Drinking Water Supplementation of trans-Cinnamaldehyde-Miglyol Microemulsions Reduces Multidrug-Resistant Salmonella Heidelberg in Turkey Poults and Augments the Antibacterial Effect of Oxytetracycline

Abstract

The use of clinically important antibiotics in U.S. poultry production has decreased drastically over the past decade. They can only be used to treat diseases under the supervision of a veterinarian. Reducing antibiotic use, even for disease treatment, can improve the long-term sustainability of the industry. In the current study, we examined the effect of supplementation of a low dose of trans-cinnamaldehyde (TC; 0.03%), a GRAS-status plant-derived compound, with or without oxytetracycline (OTC; 16 μg/mL), an anti-30S ribosomal subunit targeting antibiotic, on the multidrug-resistant (MDR) S. Heidelberg (SH) in turkey poults. Two independent experiments were conducted (N = 96). In each experiment, 48, straight-run, day-old, commercial Hybrid Converter turkey poults were randomly assigned to 6 treatments of 8 birds each: Negative Control [NC; −SH, −TC, −OTC, −0.06% Miglyol (MIG, emulsifier for TC in water)], Positive Control (PC; +SH, −TC, −OTC, −MIG), MIG Control (MIG; +SH, −TC, −OTC, +MIG), TC Group (TC; +SH, +TC, −OTC, +MIG), OTC group (OTC; +SH, −TC, +OTC, −MIG), and TC+OTC group (TC+OTC; +SH, +TC, +OTC, +MIG). OTC was supplemented from day 1 through drinking water throughout the experiment. The birds in the TC and TC+OTC groups were supplemented with TC in their drinking water for 7 days post-challenge. All birds were challenged on day 7 with 6 log₁₀ CFU of SH/bird via crop gavage. On day 14, all birds were euthanized to collect the cecum, liver, and spleen for pathogen recovery. TC at 0.03% emulsified in MIG was highly effective in reducing MDR SH colonization in turkey poults (p < 0.05) compared to the SH control (>4.5 log₁₀ CFU/g reduction) on day 14. The OTC group reduced the pathogen load by 2.5 log₁₀ CFU/g by day 14. TC enhanced the effect of OTC, reducing pathogen load by ~3.9 log₁₀ CFU/g compared to the SH control after 7 days. TC significantly reduced SH invasion into the liver and spleen compared with the SH control on day 14. The results of the study indicate that TC at 0.03% can augment OTC at 16 μg/mL for the treatment of MDR SH infection in poults and could be an industry-sustainable strategy.

Introduction

The annual occurrence of human non-typhoidal salmonellosis in the United States is approximately 1.3 million. Among these, approximately 1 million Salmonella infections are attributed to the consumption of Salmonella-contaminated food [1]. Poultry, including turkeys, are natural reservoirs of Salmonella and a constant source of infection through fecal shedding, resulting from Salmonella colonization in the ceca [2]. Pathogen colonization leads to contamination of the surrounding farm environment and contamination of poultry meat during processing [3,4,5]. This situation is a bottleneck to the sustainable advancement of the industry.

Salmonella Heidelberg (SH) is a highly invasive Salmonella serovar that frequently infects humans and is often isolated from poultry production and processing plants. SH can colonize within the cecum of poultry and be transmitted to humans through contaminated poultry [5,6]. SH is among the top five Salmonella serotypes causing clinical infections in humans [7] and has been linked to foodborne outbreaks through the consumption of contaminated ground turkey, chicken carcasses, and mechanically separated chicken meat in the US [5]. The development of antibiotic resistance in SH, high invasive capability, and association with foodborne outbreaks through poultry are serious concerns in the U.S. SH isolated from ground turkey and chicken carcasses implicated in outbreaks were resistant to multiple drugs, including the drug of choice for human salmonellosis—ceftriaxone, a third-generation cephalosporin with high activity against Gram-negative organisms [8,9].

With grave concerns about the development of antibiotic resistance among various pathogens, including the emerging MDR Salmonella, the FDA introduced the Veterinary Feed Directive (VFD), which prohibits the use of prophylactic antibiotics and necessitates veterinary oversight before using clinically important antibiotics in treating production animals [10]. This move underscores the importance of judicious antibiotic use in animal agriculture and emphasizes the need to develop natural, safe, and sustainable intervention strategies against deadly foodborne pathogens, such as Salmonella.

The use of natural alternatives against pathogens has garnered interest over the past two decades due to concerns about the development of antibiotic resistance in bacteria. Essential oils have been traditionally used to preserve foods and enhance their flavor [1,3]. The antimicrobial properties of several essential oils have been demonstrated, and a variety of active components have been identified. trans-cinnamaldehyde (TC) is the major component of cinnamon. It is classified as GRAS (generally recognized as safe) and is approved for use in foods by the Food and Drug Administration [11,12,13]. Previous studies revealed that TC is effective against S. Enteritidis in broilers [11,12,13].

Although antibiotic use in US poultry production has decreased sharply over the past decade, antibiotics should still be used judiciously under veterinary supervision to address animal health concerns [10,14]. These medically important antibiotic drugs (MIAD) include oxytetracycline (OTC), avilamycin, chlortetracycline, erythromycin, lincomycin, hygromycin B, sulfadimethoxine, sulfaquinoxaline, and virginiamycin. OTC can be effective against a range of pathogens in chickens and turkeys when combined with neomycin and certain ionophore antimicrobials added through dry animal feed [14,15]. Veterinarians can determine the most effective antibiotic for an animal health condition and administer it through injection in a hatchery or mix it into feed or drinking water [10]. Commercially available water-soluble OTC can be given to chickens and turkeys independently for up to 14 consecutive days against various bacterial organisms susceptible to the antibiotic with a required veterinary prescription.

In most studies, TC was supplemented through feed and has not been tested in water due to its limited solubility [11,12,13]. We recently reported that the direct addition of 0.08% TC to poultry drinking water resulted in a significant reduction in SH [1]. However, poor solubility of TC in water was identified as a hindrance to achieving better SH reductions. The role of miglyol (MIG), a blend of decanoyl and octanoyl glyceride-based emulsifier, was evaluated in the production of TC-MIG microemulsions. Moreover, the efficacy of plant-derived antimicrobials (PDAs), including TC, in augmenting MIADs, such as OTC, has not been studied. If found effective, TC could be a veterinarian’s aid in reducing the use of MIADs during poultry disease treatment, enhancing the sustainability of industry treatment options. Therefore, the objective of the study was to determine the anti-SH efficacy of a low dose of TC (0.03% v/v) emulsified in MIG (0.06%) alone or in combination with a low dose of OTC (16 μg/mL), administered through water, in turkey poults

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2 Materials and Methods,

2.3 Plant-Derived Antimicrobial (PDA)

trans-cinnamaldehyde (TC) (≥99%; Natural, Food Grade; Catalog # W290106-100G-K; Lot# MKBS7421V) was purchased from Sigma–Aldrich (PO Box 14508, St. Louis, MO, USA). A lower concentration of TC [0.03% (v/v)] was used in the experiment to supplement the poults through drinking water. This concentration was selected based on our previous experiments. For instance, our group recently reported the use of TC at 0.08% without solvent, delivered via drinking water to poults, resulting in anti-SH effects and a significant difference in the cecal metabolome [1,23]. Previous in vitro experiments have found that TC at a concentration as low as 0.02% could increase the sensitivity of a drug-resistant serotype, S. Typhimurium DT104, to tetracycline [24]. In this study, TC was dispersed in water using an inert diluent, miglyol (MIG; 0.06% v/v; IOI Oleo GmbH, D-58453 Witten, Germany). In our experiments, we found that 0.02% TC and 0.06% MIG interacted to produce emulsions effective against SH, while 0.06% MIG alone showed no antibacterial effects. Water containing TC and MIG was thoroughly mixed before being provided to poults. The particle size distribution of the treatments was determined using a laser diffraction particle size analyzer (Bluewave, Microtrac Inc., Montgomeryville, PA, USA) at the Minnesota Nano Center.

Nair, D.V.T.; Kollanoor Johny, A. Drinking Water Supplementation of trans-Cinnamaldehyde-Miglyol Microemulsions Reduces Multidrug-Resistant Salmonella Heidelberg in Turkey Poults and Augments the Antibacterial Effect of Oxytetracycline. Microorganisms 2025, 13, 2703. https://doi.org/10.3390/microorganisms13122703

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