publication

[…] Epidemiological investigation In Belgium, the Federal Agency for the Safety of the Food Chain (FASFC) is responsible for sampling and investigation of food, while the Health Services of the Belgian Communities collect human samples. Epidemiological information such as age, symptoms, timeline and circumstances of the outbreaks were gathered by local health inspectors and by inspectors of the FASFC using a standard questionnaire. All collected information was transmitted to the Belgian National Reference Laboratory of Foodborne Outbreaks (NRL-FBO). For each of the two outbreaks, a case was defined as an individual who consumed a meal at the respective social event and who suffered from diarrhoea. Microbiological investigation The NRL-FBO received food samples, including leftovers, of both outbreaks for detection of Salmonella, which was performed according to ISO 6579:2002. The Belgian National Reference Centre for Salmonella and Shigella (NRCSS) received Salmonella isolates from human cases of both outbreaks.Isolates of both outbreaks were serotyped by the NRCSS and phage typed by Public Health England. MLVA was performed by the Belgian NRCSS.The antimicrobial susceptibility of the Salmonella isolates was tested by determination of the minimal inhibitory concentration (MIC) of 14 antimicrobials in a Sensititre MIC plate EUVSEC with read-out on a Sensititre Vizion system. Following epidemiological cut-off values were applied: ampicillin 8 mg/l, cefotaxime 0.5 mg/l, ceftazidime 2 mg/l, chloramphenicol 16 mg/l, ciprofloxacin 0.064 mg/l, colistin 2 mg/l, gentamicin 2 mg/l, meropenem 0.125 mg/l, nalidixic acid 16 mg/l, tetracycline 8 mg/l, tigecycline 1 mg/l and trimethoprim 2 mg/l. Epidemiological cut-off values were not available for azithromycin and sulphamethoxazole. Whole genome sequencing Genomic DNA of the outbreak isolates (Table 1) was extracted with the Qiagen Genomic-tip 100/G kit. The samples were sequenced at the EMBL GeneCore facility in 40-plex on a single lane of the Illumina HiSeq 2000 using 100 bp paired-end reads. FASTQ reads from all sequences were deposited at the WIV-ISP - Salmonella BioProject at NCBI (PRJNA289069). WGS data analysis All analyses were performed on a Windows 7 platform.In CLC Genomics Workbench 8.0 the raw FASTQ reads were first trimmed to quality score limit 0.001 (Q30) with maximum 2 ambiguous nucleotides and reads with length below 15 nucleotides were discarded. These trimmed reads were then de novo assembled with automatic bubble and word size, in mapping mode ‘map reads back to contigs’ with scaffolding and a minimum contig length of 200 nucleotides.On the server of the Center for Genomic Epidemiology, the resulting contigs were uploaded to MLST 1.7 with Salmonella enterica as MLST scheme, ResFinder 2.1 and PlasmidFinder 1.2. ResFinder was used to find all available antimicrobial resistance genes with minimum 98% identity and minimum 60% of their length. In PlasmidFinder, the database of Enterobacteriaceae was searched with an identity threshold of 95%. Additionally, raw FASTQ reads were uploaded to the CSI Phylogeny 1.0a server on which the SNP calling was run with S. Enteritidis P125109 (NC_011294) as reference genome, default input parameters as described by Kaas et al. and a minimum Z-score of 1.96. The downloaded Newick file was used for visualisation of the phylogenetic tree in FigTree v1.4.2. The downloaded vcf (variant call format) files were used for investigation of the position of the SNPs on the chromosome of S. Enteritidis P125109.As PlasmidFinder results indicated that plasmids were present, trimmed reads were mapped to S. Enteritidis P125109 (NC_011294) in CLC Genomics Workbench 8.0 with default settings and unmapped reads were de novo assembled as described above. The resulting contigs were blasted to plasmids pSLA5 (NC_019002) and pSD107 (JX566770) and visualised as concentric rings with BLAST Ring Image Generator (BRIG). […]

Pipeline specifications

Software tools CLC Genomics Workbench, PlasmidFinder, CSI Phylogeny, FigTree, BRIG
Databases ResFinder
Applications Phylogenetics, WGS analysis, Genome data visualization
Organisms Salmonella enterica subsp. enterica serovar Enteritidis, Homo sapiens