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.

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.

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.

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.

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.

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.

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.

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.

Plant-derived antimicrobials reduce Listeria monocytogenes virulence factors in vitro, and down-regulate expression of virulence genes.

Listeria monocytogenes (LM) is a major foodborne pathogen causing septicemia, meningitis and death in humans. LM infection is preceded by its attachment to and invasion of human intestinal epithelium followed by systemic spread. The major virulence factors in LM include motility, hemolysin and lecithinase production. Reducing LM attachment to and invasion of host tissue and production of virulence factors could potentially control listeriosis in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) of three, generally regarded as safe (GRAS)-status, plant-derived antimicrobial compounds in reducing LM attachment to and invasion of human colon adenocarcinoma (Caco-2) and human brain microvascular endothelial cells (HBMEC). Additionally, the effect of these compounds on the aforementioned LM virulence factors was studied. The compounds and their respective SICs used relative to their MICs were trans-cinnamaldehyde (TC 0.50mM, 0.75mM with the MIC of 0.90mM), carvacrol (CR 0.50mM, 0.65mM with the MIC of 0.75mM), and thymol (TY 0.33mM, 0.50mM with the MIC of 0.60mM). All three-plant antimicrobials reduced LM adhesion to and invasion of Caco-2 and HBMEC (p<0.05). The compounds also decreased LM motility, hemolysin production and lecithinase activity (p<0.05). Real-time PCR data revealed that TC, CR, and TY down-regulated the expression of LM virulence genes by >3.0 folds compared to controls (p<0.05). Results suggest that TC, CR, and TY could potentially be used to control LM infection; however, in vivo studies are necessary to validate these results.