Indu Upadhyaya

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.

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.

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.

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.

In-Feed Supplementation of trans-Cinnamaldehyde Reduces Layer-Chicken Egg-Borne Transmission of Salmonella enterica Serovar Enteritidis

ABSTRACT 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.

Inactivation of Listeria monocytogenes on frankfurters by plant-derived antimicrobials alone or in combination with hydrogen peroxide.

Listeria monocytogenes is a significant foodborne pathogen associated with outbreaks involving contaminated ready-to-eat (RTE) products, including frankfurters. The USDA-FSIS has established a zero tolerance policy for L. monocytogenes in RTE products, thereby warranting effective post-processing interventions to control the pathogen on these foods. In the present study, the antilisterial activity of GRAS (generally recognized as safe)-status plant-derived antimicrobials (PDAs), namely β-resorcylic acid (BR), carvacrol (CR), and trans-cinnamaldehyde (TC) either alone or in combination with hydrogen peroxide (HP) as post-processing dip treatments on frankfurters was investigated. Frankfurters were surface inoculated with a five-strain mixture of L. monocytogenes (~6.0 log CFU per frankfurter), followed by dip treatment at 55 °C for 60s or 65 °C for 30s in sterile deionized water, or water containing BR (1.5%), CR (0.75%), or TC (0.75%) either alone or in combination with HP (0.1%). Treated frankfurters were vacuum-packaged, and stored at 4 °C for 70 days. Representative samples were analyzed on days 0, 1, 3, 7, 14, 28, 42, 56, and 70 of refrigerated storage for enumerating surviving L. monocytogenes on frankfurters. Six frankfurters were sampled at each time point for each treatment. On day zero, all PDAs reduced L. monocytogenes counts by >2 log CFU/frankfurter at both temperatures (P<0.05), compared to controls. From days 1 to 70, L. monocytogenes counts on PDA-treated frankfurters were consistently lower (P<0.05) and after 70 days of storage, the pathogen counts were reduced to undetectable levels on frankfurters treated with PDA-HP combinations at 65 °C, and by combinations of BR and TC with HP at 55 °C. Results suggest that PDAs alone, or in combination with HP could be effectively used as post-processing dips to reduce L. monocytogenes on frankfurters, although follow-up studies on sensory and quality characteristics of PDA-treated frankfurters are necessary.

Efficacy of Octenidine Hydrochloride for Reducing Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on Cattle Hides

ABSTRACT 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/cm2 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.

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.

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.

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.