Nanoemulsified Carvacrol as a Novel Washing Treatment Reduces Escherichia coli O157:H7 on Spinach and Lettuce.

ABSTRACT: Fresh produce continues to be the main source of foodborne illness outbreaks in the United States, implicating bacterial pathogens such as Escherichia coli O157:H7 (EHEC). The efficacy of nanoemulsified carvacrol (NCR) as a washing treatment in reducing EHEC on fresh produce was investigated. Fresh baby spinach, romaine lettuce, and iceberg lettuce leaves (2.5-cm-diameter cores) were spot inoculated with a five-strain cocktail of nalidixic acid-resistant EHEC at ∼6 log CFU/cm2. After air drying for 1 h, 20 pieces of each inoculated produce leaf were immersed in water-based treatment solutions (200 mL per group), including water alone, 25 or 50 ppm of free chlorine, and 0.25 or 0.75% NCR for 2 min. Inoculated produce leaves without any treatment served as baseline. Produce leaves were stored at 10°C, and surviving EHEC populations were enumerated on days 0, 2, 7, and 14. The viability of EHEC following NCR treatments on the fresh produce was visualized under a fluorescence microscope. NCR treatment at 0.75% immediately reduced EHEC populations on iceberg lettuce by 1.3 log CFU/cm2 as compared with the produce treated with water alone (P < 0.05). Antimicrobial activity of NCR against EHEC was comparable to chlorine treatments on day 0 for all produce (P > 0.05). After 14 days of storage at 10°C, populations of EHEC on 0.75% NCR-treated romaine lettuce were reduced by 2.3 log CFU/cm2 compared with the recovery from 50 ppm of chlorine-treated samples (P < 0.05). Microscopic images revealed that EHEC cells were observed to be clustered on the baseline samples, indicating the development of cell aggregation, compared with the scattered cells seen on NCR-treated leaf surfaces. Treatments with NCR did not significantly affect the color of the fresh produce leaves during 14 days of storage at 10°C. Results of this study support the potential use of NCR as a water-soluble natural antimicrobial wash treatment for controlling EHEC on fresh produce.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion.

Sodium Butyrate Reduces Salmonella Enteritidis Infection of Chicken Enterocytes and Expression of Inflammatory Host Genes in vitro.

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1ß, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

Draft Genome Sequences of Campylobacter jejuni Strains Isolated from Poultry.

Four wild-type Campylobacter jejuni strains isolated from the cecal contents of broiler chickens were sequenced. The average genome size was 1,622,170 bp, with 1,667 to 1,761 coding sequences and 47 to 51 RNAs. Multiple genes encoding motility, intestinal colonization, toxin production, stress tolerance, and multidrug resistance were present in all the strains.

Review of Antibiotic Resistance, Ecology, Dissemination, and Mitigation in U.S. Broiler Poultry Systems.

Since the onset of land application of poultry litter, transportation of microorganisms, antibiotics, and disinfectants to new locations has occurred. While some studies provide evidence that antimicrobial resistance (AMR), an evolutionary phenomenon, could be influenced by animal production systems, other research suggests AMR originates in the environment from non-anthropogenic sources. In addition, AMR impacts the effective prevention and treatment of poultry illnesses and is increasingly a threat to global public health. Therefore, there is a need to understand the dissemination of AMR genes to the environment, particularly those directly relevant to animal health using the One Health Approach. This review focuses on the potential movement of resistance genes to the soil via land application of poultry litter. Additionally, we highlight impacts of AMR on microbial ecology and explore hypotheses explaining gene movement pathways from U.S. broiler operations to the environment. Current approaches for decreasing antibiotic use in U.S. poultry operations are also described in this review.

Trans-Cinnamaldehyde, Eugenol and Carvacrol Reduce Campylobacter jejuni Biofilms and Modulate Expression of Select Genes and Proteins.

Campylobacter jejuni is the leading cause of human foodborne illness globally, and is strongly linked with the consumption of contaminated poultry products. Several studies have shown that C. jejuni can form sanitizer tolerant biofilm leading to product contamination, however, limited research has been conducted to develop effective control strategies against C. jejuni biofilms. This study investigated the efficacy of three generally recognized as safe status phytochemicals namely, trans-cinnamaldehyde (TC), eugenol (EG), or carvacrol (CR) in inhibiting C. jejuni biofilm formation and inactivating mature biofilm on common food contact surfaces at 20 and 37°C. In addition, the effect of phytochemicals on biofilm architecture and expression of genes and proteins essential for biofilm formation was evaluated. For the inhibition study, C. jejuni was allowed to form biofilms either in the presence or absence of sub-inhibitory concentrations of TC (0.75 mM), EG (0.61 mM), or CR (0.13 mM) for 48 h and the biofilm formation was quantified at 24-h interval. For the inactivation study, C. jejuni biofilms developed at 20 or 37°C for 48 h were exposed to the phytochemicals for 1, 5, or 10 min and surviving C. jejuni in the biofilm were enumerated. All phytochemicals reduced C. jejuni biofilm formation as well as inactivated mature biofilm on polystyrene and steel surface at both temperatures (P < 0.05). The highest dose of TC (75.64 mM), EG (60.9 mM) and CR (66.56 mM) inactivated (>7 log reduction) biofilm developed on steel (20°C) within 5 min. The genes encoding for motility systems (flaA, flaB, and flgA) were downregulated by all phytochemicals (P < 0.05). The expression of stress response (cosR, ahpC) and cell surface modifying genes (waaF) was reduced by EG. LC-MS/MS based proteomic analysis revealed that TC, EG, and CR significantly downregulated the expression of NapA protein required for oxidative stress response. The expression of chaperone protein DnaK and bacterioferritin required for biofilm formation was reduced by TC and CR. Scanning electron microscopy revealed disruption of biofilm architecture and loss of extracellular polymeric substances after treatment. Results suggest that TC, EG, and CR could be used as a natural disinfectant for controlling C. jejuni biofilms in processing areas.

Edible Coatings Fortified With Carvacrol Reduce Campylobacter jejuni on Chicken Wingettes and Modulate Expression of Select Virulence Genes.

Campylobacter jejuni, a leading cause of foodborne disease in humans, associate primarily with consumption of contaminated poultry and poultry products. Intervention strategies aimed at reducing C. jejuni contamination on poultry products could significantly reduce C. jejuni infection in humans. This study evaluated the efficacy of gum arabic (GA) and chitosan (CH) fortified with carvacrol (CR) as an antimicrobial coating treatment for reducing C. jejuni on chicken wingettes. Aforementioned compounds are generally recognized as safe status compounds obtained from gum arabic tree, crustaceans and oregano oil respectively. A total of four separate trials were conducted in which wingettes were randomly assigned to baseline, saline control (wingettes washed with saline), GA (10%), CH (2%), CR (0.25, 0.5, or 1%) or their combinations. Each wingette was inoculated with a cocktail of four wild-type strains of C. jejuni (∼7.5 log10 cfu/sample). Following 1 min of coating in aforementioned treatments, wingettes were air dried (1 h) and sampled at 0, 1, 3, 5, and 7 days of refrigerated storage for C. jejuni and total aerobic counts (n = 5 wingettes/treatment/day). In addition, the effect of treatments on wingette color was measured using a Minolta colorimeter. Furthermore, the effect of treatments on the expression of C. jejuni survival/virulence genes was evaluated using real-time quantitative PCR. Results showed that all three doses of CR, CH or GA-based coating fortified with CR reduced C. jejuni from day 0 through 7 by up to 3.0 log10 cfu/sample (P < 0.05). The antimicrobial efficacy of GA was improved by CR and the coatings reduced C. jejuni by ∼1 to 2 log10 cfu/sample at day 7. Moreover, CH + CR coatings reduced total aerobic counts when compared with non-coated samples for a majority of the storage times. No significant difference in the color of chicken wingettes was observed between treatments. Exposure of pathogen to sublethal concentrations of CR, CH or combination significantly modulated select genes encoding for energy taxis (cetB), motility (motA), binding (cadF), and attachment (jlpA). The results suggest that GA or CH-based coating with CR could potentially be used as a natural antimicrobial to control C. jejuni in postharvest poultry products.

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.

Effect of Dietary Minerals on Virulence Attributes of Vibrio cholerae.

Vibrio cholerae is a water-borne pathogen responsible for causing a toxin-mediated profuse diarrhea in humans, leading to severe dehydration and death in unattended patients. With increasing reports of antibiotic resistance in V. cholerae, there is a need for alternate interventional strategies for controlling cholera. A potential new strategy for treating infectious diseases involves targeting bacterial virulence rather than growth, where a pathogen's specific mechanisms critical for causing infection in hosts are inhibited. Since bacterial motility, intestinal colonization and cholera toxin are critical components in V. cholerae pathogenesis, attenuating these virulence factors could potentially control cholera in humans. In this study, the efficacy of sub-inhibitory concentration (SIC, highest concentration not inhibiting bacterial growth) of essential minerals, zinc (Zn), selenium (Se), and manganese (Mn) in reducing V. cholerae motility and adhesion to intestinal epithelial cells (Caco-2), cholera toxin production, and toxin binding to the ganglioside receptor (GM1) was investigated. Additionally, V. cholerae attachment and toxin production in an ex vivo mouse intestine model was determined. Further, the effect of Zn, Se and Mn on V. cholerae virulence genes, ctxAB (toxin production), fliA (motility), tcpA (intestinal colonization), and toxR (master regulon) was determined using real-time quantitative PCR. All three minerals significantly reduced V. cholerae motility, adhesion to Caco-2 cells, and cholera toxin production in vitro, and decreased adhesion and toxin production in mouse intestine ex vivo (P < 0.05). In addition, Zn, Se, and Mn down-regulated the transcription of virulence genes, ctxAB, fliA, and toxR. Results suggest that Zn, Se, and Mn could be potentially used to reduce V. cholerae virulence. However, in vivo studies in an animal model are necessary to validate these results.

Trans-Cinnamaldehyde, Carvacrol, and Eugenol Reduce Campylobacter jejuni Colonization Factors and Expression of Virulence Genes in Vitro.

Campylobacter jejuni is a major foodborne pathogen that causes severe gastroenteritis in humans characterized by fever, diarrhea, and abdominal cramps. In the human gut, Campylobacter adheres and invades the intestinal epithelium followed by cytolethal distending toxin mediated cell death, and enteritis. Reducing the attachment and invasion of Campylobacter to intestinal epithelium and expression of its virulence factors such as motility and cytolethal distending toxin (CDT) production could potentially reduce infection in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentration not inhibiting bacterial growth) of three GRAS (generally recognized as safe) status phytochemicals namely trans-cinnamaldehyde (TC; 0.005, 0.01%), carvacrol (CR; 0.001, 0.002%), and eugenol (EG; 0.005, 0.01%) in reducing the attachment, invasion, and translocation of C. jejuni on human intestinal epithelial cells (Caco-2). Additionally, the effect of these phytochemicals on Campylobacter motility and CDT production was studied using standard bioassays and gene expression analysis. All experiments had duplicate samples and were replicated three times on three strains (wild type S-8, NCTC 11168, 81-176) of C. jejuni. Data were analyzed using ANOVA with GraphPad ver. 6. Differences between the means were considered significantly different at P < 0.05. The majority of phytochemical treatments reduced C. jejuni adhesion, invasion, and translocation of Caco-2 cells (P < 0.05). In addition, the phytochemicals reduced pathogen motility and production of CDT in S-8 and NCTC 11168 (P < 0.05). Real-time quantitative PCR revealed that phytochemicals reduced the transcription of select C. jejuni genes critical for infection in humans (P < 0.05). Results suggest that TC, CR, and EG could potentially be used to control C. jejuni infection in humans.

Protective Effect of Carvacrol against Gut Dysbiosis and Clostridium difficile Associated Disease in a Mouse Model.

This study investigated the effect of carvacrol (CR), a phytophenolic compound on antibiotic-associated gut dysbiosis and C. difficile infection in a mouse model. Five to six-week-old C57BL/6 mice were randomly divided into seven treatment groups (challenge and control) of eight mice each. Mice were fed with irradiated feed supplemented with CR (0, 0.05, and 0.1%); the challenge groups were made susceptible to C. difficile by orally administering an antibiotic cocktail in water and an intra-peritoneal injection of clindamycin. Both challenge and control groups were infected with 105CFU/ml of hypervirulent C. difficile (ATCC 1870) spores or PBS, and observed for clinical signs for 10 days. Respective control groups for CR, antibiotics, and their combination were included for investigating their effect on mouse enteric microflora. Mouse body weight and clinical and diarrhea scores were recorded daily post infection. Fecal samples were collected for microbiome analysis using rRNA sequencing in MiSeq platform. Carvacrol supplementation significantly reduced the incidence of diarrhea and improved the clinical and diarrhea scores in mice (p < 0.05). Microbiome analysis revealed a significant increase in Proteobacteria and reduction in the abundance of protective bacterial flora in antibiotic-treated and C. difficile-infected mice compared to controls (p < 0.05). However, CR supplementation positively altered the microbiome composition, as revealed by an increased abundance of beneficial bacteria, including Firmicutes, and significantly reduced the proportion of detrimental flora such as Proteobacteria, without significantly affecting the gut microbiome diversity compared to control. Results suggest that CR could potentially be used to control gut dysbiosis and reduce C. difficile infection.

Application of ß-Resorcylic Acid as Potential Antimicrobial Feed Additive to Reduce Campylobacter Colonization in Broiler Chickens.

Campylobacter is one of the major foodborne pathogens that result in severe gastroenteritis in humans, primarily through consumption of contaminated poultry products. Chickens are the reservoir host of Campylobacter, where the pathogen colonizes the ceca, thereby leading to contamination of carcass during slaughter. A reduction in cecal colonization by Campylobacter would directly translate into reduced product contamination and risk of human infections. With increasing consumer demand for antibiotic free chickens, significant research is being conducted to discover natural, safe and economical antimicrobials that can effectively control Campylobacter colonization in birds. This study investigated the efficacy of in-feed supplementation of a phytophenolic compound, ß-resorcylic acid (BR) for reducing Campylobacter colonization in broiler chickens. In two separate, replicate trials, day-old-chicks (Cobb500; n = 10 birds/treatment) were fed with BR (0, 0.25, 0.5, or 1%) in feed for a period of 14 days (n = 40/trial). Birds were challenged with a four-strain mixture of Campylobacter jejuni (∼106 CFU/ml; 250 µl/bird) on day 7 and cecal samples were collected on day 14 for enumerating surviving Campylobacter in cecal contents. In addition, the effect of BR on the critical colonization factors of Campylobacter (motility, epithelial cell attachment) was studied using phenotypic assay, cell culture, and real-time quantitative PCR. Supplementation of BR in poultry feed for 14 days at 0.5 and 1% reduced Campylobacter populations in cecal contents by ∼2.5 and 1.7 Log CFU/g, respectively (P < 0.05). No significant differences in feed intake and body weight gain were observed between control and treatment birds fed the compound (P > 0.05). Follow up mechanistic analysis revealed that sub-inhibitory concentration of BR significantly reduced Campylobacter motility, attachment to and invasion of Caco-2 cells. In addition, the expression of C. jejuni genes coding for motility (motA, motB, fliA) and attachment (jlpA, ciaB) was down-regulated as compared to controls (P < 0.05). These results suggest that BR could potentially be used as a feed additive to reduce Campylobacter colonization in broilers.

Inactivation of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 on cantaloupes by octenidine dihydrochloride.

The efficacy of a new generation disinfectant, octenidine dihydrochloride (OH), as wash and coating treatments for reducing Listeria monocytogenes (LM), Salmonella spp. (SAL), and Escherichia coli O157:H7 (EC) on cantaloupe was investigated. Cantaloupe rind plugs inoculated separately with the three bacterial species (∼8 log CFU/cm(2)) were washed for 1, 3, 5 min at 25 °C in water, or chlorine (200 ppm), ethanol (1%), OH (0.01, 0.05, 0.1%) and surviving populations were measured after treatment. Additionally, inoculated cantaloupe rind plugs were coated with 2% chitosan or chitosan containing OH (0.01, 0.05, 0.1%) and sampled for surviving pathogens. Subsequently, the antimicrobial efficacy of OH wash and coating (0.1, 0.2%) on whole cantaloupes was determined. All OH wash reduced LM, SAL, and EC on cantaloupe rinds by > 5 log CFU/cm(2) by 2 min, and reduced populations to undetectable levels (below 2 log CFU/cm(2)) by 5 min (P < 0.05). Similarly, OH coating on cantaloupe rinds reduced the pathogens by 3-5 log /cm(2) (P < 0.05). Washing and coating whole cantaloupes with OH reduced the three pathogens by at least 5 log and 2 log CFU/cm(2), respectively (P < 0.05). Results suggest that OH could be used as antimicrobial wash and coating to reduce LM, SAL, and EC on cantaloupes.

In vivo efficacy of trans-cinnamaldehyde, carvacrol, and thymol in attenuating Listeria monocytogenes infection in a Galleria mellonella model.

Listeria monocytogenes is a major foodborne pathogen that causes life-threatening illnesses in humans. With emergence of antibiotic resistance in L. monocytogenes, there is considerable interest in testing the efficacy of alternative therapies for controlling listeriosis in humans. This study investigated the efficacy of three phytochemicals, namely trans-cinnamaldehyde (TC), carvacrol (CR), and thymol (TY) in reducing L. monocytogenes virulence in the recently established invertebrate model, Galleria mellonella. In addition, the effect of phytochemicals on the transcription of antimicrobial peptide genes in G. mellonella (responsible for host defense) was investigated using real-time quantitative polymerase chain reaction. G. mellonella larvae were inoculated with L. monocytogenes (10(5) CFU/larvae) either with or without the subinhibitory concentration (chemical concentration not inhibiting bacterial growth) of phytochemicals. The larvae were incubated at 37 °C for 5 days, and their mortality was scored at 24-h intervals. The transcriptional response of the defense genes was studied in inoculated and uninoculated larvae at 6 h post challenge. The experiments were repeated at least six times with replicates. All phytochemicals enhanced the survival rates of G. mellonella infected with lethal doses of L. monocytogenes (P < 0.05). CR and TC at 0.01 % concentration were found to be the most effective treatments, and increased larval survival rates by 80 % and 50 %, respectively, on day 5 (P < 0.05). The phytochemicals also upregulated the expression of antimicrobial peptide genes in G. mellonella larvae challenged with L. monocytogenes (P < 0.05). Results suggest that TC, CR, and TY could potentially be used to control listeriosis. Further investigation in an appropriate mammalian model is warranted.

Eugenol in combination with lactic acid bacteria attenuates Listeria monocytogenes virulence in vitro and in invertebrate model Galleria mellonella.

Listeria monocytogenes is a human enteric pathogen that causes severe foodborne illness in high-risk populations. Crossing the intestinal barrier is the first critical step for Listeria monocytogenes infection. Therefore, reducing L. monocytogenes colonization and invasion of intestinal epithelium and production of virulence factors could potentially control listeriosis in humans. This study investigated the efficacy of sub-inhibitory concentration (SIC) of the plant-derived antimicrobial eugenol, either alone, or in combination with five lactic acid bacteria (LAB), namely Bifidobacterium bifidum (NRRL-B41410), Lactobacillus reuteri (B-14172), Lactobacillus fermentum (B-1840), Lactobacillus plantarum (B-4496) and Lactococcus lactis subspecies lactis (B-633) in reducing Listeria monocytogenes adhesion to and invasion of human intestinal epithelial cells (Caco-2). Additionally, the effect of the aforementioned treatments on Listeria monocytogenes listeriolysin production, epithelial E-cadherin binding and expression of virulence genes was investigated. Moreover, the in vivo efficacy of eugenol-LAB treatments in reducing Listeria monocytogenes virulence in the invertebrate model Galleria mellonella was studied. Eugenol and LAB, either alone or in combination, significantly reduced Listeria monocytogenes adhesion to and invasion of intestinal cells (P < 0.05). Moreover, eugenol-LAB treatments decreased Listeria monocytogenes haemolysin production, E-cadherin binding and virulence gene expression (P < 0.05). In addition, the eugenol-LAB treatments significantly enhanced the survival rates of G. mellonella infected with lethal doses of Listeria monocytogenes (P < 0.05). The results highlight the antilisterial effect of eugenol either alone or in combination with LAB, and justify further investigations in a mammalian model.

Potentiating the Heat Inactivation of Escherichia coli O157:H7 in Ground Beef Patties by Natural Antimicrobials.

Escherichia coli O157: H7 (EHEC) is a major foodborne pathogen largely transmitted to humans through the consumption of undercooked ground beef. This study investigated the efficacy of two food-grade, plant-derived antimicrobials, namely rutin (RT), and resveratrol (RV) with or without chitosan (CH) in enhancing EHEC inactivation in undercooked hamburger patties. Further, the effect of aforementioned treatments on beef color and lipid oxidation was analyzed. Additionally, the deleterious effects of these antimicrobial treatments on EHEC was determined using scanning electron microscopy (SEM). Ground beef was inoculated with a five-strain mixture of EHEC (7.0 log CFU/g), followed by the addition of RT (0.05%, 0.1% w/w) or RV (0.1, 0.2% w/w) with or without CH (0.01% w/w). The meat was formed into patties (25 g) and stored at 4°C for 5 days. On days 1, 3, and 5, the patties were cooked (65°C, medium rare) and surviving EHEC was enumerated. The effect of these treatments on meat color and lipid oxidation during storage was also determined as per American Meat Science Association guidelines. The study was repeated three times with duplicate samples of each treatment. Both RT and RV enhanced the thermal destruction of EHEC, and reduced the pathogen load by at least 3 log CFU/g compared to control (P < 0.05). The combination of RT or RV with CH was found to be more effective, and reduced EHEC by 5 log CFU/g (P < 0.05). EHEC counts in uncooked patties did not decline during storage for 5 days (P > 0.05). Moreover, patties treated with RV plus CH were more color stable with higher a(∗) values (P < 0.05). SEM results revealed that heat treatment with antimicrobials (CH + RV 0.2%) resulted in complete destruction of EHEC cells and extrusion of intracellular contents. Results suggest that the aforementioned antimicrobials could be used for enhancing the thermal inactivation of EHEC in undercooked patties; however, detailed sensory studies are warranted.

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.

Efficacy of fumigation with Trans-cinnamaldehyde and eugenol in reducing Salmonella enterica serovar Enteritidis on embryonated egg shells.

This study investigated the efficacy of two GRAS (generally regarded as safe)-status, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde (TC) and eugenol (EUG) applied as a fumigation treatment in reducing SE on embryonated egg shells. Egg shells of day-old embryonated eggs were spot inoculated with a 4-strain mixture of SE (∼6.5 log CFU/egg) and subjected to fumigation with the aforementioned PDAs (0 or 1% concentration) for 20 minutes in a hatching incubator. SE on the shell and embryo was enumerated on days 1, 3, 6, 9, 13, 16 and 18. On day 13, the eggs were re-inoculated, followed by fumigation treatment for 20 minutes. Since the two PDAs were dissolved in ethanol (final concentration 0.04%), eggs fumigated with ethanol were included as a control.Approximately 6 log CFU/egg of SE were recovered from the shell of untreated, inoculated eggs on days 1 and 13. The fumigation of embryonated egg shells with the two PDAs was more effective in reducing SE on the shell and embryo compared to controls (P < 0.05). On day 18, the eggs fumigated with ethanol were SE positive on the shell, whereas no pathogen was detected on eggs subjected to fumigation with TC and EUG. Similarly, although the embryos of eggs subjected to fumigation with ethanol yielded 1 log CFU/egg of SE on day 18, the embryos of TC and EUG treated eggs were devoid of the pathogen. This study demonstrated that TC and EUG dissolved in 0.04% ethanol could potentially be used as a fumigation treatment for reducing SE on embryonated egg shell, however, quality traits of eggs, including the hatchability need to be ascertained.

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.

Inhibiting Microbial Toxins Using Plant-Derived Compounds and Plant Extracts.

Many pathogenic bacteria and fungi produce potentially lethal toxins that cause cytotoxicity or impaired cellular function either at the site of colonization or other locations in the body through receptor-mediated interactions. Various factors, including biotic and abiotic environments, competing microbes, and chemical cues affect toxin expression in these pathogens. Recent work suggests that several natural compounds can modulate toxin production in pathogenic microbes. However, studies explaining the mechanistic basis for their effect are scanty. This review discusses the potential of various plant-derived compounds for reducing toxin production in foodborne and other microbes. In addition, studies highlighting their anti-toxigenic mechanism(s) are discussed.