Progress 09/01/13 to 08/31/18
Outputs
Target Audience:Food safety advocates, Veterinarians, Diagnosticians, Poultry Practitioners, Producers, Researchers, Immunologists, Plant pathologists, and Food safety microbiologists, Food safety policy makers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1. Trained a Graduate student on understanding colonization of Salmonella in tomato plants under different environmental condition 2. Trained a Graduate student in performing high throughput sequencing to identify anti-Salmonella compounds 3. Trained 1 Postdoctoral Fellow in barcode sequence analysis 4. Trained 1 Research Associate in barcode sequence analysis 5. Trained 1 Graduate students in Tn-Seq pool construction, sample collection and Next-Generation sequencing library preparation How have the results been disseminated to communities of interest?Through scientific publications, fact sheets, national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective1: Determinetheinfluenceofphysicalandenvironmentalfactors,andphytopathogen infectionontheinteractionofS.Typhimuriumwithtomatoplants. 1.A. Impact of determined environmental conditions on the persistence and dissemination of Salmonella in tomato seedlings:We investigated the effects of specific environmental conditions on the persistence and dissemination of Salmonella in tomato plants following contamination. Higher environmental temperatures (30°C day/25°C night) reduced the persistence of Salmonella in the phyllosphere compared to lower temperatures (20°C day/15°C night) when plants were sprayed on the leaves with a Salmonella-contaminated solution. However, wounding cotyledons with contaminated tools improved Salmonella persistence and internalization rate (2.27%) in planta compared to sprayed inoculation (0.004%). The systemic dissemination of Salmonella to other tissues increased when contaminated plants were grown under low relative humidity condition (< 40%), independent of the inoculation method; however, Salmonella was only detected in the root systems at later sampling times (up to 98 days post-inoculation; DPI). 1.B. Impact of grafting using Salmonella-contaminated blades on the persistence and dissemination of Salmonella in tomato seedlings:After grafting tomato scions onto rootstocks using contaminated cutting tools, dissemination of Salmonella was preferentially basipetal and occasionally acropetal in the plants, with higher persistence rates and loads of Salmonella in root systems compared to foliar tissues. These results were influenced by the inoculation method and inoculum density used; however, Salmonella was not detected in the fruits harvested from contaminated plants between 90 DPI and 137 DPI. This study demonstrates that environmental temperature and relative humidity could be a good indicator for estimating the persistence of Salmonella in tomato plants. Further, root systems may represent a risk for long-term persistence of Salmonella in tomato plants. 1.C. Impact of phytopathogens on the persistence and dissemination of Salmonella in tomato seedlings:Our study showed that Clavibacter michiganensis subsp. michiganensis (Cmm) increased S. Typhimurium persistence (up to 1.8-log CFU/plant at 21 DPI) in tomato plants when both pathogens were co-inoculated compared to plants inoculated with S. Typhimurium alone, while Xanthomonas gardneri (Xg) did not affect the persistence of S. Typhimurium in planta. On the other hand, S. Typhimurium reduced the population of both phytopathogens (0.5-log Xg/plant and 1.5-log Cmm/plant at 21DPI) compared to plants inoculated with Xg or Cmm alone. However, no antagonistic effect was observed when S. Typhimurium was inoculated seven days after Cmm or Xg in the same plant tissues. Objective2:Identify, using highthroughput genomic screening, those environmental conditions and pathogen pathways required for S. Typhimuriumsurvivalin tomato plants. Here we seek to identify using high throughput genomic screening the environmental conditions and pathogen specific genetic pathways that are required for S.Typhimuriumtosurvivein tomato. Towards this end we sequenced the reference strain which will be used for transposon mutagenesis using next generation sequencing and a variety of sequence analysis routines to identify any and all polymorphismsor structural variants present in the reference strain genome. This high coverage sequence map(approximately100x) will also guide our analysis of each barcoded transposon insertion site. We have completed construction of a new barcoded transposon construct for rapid high throughput library generation.This transposon has several novel features including a 25 base pair barcode which in contrast to the 20 base pair barcodes used in previous studies offers greater than one million barcoded transposon's per strain. We received a collection of sequence-verified barcodes (as 25mers) from Dr. Steven Elledge of Harvard University as cassettes of 4 barcodes/plasmid. We transferred 100,000 of these barcodes into our S. typhimurium Tn-seq transposon and usedour barcode sequencing protocol to maximize the generation of insert containing clusters on the HiSeq2500 next-generation sequencer. We verified that over 95,000 barcodes were successfully transferred into the Tn-Seq cassette and have completed electroporation ofS. Typhimuriumto generate the Tn-Seq chemogenomic screening library. These mutants are being characterized. Objective3:EvaluateselectedcompoundsfortheireffectonSalmonellapersistenceintomato: Of the 4,182 small molecules (SMs) screenedin vitro, fourcidalSMs (SM1, SM3, SM4, and SM5) were effective at10µMand higher against several serotypes, antibiotic-resistant, and biofilm embedded Salmonella by altering cell membrane integrity. Further,no toxicity was detected on tomato seeds, plants, and fruits when tissues were treated with 200 µM of SM. Moreover, SM1 and SM4 reduced (79% and 64%, respectively) the Salmonella population in tomato plants when sprayed on the leaves with two times the minimal bactericidal concentration (MBC; SM1=100 µM, SM3 and SM5=50 µM, and SM4=20 µM) at -1 and 3 DPI. The four SMs had limited impact on the growth of beneficial plant bacteria with antagonistic effects on Salmonella (Bacillus amyloliquefaciens and subtilis, and Enterobacter sp.) when challenged in minimal growing condition at 200 µM of SM. Once SM1 and SM4 were combined with B. amyloliquefaciens or Enterobacter sp. by spraying them at -3 DPI and -1DPI and the SMs at 3 DPI, the Salmonella population was reduced by at least 98.2% in tomato plants. The four SMs also reduced (up to 95%) the Salmonella population on the surface and inside contaminated green mature and ripened tomato fruits following spraying or soaking the fruits in cold water for 30 min with 2X MBC. However, the real time in vivo imaging data showed that the stem abscission zone in fruits represent a major source of internalization and persistence of Salmonella against antimicrobials. The four SMs also displayed encouraging potential to control phytopathogens by killing them in enriched growing condition with 200 µM of SM. This study demonstrates that our four SMs could represent a safe method to control Salmonella and phytopathogens in fresh produce and compatible with the use of biocontrol agents. The antimicrobial activity of the four SMs was also tested for poultry production systems.The SMs were not pernicious to most eukaryotic cellsat 200 μMand cleared internalized Salmonella in infected Caco-2, HD11, and THP-1 cells at 6.25 µM and higher. The SMs also increased the longevity of Salmonella-infected Galleria mellonella larvae and reduced the population of internalized S. Typhimurium. Two of the SMs (SM4 and SM5) alsoreduced S. Typhimurium load in infected chicken ceca as well as its systemic translocation into other tissues, with minimal impact on the cecal microbiota. Further,the four SMs displayed synergistic effects with ciprofloxacin, meropenem and cefeprime against Salmonella.This study demonstrated that SMs are a viable source of potential antimicrobials applicable in food animal production against Salmonella. In summary, we have identified four novel anti-Salmonella compounds that are not toxic for tomato plants, effective against Salmonella in tomato seedlings, fruits, and chickens, compatible with other control methods, and effective against several plant pathogens.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
L. Deblais, R. Candelero, Y.A. Helmy, C. Vrisman, S.A. Miller, and G. Rajashekara A combined genomic and in vivo imaging platform to understanding Salmonella-tomato plant host interactions AFRI IAFP meeting (Missouri USA July 2016).
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2016
Citation:
L. Deblais, Y.A. Helmy, R. Candelero, Huang-chi Huang, Dipak Kathayat, S.A. Miller, and G. Rajashekara Small molecules, a potential novel control strategy to reduce Salmonella burden I3S meeting (Saint-malo France July 2016).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
L. Deblais, S. Miller, F.B. Gurel, and G. Rajashekara Understanding Salmonella-tomato plant host interactions and development of effective novel control stratesgies to reduce Salmonella burden in tomato production International Association of Food Protection meeting (Oregon USA - July 2015).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
L. Deblais, F. Baysal-Gurel, S.A. Miller, and G. Rajashekara Understanding Salmonella-tomato plant host interactions and development of effective novel control strategies to reduce Salmonella burden in tomato production OARDC, The Ohio State University Annual Conference (Ohio USA - Mach 2015).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
L. Deblais, R. Candelero, Y.A. Helmy, C. Vrisman, S.A. Miller, and G. Rajashekara A combined genomic and in vivo imaging platform to understanding Salmonella-tomato plant host interactions AFRI IAFP meeting (Florida USA July 2017).
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Deblais L, Helmy YA, Kathayat D, Huang HC, Miller SA, and Rajashekara G. 2018. Novel Small Molecules Affecting the Cell Envelope Integrity to Control Salmonella in Poultry. Sci Rep 8:13381.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Deblais L, Helmy YA, Testen A, Vrisman C,Jimenez Madrid AM, Kathayat D, Miller SA, and Rajashekara G. 2018. Specific environmental temperature and relative humidity conditions and grafting affect the persistence and dissemination of Salmonella in tomato plant tissues (AEM; under review).
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Deblais L, Vrisman C, Kathayat D, Helmy YA, Miller SA, and Rajashekara G. 2018. Imidazole and Methoxybenzylamine Growth Inhibitors Reduce Salmonella Persistence in Tomato Plant Tissues (Journal of food protection; under review).
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Deblais L, Miller SA, and Rajashekara G. 2018. Tomato plants and fruit contaminated with Salmonella. Fact Sheet ANR-62 (https://ohioline.osu.edu/factsheet/anr-62)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
L. Deblais, C. Virsman, D. Katayat, S.A. Miller, and G. Rajashekara Molecules(s) produced by Salmonella inhibiting the growth of several phytopathogens. Phytopathology meeting. Boston - USA - August 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara Reduction of Salmonella burden in poultry using new generation small molecules OBASM meeting (Ohio USA December 2016).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
G. Rajashekara Salmonella Interaction with Tomato Plants and Identifying Novel Control Strategies . Inviter speaker at Bejin University (China USA June 2017).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara Novel anti-Salmonella compounds reduce Salmonella burden in poultry ASM Antimicrobial Development meeting (Washington USA March 2017).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
G. Rajashekara Salmonella Interaction with Tomato Plants and Identifying Novel Control Strategies . Inviter speaker at Microbiology department seminar (Ohio USA Mai 2016).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
L. Deblais, D. Kathayat, R. Candelero-Rueda, C. Vrisman, S.A. Miller, and G. Rajashekara Thirsty tomato plants might facilitate the vascular transport of Salmonella APS meeting (accepted abstract; Tampa USA July 2016)
|
Progress 09/01/16 to 08/31/17
Outputs
Target Audience:Food safety advocates, Veterinarians, Diagnosticians, Poultry Practitioners, Producers, Researchers, Immunologists, Plant pathologists, and Food safety microbiologists, Food safety policy makers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Trained a Graduate student on understanding colonization of Salmonella in tomato plants under different environmental condition Trained a Graduate student in performing high throughput sequencing to identify anti-Salmonella compounds Trained 1 Research Associate in barcode sequence analysis How have the results been disseminated to communities of interest?The preliminary results were presented at the: L. Deblais, R. Candelero, Y.A. Helmy, C. Vrisman, S.A. Miller, and G. Rajashekara "A combined genomic and in vivo imaging platform to understanding Salmonella-tomato plant host interactions" AFRI IAFP project directors meeting (Tampa, Florida - USA - July 2017). G. Rajashekara "Salmonella Interaction with Tomato Plants and Identifying Novel Control Strategies". Inviter speaker at Bejing Agricultural research center (Beijing, China - June 2017). L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara "Novel anti-Salmonella compounds reduce Salmonella burden in poultry" ASM Antimicrobial Development meeting (Washington DC, USA - March 2017). G. Rajashekara "Salmonella Interaction with Tomato Plants and Identifying Novel Control Strategies". Invited speaker Ohio Department of Agriculture, Columbus, Ohio - USA - May 2016. L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara "Reduction of Salmonella burden in poultry using new generation small molecules" OBASM meeting (Ashland, Ohio - USA - December 2016). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: For the fifth year, given grafting is a current operation applied in tomato production, we will try to study the plant colonization after grafting with infected tools. Manuscript summary: these results are being submitted for publication. Objective 2: In the coming months we will validate the insertion sites of each of the 95,000 transposon-disruption mutants by sequencing 5 independent libraries and, based on the sequencing results, pools these to generate a collection in which each gene in the S. typhimurium genome is represented by at least 3 barcoded insertions. This collection will be screened in parallel against the 7 small molecules described in Objective 3. Objective 3: High throughput screening has shown encouraging results for the development of novel strategies against S. Typhimurium. However at this stage of the project, more analyses need to be performed before application in the field. We will confirm the antimicrobial efficacy of the selected four compounds on infected tomato fruits. Currently, we are working on the identification of the bacterial target of the four most potential compounds using insertional mutagenesis, new generation sequencing, whole genome sequencing, protein-small molecule column affinity. Based on these results, we will focus on increasing the efficacy of these compounds by downstream modification. Manuscript summary: these results are being submitted for publication.
Impacts
What was accomplished under these goals?
Objective 1: Effect of abiotic factors (temp and humidity). Environmental temperature affected S. Typhimurium (ST) survival outside the plants, while relative humidity might affect the capacity of ST to systemically colonize the host. The combination of low humidity plus high temperature seemed to be the most unfavorable growing condition for ST however it allowed a systemic colonization of the host; while the combination of high humidity plus low temperature allowed the longest and highest survival of ST outside and inside the host without systemic colonization of the host. To identify the biotic factors leading to the survival and persistence of ST in tomato plants, young tomato plants were (A) first sprayed on the leaves with a suspension of Xanthomonas gardneri (Xg; non-systemic phytopathogen), or were directly introduced in the host via cotyledons clipping with Clavibacter michiganensis michiganensis (Cmm; systemic phytopathogen) contaminated scissors; and then inoculated a week later with ST using the same inoculation method used for the phytopathogen; or (B) both the foodborne pathogen and the phytopathogens were inoculated together at the same time using the same inoculation methods as described above. For these experiments, we studied the impact of a plant pathogen on the capacity of ST to infect and survive in the sick host and understand the potential interaction between bacterial pathogens. Colonization of tomato plants was observed weekly through bacterial quantification of inoculated plants on selective media. Salmonella inhibited Xg abundance in planta when both pathogens were co-inoculated on three-week-old "Tiny Tim" seedlings using leaf spray inoculation. The presence of Xg did not affect ST survival in the tomato plant, however the presence of ST significantly reduced Xg abundance in planta at 7DPI (0.55-log) until 21DPI (1.53-log; P<0.05). Further, the alteration in Xg population in planta was not affected if Xg was introduced into the plant a week prior to the ST inoculation. In fact, Xg population was significantly increased at 21DPI when ST was present in the inoculated leaves (1.16-log), but ST population was not altered in the presence of Xg. Similar results were obtained in vitro. The agar-well diffusion assay showed that ST inhibited Xg growth on YDC agar plate (3 mm radius inhibition zone recorded after 48 hours incubation at 28°C), however no ST growth inhibition was detected with Xg on XLT-4 agar plate. The presence of ST significantly decreased Cmm population at 7DPI and 14DPI in the inoculated cotyledons (up to 1.38-log; P<0.05); while Cmm significantly increased ST population at 7DPI until 21DPI (up to 2.47-log; P<0.05) in the inoculated cotyledons. The significant increase in ST abundance was also observed in the inoculated cotyledons when Cmm was inoculated a week before ST (P<0.05); however no growth defect was observed with the Cmm population in the inoculated cotyledons. For both experiment (simultaneous or delayed inoculations), the presence of ST had no effect on the systemic transmission of Cmm in the stem. Similar results were observed when an agar-well diffusion assay was performed with Cmm and ST. ST inhibited Cmm growth on YDC agar plate (7 mm radius inhibition zone recorded after 72 hours incubation at 28°C), however, no ST growth inhibition was detected with Cmm on XLT-4 agar plate. These results suggest that 1) ST produces an antimicrobial molecule inhibiting both phytopathogens growth, 2) Cmm might enhance ST survival in planta by increasing the free-nutrients availability, and 3) plant inoculation timing seemed to impact ST and the phytopathogens' interactions and survival in tomato plants. However ST was detected only in the inoculated tissues (in the leaves with spray inoculation; in cotyledons with clip inoculation). Manuscript summary: these results are being submitted for publication Objective 2: We sequenced the reference strain which will be used for transposon mutagenesis using next generation sequencing. This high coverage sequence map (approximately 100x) will also guide our analysis of each barcoded transposon insertion site. We have completed construction of a new barcoded transposon construct for rapid high throughput library generation. This transposon has several novel features including a 25 base pair barcode which in contrast to the 20 base pair barcodes used in previous studies offers greater than 1 million barcoded transposon's per strain. We transferred 100,000 of these barcodes into our ST Tn-seq transposon and used our barcode sequencing protocol to maximize the generation of insert containing clusters on the HiSeq 2500 next-generation sequencer. We verified that over 95,000 barcodes were successfully transferred into the Tn-Seq cassette and have completed electroporation of S. Typhimurium to generate the Tn-Seq chemogenomic screening library. Currently we are validating the insertion sites of each of the 95,000 transposon-disruption mutants by sequencing 5 independent libraries and, based on the sequencing results, pools these pools to generate a collection in which each gene in the ST genome is represented by at least 3 barcoded insertions. This collection will be screened in parallel against the 7 small molecules described in Objective 3. Objective 3: A library of 4182 small molecules was tested on ST. Among this collection, a total of 21 small molecules have shown a cidal/static effect with a minimal bactericidal concentration (MBC) between 400 μM to 10 μM. This cluster was screened against nine other most prevalent (commonly associated with food poisoning) S. enterica serotypes. The five cidal SM harboring the lowest MBCs were also cidal to the nine Salmonella serotypes. Four of the selected five candidates did not induce resistance mechanisms from ST after growing the bacteria with a sub-lethal dose of compounds (0.75xMBC) or a lethal dose (2xMBC) for 15 days. These four SM were also effective against other plant pathogens such as X. cucurbitae, E. amylovora, A. citrulli, A. rhizogenes; P. syringae, and S. marcescens; and displayed restricted effects against beneficial plant associated bacteria such as Bacillus, Pseudomonas, Enterobacter. These compounds also exhibited low cytotoxicity on Caco-2 epithelial, and on HD11 macrophage cells; They were also effective in killing internalized S. Typhimurium in these cell types at concentrations between 5 μM to 100 μM. These SM did not change the phenotype of tomato fruits and seedlings, and did not reduce the tomato seed germination rate when tested at 1xMBC. These compounds reduced up to 95% of Salmonella population present outside and inside contaminated green and ripened tomato fruits when soaked for 1 hour in a cold water containing 2xMBC of SM or spray with a water solution containing 2xMBC of SM. These SM were also safe when tested on G. mellonella larvae. All four SM decreased G. mellonella mortality and internal ST survival when larvae were treated with 12.5 μg of SM before infection with Salmonella. Two out of the four SM tested on infected chickens treated daily with 200 μg of SM for five days displayed a significant reduction of ST population in ceca (up to 2.6-log reduction; Student T test; P<0.01). Further, systemic colonization of liver and spleen was also decreased. Microbiome studies showed no significant alteration of the operational taxonomic unit (OTU) abundance, diversity, and richness in ceca compared to infected chickens (Student T test; P>0.05). Only nine out of 50 OTUs identified at the genus level were significantly affected by the SM treatments (Student T test; P<0.01). In summary, we have identified four novel anti-Salmonella compounds that are not toxic for tomato plants, effective against ST in tomato fruits and chickens, and effective against several plant pathogens. Manuscript summary: these results are being submitted for publication.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
" L. Deblais, R. Candelero, Y.A. Helmy, C. Vrisman, S.A. Miller, and G. Rajashekara A combined genomic and in vivo imaging platform to understanding Salmonella-tomato plant host interactions AFRI IAFP project directors meeting (Tampa, Florida USA July 2017).
- Type:
Other
Status:
Other
Year Published:
2017
Citation:
G. Rajashekara Salmonella Interaction with Tomato Plants and Identifying Novel Control Strategies . Invited speaker at Bejing Agricultural research center (Beijing, China June 2017).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
" L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara Novel anti-Salmonella compounds reduce Salmonella burden in poultry ASM Antimicrobial Development meeting (Washington DC, USA March 2017).
- Type:
Other
Status:
Other
Year Published:
2017
Citation:
G. Rajashekara Salmonella Interaction with Tomato Plants and Identifying Novel Control Strategies . Invited speaker Ohio Department of Agriculture, Columbus, Ohio USA Mai 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
" L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara Reduction of Salmonella burden in poultry using new generation small molecules OBASM meeting (Ashland, Ohio USA December 2016).
|
Progress 09/01/15 to 08/31/16
Outputs
Target Audience:Food safety advocates, Veterinarians, Diagnosticians, Poultry Practitioners, Producers, Researchers, Immunologists, Plant pathologists, and Food safety microbiologists, Food safety policy makers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Trained a Graduate student on understanding colonization of Salmonella in tomato plants under different environmental condition Trained a Graduate student in performing high throughput sequencing to identify anti-Salmonella compounds Trained 1 Postdoctoral Fellow in barcode sequence analysis Trained 1 Research Associate in barcode sequence analysis Trained 1 Graduate students in Tn-Seq pool construction, sample collection and Next-Generation sequencing library preparation How have the results been disseminated to communities of interest?The preliminary results were presented at the: L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara "Novel anti-Salmonella compounds reduce Salmonella burden in poultry" ASM Antimicrobial Development meeting (Washington - USA - December 2016). L. Deblais, R. Candelero, Y.A. Helmy, C. Vrisman, S.A. Miller, and G. Rajashekara "A combined genomic and in vivo imaging platform to understanding Salmonella-tomato plant host interactions" AFRI IAFP meeting (Missouri - USA - July 2016). L. Deblais, Y.A. Helmy, R. Candelero, Huang-chi Huang, Dipak Kathayat, S.A. Miller, and G. Rajashekara "Small molecules, a potential novel control strategy to reduce Salmonella burden" I3S meeting (Saint-malo - France - July 2016). L. Deblais, S. Miller, F.B. Gurel, and G. Rajashekara "Understanding Salmonella-tomato plant host interactions and development of effective novel control stratesgies to reduce Salmonella burden in tomato production" International Association of Food Protection meeting (Oregon - USA - July 2015). L. Deblais, F. Baysal-Gurel,S.A. Miller, and G. Rajashekara"UnderstandingSalmonella-tomato plant hostinteractionsand development ofeffectivenovel control strategies to reduceSalmonellaburden in tomatoproduction" OARDC, The Ohio State University Annual Conference (Ohio - United States America -March 2015). What do you plan to do during the next reporting period to accomplish the goals? Objective 1: For the fourth year, we will confirm previous results at low humidity level (40%) as well as the effect of tomato pathogens (Clavibacter and Xanthomonas) on Salmonella survival in tomato plants. In addition, given grafting is a current operation applied in tomato production, we will try to study the plant colonization after grafting with infected tools. Objective 2: In the coming months we will validate the insertion sites of each of the 95,000 transposon-disruption mutants by sequencing 5 independent libraries and, based on the sequencing results, pools these to generate a collection in which each gene in the S. typhimurium genome is represented by at least 3 barcoded insertions. This collection will be screened in parallel against the 7 small molecules described in Objective 3. Objective 3: High throughput screening has shown encouraging results for the development of novel strategies against S. Typhimurium. However at this stage of the project, more analyses need to be performed before application in the field. We will provide more information about these selected compounds via testing them on infected tomato seeds and fruits. Currently, we are working on the identification of the bacterial target of the four most using insertional mutagenesis and new generation sequencing. Based on these sequencing results, we will focus on increasing the efficacy of these compounds by downstream modification.
Impacts
What was accomplished under these goals?
Objective 1: To identify abiotic factors leading to the survival and persistence of Salmonella in tomato plants, young tomato plants were sprayed on the leaves with a suspension of S. Typhimurium, or were directly introduced in the host via cotyledons clipping with contaminated scissors. For this first experiment, we studied the impact of low or high relative humidity levels (40% or 80%) and low or high temperatures (set at 20°C during days and 15°C during nights or set at 30°C during days and 25°C during nights) on the capacity of S. Typhimurium to infect and survive in the host. Following spray inoculation of the leaves, at high humidity level, the pathogen was able to survive in the phyllosphere for a longer period of time and infect the host with no effect of the environmental temperature over the first week post-inoculation. Fourteen days after inoculation, the bacterial population was stabilized at a density of 106 CFU per plant at low temperature, while the population constantly decreased over the weeks at high temperature. Finally, S. Typhimurium stayed at the inoculated tissues and didn't migrate into the stem before senescence of these tissues. At low humidity level, S. Typhimurium population showed a lower survival and infection rate than described previously. At low humidity level, the inoculated leaves displayed a shorter life time, inoculated tissues senesced one week earlier compared to plant grown at high relative humidity. Moreover, for unknown reasons S. Typhimurium was detected inside the host stems at a concentration of approximately 102 CFU per stem at 14DPI and 21DPI, and then moved to the root where it asymptomatically colonized the tissues up to 105 CFU per root system. These observations were detected for both low and high environmental temperatures as long as a low relative humidity was used. These experiments need to be repeated to confirm the observed results. Following cotyledons clipping inoculations, at high humidity level, the pathogen was able to survive outside the inoculated cotyledons and infect them during the first week post-inoculation. At 14 days post inoculation, the bacterial population was stabilized at a density of 104 CFU per plant at low temperature, while the population constantly decreased over the weeks at high temperature. Bacteria were not detected in the stem before cotyledon abscissions. At low humidity level and high temperature, S. Typhimurium population showed a lower survival rate. After 14 days post inoculation, infected tissues died due to normal senescence; however, S. Typhimurium was detected inside the host stems at a concentration of 102 CFU per stem when incubated at high environmental temperatures only. These experiments need to be repeated to confirm the observed results. In conclusion, environmental temperature affected Salmonella survival outside the plants, while relative humidity might affect the capacity of Salmonella to systemically colonize the host. The combination of low humidity plus high temperature seemed to be the most unfavorable growing condition for Salmonella however it allowed a systemic colonization of the host; while the combination of high humidity plus low temperature allowed the longest and highest survival of Salmonella outside and inside the host. To identify the biotic factors leading to the survival and persistence of Salmonella in tomato plants, young tomato plants were first sprayed on the leaves with a suspension of Xanthomonas gardneri (non-systemic phytopathogen), or were directly introduced in the host via cotyledons clipping with Clavibacter michiganensis michiganensis (systemic phytopathogen) contaminated scissors; and then inoculated a week later with S. Typhimurium using the same inoculation method used for the phytopathogen. For these experiments, we studied the impact of pre-infected tomato plant with a plant pathogen on the capacity of S. Typhimurium to infect and survive in the sick host and understand the potential interaction between bacterial pathogens. Colonization of tomato plants was observed weekly through bacterial quantification of inoculated plants on selective media. Following spray inoculation of the leaves with first X. gardneri and then S. Typhimurium, S. Typhimurium displayed an increase of survival compared to the plant only inoculated with S. Typhimurium. In plant inoculated with Salmonella only, the bacteria were detected only up to 7 DPI, while plant infected first with the plant pathogen and then with the foodborne pathogen, we could detect Salmonella up to 21 DPI. Moreover, co-inoculation increased the Salmonella abundance in the host by 2-logs at 7DPI. Following clipping inoculation of cotyledons first with Cmm and then S. Typhimurium, S. Typhimurium displayed an increased survival compared to the plant only inoculated with S. Typhimurium. In plant inoculated with Salmonella only, the bacteria were detected only up to 21 DPI; while with plant infected first with the plant pathogen and then with the S. Typhimurium, we could detect Salmonella up to 28 DPI; however the presence of Cmm did not induce significant increase in Salmonella population unlike infection with X. gardneri. For both experiments, the presence of Salmonella slightly increased (approximately 1-log) of the phytopathogen population (X. gardneri and Cmm) at 14 and 21DPI . Objective 2: Here we seek to identify using high throughput genomic screening the environmental conditions and pathogen specific genetic pathways that are required for Salmonella typhimurium to survive in tomato. Towards this end we sequenced the reference strain which will be used for transposon mutagenesis using next generation sequencing and a variety of sequence analysis routines to identify any and all polymorphisms or structural variants present in the reference strain genome. This high coverage sequence map (approximately 100x) will also guide our analysis of each barcoded transposon insertion site. We have completed construction of a new barcoded transposon construct for rapid high throughput library generation. This transposon has several novel features including a 25 base pair barcode which in contrast to the 20 base pair barcodes used in previous studies offers greater than 1 million barcoded transposon's per strain. We will validate the insertion sites of each of the 95,000 transposon-disruption mutants by sequencing 5 independent libraries and, based on the sequencing results, pools these pools to generate a collection in which each gene in the S. typhimurium genome is represented by at least 3 barcoded insertions. This collection will be screened in parallel against the selected small molecules described in Objective 3. Objective 3: As a part of objective 3, we performed high throughput screening of small molecules to discover novel effective anti-Salmonella compounds. A library of 4182 small molecules was tested on S. Typhimurium. Among this collection, a total of 21 small molecules have shown a cidal/static effect with a minimal inhibitory concentration (MIC) between 400 μM to 10 μM. This cluster was screened against 9 other Salmonella enterica serotypes (commonly associated with food poisoning) . The five cidal SM with lowest MBCs were also cidal to the nine Salmonella serotypes. Four of the selected five candidates did not induce resistance. These four SM exhibited low cytotoxicity on Caco-2 epithelial, and on HD11 and THP-1 macrophage cells; they were effective in killing internalized bacteria. These SM did not change the phenotype of tomato fruits and seedlings, and did not reduce the tomato seed germination rate when tested at 1xMIC. In summary, we have identified four novel anti-Salmonella compounds that are not toxic for tomato plants and two of them that reduced Salmonella colonization of chickens.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
- L. Deblais, R. Candelero, Y.A. Helmy, C. Vrisman, S.A. Miller, and G. Rajashekara A combined genomic and in vivo imaging platform to understanding Salmonella-tomato plant host interactions AFRI IAFP meeting (Missouri USA July 2016).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
- L. Deblais, Y.A. Helmy, R. Candelero, Huang-chi Huang, Dipak Kathayat, S.A. Miller, and G. Rajashekara Small molecules, a potential novel control strategy to reduce Salmonella burden I3S meeting (Saint-malo France July 2016).
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2016
Citation:
- L. Deblais, Y.A. Helmy, R. Candelero-Rueda, D. Kathayat, H. Huang, S.A. Miller, and G. Rajashekara Novel anti-Salmonella compounds reduce Salmonella burden in poultry ASM Antimicrobial Development meeting (Washington USA December 2016).
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:Food safety advocates, Veterinarians, Diagnosticians, Poultry Practitioners, Producers, Researchers, Immunologists, Plant pathologists, and Food safety microbiologists, Food safety policy makers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Trained a Graduate student on understanding colonization of Salmonella in tomato plants under different environmental condition Trained a Graduate student in performing high throughput sequencing to identify anti-Salmonella compounds Trained 1 Postdoctoral Fellow in barcode sequence analysis Trained 1 Research Associate in barcode sequence analysis Trained 1 Graduate students in Tn-Seq pool construction, sample collection and Next-Generation sequencing library preparation How have the results been disseminated to communities of interest?The preliminary results were presented at the: Loïc Deblais, Fulya Baysal-Gurel, Amy Lee, Sunita Sinha, Samantha Lui, Corey Nislow, Sally Miller, and Gireesh Rajashekara"UnderstandingSalmonella-tomato plant hostinteractionsand development ofeffectivenovel control strategies to reduceSalmonellaburden in tomatoproduction" Meeting International Association of Food Protection (Oregon - United States America - July 2015). Loïc Deblais, Fulya Baysal-Gurel,Sally Miller, and Gireesh Rajashekara"UnderstandingSalmonella-tomato plant hostinteractionsand development ofeffectivenovel control strategies to reduceSalmonellaburden in tomatoproduction" OARDC, The Ohio State University Annual Conference (Ohio - United States America -March 2015). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: In third year, we will confirm previous results at low humidity level (40%) and test how S. Typhimurium colonizes tomato plants under: low relative humidity and low growth temperatures (20°C days/ 15°C nights) and test how infection with tomato pathogens (Clavibacter and Xanthomonas) affect Salmonella survival in tomato plants. In addition, given grafting is a current operation applied in tomato production, we will try to study the plant colonization after grafting with infected tools. Objective 2: In the coming six months we will validate the insertion sites of each of the 95,000 transposon-disruption mutants by sequencing 5 independent libraries and, based on the sequencing results, pools these to generate a collection in which each gene in the S. typhimurium genome is represented by at least 3 barcoded insertions. This collection will be screened in parallel against the 7 small molecules described in Objective 3. Objective 3: High throughput screening has shown encouraging results for the development of novel strategies against S. Typhimurium. However at this stage of the project, more analyses need to be performed before application in the field. We will provide more information about these selected compounds via cytotoxicity tests on tomato seeds and fruits, and other human cells (using epithelial and macrophage cell lines). Based on the results from these studies, we test the most promising ones in tomato plants and fruits for their effect on Salmonella.
Impacts
What was accomplished under these goals?
Objective 1: To identify the factors leading to the survival and persistence of Salmonella in tomato plants, young tomato plants were sprayed on the leaves with a suspension of S. Typhimurium, or were directly introduced in the host via cotyledons clipping with contaminated scissors. For this first experiment, we studied the impact of low or high relative humidity levels (40% or 80%) and low or high temperatures (set at 20°C during days and 15°C during nights or set at 30°C during days and 25°C during nights) on the capacity of S. Typhimurium to infect and survive in the host. Colonization of tomato plants was observed weekly through bacterial quantification of inoculated plants on selective XLT media. Following spray inoculation of the leaves, S. Typhimurium was highly affected by the humidity level and less by the temperature settings. At high humidity level, the pathogen was able to survive in the phyllosphere and infect the host with no effect of the environmental temperature over the first week post-inoculation. Fourteen days after inoculation, the bacterial population was stabilized at a density of 106 CFU per plant at low temperature, while the population constantly decreased over the weeks at high temperature. Finally, S. Typhimurium stayed at the inoculated tissues and didn't migrate into the stem before senescence of these tissues. At low humidity level and high temperature, S. Typhimurium population showed a lower survival and infection rate than described previously. No bacteria was detected on and inside the inoculated leaves after 21 days post inoculation, however for unknown reasons, S. Typhimurium was present inside the host stems at a concentration of 102 CFU per stem. These experiments need to be repeated to confirm the observed results. Following cotyledons clipping inoculations, S. Typhimurium was highly affected by the humidity level and less by the temperature settings. At high humidity level, the pathogen was able to survive outside the inoculated cotyledons and infect them during the first week post-inoculation. At 14 days post inoculation, the bacterial population was stabilized at a density of 104 CFU per plant at low temperature and then significantly increased by1 log at 21 DPI, while the population constantly decreased over the weeks at high temperature. Bacteria were not detected in the stem before cotyledon abscissions. At low humidity level and high temperature, S. Typhimurium population showed a lower survival rate. After 21 days post inoculation, no bacteria was detected on and inside the host due to abscission of infected tissues. Despite a significant declining in survival and infection rate of S. Typhimurium at low humidity level, these conditions seemed to be more suitable for a systemic colonization of the host compared to high humidity level. On the other hand, high temperature seemed to be a significant factor in decreasing survival rate of the bacteria outside the plant only. Objective 2: Here we seek to identify using high throughput genomic screening the environmental conditions and pathogen specific genetic pathways that are required for Salmonella typhimurium to survive in tomato. Towards this end we sequenced the reference strain which will be used for transposon mutagenesis using next generation sequencing and a variety of sequence analysis routines to identify any and all polymorphisms or structural variants present in the reference strain genome. This high coverage sequence map (approximately 100x) will also guide our analysis of each barcoded transposon insertion site. Last year we completed construction of a new barcoded transposon construct for rapid high throughput library generation. This transposon has several novel features including a 25 base pair barcode which in contrast to the 20 base pair barcodes used in previous studies offers greater than 1 million barcoded transposon's per strain. In this past year we received a collection of sequence-verified barcodes (as 25mers) from Dr. Steven Elledge of Harvard University as cassettes of 4 barcodes/plasmid. We transferred 100,000 of these barcodes into our S. typhimurium Tn-seq transposon and used our barcode sequencing protocol to maximize the generation of insert containing clusters on the HiSeq 2500 next-generation sequencer. We verified that over 95,000 barcodes were successfully transferred into the Tn-Seq cassette and have completed electroporation of S. typhimurium to generate the Tn-Seq chemogenomic screening library. In the coming six months we will validate the insertion sites of each of the 95,000 transposon-disruption mutants by sequencing 5 independent libraries and, based on the sequencing results, pools these pools to generate a collection in which each gene in the S. typhimurium genome is represented by at least 3 barcoded insertions. This collection will be screened in parallel against the 7 small molecules described in Objective 3. Objective 3: As a part of objective 3, we performed high throughput screening of small molecules to discover novel effective anti-Salmonella compounds. A library of 4182 small molecules was tested on S. Typhimurium. Among this collection, a total of 21 small molecules have shown a cidal/static effect with a minimal inhibitory concentration (MIC) between 400 μM to 5 μM. This cluster was screened against 8 other most prevalent (commonly associated with food poisoning) Salmonella enterica serotypes. This screen identified 7 candidates that showed bactericidal effect on all the Salmonella enterica serotypes tested. Treatment of infected epithelial cells or macrophages by S. Typhimurium with 1xMIC small molecules was able to significantly impact their survival inside these cells. .
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Lo�c Deblais, Fulya Baysal-Gurel, Amy Lee, Sunita Sinha, Samantha Lui, Corey Nislow, Sally Miller, and Gireesh Rajashekara Understanding Salmonella-tomato plant host interactions and development of effective novel control strategies to reduce Salmonella burden in tomato production Meeting International Association of Food Protection, Oregon United States America.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Lo�c Deblais, Fulya Baysal-Gurel, Sally Miller, and Gireesh Rajashekara Understanding Salmonella-tomato plant host interactions and development of effective novel control strategies to reduce Salmonella burden in tomato production OARDC, The Ohio State University Annual Conference (Ohio United States America -March 2015).
|
Progress 09/01/13 to 08/31/14
Outputs
Target Audience: Food safety advocates, Veterinarians, Diagnosticians, Poultry Practitioners, Producers, Researchers, Immunologists, Plant pathologists, and Food safety microbiologists, Food safety policy makers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Trained a Gradaute student on understanding colonization of Salmonella in tomato plants under different environmental condition Trained a Graduate student in performing high throughput sequencing to identify anti-Salmonella compounds Trained a Postdoctoral Fellow in barcode sequence analysis Trained 2 Graduate students in Next-Generation sequencing library preparation How have the results been disseminated to communities of interest? The preliminary results were presented at the 2014 Annual Meeting Multi-State Project S-1056, “Enhancement of Microbial Food Safety by Risk Analysis” October 7-8, 2014, San Juan, Puerto Rico. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: In second year, we will test how S. typhimurium colonizes tomato plants under: high relative humidity (80%) and high growth temperatures (30°C days/ 25°C nights) and test how infection with tomato pathogens (Clavibacter and Xanthomonas) affect Salmonella survival in tomato plants. In addition, since, boluminescent stain in this study was not bioluminescent enough to study the colonization, we will try to generate a new bioluminescent S. typhimurium strain. Objective 2: Having assembled all the tools necessary to deliver on specific aim 2, we will, in the second year of this project screen three independent Salmonella typhimurium mutant libraries in 10 distinct environmental conditions. (a) Perform genome-wide screen on barcoded mutant libraries in 10 environmental conditions and (b) Optimize single strain, high throughput growth assay to confirm results from (a). Objective 3: High throughput screening have shown encouraging results for the development of novel strategies against Salmonella typhimurium. However at this stage of the project, more analyses need to be performed before application in the field. We plan to determine IC 50 for each of the 21 small molecules identified. Molecules showing bacteriostatic effect will be combined to determine possible synergestic effects. Further, we will test the selected compounds on tomato seeds and fruits, before and after Salmonell contamination in order to see the in planta effect and the phytotoxicity of these molecules will also be determined.
Impacts
What was accomplished under these goals?
Objective 1: To identify the factors leading to the survival and persistence of Salmonella in tomato plants, young tomato plants were sprayed on the leaves with a suspension of Salmonella typhimurium, or were directly introduced in the host via cotyledons clipping with contaminated scissors. For this first experiment, we also studied the impact of high relative humidity level (80%) and low temperature (set at 20°C during days and 15°C during nights) on the capacity of S. typhimurium to infect and survive in the host. Colonization of tomato plants was observed weekly through bacterial quantification of inoculated plants on selective XLT media. Following spray inoculation of the leaves, S. typhimurium was able to survive in the phyllosphere and internalize the host during the first week of the test. Fourteen days after inoculation, the bacterial population was stabilized at a density of 106 CFU per plant; however S. typhimurium stayed at the inoculated site and didn’t migrate into the stem. Following cotyledons clipping inoculations, the bacterial population was stabilized at a density of 105 CFU per plant after 14 days post inoculation. A significant increasing of internal population was observed after 21 days post inoculation, however bacteria were not detected in the stem before cotyledon abscissions. For both inoculation methods, this first investigation showed decline of the external bacterial population after 7 days post inoculation; however, bacterial population remained stable after 14 days post inoculation. High relative humidity and low temperature seemed to be not favorable for the survival of S. typhimurium on the phyllosphere and its multiplication inside the host. Objective 2: Here we seeks to identify using high throughput genomic screening the environmental conditions and pathogen specific genetic pathways that are required for Salmonella typhimurium to survive in tomato. Towards this end we sequenced the reference strain which will be used for transposon mutagenesis using next generation sequencing and a variety of sequence analysis routines to identify any and all polymorphisms or structural variants present in the reference strain genome. This high coverage sequence map (approximately 100x) will also guide our analysis of each barcoded transposon insertion site. We completed construction of a new barcoded transposon construct for rapid high throughput library generation. This transposon has several novel features including a 25 base pair barcode which in contrast to the 20 base pair barcodes used in previous studies offers greater than 1 million barcoded transposon's per strain. As before the core of this transposon is Tn5 and accordingly given the slight sequence bias of Tn5 we have tested the performance of this barcoded transposon in the mating competent E. coli strain WM 3064. Using electroporation we obtained several hundred thousand insertions. We optimized our barcode sequencing protocol to maximize the generation of insert containing clusters on the HiSeq 2500 next-generation sequencer. This empirical approach involved testing several pairs of common priming sites such that Amplicon size of each barcode is maximally distinct from non-productive amplicons. Finally we modified our robotic screening platform to adapt to bacterial growth (as opposed to yeast) and are able to collect barcoded transposon pool every five generations of growth. Objective 3: As a part of objective 3, we performed high throughput screening of small molecules to discover novel effective anti-Salmonella compounds. A library of 4182 small molecules was tested on S. typhimurium. Among this collection, a total of 21 small molecules have shown a cidal/static effect with a minimal inhibitory concentration between 400 μM to 5 μM. This cluster was screened against 8 other most prevalent (commonly associated with food poisoing) Salmonella enterica serptypes. This screen identified 7 candidates that showed bactericical effect on all the Salmonella enterica serotypes tested.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2014
Citation:
Enhancement of Microbial Food Safety by Risk Analysis 2014 Annual Meeting Multi-State Project S-1056, October 7 and 8, 2014, San Juan, Puerto Rico
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