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.

Inactivation of Escherichia coli O157:H7 in apple cider by resveratrol and naringenin.

The study investigated the efficacy of two GRAS-status phytochemicals, mega-resveratrol (RV) and naringenin (NG) to inactivate Escherichia coli O157:H7 (EHEC) in apple cider. A five-strain mixture of EHEC (∼7 log CFU/ml) was inoculated into cider, followed by the addition of RV (8.7 mM and 13.0 mM) or NG (7.3 mM and 11.0 mM). The cider samples were stored at 4 °C for 14 days and EHEC was enumerated on days 0,1,5,7 and 14. The deleterious effects of RV and NG on EHEC cells were visualized by scanning electron microscopy (SEM), and RT-qPCR was done to determine the effect of phytochemicals on three known acid resistance (AR) systems of EHEC. NG was more effective than RV and reduced EHEC counts by ∼4.5 log CFU/ml by day 14, whereas RV reduced counts by ∼2.5 log CFU/ml compared to controls (P < 0.05). SEM showed that RV and NG resulted in the destruction of EHEC cells, and surviving bacteria appeared 'lemon shaped'. RT-qPCR results revealed that RV and NG downregulated the transcription of AR associated genes in EHEC (P < 0.05). Results suggest the potential use of RV and NG as natural antimicrobial additives to enhance the microbiological safety of apple cider. However, sensory analysis studies are warranted.

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.

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.

Inactivation of Salmonella enteritidis on shell eggs by coating with phytochemicals.

Salmonella enteritidis (SE) is a major foodborne pathogen that causes human infections largely by consumption of contaminated eggs. The external surface of eggs becomes contaminated with SE from multiple sources, highlighting the need for effective egg surface disinfection methods. This study investigated the efficacy of three GRAS-status, phytochemicals, namely carvacrol (CR), eugenol (EG), and ß-resorcylic acid (BR) applied as pectin or gum arabic based coating for reducing SE on shell eggs. White-shelled eggs, spot inoculated with a 5-strain mixture of nalidixic acid (NA) resistant SE (8.0 log CFU/mL) were coated with pectin or gum arabic solution containing each phytochemical (0.0, 0.25, 0.5, or 0.75%), and stored at 4°C for 7 days. SE on eggs was enumerated on days 0, 1, 3, and 7 of storage. Approximately 4.0 log CFU/egg of SE was recovered from inoculated and pectin or gum arabic coated eggs on day 0. All coating treatments containing CR and EG, and BR at 0.75% reduced SE to undetectable levels on day 3 (P < 0.05). Results suggest that the aforementioned phytochemicals could effectively be used as a coating to reduce SE on shell eggs, but detailed studies on the sensory and quality attributes of coated eggs need to be conducted before recommending their use.

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.

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.

Antimicrobial eugenol nanoemulsion prepared by gum arabic and lecithin and evaluation of drying technologies.

The purpose of present work was to develop eugenol oil nanoemulsions using gum arabic and lecithin as food grade natural emulsifiers, and study their antimicrobial activity. In addition, our study also evaluated different drying techniques (spray drying and freeze drying) on the morphology and redispersibility of nanoemulsion powders. The optimal fabrication method, physicochemical and structural characterization, stability, and antimicrobial activity were investigated. Results showed that nanoemusions with a particle size of 103.6±7.5nm were obtained by mixing aqueous phase (0.5% gum arabic, 0.5% lecithin, w/v) and eugenol oil (1.25%, w/v), which was premixed with ethanol (as a co-surfactant), followed by high speed homogenization process. The molecular interactions among emulsifiers and eugenol were evidenced by Fourier transform infrared spectroscopy. Buchi B-90 Nano Spray Dryer was evaluated as a powerful tool to obtain ultrafine spherical powders with a size of less than 500nm, compared to flake-like aggregation obtained by freeze-drying. The dried powders exhibited excellent re-dispersibility in water and maintained their physicochemical properties after re-hydration. The nanoemulsions did not adversely affect the antimicrobial activity of eugenol against Listeria monocytogenes and Salmonella Enteritidis. Therefore, the nanoemulsions have the potential to be applied in the food industry as a food preservative or sanitizer.

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.

Characterization of Haptoglobin Isotype in Milk of Mastitis-Affected Cows.

Haptoglobin is a major acute phase protein in bovines and reportedly increases in serum and milk whey during mastitis, highlighting its potential as a diagnostic biomarker. Since haptoglobin is known to undergo tissue specific glycosylation resulting in different isoforms, this study was undertaken to characterize the isoforms of haptoglobin. Milk whey fraction and serum obtained from animals with or without clinical mastitis in Puducherry, India, were subjected to SDS-PAGE followed by western blot and immuno-detection of haptoglobin protein. All subunits (ß, α1 and α2) of haptoglobin protein were detected in serum sample obtained from clinical cases. However, only the ß-subunit was detected in milk whey fraction obtained from the respective animals. Similar results were observed with milk whey fractions from subclinical cases indicating difference in isoform of haptoglobin detected in milk whey from serum. This was further supported by RT-PCR (Reverse Transcription Polymerase Chain Reaction) analysis of haptoglobin gene (Hp) confirming the tissue specific origin of haptoglobin.

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.

Characterization of a multidrug resistant C. difficile meat isolate.

Clostridium difficile is a pathogen of significant public health concern causing a life-threatening, toxin-mediated enteric disease in humans. The incidence and severity of the disease associated with C. difficile have increased in the US with the emergence of hypervirulent strains and community associated outbreaks. The detection of genotypically similar and identical C. difficile strains implicated from human infections in foods and food animals indicates the potential role of food as a source of community associated C. difficile disease. One hundred samples each of ground beef, pork and chicken obtained from geographically distant grocery stores in Connecticut were tested for C. difficile. Positive isolates were characterized by ribotyping, antibiotic susceptibility, toxin production and whole genome sequencing. Of the 300 meat samples, only two pork samples tested positive for C. difficile indicating a very low prevalence of C. difficile in meat. The isolates were non toxigenic; however, genome characterization revealed the presence of several antibiotic resistance genes and mobile elements that can potentially contribute to generation of multidrug resistant toxigenic C. difficile by horizontal gene transfer. Further studies are warranted to investigate potential food-borne transmission of the meat isolates and development of multi-drug resistance in these strains.

Combating pathogenic microorganisms using plant-derived antimicrobials: a minireview of the mechanistic basis.

The emergence of antibiotic resistance in pathogenic bacteria has led to renewed interest in exploring the potential of plant-derived antimicrobials (PDAs) as an alternative therapeutic strategy to combat microbial infections. Historically, plant extracts have been used as a safe, effective, and natural remedy for ailments and diseases in traditional medicine. Extensive research in the last two decades has identified a plethora of PDAs with a wide spectrum of activity against a variety of fungal and bacterial pathogens causing infections in humans and animals. Active components of many plant extracts have been characterized and are commercially available; however, research delineating the mechanistic basis of their antimicrobial action is scanty. This review highlights the potential of various plant-derived compounds to control pathogenic bacteria, especially the diverse effects exerted by plant compounds on various virulence factors that are critical for pathogenicity inside the host. In addition, the potential effect of PDAs on gut microbiota is discussed.

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.

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.