Shankumar Mooyottu

Carvacrol and trans-cinnamaldehyde reduce Clostridium difficile toxin production and cytotoxicity in vitro

Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.

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.

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.

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.

Oral Supplementation of Trans‐Cinnamaldehyde Reduces Uropathogenic Escherichia coli colonization in a Mouse Model

Urinary tract infections (UTIs) in the United States result in more than 7 million hospital visits per year. Uropathogenic Escherichia coli (UPEC) is responsible for more than 80% of UTIs. Although antibiotics are the drug of choice to control UTIs, their repeated use has resulted in the emergence of antibiotic‐resistant UPEC. Thus, there is a need for effective alternate strategies to control UPEC infections. This study investigated the efficacy of trans‐cinnamaldehyde (TC), a food‐grade molecule present in cinnamon, in reducing UPEC colonization and pathogenesis in the lower UTI. Female C57BL/6 mice (6–8 weeks old) were fed ad libitum with 0, 0·1, 0·2 and 0·4% TC containing mouse chow for 10 days. Following TC supplementation, animals were experimentally infected with UPEC by transurethral catheterization. Mice were euthanized on days 1, 2 and 4 postinfection, and the bladder, urethra and urine were collected for bacterial enumeration. Prophylactic TC supplementation significantly (P ≤ 0·05) reduced UPEC colonization in the urinary bladder and urethra compared to the control. Results indicate that TC could potentially be used as an oral supplement to control UPEC‐associated lower UTIs, however, follow‐up clinical trials are warranted.

Carvacrol reduces Clostridium difficile sporulation and spore outgrowth in vitro

Purpose. Clostridium difficile is an anaerobic spore‐forming pathogen that causes a serious toxin‐mediated enteric disease in humans. Therapeutic agents that are capable of reducing C. difficile spore production could significantly minimize the transmission and relapse of C. difficile infections. This study investigated the efficacy of a food‐grade, plant‐derived compound, carvacrol (CR), in reducing C. difficile spore production, germination and spore outgrowth. Methodology. Two hyper‐virulent C. difficile isolates (ATCC BAA 1870 and 1805) were grown with or without a sub‐inhibitory concentration (SIC) of CR. Total viable counts and heat‐resistant spore counts were determined at different time intervals. Moreover, spores and vegetative cells were visualized using phase‐contrast microscopy. To determine the effect of CR on C. difficile germination and spore outgrowth, C. difficile spores were seeded in germination medium with or without the SIC and MIC of CR, and spore germination and spore outgrowth were measured by recording optical density at 600 nm. The effect of CR on C. difficile sporulation genes was also investigated using real‐time qPCR. Results. Carvacrol significantly reduced sporulation in C. difficile and down‐regulated critical genes involved in spore production (P<0.05). The SIC or MIC of CR did not inhibit C. difficile spore germination; however, the MIC of CR completely inhibited spore outgrowth. Conclusion. The results suggest that CR could potentially be used to control C. difficile by reducing spore production and outgrowth.

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.

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.