The initial steps of Salmonella pathogenesis involve adhesion and invasion of host epithelial cells. While well-studied for S. Typhimurium, the factors contributing to this process in other, host-adapted serovars remains unexplored. Here, we screened clinical isolates of serovars Gallinarum, Dublin, Choleraesuis, Typhimurium and Enteritidis for adhesion and invasion of intestinal epithelial cell lines of human, porcine, and chicken origins. 30 isolates with altered infectivity were used for genomic analyses and 14 genes and novel mutations associated with high or low infectivity were identified. The functions of candidate genes included virulence gene expression regulation, cell wall or membrane synthesis and components. The role of several of these genes in Salmonella adhesion and invasion to cells has not previously been investigated. The genes dksA (stringent response regulator) and sanA (vancomycin high-temperature exclusion protein) were selected for further analyses, and we confirmed their roles in host cell adhesion and invasion. Furthermore, transcriptomic analyses were performed for S. Enteritidis and S. Typhimurium, with two highly infective and two marginally infective isolates for each serovar. Expression profiles for the isolates with altered infection phenotypes revealed the importance of T3SS expression levels in the determination of isolate's infection phenotype. Taken together, these data indicate a new role in cell host infection for genes or gene variants previously not associated with adhesion and invasion to the epithelial cells. Importance Salmonella is a foodborne pathogen affecting over 200 million people and resulting in over 200,000 fatal cases per year. Adhesion to and invasion of Salmonella into intestinal epithelial cells is one of the first and key steps in the pathogenesis of salmonelosis. Still, around 35-40% of bacterial genes have no experimentally validated function and their contribution to the bacterial virulence, including adhesion and invasion, remains largely unknown. Therefore the significance of this study is in the identification of new genes or gene allelic variants previously not associated with adhesion and invasion. It is well established that blocking adhesion and/or invasion would stop or hamper bacterial infection, therefore the new findings from this study could be used in future developments of anti-Salmonella therapy targeting genes involved in these key processes. Such treatment could be a valuable alternative as the numbers of antibiotic-resistant bacteria is growing very rapidly.

Since the discovery of haemolysis, many studies focused on a deeper understanding of this phenotype in Escherichia coli and its association with other virulence genes, diseases and pathogenic attributes/functions in the host. Our virulence-associated factor profiling and genome-wide association analysis of genomes of haemolytic and nonhaemolytic E. coli unveiled high prevalence of adhesins, iron acquisition genes and toxins in haemolytic bacteria. In the case of fimbriae with high prevalence, we analysed sequence variation of FimH, EcpD and CsgA, and showed that different adhesin variants were present in the analysed groups, indicating altered adhesive capabilities of haemolytic and nonhaemolytic E. coli . Analysis of over 1000 haemolytic E. coli genomes revealed that they are pathotypically, genetically and antigenically diverse, but their adhesin and iron acquisition repertoire is associated with genome placement of hlyCABD cluster. Haemolytic E. coli with chromosome-encoded alpha-haemolysin had high frequency of P, S, Auf fimbriae and multiple iron acquisition systems such as aerobactin, yersiniabactin, salmochelin, Fec, Sit, Bfd and hemin uptake systems. Haemolytic E. coli with plasmid-encoded alpha-haemolysin had similar adhesin profile to nonpathogenic E. coli, with high prevalence of Stg, Yra, Ygi, Ycb, Ybg, Ycf, Sfm, F9 fimbriae, Paa, Lda, intimin and type 3 secretion system encoding genes. Analysis of HlyCABD sequence variation revealed presence of variants associated with genome placement and pathotype.

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Enterohaemolysin (Ehx) and alpha-haemolysin are virulence-associated factors (VAFs) causing the haemolytic phenotype in Escherichia coli . It has been shown that chromosomally and plasmid-encoded alpha-haemolysin are characteristic of specific pathotypes, virulence-associated factors and hosts. However, the prevalence of alpha- and enterohaemolysin does not overlap in the majority of pathotypes. Therefore, this study focuses on the characterization of the haemolytic E. coli population associated with multiple pathotypes in human and animal infectious diseases. Using a genomics approach, we investigated characteristic features of the enterohaemolysin-encoding strains to identify factors differentiating enterohaemolysin-positive from alpha-haemolysin-positive E. coli populations. To shed light on the functionality of Ehx subtypes, we analysed Ehx-coding genes and inferred EhxA phylogeny. The two haemolysins are associated with a different repertoire of adhesins, iron acquisition or toxin systems. Alpha-haemolysin is predominantly found in uropathogenic E. coli (UPEC) and predicted to be chromosomally encoded, or nonpathogenic and undetermined E. coli pathotypes and typically predicted to be plasmid-encoded. Enterohaemolysin is mainly associated with Shiga toxin-producing E. coli (STEC) and enterohaemorrhagic E. coli (EHEC) and predicted to be plasmid-encoded. Both types of haemolysin are found in atypical enteropathogenic E. coli (aEPEC). Moreover, we identified a new EhxA subtype present exclusively in genomes with VAFs characteristic of nonpathogenic E. coli . This study reveals a complex relationship between haemolytic E. coli of diverse pathotypes, providing a framework for understanding the potential role of haemolysin in pathogenesis.