In-feed supplementation of trans-cinnamaldehyde reduces layer-chicken egg-borne transmission of Salmonella enterica serovar enteritidis.

Salmonella enterica serovar Enteritidis is a major foodborne pathogen in the United States, causing gastroenteritis in humans, primarily through consumption of contaminated eggs. Chickens are the reservoir host of S. Enteritidis. In layer hens, S. Enteritidis colonizes the intestine and migrates to various organs, including the oviduct, leading to egg contamination. This study investigated the efficacy of in-feed supplementation with trans-cinnamaldehyde (TC), a generally recognized as safe (GRAS) plant compound obtained from cinnamon, in reducing S. Enteritidis cecal colonization and systemic spread in layers. Additionally, the effect of TC on S. Enteritidis virulence factors critical for macrophage survival and oviduct colonization was investigated in vitro. The consumer acceptability of eggs was also determined by a triangle test. Supplementation of TC in feed for 66 days at 1 or 1.5% (vol/wt) for 40- or 25-week-old layer chickens decreased the amounts of S. Enteritidis on eggshell and in yolk (P<0.001). Additionally, S. Enteritidis persistence in the cecum, liver, and oviduct in TC-supplemented birds was decreased compared to that in controls (P<0.001). No significant differences in feed intake, body weight, or egg production in birds or in consumer acceptability of eggs were observed (P>0.05). In vitro cell culture assays revealed that TC reduced S. Enteritidis adhesion to and invasion of primary chicken oviduct epithelial cells and reduced S. Enteritidis survival in chicken macrophages (P<0.001). Follow-up gene expression analysis using real-time quantitative PCR (qPCR) showed that TC downregulated the expression of S. Enteritidis virulence genes critical for chicken oviduct colonization (P<0.001). The results suggest that TC may potentially be used as a feed additive to reduce egg-borne transmission of S. Enteritidis.

Reducing Colonization and Eggborne Transmission of Salmonella Enteritidis in Layer Chickens by In-Feed Supplementation of Caprylic Acid.

Salmonella Enteritidis (SE) is a major foodborne pathogen responsible for causing gastrointestinal infections in humans, predominantly due to the consumption of contaminated eggs. In layer hens, SE colonizes the intestine and migrates to various organs, including the oviduct, thereby leading to egg yolk and shell contamination. This study investigated the efficacy of caprylic acid (CA), a medium-chain fatty acid, in reducing SE colonization and egg contamination in layers. Caprylic acid was supplemented in the feed at 0%, 0.7%, or 1% (vol/wt) from day 1 of the experiment. Birds were challenged with 10(10) log colony-forming units (CFU)/mL of SE by crop gavage on day 10, and re-inoculated (10(10) log CFU/mL) on day 35. After 7 days post first inoculation, eggs were collected daily and tested for SE on the shell and in the yolk separately. The birds were sacrificed on day 66 to determine SE colonization in the ceca, liver, and oviduct. The consumer acceptability of eggs was also determined by triangle test. The experiment was replicated twice. In-feed supplementation of CA (0.7% and 1%) to birds consistently decreased SE on eggshell and in the yolk (p<0.05). Supplementation of CA at 1.0% decreased SE population to ≈14% on the shell and ≈10% in yolk, when compared to control birds, which yielded ≈60% positive samples on shell and ≈43% in yolk. Additionally, SE populations in the cecum and liver were reduced in treated birds compared to control (p<0.05). No significant difference in egg production, body weight, or sensory properties of eggs was observed (p>0.05). The results suggest that CA could potentially be used as a feed additive to reduce eggborne transmission of SE.

Controlling Aspergillus flavus and Aspergillus parasiticus growth and aflatoxin production in poultry feed using carvacrol and trans-cinnamaldehyde.

Aflatoxins (AF) are toxic metabolites primarily produced by molds, Aspergillus flavus and Aspergillus parasiticus. Contamination of poultry feed with AF is a major concern to the poultry industry due to severe economic losses stemming from poor performance, reduced egg production, and diminished egg hatchability. This study investigated the inhibitory effect of 2 generally regarded as safe (GRAS), natural plant compounds, namely carvacrol (CR) and trans-cinnamaldehyde (TC), on A. flavus and A. parasiticus growth and AF production in potato dextrose broth (PDB) and in poultry feed. In broth culture, PDB supplemented with CR (0%, 0.02%, 0.04% and 0.08%) or TC (0%, 0.005%, 0.01% and 0.02%) was inoculated with A. flavus or A. parasiticus (6 log CFU/mL), and mold counts and AF production were determined on days 0, 1, 3, and 5. Similarly, 200 g portions of poultry feed supplemented with CR or TC (0%, 0.4%, 0.8%, and 1.0%) were inoculated with each mold, and their counts and AF concentrations in the feed were determined at 0, 1, 2, 3, 4, 8, and 12 weeks of storage. Moreover, the effect of CR and TC on the expression of AF synthesis genes in A. flavus and A. parasiticus (aflC, nor1, norA, and ver1) was determined using real-time quantitative PCR (RT-qPCR). All experiments had duplicate samples and were replicated 3 times. Results indicated that CR and TC reduced A. flavus and A. parasiticus growth and AF production in broth culture and chicken feed (P<0.05). All tested concentrations of CR and TC decreased AF production in broth culture and chicken feed by at least 60% when compared to controls (P<0.05). In addition, CR and TC down-regulated the expression of major genes associated with AF synthesis in the molds (P<0.05). Results suggest the potential use of CR and TC as feed additives to control AF contamination in poultry feed.

Efficacy of plant-derived compounds combined with hydrogen peroxide as antimicrobial wash and coating treatment for reducing Listeria monocytogenes on cantaloupes.

The efficacy of four plant-derived antimicrobials (PDAs), namely carvacrol, thymol, ß-resorcylic acid, and caprylic acid, with or without hydrogen peroxide (HP), as antimicrobial wash and chitosan based coating for reducing Listeria monocytogenes (LM) on cantaloupes was investigated. Cantaloupe rind plugs inoculated with LM (10(7) CFU/cm(2)) were washed for 3, 6, 10 min at 25 °C or 1, 3, 5 min at 55 or 65 °C in water, or water containing 2% PDAs with or without 2% HP. Additionally, inoculated cantaloupes (10(8) CFU/fruit) washed with 2% PDA-HP combinations at 55 or 65 °C (5 min) were cut into rindless cubical pieces, stored at 4 °C for 7 days and sampled for LM. Furthermore, inoculated plugs coated with 2% PDAs were stored for 7 days and sampled for surviving LM. Individual PDA washes reduced LM on rinds by ≥2.5 log CFU/cm(2) by 3 min (P < 0.05). PDA-HP combinations decreased LM to undetectable levels by 5 min at 55, 65 °C, and 10 min at 25 °C (P < 0.05) and reduced LM transfer from cantaloupe surface to interior (P < 0.0001). All PDA coating treatments reduced LM on cantaloupe to undetectable levels by 5 days (P < 0.05). Results indicate that PDAs alone, or with HP could be used to reduce LM on cantaloupes.

Carvacrol and trans-cinnamaldehyde reduce Clostridium difficile toxin production and cytotoxicity in vitro.

Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.

Effect of pimenta essential oil against Salmonella Agona and Salmonella Saintpaul in ground turkey meat and nonprocessed turkey breast meat.

Salmonella enterica Agona (S. Agona) and Salmonella enterica Saintpaul (S. Saintpaul) are among the emerging drug-resistant Salmonella in turkey production and processing. Rapid solutions to control emerging and uncommon serotypes such as S. Agona and S. Saintpaul are needed. This study tested pimenta essential oil (PEO) as a processing antibacterial against S. Agona and S. Saintpaul in experiments representative of different stages of turkey processing. The compound effectively reduced S. Agona and S. Saintpaul in nutrient broth studies and with mature biofilm assays. PEO was tested against a combination of S. Agona and S. Saintpaul in ground turkey meat and nonprocessed breast meat. In the first experiment with ground turkey, samples were inoculated with a mixture of S. Agona and S. Saintpaul (∼3 log10 CFU/g) and treated with PEO at different concentrations (0% PEO, 0.25% PEO, 0.5% PEO, 1% PEO, 2% PEO, and 2.5% PEO). In the second experiment with turkey breast, samples inoculated with ∼3 log10 CFU/g (SA+SP) were dipped in different concentrations of PEO with chitosan (CN) for 2 min. In both these experiments, samples were stored at 4°C, and Salmonella recovery was carried out at 0, 1, 3, 5, and 7 d. All experiments followed a completely randomized design and were repeated 6 times (n = 6). Statistical analysis was done using the PROC-ANOVA procedure of SAS. In the ground turkey meat, PEO at or above 2% reduced 2 log10 CFU/g of Salmonella by day 1. PEO at 2.5% in ground turkey meat resulted in enrichment-negative samples by 1 min, indicative of the rapid killing effect of the compound at a high concentration of PEO (P ≤ 0.05). A maximum reduction of 1.7 log10 CFU Salmonella/g of turkey breast meat was obtained after 2 min of dip treatment containing CN and 2.5% PEO. Results indicate that PEO could be used as a plant-based processing antibacterial against S. Agona and S. Saintpaul in turkey processing. Upscaling to plant-level studies is necessary before recommending its usage.

Combination of autochthonous Lactobacillus strains and trans-Cinnamaldehyde in water reduces Salmonella Heidelberg in turkey poults.

Reducing the colonization of Salmonella in turkeys is critical to mitigating the risk of its contamination at later stages of production. Given the increased susceptibility of newly hatched poults to Salmonella colonization, it is crucial to implement interventions that target potential transmission routes, including drinking water. As no individual intervention explored to date is known to eliminate Salmonella, the United States Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) recommends employing multiple hurdles to achieve a more meaningful reduction and minimize the potential emergence of resistance. Probiotics and plant-derived antimicrobials (PDAs) have demonstrated efficacy as interventions against Salmonella in poultry. Therefore, this study aimed to investigate the use of turkey-derived Lactobacillus probiotics (LB; a mixture of Lactobacillus salivarius UMNPBX2 and L. ingluviei UMNPBX19 isolated from turkey ileum) and a PDA, trans-cinnamaldehyde (TC), alone and in combination (CO), against S. Heidelberg in turkey drinking water and poults. The presence of 5% nutrient broth or cecal contents as contaminants in water resulted in S. Heidelberg growth. TC eliminated S. Heidelberg, regardless of the contaminants present. In contrast, the cecal contents led to increased survival of Lactobacillus in the CO group. Unlike TC, LB was most effective against S. Heidelberg when the nutrient broth was present, suggesting the role of secondary metabolites in its mechanism of action. In the experiments with poults, individual TC and LB supplementation reduced cecal S. Heidelberg in challenged poults by 1.2- and 1.7-log10 colony-forming units (CFU)/g cecal contents, respectively. Their combination yielded an additive effect, reducing S. Heidelberg by 2.7 log10 CFU/g of cecal contents compared to the control (p ≤ 0.05). However, the impact of TC and LB on the translocation of S. Heidelberg to the liver was more significant than CO. TC and LB are effective preharvest interventions against S. Heidelberg in poultry production. Nonetheless, further investigations are needed to determine the optimum application method and its efficacy in adult turkeys.

Antibiofilm effect of plant derived antimicrobials on Listeria monocytogenes.

The present study investigated the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) and bactericidal concentrations (MBCs) of four, generally recognized as safe (GRAS), plant-derived antimicrobials (PDAs) in inhibiting Listeria monocytogenes (LM) biofilm formation and inactivating mature LM biofilms, at 37, 25 and 4 °C on polystyrene plates and stainless-steel coupons. In addition, the effect of SICs of PDAs on the expression of LM genes critical for biofilm synthesis was determined by real-time quantitative PCR. The PDAs and their SICs used for inhibition of biofilm were trans-cinnamaldehyde (TC 0.50, 0.75 mM), carvacrol (CR 0.50, 0.65 mM), thymol (TY 0.33, 0.50 mM), and eugenol (EG 1.8, 2.5 mM), whereas the PDA concentrations used for inactivating mature biofilms were 5.0 and 10.0 mM (TC, CR), 3.3 and 5.0 mM (TY), 18.5 and 25.0 mM (EG). All PDAs inhibited biofilm synthesis and inactivated fully formed LM biofilms on both matrices at three temperatures tested (P < 0.05). Real-time quantitative PCR data revealed that all PDAs down-regulated critical LM biofilm-associated genes (P < 0.05). Results suggest that TC, CR, TY, and EG could potentially be used to control LM biofilms in food processing environments, although further studies under commercial settings are necessary.

Rapid inactivation of Salmonella Enteritidis on shell eggs by plant-derived antimicrobials.

Salmonella Enteritidis is a common foodborne pathogen transmitted to humans largely by consumption of contaminated eggs. The external surface of eggs becomes contaminated with Salmonella Enteritidis from various sources on farms, the main sources being hens' droppings and contaminated litter. Therefore, effective egg surface disinfection is critical to reduce pathogens on eggs and potentially control egg-borne disease outbreaks. This study investigated the efficacy of GRAS (generally recognized as safe) status, plant-derived antimicrobials (PDA), namely trans-cinnamaldehyde (TC), carvacrol (CR), and eugenol (EUG), as an antimicrobial wash for rapidly killing Salmonella Enteritidis on shell eggs in the presence or absence of chicken droppings. White-shelled eggs inoculated with a 5-strain mixture of nalidixic acid (NA) resistant Salmonella Enteritidis (8.0 log cfu/mL) were washed in sterile deionized water containing each PDA (0.0, 0.25, 0.5, or 0.75%) or chlorine (200 mg/kg) at 32 or 42°C for 30 s, 3 min, or 5 min. Approximately 6.0 log cfu/mL of Salmonella Enteritidis was recovered from inoculated and unwashed eggs. The wash water control and chlorine control decreased Salmonella Enteritidis on eggs by only 2.0 log cfu/mL even after washing for 5 min. The PDA were highly effective in killing Salmonella Enteritidis on eggs compared with controls (P < 0.05). All treatments containing CR and EUG reduced Salmonella Enteritidis to undetectable levels as rapidly as within 30 s of washing, whereas TC (0.75%) completely inactivated Salmonella Enteritidis on eggs washed at 42°C for 30 s (P < 0.05). No Salmonella Enteritidis was detected in any PDA or chlorine wash solution; however, substantial pathogen populations (~4.0 log cfu/mL) survived in the antibacterial-free control wash water (P < 0.05). The CR and EUG were also able to eliminate Salmonella Enteritidis on eggs to undetectable levels in the presence of 3% chicken droppings at 32°C (P < 0.05). This study demonstrates that PDA could effectively be used as a wash treatment to reduce Salmonella Enteritidis on shell eggs. Sensory and quality studies of PDA-washed eggs need to be conducted before recommending their use.

Effect of plant derived antimicrobials on Salmonella enteritidis adhesion to and invasion of primary chicken oviduct epithelial cells in vitro and virulence gene expression.

Salmonella Enteritidis (SE) is a major foodborne pathogen in the United States and one of the most frequently reported Salmonella serotypes globally. Eggs are the most common food product associated with SE infections in humans. The pathogen colonizes the intestinal tract in layers, and migrates to reproductive organs systemically. Since adhesion to and invasion of chicken oviduct epithelial cells (COEC) is critical for SE colonization in reproductive tract, reducing these virulence factors could potentially decrease egg yolk contamination. This study investigated the efficacy of sub-inhibitory concentrations of three plant-derived antimicrobials (PDAs), namely carvacrol, thymol and eugenol in reducing SE adhesion to and invasion of COEC, and survival in chicken macrophages. In addition, the effect of PDAs on SE genes critical for oviduct colonization and macrophage survival was determined using real-time quantitative PCR (RT-qPCR). All PDAs significantly reduced SE adhesion to and invasion of COEC (p < 0.001). The PDAs, except thymol consistently decreased SE survival in macrophages (p < 0.001). RT-qPCR results revealed down-regulation in the expression of genes involved in SE colonization and macrophage survival (p < 0.001). The results indicate that PDAs could potentially be used to control SE colonization in chicken reproductive tract; however, in vivo studies validating these results are warranted.

Effect of plant-derived antimicrobials, eugenol, carvacrol, and ß-resorcylic acid against Salmonella on organic chicken wings and carcasses.

Organic poultry constitutes a sizeable segment of the American organic commodities market. However, processors have limited strategies that are safe, effective, and approved for improving the microbiological safety of products. In this study, the efficacy of 3 plant-derived antimicrobials (PDAs), eugenol (EG), carvacrol (CR), and ß-resorcylic acid (BR) was evaluated against Salmonella on organic chicken wings and carcasses. Wings inoculated with Salmonella (6 log10 CFU/wing) were treated with or without the treatments (BR [0.5%, 1% w/v], EG [0.5%, 1% v/v], CR [0.5%, 1% v/v], chlorine [CL; 200 ppm v/v], or peracetic acid [PA; 200 ppm v/v]) applied for 2 min at 54°C (scalding study) or 30 min at 4°C (chilling study). Homogenates and treatment water were evaluated for surviving Salmonella. Six wings or carcasses per treatment were analyzed in each study. All treatments, except CL and 0.5% BR in the scalding study, yielded significant reductions of Salmonella on wings compared to the positive control (PC-Salmonella inoculated samples not treated with antimicrobials). To follow, carcasses inoculated with Salmonella (higher inoculum [106 CFU/carcass] or lower inoculum [104 CFU/carcass]) and immersed in antimicrobials (CR 1% [v/v] and industry controls [CL {200 ppm}, or PA [200 ppm]) for 30 min at 4°C were stored until analysis. For the higher inoculum study, 1% CR resulted in a 3.9 log10 CFU/g reduction of Salmonella on the carcass on d 0 compared to PC (P < 0.05); however, CL yielded no reduction. On d 3, CR and PA resulted in 0.9 and 1.2 log10 CFU/g reduction of Salmonella, respectively (P < 0.05). For the lower inoculum study, consistent Salmonella reductions were obtained with CR and PA (1.4-2.1 log10 CFU/g) on d 0 and 7. High reductions of Salmonella in processing water were obtained in all studies. CR effectively controls Salmonella on wings and carcasses and in processing water immediately after application. Follow-up studies on the organoleptic characteristics of PDA-treated chicken carcasses are necessary.

Reduction of Salmonella enterica serovar enteritidis colonization in 20-day-old broiler chickens by the plant-derived compounds trans-cinnamaldehyde and eugenol.

The efficacies of trans-cinnamaldehyde (TC) and eugenol (EG) for reducing Salmonella enterica serovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks (n = 75/experiment) were randomly assigned to five treatment groups (n = 15/treatment group): negative control (-ve S. Enteritidis, -ve TC, or EG), compound control (-ve S. Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+ve S. Enteritidis, -ve TC, or EG), low-dose treatment (+ve S. Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+ve S. Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherent Salmonella (n = 5/experiment). On day 8, birds were inoculated with ∼8.0 log(10) CFU S. Enteritidis, and cecal colonization by S. Enteritidis was ascertained (n = 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecal S. Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced (P < 0.05) S. Enteritidis in the cecum (≥3 log(10) CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments (P > 0.05); however, EG supplementation led to significantly lower (P < 0.05) body weights. Follow-up in vitro experiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibit Salmonella growth) of TC and EG reduced the motility and invasive abilities of S. Enteritidis and downregulated expression of the motility genes flhC and motA and invasion genes hilA, hilD, and invF. The results suggest that supplementation with TC and EG through feed can reduce S. Enteritidis colonization in chickens.

Effect of plant-derived antimicrobials against multidrug-resistant Salmonella Heidelberg in ground Turkey.

Salmonella Heidelberg (SH) is a highly invasive human pathogen for which turkeys can serve as reservoir hosts. Colonization of turkeys with SH may result in potential contamination and is a greater challenge to prevent in comminuted products. Antimicrobial efficacy of 3 GRAS-status plant-derived antimicrobials (PDAs), lemongrass essential oil (LG), citral (CIT), and trans-cinnamaldehyde (TC), against SH in ground turkey, a comminuted product implicated in several outbreaks, was evaluated in this study. Ground turkey samples inoculated with ∼3.50 log10 CFU/g of a three-strain SH cocktail were treated with either LG, CIT, or TC at either 0.5, 1, or 2% (vol/wt). Samples were stored at 4°C, and bacterial enumeration was performed on d 0, 1, 3, and 5. Appropriate controls were included alongside all treatments. Fluorescence microscopy was performed to evaluate the direct impact of the PDAs against SH in vitro. Appearance and aroma difference testing of raw patties was also performed for select treatments with trained sensory panelists. Treatment with 2% TC yielded a 2.5 log10 CFU/g reduction by d 1 and complete reduction by d 5 (P < 0.05). By d 3, 2% CIT and 2% LG resulted in SH reduction of at least 1.7 log10 CFU/g (P < 0.05). Addition of 1% TC resulted in reduction of at least 1.8 log10 CFU/g by d 3 (P < 0.05). Participants could distinguish PDA-treated raw patties by aroma. Most participants (7/11) could not distinguish patties treated with 0.5% TC based on appearance. Microscopic images indicate that all PDAs resulted in disruption of the SH membrane. Results of the present study indicate that the three tested PDAs, LG, CIT, and TC are effective against SH in ground turkey, indicating their potential use as interventions to mitigate Salmonella contamination in comminuted turkey products.

Effect of lemongrass essential oil against multidrug-resistant Salmonella Heidelberg and its attachment to chicken skin and meat.

Salmonella Heidelberg (S. Heidelberg) is a major pathogen implicated in foodborne outbreaks for which poultry products can serve as an epidemiological source. This study determined the efficacy of GRAS-status lemongrass essential oil (LGEO) against S. Heidelberg in vitro and on the pathogen's attachment to skin and meat. At first, employing in vitro assays, the effect of LGEO on multidrug-resistant S. Heidelberg multiplication and motility was examined. Biofilm inhibition and inactivation assays were also performed. The quorum-sensing modulating effect of LGEO was determined. In follow-up experiments, chicken skin or meat samples inoculated with S. Heidelberg were treated with various concentrations of LGEO at different time points at simulated scalding (54°C) and chilling (4°C) temperatures. The samples were incubated, and the surviving populations of S. Heidelberg were enumerated to determine if LGEO could be a potential processing aid in poultry operations. Duplicate samples were included in each treatment, and the experiments were repeated at least 3 times. Significant reductions of S. Heidelberg of at least 4.0 log10 CFU/mL after 24 h in nutrient broth and poultry cecal contents was observed with 0.5% LGEO. Complete inhibition of motility, biofilm formation, and inactivation of pre-formed biofilms was observed with 0.15% LGEO (P ≤ 0.05). Concentrations of LGEO at 0.5% and 1% affected violacein production (P ≤ 0.05). On skin samples, all concentrations significantly reduced S. Heidelberg by 1.2 to 3.9 log10 CFU/sample after 2 min at 54°C. We obtained a significant reduction of the pathogen in meat samples at 54°C and skin samples at 4°C with 2% LGEO. All concentrations significantly reduced S. Heidelberg from the treatment water kept at 4°C and 54°C (P ≤ 0.05). In conclusion, LGEO could potentially serve as a natural antimicrobial strategy in scalding and chilling waters to reduce S. Heidelberg during processing. However, additional studies are warranted before recommending its commercial use.

Effect of caprylic acid alone or in combination with peracetic acid against multidrug-resistant Salmonella Heidelberg on chicken drumsticks in a soft scalding temperature-time setup.

The antimicrobial efficacy of caprylic acid (CA), a medium-chain fatty acid, against multidrug-resistant Salmonella Heidelberg (MDR SH) on chicken drumsticks in a soft-scalding temperature-time setup was investigated. Based on the standardization experiments in nutrient media and on chicken breast fillet portions, intact chicken drumsticks were spot inoculated with MDR SH and immersed in water with or without antimicrobial treatments at 54°C for 2 min. The treatments included 0.5% CA, 1% CA, 0.05% peracetic acid (PAA), 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA. Additionally, the efficacy of the potential scald treatments against MDR SH survival on drumsticks for a storage period of 48 h at 4°C was determined. Furthermore, the effect of these treatments on the surface color of the drumsticks was also evaluated. Appropriate controls were included for statistical comparisons. The antimicrobial treatments resulted in a significant reduction of MDR SH on drumsticks. For the lower inoculum (∼2.5 log10 CFU/g) experiments, 0.5% CA, 1% CA, 0.05% PAA, 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA resulted in 0.7-, 1.0-, 2.5-, 1.4-, and 1.5- log10 CFU/g reduction of MDR SH on drumsticks, respectively (P < 0.05). The same treatments resulted in 0.9-, 1.3-, 2.5-, 2.2-, and 2.6- log10 CFU/g reduction of MDR SH when the drumsticks were contaminated with a higher inoculum (∼4.5 log10 CFU/g) level (P < 0.05). Moreover, the antimicrobial treatments inactivated MDR SH in the treatment water to undetectable levels, whereas 2.0- to 4.0- log10 CFU/mL MDR SH survived in the positive controls (P < 0.05). Also, the treatments were effective in inhibiting MDR SH on the drumsticks compared to the respective controls during a storage period of 48 h at 4°C; however, the magnitude of reduction remained the same as observed during the treatment (P < 0.05). Additionally, none of the treatments affected the color of the drumsticks (P > 0.05). Results indicate that CA could be an effective natural processing aid against MDR SH on chicken products.

Trans-Cinnamaldehyde and Eugenol Increase Acinetobacter baumannii Sensitivity to Beta-Lactam Antibiotics.

Multi-drug resistant (MDR) Acinetobacter baumannii is a major nosocomial pathogen causing a wide range of clinical conditions with significant mortality rates. A. baumannii strains are equipped with a multitude of antibiotic resistance mechanisms, rendering them resistant to most of the currently available antibiotics. Thus, there is a critical need to explore novel strategies for controlling antibiotic resistance in A. baumannii. This study investigated the efficacy of two food-grade, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde (TC) and eugenol (EG) in decreasing A. baumannii's resistance to seven ß-lactam antibiotics, including ampicillin, methicillin, meropenem, penicillin, aztreonam, amoxicillin, and piperacillin. Two MDR A. baumannii isolates (ATCC 17978 and AB 251847) were separately cultured in tryptic soy broth (∼6 log CFU/ml) containing the minimum inhibitory concentration (MIC) of TC or EG with or without the MIC of each antibiotic at 37°C for 18 h. A. baumannii strains not exposed to the PDAs or antibiotics served as controls. Following incubation, A. baumannii counts were determined by broth dilution assay. In addition, the effect of PDAs on the permeability of outer membrane and efflux pumps in A. baumannii was measured. Further, the effect of TC and EG on the expression of A. baumannii genes encoding resistance to ß-lactam antibiotics (blaP), efflux pumps (adeABC), and multi-drug resistant protein (mdrp) was studied using real-time quantitative PCR (RT-qPCR). The experiment was replicated three times with duplicate samples of each treatment and control. The results from broth dilution assay indicated that both TC and EG in combination with antibiotics increased the sensitivity of A. baumannii to all the tested antibiotics (P < 0.05). The two PDAs inhibited the function of A. baumannii efflux pump, (AdeABC), but did not increase the permeability of its outer membrane. Moreover, RT-qPCR data revealed that TC and EG down-regulated the expression of majority of the genes associated with ß-lactam antibiotic resistance, especially blaP and adeABC (P < 0.05). The results suggest that TC and EG could potentially be used along with ß-lactam antibiotics for controlling MDR A. baumannii infections; however, their clinical significance needs to be determined using in vivo studies.

Inhibition and Inactivation of Escherichia coli O157:H7 Biofilms by Selenium.

The present study investigated the efficacy of selenium (Se) in reduction of enterohemorrhagic Escherichia coli (EHEC) exopolysaccharide (EPS) synthesis, inhibition of biofilm formation at 25 and 4°C on polystyrene surface, and inactivation of mature EHEC biofilms in combination with hot water. Sterile 96-well polystyrene plates inoculated with EHEC (∼6.0 log CFU per well) were treated with a subinhibitory concentration (SIC) of Se, and biofilms were allowed to mature at 4 and 25°C for 96 h. Biofilm-associated bacterial population was determined by scraping and plating, whereas the extent of EPS production was determined using ruthenium red staining assay. Solid surface assay was used to study the effect of Se on early attachment of EHEC cells to polystyrene. The efficacy of Se in rapid inactivation of preformed, mature EHEC biofilm was investigated by treating biofilms on polystyrene plates with the MBC of Se in combination with hot water at 80°C with a contact time of 0 min, 30 s, 2 min, and 5 min. Furthermore, the effect of Se on EHEC biofilm architecture was visualized using confocal microscopy, whereas the effect of Se on EHEC biofilm genes was determined using real-time quantitative PCR (RT-qPCR). Finally, the potential feasibility of coating stainless steel surfaces with Se nanoparticles to inhibit EHEC biofilm formation was studied. Se reduced early attachment of planktonic cells, biofilm formation, and EPS synthesis in EHEC ( P < 0.05). Se in combination with hot water reduced biofilm-associated bacterial counts by 3 to 4 log CFU/mL at 5 min of exposure compared with the control ( P < 0.05). However, hot water treatment alone decreased biofilm-associated bacterial counts by only 1.0 log CFU/mL. RT-qPCR results revealed that Se down-regulated the transcription of critical genes associated with biofilm synthesis in EHEC ( P < 0.05). The results collectively suggest that Se could potentially be used to control EHEC biofilms in food processing environments, but appropriate applications need to be validated.

Characterizing the Antimicrobial Function of a Dairy-Originated Probiotic, Propionibacterium freudenreichii, Against Multidrug-Resistant Salmonella enterica Serovar Heidelberg in Turkey Poults.

Antimicrobial potential of a dairy-origin probiotic bacteria, Propionibacterium freudenreichii, against multidrug-resistant Salmonella Heidelberg (SH) in turkey poults was determined in the current study. Employing in vitro experiments, two strains (subsp.) of P. freudenreichii: P. freudenreichii freudenreichii B3523 (PF) and P. freudenreichii shermanii B4327 (PS) were tested for their ability to resist low pH (2.5) and bile salts (0.3%). In addition, the ability of the strains to adhere to and invade avian epithelial cells was determined after exposure to Propionibacterium strains followed by SH challenge. Moreover, the antibacterial activity of the strains' cell-free culture supernatants (CFCSs) were tested against three major foodborne pathogens, including SH. Furthermore, the susceptibility of the strains to common antibiotics used for human therapy was determined. The hemolytic properties of the strains were determined in comparison to Streptococcus pyogenes, a known hemolysis-causing pathogen. Appropriate controls were kept in all studies. Using two in vivo experiments, PF was tested against SH colonization of poult ceca and dissemination to liver and spleen. The four treatment groups were: negative control, PF control (PFC), SH control (SC), and a test group (PFS; PF + SH). The poults in the PFC and PFS groups were inoculated with 1010 CFU ml-1 PF on day 1 through crop gavage and subsequently supplemented through drinking water. On day 7, SC and PFS groups were challenged with SH at 106 CFU ml-1, and after 7 days, cecum, liver, and spleen were collected for determining surviving SH populations. Results indicated that both PF and PS resisted pH = 2.5 and 0.3% bile salts with surviving populations comparable to the control and adhered well onto the avian epithelial cell lines. The strains were susceptible to antibiotics and did not invade the epithelial cells or exhibit hemolytic properties. The CFCSs were highly bactericidal against all tested pathogens. In turkey poults, PF significantly reduced cecal colonization of SH and the dissemination of the pathogen to the liver, compared to the SH challenge controls (P < 0.05). Results revealed that PF, a non-host gastrointestinal tract-derived probiotic, could be an antibiotic alternative to prevent the early colonization of SH in poults, improving the preharvest safety of turkeys.

Antibiotic-Resistant Salmonella in the Food Supply and the Potential Role of Antibiotic Alternatives for Control.

Salmonella enterica is one of the most ubiquitous enteropathogenic bacterial species on earth, and comprises more than 2500 serovars. Widely known for causing non-typhoidal foodborne infections (95%), and enteric (typhoid) fever in humans, Salmonella colonizes almost all warm- and cold-blooded animals, in addition to its extra-animal environmental strongholds. The last few decades have witnessed the emergence of highly virulent and antibiotic-resistant Salmonella, causing greater morbidity and mortality in humans. The emergence of several Salmonella serotypes resistant to multiple antibiotics in food animals underscores a significant food safety hazard. In this review, we discuss the various antibiotic-resistant Salmonella serotypes in food animals and the food supply, factors that contributed to their emergence, their antibiotic resistance mechanisms, the public health implications of their spread through the food supply, and the potential antibiotic alternatives for controlling them.

Food Grade Pimenta Leaf Essential Oil Reduces the Attachment of Salmonella enterica Heidelberg (2011 Ground Turkey Outbreak Isolate) on to Turkey Skin.

Salmonella attached to the poultry skin is a major source of carcass contamination during processing. Once attached to the poultry skin, it is difficult to detach and inactivate Salmonella by commonly used antimicrobial agents since the pathogen is entrapped deeply in the feather follicles and the crevices on the skin. Essential oils could be natural, safe, and effective alternatives to synthetic antimicrobial agents during commercial and organic processing setup. The present study evaluated the efficacy of pimenta (Pimenta officinalis Lindl.) leaf essential oil (PEO), and its nanoemulsion in reducing Salmonella Heidelberg attachment on to turkey (Meleagris gallopavo) skin during simulated scalding (65°C) and chilling (4°C) steps in poultry processing. A multidrug resistant S. Heidelberg isolate from the 2011 ground turkey outbreak in the United States was used in the study. Results showed that PEO and the nanoemulsion resulted in significant reduction of S. Heidelberg attachment on turkey skin. Turkey skin samples treated with 1.0% PEO for 5 min resulted in >2 log10 CFU/sq. inch reduction of S. Heidelberg at 65 and 4°C, respectively (n = 6; P < 0.05). Similarly, skin samples treated with 1.0% pimenta nanoemulsion (PNE) for 5 min resulted in 1.5- and 1.8- log10 CFU/sq. inch reduction of S. Heidelberg at 65 and 4°C, respectively (n = 6; P < 0.05). In addition, PEO and PNE were effective in reducing S. Heidelberg on skin during short-term storage at 4 and 10°C (temperature abuse) (n = 6; P < 0.05). No Salmonella was detected in the dipping solution containing 0.5 or 1.0% PEO or PNE, whereas a substantial population of the pathogen survived in the control dipping solution. The results were validated using scanning electron -, and confocal - microscopy techniques. PEO or PNE could be utilized as an effective antimicrobial agent to reduce S. Heidelberg attachment to turkey skin during poultry processing.