**3. Future directions**

*Microorganisms*

in mice [118].

swimming motility agar [115].

*phase of salmonellosis*

O-antigen and the mutant displayed enhanced susceptibility to complement-mediated killing and was highly attenuated in mice [113]. The transcription of *wzzST* gene is independently activated by two-component systems of Typhimurium, PmrA/PmrB (PmrA, sensor; PmrB, response regulator) and RcsC/YojN/RcsB (RcsC, sensor; YojN, intermediate phosphotransfer protein; RcsB, response regulator) [114]. PmrA/PmrB is activated through two pathways: one is directly activated through its cognate sensor PmrB in response to Fe3+ and the other is dependent on the PhoP/PhoQ two-component system in response to low Mg2+. The RcsC/YojN/ RcsB is activated in the presence of low Mg2+ plus Fe3+ [114]. In addition, mutants in a number of genes (*rfaG*, *rfaI*, *rfaL*, *rfaQ*, *rfaP*, *rfbC*, *rfbD*, *rfbJ*, *rfbM*, *rfbP*, *yibR*) necessary for LPS biosynthesis/assembly had severely impaired movement on

*2.4.2 PagC and Rck confer resistance to the complement-mediated bacterial activity*

*2.4.3 Siderophores are important for bacterial growth in serum in the extracellular* 

Iron is an essential element for the growth of most bacteria through its involvement in a variety of metabolic and regulatory functions [119]. Studies with different iron concentrations in growth media demonstrated an effect on gene expression of the iron acquisition systems encoded both on the chromosome and plasmids at both transcriptional and translational levels [120]. Siderophores which are bacterial molecules that bind and transport iron are important for bacterial growth in serum in the extracellular stage of *Salmonella* systemic infection. They are not required after bacteria reside in SCV where siderophore-independent iron acquisition systems are sufficient for iron uptake during intracellular stage. *Salmonella* produce two major types of siderophores, high-affinity catecholate consisting of salmochelin and enterobactin the latter also known as enterochelin and a lowaffinity hydroxamate known as aerobactin which is expressed under iron-restricted conditions [121]. The synthesis, secretion, and uptake of salmochelin requires genes clustered at two genetic loci, the *fepA* gene cluster and *iroBCDEN* operon. The *fepA* gene cluster includes most *ent* genes for synthesis and export [122]. The *iroBCDEN* operon encodes gene products for enterobactin glycosylation (IroB, glycosyltransferase), export (IroC, ABC transporter protein), and utilization (IroD, esterase; IroE, hydrolase; IroN, outer membrane receptor) [122]. Mutants deficient in *iroB* or *iroC* exhibit reduced virulence during systemic infection of mice via intraperitoneal route, as indicated by lower bacterial load in liver and a delayed time of death [122]. Moreover, the enterobactin metabolite, 2, 3-dihydroxybenzoyl serine (DHBS), can also be used by *Salmonella* as sources of iron, albeit at much lower affinities, by recognizing the three catechelate receptors, FepA, IroN and Cir. The three receptors demonstrate a significant degree of functional redundancy. The Typhimurium

In addition to LPS, two outer membrane proteins, the 18-kDa PagC [116] and the 17-kD Rck [117], confer a high level of resistance to the complement-mediated bactericidal activity. These two proteins share homology with virulence-associated outer membrane protein Ail from *Yersinia* that blocks formation of the complement membrane attack complex on the bacterial surface. Similarly, complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes [117]. One strain of Typhimurium which contains a single mutation in *pagC* had a virulence defect and decreased survival in cultured murine macrophages and 100-fold reduction in intraperitoneal virulence

**180**

The advent of next generation sequencing (NGS) has provided an opportunity to verify or improve on knowledge gained from *in vitro* and *in vivo* analyses of *Salmonella* mutants which were designed for the purpose of understanding gene function and mechanism of action. Recently, Rakov et al. [14] carried out bioinformatics analysis of 500 *Salmonella* genomes and identified 70 allelic variants virulence factors which were associated with different pathogenesis outcomes, i.e. gastrointestinal vs. invasive disease. However, the causative relationship between a putative virulence factor and disease outcome using a genomics based tool is yet to be attained. To that end, we propose the development of a comprehensive genome based tool such as a NGS AmpliSeq assay that can be used to simultaneously interrogate the presence and potential expression of over 200 virulence genes of *Salmonella* identified in this communication. The tool can be used to evaluate differences in strains and correlate the output with virulence phenotype derived from epidemiological or experimental observations which can be developed simultaneously or based on historical documentation. The tool could be used in assessing the potential risk posed by a strain of *Salmonella* given the fact that the serovars obtained from the environment are often distinct with those involved in human diseases. The technology appears suitable for dissecting the complexity associated with the redundancy and pleiotropic nature of some of the currently known virulence genes. In addition, NGS based analysis of virulence genes should provide new insights on *Salmonella* evolution and a better tool for analyzing epidemiological data that could translate to a reduction in the burden on human health posed by this important foodborne and zoonotic pathogen.

### **4. Conclusions**

This review provides an outline of over 200 identified virulence determinants and details of their involvement in the four steps of *Salmonella* pathogenesis, namely: attachment, invasion, intramacrophage survival/replication and systemic dissemination. The genetic regulation of only some of the virulence determinants have been elucidated in live animal models such as mice and cattle, and this has enriched our understanding of the pathogenesis and mechanism of diarrhea and systemic disease. The majority of the current evidence on pathogenesis

and virulence determinants of NTS was derived from murine model of serovar Typhimurium infection with and only a few studies focused on NTS infection in humans. For this reason, the relevance of published observations is often called into question. Linking clinical, epidemiological and experimental observations on the nature and severity of diseases caused by *Salmonella* organisms with the presence of a large number of virulence genes currently may not garner enough predictive ability to infer virulence or pathogenetic potential of a strain. Still, the increasing availability of a large number of *Salmonella* genomes in the public databases is proving to be a timely resource. Next generation sequencing and the twin subject of bioinformatics represent an unprecedented opportunity to verify past observations and help improve our understanding of *Salmonella* virulence towards a coherent and comprehensive understanding of the mechanism of *Salmonella* pathogenesis. What is required is a robust laboratory tool that can be used to analyze the large number of virulence genes in an isolate using the tools of whole genome sequencing. We expect that a tool such as an AmpliSeq assay for *Salmonella* virulence could be developed to generate accurate and reliable information that can be fed into a quantitative risk assessment framework. This could usher a new era of risk management customized for a *Salmonella* strain involved in an outbreak and should translate to impactful outcomes in the areas of improved food safety, evaluation of zoonotic diseases and reducing the burden of human salmonellosis.
