Efficacy of Plant-Derived Antimicrobials in Controlling Enterohemorrhagic Escherichia coli Virulence In Vitro.

Escherichia coli O157:H7 is a major foodborne pathogen that can cause serious human illness characterized by hemorrhagic diarrhea and kidney failure. The pathology of enterohemorrhagic E. coli O157:H7 (EHEC) infection is primarily mediated by verotoxins, which bind to the globotriaosylceramide receptor on host cells. Antibiotics are contraindicated for treating EHEC infection because they lead to increased verotoxin release, thereby increasing the risk of renal failure and death in patients. Thus, alternative strategies are needed for controlling EHEC infections in humans. This study investigated the effect of subinhibitory concentrations of five plant-derived antimicrobial agents (PDAs) that are generally considered as safe, i.e., trans-cinnamaldehyde, eugenol, carvacrol, thymol, and ß-resorcylic acid, on EHEC motility, adhesion to human intestinal epithelial cells, verotoxin production, and virulence gene expression. All tested PDAs reduced EHEC motility and attachment to human intestinal epithelial cells (P < 0.05) and decreased verotoxin synthesis by EHEC. The reverse transcription real-time PCR data revealed that PDAs decreased the expression of critical virulence genes in EHEC (P < 0.05). The results collectively suggest that these PDAs could be used to reduce EHEC virulence, but follow-up studies in animal models are necessary to validate these findings.

Anticarcinogenic properties of medium chain fatty acids on human colorectal, skin and breast cancer cells in vitro.

Colorectal cancer, breast cancer and skin cancer are commonly-reported cancer types in the U.S. Although radiation and chemotherapy are routinely used to treat cancer, they produce side effects in patients. Additionally, resistance to chemotherapeutic drugs has been noticed in cancers. Thus, there is a need for effective and safe bioprophylactics and biotherapeutics in cancer therapy. The medicinal value of goat milk has been recognized for centuries and is primarily attributed to three fatty acids, namely capric, caprylic and caproic acids. This research investigates the anticancer property of these fatty acids on human colorectal, skin and mammary gland cancer cells. The cancer cells were treated with various concentrations of fatty acids for 48 h, and cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Additionally, real-time quantitative PCR (RT-qPCR) was performed to elucidate the potential anti-cancer mechanisms of the three fatty acids under investigation. Capric, caprylic and caproic acids reduced cancer cell viability by 70% to 90% (p < 0.05) compared to controls. RT-qPCR data indicated that these natural molecules produced anticancer effects by down-regulating cell cycle regulatory genes and up-regulating genes involved in apoptosis. Future research will validate the anticancer effect of these fatty acids in an appropriate in vivo model.

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.

Efficacy of plant-derived antimicrobials as antimicrobial wash treatments for reducing enterohemorrhagic Escherichia coli O157:H7 on apples.

This study investigated the efficacy of 3 GRAS-status, plant-derived antimicrobials (PDAs), trans-cinnamaldehyde (TC), carvacrol (CR), and ß-resorcylic acid (BR) applied as an antimicrobial wash for killing Escherichia coli O157:H7 on apples. "Red delicious" apples inoculated with a 5 strain mixture of E. coli O157:H7 were subjected to washing in sterile deionized water containing 0% PDA (control), 0.15% TC, 0.35% TC, 0.15% CR, 0.30% CR, 0.5% BR, or 1% BR for 1, 3, and 5 min at 23 °C in the presence and absence of 1% soil, and surviving pathogen populations on apples were enumerated at each specified time. All PDAs were more effective in reducing E. coli O157:H7 compared to the water wash treatment (P < 0.05) and reduced the pathogen by 4- to 5-log CFU/apple in 5 min. Chlorine (1%) was the most effective treatment reducing the pathogen on apples to undetectable levels in 1 min (P < 0.05). Moreover, the antimicrobial effect of CR and BR was not affected by the presence of soil, whereas the efficacy of TC and BR was decreased in the presence of soil. Further, no bacteria were detected in the wash solution containing CR and BR; however, E. coli O157:H7 was recovered in the control wash water and treatment solutions containing TC and chlorine, in the presence of 1% soil (P < 0.05). Results suggest that the aforementioned PDAs, especially CR and BR could be used effectively to kill E. coli O157:H7 on apples when used as a wash treatment. Studies on the sensory and quality characteristics of apples treated with PDAs are needed before recommending their usage.

Inactivation of Escherichia coli O157:H7 on cattle hides by caprylic acid and ß-resorcylic acid.

Two naturally occurring, generally recognized as safe compounds, namely, caprylic acid (CA) (1%) and b -resorcylic acid (BR) (1%), and their combination, applied at 23 and 60°C were evaluated for their antimicrobial effects against Escherichia coli O157:H7 on cattle hides in the presence and absence of bovine feces. Fresh cleaned cattle hides were cut into pieces (5 cm(2)), air dried, and inoculated with a five-strain mixture of nalidixic acid-resistant (50 µg/ml) E. coli O157:H7 (∼8.0 log CFU). The hide samples were air dried under a biosafety hood for 2 h and sprayed with 95% ethanol, 1% CA, 1% BR, or a mixture of 1% CA and 1% BR at 23 or 60°C. The hide samples were kept at 23°C, and E. coli O157:H7 populations were determined at 2 and 5 min after treatment. Both CA and BR were effective in decreasing E. coli O157:H7 populations on hides by 3 to 4 log CFU/cm(2) (P < 0.05). Sterile bovine feces had no effect on the decontaminating property of CA and BR on cattle hides (P > 0.05). Results of this study indicate that CA and BR could potentially be used to decontaminate cattle hides, but follow-up research under slaughterhouse conditions is warranted.

Efficacy of octenidine hydrochloride for reducing Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on cattle hides.

The efficacy of octenidine hydrochloride (OH; 0.025, 0.15, and 0.25%) for inactivating Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on cattle hides was investigated at 23°C in the presence and absence of bovine feces. All tested concentrations of OH were effective in decreasing more than 5.0 log CFU of bacteria/cm(2) in 5 min (P < 0.01). The results suggest that OH could be used to decontaminate cattle hides; however, further studies under commercial settings are necessary to validate these results.

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.

Enhancing the thermal destruction of Escherichia coli O157:H7 in ground beef patties by trans-cinnamaldehyde.

The effect of trans-cinnamaldehyde (TC) on the inactivation of Escherichia coli O157:H7 in undercooked ground beef patties was investigated. A five-strain mixture of E. coli O157:H7 was inoculated into ground beef (7.0log CFU/g), followed by addition of TC (0, 0.15, and 0.3%). The meat was formed into patties and stored at 4 degrees C for 5 days or at -18 degrees C for 7 days. The patties were cooked to an internal temperature of 60 or 65 degrees C, and E. coli O157:H7 was enumerated. The numbers of E. coli O157:H7 did not decline during storage of patties. However, cooking of patties containing TC significantly reduced (P<0.05) E. coli O157:H7 counts, by >5.0log CFU/g, relative to the reduction in controls cooked to the same temperatures. The D-values at 60 and 65 degrees C of E. coli O157:H7 in TC-treated patties (1.85 and 0.08min, respectively) were significantly lower (P<0.05) than the corresponding D-values for the organism in control patties (2.70 and 0.29min, respectively). TC-treated patties were more color stable and showed significantly lower lipid oxidation (P<0.05) than control samples. TC enhanced the heat sensitivity of E. coli O157:H7 and could potentially be used as an antimicrobial for ensuring pathogen inactivation in undercooked patties. However detailed sensory studies will be necessary to determine the acceptability to consumers of TC in ground beef patties.

Antibiofilm effect of trans-cinnamaldehyde on uropathogenic Escherichia coli.

PURPOSE: Urinary tract infections are the most common hospital acquired infections in humans, caused primarily by uropathogenic Escherichia coli. Indwelling urinary catheters for bladder drainage in humans become encrusted with uropathogenic E. coli biofilms that are resistant to common antibiotics, resulting in chronic infections. We studied the efficacy of the cinnamon ingredient trans-cinnamaldehyde (Sigma) for preventing uropathogenic E. coli biofilm. We also determined the efficacy of trans-cinnamaldehyde as an ingredient in catheter lock solution to inactivate preformed uropathogenic E. coli biofilm. MATERIALS AND METHODS: Polystyrene plates and urinary catheters inoculated with uropathogenic E. coli (5 to 6.0 log cfu) were treated with trans-cinnamaldehyde (0%, 0.1%, 0.25% or 0.5%) at 37C. Catheters with uropathogenic E. coli biofilm were also treated with lock solution containing trans-cinnamaldehyde (0%, 1%, 1.25% or 1.5%). Uropathogenic E. coli biofilm on control and trans-cinnamaldehyde treated plates and catheters was determined on incubation days 0, 1, 3 and 5. Trans-cinnamaldehyde potential cytotoxity, if any, was determined in HTB-4 bladder epithelial cells (ATCC). RESULTS: At all concentrations trans-cinnamaldehyde effectively prevented uropathogenic E. coli biofilm on plates and catheters. As a constituent in catheter lock solution, it inactivated uropathogenic E. coli biofilm on catheters. Trans-cinnamaldehyde produced no cytotoxic effects on human bladder epithelial cells at the tested concentrations. CONCLUSIONS: Results suggest that trans-cinnamaldehyde may be applied as a catheter surface coating or as an ingredient in catheter lock solution to prevent urinary tract infection in humans.

Inactivation of Escherichia coli O157:H7 in apple juice and apple cider by trans-cinnamaldehyde.

This study investigated the antimicrobial effect of low concentrations of trans-cinnamaldehyde (TC) on Escherichia coli O157:H7 in apple juice and apple cider. A five-strain mixture of E. coli O157:H7 was inoculated into apple juice or cider at approximately 6.0 log CFU/ml, followed by the addition of TC (0%v/v, 0.025%v/v, 0.075%v/v and 0.125%v/v). The inoculated apple juice samples were incubated at 23 degrees C and 4 degrees C for 21 days, whereas the cider samples were stored only at 4 degrees C. The pH of apple juice and cider, and E. coli O157:H7 counts were determined on days 0, 1, 3, 5, 7, 14 and 21. TC was effective (P<0.05) in inactivating E. coli O157:H7 in apple juice and apple cider. At 23 degrees C, 0.125 and 0.075%v/v TC completely inactivated E. coli O157:H7 in apple juice (negative by enrichment) on days 1 and 3, respectively. At 4 degrees C, 0.125 and 0.075%v/v TC decreased the pathogen counts in the juice and cider to undetectable levels on days 3 and 5, respectively. Results indicate that low concentrations of TC could be used as an effective antimicrobial to inactivate E. coli O157:H7 in apple juice and apple cider.

Prophylactic supplementation of caprylic acid in feed reduces Salmonella enteritidis colonization in commercial broiler chicks.

Salmonella Enteritidis is a major foodborne pathogen for which chickens serve as reservoir hosts. Reducing Salmonella Enteritidis carriage in chickens would reduce contamination of poultry meat and eggs with this pathogen. We investigated the prophylactic efficacy of feed supplemented with caprylic acid (CA), a natural, generally recognized as safe eight-carbon fatty acid, for reducing Salmonella Enteritidis colonization in chicks. One hundred commercial day-old chicks were randomly divided into five groups of 20 birds each: CA control (no Salmonella Enteritidis, CA), positive control (Salmonella Enteritidis, no CA), negative control (no Salmonella Enteritidis, no CA), and 0.7 or 1% CA. Water and feed were provided ad libitum. On day 8, birds were inoculated with 5.0 log CFU of Salmonella Enteritidis by crop gavage. Six birds from each group were euthanized on days 1, 7, and 10 after challenge, and Salmonella Enteritidis populations in the cecum, small intestine, cloaca, crop, liver, and spleen were enumerated. The study was replicated three times. CA supplementation at 0.7 and 1% consistently decreased Salmonella Enteritidis populations recovered from the treated birds. Salmonella Enteritidis counts in the tissue samples of CA-treated chicks were significantly lower (P < 0.05) than those of control birds on days 7 and 10 after challenge. Feed intake and body weight did not differ between the groups. Histological examination revealed no pathological changes in the cecum and liver of CA-supplemented birds. The results suggest that prophylactic CA supplementation through feed can reduce Salmonella Enteritidis colonization in day-old chicks and may be a useful treatment for reducing Salmonella Enteritidis carriage in chickens.

Reduction of Escherichia coli O157:H7 in cattle drinking-water by trans-cinnamaldehyde.

Cattle serve as a major reservoir of E. coli O157:H7 and excrete the pathogen in feces. Environmental persistence of E. coli O157:H7 plays a vital role in its epidemiology on farms, and cattle water troughs are a demonstrated long-term reservoir of E. coli O157:H7 for animals. The objective of this study was to investigate the potential of low concentrations of trans-cinnamaldehyde for killing E. coli O157:H7 in cattle drinking-water. A five-strain mixture of E. coli O157:H7 was inoculated (at approximately 8.0 log colony-forming units [CFU]/mL) into 100 mL samples of well water containing 0, 0.03, 0.05, 0.07, or 0.1% trans-cinnamaldehyde. Additionally, water samples containing (1% w/v) bovine feces or feed were also included. The samples were incubated at 21 degrees , 8 degrees , or 4 degrees C for 7 days and tested for viable E. coli O157:H7 on days 0, 1, 3, 5, and 7. Triplicate samples of each treatment and control were included and the study was replicated twice. All concentrations of trans-cinnamaldehyde were effective in killing E. coli O157:H7 in water, but the magnitude of killing significantly increased with increase in trans-cinnamaldehyde concentration and storage temperature (p < 0.05). The presence of feed or feces in water decreased the antibacterial effect of trans-cinnamaldehyde on E. coli O157:H7 (p < 0.05). This study indicated that trans-cinnamaldehyde is effective in killing E. coli O157:H7 in cattle drinking-water, but detailed palatability studies on cattle intake of water containing the antimicrobial are needed.