Workbench 8

A combination of PHAST [32 (link)] and PHASTER [33 (link)] was used to identify putative prophages in the complete genome of CFSAN029793. PHAST was used to identify insertion sites and PHASTER was used to identify the start and end of each prophage. Then the presence/absence of these putative prophages in draft genomes was determined by a combination of two approaches: 1) the insertion sites of the CFSAN029793 prophages were identified in the CLC Genomics WorkBench 8.5.1 (Aarhus, Denmark)-assembled draft genomes by BLAST and the sequences adjacent to the insertion sites were compared to the prophages of CFSAN029793, and 2) the CFSAN029793 prophages were searched against draft genomes by BLAST and a threshold of ≥ 60% query coverage with ≥80% sequence identity [34 (link), 35 (link)] of BLAST alignment indicated the presence of a CFSAN029793 prophage in a draft genome.

Nucleic acid and deduced amino acid products were analyzed using CLC Genomics Workbench 8.5.1. The GaTLV nucleotide and deduced protein sequences were compared with other viral sequences from GenBank and EMBL databases using the FASTA (Pearson and Lipman, 1988 (link)) and BLAST (Altschul et al., 1990 (link)) programs. Identity and similarity percentages were obtained using mean length of sequences, BLOSUM62 matrix with gap cost of 10 and gap extension of 0.5. Alignment analysis and tentative Maximum Likelihood-based phylogenetic trees of amino acid sequences were performed using the MUSCLE (Edgar, 2004 (link)) and MEGA7 (Kumar et al., 2016 (link)) softwares. The best ML-fitted model for each sequence alignments was used and bootstrapping analyses of 100 replicates were performed. Trees were visualized using iTOL (Letunic and Bork, 2016 (link)).
Protein structure, modeling, and structural similarity match to Protein Data Bank (PDB) were performed using the I-TASSER suite (Yang et al., 2015 (link)) (http://zhanglab.ccmb.med.umich.edu/I-TASSER/, last visited 05/2018). Correlation coefficients were determined using the statistical software package Statgraphics Centurion version 15.1.02 (StatPoint technologies, Inc., Warrenton, VA, USA).

In silico MLST was performed using CLC Genomics WorkBench 8.5.1 (Aarhus, Denmark) and the MLST profiles defined in the PasteurMLST L. monocytogenes database (now in BIGSdb-Lm, http://bigsdb.pasteur.fr/listeria). The CFSAN SNP Pipeline was used to construct a phylogeny of a subset of isolates taken from this ice cream outbreak as well as isolates from previous CC5-associated outbreaks/incidents: 2011 U.S. cantaloupe outbreak [17 (link)], 2013 U.S. Hispanic-style cheese outbreak [16 ], and serotype 1/2b isolates from the 2014 U.S. stone fruit recall [36 ]. We chose isolates exhibiting different PFGE profiles observed in those incidents. To avoid using only ST5 to represent CC5, we included a CC5 isolate (CFSAN028312) of ST745 in the analysis. Four non-CC5 serotype 1/2b isolates were chosen for comparison: the environmental isolate from Facility III, CFSAN032502 (singleton ST489, the environmental isolate from Facility III), F4233 (ST3 of CC3), LM07-01067 (ST386 of CC224), CFSAN003423 (ST379 of CC379) and CFSAN003438 (ST379 of CC379); these STs differed from ST5 by 4, 5, 6, 2 and 2 MLST alleles, respectively.