NutriClay Zn Harbors Bioavailable Zinc and Suppresses Multidrug-Resistant Salmonella Choleraesuis

Abstract

Pharmacological doses of zinc oxide (ZnO), far in excess of the nutritional requirement for zinc, are commonly added to weanling swine feed to suppress enterotoxigenic bacteria and thereby support piglet weight gains. However, excessive ZnO in the diet has come under scrutiny due to concerns that excreted zinc may accumulate beyond safe levels within topsoil and water supplies, as well as foster antibiotic drug resistance in bacterial pathogens that could then infect livestock and/or humans. Indeed, multidrug-resistant (MDR) Salmonella have been isolated from swine feces, focusing attention on new technologies to protect swine (and ultimately humans) while reducing dietary zinc toward the nutritional requirement. We hypothesized that NutriClay^Zn (an edible Generally Recognized As Safe substance consisting of low-dose ZnO immobilized on montmorillonite clay) could suppress MDR Salmonella, provide bioavailable zinc, and serve as a novel feed ingredient for weanling swine. NutriClay^Zn dose–response efficacy and thermal stability were assessed in cultures of MDR Salmonella Choleraesuis. Dissociated elemental zinc was measured under simulated gastrointestinal conditions. NutriClay^Zn consumption was assessed via ad libitum feeding, with bioavailable zinc calculations according to weanling swine treatment group intakes. Salmonella Choleraesuis (resistant to quinolone, sulfonamide, and aminoglycoside antibiotics) was susceptible to growth inhibition by heat-stable NutriClay^Zn. Under simulated gastrointestinal conditions, 22.8 µg of elemental zinc were released per milligram NutriClay^Zn. Furthermore, the rate at which weanling swine consumed NutriClay^Zn-containing feed (0.6 kg/day) supports fulfillment of the nutritional zinc requirement. These findings encourage future MDR Salmonella pathogen challenge trials, designed with NutriClay^Zn replacing pharmacological ZnO for control of enterotoxigenic bacteria in swine, as well as potentially eliminating the zinc component of mineral premixtures that are currently added to feed for nutritional purposes.

Introduction

Nontyphoidal Salmonella infections lead to 1.35 million cases of foodborne illness each year in the United States, including 26,500 hospitalizations and 420 deaths (CDC, 2023). These illnesses, often characterized by severe gastroenteritis (Saphra and Winter, 1957), come with a staggering 4.1 billion dollar negative impact on the U.S. economy in the form of lost wages and premature deaths (Hoffman and Ahn, 2021; Hoffman et al., 2015). Although foods are vulnerable to contamination at many points along the supply chain from farm to consumer (CDC, 2024), Salmonella carried by swine (and found in pork products) are thought to be responsible for 8–20% of human salmonellosis cases across the United States and Europe (Berri et al., 2020; USDA, 2020). Moreover, among 2659 Salmonella serotypes (Monte and Sellera, 2020), Salmonella enterica serotype Choleraesuis (hereinafter referred to as Salmonella Choleraesuis) stands out as having its primary reservoir in production swine (Ferrari et al., 2019), while potentially causing sickness in both the porcine host and the human consumer of pork (Soliani et al., 2023). Indeed, Salmonella Choleraesuis has been linked to invasive bloodstream infections and sepsis in humans, with endocarditis and osteomyelitis as the most common inflammatory tissue manifestations (Bäumler and Fang, 2013; Saphra and Winter, 1957; Threlfall et al., 1992).

Historically, prophylactic antibiotics were used in swine feed to prevent bacterial infections and thereby promote animal growth. However, such indiscriminate use of antibiotics is no longer allowed in the United States and Europe because this practice may foster the development of antibiotic-resistant strains of bacteria. Ensuing infections in humans and/or animals would then be especially difficult to treat under existing antibiotic regimens (Armbruster and Roberts, 2018; Dewulf et al., 2022). Strains of Salmonella Choleraesuis (Chiu et al., 2005), as well as other pathogenic microbes derived from swine (Bearson, 2022; Rodrigues et al., 2020), are known to express various forms of resistance to antibiotic drugs. Over the past 5 years, Salmonella Choleraesuis has been identified in human blood samples and found to be resistant to colistin, ampicillin, cephalosporins, sulfonamides, chloramphenicol, ciprofloxacin, nalidixic acid (quinolone), fluoroquinolone, streptomycin, and/or tetracycline (Dos Santos et al., 2020; Oransathid et al., 2022).

Following the regulatory bans on prophylactic antibiotics, industry began relying on pharmacological doses of zinc oxide (ZnO) to prevent infections and promote the growth of weanling production animals. ZnO (as a free chemical) suppresses the propagation of various bacteria in vitro (Pasquet et al., 2014) and has proven generally safe in vivo at levels up to 3000 ppm in feed for weanling pigs (Burrough et al., 2019). Although feed admixtures containing ZnO remain allowable in the United States, pharmacological ZnO (beyond the animal’s nutritional requirement for zinc) has recently been banned in Europe. The European ban was primarily due to concerns that excreted zinc (within manure) could accumulate in topsoil and ultimately exceed environmental compartment safety limits (EMA, 2017). A secondary concern was that excess zinc, unabsorbed from the gastrointestinal tract and excreted in feces, could impact microbes in soil and groundwater and thereby contribute to the threat posed by antibiotic-resistant bacteria (EMA, 2017; Langer, 2022).

In light of concerns around both prophylactic antibiotics and pharmacological doses of free chemical ZnO, swine producers have incorporated enhanced biosecurity measures (Fangman and Zulovich, 2003), while researchers explore alternative interventions (e.g., zinc nanoparticles, probiotics, essential oils, polyphenols) aimed at preventing bacterial infections and supporting normal animal growth. Although some alternatives appear promising, they often do not match the efficacy and/or feasibility attributed to pharmacological ZnO in weanling swine diets (Bonetti et al., 2021). Given that the primary role of ZnO in the diet is to provide zinc nutrition, and antibacterial efficacy is only achieved at levels far above the nutritional requirement (Sampath et al., 2023), we hypothesized that a relatively low dose of immobilized ZnO could satisfy both requirements. Herein, we describe an innovative hybrid of ZnO and montmorillonite clay (NutriClay^Zn) that is shelf-stable at high ambient temperature yet liberates modest amounts of free zinc under physiological conditions. Moreover, NutriClay^Zn is lethal against Salmonella Choleraesuis, and it is readily consumed as a feed admixture by weanling swine.

Methods

Montmorillonite clay and chemical reagents

Sodium-rich montmorillonite (MMT) clay originated from the Newcastle Formation (State of Wyoming, USA), and was curated by The Clay Minerals Society (Chantilly, Virginia, USA) with cation exchange capacity of 76.4 mEq/100g. Unless otherwise noted below, chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

NutriClay^Zn synthesis

NutriClay^Zn was synthesized using Generally Recognized As Safe (GRAS) ingredients via a four-step process, as previously described (Jackson et al., 2025). All experiments below were carried out with NutriClay^Zn finished by mortar and pestle to a particle size of <212 µm.

Bacterial culture and antimicrobial susceptibility

The isolated Gram-negative swine pathogen, Salmonella Choleraesuis (var kunzendorf 3246 pp), was selected for resistance to novobiocin (NO) and nalidixic acid (NA) and placed into stock culture by the USDA Agricultural Research Service (College Station, TX, USA). Bacteria were maintained under standard protocols governing BSL-2 pathogen research within USDA. Salmonella Choleraesuis was assessed for antimicrobial susceptibility by Sensi-Disc^TM Antimicrobial Susceptibility Test Discs (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) according to kit instructions and accompanying interpretive chart (see Table 1). In experiments assessing NutriClay^Zn efficacy against Salmonella Choleraesuis, the bacteria were grown in Mueller–Hinton broth (pH 7.4), using stock cultures with starting concentration of 10² colony-forming units (CFU) per milliliter (mL). All experiments were carried out in 10 mL glass culture tubes (n = 3/group, plus concurrent controls devoid of NutriClay^Zn). Treatment group dosages of NutriClay^Zn were chosen based upon theoretical ZnO contents as a function of the NutriClay^Zn synthesis reaction at 50% of MMT cation exchange capacity. These dosages (2620 and 7850 ppm NutriClay^Zn) align, respectively, with 1 mM and 3 mM ZnO concentrations in the culture broth if the entire amounts of synthesized ZnO were released from MMT clay.

Table 1.

Multidrug Resistance Profile for Salmonella Choleraesuis

Antimicrobial agent	Antimicrobial mechanism of action	Zone diameter^a (mm)
Nalidixic acid	DNA gyrase	≤13
Sulfisoxazole	Folic acid metabolism	≤12
Streptomycin	Protein synthesis (30S)	≤11

National Committee for Clinical Laboratory Standards: Performance standards for antimicrobial disk susceptibility tests (BD BBL Sensi-Disc).

Upon NutriClay^Zn administration, samples were maintained at 37°C with agitation for 6 h, followed by serial dilutions in phosphate-buffered saline and plating on Brilliant Green Agar containing 25 µg/mL NO and 20 µg/mL NA. Plated cultures were allowed to incubate (37°C) overnight prior to colony counts and imaging. Each experiment produced colony count data for mean comparisons. In the case of experiments comparing two means, independent t-test (and accompanying test for homogeneity of variance) was performed. Alternatively, in experiments generating three group means, ANOVA was performed, followed by Tukey’s Studentized Range (Honestly Significant Difference) for pairwise comparisons. Statistical calculations were carried out using JMP IN (SAS Institute; Cary, NC). Significant differences were established at p < 0.05. Representative culture plates were imaged on a Bencher M2 Copy Stand with a mounted Nikon D7200 camera under 3200 Kelvin lighting conditions. All plate images were captured using identical exposure settings.

NutriClay^Zn dissociation under culture conditions

Zinc dissociation, away from NutriClay^Zn and into Mueller–Hinton broth, was measured using the above experimental design (except that no pathogenic bacteria were present in the broth, and n = 4/group), with analysis by inductively coupled plasma mass spectrometry (ICP-MS) as previously described (Jackson et al., 2025).

NutriClay^Zn dissociation under gastrointestinal conditions

Since NutriClay^Zn is intended for use as a feed additive, physiological media simulating the fed state were used, as previously described (Klein, 2010). Increasing dosages of NutriClay^Zn (2.62, 5.24, 7.85, 10.48, and 13.10 mg/mL) were exposed to Simulated Gastric Fluid (Ricca Chemical; Arlington, TX, USA) supplemented with 14 mg/mL whole milk powder (Hoosier Hill Farm; Fort Wayne, IN, USA) and 3.2 mg/mL pepsin from porcine gastric mucosa (≥2500 units/mg protein). Samples (n = 4/group) were allowed to incubate (37°C) with agitation for 2 h prior to pelleting of NutriClay^Zn by centrifugation and analysis of supernatants for elemental zinc by ICP-MS. Residual supernatants were siphoned and discarded. NutriClay^Zn pellets (n = 4/group) were then resuspended in Fed State Simulated Intestinal Fluid (Marques, 2004) and allowed to incubate (37°C) with agitation for 6 h prior to centrifugation and analysis of supernatants for elemental zinc by ICP-MS. Data from gastric and intestinal conditions were individually fit to Generalized Linear Models (using R statistical software) to predict bioavailable zinc across the dose range.

In vivo consumption of NutriClay^Zn

In accordance with an approved Institutional Animal Care and Use protocol (Agricultural Research Service, USDA, College Station, TX, USA), NutriClay^Zn consumption was assessed in weanling swine. Male piglets (21 days of age upon study initiation; n = 3/group) received Weaned Piglet Starter Ration [control mash diet, consisting of the following ingredients by weight: 34.36% corn, 15.06% soybean meal, 31.38% whey, 6.53% plasma proteins, 6.79% fish meal, 0.16% L-lysine hydrochloride, 0.04% L-threonine, 0.16% DL-methionine, 4.05% soy oil, 0.31% monocalcium phosphate, 0.50% limestone, 0.25% salt, 0.25% KSU swine vitamin premixture with phytase (Nutra Blend; Neosho, MO, USA), 0.15% TRA-MIN swine/poultry trace mineral premixture (Animal Science Products; Nacogdoches, TX, USA)], or the same diet supplemented with 3000 ppm NutriClay^Zn as admixture, for 10 days. All animals were housed within a climate-controlled facility, maintaining thermoneutral conditions based on piglet body weight. Animals were individually caged, yet side wall partitions (galvanized wire) allowed for socialization, with food and water ad libitum. Body weights were recorded upon study initiation (Day 0) and again on Day 9. Feed intake was assessed to determine whether the NutriClay^Zn admixture impedes consumption of the base (control) diet. Feed intakes and body weights (Control vs. NutriClay^Zn treatment) were compared by independent t-test (and accompanying test for homogeneity of variance) using JMP IN (SAS Institute; Cary, NC), with statistical significance requiring p < 0.05.

Results

NutriClay^Zn suppresses multidrug-resistant Salmonella Choleraesuis

As outlined in Table 1, Salmonella Choleraesuis was resistant to antibiotic drugs having three distinct mechanisms of action (i.e., DNA gyrase, folic acid metabolism, and protein synthesis). Conversely, upon exposure to NutriClay^Zn, growth of Salmonella Choleraesuis was dose-dependently and significantly (p < 0.05) suppressed. The minimum threshold antiproliferative dose was 2620 ppm, while 7850 ppm NutriClay^Zn in culture broth led to 98% bacterial growth reduction (Fig. 1). If all synthesized ZnO within NutriClay^Zn were released, these NutriClay^Zn dosages could generate, respectively, 65 and 196 ppm elemental zinc within bacterial culture broth. However, NutriClay^Zn dosages released only portions of their synthesized ZnO, generating 47 and 91 ppm elemental zinc within culture broth.

FIG. 1.

NutriClay^Zn suppresses propagation of Salmonella Choleraesuis. Bacterial cultures suspended in Mueller–Hinton broth were exposed to increasing concentrations of NutriClay^Zn that released up to 91 ppm elemental zinc within 6 h at 37°C (with agitation). Cultures were plated on Brilliant Green Agar and incubated at 37°C for 18 h prior to counting bacterial colonies. Data are expressed as colony-forming units per milliliter (CFU/mL) for documentation of logarithmic growth. Values are means ± SEM, n = 3. Means without a common letter differ, p < 0.05.

NutriClay^Zn is heat-stable, with consistent antibacterial efficacy and zinc dissociation

Following 70°C storage for 6 months, NutriClay^Zn remained effective, with the 7850 ppm concentration suppressing the growth of Salmonella Choleraesuis by 97% (Figs. 2 and 3). Moreover, the proportion of synthesized ZnO being released did not change, as both pre- and poststorage NutriClay^Zn samples generated 91 ppm elemental zinc within the culture broth.

FIG. 2.

NutriClay^Zn suppresses propagation of Salmonella Choleraesuis and is stable following thermal stress storage. Bacterial cultures suspended in Mueller–Hinton broth were exposed to either freshly synthesized NutriClay^Zn or NutriClay^Zn stored at 70°C for 6 months. Following 6 h at 37°C with agitation, cultures were plated on Brilliant Green Agar. Plates were then incubated at 37°C for 18 h, followed by counting of bacterial colonies. Data are expressed as colony-forming units per milliliter (CFU/mL) for documentation of logarithmic growth. Values are means ± SEM, n = 3. Means without a common letter differ, p < 0.05. Pre-Storage and Post-Storage NutriClay^Zn samples released equal mean amounts of elemental zinc (91 ppm) into the culture broth.

FIG. 3.

Salmonella Choleraesuis growth on Brilliant Green Agar following 6-h exposure of the bacteria to Mueller–Hinton broth only (Control) or Mueller–Hinton broth containing 7850 ppm NutriClay^Zn.

NutriClay^Zn liberates elemental zinc under gastrointestinal conditions

Release of zinc from NutriClay^Zn was measured under gastric and downstream intestinal conditions. Incremental dosages of NutriClay^Zn accounted for a range of potential maximum concentrations spanning from 1 mM ZnO (derived from 2.62 mg/mL NutriClay^Zn) to 5 mM ZnO (derived from 13.10 mg/mL NutriClay^Zn) within fed-state gastric fluid. As shown in Figure 4, release of zinc into gastric fluid followed a linear pattern, described by the equation y = 0.022(x) + 0.001. Residual zinc release into fed-state intestinal fluid was also linear [y = 0.001(x) − 0.003]. If given a 10 mg dose of NutriClay^Zn, 221 µg of elemental zinc would be released into gastric fluid over 2 h, with an additional 7 µg released into intestinal fluid over the subsequent 6 h.

FIG. 4.

NutriClay^Zn liberates zinc under gastric and intestinal conditions. Increasing dosages of NutriClay^Zn were exposed to (A) simulated fed-state gastric fluid (2 h, pH 2) followed by (B) simulated fed-state intestinal fluid (6 h, pH 7.4). Liberated zinc was measured by ICP-MS. Values are means ± SEM, n = 4. Data were modeled to generate linear equations for extrapolation of zinc release within the examined NutriClay^Zn dose range.

NutriClay^Zn is readily consumed by weanling swine

Swine consumed nutritionally complete feed supplemented with 3000 ppm NutriClay^Zn while displaying average body weight gains that modestly (yet not significantly) exceeded control animals. As outlined in Table 2, NutriClay^Zn admixture did not adversely impact the consumption of control feed. Average daily feed consumption was 0.5 kg and 0.6 kg for control and NutriClay^Zn-supplemented diets, respectively.

Table 2.

Feed Consumption by Weanling Swine: Control Diet Versus 3000 ppm NutriClay^Zn Admixture (n = 3 Pigs/Group)

	Control $(\bar{x} \pm SEM)$	NutriClay^Zn $(\bar{x} \pm SEM)$
Body weight (kg), Day 0	6.6 ± 0.2	6.6 ± 0.1
Body weight (kg), Day 9	10.1 ± 0.4	10.8 ± 0.5
Feed intake (kg), Days 1 through 9	4.4 ± 0.6	5.7 ± 0.1

Discussion

Due to an immature gastrointestinal tract, weanling swine are especially vulnerable to Salmonella, strains of which are often resistant to the aminoglycoside streptomycin (Heo et al., 2013; Tassinari et al., 2019). Treatment for suspected Salmonella infection in swine often involves other aminoglycosides and/or broad-spectrum antibiotics (Burrough, 2021), yet these interventions can fail if the disease-causing bacterial strain (Chiu et al., 2005) has become multidrug-resistant (MDR). From the standpoint of disease prevention, administering pharmacological doses of free chemical ZnO in weanling swine feed can help control Salmonella and other gastrointestinal pathogens (Fangman and Zulovich, 2003; Langer, 2022), yet this status quo practice may impact the environment and/or promote antimicrobial resistance over time (EMA, 2017). Given limited alternatives to support swine health and ultimately protect human consumers, we questioned how to combat Salmonella that are resistant to antibiotics with disparate mechanisms of action.

Salmonella Choleraesuis was shown to be resistant to streptomycin (aminoglycoside), as well as resistant to nalidixic acid (quinolone) and the broad-spectrum antibiotic sulfisoxazole (sulfonamide). As such, this MDR Salmonella was not susceptible to three mechanistic targets (i.e., DNA gyrase, folic acid metabolism, and protein synthesis) of traditional antibiotic therapy (Pancu et al., 2021). Salmonella Choleraesuis was, however, susceptible to growth inhibition by NutriClay^Zn. The minimum threshold inhibitory dose of NutriClay^Zn liberated 47 ppm elemental zinc (Fig. 1), which is less than 2% of zinc from the standard 3000 ppm ZnO feed admixture currently given to swine for control of enterotoxigenic Salmonella. Even the higher dose of NutriClay^Zn, inhibiting MDR Salmonella Choleraesuis growth by 98%, generated only 91 ppm elemental zinc (i.e., less than 4% of the status quo). These findings suggest that NutriClay^Zn could serve as an alternative to pharmacological ZnO for control of MDR Salmonella, yet with far less zinc discharge that might impact the environment or otherwise exacerbate antimicrobial resistance.

NutriClay^Zn thermal stability (Fig. 2) encourages practical applications. As many swine producers work throughout the heat of summer, as well as on narrow profit margins, it is important to develop feed ingredients that do not require refrigeration or other specialized storage conditions. Our findings with NutriClay^Zn align with literature reporting thermal stability of MMT clays (up to 500°C) that have been pillared with metal oxides for use as industrial catalysts and adsorbents (Mishra et al., 1996). Furthermore, the remarkable thermal stability of NutriClay^Zn inspires use in cooking by humans at high risk for enterotoxigenic bacterial infection. Such human populations include military personnel and people displaced by natural disasters and geopolitical conflicts (Mao et al., 2023; Ziadeh et al., 2020). Indeed, MMT clay (without amended zinc or other nutritional components) has been investigated and successfully incorporated into cooking practices of the native peoples of Africa as dietary adsorbents against aflatoxin (Elmore et al., 2014).

Under gastrointestinal conditions (Fig. 4), NutriClay^Zn liberates elemental zinc in a linear, dose-dependent fashion. Moreover, to provide a foundation for future animal trials (investigating in vivo efficacy of NutriClay^Zn to protect against Salmonella challenge), we conducted a preliminary safety assessment in weanling swine. It should be noted that the control diet was minimally zinc-adequate (75 ppm ZnO in the feed), and no additional free chemical zinc was added to either the control or treatment group diets. The concentration of NutriClay^Zn chosen for the treatment group diet was 3000 ppm, a metric slightly higher than the minimum threshold inhibitory dose (i.e., 2620 ppm) against Salmonella Choleraesuis. Given that treatment group animals consumed the diet at a rate slightly greater than controls (Table 2), there was no indication that NutriClay^Zn imparted any sensory aversion or other impediment to feed consumption. Control group animals (consuming 0.5 kg feed per day, without NutriClay^Zn) obtained nutritional zinc only from the 75 ppm ZnO feed admixture, thereby receiving 30 mg/day free elemental zinc. Based on our in vitro gastrointestinal bioavailability data, treatment group animals (consuming 0.6 kg feed per day) could receive up to 77 mg/day of free elemental zinc, including 41 mg/day liberated from NutriClay^Zn and 36 mg/day from the 75 ppm ZnO feed admixture.

Back calculating from the treatment group’s total free zinc (77 mg/day), if all had been provided as free ZnO (which is, by mass, 80% zinc) without any NutriClay^Zn, the concentration of ZnO in feed would be 160 ppm. This is less than 6% of the 3000 ppm ZnO feed admixture currently used to control enterotoxigenic bacteria in weanling swine. Furthermore, if we consider the higher dose of NutriClay^Zn suppressing the growth of Salmonella Choleraesuis by 98% (i.e., 7850 ppm), and round up to 8000 ppm NutriClay^Zn in the diet, a treatment group eating 0.6 kg feed would obtain a total of 145 mg/day free elemental zinc. Back calculating (as above), from 145 mg free elemental zinc, yields 303 ppm ZnO in feed. This is still less than 11% of the status quo 3000 ppm ZnO in feed for controlling bacterial pathogens. Hence, our data suggest that NutriClay^Zn could serve to reduce dietary zinc toward the nutritional requirement and thereby lessen the environmental burden of pharmacological oral administration of ZnO in weanling swine.

Our findings establish the foundation for developing NutriClay^Zn as a safe and effective feed additive to replace pharmacological ZnO (complementing rigorous biosecurity management practices), while likely also providing nutritional zinc. The linear release of elemental zinc from NutriClay^Zn under in vitro gastrointestinal conditions allows for predicting zinc bioavailability. In turn, unimpeded feed intake by swine (0.6 kg/day containing 3000 ppm NutriClay^Zn) argues that NutriClay^Zn alone could provide 41 mg/day elemental zinc, which is somewhat higher than the 30 mg/day elemental zinc provided by 75 ppm ZnO in the control diet. Future Salmonella challenge trials should be designed to characterize NutriClay^Zn efficacy in vivo, alongside concurrent study arms examining outcomes of diets deficient in the zinc component of mineral premixtures that traditionally provide nutritional zinc for weanling swine.

Footnotes

Authors’ Contributions

S.J.T.J., K.A., W.J.B., G.A.A., R.C.A., R.B.H., and T.D.P. designed research. S.J.T.J., K.A., R.E.D., K.J.R., M.W., and R.B.H. conducted research. S.J.T.J., R.C.A., R.B.H., and T.D.P. provided essential reagents, materials, and/or equipment. S.J.T.J. and R.B.H. analyzed the data. S.J.T.J. wrote the article. S.J.T.J. and T.D.P. had primary responsibility for final content. All authors have read and approved the final article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was funded by the U.S. Department of Agriculture (Agricultural Research Service, Texas A&M AgriLife IHA, and Texas A&M University Hatch Project TEX0-2-6215) and the U.S. National Institute of Environmental Health Sciences (P42 ES027704). Supporting sources had no restrictions regarding the publication of this work.

References

Armbruster

, Roberts

. The political economy of US antibiotic use in animal feed. Food Safety Economics, 2018:293–322; doi: 10.1007/978-3-319-92138-9_15

Bäumler

, Fang

. Host specificity of bacterial pathogens. Cold Spring Harb Perspect Med, 2013; 3(12):a010041; doi: 10.1101/cshperspect.a010041

Bearson

SMD

. Salmonella in swine: Prevalence, multidrug resistance, and vaccination strategies. Annu Rev Anim Biosci, 2022; 10(1):373–393; doi: 10.1146/annurev-animal-013120-043304

Berri

, Hogan

, Saade

, et al. IPEC-1 variable immune response to different serovars of Salmonella enterica subsp. enterica. Vet Immunol Immunopathol, 2020; 220:109989; doi: 10.1016/j.vetimm.2019.109989

Bonetti

, Tugnoli

, Piva

, et al. Towards zero zinc oxide: Feeding strategies to manage post-weaning diarrhea in piglets. Animals (Basel), 2021; 11(3); doi: 10.3390/ani11030642

Burrough

. Intestinal Salmonellosis in Pigs. Merck Veterinary Manual; 2021. Available from: https://www.merckvetmanual.com/digestive-system/intestinal-diseases-in-pigs/intestinal-salmonellosis-in-pigs

Burrough

, De Mille

, Gabler

. Zinc overload in weaned pigs: Tissue accumulation, pathology, and growth impacts. J Vet Diagn Invest, 2019; 31(4):537–545; doi: 10.1177/1040638719852144

CDC. U.S. Department of Health & Human Services. Salmonella; 2023. (Available from: https://www.cdc.gov/salmonella/index.html).

CDC. U.S. Department of Health & Human Services. How Food Gets Contaminated - The Food Production Chain, 2024. (Available from: https://www.cdc.gov/foodborne-outbreaks/foodproductionchain/?CDC_AAref_Val=https://www.cdc.gov/foodsafety/production-chain.html).

10.

Chiu

, Tang

, Chu

, et al. The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen. Nucleic Acids Res, 2005; 33(5):1690–1698; doi: 10.1093/nar/gki297

11.

Dewulf

, Joosten

, Chantziaras

, et al. Antibiotic use in european pig production: Less is more. Antibiotics (Basel), 2022; 11(11); doi: 10.3390/antibiotics11111493

12.

Dos Santos

, Cunha

MPV

, Bertani

AMJ

, et al. Detection of multidrug- and colistin-resistant Salmonella Choleraesuis causing bloodstream infection. J Antimicrob Chemother, 2020; 75(7):2009–2010; doi: 10.1093/jac/dkaa076

13.

Elmore

, Mitchell

, Mays

, et al. Common African cooking processes do not affect the aflatoxin binding efficacy of refined calcium montmorillonite clay. Food Control, 2014; 37:27–32; doi: 10.1016/j.foodcont.2013.08.037

14.

EMA, European Medicines Agency. 2017. European Commission final decision: Zinc oxide Article-35 referrral.(Available from: https://www.ema.europa.eu/en/medicines/veterinary/referrals/zinc-oxide#all-documents).

15.

Fangman

, Zulovich

. Alternative Management Practices in Pork Production. In: Swine Housing II. American Society of Agricultural Engineers: St. Joseph, MI, 2003.

16.

Ferrari

, Rosario

DKA

, Cunha-Neto

, et al. Worldwide Epidemiology of Salmonella Serovars in Animal-Based Foods: A Meta-analysis. Appl Environ Microbiol, 2019; 85(14); doi: 10.1128/aem.00591-19

17.

Heo

, Opapeju

, Pluske

, et al. Gastrointestinal health and function in weaned pigs: A review of feeding strategies to control post-weaning diarrhoea without using in-feed antimicrobial compounds. J Anim Physiol Anim Nutr (Berl), 2013; 97(2):207–237; doi: 10.1111/j.1439-0396.2012.01284.x

18.

Hoffman

, Ahn

J-W

. Updating Economic Burden of Foodborne Diseases Estimates for Inflation and Income Growth. U.S. Department of Agriculture Economic Research Service [ERR 297] (Available from: https://www.ers.usda.gov/publications/pub-details/?pubid=102639), November 2021.

19.

Hoffman

, Maculloch

, Batz

, et al. Economic Burden of Major Foodborne Illnesses Acquired in the United States. U.S. Department of Agriculture Economic Research Service [EIB 140]; (Available from: https://www.ers.usda.gov/webdocs/publications/43984/52807_eib140.pdf), May 2015.

20.

Jackson

SJT

, Andrews

, Droleskey

, et al. NutriClay^Zn Binds Aflatoxin B1 and Suppresses Enterotoxigenic Salmonella and Escherichia coli . J Food Prot, 2025; 88(5):100486; doi: 10.1016/j.jfp.2025.100486

21.

Klein

. The use of biorelevant dissolution media to forecast the in vivo performance of a drug. AAPS J, 2010; 12(3):397–406; doi: 10.1208/s12248-010-9203-3

22.

Langer

. How the Ban of Zinc Oxide Changes Swine Health Management. 2022 (Available from: https://www.zinpro.com/how-the-ban-of-zinc-oxide-changes-swine-health-management/#:∼:text=Minimize%20Change%20of%20the%20Pig,essential%20for%20normal%20gut%20function).

23.

Mao

, Zeineldin

, Usmani

, et al. Local and environmental reservoirs of salmonella enterica after hurricane florence flooding. Geohealth, 2023; 7(11):e2023GH000877; doi: 10.1029/2023gh000877

24.

Marques

. Dissolution media simulating fasted and fed states. Dissolution Technol, 2004; 11(2):16–16; doi: 10.14227/DT110204P16

25.

Mishra

, Parida

, Rao

. Transition Metal Oxide Pillared Clay: 1. A Comparative Study of Textural and Acidic Properties of Fe(III) Pillared Montmorillonite and Pillared Acid Activated Montmorillonite. J Colloid Interface Sci, 1996; 183(1):176–183; doi: 10.1006/jcis.1996.0532

26.

Monte

, Sellera

. Salmonella. Emerg Infect Dis, 2020; 26(12):2955; doi: 10.3201/eid2612.et2612

27.

Oransathid

, Sukhchat

, Margulieux

, et al. First report: Colistin resistance gene mcr-3.1 in salmonella enterica serotype choleraesuis isolated from human blood sample from Thailand. Microb Drug Resist, 2022; 28(1):102–105; doi: 10.1089/mdr.2020.0553

28.

Pancu

, Scurtu

, Macasoi

, et al. Antibiotics: Conventional therapy and natural compounds with antibacterial activity-a pharmaco-toxicological screening. Antibiotics (Basel), 2021; 10(4); doi: 10.3390/antibiotics10040401

29.

Pasquet

, Chevalier

, Couval

, et al. Antimicrobial activity of zinc oxide particles on five micro-organisms of the Challenge Tests related to their physicochemical properties. Int J Pharm, 2014; 460(1–2):92–100; doi: 10.1016/j.ijpharm.2013.10.031

30.

Rodrigues

, Panzenhagen

, Ferrari

, et al. Antimicrobial resistance in nontyphoidal salmonella isolates from human and swine sources in brazil: A systematic review of the past three decades. Microb Drug Resist, 2020; 26(10):1260–1270; doi: 10.1089/mdr.2019.0475

31.

Sampath

, Sureshkumar

, Seok

, et al. Role and functions of micro and macro-minerals in swine nutrition: A short review. J Anim Sci Technol, 2023; 65(3):479–489; doi: 10.5187/jast.2023.e9

32.

Saphra

, Winter

. Clinical manifestations of salmonellosis in man. N Engl J Med, 1957; 256(24):1128–1134; doi: 10.1056/nejm195706132562402

33.

Soliani

, Rugna

, Prosperi

, et al. Salmonella infection in pigs: Disease, prevalence, and a link between swine and human health. Pathogens, 2023; 12(10); doi: 10.3390/pathogens12101267

34.

Tassinari

, Duffy

, Bawn

, et al. Microevolution of antimicrobial resistance and biofilm formation of Salmonella Typhimurium during persistence on pig farms. Sci Rep, 2019; 9(1):8832; doi: 10.1038/s41598-019-45216-w

35.

Threlfall

, Hall

, Rowe

. Salmonella bacteraemia in England and Wales, 1981-1990. J Clin Pathol, 1992; 45(1):34–36; doi: 10.1136/jcp.45.1.34

36.

USDA. Food Safety and Inspection Service, Office of Public Health Science, Risk Assessment and Analytics Staff. Public Health Effects of Performance Standards for Comminuted Pork and Pork Cuts, April 2020. (Available from: https://www.fsis.usda.gov/sites/default/files/media_file/2022-02/Pork_Salmonella_Performance_Standards_Risk_Assessment_April_8_2020_Feb_8_2022.pdf).

37.

Ziadeh

, Clark

, Williams

, et al. Update: Incidence of acute gastrointestinal infections and diarrhea, active component, U.S. Armed Forces, 2010-2019. Msmr, 2020; 27(8):9–14.