Progress 12/15/13 to 12/14/20
Outputs
Target Audience:Seed producers and processors, vegetable and sprout growers, researchers in the food industry, government and academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has graduated two Ph.D. students and one MS student, and is currently training 1 Ph.D. students and 1 laboratory helper. One of the graduated Ph.D. students has become a faculty member in a Chinese university, and another Ph.D. graudate is now working in a company in California. The MS is currently working towards her doctoral degree in another institution. This project provides an excellent opportunity for the students to gain research training and laboratory experience. Meanwhile, they were/are given the responsibility to supervise the laboratory helper, which is an opportunity for them to learn management and supervisory skills. In addition to laboratory research, the graduate students are encouraged to get involved in food safety professional societies. The involvement will allow them to develop professional networks and get connected with their peers. How have the results been disseminated to communities of interest?Results of the research have been presented at the virtual annual meetings of International Association for Food Protection and GA Bioscience Summit. Abstracts have also been submitted to IUFoST and Food Micro 2020 for poster presentation, but both meeting have been postponed till 2021. In addition to the paper published in Applied and Environmental Microbiology in 2017, two papers published in Applied and Environmental Microbiology and Food Control in 2018, one paper published in Food Control in 2019, and two mansicrpts published in International Journal of Food Microbiology and Journal of Food Protection in 2020, one additional manuscript has been submitted to MDPI Food for review and publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Part of the objective 3 of the project (Identify the genes and gene products that are critical for Salmonella and E. coli O157:H7 attachment to vegetable seeds) has been addressed. Mini-Tn10 transposon mutagenesis was used to randomly interrupt the genes of Salmonella enterica serotype Tennessee. The ability of selected Salmonella mutants in forming biofilms were compared with their wild type parent in a 24-well polystyrene tissue culture plate. Biofilm mass was quantified using the crystal violet binding assay. Mutants forming significantly less (P<0.05) biofilm mass in comparison to their wild type parent were selected. Genomic DNA of mutant cells were extracted and subjected to deep DNA sequencing. Specific gene in each mutant that was interrupted by the mini-Tn10 insertion was identified by comparing the obtained DNA sequencing data with those deposited in the Genbank using BLAST search. A total of 56 colonies of S. Tennessee mutants were obtained, and among them, 5 colonies were selected for further analysis according to the results of biofilm assay. Cells of the 5 mutants formed significantly less (P<0.05) biofilm mass than the parent strain. Sequencing analysis revealed that the interrupted genes in collected mutants encode for bacterial cell membrane lipoprotein, DNA topoisomerase III, attachment invasive locus protein, bacteriocin immunity protein, or cell division protein.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chen, J., and Y. Wang. 2020. Salmonella genes critical for attachment and biofilm formation. Int. J. Food Microbiol. 320:108524.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Cui, Y., D. Liu, and J. Chen. 2020. Fate of Salmonella enterica and enterohemorrhagic Escherichia coli on vegetable seeds, contaminated by direct contact with artificially inoculated soil, during germination. J. Food Prot. 7:1218-1226.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Liu, D., Y. Cui, R. Walcott, and J. Chen. 2021. Influence of bacterial competitors on Salmonella enterica and enterohemorraghic Escherichia coli growth in microbiological media and attachment to vegetable seeds. MDPI Food.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Lee, S., and J. Chen. 2020. Genes of Salmonella enterica serotype Typhimurium involving in attachment and biofilm formation on almond and peanut seeds. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 2-8 August. Cleveland OH.
|
Progress 12/15/18 to 12/14/19
Outputs
Target Audience:Seed producers and processors, vegetable and sprout growers, researchers in the food industry, government and academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has graduated two Ph.D. students and is currently training 2 graduate students and 1 laboratory helper. One of the graduated Ph.D. students has become a faculty member in a Chinese university, and the other student is working in a company in California. The project provides an excellent opportunity for the students to gain research training and laboratory experience. Meanwhile, they were/are given the responsibility to supervise the laboratory helper, which is an opportunity for them to learn management and supervisory skills. In addition to laboratory research, the graduate students are encouraged to get involved in food safety professional societies. The involvement will allow them to develop professional networks and get connected with their peers. How have the results been disseminated to communities of interest?Results of the research have been presented at the annual meetings of International Association for Food Protection, Southeastern Regional Fruits and Vegetable Conferences, and USDA NIFA Project PD meetings. In addition to the paper published in Applied and Environmental Microbiology in 2017, and two papers published in Applied and Environmental Microbiology and Food Control in 2018, one more mansicrpt was published in Food Control in 2019. Two additional manuscripts have been submitted to International Journal of Food Microbiology and Food Control in 2019. What do you plan to do during the next reporting period to accomplish the goals?We will continuously work on Objective 3 of the proposed project (Identify the genes and gene products that are critical for Salmonella and E. coli O157:H7 attachment to vegetable seeds and transmission from seeds to sprout and seedling tissues) and identify additional genes whose products are important for Salmonella and enterohemorrhagic E. coli attachment to seed surfaces. Specific details of the research activities are descried below. The sites of Tn10 insertion in 10 remaining Salmonella mutants will be determined. The role of mutated Salmonella genes will be experimentally verified. The physiological role of the identified genes in selected E. coli O157:H7 mutants will be verified. The graduate students working on the project will be graduated in the second, 12-month no-cost extension period.
Impacts
What was accomplished under these goals?
Part of the objective 3 of the project (Identify the genes and gene products that are critical for Salmonella and E. coli O157:H7 attachment to vegetable seeds) has been addressed. Materials and methods Additional genes that are important for Salmonella and E. coli O157:H7 attachment and colonization are identified. Using a new donor strain, E. coli BW20767 (pir+) harboring plasmid pJA1 (ampr and mini-Tn10:lacIq:kan), additional conjugation experiments were conducted in order to obtain additional Salmonella mutant colonies. The isolated colonies were tested for their susceptibility to ampicillin in order to ascertain that the plasmid that delivered the mini-Tn10 was not expressed and acquired kanamycin resistance in mutant cells was due to the results of transposon mutagenesis. At the following, biofilm forming ability of the colonies was tested on an abiotic surface. Mutant cells with significantly altered ability in attachment and biofilm formation, in comparison to their wild type parents, were examined to identify the site of mini-Tn10 insertion by using whole genomic sequencing. Experimental approach described above was also used in the mutagenesis studies using E. coli O157:H7 as recipient cells. Results A total of 56 Salmonella mutants were selected, 18 from S. Baildon, 20 from S. Cubana, and 18 from S. Montevideo. The sites of Tn10 insertion in 6 Salmonella mutants have been identified using the above approach. Results showed that the mini-Tn10 had inserted into the 6 different genes on S. Cubana or S. Montevideo genome. Furthermore, a total of 54 E. coli O157:H7 mutants were selected, 18 each from E. coli O157:H7 F4546, K4492, and H1730, respectively. The sites of Tn10 insertion in 19 E. coli mutants have been identified using the aforementioned approach. Results showed that the mini-Tn10 had inserted into the genes encoding 19 different products.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Liu, D., Y. Cui, R. Walcott, J.-C. Diez-Perez, and J. Chen. 2019. Transmission of human enteric pathogens form artificially inoculated flowers to vegetable sprouts/seedlings developed from seeds produced by the contaminated flowers. Food Control. 99:21-27.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Chen, J., and Y. Wang. Salmonella genes critical for biofilm formation on polystyrene surface and attachment to vegetable seeds. Int. J. Food Microbiol. Submitted.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Cui, Y., D. Liu, and J. Chen. Fate of Salmonella enterica and enterohemorrhagic Escherichia coli on vegetable seeds, contaminated by direct contact with artificially inoculated soil, during germination. Food Control Submitted.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
Liu, D. and J. Chen. Influence of bacterial competitors on Salmonella enterica growth in microbiological media and attachment to vegetable seeds. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 21-24 July. Louisville KT. P2-113.
|
Progress 12/15/17 to 12/14/18
Outputs
Target Audience:Seed producers and processors, vegetable and sprout growers, researchers in the food industry, government and academia. Changes/Problems:In addition to sequencing degenerated PCR products, whole genomic sequencing will also be used to identify the sites of mini-Tn10 insertion on Salmonella and E. coli chromosomes in Objective 3 of the project. What opportunities for training and professional development has the project provided?The project has graduated two Ph.D. students and is currently training 2 graduate students and 1 laboratory helper. One of the graduated Ph.D. students has become a faculty member in a Chinese university, and the other student is working in a company in California. The project provides an excellent opportunity for the students to gain research training and laboratory experience. Meanwhile, they are given the responsibility to supervise the laboratory helper, which is an opportunity for them to learn management and supervisory skills. In addition to laboratory research, the graduate students are encouraged to get involved in food safety professional societies. The involvement will allow them to develop professional networks and get connected with their peers. How have the results been disseminated to communities of interest?Results of the research have been presented at the annual meeting of International Association for Food Protection, and USDA NIFA Project PD meeting. In addition to the paper published in Applied and Environmental Microbiology in 2017, two additional papers were published in Applied and Environmental Microbiology and Food Control, respectively in 2018. Furthermore, another manuscript was recently submitted to Food Control. What do you plan to do during the next reporting period to accomplish the goals?We will continuously work on Objective 3 of the proposed project (Identify the genes and gene products that are critical for Salmonella and E. coli O157:H7 attachment to vegetable seeds and transmission from seeds to sprout and seedling tissues) and identify additional genes whose products are important for Salmonella and enterohemorrhagic E. coli attachment to seed surfaces.
Impacts
What was accomplished under these goals?
Objective 4 of the project (Determine whether the presence of plant pathogens and biological control agents will change the attachment and colonization behaviors of Salmonella and E. coli O157:H7) was addressed in 2018. Materials and Methods We evaluated whether a selected plant pathogen (Pseudomonas syringae pv. Tomato DC3000 [Pst DC3000]) and a probiotic bacterium (Lactobacillus rhamnosus GG), as well as bacterial strains previously used as biocontrol agents in plant science (P. fluorescence A506, B. mojavensis RRC 101, and B. subtilis ATCC 6051) could compete with human pathogens, such as Salmonella and EHEC, for growth in microbiological media and attachment to the seeds of alfalfa, fenugreek, lettuce and tomato and to determine whether the metabolites in cell-free supernatants of competitive bacterial spent cultures could inhibit the growth of Salmonella and EHEC. Results The presence of competitive bacteria, especially L. rhamnosus GG significantly inhibited the growth of Salmonella and EHEC (P < 0.05). The mean population of Salmonella in co-culture with L. rhamnousus GG at 37°C was 5.36-log units lower than the population in the control and 3.53-log units lower than the Salmonella population in the control at 25°C, while the mean population of EHEC in co-culture with L. rhamnousus GG at 37°C was 1.49-log units lower than the population in the control and 1.33-log units lower than the EHEC population in the control at 25°C. The mean attachment ratios of Salmonella or EHEC from the four types of vegetable seeds were significantly lower (P < 0.05) when the competitive bacterial strains were present. When no competitive bacterial strains were present, the mean attachment ratios of Salmonella and EHEC cells to the four types of seeds were 10.5% and 3.9%, respectively. In the co-cultures with P. fluorescence A506, both Salmonella (7.0 %) and EHEC (2.4%) strains had lower attachment ratios. The addition of L. rhamnousus GG cell-free supernatants to the Salmonella and EHEC cultures resulted in significant reductions in the pathogen populations. Salmonella cells became undetectable (< 10 CFU/ml) at the 12 h sampling point and forward, and a total of 5 log unit reduction was achieved after the 24 h incubation period. The mean population of the four EHEC strains decreased 2.4 log CFU/ml from the 4 h to the 8 h sampling points and a total of 4 log CFU/ml reduction was observed after the 24 h incubation period.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Cui, Y. 2018. Salmonella enterica and enterohemorrhagic Escherichia coli on vegetable seeds mechanism of attachment, fate during germination, and control.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Liu, D. 2018. Fate of Salmonella enterica and enterohemorrhagic Escherichia coli during seed germination and in the presence of bacterial competitors.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Cui, Y., D. Liu, and J. Chen. 2018. Fate of Salmonella enterica and enterohemorrhagic Escherichia coli cells attached to vegetable seeds during germination. Food Control. 88:229-235.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Liu, D., Y. Cui, R. Walcott, and J. Chen. 2018. Fate of Salmonella enterica and pathogenic E. coli cells artificially internalized into vegetable seeds during germination. Appl. Environ. Microbiol. 84:e01888-17. https://doi.org/10.1128/AEM. doi:10.1128/AEM.01888-17.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Liu, D., Y. Cui, R. Walcott, J.-C. Diez-Perez, and J. Chen. Transmission of human enteric pathogens form artificially-inoculated flowers to vegetable sprouts/seedlings developed from seeds produced by the contaminated flowers. Food Control. Submitted.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Chen, J., and Y. Wang. 2018. Genetic determinants of Salmonella enterica critical for biofilm formation on abiotic surface and attachment to vegetable seeds. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 8-11 July. Salt Lake City UT p.87.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Liu, D., Y. Cui, R. Walcott, and J. Chen. 2018. Transmission of human enteric pathogens from artificially-inoculated flowers to vegetable sprouts/seedlings developed via contaminated seeds. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 8-11 July. Salt Lake City UT p.87.
|
Progress 12/15/16 to 12/14/17
Outputs
Target Audience:Seed producers and processors, vegetable and sprout growers, researchers in the food industry, government and academia. Changes/Problems:EHEC mutants with decreased ability in biofilm formation have been selected. However, technical difficulty has been encountered in generating degenrate PCR products that can be sequenced by the Sanger Sequencing Technology. We are in the process of solving the technical issues. What opportunities for training and professional development has the project provided?The project is currently training 4 graduate students and 1 laboratory helper. All graduate students have the interest to become future faculty members. The project provides an excellent opportunity for the students to gain research training and laboratory experience. Meanwhile, they are given the responsibility to supervise the laboratory helper, which is an opportunity for them to learn management and supervisory skills. In addition to laboratory research, the graduate students are encouraged to get involved in food safety professional societies. The involvement will allow them to develop professional networks and get connected with their peers. How have the results been disseminated to communities of interest?Results of the research have been presented at the annual meeting of International Association for Food Protection, Southeast Regional Fruits and Vegetable Conference, and Georgia Bio Innovation Summit, as well as the USDA NIFA Project PD meeting. Two peer-reviewed publications have been published in Applied and Environmental Microbiology. A third manuscript in currently under review by Food Control. What do you plan to do during the next reporting period to accomplish the goals?We will work on Objective 4 of the proposed project (Determine whether the presence of plant pathogens and biological control agents will change the attachment and colonization behaviors of Salmonella and E. coli O157:H7). We will continue the mutagenesis study with E. coli O157:H7.
Impacts
What was accomplished under these goals?
Objective 3 of the proposal was addressed in 2017. Materials and Methods Bacterial strains used E. coli SM10 (streps) with plasmid pLBT (ampr; mini-Tn10:lac:kanr) was used as a donor strain in the mutagenesis study. Three Salmonella strains (kans, amps) that were isolated from fresh produce-related outbreaks of human gastrointestinal infections were used in the study. The Salmonella strains were made resistant to streptomycin (kans, amps, strepr) before being used as recipients. All bacterial cultures used in the study were from our laboratory culture collection. Transposon mutagenesis Cells of individual donor and recipient strains were grown for 18 h in Luria-Bertani (LB) broth at 37°C. The resulting cultures were diluted to an optical density at 600 nm of 0.2, and the donor and a recipient culture were mixed at a 1:1 ratio. Twenty-five μl of the mixtures were spotted on the surface of LB agar, and the cultures were incubated at 37°C for 16 h. Salmonella colonies resistant to kanamycin and streptomycin were selected. The identities of selected Salmonella colonies were confirmed by growth on XLT4 agar, slide agglutination with Poly A-I & Vi antiserum, and polymerase chain reaction using the invA & invE primers. The susceptibility of the identified Salmonella colonies to ampicillin was examined. Selection of mutants deficient in biofilm formation The ability of selected mutant cells in forming biofilms were assessed using 24 well polystyrene tissue culture plates as contact surface. Diluted (1:40) overnight cultures of Salmonella mutants (2 ml) were inoculated into individual wells of the tissue culture plates. Plates which were inoculated with the wild type parental cultures and un-inoculated LB broth were used as controls. After incubation for 7 days at 28oC, the broth cultures were withdrawn, and bacterial cells loosely attached to the surface were removed by washing the plates twice, with 2 ml of sterile water each time. The plates with biofilms were air dried at 60oC for 2 h, and the biofilm mass was quantified using the crystal violet binding assay previously described by Pawar et al. (2005). Attachment to vegetable seeds The attachment of Salmonella mutants to vegetable seeds was conducted according to the method described by Cui et al. (2017). Two grams of alfalfa and fenugreek seeds were sanitized with 10 ml of 20,000 ppm sodium hypochlorite solution (pH 6.8) at room temperature for 15 min and then neutralized with 10 ml of Dey-Engley neutralizing broth for 10 min followed by rinsing with sterile water. An overnight culture of each Salmonella mutant in LB no salt broth (103 CFU/ml) was mixed with sanitized seeds. Vegetable seeds were allowed to attach to the surface of vegetable seeds at 20 °C for 5 h with gentle mixing. The inocula were then decanted, and seeds were rinsed twice, each with 10 ml of sterilized water for 30 s with gentle mixing. Seeds were then soaked overnight at 4 °C in 5 ml of phosphate-buffered saline (pH 7.4) to release attached bacterial cells. Each sample in the experiment was duplicated and all experiments were conducted twice. The ratio of number of attached cells to the number of cells in an inoculum was reported as attachment ratio. Extraction of total cellular DNA Overnight cultures of mutant cells with altered ability in formation of biofilm and attachment to vegetable seeds due to transposon mutagenesis were centrifuged for 10 min at 5,000 g and resulting pellets were re-suspended in 200 µl of TE buffer. Genomic DNA was extracted and purified using a commercial kit from Thermo Scientific by following the manufacturer's instructions. Identification of genes whose products are critical for pathogen attachment and biofilm formation Extracted DNA of Salmonella mutants was used as templates, and an oligonucleotide sequence near the end of mini-Tn10 and EcoRI or PstI recognizing sequence were used as forward and backward primer, respectively in PCR reactions to amplify a single degenerate PCR product from each mutant. Amplified PCR products were purified using the PureLink PCR Purificatio Kit (Invitrogen) by following the manufacturer's instructions. Purified products were submitted to Eurofins Genomics, a Eurofins MWG Operon company (Louisville, KY) for sequencing using the cycle sequencing technology (dideoxy chain termination/cycle sequencing) on a ABI 3730XL sequencing machine. The sequencing results were compared to the published sequences using BLAST search. Site of mini-Tn10 insertion was subsequently identified and confirmed. Results Over 1,000 mutant colonies were screened, and a total of 484 mutant colonies were selected from the experiment based on colony morphology and hydrophobicity. The selected colonies were confirmed as Salmonella by the biochemical, serological, and PCR tests stated previously. Colonies that were sensitive to ampicillin, an antibiotic marker on pLBT that delivered the transposons from the donor to recipient cells, were selected for biofilm formation on polystyrene surface and attachment to vegetable seeds. Mutant colonies of Salmonella accumulated either significantly more or less biofilm mass on polystyrene surface compared to the biofilm mass formed by their parent cells. Five Salmonella mutant colonies, M13 and M36 (from S. Baildon), M370 (from S. Enteritidis), and M470 (from S. Cubana) with decreased biofilm-forming ability and mutant colony M484 (from S. Enteritidis) with increased biofilm-forming ability were selected for further study. The biofilm mass accumulated by the mutant cells correlated positively with their attachment ratios from fenugreek and alfalfa seeds with a R2 value of 0.97 and 0.67, respectively. DNA sequence of amplified products has been identified. Sequencing analysis of the degenerate PCR products and subsequent BLAST search revealed that the mini-Tn10 on pLBT has inserted into the cdg, trx, fadI, or rxt on Salmonella chromosomes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Cui, Y, R. Walcott, and J. Chen. 2017. Differential attachment of Salmonella enterica and enterohemorrhagic Escherichia coli to alfalfa, lettuce, tomato and fenugreek seeds. Appl. Environ. Microbiol. 83:e03170-e03176.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Liu, D., Y. Cui, R. Walcott, and J. Chen. 2018. Fate of Salmonella enterica and pathogenic E. coli cells artificially internalized into vegetable seeds during germination. Appl. Environ. Microbiol. doi: 10.1128/AEM.01888-17.
- Type:
Journal Articles
Status:
Other
Year Published:
2017
Citation:
Liu, D., Y. Cui, R. Walcott, and J. Chen. Transmission of human enteric pathogens from artificially-inoculated flowers to vegetable sprouts/seedlings developed via contaminated seeds. (Completed, not yet submitted).
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Cui, Y., D. Liu, and J. Chen. Fate of Salmonella enterica and enterohemorrhagic Escherichia coli cells attached to vegetable seeds during germination. Food Control (Under review)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Liu, D., Y. Cui., R. Walcott, and J. Chen. 2017. Migration of pathogenic E. coli, artificially internalized into vegetable seeds, to different sections of sprouts/seedlings during germination. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 9-12 July. Tampa, FL. p.70.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Cui, Y., D. Liu, R. Walcott, and J. Chen. 2017. Migration and growth of enterohemorrhagic Escherichia coli from inoculated and accompanying contaminated vegetable seeds to sprouts or seedlings. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 9-12 July. Tampa, FL p. 94.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Liu, D., Y. Cui., R. Walcott, and J. Chen. 2017. Migration of pathogenic E. coli, artificially internalized into vegetable seeds, to different sections of sprouts/seedlings during germination. GA Bio Innovation Summit. 24 October. Atlanta, GA p. 39.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Cui, Y., D. Liu, R. Walcott, and J. Chen. 2017. Migration and growth of enterohemorrhagic Escherichia coli from inoculated and accompanying contaminated vegetable seeds to sprouts or seedlings. GA Bio Innovation Summit. 24 October. Atlanta, GA p. 39.
|
Progress 12/15/15 to 12/14/16
Outputs
Target Audience:Seed producers and processors, vegetable and sprout growers, researchers in the food industry, government and academia. Changes/Problems:Changes or modifications have been made on some steps of experimental approaches under Objective 3. Instead of using one E. coli donor as proposed in the grant application, a second donor was identified and used in the study since the first donor contains LacZ and might not be a good candidate for mutagenesis involving E. coli. Both donors were used in the Salmonella conjugation experiment and only 1 was used in the E. coli experiment. Instead of using cloning and sequencing as an experimental approach to identify site of transposon insertion as outlined in our grant application, we used degenerate PCR and DNA sequencing, and sequence alignment as a preliminary step to identify the site of transposon insertion followed by a secondary, specific PCR amplification and DNA sequencing. The protocol is proven successful since the site of transposon insertion has been identified in four mutants of Salmonella. Instead of using seeds as matrix to assess the attachment and biofilm-forming ability of mutant colonies, polystyrene surfaces were used. This is because vegetable seeds have different and uneven surface which brought undesirable variation to the experiment. What opportunities for training and professional development has the project provided?The project is currently training 4 graduate students and 1 laboratory helper. All graduate students have the interest to become future faculty members. The project provides an excellent opportunity for the students to gain research training and laboratory experience. Meanwhile, they were given the responsibility to supervise the laboratory helper, which is an opportunity for them to learn management and supervisory skills. In addition to laboratory research, the graduate students were encouraged to get involved in food safety professional societies. The involvement will allow them to develop professional networks and get connected with their peers. How have the results been disseminated to communities of interest?Results of the research have been presented at the annual meeting of International Association for Food Protection and USDA NIFA Project PD annual meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 3 (Identify the genes and gene products that are critical for Salmonella and E. coli O157:H7 attachment to vegetable seeds and transmission from seeds to sprout and seedling tissues) will continue. We will also start working on Objective 4 of the proposed project (Determine whether the presence of plant pathogens and biological control agents will change the attachment and colonization behaviors of Salmonella and E. coli O157:H7) in the second half of 2017.
Impacts
What was accomplished under these goals?
The remaining portion of Objective 2 and initial portion of Objective 3 in the research grant application were addressed in 2016. Objective 2 Methodology Lettuce, tomato, alfalfa and fenugreek flower inoculation and seed collection Three lettuce plants (cultivar Iceberg) were grown in 10-L plastic pots containing commercial potting mix (Fafard 3B mix, Sungro, Agawam, MA). Pots were placed in an environmental chamber under the condition of 24 ºC, mean relative humidity of 76% and 12-h of natural daily light. Each of the three plants was retained for Salmonella and E. coli O157:H7 inoculation or as un-inoculated controls. At least 50 flowers on each plant were inoculated with a cell suspension of S. Baildon or E. coli K4492 at 107 CFU/ml using sterile cotton swabs. Flowers in the control plants were inoculated with PBS (pH 7.4). Following the inoculation, the plants were left to mature for 20 days before seeds were collected. Fourteen tomato plants (cultivar Roma) were grown in environmental chambers under the conditions similar to those used for growing lettuce. At least 20 flowers on each of the six plants were inoculated with S. Montevideo or E. coli K4492. Similar number of flowers on each of the two remaining plants was inoculated with PBS buffer. The plants were left to mature for 40-45 days before seeds were collected. Twelve alfalfa plants were grown in the environmental chambers described above. About 40-80 flower clusters on each of the five plants were inoculated with S. Stanley or E. coli 4492. Same number of flower clusters on each of the remaining two plants was inoculated with PBS buffer. Seed pots were allowed to mature for 42-48 days before seeds were collected. Twenty-seven fenugreek plants were cultivated under the conditions described above and about 10-14 flowers on 9 different plants were inoculated with S. Baildon, E. coli O104:H4 ATCC BAA-2326 and PBS buffer, respectively. Seed pots were allowed to mature for 35 days before seeds were collected. Nalidixic acid resistant cells of Salmonella and E. coli O157:H7 were used as inocula in the study. Sample preparation and pathogen isolation Harvested seeds were surface disinfected and disinfected seeds were dried overnight in a laminar flow hood. The seeds were then germinated on 1.0% water agar in a germination box at 25oC for 7 days. Seedlings/sprouts were homogenized in 2.0 ml of PBS buffer and plated on bismuth sulfite agar or sorbitol MacConkey agar supplemented with 50 µg/ml of nalidixic acid. Objective 2 Results Lettuce seeds were harvested from 50 Salmonella inoculated flowers, E. coli inoculated flowers and control flowers and each flower produced ca. 10-25 seeds. Tomato seeds were harvested from 91 Salmonella inoculated fruits, 81 E. coli inoculated fruits and 25 control fruits, and each fruit generated 2-17 seeds. With regard to alfalfa, 3-11 pods were harvested from each inoculated flower cluster. Salmonella inoculated cluster had an average 1.8 seeds per pod, E. coli inoculated cluster has an average of 2.4 seeds per pod, and the control sample had an average of 2.2 seeds per pod. The control fenugreek flowers generated 11.6 seeds per pot, whereas Salmonella and E. coli inoculated fenugreek flowers generated 10.4 and 11 seeds per pot, respectively. One tomato sample had 4 CFU/seed of Salmonella and 6 additional samples tested positive for Salmonella after enrichment. Furthermore, two alfalfa samples and 1 fenugreek sample tested positive for Salmonella. All samples tested negative for the inoculated E. coli and no lettuce samples tested positive for Salmonella. Objective 3 Methodology Transposon mutagenesis and selection of attachment mutants E. coli (pLBT; mini-Tn10:lac:kan) and E. coli (PUT; mini-Tn5:LuxAB:Tc) were used as donor strains in the mutagenesis studies involving Salmonella but only the latter was used in the experiment involving EHEC. Three strains of Salmonella (kans or Tcs) and three strains of E. coli O157:H7 (Tcs), that were recovered from fresh produce-related outbreaks of human gastrointestinal infections, were used as recipients in the mutagenesis study. The Salmonella strains were made resistant to streptomycin when E. coli (pLBT; mini-Tn10:lac:kan) was used as a donor and to nalidixic acid when E. coli (PUT; mini-Tn5:LuxAB:Tc) was used as a donor. The EHEC recipients were only made resistant to nalidixic acid since only E. coli (PUT; mini-Tn5:LuxAB:Tc) was used as a donor strain. Individual donor and recipient were grown for 18 h in Luria Bertani (LB) broth at 37°C. The resulting cultures were diluted to an optical density at 600 nm of 0.2, and mixed at a 1:1 ratio. Twenty-five μl of the mixtures were spotted on LB agar, and the cultures were incubated at 37°C for 16 h. Following the incubation, non-attachment mutants were selected. Identification of genes whose products are critical for pathogen attachment and biofilm formation Total cellular DNA was extracted from selected mutant colonies with altered ability in biofilm formation due to transposon mutagenesis. Extracted DNA of Salmonella and EHEC was used as templates, and an oligonucleotide sequence near the end of mini-Tn10 and EcoRI or PstI recognizing sequence were used as forward and backward primer, respectively in PCR reactions to identify the potential sites of mini-Tn10 insertion in Salmonella. In E. coli, the potential sites of transposon insertion was identified using selected sequences near the end of min-Tn5 and EcoRI or PstI recognizing sequence as primers. Amplified PCR products were sequenced. Obtained DNA sequences were compared to the published sequences using BLAST search. Site of transposon insertion was subsequently identified and confirmed. Objective 3 Results In the mutagenesis experiment involving the Salmonella strains, over 1,000 mutant colonies were screened, and a total of 484 mutant colonies were selected from the experiment involving one of the E. coli donors (pLBT; mini-Tn10:lac:kan). From the conjugation experiment between E. coli (pUT; mini-Tn5:LuxAB:Tc) and Salmonella, approximately the same number of potential colonies were screened and a total of 158 colonies were selected for further analysis. The selected colonies were confirmed using biochemical tests, slide agglutination assay, as well as PCR using the inv gene primers. Most importantly, all colonies were sensitive to ampicillin, an antibiotic marker on pLBT and pUT plasmids that delivered the transposons from the donor to recipient cells. In the mutagenesis experiment involving the EHEC strains, close to 2,000 colonies were screened and approximately 15 mutant colonies were sensitive to ampicillin, the antibiotic resistance marker on transposon-bearing plasmid. The colonies were also confirmed using biochemical tests, slide agglutination assay, as well as PCR and primers derived from the Shiga toxin genes. Mutant colonies of Salmonella and EHEC accumulated either significantly more or less biofilm mass on polystyrene surface compared to the biofilm mass formed by their parent cells. Ten Salmonella mutants and 5 EHEC mutants, some with decreased biofilm forming ability and others with increased biofilm forming ability were selected for further study. Degenerated PCR products have been obtained from selected Salmonella mutants using the oligonucleotide primer near the end mini-Tn10 or min-Tn5 and EcoRI recognizing sequence as primers. DNA sequence of amplified products has been identified through DNA sequencing and BLAST search. Based on obtained sequences, specific primers have been designed, and secondary PCR assays have been conducted using the new primers. Site of transposon insertion in four of the mutant colonies has been identified and we are in the process of confirming the results. We are also in the process of analyzing the site of transposon insertion in EHEC and other Salmonella mutants.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Cui, Y. R. Walcott, and J. Chen. 2016. Migration of Salmonella enterica from inoculated and accompanying contaminated vegetable seeds to sprouts or seedlings. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 31 July 3 August. St. Louis, MO. p. 174.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Liu, D. Y. Cui, R. Walcott, and J. Chen. 2016. Migration of Salmonella enterica, artificially internalized into vegetable seeds, to different sections of sprouts/seedlings during germination. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 31 July 3 August. St. Louis, MO. p. 173.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Cui, Y, R. Walcott and J. Chen. Differential attachment of Salmonella enterica and enterohemorrhagic Escherichia coli to alfalfa, lettuce, tomato and fenugreek seeds. Appl. Environ. Microbiol. (Submitted).
- Type:
Journal Articles
Status:
Other
Year Published:
2017
Citation:
Liu, D., Y. Cui, R. Walcott, and J. Chen. In preparation. Migration of Salmonella enterica and pathogenic E. coli, artificially internalized into vegetable seeds, to different sections of sprouts/seedlings during germination. Appl. Environ. Microbiol. (In preparation).
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Progress 12/15/14 to 12/14/15
Outputs
Target Audience:Seed producers and processors, vegetable and sprout growers, reseachers in the food industry, government and academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project is currently training 4 Ph.D. level graduate students and 1 laboratory helper. All gradaute students have the interest to become future faculty members. The project provides an excellent opportunity for the students to gain research training and laboratory experience. Meanwhile, they were given the responsbility to supervise the laboratory helper, which is an opportunity for them to learn mamngement and supervisory skills. In addition to laboratory research, the graduate students were encouraged to get involved in food safety professional societies. The involvement will allow them to develop professional networks and get connected with their peers. How have the results been disseminated to communities of interest?Results of the research have been presented at the annual meeting of two professional societies, International Association for Food Protection and Georgia Bio Innovation Summit. What do you plan to do during the next reporting period to accomplish the goals?We will address Objective 3 of the proposal "Identify the genes and gene products that are critical for Salmonella and E. coli O157:H7 attachment to vegetable seeds and transmission from seeds to sprout and seedling tissues".
Impacts
What was accomplished under these goals?
The project proceeds as what was proposed. Objective 2 (Observe the migration of selected human enteric pathogens from contaminated seeds to sprouts and different areas of seedlings) is addressed in 2015. Output: Alfalfa, fenugreek, tomato and lettuce seeds (2 g) were sanitized with 3% calcium hypochlorite followed by neutralization with Dey-Engley buffer. Sanitized seeds were rinsed twice with sterile water. Each type of vegetable seeds were artificially contaminated (by simulating infestation, accompanying contamination and infiltration) with 2 or 4 log units of 4 individual strains of Salmonella (S. Stanley from a sprout associated outbreak, S. Baildon from a tomato associated outbreak, S. Cubana from a spout associated outbreak and S. Montevideo from a tomato associated outbreak) and 4 individual strains of E. coli (F4546 from a sprout associated outbreak, K4492 from a spinach associated outbreak, H1730 from a lettuce associated outbreak and ATCC BAA-2326 from a fenugreek associated outbreak). For infestation, seeds were exposure to 20 ml of 102 or 104 CFU/ml of Salmonella or E. coli which were from 24-h cultures grown in LB no salt broth supplemented with nalidixic acid (NA) at 50 μg per ml. For accompanying contamination, seeds were mixed with 20 g of autoclaved sandy soil containing 102 or 104 CFU/ml of Salmonella or E. coli. For pathogen infiltration, seeds were exposed under negative pressure to 20 ml of 102 or 104 CFU/ml of Salmonella or E. coli and following the contamination, a 2nd step chlorine treatment and neutralization were applied to the contaminated seeds in order to remove the bacterial cells that might be located on seed surface. Precise inoculation levels were determined using standard plating count assay. Contaminated seeds were then placed on 1% water agar in square Petri dishes for germination, and 5-10 each type of seeds that were inoculated with each pathogen were selected at day 1, 3, 5, 7 and 9 and sampled on tryptic soy agar (TSA) for total aerobic counts, NA-TSA and bismuth sulfite agar for inoculated Salmonella counts, and NA-TSA, NA-MacConkey/NA-sorbitol MacConkey agar for inoculated E. coli counts. Each sample had a duplicate, the each experiment was repeated once. Fisher's Least Significance Design of General Linear Model of SAS program was used to determine the significance of difference among the average populations of each tested pathogen on different vegetable seeds or different sections of seedlings. The same statistical test was also used to analyze the influence of sample time on bacterial counts on seeds and seedlings. Outcome: In general, pathogen infestation resulted in the highest incidence/number of contaminated seeds and seedlings, followed by accompanying contamination and pathogen infiltration. Detailed results are summarized below. Seed Contamination by Infestation: Salmonella: Fenugreek seeds and seedlings had the highest Salmonella counts followed by alfalfa and lettuce seeds and seedlings. Tomato seeds and seedlings had average cell counts that were 4.61-4.89 log units lower than those on the other types of seeds and seedlings. More Salmonella was recovered from cotyledon, followed by seed coat, seedling root, seedling stem, seed-seed coat, and initially inoculated seeds. The populations of Salmonella increased with germination time from Day 1 to Day 9. Seeds and seedlings contaminated with S. Cubana had significantly higher cell counts than those that were contaminated with S. Stanley and S. Montevideo. Seeds and seedlings contaminated with S. Baildon had the 2nd highest cell counts which were not significantly different from those that were inoculated with the other 3 Salmonella strains. E. coli: Similar to the Salmonella experiment, fenugreek seeds and seedlings had the highest cell counts followed by alfalfa and lettuce seeds and seedlings. Tomato seeds and seedlings had average cell counts that were 3.53-3.99 log units lower than those on the other types of seeds and seedlings. The populations of E. coli increased with germination time from Day 1 to Day 9. Different from the Salmonella experiment, more E. coli cells were recovered from seed coat followed by seedling root, seed-seed coat, cotyledon, seedling stem and initially inoculated seeds. Seeds and seedlings inoculated with F4546 and H1730 had significantly higher average cell counts than those inoculated with the other two E. coli strains. Seeds and seedlings contaminated with K4492 had significantly higher cell counts than those inoculated with BAA-2326. Accompanying Contamination Salmonella: Seeds and seedlings inoculated with the 4 Salmonella strains had significantly different cell counts. Seeds inoculated with S. Baildon had the highest Salmonella counts than those that were inoculated with S. Stanley, S. Cubana and S. Montevideo. Seed coat had the highest number of Salmonella followed by seedling root, cotyledon, seedling stem, seed-seed coat, and initially inoculated seeds. The populations of the pathogens on inoculated seeds and seedlings increased as germination time increased, but the cell counts from samples assayed at Day 5 and 7 were not significantly different. Seeds and seedlings of fenugreek had the highest Salmonella counts followed by lettuce, and alfalfa seeds and seedlings. The contamination rates of tomato seeds and seedlings were1.41 to 4.07 log units lower than the other types of seeds and seedlings. Salmonella cell counts on the 4 types of seeds and seedlings were significantly different. E. coli: Seeds and seedlings inoculated with the 4 E. coli strains also had significantly different cell counts. Those inoculated with F4546 had the highest cell counts, followed by K4492, H1730 and BAA-2326. Seed coat had the highest number of E. coli followed by seedling root, seed-seed coat, cotyledon, seedling stem, and initially inoculated seed. The populations of pathogens on inoculated seeds and seedlings increased during germination with the exceptions of Day 3 and Day 5. Seeds and seedlings of fenugreek had the highest E. coli counts, followed by lettuce seeds and seedlings. Average cell counts on tomato and alfalfa seeds and seedlings were 1.27 and 0.87 log CFU/g, respectively. E. coli cell counts on the 4 types of seeds and seedlings were significantly different. Infiltration: Salmonella: Seeds and seedlings inoculated with S. Stanley, S. Cubana and S. Baildon had significantly higher cell counts than those inoculated with S. Montevideo. The populations of Salmonella recovered from cotyledon and seed coat were significantly higher than those on seedling root, seed-seed coat, seedling stem and initially contaminated seed. The populations of inoculated E. coli on day 5 to day 9 were not significantly different, but these cell populations were significantly higher than the pathogen populations at the earlier stage of the germination. Fenugreek and alfalfa seeds and seedlings had the highest populations of E. coli. The lettuce and tomato seeds and seedlings had much lowered average cell counts, and were not significantly different from each other. E. coli: Seeds and seedlings inoculated with K4492, H1730 and BAA-2326 had significantly higher cell counts than those inoculated with F4546. The populations of E. coli recovered from seed coat and cotyledon were significantly higher than those on the initially contaminated seed. But pathogens recovered from other sections of the seedlings were not significantly different. In general, the populations of inoculated E. coli increased as germination time increased except day 5. Alfalfa and fenugreek seeds and seedlings had the highest populations of E. coli. The lettuce and tomato seeds and seedlings had much lowered average cell counts, ranging from 0.20-0.34 log CFU/seed.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Cui, Y., R. Walcott, and J. Chen. 2015. Attachment of various serovars of Salmonella enterica to vegetable seeds with different surface characteristics. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 25-29 July. Portland, OR. p. 52.
Cui, Y., R. Walcott, and J. Chen. 2015. Attachment ability of Shiga toxin-producing Escherichia coli to alfalfa, lettuce, tomato and fenugreek seeds. Int. Assn. Food Prot. Annu. Mtg. Prog. Abstr. Book. 25-29 July. Portland, OR. p. 74.
Cui, Y., R. Walcott, and J. Chen. 2015. Attachment of various serovars of Salmonella enterica to vegetable seeds with different surface characteristics. GA Bio Innovation Summit Prog. Abstr. Book. 2 Nov. Atlanta GA. p. 39.
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Progress 12/15/13 to 12/14/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project is currently training three graduate students and 1 undergraduate student. The gradaute students have the interest to become faculty members and the undergradaute student wants to enroll phamacy school next year. The project provides an excellent opportunity for the students to gain research training and laboratory experience. The graduate students were given the responsbility to supervise the undergrauate student, which will provide the opportunity for them to learn mamngement skills. In addition to laboratory research, the graduate students were encouraged to get involved in food safety professional societies. The involvement in professional society will allow them to develop their professional networks and get connected with their peers. How have the results been disseminated to communities of interest? The results obtained are currently being analyzed. We plan to present the results at the annual meeting of several professional societies, including the annual meeting of IAFP and Georgia Life Science Summit. What do you plan to do during the next reporting period to accomplish the goals? We plant to analyze the results obtained in this report period and present them at the annual meeting of professional societies. We also plan to address Objective 2 of the project (Observe the migration of selected human enteric pathogens from contaminated seeds to sprouts and different areas of seedlings)in the next reporting period.
Impacts
What was accomplished under these goals?
Objective 1 of the project was addressed in the first year of the project. Methodology used: Chemically treated vs. untreated and intact vs. mechanically damaged seeds of alfalfa, fenugreek, tomato and lettuce were artificially contaminated with 2 or 4 log of 3 individual strains of Salmonella (S. Stanley from a sprout associated outbreak, S. Baildon from a tomato associated outbreak, S. Cubana from a spout associated outbreak and S. Montevideo from a tomato associated outbreak) and 3 individual strain of E. coli (F4546 from a sprout associated outbreak, K4492 from a spinach associated outbreak, H1730 from a lettuce associated outbreak and ATCC BAA-2326 from a fenugreek associated outbreak). Each type of vegetable seeds (2 g) was sanitized with 3% calcium hypochlorite which was neutralized with Dey-Engley buffer after the decontamination. Sanitized seeds were rinsed twice with sterile water and then inoculated with 20 ml of 102 or 104 CFU/ml of Salmonella or EHEC which were from 24-h cultures grown on LB no salt broths supplemented with nalidixic acid at 50 μg per ml. Precise inoculation levels were determined by plating 100 μl of appropriately diluted cultures on tryptic soy agar containing nalidixic acid. Contaminated vegetable seeds were shaken at 50 rpm on a platform shaker at 21°C for 5 h. The inocula were then decanted to a sterile test tube, and seeds were rinsed by adding 10 ml of sterile water and then decanting the liquids into sterile test tubes. Contaminated seeds were rinsed twice with sterile water with nalidix acid. Each type of vegetable seeds were then soaked overnight at 4°C in 5 ml of phosphate buffered saline (pH 7.4). The samples were then removed from refrigeration storage and vortexed for 30 s at maximal speed. Every sample and appropriate dilutions were plated onto bismuth sulfite agar,MacConkey or Sorbitol MacConkey agar with nalidixic acid. All samples were also plated on tryptic soy agar (TSA) and TSA with nalidixic acid. The CFU per ml of rinse and CFU per gram of seed were reported. The ratio of the number of attached cells to the number of inoculated cells was reported as irreversible adhesion capacities. Student t tests were performed to determine the significance of difference among the average populations of each tested pathogen on vegetable seeds. The same statistical test will be used to analyze the differences in pathogen attachment to intact vs. mechanically damaged seeds as well as chemically treated vs. untreated seeds. Results Obtained: Results of statistical analysis revealed that on average, Salmonella and E. coli adhesion to the mechanically damaged seeds was significantly higher (p<0.05) than to the intact seeds. Similarly, pathogen attachment to the untreated seeds was either significantly (p<0.05) or numerically higher than to the chemically treated seeds. On average, S. Baidon had the greatest attachment capacity to the vegetable seeds used in the study, which was followed by S. Cubana, S. Stanley, and then S. Montevideo. E. coli K4492 had the greatest adhesion capacity, which was followed by E. coli F4546, and then H1730 and ATCC BAA-2326. On average, Salmonella adhered to lettuce seeds at the highest rate among the four strains used in the present study, followed by other types of seeds. E. coli strains used in the study also had the greatest adherence on lettuce seeds, followed by tomato seeds, alfalfa seeds and fenugreek seeds. On average, E. coli strains used in the study had relatively lower irreversible adhesion ratios comparing to the Salmonella strains. However, E. coli K4492 has relatively higher irreversible adhesion ratios than some of the Salmonella strains used in the study.
Publications
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