Several members of the genus Salmonella are major foodborne pathogens [77]. It comprises two species: S. bongori and S. enterica. Almost all Salmonella organisms that cause disease in humans and domestic animals are serovars belonging to S. enterica subspecies enterica. Broadly, the diseases caused by Salmonella in humans are of two kinds: 1) systemic febrile illness termed typhoid/enteric fever, and 2) acute self-limiting gastroenteritis. The serovars that cause typhoid are referred to as typhoidal Salmonella and include S. enterica subsp. enterica serovar Typhi and S. enterica subsp. enterica serovar Paratyphi A, B, and C. S. Typhi causes approximately 76.3% of global enteric fever cases [78]. S. Typhi and S. Paratyphi are restricted to humans and higher primates, and clinical manifestations of the infection include sustained high fever, abdominal pain, headache, weakness, malaise, and transient diarrhea/constipation. Without appropriate and effective antibiotic therapy, the infection may lead to gastrointestinal bleeding, intestinal perforation, septic shock, and death [79,80]. The Non-Typhoidal Salmonella (NTS) serovars cause self-limiting gastroenteritis and include S. enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Enteridis, which are the most prevalent clinical isolates, according to the World Health Organization (WHO). These pathogens are broader in host range and infect humans and animals such as poultry, cattle, reptiles, and amphibians. Infections with NTS typically involve self-limiting diarrhea, stomach cramps, headache, vomiting, and fever that resolve on their own; however, the infection can be severe in children and the elderly and can sometimes be fatal [81]. NTS serovars have been reported to cause an invasive infection similar to typhoid, particularly in sub-Saharan Africa, predominantly in children and HIV-positive adults, with several co-morbidities such as ongoing or recent malaria infection and malnutrition contributing to higher mortality [82,83,84]. Salmonella infections represent a considerable economic burden and public health concern in both developing and developed countries. The Center for Disease Control and Prevention (CDC), estimates that 1.35 million infections by Salmonella occur in the United States. NTS causes more than 93 million global infections per year, with 155,000 deaths [85]. A study examining the global burden of diseases, injuries, and risk factors in 2017 reported 14.3 million cases of enteric fever globally, with a case fatality of 0.95% resulting in approximately 135,000 deaths [78]. Children, the elderly, and those residing in lower-income countries account for the greatest incidences [86,87].

 

Salmonella is acquired via contaminated food and water. Luminal bacteria invade M cells and absorptive enterocytes via a specialized apparatus called the type three secretion system (T3SS) [88] encoded on the Salmonella Pathogenicity Islands (SPIs) [89]. The T3SS injects bacterial proteins into host cells allowing the bacteria to essentially commandeer host cellular processes to induce cytoskeletal rearrangements that engulf the bacteria into specialized vesicles called the Salmonella containing vacuoles (SCVs) [90]. This invasion process, with subsequent translocation across the epithelium, is followed by the uptake of the bacteria by macrophages and dendritic cells in the intestinal submucosa, where bacterial proteins interfere with phagolysosomal maturation and allow the bacteria to survive inside the cells [91,92,93]. NTS serovars undergo prolific growth in the intestine, while T3SS effectors induce fluid secretion and promote inflammation. Immune signaling via recognition of pathogen-associated molecular patterns such as lipopolysaccharide (LPS) and flagella also induce a robust inflammatory response, which actually provides Salmonella with a growth advantage over resident microflora [81,94,95,96]. The immune response eventually limits Salmonella growth; nevertheless, the short-term proliferation is sufficient to ensure propagation. Typhoidal Salmonella strains elicit a more attenuated inflammatory response in the intestine, especially in terms of limited neutrophil recruitment [79,97,98]. Bacteria migrate to the mesenteric lymph nodes (MLN) and systemically within the reticuloendothelial cells, and as free bacteria in the blood or lymph, to establish new foci of infection in the liver, spleen, bone marrow, and gallbladder [99]. At these new sites, the bacteria replicate and re-enter the intestinal lumen via secretion in bile, promoting the shedding of the bacteria to continue the cycle of new infections by contaminated food and water. In 3%–5% of cases, the bacteria can persist for long durations in the gallbladder, which serves as a reservoir of chronic infection [100]. Chronic infection with Salmonella has been found to be a risk factor for the development of malignant neoplasms, including gallbladder cancer [101,102] and colorectal cancer [102,103].

 

The emergence of multi-drug resistance to conventional antibiotics complicates the treatment of Salmonella infection [104,105,106,107]. Antibiotic treatment destroys the resident microflora, provides a niche for Salmonella to proliferate, and may lead to increased levels of bacterial shedding [108]. Additionally, bacterial populations that express an antibiotic-tolerant phenotype can evade treatment and persist, causing relapses of the infection as well as the evolution of bacterial virulence [109,110]. Asymptomatic carriers act as reservoirs, contribute to the continued propagation of the pathogen, and are particularly important for food safety considerations. Currently, there are no effective vaccines against gastrointestinal Salmonella infections. Several typhoid vaccines are available and licensed in many countries; however, robust protection is limited and has been associated with injection site reactions. Furthermore, the vaccines have not been widely adopted by public health programs [111]. With the significant aging populations in both developed and developing countries [112,113], more people are at risk for severe consequences of Salmonella infections. Advances in the mechanistic understanding of Salmonella infections will facilitate the development of improved control strategies, particularly, safe and effective vaccines. The broad conservation of host responses as well as the molecular machinery used by Salmonella strains during infection of various hosts, namely T3SSs encoded by SPI1 and SPI2 that enable invasion of epithelial cells and subsequent intracellular survival, allows several model systems to be applied for Salmonella research [114]. Each non-human model has pros and cons and varies in the ability to recapitulate natural infection. Given the host-specific aspects of infection physiology, it is necessary to be cautious in applying the results from these model systems to human patients. Additionally, with Salmonella being an important tool to understand host physiology, metabolism, immune function, and interactions with microbes, human-specific investigations are valuable to the research community.

Transformed cell lines such as Cos-1, MDCK, HeLa, HepG2, CaCo2, and T-84 have been used to carry out several fundamental studies on Salmonella pathogenicity, such as the identification of the T3SS and the SPIs [88,115]. However, these cells have drawbacks in that the cells do not adequately represent the physiological characteristics of normal human tissue [116,117]. Explant tissue cultures have organotypic properties that can be vital for studies on development and physiology but are limited by culturing difficulties and short life span [118,119]. Classically, animal models have offered solutions for several of these limitations, and have been used to corroborate data obtained from other model systems as well as to investigate the deeper molecular mechanism of infection. For example, S. Typhimurium is a natural pathogen of calves and causes gastroenteritis with clinical and pathological manifestations similar to humans, namely diarrhea, anorexia, fever, localized infection, and neutrophil infiltration [120,121]. Meanwhile, the bovine ligated loop was instrumental in characterizing fluid accumulation and host inflammatory responses following S. Typhimurium infection. For example, mutants lacking the invasion proteins SipA, SopA, and SopD were shown to have little to no effect on the ability of S. Typhimurium to invade epithelial cells, but were shown to reduce the fluid accumulation and neutrophil immigration in bovine loops [122,123].

 

More importantly, the vast majority of studies on Salmonella pathogenesis have been conducted in the murine model, including studies for the development of new vaccines. This model has been useful in clarifying various aspects of in vivo Salmonella pathogenesis; however, it does have limitations with respect to its ability to faithfully reproduce all aspects of Salmonella infection in humans. S. Typhimurium causes two very different types of diseases in human and mice. In humans, the infection is localized, dominated by the infiltration of neutrophils and self-limiting. In mice, S. Typhimurium spreads systemically, with slow infiltration of mononuclear inflammatory cells and little or no localized intestinal tissue injury. The host responses involved are also dramatically different. Since the clinical manifestations and pathology of mice infected with S. Typhimurium resemble those observed in humans with S. Typhi infection, the model has been used as a surrogate to study typhoid pathogenesis [124]. A mouse model of S. Typhimurium-induced enterocolitis has been developed and involves pre-treatment of mice with a single dose of streptomycin. This procedure diminishes the colonization resistance by the commensal microbiota, allowing Salmonella (that carry the gene for resistance to streptomycin) to grow to high densities in the cecum and large intestine and trigger acute gastroenteritis [125]. Mouse models have also been developed to mimic chronic infections of Salmonella observed in certain carrier individuals, which typically involve infecting susceptible mice with avirulent strains, or sub-lethal doses of S. Typhimurium in resistant mice. Experimental interpretations from the mouse model may not translate to human disease. S. Typhi and S. Typhimurium share about 89% of their genes, with approximately 500 genes unique to S. Typhimurium and 600 genes unique to S. Typhi, including the genes encoding typhoid toxin and the immunoprotective capsule [126,127]. S. Typhi and S. Paratyphi also have pseudogenes as well as small sequence differences in genes encoding the T3SS apparatuses and related effectors that may have important implications in pathogenesis. It is possible that virulence factors that may have no role in S. Typhi-mediated infection in humans may be important for mouse infection by S. Typhimurium and vice versa. Finally, S. Typhi genes required for causing typhoid but absent in S. Typhimurium cannot be studied easily using the latter. Typhoid fever can be induced experimentally by oral infection in higher primates or human volunteers, but these studies come with their own set of difficulties and ethical objections. Researchers have attempted to compensate for the discrepancies by developing humanized mice, i.e., immunodeficient mice engrafted with human hematopoietic cells [128,129], but these models are cumbersome, expensive, and still do not guarantee that mouse-specific factors will not add complexities or variations to the data generated. In addition, host genetic background has been found to play a role in susceptibility to invasive NTS infections, a concept that borders on the realm of personalized medicine [130]. Thus, organoids offer a promising model to mechanistically study host-specific aspects of infection.

One of the earliest studies that utilized organotypic 3D structures to investigate Salmonella pathology was carried out by Nickerson, CA et al., in 2001 [26]. The authors generated 3D organotypic cultures by growing human embryonic intestinal cell line Int-407 in rotating wall vessel (RWV) bioreactors and subsequently infected the cells with S. Typhimurium. The resulting infection was quite different from what had previously been observed in monolayer cultures. There was minimal loss of structural integrity, lower ability of the bacteria to adhere to and invade epithelia, and lowered expression of cytokine in 3D Int-407 aggregates as compared to infected Int-407 monolayers. Since the authors observed that the 3D Int-407 aggregates more closely resembled in vivo characteristics (tissue organization, tight junctions, apical-to-basal polarity, microvilli development, expression of extracellular and basement membrane proteins, and greater M cell glycosylation pattern), the authors concluded that the infection phenotypes observed in the 3D aggregates were likely representative of an in vivo infection. This study laid the groundwork for the use of 3D organotypic cultures to study Salmonella biology. The following section will highlight research performed in both mouse and human organotypic models that have improved our understanding of Salmonella pathogenesis (Figure 3).