Summary: We have developed a website, www.in-silico.com, which runs a software program that performs three basic tasks in completely sequenced bacterial genomes by in silico analysis: PCR amplification, amplified fragment length polymorphism (AFLP–PCR) and endonuclease restriction. For PCR, after selection of the genome and introduction of primers, fragment size, DNA sequence and corresponding open reading frame (ORF) identity of the resulting PCR product is computed. Plasmids of sequenced species may be included in the analysis. Theoretical AFLP–PCR analyzes similar parameters, and includes a suggestion tool providing a list of commercial restriction enzyme pairs yielding up to 50 amplicons in the selected genome. Endonuclease restriction analysis of complete genomes and plasmids calculates the number of restriction sites for endonucleases in a given genome. If the number of fragments is 50 or fewer, pulsed field gel electrophoresis image and restriction maps are illustrated. Other tools that have been included in this site are ORF search by name and DNA to protein translation as well as restriction digestion of user-defined DNA sequences. Availability: This is a new molecular biology resource freely available over the Internet at http://www.in-silico.com

Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism of PCR products (PCR-RFLP) are extensively used molecular biology techniques. An exercise for the design and simulation of PCR and PCR-RFLP experiments will be a useful educational tool.

We analyzed a collection of 60 Salmonella enterica 4,5,12:i:- phage type U302 multidrug-resistant monophasic variant strains, isolated in Spain between 2000 and 2007. Most strains showed resistance to ampicillin (A), chloramphenicol (C), sulfamethoxazole (Su), gentamicin (G), streptomycin (S), tetracycline (T), and co-trimoxazole (SxT) (an ACSuGSTSxT resistance pattern). Only one pulsed-field gel electrophoresis (PFGE) type was detected, with 19 subtypes (Simpson's index of diversity [SID] = 0.89). Multiple-locus variable-number tandem-repeat analysis (MLVA) showed more variability, with 32 profiles (SID = 0.97), but only showed diversity at the STTR5 and STTR6 loci. PCR and sequencing demonstrated all strains contained the same allantoin-glyoxylate pathway deletion. Four types of deletions were detected in the fljAB operon, all starting at the same position, at the STM2758 gene, and followed by an IS 26 insertion. Furthermore, a representative set of strains of the four deletion types harbored plasmids with IS 26 . We propose that a Salmonella enterica serotype Typhimurium U302 multidrug-resistant (ACSuGSTSxT) strain, defective for the allantoin-glyoxylate pathway and containing IS 26 at plasmid pU302L, could be the ancestor of the variant in Spain.

The use of whole-genome sequencing (WGS) for bacterial characterisation has increased substantially in the last decade. Its high throughput and decreasing cost have led to significant changes in outbreak investigations and surveillance of a wide variety of microbial pathogens. Despite the innumerable advantages of WGS, several drawbacks concerning data analysis and management, as well as a general lack of standardisation, hinder its integration in routine use. In this work, a bioinformatics workflow for (Illumina) WGS data is presented for bacterial characterisation including genome annotation, species identification, serotype prediction, antimicrobial resistance prediction, virulence-related genes and plasmid replicon detection, core-genome-based or single nucleotide polymorphism (SNP)-based phylogenetic clustering and sequence typing. Workflow was tested using a collection of 22 in-house sequences of Salmonella enterica isolates belonging to a local outbreak, coupled with a collection of 182 Salmonella genomes publicly available. No errors were reported during the execution period, and all genomes were analysed. The bioinformatics workflow can be tailored to other pathogens of interest and is freely available for academic and non-profit use as an uploadable file to the Galaxy platform.