Non-Typhoidal Salmonella: Decoding Mechanisms for Effective Disease Control

1.1 Overview and prevalence

Salmonella enterica (S. enterica) is a Gram-negative facultative intracellular bacterial pathogen that is a member of the Enterobacteriaceae family [1]. S. enterica consists of more than 2600 serovars of which S. enterica subspecies enterica is composed of serovars like S. Typhi that are host-adapted serovars restricted to humans, while other serovars like S. Enteritidis and S. Typhimurium are broad host range serovars affecting multiple species [2]. Typically, S. Typhi human infections are characterized by typhoid fever and invasive disease [3]. However, non-typhoidal Salmonella (NTS) species are one of the leading causes of self-limiting bacterial gastrointestinal disease in humans [4]. Interestingly, NTS species have been associated with invasive disease (iNTS) in sub-Saharan Africa that manifests as bacteremia leading to a case fatality rate of 20.6% [5]. The consumption of contaminated poultry products has been attributed to serve as the main source of human NTS infections, though fresh produce has emerged as a significant contributor in recent outbreaks [6]. NTS human infections result in an estimated annual global burden of 93.8 million cases leading to 155,000 deaths [7]. According to the Centers for Disease Control and Prevention (CDC), NTS species are responsible for an estimated 1.35 million cases of Salmonellosis annually, making them the second most common cause of foodborne illness [8]. This accounts for approximately 19,336 hospitalizations, a loss of 17,000 quality-adjusted life years (QALYs), and an estimated USD $3.3 billion in annual healthcare costs and productivity losses [9]. Additionally, NTS serotypes Enteritidis and Typhimurium are the predominant serotypes associated with human infections in Africa, Asia, Europe, Latin America and the Caribbean, and in North America [10].

1.2 Sources and transmission

According to data from the CDC’s National Outbreak Reporting System (NORS), 3115 outbreaks were reported from 2009 to 2023 in the United States that were attributed to NTS species, resulting in 70,458 illnesses, 11,866 hospitalizations and 108 deaths [11]. Moreover, states with the highest number of Salmonella outbreaks in the U.S. over the 14-year period included Minnesota (324), Texas (356), Ohio (363), Illinois (378), California (416), New York (436) and Pennsylvania (436) (Table 1) [11]. Over 75% of the described outbreaks were linked to foodborne transmission, originating from a variety of sources including seeded vegetables, chicken, fruits, pork, beef, eggs and other produce [12]. Additionally, recent Salmonella outbreaks in 2024-2025 (Table 2) include those that are associated with charcuterie meats, fresh basil, backyard poultry, pet bearded dragons, cucumbers, small turtles, eggs, geckos, cucumbers, pistachio cream, and frozen sprouted beans [13], implying that Salmonella surveillance and control are critical steps in ensuring a safer food supply that will ultimately lead to lower human infections [14, 15, 16, 17]. Taken together, the need to further understand molecular mechanisms of Salmonella pathogenesis and virulence are vital since the incidence of NTS and iNTS, along with the associated Multidrug Resistance (MDR) are on the rise [18].

2.1 Introduction to NTS virulence mechanisms

NTS serovars, such as S. Typhimurium and S. Enteritidis, are major causes of foodborne illness worldwide, typically resulting in self-limiting gastroenteritis in immunocompetent hosts. However, in vulnerable populations, including infants, the elderly, and the immunocompromised, NTS can breach the intestinal barrier, resulting in invasive bloodstream infections [5]. The pathogenesis of NTS involves a coordinated series of virulence mechanisms that enable intestinal invasion, immune evasion, and intracellular survival [19]. These mechanisms are largely encoded on chromosomal Salmonella pathogenicity islands (SPIs) and serovar-specific virulence plasmids, which together orchestrate the bacterium’s ability to colonize, invade, and persist within the host [20, 21]. Therefore, an in-depth understanding of molecular virulence determinants is crucial for developing effective strategies aimed at controlling this pathogen.

2.2 Salmonella pathogenicity islands

SPI are clusters of genes coding for virulence factors [22]. These operons are distinct from other regions of DNA due to their high content of Cytosine and Guanine [23]. The proteins coded for within these operons facilitate the survival of the bacterium within various hosts and environments. Twenty-four such SPIs have been identified in Salmonella spp. (Table 3), with SPI-1 and SPI-2 having been the best characterized [24].

SPI-1 is conserved across all Salmonella spp. [25]. It encodes a Type 3 Secretion System (T3SS) and several effector molecules which are then translocated into host cells via a needle-like injectisome to help invade the epithelium [26]. Major effector molecules include SipA, which plays a role in actin rearrangement in host cells, SipB and SipC, which help with adhesion to epithelial cells [26, 27]. AvrA helps prevent apoptosis of infected macrophages to allow bacterial dissemination in the host [28].

SPI-2 is found in Salmonella enterica that is primarily expressed once the bacterium is inside the host cell within a membrane bound vacuole [29]. It encodes a second T3SS (T3SS-2), as well as secreted effector proteins which play a major role in bacterial survival and replication within host cells [30]. These effector proteins help regulate the positioning of the Salmonella-containing vacuole (SCV), modulating cytoskeletal rearrangements, inhibiting the host inflammatory cytokines and delaying macrophage cytotoxicity [31].

2.3 Type 3 secretion systems

The T3SS is a nanomachine complex found in a number of Gram-negative bacteria, including Yersinia, Shigella, Enteropathogenic Escherichia coli, and Salmonella [32]. These injectisomes allow bacterial effector proteins to be injected directly into host cells [33]. Typically, the T3SS is comprised of a translocon, tip complex, needle, basal body, and cytoplasmic ATPase complex [34]. The basal body is made up of two rings, which help anchor the complex to the inner and outer membrane of the bacterial cell wall [35], while the needle is a hollow polymeric rod that allows effector proteins to be secreted [36]. Additionally, the tip complex, on top of the needle, interacts with the host cells [37] and the translocon forms pores in the host cell membrane to allow for the passage of effector molecules [38]. Salmonella enterica possesses two distinct T3SS, with T3SS-1 being functional during the initial invasion stage and T3SS-2 aiding in intracellular survival within the SCV. However, both are structurally analogous (Table 4) [39].

2.4 Virulence plasmids

Virulence plasmids are extrachromosomal DNA elements found in certain serovars of Salmonella, including Choleraesuis, Dublin, Enteritidis, and Typhimurium [40]. The gene products of these plasmids are not essential for the initial phase of intestinal colonization; however, they have been implicated in enhancing the intracellular survival of the pathogen [41]. All serovars with the plasmid carry the highly conserved, spvRABCD locus, yet the plasmid itself varies in size across the different serovars, ranging anywhere from 50 to 95 kb [42]. spvR encodes a transcriptional regulator that upregulates the operon in the intracellular environment while SpvA downregulates the operon. Taken together, the action of both of these regulators allows for tight transcriptional control [43]. SpvB is a cytotoxin with ADP-ribosyltransferase activity that blocks host cell actin polymerization, aiding in the proliferation of Salmonella within macrophages [44]. SpvC blocks MAP kinase signaling pathways and has potent anti-inflammatory effects shown to promote bacterial dissemination in murine models [45]. Additionally, SpvD is a hydrolase that inhibits the NF-κB pathway [46].

3.1 Introduction and scope of the problem

Antibiotic-resistant Salmonella presents a pressing challenge in modern food safety and public health [47]. The magnitude of this problem is significant and on the rise. In 2013, the CDC published an antibiotic resistance threat list, which categorized both NTS and Salmonella Typhi as “serious threats”. That year, drug-resistant NTS and Salmonella Typhi accounted for 100,000 (8% of total NTS infections that year; 3% resistant to ciprofloxacin, 3% resistant to ceftriaxone; 5% resistant to 5 or more antibiotics) and 3800 (67% of total infections that year; 67% resistant to ciprofloxacin) infections across the United States, respectively [48]. In just 6 years, these numbers rose to approximately 255,000 (9% resistance to ciprofloxacin in 2020) and 6000 (85% resistance to ciprofloxacin in 2020), respectively [49].

However, the global nature of this crisis extends far beyond American borders. In Bangladesh, the combined prevalence of Salmonella in livestock and poultry is 37%, with tetracycline showing the highest resistance rates at 81% [50]. In China, a comprehensive meta-analysis revealed that the pooled prevalence of Salmonella isolated from pork was 17%, with high antibiotic resistance rates observed for tetracycline (68%), sulfisoxazole (65%), ampicillin (43%), streptomycin (42%), and sulfamethoxazole (42%) [51]. Table 5 displays an increasing trend in Salmonella antibiotic resistance to commonly used antibiotics in humans, livestock, poultry, and pork.

3.2 Origins and drivers of resistance

The development of antibiotic resistance in Salmonella is linked to agricultural practices and the extensive use of antibiotics in food animal production. The global demand for high-value animal protein has driven countries to adopt highly profitable production systems that rely heavily on antimicrobials to maintain productivity and animal health [53]. The intensive nature of modern animal production systems has created conditions that promote the development and dissemination of antimicrobial resistance. Antibiotics are routinely employed in livestock operations for multiple purposes: growth promotion, prophylactic prevention of disease, therapeutic treatment of infections, and metaphylactic control of disease outbreaks [54]. The scale of this usage is remarkable, with previous studies indicating that antibiotic consumption for non-therapeutic purposes in poultry, swine, and cattle outweighs human antibiotic use by several-fold [55]. This approach, while economically advantageous, has contributed significantly to the antimicrobial resistance crisis.

3.3 Molecular mechanisms of resistance

The molecular basis of antibiotic resistance in Salmonella is complex and multifaceted. Resistance is primarily driven by horizontal gene transfer through plasmids, integrons, and transposons [56]. These mobile genetic elements carry genes that encode various antibiotic resistance mechanisms, including efflux pumps, mutations in target genes, and the production of β-lactamases such as TEM, SHV, and CTX-M [57, 58, 59]. Major resistance genes have been identified in various Salmonella serovars isolated from the food supply, including resistance to β-lactams, extended-spectrum β-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and chloramphenicol [60]. This expanding resistance profile makes treatment of Salmonella increasingly challenging by limiting therapeutic options for clinicians. A particularly concerning aspect of Salmonella resistance is the role of biofilm formation. Biofilm production is a major factor contributing to antibiotic resistance, as it can directly and indirectly shield bacteria from the effects of antibiotic action [61]. Salmonella can form biofilms on both abiotic and biotic surfaces, including gallstones and epithelial cells, leading to persistent infections and reinfection cycles [61]. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of antibiotics can increase 100- to 1000-fold due to biofilm formation, making treatment significantly more difficult [62].

3.4 Environmental reservoirs and transmission pathways

The environmental dimension of antibiotic resistance adds another layer of complexity to the Salmonella resistance problem. Environmental microorganisms serve as excellent sources of antimicrobial resistance genes, collectively termed the ‘environmental resistome’ [63]. This resistome plays a critical role in transferring antimicrobial resistance to pathogenic microorganisms and directly affects human health by entering the food chain [63]. The transmission pathways for antibiotic-resistant Salmonella are numerous and interconnected. Lactic acid bacteria have been identified as carriers of resistance genes that may serve as reservoirs for enteropathogenic bacteria like Salmonella [54]. Erythromycin, vancomycin, and tetracycline resistance genes have been identified in lactic acid-producing bacteria isolated from fermented dairy products, sausages, and raw meat products, including poultry, beef, and pork [64]. Foodborne pathogens can enter farms through various sources, including contaminated water, fecal matter, personnel, equipment, vehicles, insects, rodents and pets [65]. The movement of portable equipment and vehicles can act as vectors for carrying pathogens to farms and slaughterhouses [66]. Furthermore, antibiotic-resistant bacteria spread through washing systems, floors, flush systems, and holding pens, potentially ending up in animal carcasses [67]. Particularly noteworthy is the finding that antibiotic-resistant pathogens such as S. Typhimurium have been recovered from swine and poultry housed in antibiotic-free production systems, highlighting the important role of ‘environmental ressistomes’ in spreading resistance [63, 68].

3.5 Regional variations and emerging patterns

The prevalence and patterns of NTS antibiotic resistance show significant regional variations, reflecting differences in agricultural practices, regulatory frameworks, and surveillance systems. Studies from commercial poultry farms revealed concerning levels of multidrug resistance with Salmonella isolates showing resistance to streptomycin (30.9%), gentamicin (12.6%), sulfadimethoxine (20.9%), tetracycline (13.9%), and trimethoprim-sulfamethoxazole combination (8.6%) [52]. Among these isolates, 20% were resistant to three or more antibiotics, while 67% of S. Heidelberg and 54% of S. Kentucky isolates showed resistance to five or more antibiotics [52]. The global trends in Salmonella antibiotic resistance are concerning. As previously stated, the United States experienced a doubling in the prevalence of drug-resistant Salmonella in less than 6 years [48, 49]. Similarly, in Bangladesh, meta-analysis revealed a significant positive correlation between Salmonella prevalence and time [50]. A recent Chinese meta-analysis also detected a growing trend in Salmonella prevalence in pork over time [51].

3.6 Public health implications and future outlook

The threat of antibiotic resistance has far-reaching consequences for human health and well-being, including increased healthcare costs, productivity losses, and higher susceptibility to other serious illnesses [69]. The long-term projections are sobering. If no further political measures are taken to address the spread of antimicrobial resistance, it is expected to cause over 10 million deaths per year by 2050, exceeding cancer-related deaths [70]. Given the limited pipeline for new antimicrobials, expert management of currently available drugs has become critical [53]. In response to these challenges, the World Health Organization (WHO), in collaboration with the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (WOAH), has declared antimicrobial resistance an issue of global concern that requires a holistic and multi-sectoral approach [53]. Regulatory agencies have begun implementing measures to address agricultural antibiotic use, with the Food and Drug Administration (FDA) issuing rules to phase out antibiotics from agriculture and require veterinary oversight for therapeutic antibiotic use in food animals and poultry [71].

3.7 Alternative approaches and future directions

The recognition of this growing antibiotic resistance challenge has sparked interest in alternative approaches to pathogen control. Alternatives such as probiotics, prebiotics, and phytobiotics are being tested against drug-resistant pathogens due to their broad spectrum of antimicrobial activity [72]. Ideally, these alternatives should be non-toxic, not result in residue buildup, palatable to animals, stable in the gut, capable of augmenting beneficial flora, and effective at inactivating harmful pathogens [54]. Another promising method of combating antimicrobial resistance is the development of efflux pump inhibitors. Inhibition of efflux systems and interruption of biofilm formation may reverse antibiotic resistance in bacteria and potentiate antibiotic efficiency [61]. Additionally, research has shown that the ability of pathogens to form biofilms decreases after deletion of various efflux pumps, including acrD, acrEF, emrAB, macAB, mdfA, mdsABC, mdtBC, mdtK, and tolC from Salmonella enterica serovars [73].

3.8 Challenges with antibiotic resistance

The challenge of antibiotic-resistant Salmonella in the food supply represents a complex, multifaceted problem that requires coordinated global action. Extensive use of antibiotics, combined with an intricate web of environmental reservoirs and transmission pathways, has created a situation where resistance continues to grow despite various control efforts. The regional variations, yet consistent global rise in resistance patterns, underscore the need for global action tailored to local agricultural practices and regulatory environments. Moving forward, success in controlling Salmonella antibiotic resistance will depend on the implementation of comprehensive strategies that combine judicious antibiotic use, enhanced surveillance systems, improved biosecurity measures, and the development of effective alternatives to traditional antimicrobials [60]. The stakes continue to rise as the continued spread of resistance threatens food supply safety and the effectiveness of antimicrobial therapy for treating human infections.

4.1 Surveillance and control

Strong surveillance and response systems are key to managing NTS infections around the world. In the U.S., the CDC created a PulseNet network that helps track and respond to foodborne outbreaks by connecting public health labs across the country. Using tools like whole genome sequencing (WGS) and pulsed-field gel electrophoresis (PFGE), PulseNet can quickly identify clusters of related Salmonella cases. This allows health officials to trace sources, contain outbreaks, and implement targeted public health measures more efficiently [74, 75].

Working alongside PulseNet is FoodNet, the Foodborne Diseases Active Surveillance Network, which tracks trends in foodborne illnesses at selected sites across the country. FoodNet plays a key role in providing incidence data, assessing the impact of food safety policies, and detecting emerging pathogens. Together, PulseNet and FoodNet form the foundation of the country’s efforts to detect, investigate, and reduce NTS infections, shaping national public health strategies [76]. Globally, similar surveillance efforts are driven by international collaborations like the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC). Together, they publish annual zoonosis reports that track Salmonella in food, animals, and humans across Europe [77].

Food recalls are one of the most visible outcomes of strong surveillance systems. When NTS is detected in food products, agencies such as the FDA or USDA can issue recalls to stop the spread and protect public health [78]. Early detection and a coordinated response play a key role in limiting the impact of outbreaks. Beyond removing contaminated products from the market, these recalls also highlight gaps in food safety practices, which encourage broader improvements across the supply chain [79].

Another key part of controlling NTS infections is the careful use of antibiotics, especially for high-risk groups. While most NTS infections resolve on their own, treatment is recommended for people who are more vulnerable, such as infants, older adults, and those with weakened immune systems [80]. Antibiotics may be needed when symptoms include high fever, persistent diarrhea, bacteremia, or other signs of a more serious, invasive infection [81]. However, the growing issue of antimicrobial resistance is a major concern. Multidrug-resistant Salmonella strains are on the rise, complicating treatment and reinforcing the need for responsible antibiotic use [82, 83].

4.2 Prevention and vaccination

Public education and strong food safety regulations play crucial roles in preventing NTS infections. Key strategies include promoting safe food handling and hygiene practices, as well as enforcing regulations throughout the food production and distribution process. These efforts aim to reduce infection rates by limiting the spread of the bacteria at both the individual and system-wide levels [84].

Public education focused on hygiene and safe food preparation serves as a critical method of primary prevention by reducing the risk of the initial NTS infection and is represented as the bottom tier of the prevention pyramid in Figure 1. Handwashing is a simple but powerful tool, especially given the zoonotic and fecal-oral transmission of Salmonella. The CDC’s Life is Better with Clean Hands and #KeepHandsClean campaigns are a national effort aimed at raising awareness about the importance of handwashing at homes and in public settings [84]. By offering clear, practical guidance on when and how to wash hands, the campaign helps prevent not just Salmonella infections but a range of gastrointestinal and respiratory illnesses, including but not limited to Norovirus, Shigella, E. coli, Rotavirus, Influenza, and the common cold [85, 86].

In addition to hand hygiene, everyday food safety practices at the consumer level are essential for preventing NTS infections. These include cooking poultry and eggs to safe internal temperatures, avoiding raw or undercooked high-risk foods, preventing cross-contamination in the kitchen, and keeping perishable items properly refrigerated, consistent with CDC’s Clean, Separate, Cook & Chill framework [87]. Moreover, public health messages promoting these habits are disseminated via public service announcements, food packaging labels, and local education efforts.

Educational campaigns in schools, childcare centers, and food service establishments build lifelong food safety habits. CDC partners with initiatives such as the Partnership for Food Safety Education’s Fight BAC! campaign, which delivers consumer-focused food safety education grounded in the “Clean–Separate–Cook–Chill” model [88]. Additionally, educational campaigns in schools, childcare centers, and food service establishments play a vital role in building lifelong food safety habits. In food service environments, certification programs are often required by local health departments to educate workers on Salmonella prevention, safe food handling, and sanitation [89]. These training courses emphasize the importance of personal hygiene, proper temperature control, and thorough cleaning practices, all of which are critical for interrupting the transmission of foodborne pathogens. Numerous studies confirm that following basic hygiene and food-handling guidelines significantly lowers the risk of NTS transmission [86, 89].

At the policy level, food safety regulations are the foundation of large-scale prevention efforts. One of the most important frameworks is the Hazard Analysis Critical Control Point (HACCP) system. Originally developed for NASA’s space food program, HACCP has since become a global standard and is now required across many parts of the food industry. The system focuses on controlling critical points in the production process where contamination is most likely to occur. HACCP is widely implemented in poultry processing facilities to reduce microbial contamination and maintain consistent safety standards [90, 91]. In addition, agencies like the USDA and FDA enforce strict Salmonella performance standards in meat and poultry processing facilities. Regular inspections are carried out to monitor compliance, including microbial testing, audits of sanitation practices, and reviews of facility HACCP plans. The use of WGS data from surveillance programs like PulseNet, help these efforts by enabling regulators to trace outbreaks and quickly identify weaknesses in food safety systems [76, 87]. In summary, secondary prevention efforts, as depicted by the second tier of the prevention pyramid in Figure 1, includes early detection and rapid response measures such as PulseNet’s WGS surveillance, HACCP reviews, and public health education to detect and address risks early.

Finally, tertiary prevention, represented as the third tier of the prevention pyramid in Figure 1, focuses on the clinical management of NTS infections and the prevention of complications such as dehydration, bacteremia, endovascular infections, and osteomyelitis. It also includes antimicrobial stewardship, particularly in cases involving multidrug-resistant strains (as discussed in the Salmonella Antibiotic Resistance section of this chapter). Tertiary prevention is especially important for high-risk populations, including immunocompromised individuals, such as those with HIV, and groups at risk for invasive infection, such as neonates under 3 months of age, adults over 50 with suspected atherosclerosis, and individuals with cardiac conditions, immunosuppression, or significant joint disease [86].

Efforts to control NTS through public education and food safety regulations have led to important progress. However, these measures alone are unlikely to achieve sustained global control, especially in high-burden areas like sub-Saharan Africa where vulnerable populations face the greatest risk [92]. In these regions, vaccination is emerging as a critical next step, particularly given the growing threat of multidrug-resistant NTS strains that limit treatment options [93]. Although no NTS vaccine has been officially licensed for human use as of 2025, several promising candidates are moving through the development pipeline [93]. These vaccines, their serovar coverage and stage of development are shown in Table 6.

Beyond these, innovative strategies such as subunit vaccines targeting conserved proteins like InvH, small extracellular vesicle-based platforms, and antigens identified through reverse vaccinology have demonstrated encouraging results in animal models [100, 101]. While these approaches show promise, they face practical hurdles including limited strain coverage, high production costs, and the challenge of integrating new vaccines into existing immunization programs. Addressing these barriers will be essential to ensure that future NTS vaccines are both effective and accessible where they are needed most.