Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments. Approach (from AD-416): Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and peptides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In collaboration with Dr. Sadowsky (U. Minisoda), natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches. During the course of the project, researchers at Beltsville, Maryland, conducted research to understand the molecular mechanisms of foodborne bacterial pathogens in interacting with food and environmental matrices. For Objective 1, researchers determined the roles of bacterial cell surface structures and proteins, including flagella and several fimbriae from E. coli O157:H7 and O104:H4 in interacting with plant and abiotic surfaces in biofilm formation. Special effort was placed in characterizing the interactions of foodborne bacterial pathogens with natural microflora in forming multispecies biofilms. This research greatly advanced our understanding of the survival of foodborne bacterial pathogens in environmental matrices and the importance of multispecies biofilm formation. For Objective 2, researchers characterized the proteome of Salmonella grown under conditions simulating irrigation and wash water, determined the roles of bacterial osmoregulated periplasmic glucans (OPGs) in detergent tolerance and swarm motility. Accomplishments 01 Understanding biofilm formation by Escherichia coli O104:H4. E. coli 104:H4 is a newly emerged special type of foodborne pathogenic E. coli, which was the causative agent for one of the most deadly foodborne outbreaks a few years ago. In comparison to E. coli O157:H7, O104:H4 has a strong tendency of biofilm formation (a process that microorganisms form a film-like community that is more resistant to environmental stresses) on intestinal epithelial cells as well as on other surfaces such as stainless steel. ARS scientists in Beltsville, Maryland determined that the deletion of the gene encoding the aggregative adherence fimbriae (a special cell surface filament that enable its adherence to gut surface) led to significant reduction of biofilm formation on plant and glass surfaces, but had no effect on the initial attachment. This observation indicated that the aggregative adherence fimbriae is not only critical for pathogenesis, but also for survival in the environments outside animal hosts, including food matrices. These observations indicated that these two prominent foodborne pathogens have very different mechanisms for interacting with environmental matrices. This information will be useful for further understanding the survival mechanisms of these foodborne pathogens. 02 Understanding the interaction of environmental bacteria and foodborne pathogens in biofilm formation. Ralstonia insidiosa is an opportunistic pathogen that often contaminates water supply systems. ARS scientists in Beltsville, Maryland previously isolated R. insidiosa from the native microflora in produce packing facilities and showed it promoted the incorporation of E. coli O157:H7 into dual species biofilms. It was also shown to enhance biofilm formation by other pathogenic E. coli, Salmonella, and Listeria monocytogenes strains in dual species cultures and it appears to play a role of �bridge bacteria� in multispecies biofilm formation. R. insidiosa was shown to induce aggregation, a key step in biofilm formation, by L. monocytogenes in mixed cultures. This observation highlights the importance of contact dependent interactions among native microflora and foodborne pathogens. 03 Inhibition of bacterial pathogens by compounds isolated from agricultural wastes. Controlling spread of human pathogens on fresh produce is a top priority for public health reasons. Isolation of compounds from agricultural waste that would stop or retard the spread of human pathogens was explored using Salmonella enterica serovar Typhimurium as a model organism. Compounds extracted from various agricultural wastes such as soybean husk, peels of orange, pineapple, avocado, and pomegranate were studied for inhibiting pathogen spread. Pomegranate peel extract inhibited pathogen spread on moist surfaces and was examined in further detail. Several key Salmonella motility regulatory genes were down regulated upon exposure to pomegranate extract. The research will benefit the fresh produce industry and increase the microbial food safety of the American food supply.
Impacts
(N/A)
Publications
- Liu, N., Bauchan, G.R., Francoeur, C.B., Shelton, D.R., Lo, M., Nou, X. 2016. Ralstonia insidiosa serves as bridges in biofilm formation by foodborne pathogens Listeria monocytogenes, Salmonella enterica, and enterohemorrhagic Escherichia coli. Food Control. 65:14-20.
- Gou, A., Xu, Y., Mowery, A.K., Nagy, A., Bauchan, G.R., Nou, X. 2016. Ralstonia insidiosa induces cell aggregation by Listeria monocytogenes. Food Control. 67:303-309.
- Islam, N., Nagy, A., Garrett, W.M., Shelton, D.R., Cooper, B., Nou, X. 2016. Different cellular origins and functions of extracellular proteins from Escherichia coli O157:H7 and O104:H4 as determined by comparative proteomic analysis. Applied and Environmental Microbiology. doi: 10.1128/ aem.00977-16.
- Nagy, A., Bauchan, G.R., Shelton, D.R., Nou, X., Xu, Y. 2016. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4. International Journal of Food Microbiology. 229:44-51.
|
Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments. Approach (from AD-416): Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and peptides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In collaboration with Dr. Sadowsky (U. Minisoda), natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches. Cell surface and secreted proteins may play a role in bacterial attachment to biotic and abiotic surfaces. Secreted proteins were prepared from E. coli O157:H7 and O104:H4 strains and the secretomes analyzed using Mud-Pit GC/MS. There were significant differences between the secretomes of these two E. coli strains. Efforts are being made to understand the biological significance of these differences. E. coli O104:H4 represents a new class of EHEC (Enterohemorrhagic E. coli) that also belongs to EAEC (Enteroaggregative E. coli) pathotype. In addition to shigatoxin production, E. coli O104:H4 produce aggregative adherence fimbriae (AAF/I), which promote cell aggregation and biofilm formation, and other virulence factors. The gene encoding the major units of AAF/I was deleted using lambda-red recombinase mutagenesis and the biofilm formation by the knock-out mutant on abiotic surfaces was significantly reduced. Currently Environmental Microbial and Food Safety Laboratory (EMFSL) scientists are examining the effect of the mutation of the ability of the organism to attach to fresh produce surfaces. EMFSL scientists have previously determined that an environmental bacterium frequently isolated in produce-processing plants with strong biofilm formation potential, Ralstonia insidiosa, promoted the incorporation of foodborne bacterial pathogens, including E. coli O157:H7, into dual-species biofilms. We demonstrated that R. insidiosa induced the aggregation of Listeria monocytogenes in dual species culture, suggesting a potential mechanism for the enhanced biofilm formation by L. monocytogenes. Current research is examining the interactions between R. insidiosa and L. monocytogenes in forming polymicrobial biofilms. Accomplishments 01 Role of E. coli cell surface proteins in attachment to produce and abiotic surfaces. The genes for eight major cell surface proteins in E. coli O157:H7 were deleted by lambda-red recombinase mutagenesis and the corresponding effects on the organism�s ability to attach to produce and abiotic surfaces were examined. Among the major cell surface protein genes, the deletion of the flagellum (FliC) and Outer Membrane Protein A (OmpA) genes resulted in the most significant reduction in cellular attachments. This information will be useful to the scientific community and food quality regulatory agencies. 02 Interactions of environmental bacteria and foodborne pathogens in biofilm formation. Biofilm-forming bacteria, resident to food processing facilities, are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. Scientists in Beltsville, Maryland, previously showed that Ralstonia insidiosa, a strong biofilm former frequently isolated from produce-processing environments, promoted the incorporation of Escherichia coli O157:H7 into dual-species biofilms. Scientists in Beltsville, Maryland, examined the interactions between E. coli O157:H7 and R. insidiosa under different incubating conditions and found the interactions were not significantly affected by either low incubating temperature (10oC) or limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions, E coli O157:H7 cells showed a strong tendency to co-localize with R. insidiosa on glass surfaces in the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found in the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms. This information will be useful to other scientists, food processors and regulatory agencies. 03 Inhibition of bacterial pathogens by compounds isolated from agricultural wastes. Controlling spread of human pathogens on fresh produce is a top priority for public health reasons. Isolation of compounds from agricultural waste that would stop or retard the spread of human pathogens was explored using the Salmonella enterica serovar Typhimurium as a model organism. Compounds extracted from various agricultural wastes, such as soybean husks, orange peels, pineapples, avocados, and pomegranates are used for inhibiting pathogen spread. Pomegranate peel extract inhibited pathogen spread on moist surfaces and was examined at the molecular level in further detail. Several key Salmonella motility regulatory genes were down-regulated upon exposure to pomegranate extract. Pomegranate peel offers great potential as a bio-active repellent for pathogenic microorganisms. This research will benefit the fresh produce industry, as well as increasing the microbial food safety of the Americans food supply. 04 Survival and distribution of E. coli 157:H7 in a microgreen production system. Microgreens are a new class of fresh produce that have an expanding market. Scientists in Beltsville, Maryland, examined the survival/proliferation of E. coli O157:H7 in different microgreen production systems and explored the spatial-temporal distributions of E. coli O157:H7 during the course of microgreen growth. Scientists demonstrated that E. coli O157:H7 inoculated on radish seeds could result in systematic contaminations on all parts of the plant and growth media, but that seed coats remained the focal point of contamination throughout the microgreen growth period. This information will be useful to producers and the FDA in assessing the food safety risks of microgreens.
Impacts
(N/A)
Publications
- Mahadwar, G., Chauhan, K.R., Bhagavathy, G., Murphy, C.F., Smith, A.D., Bhagwat, A.A. 2015. Swarm motility of Salmonella enterica serovar Typhimurium is inhibited by compounds from fruit peel extracts. Letters in Applied Microbiology. 60:334-340.
- Liu, N.T., Nou, X., Bauchan, G.R., Murphy, C.F., Lefcourt, A.M., Shelton, D.R., Lo, Y. 2015. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut processing plant. International Journal of Food Microbiology. 78:121-127.
- Huang, J., Luo, Y., Nou, X. 2015. Growth of Salmonella and Listeria monocytogenes on fresh-cut cantaloupe under different temperature abuse scenarios. Journal of Food Protection. 78:1125-135.
- Nagy, A., Mowery, J.D., Bauchan, G.R., Wang, L., Nichols-Russell, L., Nou, X. 2015. Role of major surface structures of Escherichia coli O157:H7 in initial attachment to biotic and abiotic surfaces. Applied and Environmental Microbiology. 81:4720-4727.
- Xiao, Z., Bauchan, G.R., Nichols-Russell, L.K., Luo, Y., Wang, Q., Nou, X. 2015. Proliferation of Escherichia coli O157:H7 in soil and hydroponic microgreen production systems. Journal of Food Protection. DOI:10.4315/ 0362-028X.JFP-15-063.
|
Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments. Approach (from AD-416): Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and peptides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In collaboration with Dr. Sadowsky (U. Minisoda), natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches. Cell surface appendages may facilitate bacterial attachment to biotic and abiotic surfaces. To determine the functions of various cell surface components in these processes, procedures for proteomic analyses of bacterial cell surface appendages are being developed. E. coli O157:H7 cell surface proteins are labeled and purified using a biotin- streptavidin cell surface labeling kit. Mass spectrometric profile of over 200 proteins has been determined in collaboration with scientist at University of Maryland Institute of Bioscience and Biotechnology Research. However, major procedure optimization to reduce cytoplasmic protein contamination and to increase coverage of outer membrane proteins still needs to be achieved. Currently controlled trypsin digestion (shaving) of whole bacterial cells is being tested as an alternative for cell surface protein preparation. DNA and RNA aptamers have been developed in recent years for identifying specific bacterial pathogens and other environmental contaminants. We have attempted to select DNA aptamers that specifically bind to E. coli O104:H4, an emerging type of enterohemorrhagic E. coli. Several potential aptamer sequences have been selected by positive-selection with E. coli O104:H4 and counter-selection with E. coli K12. The binding affinity and specificity of these potential aptamers for E. coli O104:H4 are being evaluated. Multiple E. coli O157:H7 mutants for cell surface proteins have been generated. These mutants are being tested for interactions with fresh produce and abiologic surfaces, including attachment and biofilm formation. We have also constructed a plasmid that will be needed for generating site directed mutations in E. coli O104:H4. Pathogen strains exhibiting enhanced virulence and infectivity are frequently isolated in food-borne diarrheal outbreaks. Citrobacter rodentium, a mouse pathogen that mimics many aspects of enterohemorrhagic E. coli O157:H7 infection of humans, can serve as a useful model for studying virulence mechanisms. C. rodentium developed a hyperinfectious state once it was passed through the mouse gastrointestinal tract and its LD50 decreased by ~2-logs. C. rodentium cells lost their hyperinfectious state once cultured on laboratory media. To fully understand molecular events behind superinfectious state of this pathogen, entire RNA population (transcriptome) of C. rodentium cells excreted in mice feces was without subculturing the cells. Computational techniques were utilized to eliminate reads corresponding to fecal microflora and mouse intestinal cells. C. rodentium gene expression patterns specifically corresponding to anaerobic growth conditions, mouse fecal contents as well as pathogenesis were identified. Developing tools to identify traits of hyper-virulent outbreak strains will provide a better measure of causality and food safety risk. Accomplishments 01 Improved bacterial mRNA preparations for RNA sequencing. Next generation sequencing is a powerful technology and its application to sequencing entire RNA populations of food-borne pathogens will provide valuable insights. Due to the abundance of rRNA and the lack of mRNA polyadenylation in prokaryotes, effective rRNA removal is required for bacterial mRNA enrichment and sequencing. We evaluated three commercially available rRNA-depletion kits to determine the efficiency of rRNA removal and enrichment of mRNA from Salmonella enterica serovar Typhimurium strain SL1344. The three protocols achieved varying degrees of rRNA depletion and resulted in 8 to 1000-fold enrichment of mRNA. rRNA removal probes from two kits were unable to titrate out 23S rRNA species, showed bias, and were less efficient at 3�-end of 16S rRNA. The Ribo-Zero kit was most efficient in eliminating 16S, 23S and 5S ribosomal RNA species from the transcriptome of S. enterica serovar Typhimurium strain SL1344. The information provided by this study will be useful to other scientists interested in next generation sequencing analyses of RNA. 02 Role of Osmoreguoated Periplasmic Glucans in swarm motility of Salmonella enterica serovar Typhimurium. Salmonella spp. strains exhibit unique motility when placed on moist growth surfaces known as swarming motility. Here we report Salmonella sp. carrying a mutation in opgGH genes are unable to exhibit swarm motility in spite of the fact that osmolarity of swarm medium exceeds 400 mos mol l-1. Plasmid carrying opgGH genes with site-directed mutations at the active site amino acid residues failed to restore swarm motility. Full length opgGH gene products were needed to support swarm motility even though no OPG synthesis was detected in media with osmolarity > 400 mos mol l- 1. Since swarm motility promoting media are iso- or hyperosmotic (> 400 mos mol l-1), the requirement of opgGH genes for swarm motility appears to be indirect. Although OPG synthesis is expected to be repressed in media favoring swarm motility, transcription of opgGH was not repressed. Catalytically functional opgGH proteins are needed to support swarm motility. 03 Biofilm formation affected by native microflora. Biofilm formation is an important mechanism for bacterial survival in stressful environments. We further investigated the biofilm formation by foodborne bacterial pathogens in the presence of certain species of native microflora. The incorporation of E. coli O157:H7, Salmonella, and Listeria monocytogenes all increased significantly when co-cultured with Ralstonia insidiosa, a strong biofilm producer isolated previously from produce processing plant. This interaction was also observed under different conditions, including different temperatures, nutrient contents, and substratum surfaces. This suggests that native microflora- based biofilms may play an important role in the survival of bacterial pathogens in produce processing environments.
Impacts
(N/A)
Publications
- Liu, N.T., Nou, X., Lefcourt, A.M., Shelton, D.R., Lo, M. 2013. Dual- species biofilms formation by Escherichia coli O157:H7 and environmental bacteria isolated from fresh-cut processing plants. International Journal of Food Microbiology. 171:15-20.
- Bhagwat, A., Z Ying, I., Smith, A. 2013. Evaluation of ribosomal RNA removal protocols for Salmonella RNA-Seq projects. Advances in Microbiology. 4:25-32.
- Bhagwat, A.A., Ying, I., Karns, J.S., Smith, A.D. 2013. Determining RNA quality for NextGen sequencing: some exceptions to the gold standard rule of 23S to 16S rRNA ratio. Advances in Microbiology. 1:10.
|
Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments. Approach (from AD-416): Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and pipetides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In collaboration with Dr. Sadowsky (U. Minisoda), natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches. In order to gain knowledge of genetic and molecular activities of Salmonella sp. a transcriptome analysis approach was taken. High quality RNA isolation was found to be critical for sequencing entire messenger RNA populations at any given time point. Methods were designed to generate reliable sequencing libraries avoiding artifacts which could be generated by high-throughput RNA analysis. Substantial progress has been achieved in obtaining 10-20-fold coverage of Salmonella genome in RNA-Seq reactions. Pathogen strains exhibiting enhanced virulence and infectivity are frequently isolated in food-borne diarrheal outbreaks. Citrobacter rodentium, a mouse pathogen that mimics many aspects of enterohemorrhagic E. coli O157:H7 infection of humans, can serve as a useful model for studying virulence mechanisms. C. rodentium developed a hyperinfectious state once it was passed through the mouse gastrointestinal tract. Hyperinfectious C. rodentium cells were extremely acid sensitive compared to cells grown in laboratory media. Growth under anaerobic environment or on fecal components did not induce this hyperinfectious state. Developing tools to identify traits of hyper-virulent outbreak strains will provide a better measure of causality and food safety risk. Cell surface appendages facilitate bacterial attachment to biotic and abiotic surfaces. Procedures for proteomic analyses of bacterial cell surface appendages are being developed, including trypsin digestion(shaving) and selective labeling and analysis using LC-MC. E. coli and Salmonella mutants for cell surface proteins have been generated. These mutants will be tested for interactions with fresh produce and abiologic surfaces. Accomplishments 01 Determination of detergent tolerance of food-borne pathogens. Osmoregulated periplasmic glucans (OPGs) are synthesized by members of the family Enterobacteriaceae when grown under low osmotic conditions. Enteropathogens such as Shigella flexneri spend considerable time outside the host intestinal environment, such as in irrigation waters where low nutrient low osmolarity conditions normally exist. We demonstrated that OPGs of S. flexneri are required for optimal growth under low osmolarity low nutrient conditions. OPGs of S. flexneri are anionic. Based on homology of the OPG biosynthesis genes to those of Escherichia coli, the most likely function of opgC and opgB genes is to add succinate and phosphoglycerol residues respectively onto OPGs. We constructed opgB, opgC and opgBC mutants of S. flexneri. The mutant strain defective in opgC and opgB genes synthesized neutral OPGs which, although were beneficial for the organism�s growth in hypoosmotic media, were ineffective in combating stress caused by anionic detergents. Cloned wild type genes opgB, opgC, and opgBC upon mobilization to respective opg mutants, simultaneously restored anionic charges to OPGs and tolerance to detergents to wild type levels. It appears that anionic charges on the OPGs contribute towards overcoming the stress by anionic detergents such as sodium dodecyl sulfate (SDS) and sodium deoxycholate. 02 Native microflora influences biofilm formation by food-borne pathogens. Biofilm formation is an important mechanism for bacterial survival in stressful environments. Many foodborne bacterial pathogens are poor biofilm formers. However they are capable of interacting with other bacteria with strong biofilm forming potential. Ralstonia insidisa, a bacterial species that was frequently isolated from fresh produce processing plants was shown to be a strong biofilm producer and strongly enhanced the incorporation of E. coli O157:H7 cells in the dual-species biofilms. This suggests that native microflora-based biofilms may play an important role in the survival of bacterial pathogens in produce processing environments.
Impacts
(N/A)
Publications
- Bhagwat, A.A., Yi Ning, L., Liu, L., Mahesh, D., Porteen, K. 2012. Role of anionic charges of periplasmic glucans of Shigella flexneri in overcoming detergent stress. Foodborne Pathogens and Disease. 9(7):632-637.
- Smith, A.D., Bhagwat, A.A. 2013. Hypervirulent- host-associated Citrobacter rodentium cells have poor acid tolerance. Current Microbiology. 66(5):522-526.
- Liu, N., Lefcourt, A.M., Nou, X.A., Shelton, D.R., Zhang, G., Lo, Y. 2013. Native microflora in fresh-cut processing plants and their potentials of biofilm formation. Journal of Food Protection. 76(5):827-832.
|
Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments. Approach (from AD-416): Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and pipetides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In a recent collaboration natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches. Several techniques for preparing bacterial cell surface proteins have been tested. Cell surface protein sheering and concentration protocols have been optimized using ultrasonic blending, high frequency vortex, and microfiltration. A membrane-impermeable cleavable biotinylation reagent (Sulfo-NHS-SS-Biotin) was used to selectively label bacterial cell surface proteins. Labeled proteins are concentrated using neutoavidin affinity binding. Improvement to minimize cytoplasmic protein contamination is continuing. The interaction of E. coli O157:H7 with bacterial strains isolated from fresh produce processing plants, resulting in the formation of heterospecies biofilms, was assessed. Certain Gram-negative phytopathogenic bacteria, such as Burkholderia caryophylli and Rhizobium radiobacter, enhanced E. coli O157:H7 presence in dual-species biofilms. Two soil bacteria, Ralstonia insidiosa and Sphingomonas rhizophila, were able to significantly promote the presence of E. coli O157:H7 with a total increase of 0.7 log CFU/cm2 compared to its monoculture biofilm. Research is being conducted to understand this apparent synergetic process and the implications to sanitization practices. The effect of a new wash aid (T128) in combination with chlorine on Salmonella survival and infiltration in tomato was examined. As the organic load increased and free chlorine level decreases in chlorinated wash water, Salmonella were able to survive and infiltrate into the fruits through the stem scars. Application of T128 was found to significantly decrease this risk. The ability to survive under environmental stress conditions enables Salmonella spp. to successfully enter the food chain. Survival of human pathogens in vegetable wash waters is a persistent cause of food borne infections. We have identified two genes in the glucans gene family, opgB and opgC, which are essential to overcome detergent and sanitation agents which are used in produce wash steps. Genetic mutation in the corresponding gene rendered Salmonella strains incapable of rapid growth in the presence of detergents, however they remained fully virulent. Identification of genes essential for stress tolerance is crucial in designing specific sanitation agents to wash vegetables. The development of better cleaning agents to eliminate Salmonella spp. in food would be extremely beneficial to the food processing industry. Accomplishments 01 Identification of entire cellular proteins of Salmonella under condition similar to irrigation- and wash waters. The ability of Salmonella spp. survive in irrigation waters enables them to enter the food chain. Vegetable wash waters and irrigation waters have been implicated in rece outbreaks of infections caused by Salmonella spp. In this study, ARS researchers at Beltsville analyzed the entire protein component of Salmonella during its growth in low osmotic media resembling irrigation waters. The study identified several cellular proteins which were essential for optimal growth of Salmonella in low osmotic conditions. Characterizing human pathogens grown under conditions mimicking fresh produce handling and washing practices will advance our knowledge of how enteric human pathogens enter and survive in our food chain.
Impacts
(N/A)
Publications
- Bhagwat, M., Bhagwat, A.A. 2011. Microbial Genome Analysis and Comparisons: Web-based Protocols and Resources. Methods in Molecular Biology. 765:297-307.
- Bhagwat, A.A., Kannan, P., Leow, Y., Dharne, M., Smith, A.D. 2012. Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica Serovar Typhimurium SL1344 in mice virulence. Archives Of Microbiology. 194(6):541-548.
- Cooper, B., Chen, R., Garrett, W.M., Chang, C., Tucker, M.L., Bhagwat, A.A. 2012. Proteomic pleiotropy of OpgGH, an operon necessary for efficient growth of Salmonella enterica serovar Typhimurium under low-osmotic conditions. Journal of Proteome Research. 11:1720-1727.
|
Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments. Approach (from AD-416) Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and pipetides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In collaboration with Dr. Sadowsky (U. Minisoda), natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches. Considerable progress has been made towards meeting the sub-objective 1.1 i.e. to develop methods for profiling and characterizing bacterial cell surface structures. Initial trials have indicated that we can harvest cell surface protein without having any adverse effects on cell viability. This is an important step in order to prevent contamination of cytoplasmic proteins into surface protein fraction. Further tests are being conducted to identify surface proteins. The research team has also made progress toward meeting goals of sub-objective 1.3 in constructing mutants with altered cell surface properties.
Impacts
(N/A)
Publications
|
|