Prevalence of Salmonella in Free-Range Pigs: Risk Factors and Intestinal Microbiota Composition

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.info:eu-repo/semantics/publishedVersio

Salmonella enterica 4,5,12:b:- serotipo berriaren karakterizazioa eta etxeko dortokekin erlazionatutako arriskua

Salmonellosis, is one of the most common toxinfection caused by bacteria of the genus Salmonella in developed countries. In general, human salmonellosis is associated with contaminated food. However, contact with animals can be an important transmission route for Salmonella. Turtles have been shown to be reservoirs of different Salmonella serotypes. Considering that these reptiles are very common domestic pets, they can be a risk for public health, especially among children. In this work, a new monophasic serotype with the antigenic formula 4,5,12:b:- has been characterized. Most of the isolates analyzed have been isolated from children under 5 years of age in different regions of Spain. By means of different molecular techniques, it has been confirmed that 4,5,12:b:- isolates are genetically related to the biphasic isolates of Paratyphi B Java and that this serotype is their possible ancestor. The great homogeneity between the studied strains suggests that the origin of the turtles infected by these strains is closely related and that in Spain turtle shops have a common distribution point that contributes to the spread of this pathogen.; 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

Intestinal microbiota composition in free-range pigs is associated with the presence of Salmonella

Extensive pig systems are gaining importance as quality production systems and as stand ard for sustainable rural development and animal welfare. However, the effect of natural food on Salmonella epidemiology remains unknown. Here we assessed the presence of Salmonella in the in testinal content, the risks factors associated, and the gut microbiota composition in pigs selected from Salmonella-free and high prevalence farms. 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%), exhibiting multi-drug resistance (58.6%). Risk factor analysis identified feed composition, type/variety of vegetation available, and silos' cleaning/disinfection, as main factors associated with Salmonella prevalence. Clear differences in the intestinal microbiota were found between Salmonella-positive and Salmonella-negative popu- lations, showing the former increasing Proteobacteria and decreasing Bacteroides populations. Butyr ate and propionate producers, including Clostridium, Turicibacter, Bacteroidaceae_uc, and Lactoba- cillus were enriched 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' gut is directly related to the natural veg- etation accessible, determining the composition of the intestinal microbiota.This work was funded by the Caja Navarra Foundation (project reference 70628)

Genotyping Study of Salmonella 4,[5],12:i:- Monophasic Variant of Serovar Typhimurium and Characterization of the Second-Phase Flagellar Deletion by Whole Genome Sequencing

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

Multidrug resistance in Salmonella isolates of swine origin: mobile genetic elements and plasmids associated with cephalosporin resistance with potential transmission to humans

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 blaCMY-2 or blaCTX-M-9 genes responsible for cefotaxime resistance. Filter mating experiments showed that the two plasmids carrying blaCTX-M-9 were conjugative while that carrying blaCMY-2 was self-transmissible by transformation. Whole-genome sequencing and comparative analyses were performed on the isolates and plasmids. The IncC plasmid pSB109, carrying blaCMY-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 blaCTX-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.IMPORTANCEThe 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.The work was co-financed by Departamento de Innovación, Empresa y Empleo de Gobierno de Navarra (reference IIQ14061.RI1), the CERCA program from the Generalitat de Catalunya, and Basque Government agreement (PA20/03). A.A.-G.'s predoctoral contract is supported by UPV/EHU (PIF 19/290). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Genotyping Study of Salmonella 4,[5],12:i:- Monophasic Variant of Serovar Typhimurium and Characterization of the Second-Phase Flagellar Deletion by Whole Genome Sequencing

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

Biofilm formation of Salmonella enterica serovar 4,[5],12:i:- and Pan-Genome-Wide Association Study

Trabajo presentado en la 2nd FEMS Conference on Microbiology, celebrada en Belgrado (Serbia), del 30 de junio al 2 de julio de 202

Pan-Genome-Wide Association Study reveals a key role of the salmochelin receptor IroN in the biofilm formation of Salmonella Typhimurium and its monophasic variant 4,[5],12:i:-

In Press, Journal Pre-proofSalmonella enterica subsp. enterica serovar Typhimurium variant 4,[5],12:i:- (so called S. 4,[5],12:i:-) has rapidly become one of the most prevalent serovars in humans in Europe, with clinical cases associated with foodborne from pork products. The mechanisms, genetic basis and biofilms relevance by which S. 4,[5],12:i:- maintains and spreads its presence in pigs remain unclear. In this study, we examined the genetic basis of biofilm production in 78 strains of S. 4,[5],12:i:- (n = 57) and S. typhimurium (n = 21), from human gastroenteritis, food products and asymptomatic pigs. The former showed a lower Specific Biofilm Formation index (SBF) and distant phylogenetic clades, suggesting that the ability to form biofilms is not a crucial adaptation for the S. 4,[5],12:i:- emerging success in pigs. However, using a pan-Genome-Wide Association Study (pan-GWAS) we identified genetic determinants of biofilm formation, revealing 167 common orthologous groups and genes associated with the SBF. The analysis of annotated sequences highlighted specific genetic deletions in three chromosomal regions of S. 4,[5],12:i:- correlating with SBF values: i) the complete fimbrial operon stbABCDE widely recognized as the most critical factor involved in Salmonella adherence; ii) the hxlA, hlxB, and pgiA genes, which expression in S. typhimurium is induced in the tonsils during swine infection, and iii) the entire iroA locus related to the characteristic deletion of the second-phase flagellar genomic region in S. 4,[5],12:i:-. Consequently, we further investigated the role of the iro-genes on biofilm by constructing S. typhimurium deletion mutants in iroBCDE and iroN. While iroBCDE showed no significant impact, iroN clearly contributed to S. typhimurium biofilm formation. In conclusion, the pan-GWAS approach allowed us to uncover complex interactions between genetic and phenotypic factors influencing biofilm formation in S. 4,[5],12:i:- and S. typhimurium.This work was financially supported by the Basque Government (grant number PA20/03) and the University of the Basque Country (UPV/EHU) (grant number GIU21/021). A. Arrieta-Gisasola is recipient of a Ph.D. fellowship from the University of the Basque Country (UPV/ EHU) (PIF 19/290).Peer reviewe

A Practical Bioinformatics Workflow for Routine Analysis of Bacterial WGS Data

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

Estudio de tolerancia a metales en Salmonella enterica subsp . enterica serotipo 4,[5],12:i:

Trabajo presentado en el XXII Congreso Nacional de Microbiología de los Alimentos de la Sociedad Española de Microbiología, celebrado en Jaén (España), del 12 al 15 de septiembre de 2022La salmonelosis es la segunda zoonosis de transmisión alimentaria más reportada en humanos en Europa, siendo la variante monofásica de Salmonella Typhimurium 4 5 12 i el principal serotipo detectado en España 1 La prevalencia creciente que este serotipo ha experimentado desde 2015 en el ganado porcino, podía estar asociada al uso de metales pesados como desinfectantes y/o aditivos alimentarios presentes en los piensosArrieta Gisasola A agradece a la UPV/EHU por su contrato como personal investigador predoctoral en formación PIF 19 29