Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States.

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

Evaluation of the Illumina iSeq whole genome sequencing system for enteric disease surveillance and outbreak detection.

The Illumina iSeq low-capacity sequencing platform was evaluated for use in foodborne disease surveillance and outbreak detection. The platform produced high quality sequence data comparable to that of the Illumina MiSeq and was cost-effective with fast turn-around time in low sample volume environments.

Sequencing of Enteric Bacteria: Library Preparation Procedure Matters for Accurate Identification and Characterization.

Enzymatic library preparation kits are increasingly used for bacterial whole genome sequencing. While they offer a rapid workflow, the transposases used in the kits are recognized to be somewhat biased. The aim of this study was to optimize and validate a protocol for the Illumina DNA Prep kit (formerly Nextera DNA Flex) for sequencing enteric pathogens and compare its performance against the Nextera XT kit. One hundred forty-three strains of Campylobacter, Escherichia, Listeria, Salmonella, Shigella, and Vibrio were prepared with both methods and sequenced on the Illumina MiSeq using 300 and/or 500 cycle chemistries. Sequences were compared using core genome multilocus sequence typing (cgMLST), 7-gene multilocus sequence typing (MLST), and detection of markers encoding serotype, virulence, and antimicrobial resistance. Sequences for one Escherichia strain were downsampled to determine the minimum coverage required for the analyses. While organism-specific differences were observed, the Prep libraries generated longer average read lengths and less fragmented assemblies compared to the XT libraries. In downstream analysis, the most notable difference between the kits was observed for Escherichia, particularly for the 300 cycle sequences. The O group was not predicted in 32% and 4% of XT sequences when using blast and kmer algorithms, respectively, while the O group was predicted from all Prep sequences regardless of the algorithm. In addition, the ehxA gene was not detected in 6% of XT sequences and 34% were missing one or more of the type III secretion systems and/or plasmid-associated genes, which were detected in the Prep sequences. The coverage downsampling revealed that acceptable assembly quality and allele detection was achieved at 30 × coverage with the Prep libraries, whereas 40-50 × coverage was required for the XT libraries. The better performance of the Prep libraries was attributed to more even coverage, particularly in genome regions low in GC content.

Comparison of four enzymatic library preparation kits for sequencing Shiga toxin-producing Escherichia coli for surveillance and outbreak detection.

Four enzymatic DNA library preparation kits were compared for sequencing Shiga toxin-producing E. coli. All kits produced high quality sequence data which performed equally well in the downstream analyses for surveillance and outbreak detection. Important differences were noted in the workflow user-friendliness and per sample cost.