Progress 01/08/15 to 09/30/19
Outputs
Target Audience:Research community: We have developed novel animal models and a strain culture library that advances the fundamental understanding of gut microbiome function and enteric pathogen invasion. For example, we developed a germfree chicken model that can be used to study the gut microbiota function in poultry. This model is also helpful in establishing safety of new probiotic strains in poultry. We have also isolated a large number of gut bacterial species from swine, poultry and swine. Genomes of these strains have been sequenced and we have also characterized these strains using a number of phenotypic assays. The availability of this strain library is helpful for the research community to understand the role of commensal bacteria in suppressing the invasion of enteric pathogens such as C. difficile and S. enterica. Swine Industry: A new strain of Salmonella enterica has caused several outbreaks in the US. We analyzed the prevalence of this strain in pork processing plants and found that the type of carcass chilling may have an impact on the prevalence of this strain pork products. This information is useful for the swine industry to develop better Salmonella control measures in the post-harvest stage. Beef industry: We conducted surveillance studies on Salmonella prevalence in beef cattle. Our analysis could find new mechanisms behind the spread of Salmonella in beef cattle. For example, our analysis found that Salmonella Heidelberg, a serotype of Salmonella commonly found in poultry caused outbreaks in beef cattle by acquiring new genes that enhanced the adherence of Salmonella to the host cells. Furthermore, our analysis of Salmonella Dublin found that the strains prevalent in United States have significantly higher number of antibiotic resistance genes when compared to strains from Europe. These findings are helpful for developing better treatment and control measures against Salmonellosis in beef cattle. Poultry Industry: Colonization of pathogenic Salmonella in poultry and subsequent contamination of poultry products by Salmonella is a major problem faced by the poultry industry. Development of defined bacterial mixes that can reduce or prevent on-farm Salmonella colonization could reduce the incidence of Salmonellosis through poultry products. If successful when tested in production conditions, this approach could avoid the use of antibiotics to control Salmonella. This will be of particular interest to poultry producers who are interested in raising chicken using "non-antibiotic" methods. Food industry: We have optimized whole genome sequencing as an improved method of tracking Salmonella in food matrices. Our results from whole genome sequencing based Salmonella testing will help food industry to adopt this new method in packaging and food processing plants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Four Postdoctoral fellows, two visiting scientists, six graduate students and five undergraduate students were provided training on anaerobic bacteriology, bacterial genomics, and computational biology. Milton Thomas, PhD, Postdoctoral fellow: Was trained on next generation genome sequencing methods, comparative genomic analysis of microbial genomes and metagenomes. Chayan Roy, PhD, Postdoctoral fellow: Was trained in anaerobic bacteriology and germfree animal management protocols. Abhijit Maji, PhD, Postdoctoral fellow: Was trained in anaerobic bacteriology and bacterial genomics and computational biology. Roshan Kumar, PhD, Postdoctoral fellow: Performed whole genome sequencing, genome annotation and comparative genome analysis to define new bacterial species. Samara Mattiello, visiting scientist: Was trained in antimicrobial resistance tracking using whole genome sequencing based methods. Sirima Mingmongkolchai, visiting scientist: Performed in vitro screening of gut bacteria to identify beneficial strains that improve gut health and provide colonization resistance to C. difficile. Sudeep Ghimire, PhD student: Optimized methods for isolating obligate anaerobes and identified three new species of gut bacteria. Linto Antony, PhD student: Identified Salmonella transmission patterns using whole genome sequencing. Gavin Fenske, PhD student: Learned skills in comparative genomics using Python and R. Supapit Wongkuna, PhD student: Used the gnotobiotic chicken model to identify the colonization dynamics of chicken gut microbiota in germfree chicks. Maria Antony, MS Student: Learned skills in anaerobic bacteriology, whole genome sequencing and bioinformatics Mitchel Keena, Andrew Foley, Alex Fonder, Garrett Francis and Sydney Bormann, BS students: Learned whole genome sequencing based typing of new bacterial species. How have the results been disseminated to communities of interest?Scientific community: Seventeen peer reviewed journal articles have been published during this project period. We sequenced over 4,000 Salmonella genomes and made the raw genome data freely available to research community through NCBI sequence read archive (NCBI SRA). Stakeholders: PI has presented the results on the genomics based surveillance of Salmonella enterica in the annual meetings of South Dakota Poultry council and the SD Beef Industry council. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis (100% Accomplished) The initial work plan under this objective was to develop a gnotobiotic miniature pig model to study enteric disease pathogenesis. However, we had difficulty in maintaining piglets under sterile conditions. Furthermore, several piglets died when they were implanted with human gut microbiota. Therefore, we established two alternative models. As the first alternative, we set up a gnotobiotic mouse model. A breeding protocol was submitted to SDSU IACUC for rearing and breeding of germfree mice. Personnel were provided hands-on training for managing gnotobiotic mice. Four germfree mouse isolators were purchased and set up for breeding. This facility was used for studying Clostridium difficile pathogenesis. As a second alternative, we established a mini-bioreactor model to study gut microbiota function. This system allows the cultivation and functional analysis of complex microbiota. Furthermore, the gut microbiota community established in the mini-bioreactor system was stable and recapitulated major phenotypic features such as the ability to exclude enteric pathogens. We also expanded the scope of this objective to study enteric disease pathogenesis in chicken. To this end, a germfree chicken model was developed to study Salmonella pathogenesis. We standardized methods to hatch chickens that were germfree by modifying the germfree pig isolators. Protocols were developed for sterilizing hatching eggs and for incubating them in germfree isolators. We then tested whether this germfree model was suitable for bacterial colonization studies by administering adult chicken microbiome to 3 day old germ-free chicks. Chicks were enthanized on 0, 9 and 18 days of age and intestinal samples were collected and subjected to genomic analysis. The five most prevalent phyla that colonized the germfree chicks were Bacteroidetes (45.73±3.35%), Firmicutes (36.47±2.60%), Proteobacteria (8.28±0.91%), Actinobacteria (5.09±0.52%), and Spriochetes (2.10±0.38%). Principle coordinate analysis indicated the 0, 9, and 18 day variables clustered together and the microbial communities changed temporally. The Morista-Horn index values ranged from 0.72 to 1, indicating the communities at 0, 9, or 18 days were more similar than dissimilar. The predicted functional profiles of the microbiomes of 0, 9, and 18 days were also similar. These results indicate the gnotobiotic chicks stably maintain the phylogenetic diversity and predicted metabolic functionality of the inoculum community. Furthermore, we developed a chicken gut microbiota culture library that is composed of more than 1,100 isolates. We characterized the strain library by sequencing the strain genomes and also defining important phenotypes that determine colonization resistance against enteric pathogens. These phenotypic screens included the ability to inhibit Salmonella enterica, production of volatile fatty acids, tolerance to bile acids and the production of secondary metabolites. A defined consortium of strains was developed that could reduce the colonization of Salmonella when tested using the germfree chicken model. A provisional patent application has been filed based on this data. Objective 2. Study the transmission dynamics and evolution of enteric diseases. (100% Accomplished) Salmonella enterica is the major cause of foodborne infections in the United States. The US Center for Disease Control estimates that the genus Salmonella is responsible for 1.2 million illnesses per year in the US alone. Therefore, under this objective we focused our efforts to understand the mechanisms behind the transmission and persistence of Salmonella in pre-harvest and post-harvest level. We used whole genome sequencing as the primary method to characterize the strains. In the last five years, we isolated and over 4,000 Salmonella strains from over 25 states in the US. Since Salmonella pathogenesis depends on the serotype of the strains, data was analyzed based on the serotype classification. First serotype we analyzed was Salmonella enterica Dublin which is of interest to cattle production and human health. The Dublin serotype primarily is a cattle associated strain. However, the serotype is a zoonotic agent and elicits significant pathology in human patients. To better understand the genomic patterns associated with dissemination, host preference, and virulence, 82 Salmonella enterica Dublin strains isolated from cattle were sequenced on the Illumina MiSeq platform. Additionally, genomes for 856 other S. enterica Dublin strains were gathered from public databases. The antibiotic resistance and virulence gene catalogues for the serotype were characterized through homology searching within the genome assemblies. Additionally, the core genome of the serotype was elucidated allowing for high-resolution taxonomic clustering of the isolates. Whole genome taxonomy methods revealed that strains from the US are highly clonal and distinct from strains isolated in the United Kingdom, Brazil, and Africa. Resistance to antimicrobial agents was observed as widespread among the United States strains including many multidrug resistant clones. The second serotype we analyzed was Salmonella Heidelberg which is primarily a poultry adapted serotype of Salmonella that can also colonize other hosts and cause human disease. In this study, we compared the genomes of outbreak associated non-outbreak causing Salmonella Heidelberg strains from diverse hosts and geographical regions. Human outbreak-associated strains in this study were from a 2015 multistate outbreak of Salmonella Heidelberg involving 15 states in the US which originated from bull calves. Our clinicopathologic examination revealed that cases involving Salmonella Heidelberg strains were predominantly young, less than weeks-old, dairy calves. Pre-existing or concurrent disease was found in the majority of the calves. Detection of Salmonella Heidelberg correlated with markedly increased death losses clinically, comparable to those seen in herds infected with S. enterica Dublin, a known serious pathogen of cattle. Whole genome single nucleotide polymorphism based analysis revealed that these calf isolates formed a distinct cluster along with outbreak associated human isolates. The defining feature of the outbreak associated strains, when compared to older isolates of S. Heidelberg, is that all isolates in this cluster contained Saf fimbrial genes which are generally absent in S. Heidelberg. The acquisition of several single nucleotide polymorphisms and the gain of Saf fimbrial genes may have contributed to the increased disease severity of these Salmonella Heidelberg strains.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Fenske G, Thachil A, McDonough P, Glaser, Scaria J. 2019. Geography shapes the population genomics of Salmonella enterica Dublin. Genome Biol Evol. Jul 22. pii: evz158. PMID:31329231
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Ceric O, Tyson GH, Goodman LB, Mitchell PK, Zhang Y, Prarat M, Cui J, Peak L, Scaria J, Antony L, et al. 2019. Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada. BMC Veterinary Research. 15,1,130. PMID: 31060608
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Christopher?Hennings J, Erickson GA, Hesse RA, Nelson EA, Rossow S, Scaria J, Slavic D. 2019. Diagnostic tests, test performance, and considerations for interpretation. Diseases of Swine. Pp 75-97. John Wiley & Sons.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Bessire BC, Thomas M, Gehring KB, Savell JW, Griffin DB, Taylor TM, Mikel WB, Campbell JA, Arnold AN, Scaria J. 2019. Salmonella prevalence in lymph nodes of sows and market hogs in the United States. Meat and Muscle Biology. 2, 2, 129-130.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Haberecht HB, Nealon NJ, Gilliland JR, Holder AV, Runyan C, Oppel RC, Ibrahim HM, Mueller L, Schrupp F, Vilchez S, Antony L, Scaria J, Ryan EP. 2019. Antimicrobial-resistant Escherichia coli from environmental waters in Northern Colorado. J Environ Public Health. Feb 18:3862949 PMID : 30906330
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Dhakan DB, Maji A, Sharma AK, Saxena R, Pulikkan J, Grace T, Gomez A, Scaria J, Amato KR, Sharma VK. 2019. The unique composition of Indian gut microbiome, gene catalogue, and associated fecal metabolome deciphered using multi-omics approaches. Gigascience. Mar 1, 8(3). PMID : 30698687
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Dhakal S, Gourapura RJ, Wang L, Antony L, Rank J, Bernardo P, Ghimire S, Bondra K, Siems C, Lakshmanappa YS, Sankar R, Hogshead B, Krakowka S, Kauffman M, Scaria J, LeJeune J, Zhongtang Y. 2019. Amish (rural) versus non-Amish (urban) infant fecal microbiotas are highly diverse and their transplantation lead to differences in mucosal immune maturation in a humanized germfree piglet model. Frontiers in Immunology, 10, 1509. PMID:31379808
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Thomas M, Wongkuna S, Ghimire S, Kumar R, Antony L, Doerner KC, Singery A, Nelson E, Woyengo T, Chankhamhaengdecha S, Janvilisri T, Scaria J. 2019. Gut microbial dynamics during conventionalization of germfree chicken. mSphere, 4, 2,e00035-19, PMID: 30918057
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Poultry Industry: Colonization of pathogenic Salmonella in poultry and subsequent contamination of poultry products by Salmonella is a major problem faced by the poultry industry. Development of defined bacterial mixes that can reduce or prevent on-farm Salmonella colonization could reduce the incidence of Salmonellosis through poultry products. If successful when tested in production conditions, this approach could avoid the use of antibiotics to control Salmonella. Therefore, this will be of particular interest to poultry producers who are interested in raising chicken using "non-antibiotic methods. Research community: We are targeting food diagnostic testing scientists. Our results from whole genome sequencing based Salmonella testing will help them to adopt this new method in their laboratories. Changes/Problems:Since we found that maintaining gnotobiotic pig model is expensive and laborious, we standardized a germfree mouse model to study C. difficile pathogenesis and a germfree chicken model to study Salmonella enterica pathogenesis. Therefore to achieve the goals of objective 1, we will be using these alternative animal models. What opportunities for training and professional development has the project provided?Three Postdoctoral fellows, two visiting scientists, five graduate students and two undergraduate students were provided training on anaerobic bacteriology, bacterial genomics and computational biology. Chayan Roy, PhD, Postdoctoral fellow: Was trained in anaerobic bacteriology and germfree animal management protocols. Abhijit Maji, PhD, Postdoctoral fellow: Was trained in anaerobic bacteriology and bacterial genomics and computational biology. Roshan Kumar, PhD, Postdoctoral fellow: Performed whole genome sequencing, genome annotation and comparative genome analysis to define new bacterial species. Samara Mattiello, visiting scientist: Was trained in antimicrobial resistance tracking using whole genome sequencing based methods. Sirima Mingmongkolchai, visiting scientist: Performed in vitro screening of gut bacteria to identify beneficial strains that improve gut health and provide colonization resistance to C. difficile. Sudeep Ghimire, PhD student: Optimized methods for isolating obligate anaerobes and identified three new species of gut bacteria. Linto Antony, PhD student: Identified Salmonella transmission patterns using whole genome sequencing. Gavin Fenske, PhD student: Learned skills in comparative genomics using Python and R. Supapit Wongkuna, PhD student: Used the gnotobiotic chicken model to identify the colonization dynamics of chicken gut microbiota in germfree chicks. Garrett Francis and Sydney Bormann, BS students: Learned whole genome sequencing based typing of new bacterial species. How have the results been disseminated to communities of interest?Scientific community: Seven journal articles have been published as pre-prints or as peer-reviewed articles during this reporting period. Stakeholders: PI has presented the results on the genomics based surveillance of Salmonella enterica in South Dakota Beef producers meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis In the remainder of this project duration we will further standardize the mouse model to study C. difficile pathogenesis. Additionally, we will organize the unpublished data from the gnotobiotic chicken model and prepare peer reviewed manuscripts for publication. Objective 2. Study the transmission dynamics and evolution of enteric diseases. We will continue to collect Salmonella enterica samples through ADRDL and through collaborating labs in the Midwest region. To understand the mechanisms behind the evolution and virulence, genomes of the collected strains will be sequenced and compared against other available genomes of same Salmonella serotype.
Impacts
What was accomplished under these goals?
Objective 1: Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis (90% Accomplished) As reported in the previous year, we faced significant challenges in managing gnotobiotic pigs as a model for enteric disease pathogenesis. To circumvent this problem, we standardized two different animal models. In the first project, we set up a gnotobiotic mouse model. A breeding protocol was submitted to SDSU IACUC for rearing and breeding of germfree mice. Personnel were provided hands-on training for managing gnotobiotic mice. Four germfree mouse isolators were purchased and set up for breeding. This facility will be used for studying the Clostridium difficile pathogenesis. A second animal model was developed to study Salmonella pathogenesis. In this project, we standardized methods to hatch chickens that are germfree by modifying the germfree pig isolators. Protocols were developed for sterilizing hatching eggs and for incubating them in germfree isolators. We then tested whether this germfree model is suitable for bacterial colonization studies by administering adult chicken microbiome to 3 day old germ-free chicks. Chicks were enthanized on 0, 9 and 18 days of age and intestinal samples were collected and subjected to genomic analysis. The five most prevalent phyla that colonized the germfree chicks were Bacteroidetes (45.73±3.35%), Firmicutes (36.47±2.60%), Proteobacteria (8.28±0.91%), Actinobacteria (5.09±0.52%), and Spriochetes (2.10±0.38%). Principle coordinate analysis indicated the 0, 9 day and 18 day variables clustered together and the microbial communities changed temporally. The Morista-Horn index values ranged from 0.72 to 1, indicating the communities at 0, 9 or 18 days were more similar than dissimilar. The predicted functional profiles of the microbiomes of 0, 9 and 18 days were also similar. These results indicate the gnotobiotic chicks stably maintain the phylogenetic diversity and predicted metabolic functionality of the inoculum community. The domestic chicken is the cornerstone of animal agriculture worldwide with a flock population exceeding 40 billion birds/year. It serves as the economically valuable source of protein globally. Microbiome of poultry has important effects on chicken growth, feed conversion, immune status, and pathogen resistance. The significance of this study is in developing a gnotobiotic chicken model to study chicken gut microbiota function. Our experimental model shows that young germfree chicks are able to colonize diverse set of gut bacteria. Therefore, besides using this model to study mechanisms of gut microbiota interactions in the chicken gut, our model could be also used for applied aspects such as determining the safety and efficacy of new probiotic strains derived from chicken gut microbiota that could exclude Salmonella. Objective 2. Study the transmission dynamics and evolution of enteric diseases. (90% Accomplished) Salmonella infections continue to rise in the United States and globally. The United States Center for Disease Control estimates that the genus Salmonella is responsible for 1.2 million illnesses per year in the United States alone. In this reporting period, we focused our efforts to understand transmission and evolution of two Salmonella serotypes. First serotype we analyzed was Salmonella enterica Dublin which of interest to cattle production and human health. The Dublin serotype primarily is a cattle associated strain. However, the serotype is a zoonotic agent and elicits significant pathology in human patients. To better understand the genomic patterns associated with dissemination, host preference, and virulence, 82 Salmonella enterica Dublin strains isolated from cattle were sequenced on the Illumina MiSeq platform. Additionally, genomes for 856 other S. enterica Dublin strains were gathered from public databases. The antibiotic resistance and virulence gene catalogues for the serotype were characterized through homology searching within the genome assemblies. Additionally, the core genome of the serotype was elucidated allowing for high-resolution taxonomic clustering of the isolates. Whole genome taxonomy methods revealed that strains from the United States are highly clonal and distinct from strains isolated in the United Kingdom, Brazil, and Africa. Resistance to antimicrobial agents was observed as widespread among the United States strains including many multidrug resistant clones. The second serotype we analyzed was Salmonella heidelberg which is primarily a poultry adapted serotype of Salmonella that can also colonize other hosts and cause human disease. In this study, we compared the genomes of outbreak associated non-outbreak causing Salmonella heidelberg strains from diverse hosts and geographical regions. Human outbreak-associated strains in this study were from a 2015 multistate outbreak of Salmonella heidelberg involving 15 states in the United States which originated from bull calves. Our clinicopathologic examination revealed that cases involving Salmonella heidelberg strains were predominantly young, less than weeks-old, dairy calves. Pre-existing or concurrent disease was found in the majority of the calves. Detection of Salmonella heidelberg correlated with markedly increased death losses clinically, comparable to those seen in herds infected with S. enterica Dublin, a known serious pathogen of cattle. Whole genome single nucleotide polymorphism based analysis revealed that these calf isolates formed a distinct cluster along with outbreak associated human isolates. The defining feature of the outbreak associated strains, when compared to older isolates of S. heidelberg, is that all isolates in this cluster contained Saf fimbrial genes which are generally absent in S. heidelberg. he acquisition of several single nucleotide polymorphisms and the gain of Saf fimbrial genes may have contributed to the increased disease severity of these Salmonella heidelberg strains.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Thomas M, Wongkuna S, Ghimire S, Doerner K, Singery A, Nelson EA, Woyengo T, Chankhamhaengdecha S, Janvilisri T , Scaria J. (2018) Gut Microbial succession during conventionalization of germfree chicken. bioRxiv doi: https://doi.org/10.1101/360784
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
Ghimire S, Kumar R, Nelson EA, Hennings J, Scaria J. (2018) Genome sequence and description of Blautia brookingsii str SG772 nov., a new species of anaerobic bacterium isolated from healthy human gut. bioRxiv doi: https://doi.org/10.1101/327007
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Antony L, Behr M, Sockett D, Miskimins D, Aulik N, Christopher-Hennings J, Nelson E, Allard MW, and Scaria J. (2018) Genome Divergence and Increased Virulence of Outbreak Associated Salmonella enterica subspecies enterica serovar Heidelberg. Gut Pathogens (under review)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Kolady, D., Kattelmann, K. K., Vukovich, C., Scaria, J. (2018) Awareness and use of probiotics among the millennials in the U.S.: Drivers and implications. Functional Foods in Health and Disease (in press)
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Deblais, L., Lorentz, B., Scaria, J., Nagaraja, K. V., Nisar, M., Lauer, D., Voss, S., Rajashekara, G. (2018). Comparative Genomic Studies of Salmonella Heidelberg Isolated From Chicken- and Turkey-Associated Farm Environmental Samples. Frontiers in microbiology, 9, 1841.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Bessire BC, Thomas M, Gehring KB, Savell JW, Griffin DB, Taylor TM, Mikel WB, Campbell JA, Arnold A, Scaria J. (2018) National survey of Salmonella prevalence in lymph nodes of sows and market hogs, Journal of Translational Animal Science. Volume 2, Issue 4, 1 October 2018, Pages 365371.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Erickson, A. K., Murray, D. L., Ruesch, L. A., Thomas, M., Lau, Z., Scaria, J. (2018). Genotypic and Phenotypic Characterization of Salmonella Isolated from Fresh Ground Meats Obtained from Retail Grocery Stores in the Brookings, South Dakota Area. J. of Food Protection, 81(9), 1526-1534.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Beef producers: Colonization of pathogenic Salmonella in beef cattle is a major problem faced by the beef producers. High grain diet in beef cattle results in higher incidence of Salmonella colonization and increases the risk of beef contamination. This project studies the transmission dynamics of Salmonella in feedlots, beef cattle and people. Results obtained in this project will help beef producers in designing better herd management methods to control the colonization of Salmonella in beef cattle. Food processing and packaging industry: We are targeting professionals in this sector because we are developing new test methods to detect the presence and source of Salmonella in processing plants handling beef and pork. Better trace back of contamination could be achieved using whole genome sequencing. We targeted this industry by presenting our results in professional meetings attended by food processing and packing industry members. Research community: We are targeting food diagnostic testing scientists. Our results from whole genome sequencing based Salmonella testing will help them to adopt this new method in their laboratories Changes/Problems:Under objective 1, we planned to develop a miniature piglet model to study C. diffiicle pathogenesis. However, the cost of piglet model has been very high. To keep the experimental cost manageable, we are using a mouse model for pilot studies. Positive results from the mouse studies then will be further verified using the piglet model. This modified approach is expected to reduce the number of piglets needed for the study and to reduce the experimental cost. What opportunities for training and professional development has the project provided?One Postdoctoral fellow, two visiting scientists, four graduate students and two undergraduate students were provided training on anaerobic bacteriology, bacterial genomics and computational biology. Milton Thomas, PhD, Postdoctoral fellow: Was trained in bacterial genomics and sequenced the genomes of Salmonella enterica isolates and performed the computational analysis of the sequence data. Samara Mattiello, visiting scientist: Was trained in performing genomics based detection of antibiotic resistance in enteric bacterial pathogens. Sirima Mingmongkolchai, visiting scientist: Performed in vitro screening of gut bacteria to identify beneficial strains that improve gut health and provide colonization resistance to C. difficile. Sudeep Ghimire, PhD student: Developed a method to protect beneficial bacteria from bile acid toxicity. This method provides a new means of using bile sensitive bacteria as direct fed microbials. Linto Antony, MS student: Performed comparative genomics of Salmonella enteric to determine the mechanisms behind the emergence of more virulent strain types. Gavin Fenske, MS student: Learned skills in gas chromatography and mass spectroscopy. Supapit Wongkuna, PhD student: Developed a gnotobiotic chicken model to determine the role of gut bacteria in preventing colonization of enteric pathogens. Garrett Francis, BS student: Learned anaerobic bacterial culturing and MALDI-TOF based bacterial identification. Sydney Bormann, BS student: Learned molecular biology methods such as bacterial genomics DNA isolation and preparation of sequencing libraries. How have the results been disseminated to communities of interest?Scientific community: We have published four peer reviewed manuscripts during this reporting period. Stakeholders: PI has presented the results on the genomics based surveillance of Salmonella enterica in South Dakota food safety summit and SD Beef producers meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 1.Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis We plan to use high-throughput culturing of gut bacteria followed by genomics based characterization to identify beneficial strains that could improve gut health and to prevent enteric infections. Objective 2.Study the transmission dynamics and evolution of enteric diseases. We plan to continue the genomic epidemiology based surveillance of S. enterica in beef cattle and poultry. Samples will be collected from several regions of the United States. Objective 3.Develop new a generation diagnostic tests for enteric diseases. We plan to publish at least 3 peer reviewed manuscripts based on the current results.
Impacts
What was accomplished under these goals?
Objective 1.Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis We tested whether different types of diets could change susceptibility of the host to C. difficile infection. Since the cost of the miniature pig model is very high, we conducted a preliminary study using a mice model. The goal of this study was to characterize biochemical profiles manifested in mouse cecal content samples originating from a study investigating the potential attentuative influence of diet composition on Clostridium difficile infection. Experimental groups of five mice were maintained on three different isocaloric diets for 30 days, further segregated into three treatment groups; control untreated mice, mice treated with antibiotic cocktail alone and mice treated with antibiotic cocktail plus C. difficile spore infection (intragastric administration of 1000 CFU). All mice including the control group received a single intra-peritoneal dose of clindamycin one day prior to initiation of the antibiotic/C. difficile challenge. We used metabolomics to analyze the impact of dietary difference in susceptibility to C. difficile infection. The extracted samples were split into equal parts for analysis on the GC/MS and LC/MS/MS platforms. The findings in this study suggest that antibiotic treatment has a significant impact on microbe-related metabolic processes in the gut. Specifically, decreased levels of secondary bile acids and amino acid catabolites may reflect an altered micro flora composition and ultimately contribute to diminished lipid hydrolysis, food absorption, and protein degradation. Consequently, altered glycolytic and mitochondrial metabolism in the gut coupled with enhanced nucleotide catabolism may promote the generation of free radicals and oxidative stress. Notably, C. difficile infection in the presence of antibiotic yielded a similar biochemical phenotype as antibiotic alone in high protein-low fiber diet and low protein-high fiber diet samples as suggested by PCA analysis and hierarchical clustering. In contrast, many of the metabolic abnormalities described above were greatest in animals consuming low protein-high fiber diet in the presence of antibiotic cocktail alone and subsequently were dampened upon C. difficile infection. Though multiple trends were shared between each dietary intervention, these findings suggest that a low protein-high fiber diet may warrant further investigation to determine its direct impact on the microbiome community, while the small number of differences between the antibiotic cocktail alone and infection in the presence of a high protein-low fiber diet may be of interest inpotentially reducing the effects of C. difficile infection. Based on these results, a manuscript is under preparation. Objective 2. Study the transmission dynamics and evolution of enteric diseases. We used NGS based genomic epidemiology to track the source of a multistate outbreak of Salmonella heidelberg. Our analysis determined that the outbreak causing strains have acquired virulence genes related to host cell adhesion which could have contributed the increased invasiveness of the outbreak causing isolates. We further identified that the outbreak causing strains could have been circulating in the mid-west region 3-5 years before the onset of the outbreak. A manuscript based on these results is under preparation. Objective 3. Develop new a generation diagnostic tests for enteric diseases. Salmonellosis is a major problem affecting food safety and is the leading cause of foodborne illness in the United States accounting for approximately 1.2 million infections, 23,000 hospitalizations and 450 deaths annually. Wildlife has been considered as a potential reservoir of antibiotic resistant Salmonella that may be transmitted to humans through its contact with food animals. To analyze this possibility and togenerate a reference set of Salmonella enterica genomes isolated from wildlife from the United States as reference for traceback studies, we sequenced the whole genomes of 103 Salmonella isolates sampled between 1988 and 2003 from wildlife and exotic pets. Among 103 isolates, 50.48% were from wild birds, 0.9% was from fish, 24.27% each were from reptiles and mammals. Resistance against the aminoglycoside streptomycin was most common while 9 isolates were found to be multi-drug resistant having resistance against more than three antibiotics. Determination of virulence gene profile revealed that the genes belonging to csg operons, the fim genes that encode for type 1 fimbriae and the genes belonging to type III secretion system were predominant among the isolates. The universal presence of fimbrial genes and the genes encoded by pathogenicity islands 1-2 among the isolates we sequenced indicates that these isolates could potentially cause disease in humans. Therefore, the genomes we sequenced could be a valuable reference point for future traceback investigations when wildlife is considered to be the potential source of human Salmonellosis. A peer reviwed manuscript has been published based on this study.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Hennings, J., Erickson, G., Hesse, R., Nelson, E. A., Rossow, S., Scaria, J., Slavic, D. (in press). Diagnostic Tests, Test Performance, and Considerations for Interpretation. Diseases of Swine, 11th Edition (vol. Chapter 7). Wiley-Blackwell.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Palanisamy, S., Chang, Y., Scaria, J., Penha Filho RAC, Peters, K. E., Doiphode, S. H., Sultan, A., Mohammed, H. O. (2017). Genetic Relatedness Among Shiga Toxin-Producing Escherichia coli Isolated Along the Animal Food Supply Chain and in Gastroenteritis Cases in Qatar Using Multilocus Sequence Typing. Foodborne pathogens and disease, 14(6), 318-325.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Thomas, M., Webb, M. J., Ghimire, S., Blair, A. D., Olson, K. C., Fenske, G. J., Fonder, A. T., Hennings, J., Brake, D., Scaria, J. (2017). Metagenomic characterization of the effect of feed additives on the gut microbiome and antibiotic resistome of feedlot cattle. Scientific reports, 2017 Sep 25;7(1):12257. doi: 10.1038/s41598-017-12481-6. PMID:28947833
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Thomas, M., Fenske, G. J., Ghimire, S., Welsh, R., Ramachandran, A., Scaria, J. (2017). Whole genome sequencing-based detection of antimicrobial resistance and virulence in non-typhoidal Salmonella enterica isolated from wildlife. 2017 Nov 21;9:66. doi: 10.1186/s13099-017-0213-x. eCollection 2017. PMID:29201148
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Beef, swine and poultry producers: Colonization of pathogenic Salmonella in beef cattle is a major problem faced by the beef, swine and poultry producers. High grain diet in beef cattle results in higher incidence of Salmonella colonization and increases the risk of beef contamination. This project studies the transmission dynamics of Salmonella in feedlots, beef cattle and people. Outbreaks caused by variant strains of Salmonella is problem for swine and poultry producers. Results obtained in this project will help beef, swine and poultry producers in designing better management methods to control Salmonella . Food processing and packaging industry: We are targeting professionals in this sector because we are developing new test methods to detect the presence and source of Salmonella in processing plants handling beef and pork. Better trace back of contamination could be achieved using whole genome sequencing. We targeted this industry by presenting our results in professional meetings attended by food processing and packing industry members. Research community: We are targeting food diagnostic testing scientists. Our results from whole genome sequencing based Salmonella testing will help them to adopt this new method in their laboratories Changes/Problems:We have now expanded the scope of objective 1 to include culturing of gut microbiota from swine, chicken, and humans, and studying the mechanisms of gut microbiota in providing colonization resistance to enteric pathogens. It is a natural progression of the work from using pig as a model to study the role of gut microbiome in enteric diseases. What opportunities for training and professional development has the project provided?One Postdoctoral fellow, one visiting scholar, three graduate students and two undergraduate students were provided training on microbiology, diagnostics, genomics, bioinformatics Milton Thomas, PhD, Postdoctoral fellow: Performed sampling of feedlots in South Dakota to determine the prevalence of Salmonella enterica. Sequenced the genomes of Salmonella enterica isolates and performed the computational analysis of the sequence data. Sudeep Ghimire, DVM, PhD student: Developed a culturomics method for isolating beneficial bacteria from mammalian gut. Identified bacteria from gut that suppress the colonization of Clostridium difficile. Linto Antony, DVM, MS student: Performed experiments using gnotobiotic pigs to identify the colonization dynamics of beneficial bacteria in the hind gut. Gavin Fenske, MS student: Learned skills in anaerobic bacteriology and molecular biology. Specific skills learned includes PCR, identification of anaerobes using MALDI-TOF and 16s rRNA sequencing. Supapit Wongkuna, visiting scholar: Isolated several new species of anaerobic bacteria from gut microbiota of healthy chicken and identified species that suppress the colonization of Salmonella enterica. Alex Thomas Fonder, BS student: Learned to set up and run mini-bioreactor arrays to enrich beneficial bacteria from healthy mammalian gut microbiota. How have the results been disseminated to communities of interest?Scientific community: We have published three peer reviewed manuscripts and a conference abstract during this reporting period. Stakeholders: PI has presented the results on the Salmonella genome sequencing and identification of beneficial bacteria that reduced Salmonella colonization at a SD Beef producers meeting and at a SD poultry producers meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis and to study the role of gut microbiome in gut health. In the past year, we have developed a gut microbiome library from swine and chicken. We plan to use mini-bioreactor array based continuous flow culture model to further characterize the strain mixes and determineefficacy in suppressing S. enterica and C. difficile growth. We will also determine the mechanisms of pathogen inhibition by metabolite profiling and metagenome sequencing of the samples. Objective 2. Study the transmission dynamics and evolution of enteric diseases.We plan to conduct genomic epidemiology based surveillance of S. enterica in beef cattle and poultry. Samples will be collected from the United States and South America. New collaborations will be established to collect samples from South America. We also plan to publish at least 3 peer reviewed manuscripts based on the current results.
Impacts
What was accomplished under these goals?
Objective 1. Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis and to study the role of gut microbiome in gut health: A major focus of the work in this reporting period was to develop gut microbiota libraries from agriculturally important animals. This goal has basic and applied aspects. Developing a comprehensive library of bacteria from agriculturally important species such as swine and poultry would advance the knowledge about the role of these species in gut ecology. On the applied front, many of these species could be beneficial because they suppress the colonization of Salmonella in the gut. Our main accomplishment in this area is the culturing of 1,300 isolates of bacteria from healthy swine gut and 1,200 isolates from healthy chicken gut. Besides known species, we identified 25 new species of bacteria. This is of fundamental importance in gut ecology because we can now usecultures of these isolates to testits colonization dynamics in the host and whether these isolates could inhibit pathogen colonization. We also screened the gut microbiota culture library to identify species that could inhibit the growth of Clostridium difficile and Salmonella enterica. To identify these strains, we co-cultured each individual isolate with C. difficile or S. enterica. Reduction of growth was determined by calculating the colony forming unit (CFU) of C. difficile or S. enterica. We have so far identified more than 10 species that inhibit pathogen growth. Objective 2. Study the transmission dynamics and evolution of enteric diseases. We used whole genome sequencing (WGS) to characterize Salmonella enterica strains isolated from retail market samples in South Dakota. Samples were collected from grocery stores and included ground pork, beef, and turkey. After screening several hundred samples, we determined that about 10% samples were Salmonella positive. We sequenced genomes of 37 isolates from this surveillance testing. This testing helped us to improve the protocols involved in WGS based trace back studies to detect Salmonella in retail food samples.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Genome Sequences of Salmonella enterica subsp. enterica Serovar Lubbock Isolated from Liver Abscesses of Feedlot Cattle, Amachawadi RG, Thomas M, Nagaraja TG and Scaria J*. GenomeA. 2016 May 5;4(3). pii: e00319-16 PMID: 27151794
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Thomas M, Thachil A, Ghimire A, Glaser A, Pillatzki A, Nelson EA, Christopher-Hennings J, Daly M, and Scaria J. Genotypic and phenotypic characterization of Salmonella enterica serovar Dublin in cattle. AAVLD meeting, Greensboro, October 13-16, 2016
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Progress 01/08/15 to 09/30/15
Outputs
Target Audience:We mainly worked on defining the risk factors that cause Salmonella persistence in swine and cattle. Consquently, the target audience for this project was swine and cattle producer groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two undergraduate students, one gradaute student and one Postdoctoral fellow was trained in microbial genomics and bioinformatics methods How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?1. We will collect Escherichia coli 0157 strains and Salmonella from feedlots in South Dakota in and other mid-western states and sequence the whole genomes for analyszing antibiotic resistance pattern and the outbreak potential of those strains. 2. We will develop defined bacterial mixes that could suppress enteric pathogens such as Salmonella and Clostridium difficile in swine.
Impacts
What was accomplished under these goals?
During this reporting period, most efforts were directed towards establishing genomics based surveillance program with the focus on Salmonella enterica. We optimized methods and protocols for sequencing whole genomes of Salmonella using Illumina MiSeq platform. Additional funding for the sequencing program was obtained from the Food and Drug Administration (FDA). We used 2x250 paired end chemistry for sequencing Salmonella genomes. Over 350 Salmonella genomes were sequenced. The complete genome sequencing data was deposited in the Sequence Read Archive (SRA), National Center for Biotechnology Information (NCBI). Complete data can be accessed through the following web link at SRA http://www.ncbi.nlm.nih.gov/bioproject/PRJNA280335 . Our samples were collected from 15 different states in United States. The primary host species for these samples are swine and cattle. The Salmonella genomes we sequenced is being used by FDA as reference genomes for tracking any outbreak causing strains and also for developing data models to predict the outbreak potential of a Salmonella strain. Availability of whole genome sequences of Salmonella from South Dakota and the mid west region also will facilitate the tracking of antibiotic resistance in Salmonella.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Thachil A, Suzuki H, Glaser A, Thomas M, Das S, Gopinath G, Beaubrun JG, Addy N, Chase H, Jayaram A, Yoo Y, Chung T, Grim C, Hanes D, and Scaria J. Genomic Epidemiology of Salmonella enterica serotype Cerro; a newly emerged Salmonella serotype in the United States. ASM Conference on Rapid Next-Generation Sequencing and Bioinformatic Pipelines for Enhanced Molecular Epidemiologic Investigation of Pathogens
September 24 - 27, 2015, Washington DC, Abstract#112
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Useh NM, Suzuki H, Akange N, Thomas M, Foley M, Keena M, Nelson E, Hennings J, and Scaria J
Molecular and genomic typing of poultry associated Salmonella enterica strains from Nigeria. ASM Conference on Rapid Next-Generation Sequencing and Bioinformatic Pipelines for Enhanced Molecular Epidemiologic Investigation of Pathogens. September 24 - 27, 2015, Washington DC, Abstract#82
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