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.

After Salmonella Enteritidis and S. Typhimurium, S. 4,[5],12:i:- is the most reported serovar in human clinical cases. During the past 20 years, many tools have been used for its typing and second-phase flagellar deletion characterization. Currently, whole genome sequencing (WGS) and different bioinformatic programs have shown the potential to be more accurate than earlier tools. To assess this potential, we analyzed by WGS and in silico typing a selection of 42 isolates of S. 4,[5],12:i:- and S. Typhimurium with different in vitro characteristics. Comparative analysis showed that SeqSero2 does not differentiate fljB-positive S. 4,[5],12:i:- strains from those of serovar Typhimurium. Our results proved that the strains selected for this work were non-clonal S. 4,[5],12:i:- strains circulating in Spain. Using WGS data, we identified 13 different deletion types of the second-phase flagellar genomic region. Most of the deletions were generated by IS26 insertions, showing orientation-dependent conserved deletion ends. In addition, we detected S. 4,[5],12:i:- strains of the American clonal line that would give rise to the Southern European clone in Spain. Our results suggest that new S. 4,[5],12:i:- strains are continuously emerging from different S. Typhimurium strains via different genetic events, at least in swine products.

The emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context requiring continuous surveillance. Resistance to ciprofloxacin and cephalosporins is of particular concern. Since pigs are a relevant source of foodborne Salmonella for human beings, we studied transmissible AMR genes and MGE in a collection of 83 strains showing 9 different serovars and 15 patterns of multidrug resistant (MDR) previously isolated from pigs raised in the conventional breeding system of Northern Spain. All isolates were susceptible to ciprofloxacin and three isolates carried bla _CMY-2 or bla _CTX-M-9 genes responsible for cefotaxime resistance. Filter mating experiments showed that the two plasmids carrying bla _CTX-M-9 were conjugative while that carrying bla _CMY-2 was self-transmissible by transformation. Whole-genome sequencing and comparative analyses were performed on the isolates and plasmids. The IncC plasmid pSB109, carrying bla _CMY-2 , was similar to one found in S . Reading from cattle, indicating potential horizontal transfer between serovars and animal sources. The IncHI2 plasmids pSH102 in S . Heidelberg and pSTM45 in S . Typhimurium ST34, carrying bla _CTX-M-9 , shared similar backbones and two novel “complex class 1 integrons” containing different AMR and heavy metal genes. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food. IMPORTANCE: The emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context. Since pigs are a relevant source of foodborne Salmonella for humans, in this study, we investigate different aspects of AMR in a collection of 83 Salmonella showing nine different serovars and 15 patterns of multidrug resistant (MDR) isolated from pigs raised in the conventional breeding system. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food.

Extensive pig systems are gaining importance as quality production systems and as the standard for sustainable rural development and animal welfare. However, the effects of natural foods on Salmonella epidemiology remain unknown. Herein, we assessed the presence of Salmonella and the composition of the gut microbiota in pigs from both Salmonella-free and high Salmonella prevalence farms. In addition, risk factors associated with the presence of Salmonella were investigated. The pathogen was found in 32.2% of animals and 83.3% of farms, showing large differences in prevalence between farms. Most isolates were serovars Typhimurium monophasic (79.3%) and Bovismorbificans (10.3%), and exhibited a multi-drug resistance profile (58.6%). Risk factor analysis identified feed composition, type/variety of vegetation available, and silos’ cleaning/disinfection as the main factors associated with Salmonella prevalence. Clear differences in the intestinal microbiota were found between Salmonella-positive and Salmonella-negative populations, showing the former with increasing Proteobacteria and decreasing Bacteroides populations. Butyrate and propionate producers including Clostridium, Turicibacter, Bacteroidaceae_uc, and Lactobacillus were more abundant in the Salmonella-negative group, whereas acetate producers like Sporobacter, Escherichia or Enterobacter were more abundant in the Salmonella-positive group. Overall, our results suggest that the presence of Salmonella in free-range pigs is directly related to the natural vegetation accessible, determining the composition of the intestinal microbiota.

Salmonelosia, herrialde garatuetako toxiinfekzio ohikoenetariko bat da, Salmonella generoko bakterioek eragindakoa. Oro har, giza salmonelosia elikagai kutsatuekin lotzen da. Hala ere, animaliekin izandako kontaktua Salmonella generoaren transmisio bide garrantzitsua izan daiteke. Dortokak Salmonella serotipo desberdinen gordailuak direla frogatu da. Narrasti hauek etxeko maskota oso ohikoak direla kontuan izanda, osasun publikorako arriskutsuak izan daitezke, batez ere haurren artean. Horregatik, lan honetan 4,5,12:b:- formula antigenikoa duen serotipo monofasiko berri bat karakterizatu da. Aztertutako isolatu gehienak 5 urtetik beherako haurretan isolatu dira, Espainiako eskualde desberdinetan. Teknika molekular ezberdinen bidez, baieztatu da 4,5,12:b:- isolatuak Paratyphi B Javako iso- latu bifasikoekin genetikoki erlazionatuta daudela eta serotipo hori dela haren arbaso posiblea. Aztertutako anduien arteko homogeneotasun handiak iradokitzen du, andui horietaz infektatutako dortoken jatorria estuki lotuta dagoela, Espainian dortoken saltokiek banaketa-puntu komuna dutela, eta horrek patogeno honen sakabanaketan laguntzen duela.