Progress 07/01/16 to 07/09/18
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop protocols to isolate and identify bioactive compounds from agricultural products such as fruit peels and rinds. Objective 2: Investigate mode of action of bioactive compounds that are able to mitigate virulence in the gut (mouse model), or restrict environmental dissemination and survival of pathogens, utilizing metagenomic, transcriptomic and proteomic analyses. Sub-objective 2.1. Determine influence of bioactive compounds on pathogens during in vitro (i.e., biofilm formation, swarm motility, and survival in harsh environments such as low water activity and low pH) and ex vivo assays (i.e. invasion of cultured human intestinal cells). Sub-objective 2.2. Determine how bioactive compounds impact host microbiome and pathogens during infection in animal models by acquiring and interpreting genomic and transcriptomic data. Approach (from AD-416): Objective 1: Several plant-derived bioactive compounds, from the non- edible portions of pomegranate, orange and avocado, have been detected and are currently being investigated for their antimicrobial activity. A more systematic approach will be adopted for screening bioactive compounds. To begin with screening protocols will be focused on evaluating pathogen responses under different conditions, including migration on wet surfaces, biofilm formation, survival in stressful environments (e.g. low pH and reduced water activity), and competitiveness in the gastrointestinal tract. We will investigate synergistic effects which might render antimicrobial resistant isolates sensitive to conventional antibiotics. Identification of such compounds will aid in developing environmentally compatible strategies to eliminate/ control pathogens. Objective 2: We have previously documented that pomegranate peel extracts restrict Salmonella migration on wet surfaces by down-regulating the genes required for motility. We will examine if these extracts have potential to significantly restrict the infectivity of Citrobacter in the mouse gut (model system for enterohemorrhagic infections). Transcriptomics approaches will be utilized to determine the impact of bioactive compounds on virulence of pathogens inside and outside the host environment; while metagenomic profiling will be conducted to assess the impact of bioactive compounds on composition and competitiveness of host gut microflora on disease progression. Developing a better understanding of the mode of action of promising plant-derived bioactive compounds against food-borne pathogens will significantly aid in developing strategies to reduce antibiotic use. This is the final report for the Project 8042-42000-007-00D that will be terminated in FY2018. Key findings of the project were: 1) the metabolic distribution of compounds acting to inhibit bacterial motility was examined in pomegranate peel, inner membrane, and edible aril portion to establish that the concentration of polyphenolic compounds (i.e., punicalagins, punicalins, compounds with anti-swarming activity) in peels were significantly higher than the edible aril portion; 2) bioactivity of polyphenolic compounds, obtained from crude extracts of pomegranate rind, against food-borne pathogens on wet surfaces was established based on their ability to inhibit spread of pathogens on wet surfaces; and 3) it was demonstrated that pomegranate peel extract has an effect on infectious colitis induced by C. rodentium in a mouse model by altering the colonic pathology induced by C. rodentium in vivo. In FY 2018, in collaboration with ARS scientists at the Diet, Genomics, and Immunology laboratory at the Beltsville Human Nutrition Research Center, the effect of pomegranate peel extracts (POM) on infectious colitis induced by Citrobacter rodentium in a mouse model was tested. The results demonstrate that the pomegranate peel extract reduces colonic pathology and development of a systemic infection by C. rodentium in vivo. POM peel extract treatment completely prevents infection-induced mortality and significantly reduces damage to the colonic mucosa, thus preventing translocation of bacteria to the systemic compartment that is characteristic of untreated mice. POM treatment does not alter peak C. rodentium load, clearance, or the cytokine response to infection indicating that the effect is on barrier function. One gene, Ang4, which is important for control of enteric bacteria and barrier function, is increased in POM-treated mice. In addition, POM treatment helps decrease the loss of goblet cells and mucin production as determined by Alcian blue and lectin staining. Thus, by preserving both the colonic crypts and at least some mucus production, POM treatment ablates the severity of the C. rodentium infection. Additionally, studies looking at the effects of POM treatment on the microbiome in both uninfected and infected mice were conducted; these results are currently being analyzed and appear to confirm that POM treatment caused significant changes to the microbiome. Changes in specific taxa, including an increase in the class Bacteroidia and a decrease in Bacilli (Firmicutes), were observed while Clostridia abundance was stable. In general, decreasing the Firmicute/Bacteriodia ratio is considered beneficial, possibly by altering short-chain fatty acid production. Other lower abundance operational taxonomic units also changed. Manuscripts describing the effects of POM on C. rodentium infections and the microbiome are in preparation and will be submitted in FY2018.
Impacts
(N/A)
Publications
- He, A., Penix, S.R., Basting, P.J., Griffith, J.M., Creamer, K.E., Camperchioli, D., Clark, M.W., Gonzales, A.S., Jorge, S., George, N.S., Bhagwat, A.A., Slonczewski, J.L. 2017. Acid evolution deletes amino-acid decarboxylases and reregulates catabolism of Escherichia coli K-12. Applied and Environmental Microbiology. 83(12):1-13.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop protocols to isolate and identify bioactive compounds from agricultural products such as fruit peels and rinds. Objective 2: Investigate mode of action of bioactive compounds that are able to mitigate virulence in the gut (mouse model), or restrict environmental dissemination and survival of pathogens, utilizing metagenomic, transcriptomic and proteomic analyses. Sub-objective 2.1. Determine influence of bioactive compounds on pathogens during in vitro (i.e., biofilm formation, swarm motility, and survival in harsh environments such as low water activity and low pH) and ex vivo assays (i.e. invasion of cultured human intestinal cells). Sub-objective 2.2. Determine how bioactive compounds impact host microbiome and pathogens during infection in animal models by acquiring and interpreting genomic and transcriptomic data. Approach (from AD-416): Objective 1: Several plant-derived bioactive compounds, from the non- edible portions of pomegranate, orange and avocado, have been detected and are currently being investigated for their antimicrobial activity. A more systematic approach will be adopted for screening bioactive compounds. To begin with screening protocols will be focused on evaluating pathogen responses under different conditions, including migration on wet surfaces, biofilm formation, survival in stressful environments (e.g. low pH and reduced water activity), and competitiveness in the gastrointestinal tract. We will investigate synergistic effects which might render antimicrobial resistant isolates sensitive to conventional antibiotics. Identification of such compounds will aid in developing environmentally compatible strategies to eliminate/ control pathogens. Objective 2: We have previously documented that pomegranate peel extracts restrict Salmonella migration on wet surfaces by down-regulating the genes required for motility. We will examine if these extracts have potential to significantly restrict the infectivity of Citrobacter in the mouse gut (model system for enterohemorrhagic infections). Transcriptomics approaches will be utilized to determine the impact of bioactive compounds on virulence of pathogens inside and outside the host environment; while metagenomic profiling will be conducted to assess the impact of bioactive compounds on composition and competitiveness of host gut microflora on disease progression. Developing a better understanding of the mode of action of promising plant-derived bioactive compounds against food-borne pathogens will significantly aid in developing strategies to reduce antibiotic use. The metabolic distribution of compounds acting to inhibit bacterial motility were examined in pomegranate peel, inner membrane, and edible aril portion. Compounds were isolated from peels dried under three different drying treatments (freeze drying, ambient temperature, and oven drying). Human and mouse pathogens, Salmonella enterica Serovar Typhimurium and Citrobacter rodentium (respectively) were used as test organisms. Based on the multivariate analysis, 29 metabolites discriminated the pomegranate peel, inner membrane, and edible aril portion as well as the three different drying methods. Punicalagins were detected in higher quantities in all fractions as compared to ellagic acid and punicalins. The concentration of polyphenolic compounds (i.e., punicalagins, punicalins, compounds with anti-swarming activity) in peels were significantly higher than the edible aril portion. Significant metabolite changes in profiles measured in terms of total phenolic content, radical scavenging and anti-swarming activity, were observed in response to different temperatures during extraction process. In collaboration with ARS scientists from the Beltsville Human Nutrition Research Center (BHNRC) and the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL), we have tested the effect of pomegranate peel extracts on infectious colitis induced by Citrobacter rodentium (Cr). The results clearly indicate that the pomegranate peel extract (POM) alters the colonic pathology induced by Cr in vivo. POM treatment completely prevents infection induced mortality, reduces damage to the colonic mucosa, and prevents translocation of bacteria to the systemic compartment that is characteristic of untreated mice. Interestingly, POM treatment does not alter peak Cr load, clearance, or the cytokine response to infection indicating that the effect is on barrier function. One gene, Ang4, which is important for control of enteric bacteria and barrier function, is increased in POM treated mice. POM is also known to affect the ability of bacteria to swarm, a characteristic associated with virulence. To gain further insight into the mechanisms by which POM affected swarming, RNAseq data were generated for Cr grown under swarming conditions in the presence or absence of POM. Significant differences in gene expression have been identified and the analysis is continuing. Studies examining the effect of feeding POM on the microbiome have also been undertaken and notably, Akkermansia muciniphila levels were increased by POM feeding. Accomplishments 01 Whole genome sequence of lytic Pasteurella multocida phage PMP-GAD-IND. The lytic Pasteurella bacteriophage was isolated from sewage of dairy farm of GADVASU, India, using Pasteurella multocida B:2 as the host strain. The lytic activity of the Pasteurella phage was tested against 10 field isolates of P. multocida B:2. The phage exhibited lytic activity against 90% of multidrug resistant field isolates of P multocida B:2. Haemorrhagic Septicaemia (HS) caused by Pasteurella multocida serotypes B:2 and E2:is an acute, fatal disease of cattle and buffaloes with high morbidity and mortality rates. This fully sequenced and annotated complete genomic sequence of lytic Pasteurella multocida phage PMP-GAD-IND is the first bacteriophage having lytic activity against P. multocida type A (fowl cholera), as well as multiple multidrug resistant field isolates of P multocida B:2 and will provide data for a better understanding to identify the bacteria-killing characteristics of lytic enzymes instead of antibiotics.
Impacts
(N/A)
Publications
- Bhagwat, A.A., Young, L., Smith, A.D., Bhagwat, M. 2017. Transcriptomic analysis of swarm motility phenotype of Salmonella enterica serovar Typhimurium mutant defective in periplasmic glucan synthesis. Current Microbiology. doi:10.1007/s00284-017-1267-1.
- John, K.M.A., Bhagwat, A.A., Luthria, D.L., 2017. Swarm motility inhibitory and antioxidant activities of pomegranate peel processed under three drying conditions. Food Chemistry. 235:145-153.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop protocols to isolate and identify bioactive compounds from agricultural products such as fruit peels and rinds. Objective 2: Investigate mode of action of bioactive compounds that are able to mitigate virulence in the gut (mouse model), or restrict environmental dissemination and survival of pathogens, utilizing metagenomic, transcriptomic and proteomic analyses. Sub-objective 2.1. Determine influence of bioactive compounds on pathogens during in vitro (i.e., biofilm formation, swarm motility, and survival in harsh environments such as low water activity and low pH) and ex vivo assays (i.e. invasion of cultured human intestinal cells). Sub-objective 2.2. Determine how bioactive compounds impact host microbiome and pathogens during infection in animal models by acquiring and interpreting genomic and transcriptomic data. Approach (from AD-416): Objective 1: Several plant-derived bioactive compounds, from the non- edible portions of pomegranate, orange and avocado, have been detected and are currently being investigated for their antimicrobial activity. A more systematic approach will be adopted for screening bioactive compounds. To begin with screening protocols will be focused on evaluating pathogen responses under different conditions, including migration on wet surfaces, biofilm formation, survival in stressful environments (e.g. low pH and reduced water activity), and competitiveness in the gastrointestinal tract. We will investigate synergistic effects which might render antimicrobial resistant isolates sensitive to conventional antibiotics. Identification of such compounds will aid in developing environmentally compatible strategies to eliminate/ control pathogens. Objective 2: We have previously documented that pomegranate peel extracts restrict Salmonella migration on wet surfaces by down-regulating the genes required for motility. We will examine if these extracts have potential to significantly restrict the infectivity of Citrobacter in the mouse gut (model system for enterohemorrhagic infections). Transcriptomics approaches will be utilized to determine the impact of bioactive compounds on virulence of pathogens inside and outside the host environment; while metagenomic profiling will be conducted to assess the impact of bioactive compounds on composition and competitiveness of host gut microflora on disease progression. Developing a better understanding of the mode of action of promising plant-derived bioactive compounds against food-borne pathogens will significantly aid in developing strategies to reduce antibiotic use. Polyphenolic compounds have received much attention lately due to their potential health benefits. Since non-edible portions of certain fruit peels and rinds are rich in polyphenolic compounds, we are investigating their bioactivity against food-borne pathogens. We initiated experiments to study the effect of polyphenolic compounds on migration of food-borne pathogens on wet surfaces. Based on the ability to inhibit spread of pathogens on wet surfaces, crude extracts of pomegranate rind were separated into several sub-fractions. These various fractions are currently being tested against model pathogens, namely Salmonella and Citrobacter, to identify the active compounds. This data will provide insight into the potential mode(s) of action of polyphenolic compounds.
Impacts
(N/A)
Publications
- Smith, A.D., Yan, X., Chen, C.T., Dawson, H.D., Bhagwat, A.A. 2016. Understanding the host-adapted state of Citrobacter rodentium by transcriptomic analysis. Archives Of Microbiology. 198:353-362.
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