Gene-by-gene approaches are becoming increasingly popular in bacterial genomic epidemiology and outbreak detection. However, there is a lack of open-source scalable software for schema definition and allele calling for these methodologies. The chewBBACA suite was designed to assist users in the creation and evaluation of novel whole-genome or core-genome gene-by-gene typing schemas and subsequent allele calling in bacterial strains of interest. chewBBACA performs the schema creation and allele calls on complete or draft genomes resulting from de novo assemblers. The chewBBACA software uses Python 3.4 or higher and can run on a laptop or in high performance clusters making it useful for both small laboratories and large reference centers. ChewBBACA is available at https://github.com/B-UMMI/chewBBACA.

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In response to the EFSA call New approaches in identifying and characterizing microbial and chemical hazards, the project INNUENDO (https://sites.google.com/site/theinnuendoproject/) aimed to design an analytical platform and standard procedures for the use of whole‐genome sequencing in surveillance and outbreak investigation of food‐borne pathogens. The project firstly attempted to identify existing flaws and needs, and then to provide applicable cross‐sectorial solutions. The project focused in developing a platform for small countries with limited economical and personnel resources. To achieve these goals, we applied a user‐centered design strategy involving the end‐users, such as microbiologists in public health and veterinary authorities, in every step of the design, development and implementation phases. As a result, we delivered the INNUENDO Platform V1.0 (https://innuendo.readthedocs.io/en/latest/), a stand‐alone, portable, open‐source, end‐to‐end system for the management, analysis, and sharing of bacterial genomic data. The platform uses Nextflow workflow manager to assemble analytical software modules in species‐specific protocols that can be run using a user‐friendly interface. The reproducibility of the process is ensured by using Docker containers and throught the annotation of the whole process using an ontology. Several modules, available at https://github.com/TheInnuendoProject, have been developed including: genome assembly and species confirmation; fast genome clustering; in silico typing; standardized species‐specific phylogenetic frameworks for Campylobacter jejuni, Yersinia enterocolitica, Salmonella enterica and Escherichia coli based on an innovative gene‐by‐gene methodology; quality control measures from raw reads to allele calling; reporting system; a built‐in communication protocols and a strain classification system enabling smooth communication during outbreak investigation. As proof‐of‐concepts, the proposed solutions have been thoroughly tested in simulated outbreak conditions by several public health and veterinary agencies across Europe. The results have been widely disseminated through several channels (web‐sites, scientific publications, organization of workshops). The INNUENDO Platform V1.0 is effectively one of the models for the usage of open‐source software in genomic epidemiology.

BackgroundCampylobacter jejuni and C. coli share a multitude of risk factors associated with human gastrointestinal disease, yet their phylogeny differs significantly. C. jejuni is scattered into several lineages, with no apparent linkage, whereas C. coli clusters into three distinct phylogenetic groups (clades) of which clade 1 has shown extensive genome-wide introgression with C. jejuni, yet the other two clades (2 and 3) have less than 2% of C. jejuni ancestry. We characterized a C. coli strain (76339) with four novel multilocus sequence type alleles (ST-5088) and having the capability to express gamma-glutamyltranspeptidase (GGT); an accessory feature in C. jejuni. Our aim was to further characterize unintrogressed C. coli clades 2 and 3, using comparative genomics and with additional genome sequences available, to investigate the impact of horizontal gene transfer in shaping the accessory and core gene pools in unintrogressed C. coli.ResultsHere, we present the first fully closed C. coli clade 3 genome (76339). The phylogenomic analysis of strain 76339, revealed that it belonged to clade 3 of unintrogressed C. coli. A more extensive respiratory metabolism among unintrogressed C. coli strains was found compared to introgressed C. coli (clade 1). We also identified other genes, such as serine proteases and an active sialyltransferase in the lipooligosaccharide locus, not present in C. coli clade 1 and we further propose a unique scenario for the evolution of Campylobacter ggt.ConclusionsWe propose new insights into the evolution of the accessory genome of C. coli clade 3 and C. jejuni. Also, in silico analysis of the gene content revealed that C. coli clades 2 and 3 have genes associated with infection, suggesting they are a potent human pathogen, and may currently be underreported in human infections due to niche separation.

The occurrence of Mycobacterium spp. in freshwater and marine ornamental fish was studied in Italy from June 2002 to May 2005. Two surveys were carried out, one of aquarium fish sent to the Laboratory for diagnosis, and the other of prevalence of infection by mycobacteria in ornamental fish imported into Italy. Bacterial isolation was carried out from the spleen, kidney and liver, and the isolates were subsequently identified by biochemical tests. In the first survey, 387 fish were examined and Mycobacterium spp. were isolated from 181 (46.8%) fish. In the second survey 127 batches of ornamental fish from different countries were examined. Mycobacterium spp. were isolated from 38 (29.9%) batches. The following species were found: M. fortuitum, M. peregrinum, M. chelonae, M. abscessus, M. marinum, M. gordonae, M. nonchromogenicum and M. interjectum. There was a high prevalence of infection independent of the presence of macroscopic lesions. Mycobacterium fortuitum and M. chelonae were more prevalent than M. marinum in the samples examined.

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This Opinion considers the application of whole genome sequencing (WGS) and metagenomics for outbreak investigation, source attribution and risk assessment of food-borne pathogens. WGS offers the highest level of bacterial strain discrimination for food-borne outbreak investigation and sourceattribution as well as potential for more precise hazard identification, thereby facilitating more targeted risk assessment and risk management. WGS improves linking of sporadic cases associated with different food products and geographical regions to a point source outbreak and can facilitate epidemiological investigations, allowing also the use of previously sequenced genomes. Source attribution may be favoured by improved identification of transmission pathways, through the integration of spatial-temporal factors and the detection of multidirectional transmission and pathogenhost interactions. Metagenomics has potential, especially in relation to the detection and characterisation of non-culturable, difficult-to-culture or slow-growing microorganisms, for tracking of hazard-related genetic determinants and the dynamic evaluation of the composition and functionality of complex microbial communities. A SWOT analysis is provided on the use of WGS and metagenomics for Salmonella and Shigatoxin-producing Escherichia coli (STEC) serotyping and the identification of antimicrobial resistance determinants in bacteria. Close agreement between phenotypic and WGSbased genotyping data has been observed. WGS provides additional information on the nature and localisation of antimicrobial resistance determinants and on their dissemination potential by horizontal gene transfer, as well as on genes relating to virulence and biological fitness. Interoperable data will play a major role in the future use of WGS and metagenomic data. Capacity building based on harmonised, quality controlled operational systems within European laboratories and worldwide is essential for the investigation of cross-border outbreaks and for the development of international standardised risk assessments of food-borne microorganisms.

h Helicobacter heilmannii naturally colonizes the stomachs of dogs and cats and has been associated with gastric disorders in humans. Nine feline Helicobacter strains, classified as H. heilmannii based on ureAB and 16S rRNA gene sequences, were divided into a highly virulent and a low-virulence group. The genomes of these strains were sequenced to investigate their phylogenetic relationships, to define their gene content and diversity, and to determine if the differences in pathogenicity were associated with the presence or absence of potential virulence genes. The capacities of these helicobacters to bind to the gastric mucosa were investigated as well. Our analyses revealed that the low-virulence strains do not belong to the species H. heilmannii but to a novel, closely related species for which we propose the name Helicobacter ailurogastricus. Several homologs of H. pylori virulence factors, such as IceA1, HrgA, and jhp0562-like glycosyltransferase, are present in H. heilmannii but absent in H. ailurogastricus. Both species contain a VacA-like autotransporter, for which the passenger domain is remarkably larger in H. ailurogastricus than in H. heilmannii. In addition, H. ailurogastricus shows clear differences in binding to the gastric mucosa compared to H. heilmannii. These findings highlight the low-virulence character of this novel Helicobacter species. Helicobacter pylori is considered one of the most successful human pathogens. Infection with this agent has been associated with a wide range of gastric disorders. However, H. pylori is not the only Helicobacter species causing gastric disease in humans. Helicobacter heilmannii (sensu stricto), a zoonotic bacterium naturally colonizing the stomachs of cats and dogs, has been associated with gastritis, peptic and duodenal ulcers, and mucosa-associated lymphoid tissue (MALT) lymphoma in humans (1-6). This Helicobacter species is highly prevalent in the stomachs of clinically healthy cats and dogs as well as in those of animals showing chronic active gastritis (1, 4). Its pathogenic significance in these animals remains unclear and is probably strain dependent or related to host differences (1).Little information is available regarding the pathogenesis of H. heilmannii infections in humans (1, 7). A recent experimental infection study, using Mongolian gerbils as an in vivo model to study Helicobacter-related gastric pathology in humans, investigated the colonization capacities and virulence of nine different Helicobacter strains (8). These helicobacters had been isolated from the gastric mucosae of stray cats and had been classified as H. heilmannii on the basis of the ureAB and 16S rRNA gene sequences (9). At 9 weeks postinfection, the induction of an antrum-dominant chronic active gastritis associated with the formation of lymphocytic aggregates and upregulation of the proinflammatory cytokine interleukin 1␤ (IL-1␤) was shown for seven strains. However, differences in the expression of IL-1␤ were noted, together with differences in the intensity of the obser...