Salmonella: Role in Internal and External Environments and Potential as a Therapeutic Tool

Abstract

Salmonella has had a long and intimate relationship with humans and continues to raise concerns for human health, but this close bond also provides opportunities for new therapeutics and treatments. Although Salmonella enterica serovar Typhi is the principal organism that comes to mind in terms of death and morbidity, it is the non-typhoidal Salmonellae that have the most health and economic implications. The developed world has had a challenging relationship with Salmonella, particularly in the UK and the EC/EU, experiencing significant Salmonella outbreaks in the 1980s and 1990s. As a consequence, the research focus was on understanding the nature of infection in food animals and on developing ways and means of controlling zoonotic infections. This led to the development of numerous model systems for the study of Salmonella both in vitro and in vivo. The introduction of vaccination has all but eliminated Salmonella in eggs and reduced perceived risk held by the general public. At the same time as Salmonella in eggs was being brought under control in the UK and EU, the danger posed by antibiotic resistance was beginning to emerge. In the past, with the efficacy of antibiotics against Gram-negative bacteria being unchallenged, there was limited focus on the threat posed by antibiotic resistance in non-typhoidal Salmonella. However, the identification of Salmonella as the first ‘multidrug-resistant’ organism, the presence of invasive non-typhoidal Salmonella in North Africa and the emergence of monophasic Salmonella enterica serovar Typhimurium across Europe, Asia and the Americas have prompted renewed interest in Salmonella research, particularly in the context of non-infectious disease, biofilm studies and antibiotic resistance. At the same time, research has continued to develop ways of taking advantage of what Salmonella offers in the way of pathogenic factors and the therapeutic and treatment applications in areas such as vaccine development, cancer therapeutics and drug delivery and the role of Salmonella in non-infectious diseases supported by developments in molecular and genomic methods.

1. Introduction

The focus of Salmonella research has ebbed and flowed in the last three decades. The challenge of Salmonella zoonosis in the 1990s, required better isolation, detection and identification methods for non-typhoidal Salmonella (NTS). The advent and development of molecular methods have meant the successful replacement of the gold standard pulse-field electrophoresis (PGGE) method with the adoption of whole-genome systems including multilocus sequence typing (MLST) [1,2]. Whole-genome sequencing (WGS) can be used to detect and control Salmonella [3] in a number of ways, including the following: serotyping [4] (the SeqSero web-based tool can predict Salmonella serotypes using WGS data [5]); AMR (WGS is a rapid and accurate tool for AMR surveillance [6]); subtyping (WGS can be used with single-nucleotide polymorphism (SNP) and multilocus sequence type (MLST) subtyping methods to identify genetically related genomes); outbreak surveillance (WGS can be used in combination with real-time epidemiological investigation to detect and investigate outbreaks, e.g., SIEGA) [7,8]. The work on Salmonella enterica serovar Typhi has been particularly important both in terms of understanding the genetic separation of NTS (reviewed in detail [9]) and the potential of S. Typhi as a vaccine tool. The observation that typhoidal and NTS infections induce such different clinical presentations and immune responses in humans may be attributed to a degree of metabolic and virulence gene degradation that exists in the genomes of typhoidal serovars, perhaps explaining the restricted host tropism of these pathogens [10]. It has been suggested that NTS serovars have evolved to flourish in the inflamed gut environment and use inflammation to outcompete the normal microbiota [11]. It has been proposed that typhoidal strains may have lost this ability and so do not induce inflammation but act systemically instead. In addition, the move by the statutory agencies in the UK [3] and elsewhere towards whole-genome sequencing (WGS) analysis with both SNP- and allelic-based methods for the detection and identification of Salmonella means the potential is now there for very detailed and precise information that previous conventional methods were unable to provide. The complications of antimicrobial resistance (AMR) have also resulted in fluoroquinolone-resistant NTS being included in an unenviable club of bacteria, the WHO’s list of antibiotic-resistant high-priority pathogens [12], and considered a research priority as part of the WHO global research agenda [13]. This review seeks to bring together elements of Salmonella research that have tended to be looked at in isolation in the past. There is no doubt that a more holistic approach to Salmonella science is required. The One-Health initiative adopted in the face of the unrelenting increase in global antimicrobial resistance is a possible approach for integrating some of the currently more disparate topics. The objectives of this review are as follows: to highlight the importance of newly emerging variants and serovars; to detail the impact of Salmonella biofilms in the internal and external environments; to focus attention on the potential of Salmonella as an anti-viral and anti-cancer tool; to highlight Salmonella as a platform for vaccines and in particular anti-cancer vaccines.

This review is not exhaustive; it focusses on aspects of Salmonella and applied research that impact discrete areas of health and the environment. This review considers the current state of Salmonella research in terms of the fight against the disease and the potential therapeutic applications of Salmonella (Figure 1).

2. Emerging Salmonella Variants

Focus on WGS tools has been spurred on by newly emerging variants including the occurrence of invasive non-typhoidal Salmonellosis (iNTS). The presence of iNTS was first recognised in West Africa associated with malaria and East Africa with malnourishment; interest in iNTS has increased because of the levels of multidrug resistance associated with these strains, and the implications for the very young, old, malnourished and immunocompromised are significant. Whereas their function as a reservoir of disease and human transmission is unclear, those strains causing disease are the most notable because of their invasive phenotype accompanied by genome degradation. A review by Reddy and co-workers [11] found NTS to be responsible for 29.1% of all bloodstream infections, and NTS bacteraemia is an important cause of morbidity and death in Africa. Van Puyvelde and colleagues [14], investigating invasive S. Typhimurium (iNTS) in sub-Saharan Africa, found specific risk factors for iNTS infection (malnutrition, HIV and malaria coinfections) to be significant in the human population. Water, sanitation and hygiene, nutrition and control of malaria and HIV are all confounders in endemic regions. Unfortunately, the data on NTS bacteraemia in Africa remain limited, so it has been difficult to estimate the true burden. Nonetheless, these infections are a significant obstacle to economic progress in developing countries, hence the need for efficacious and cost-efficient vaccines for Africa. The emergence of the iNTS variant has been closely followed by monophasic S. Typhimurium (for a detailed review, see [15]. It lacks one or more flagellar assemblies [16], i.e., the fljB-encoded second-phase H antigen. Moreover, the rise in monophasic cases has been associated with disease in humans, particularly the young and the elderly, compounded by the multidrug resistance associated with this variant.

The monophasic variant of Salmonella enterica serovar Typhimurium (mST), namely S. Typhimurium (4, [5], 12:i:-), was initially associated with pork zoonosis in Europe [17] and, though rarely reported before the 2000s, has now become the third most common serovar in the EU and UK [18]. It is placed in the top five serovars in human infections according to the most recent EFSA and ECDC report [19] and was most recently associated with a pan-European outbreak associated with chocolate intake in children [20]. ASSuT, known as the ‘European Clone’, is the predominant MDR pattern found in mST in China [21], with a Spanish clone exhibiting a unique ACSuGSTTM profile. Indeed, mST and Salmonella enterica serovar Infantis are amongst the five serovars most associated with human cases of Salmonellosis in the EU in 2021 [22]. A systematic review of studies published in China found a high multidrug resistance rate (86%) associated with the detection prevalence of mST, higher in Southern China compared to Spain and the USA but lower than in Italy [23,24,25,26]. An analysis of isolates sequenced (2015–2018) in the USA found that 63% of human cases were associated with pork consumption/contact with swine and had the ASSuT profile [27]. It is thought that the level of multidrug resistance in mST may have contributed to the rapid spread of the serovar.

Amongst the Salmonella serovars in circulation, Salmonella entericia serovar Rissen (S. Rissen) [28,29] is now frequently reported worldwide and has a significant association with the intensive pig industry. The most recent ECDC report [30] places S. Rissen in the top 20 of the most frequent Salmonella serovars associated with human Salmonellosis. S. Rissen is the predominant serotype associated with swine production in Thailand [31]. Elbediwi and colleagues [32] reported that the majority of S. Rissen isolates from across China were derived from humans (63.4%; 749/1182). In China, pork continues to dominate the diet, and significant cases of S. Rissen have been reported [32]. Wang and colleagues [33] have confirmed earlier work dividing S. Rissen into two clusters, Clusters A and B. Wang and co-workers further subdivided Cluster B into Clusters B-1 and B-2, with B-2 showing additional profiles of quinolone (qnrS1), chloramphenicol (cmlA1) and tetracycline (tet(M)) resistance. The authors suggest that this continuing divergence demonstrates the ability of S. Rissen to continually evolve through pork production based on its evolving MDR profile. More recently, significantly higher resistance rates have been identified in strains isolated from humans compared to non-human sources [34], with higher frequencies of multidrug resistance and probable clonal dissemination in this serovar. In common with these studies, work on Portuguese isolates [35] has found a low degree of genetic diversity but a high degree of genetic relatedness in this serovar related to pork production. Their analysis identified the presence of two major clusters composed of MDR isolates, with the majority being resistant to at least five antibiotics. This may go some way towards explaining the continuing spread of this serovar.

The emergence of new variants is not restricted to bacterial competition or antibiotic pressure; several workers have already identified a number of the seasonal influences on Salmonella outbreaks [36,37,38], both in the environment and the inevitable clinical cases. There is also, with climate change [39], the potential for increases in susceptibility to plant fungal infections making vegetable crops more sensitive to Salmonella carriage [40,41]. Temperature fluctuations [42] and extreme weather events can impact Salmonella transmission in a number of ways, including faster replication (for every 5 °C rise, there can be a 5–10% increase in infections), contaminated crops (floodwaters can carry Salmonella bacteria from sewage, animal waste and soil into crops) and contaminated water (floodwaters can contaminate drinking water and also recreational waters (including seawater)), all with significant implications for human health (reviewed in detail by [22,43]). Without a doubt, it is low- to middle-income countries that will be the least well prepared to deal with the impact of climate-induced increases in Salmonella outbreaks [44]. There are also agricultural developments in response to sustainability, e.g., the increased utilisation of animal manures and soil supplementation [45,46]. These inputs may have implications for an increase in pathogenic organisms, including Salmonella, in fruits, grains and vegetables, and a subsequent increase in clinical cases.

3. Salmonella in the External Environment

In the natural environment as well as the host, biofilms are important for microbial survival. The main challenges associated with Salmonella biofilm formation in the clinical and food environments are detection and removal. S. Infantis causes particular issues in the poultry industry [47]. Despite being an intestinal pathogen, S. Infantis can readily persist outside the organism’s host by forming biofilm as an important survival adaptation mechanism. Cwiek and colleagues [48], investigating Salmonella enterica serovar Enteritidis, found that those isolated from poultry exhibited better biofilm growth than strains isolated from humans. Furthermore, studies have shown that biofilm formation provides tolerance to various stressors (temperature, pH, disinfection, antibiotics, etc.) encountered in both host and non-host environments (reviewed in detail by [49]). The expression of curli and cellulose, well-recognised adhesins for both biotic and abiotic surfaces, is strongly dependent on temperature, leading to an increase in persistence [50]. Antibiotic treatment is only recommended in vulnerable patients, such as the young, the elderly and patients with weakened immunity with biofilms identified in key areas of pathogenicity of disease [51]. Typhoid fever is an acute food-borne illness, predominantly caused by Salmonella enterica serovar Typhi, that is often characterised by high fever, weakness, headache, abdominal pain and constipation. If it is untreated, serious complications may arise, including intestinal bleeding, bowel perforation, septicaemia, meningitis and death.

S. Typhimurium has the ability to form biofilms on the many materials we associate with the built environment, including products in everyday use such as glass, stainless steel and plastic [52]. This has implications both for the food and clinical environments. Given its ability to colonise these everyday surfaces, it follows that S. Typhimurium biofilm can survive on food contact surfaces for significant periods with the added complication of it being difficult to remove [53]. Along with contact surfaces, Salmonella have the ability to form biofilms on food under favourable conditions [54,55,56]. The formation of Salmonella biofilm increases the risk of microbial contamination in the food chain and consequently impacts clinical health; it is a complex process regulated by different genes whose contributions are just beginning to be elucidated [57]. The genes adrA, csgD, fljB and flhD and sRNAs (ArcZ, CsrB and SroC) may play significant roles in S. Typhimurium biofilm formation [58,59,60]. For instance, adrA regulates cellulose synthesis through c-di-GMP [61]; CsgD regulates the expression of matrix compounds [62,63,64]. Cell motility is important in the initial attachment to the contact surfaces and hence biofilm production [65], with fljB and flhD genes being critical for flagellar regulation. Simulated gastrointestinal conditions (pH, agitation, enzymatic activity) have an effect on biofilm formation [66], with biofilm formation in mST thought to be beneficial in its dissemination. Biofilm formation enables infection and persistence in the environment [61,67], with a range of genes having already been identified as being involved (

Table 1. Examples of genes in NTS identified with a role in biofilm formation.

Gene Responsible	Function	Reference
flgK, rfbA	Flagella, lipopolysaccharide production	[72]
bcsABZC, bcsEFG	Synthesis of exopolysaccharide cellulose	[73]
EutE, Sufs/SufE, OmpL CopG	Biosynthesis of aldehyde; dehydrogenase; cysteine desulfurase transporter protein; ribbon helix protein	[74]
Cdg, trx, rtx	Decrease in biofilm mass	[75]
fadI	Rate of biofilm formation	[75]
marT	General regulation of biofilm-related genes	[76]
fimA, fimH	Adherence-mediated biofilm formation	[77]
bapA	Biofilm mass formation	[78,79]
rspA	Temperature regulation of biofilm formation	[80]

). Although traditionally associated with poultry, meat and dairy products, there has been a renewed interest in Salmonella biofilms because of outbreaks associated with low-moisture conditions [68] and ready-to-eat products which now dominate Western supermarkets [69,70]. Salmonella have been shown to survive for long periods of time in low-moisture foods [71]; hence, it is important that we understand the genes and regulatory processes involved and be in a position in the future to introduce appropriate control measures.

4. Salmonella Biofilm in Body Systems (Internal)

Whereas the external environment sees NTS in the ascendancy, the internal human environment is more impacted by S. Typhi (reviewed in detail [49]). It was initially unclear whether biofilm could form in vivo due to a lack of evidence of csgD expression in NTS isolates tested at temperatures ≥37 °C [62]. In the case of S. Typhi, we are more familiar with the acute disease (typhoid fever). However, it is now clear that Salmonella produces Extracellular Polymeric Substances (EPSs) in vivo and that there is an association with chronic infections [81], and environmental signals other than temperature may be able to counteract its effects on csg expression [82]. Notwithstanding, there is evidence of Salmonella biofilm formation in various in vivo systems, including chronic gallbladder carriage of the human-specific S. Typhi [83].

Gallstones provide a suitable surface for the attachment of S. Typhi biofilms [84,85] and the development of similar disorders. Flagella may play a role in the initial attachment of S. Typhi to gallstone cholesterol surfaces mediated by FliC [86]. S. Typhi can also secure itself to the gallbladder epithelium [85,87,88]. Biofilm may provide protection to Salmonella from bile, bile salts and antibiotic therapy in the gallbladder environment, facilitating long-term survival and further transmission [81,89]. During chronic infections, the immune system interacts with Salmonella biofilms in a number of ways, including the following: inhibiting apoptosis; inducing an inappropriate or skewed immune response [90]; tolerance to innate immunity; host cell damage and establishing itself in M2 macrophages [49,81] (see [91] for a detailed review on chronic infections). Recently, [92], investigating starvation, biofilm production in S. Typhi and adherence, identified a central role for rpoS in regulating this process.

Chronic gallstone biofilm carriers are the only known reservoir of S. Typhi, and individuals have been shown to continue shedding for 12 months or more [83]. There is a strong correlation between the carriage of chronic S. Typhi and gallstones, with the latter considered a major risk factor for gallbladder cancer (GBC), [93]. While GBC is a (relatively) rare malignancy in Western countries, its incidence is higher in countries with endemic S. Typhi, with typhoid toxin (CDT) having a possible role in long-term infection. CDT induces DNA double-stranded breaks and the activation of the MAPK and AKT pathways, leading to the transformation of pre-transformed cells [93,94]. The association between S. Typhi infection and/or chronic carriage and cancer has become well recognised along with the role of Salmonella in other chronic diseases [95].

There is limited evidence in vitro of NTS infection being accompanied by biofilm formation [96,97,98,99,100], and that evidence is restricted to adherence to human and chicken epithelial cells. Work with HT-29 cells and S. Typhimurium biofilm-forming strains suggested a reduction in virulence. In contrast, Salmonella enterica serovar Enteritidis biofilm formation on Hep-2 and Caco-2 cells was correlated with virulence, although the comparison was of different serovars in different cell lines [96,99]. S. Typhimurium biofilms may also be involved in the formation of extracellular aggregates in vivo. In the Caenorhabditis elegans gut model, CsgD-dependent formation of aggregates was associated with bacterial persistence and host survival, possibly linked to transmission of similar populations in the mouse large intestine [101,102,103]. However, evidence of NTS infections in the human intestine involving biofilms is limited [10,104]. Salmonella curli are thought to be a major mechanism in biofilm formation [105,106,107], and the production of curli amyloids has been shown in the gastrointestinal (GI) tract of orally infected mice. So, biofilm genes may be expressed during a human infection, but the determinant genes required to establish biofilm are still unclear [106,107,108].

The ability of Salmonella to form biofilm in vivo likely leads to bone disease in addition to diseases of organs and soft tissue. Though rare, Salmonella osteomyelitis does occur [109]. In Africa, it is commonly associated with sickle cell anaemia including spleen damage, defects in complement activation, genetic factors, deficiencies in micronutrients and the presence of infarcted or necrotic bone (for a detailed systematic review, see [110]). In children with haemoglobinopathies, it is a significant cause of morbidity and death [110]. In these situations, gut-borne bacteria may enter the blood stream, and blood-borne bacteria may enter damaged bone. Failure and near-failure of the liver and spleen may also reduce the clearance of blood-borne bacteria, and this may be particularly damaging in the young [111,112]. Immunocompromised adults may have Salmonella-associated osteomyelitis despite the common risk factors for infection being absent [113,114].

Much of the evidence identifying biofilm formation with persistence in NTS is from zoonotic work [115]. However, there is increasing evidence of similar findings in humans [49,116]. It appears that the ability to form a biofilm is a conserved trait found in numerous serovars of S. enterica, with biofilm formation allowing Salmonella to persistently colonise sites both inside and outside of the animal host, which in combination with AMR (Figure 2A) is the ultimate enhancement of both bacterial survival and transmission.

5. Salmonella Combatting Non-Infectious Diseases Including Cancer

S. Typhimurium is suitable for oral administration [117] and can induce mucosal and systemic immune responses [118,119]. The early Salmonella aroA, aroC or aroD mutations prevented the development of infection but persisted enough for an immune response. Genetically modified recombinant attenuated Salmonella vaccine (RASV) strains can display a hyperinvasive phenotype to maximise Salmonella host entry and allow for host cell internalisation. This has resulted in a strain capable of evading apoptosis, prompting DNA to more efficiently enter the target cell [120], and the manipulation of lipid A (as an immunogen) has resulted in an improved vaccine carrier [121]. The potential of Salmonella as/in vaccines against non-infectious diseases is of particular interest [122,123], though there are few examples that have progressed even to animal trials; e.g., Hauer and colleagues [124] used an S. Typhimurium encoding VEGFR2 to reduce atherosclerosis in mice.

The suggestion that bacterial systems could be used to treat cancers is not new. In the late 1800s, Coley improved patient outcomes using a Streptococcus pyogenes injected into cancers (cited in [125]). Salmonella was not originally included in the scenario of the Coley vaccines, but it was later recognised that Salmonella not only could be a potential therapy [126] in the fight against cancer but also may be a risk factor in the development of cancer/cancer risk, and S. Typhi itself may have a role in some cancers [127]. Duijster and colleagues [128] identified 10 bacteria including NTS and S. Typhi associated with gastrointestinal cancers. Notwithstanding the clear linkages between S. Typhi and GBC, there is evidence of Salmonella-mediated anti-tumour therapy promoting significant tumour suppression and prolonged survival in many studies [129]. Salmonella’s potential as a therapeutic tool (Figure 2B) has been reviewed in detail [130], and work in experimental models [131] has shown that repeated exposure to NTS increases the risk of colon cancer. Shanker and Sun [132], in their review of Salmonella as an environmental risk factor, identified repetitive NTS infections as significant in the proliferation of transformed cells in tissue cultures.

Salmonella bacteria have been studied as a potential treatment for cancer in a process called bacterial-mediated cancer therapy (BMCT), [133]. These therapies include tumour targeting, drug delivery, combination therapies, immune response stimulation, genetic modification and the ability to utilise a hypoxic environment [134]. The potential benefits of Salmonella-mediated therapy include stronger tumour suppression, improved side effects, inhibition of tumour growth and prolonged survival (in mice) [135]. To combat cancer, a number of approaches have been suggested [136,137,138], among which the anti-tumour effects of S. Typhimurium LT2 CRC2631 are probably the most studied [139]. Other Salmonella serovars have been investigated as models, e.g., Salmonella entericia serovar Choleraesuis [140,141]; however, VNP20009 [142] and A1-R1 [143], e.g., the prevention of tumour angiogenesis in a mouse model by VNP20009 along with endostatin [144], remain the most prominent in the literature (see

Table 2. Salmonella in approaches to cancer treatment.

Approach	Method	Reference
Delivery of oncolytic viruses (OVs)
	Intracellular-delivering Salmonella to carry OVs into cancer cells	[148]
Immune system regulating properties
	Intracellular infection of melanoma	[149]
Combination treatments
	Deliver cytolysin to tumours	[150,151]
	Salmonella bioimaging to target metastatic tumours	[146]
Genetically engineered Salmonella
	Immune cytokines	[147]
	Anti-cancer drugs	[152]
	Expressing enzymes to activate anti-cancer pro-drugs	[153]
	Expressing tumour-specific antibodies	[154]
	Expressing oncogene silencing RNA	[155]

). The oncolytic capacity of Salmonella and its ability to reduce tumour growth are clearly an advantage, but these tumours can grow back following treatment; therefore, attention has been given to direct killing by immune molecules [145]. The outer membrane vesicles (OMVs) of Salmonella (reviewed in detail [146]) may have potential over attenuated strains in terms of safety, and their immunogens can activate the immune system. The intrinsic mechanisms of Salmonella crucial in combating cancer are still being elucidated (reviewed in detail [147]).

The ability to target tumour regions is important [152,153]. The natural toxicity of S. Typhimurium towards tumours and its ability to cope in an anoxic environment coupled with cytokine involvement make it the ideal tool [154,155]. Salmonella, protected as it is by the immune system [156,157,158], may provide efficacy in terms of an adjuvant effect or in tumour colonisation [159]. The balance of safety and therapeutic efficacy is crucial and, as mentioned previously, has resulted in two prominent Salmonella vector strains, VNP20009 and A1-R [160]. Over-manipulation of VNP20009 has resulted in it being less effective in some instances [141], with the loss of anti-tumour properties, whereas the variant A1-R has shown potential in murine tumours and patient-derived orthotropic xenografts [143,144]. In contrast, SF200, which contains mutations affecting LPS and flagella synthesis has been shown to increase immune stimulation [143,144]. However, despite these advances, challenges remain in the selection of an appropriate model coupled with the approaches to developing Salmonella as a tool against cancer. Despite the number of high-quality publications in the area [130], evidence of efficacy in humans is limited to a few clinical trials where there has been some success (for an extensive review, see [161]), including an IL-2-expressing, attenuated S. Typhimurium in the unresectable hepatic spread of a solid tumour (https://www.clinicaltrials.gov/study/NCT01099631, [162], accessed on 28 October 2024).

Salmonella shows promise as a live attenuated agent, with workers investigating a range of effectors including interleukins and cytokines. The anti-tumour effects of S. Typhimurium expressing proinflammatory cytokines and monocytes have also been identified in melanoma-bearing mice. More recently, it has been shown that tumour antigen coating promoted antigen-specific CD8+ T immunity with increased Type 1 interferon anti-tumour activity [163], indicating that Salmonella does harness immunological processes, albeit indirectly. Zha and colleagues [164] found that AvrA suppressed intestinal inflammation and inhibited the secretion of cytokines IL-12, IFN-γ and TNF-α. AvrA expression in Salmonella enhanced its invasiveness. Liver abscesses and Salmonella translocation to the gallbladder were observed. Further studies have emphasised the importance of AvrA in intestinal inflammation, bacterial translocation and chronic infection associated with colon cancer in vivo. That being said, there are also some disadvantages to the BMCT platform in cancer therapy, including safety issues. Salmonella-mediated cancer therapy (SMCT) has faced specific problems, with most phase I clinical trials having unsatisfactory outcomes. For a successful clinical outcome, the intrinsic and extrinsic factors associated with tumours [130] and the ability to clear Salmonella from the area around tumours are crucial to the success of SMCT in clinical applications. Moreover, efforts to balance attenuation may increase the risk of septic shock, and pre-exposure to these pathogens in the environment may have induced immunity against these pathogens, so vector-specific immunity may ultimately interfere with actual vaccine delivery.

5.1. Drug with a Pro-Drug Activating Enzyme

This innovative approach entails a systemic pro-drug being converted to a cytotoxic drug and resulting in a localised anti-tumour effect; for instance, CD-20-targeting antibodies [165] and an engineered VNP20009 [166]. S. Typhimurium expressing HSVtk showed GCV-mediated, dose-dependent suppression of tumour growth in mice bearing B16F10 melanoma [167,168]. Using S. Infantis as an HSVtk-expression vector has resulted in similar anti-tumour activity in a rat model of bladder cancer [169], with Zhou and co-workers [170] showing the suppression of metastasis is a range of tumour models.

5.2. Induction of Cell Death

S. Typhimurium expressing FasL can initiate an apoptotic signal in Fas-sensitive cells [171,172] and has been shown to inhibit tumour growth in breast and colon carcinoma models [173], and recombinant VPN20009 can extend survival in tumour-bearing mice by inducing more apoptosis [174]. Salmonella incorporating hypoxic-inducible NirB and radiation-inducible RecA reduced tumour growth and prolonged survival [175], with Chen and co-workers [176] showing that triptolide (TPL) could lead to enhanced apoptosis in B16F10 cells in vitro. Attenuated S. Typhimurium carrying a cytotoxic protein (HlyE) has been shown to increase tumour necrosis and growth retardation [177]. Moreover, Guan and co-workers [178] showed that using an apoptin-expressing S. Typhimurium extended tumour growth retardation and reduced micro-vessel density systemically. More recently, SPI-1 and SPI-2 have been shown to affect host inflammatory pathways to induce cytotoxicity and cellular apoptosis in RAW264.7 macrophages [179].

5.3. Post-Translational Gene Silencing

Salmonella may be advantageous in overcoming some of the difficulties involved in gene silencing in eukaryotic cells. Small interfering RNA (siRNA) breaks down quickly in the extracellular environment [180,181], restricting entry to eukaryotic cells. An attenuated S. Typhi for multidrug-resistance gene (MDR1) siRNA showed the potential of this approach [182], with Xiang and co-workers [181] showing the transfer of RNAi effector molecules from bacterial vectors to mammals in vivo. Furthermore, work using S. Typhimurium against tumour transcription factor STAT3 showed that combined therapy performed better than the vector alone [183], with further work [184] illustrating the silencing of targeted gene VNP (PhoP/Q-) with the release of an shRNA-expressing plasmid into the cytoplasm of host cells.

Delayed tumour growth and prolonged survival in a murine melanoma model were observed in the development of an orally administered S. Typhimurium incorporating a short-hairpin RNA targeting bcl-2. S. Typhimurium transformed with an shRNA plasmid was effective against indoleamine 2,3-dioxygenase 1 (IDO) in tumour-bearing mice. Silencing host IDO expression [185] resulted in intra-tumoral cell death with significant tumour infiltration by polymorphonuclear neutrophils. This silencing also enhanced S. Typhimurium colonisation, potentially mediating the immune response to S. Typhimurium. An S. Typhimurium VNP20009 targeting IDO [186] was shown to activate innate immunity and mitigate colorectal cancer growth. An S. Typhimurium strain delivering antisense RNA was also effective in silencing RCAS1 mRNA expressed in cancer cells and tumour tissues [187]. Hence, some promising RNAi-mediated anti-tumour therapeutic strategies have been developed, and further investigation will expand potential applications.

6. Salmonella as an Anti-Viral

The potential of bacteria-based therapies and Salmonella in particular as anti-virals gained renewed interest during and following the SARS-CoV-2 pandemic. Salmonella has some natural advantages over other therapy platforms in terms of its potential as an oral vaccine and the advantages that might afford in developing countries. Bacteria-based orally administered vaccines have attracted attention as a safer vaccine technology compared with vascular or intramuscular injection approaches (reviewed in detail [161]). Karpenko and colleagues [188] tested an orally administered S. Enteriditis vector (E23) harbouring genes encoding immune-dominant HIV antigens in mice, generating both humoral and T-cellular responses against HIV-1, and Chen and colleagues [189] showed that a plasmid carrying attenuated Salmonella could induce the production of HPV16-L1 antibodies and IL-2 and INF-y in mice serum. Furthermore, Yoon and colleagues [190] developed engineered spike proteins of SARS-CoV-2 which triggered SARS-CoV-2-specific antibody production and activated COVID-19-specific cellular immune responses. This orally administrated Salmonella BRD509 was shown to have reduced cytotoxic effects in vivo. Hence, these in vitro and in vivo data suggest that Salmonella strains may represent a valuable new delivery system for COVID-19 vaccines. Notwithstanding the potential benefits, it is also important to be mindful that Salmonella has been seen to interact with COVID-19 infections with an as yet uncharacterised role in long COVID-19 [191]

7. Future Developments

Combatting multidrug-resistant Salmonella in the context of an ever-dwindling number of effective antibiotics will be a focus of future work. Current work on therapeutic vaccines, particularly mRNA delivery, would appear to hold particular promise. Increasing the efficacy of current antibiotics [192,193] together with the development of prophylaxis or therapeutic vaccines to fight Salmonella infection will be paramount. There is the potential to develop therapeutic vaccines, particularly in terms of new and emerging variants [194], their zoonotic origin [195,196] and outbreaks [197]. A stumbling block is the lack of translation of the published research, so perhaps a consolidation of platforms would speed up the development of real-world outcomes. Vaccine development is one area where this consolidation of platforms and adoption of BMCT would be advantageous [133]. An increased understanding of biofilms, particularly in the external environment, could be key not only in the control of Salmonella but also in the spread of antibiotic resistance. There is no doubt that climate change will be the most challenging for society in the near to long term. One of the most significant challenges will be how research will address potential increases in newly emerging variants in the face of Salmonella fitness and antibiotic resistance in low- to middle-income countries in the Global South.

8. Conclusions

While researchers continue to push forward their ideas on the clear advantages of Salmonella as a worthwhile tool in the fight against both infectious and non-infectious diseases, the reality of disease-causing Salmonella coupled with the rise in multidrug resistance continues to confound them. Ultimately, a breakthrough application in targeted molecular tools will be required to take full advantage of this remarkable organism.