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Summary Heterogeneity of infection and extreme shedding patterns are common features of animal infectious diseases. Individual hosts that are super‐shedders are key targets for control strategies. Nevertheless, the mechanisms associated with the emergence of super‐shedders remain largely unknown. During chicken salmonellosis, a high heterogeneity of infection is observed when animal‐to‐animal cross‐contaminations and reinfections are reduced. We hypothesized that unlike super‐shedders, low‐shedders would be able to block the first Salmonella colonization thanks to a different gut microbiota. The present study demonstrates that (i) axenic and antibiotic‐treated chicks are more prone to become super‐shedders; (ii) super or low‐shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low‐ and super‐shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics., The present study demonstrates that (i) axenic and antibiotic‐treated chicks are more prone to become super‐shedders; (ii) super or low‐shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low‐ and super‐shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics.

International audience; Numerous genomes are available in databases and one of the biggest challenges is to understand the function of each gene. In bacteria, transposon mutant libraries have long been used to identify genes important for bacterial survival, growth or virulence. Recent advances in high throughput sequencing now allow the screening of millions of mutants in a single or few experiments using methods such as TraDIS or Tn-seq. These methods are sensitive enough to detect small defects in mutant fitness and allow to study genes but also intergenic regions, promoter regions and essential protein domains in coding sequences if the library is saturated with transposon insertions. Here, we present the construction and the characterization of a saturated library in Salmonella Typhimurium ATCC 14028 virulent strain, which is commonly used in research laboratories. This library was obtained using the Himar1 transposon that randomly inserts into TA dinucleotides sites onto the genome. Transposon mutant library was sequenced with an Illumina NextSeq 500. After read mapping, further analyses were carried out with the TRANSIT software. These analyses confirmed saturation of the transposon mutant library both on the chromosome and the virulence plasmid. This allowed us to determine the essential genes and those providing advantage or disadvantage for bacterial growth. This Tn-seq library is now used in several research projects and is available to the scientific community through collaboration

International audience; Introduction and objectives: Salmonella Typhimurium is a facultative intracellular entero-pathogen that remains a risk to public health worldwide. The tools and strategies allowing it to invade and survive in host cells are numerous. Nowadays, it is the only pathogen able to invade host cells using both a trigger and a zipper mechanism, respectively depending on the Type 3 secretion system-1, encoded mainly on Salmonella pathogenicity island I, and of the two invasins Rck (Wiedemann et al. 2016) and PagN (Barilleau et al. 2021). Intracellularly, Salmonella are contained in a Salmonella containing vacuole (SCV) whose maturation leads to an acidic and low divalent-cation environment. Nevertheless, in the last decades several studies gave some evidence for a Salmonella escape from the early SCV, leading to cytosolic hyper replication of the pathogen. As PagN expression was previously shown to dependent on acidic pH and low divalent cation concentration (Lambert and Smith 2008), we explored the potential role of PagN in the SCV.Materials and methods: Gentamicin protection assays to determine adhesion, invasion and multiplication were performed in chinese hamster ovary epithelial cell line. PagN expression was evaluated at several time post-infection by western blot. SCV escaping was considered by two ways to discriminate cytosolic vs vacuolar Salmonella: 1) a gentamicine / chloroquine assay, 2) cytometry and microscopy based on a localization-reporting dual color fluorescent plasmidexpressed by Salmonella (Noster et al 2019). Salmonella wild-type, pagN mutant and the complemented strain were used. Lastly, intracellular expression of PagN was imaged through confocal microscopy.Results, discussion and conclusion: PagN is highly expressed by Salmonella Typhimurium in the SCV and a reduced escape of the pagN mutant was observed compared to the wild-type strain leading to a reduced replication of the mutant in the cytosol. The impact of these cytosolic replicative bacteria on reinfection in vivo remains to be evaluated as described recently (Chong et al 2021).

OHEJP Project MoMIR-PPC This version only explain the confidential Deliverable D1.1, which has been uploaded on the OHEJP website. The list of genes will be available, after the publication of the results.

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International audience; Aim: Many studies have revealed the importance of heterogeneous shedding levels in the context of infectious diseases. Individual hosts that excrete higher levels of pathogens (so-called super-shedders) are key targets for control strategies. However, the mechanisms associated with the emergence of super-shedders remain largely unknown. The development of a new infection model for chickens reared in isolator, where animal reinfections are greatly reduced, has allowed us to demonstrate that two main Salmonella Enteritidis shedding phenotypes can emerge within the same chicken genetic background. These phenotypes were designated as super- and low-shedder on the levels of Salmonella faecal excretion and caecal colonization. Methods and results: In this project, we analysed the parameters that could be responsible for these phenotypes, namely: 1- the modification of the virulence of the Salmonella strain during the asymptomatic carrier state ; 2- the faecal and caecal microbiota composition ; 3- the immune status of chicks measured in kinetic in blood. We also analysed the impact on these parameters of four commensal bacteria inoculated before infection. Conclusions: The data obtained showed the protective effect of the four commensal bacteria on Salmonella excretion and their major impact on gut microbiota composition and immune response.

International audience; The MoMIR-PPC project:The project aims to develop new approaches to predict, identify and prevent the appearance of animal and human super-shedders based on immune response and gut microbiota (GM) composition. The project focuses on four objectives. 1-Decipher why some animals become super-shedders or human become chronic carriers. 2-Identify immune and microbiota biomarkers to detect super-shedders and/ or prolonged carriers. 3-Define preventive and control measures by the characterisation of prebiotics, probiotics and nutraceutical products. 4-Develop mathematical models to provide new risk management tools and a pool of biosecurity measures at the farm levels. For this purpose, we have focused our research on Salmonella infections, which are an important economic and public health problem worldwide. Our INRA project:The development of a new infection model in isolator, where animal reinfections are greatly reduced, demonstrated that two main shedding phenotypes may emerge within a same chicken genetic background. Depending on the levels of Salmonella faecal excretion and caecal colonization levels, we may define the super- and low-shedder categories. In this project, we analysed the role of 1-gut microbiota, 2-immune status of chicks, 3-virulence of Salmonella strains.Metabarcoding characterization showed that GM composition before infection partly determined the levels of Salmonella colonization. Consistent with this idea, the transfer of GM, collected before infection from individuals that later developed the super-shedding syndrome yielded to the development of the super-shedder phenotype. In the same way, the transplantation of GM taken from adults, which are more resistant than chicks, can transfer resistance to Salmonella colonization. The analysis of GM composition before and after infection revealed significant differences among super and low-shedder chicks.In conclusion, some gut bacteria present before infection in low-shedder animals could be used as protective probiotics or as biomarkers. These results also suggest that Salmonella colonization is inhibited and/or promoted by a subset of microbes naturally found, before Salmonella colonization, in varying abundances within the GM.

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International audience; Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens

Defensins are natural antimicrobial peptides. The avian beta-defensin AvBD7 isolated from the chicken bone marrow possess broad antibacterial spectrum and strong resistance to proteolysis. However, its ability to fight systemic infections of major concern for public health, such as salmonellosis, is unknown. As a first approach, fluorescence labeling of AvBD7 allowed to track its systemic distribution after intraperitoneal injection in mice using whole body live imaging. It was associated to peritoneal cells and to deeper organs such as the liver. In the next step, the use of labeled AvBD7 allowed to observe its interaction with murine macrophages in culture. After incubation, it was able to penetrate inside the cells through an endocytosis-like mechanism. Furthermore, natural AvBD7 contributed to the control of intracellular multiplication of a multidrug resistant Salmonella strain, after incubation with infected macrophages. Finally, administration in a model of systemic lethal Salmonella infection in mice led to significant improvement of mouse survival, consistently with significant reduction of the liver bacterial load. In conclusion, the results reveal a hitherto unknown intracellular antibacterial effect of AvBD7 in Salmonella target cells and support AvBD7 as a candidate of interest for the treatment of infectious diseases caused by multidrug-resistant pathogenic Enterobacteriaceae.

Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens

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