Progress 09/15/15 to 09/30/17
Outputs Target Audience:Poultry producers: Infection from contaminated poultry products is the major source of Salmonellosis in the United States. WGS based trace back methods we are developing will help poultry industry to formulate better decontamination protocols in the supply system. Swine producers: Improving pre-harvest food safety is a major focus for swine producers. Our work on WGS based Salmonella detection and trace back will help swine producers to implement better farm management practices to reduce Salmonella incidence in the production system. 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:
Nothing Reported
What opportunities for training and professional development has the project provided?One Postdoctoral fellow, two visiting scientists, three graduate students and two undergraduate students were provided training in 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, DVM, 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, DVM, 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, visiting scientist: 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??Dr. Scaria, NC1202 PI at South Dakota State University gave the keynote address for the South Dakota Food Safety Summit held at Brookings SD on October 12, 2017. The talk entitled "Emerging Pathogens and Antimicrobial Effectiveness for Pathogen Reduction in Beef Slaughter" discussed the use of whole genome sequencing as a means of tracking antimicrobial resistance in beef cattle. Members of the academic community, regulatory agencies and the processing industry were participants. To disseminate our results, we published two peer reviewed manuscripts in this reporting period. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts What was accomplished under these goals?
In this multi-state project, our laboratory focused on objectives 2 and 4. Objective 2. In North America, antibiotic feed additives such as monensin and tylosin are added to the finishing diets of feedlot cattle to counter the ill-effects of feeding diets with rapidly digestible carbohydrates. While these feed additives have been proven to improve feed efficiency and reduce liver abscess incidence, how these products impact the gastrointestinal microbiota and antibiotic resistance is not completely understood. To determine this, we analyzed the impact of providing antibiotic feed additives to feedlot cattle using metagenome sequencing of treated and control animals. This study compared the gut microbiome and resistome of steers provided antibiotic feed additives, monensin and tylosin, with control animals that were fed the same ration but not provided antibiotics. The treated steers also received growth promoting implants and a beta-agonist during the last 31 days of the trial, both of which are common practices in conventional feedlot management. Monensin, an anti-bacterial ionophore, is widely used in the feedlot industry to enhance production. It reduces the average feed intake and increases weight gain by enhancing propionic acid production in the rumen thereby resulting in improved energy efficiency. This feed additive also selectively reduces the gram-positive bacterial population. Tylosin is also an antimicrobial agent that acts against gram-positive bacteria. The major role of tylosin is to reduce the incidence of liver abscesses when steers are fed a rapidly fermentable carbohydrate-rich diet during the finishing phase of beef production. In the rumen, use of antibiotic feed additives was inversely associated with species diversity and richness as indicated by lower Chao1, ShannonH, and inverse Simpson indices for antibiotic treated steers. Previous research also had shown that monensin decreased rumen bacterial diversity both in vitro and in vivo . Contrary to the findings in the rumen, the microbial population in colon and cecum samples had similar diversity for both treatment groups. This suggests that antibiotic feed additives had only a limited effect on the distal gut microbiota of steers. A primary reason could be that monensin is actively absorbed from the rumen, metabolized in the liver, and excreted through bile and only <10% of unmetabolized monensin reaches the distal gut. This study also would be the first to report antibiotic resistance genes in the GIT of feedlot animals. We detected antibiotic resistance genes coding for 8 classes of drugs in the various compartments of beef cattle gastrointestinal tract. One of the major findings our study was that use of tylosin did not increase the presence of antibiotic resistance genes in the antibiotic treated steers. Tylosin resistance genes tlr, ermB, and ermX were not detected in any of the samples. Although macrolide resistance genes ermF and ermG were present only in the rumen of antibiotic treated steers, the gene encoding for macrolide efflux pump, mefA, was ubiquitously found in all three compartments of both treatment groups. This is in contrast to the earlier finding where the use of tylosin increased the presence of macrolide resistance genes in feedlot steers.. Another finding was that the distribution of cfxA and nimJ genes, which provide resistance to beta-lactams and metronidazole respectively, differed between forestomach and hind-gut. This could indicate that sampling feces alone might not reveal the whole picture on antibiotic resistance gene in the case of ruminants. Sampling forestomach along with fecal samples would be more effective in predicting a comprehensive antibiotic resistance gene profile although collecting rumen samples would be difficult in farm conditions. Overall, our results did not show significant difference in antibiotic resistance gene profile of steers in control and antibiotic treated groups. Since the total duration of antibiotic feed additive in this study was only 74 days, the antibiotic resistance gene profile we discovered here may not be representation of longer term effect of such feed additives. Objective 4. To understand the transmission of antibiotic resistance genes, we determined the antibiotic resistance and virulence gene profile of Salmonella enterica isolated from wildlife. Multiple serovars of S. enterica originating from mammalian, reptilian and avian hosts have been reported to cause infections in humans. Wildlife and exotic pets harboring Salmonella are potential sources for human infections. Transmission of Salmonella from wildlife and exotic animals to humans occurs through multiple pathways. To determine whether Salmonella from wildlife could act as source of antibiotic resistant strain infections in humans, a total of 103 Salmonella isolates sampled between 1988 and 2003 from wildlife and exotic pets were included in the study to determine the antimicrobial susceptibility using whole genome sequencing. Among 103 isolates, 52 isolates (50.48%) were from wild birds, 1 isolate (0.9%) was from fish, 25 isolates each (24.27%) were from reptiles and mammals. The serovars of 96 isolates in this study were determined at the National Veterinary Service Laboratory, Ames, IA, and the remaining 6 serovars were predicted using Seqsero . The serovar of one isolate (ADRDL-093) was not identified under Kauffmann-White classification. A total of 45 serovars were identified among the 103 isolates, of which Typhimurium (12.62%) was the most frequent serovar. Other serovars that had higher prevalence were Enteritidis (6.8%), Anatum (5.8%), Arizona (5.8%), Bredeney (3.9%) and Montevideo (3.9%). An antimicrobial susceptibility test of 103 Salmonella bacterial isolates was performed using Sensititre NARMS gram-negative plates. Fifty-two out of the 103 isolates (50.48%) showed resistance to at least one antibiotic. Resistance against theaminoglycoside streptomycin was most commonly observed. However, only three isolates (2.9%) showed resistance to gentamicin which also belonged to the aminoglycoside class of antibiotics. In the beta-lactam group, ampicillin resistance was the most common phenotype and was seen in 11 of the isolates (10.67%). All the isolates were susceptible to ceftriaxone except one with intermediate resistance. Chloramphenicol resistance was observed for seven isolates (6.7%), trimethoprimsulfamethoxazole resistance in 4 (3.88%) and tetracycline resistance in 19 (18.44%) of the isolates. The presence of antibiotic resistance genes that code for the resistance was identified from the whole whole genome sequence data of the strains. Twenty-two genes that provided resistance to aminoglycosides were detected and the genes were present in 100 isolates. The sensitivity was 100% and specificity was 5.45% for resistance against aminoglycosides. Genes responsible for resistance to beta-lactam antibiotics were detected in 11 isolates which were also resistant by antimicrobial susceptibility test. The plasmid-mediated cephalosporinase gene blaLAT-1, plasmid-borne class C betalactamase gene blaBIL-1, and blaCMY (Class C) genes were found together and were detected in three isolates. Genes belonging to blaTEM (class A) were found in eight isolates. Collectively, there were 280 beta-lactamase genes present in those 11 isolates. Tetracycline resistance encoded by tet(A), tet(B), tet(C), and tet(D) genes for tetracycline efflux pumps were detected in 18 samples all of which were also resistant by antimicrobial susceptibility test. The sensitivity was 94.74% and specificity was 100% for tetracycline resistance. Two isolates carried the mph(A) gene which confers resistance to macrolides. NC1202 group members Dr. Scaria (SDSU) and Dr. Orhan (Iowa state U) collaborated on a project to track antimicrobial resistance in food animals. Total funding $385,000, Agency - FDA, Project duration 10/01/2017 - 09/30/2022
Publications
- 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 sequencingbased
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:Swine producers: Improving pre-harvest food safety is a major focus for swine producers. Our work on WGS based Salmonella detection and trace back will help swine producers to implement better farm management practices to reduce Salmonella incidence in the production system. Poultry producers: Infection from contaminated poultry products is the major source of Salmonellosis in the United States. WGS based trace back methods we are developing will help poultry industry to formulate better decontamination protocols in the supply system. Scientific 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:
Nothing Reported
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 foodborne pathogen detection, trace back and bioinformatics analysis. Milton Thomas, PhD, Postdoctoral fellow: Performed Salmonella and E. coli genome sequencing library preparation, whole genome sequencing and bioinformatics analysis of WGS data. Gavin Fenske, MS student: Learned skills in Salmonella isolation, PCR, MALDI-TOF and 16s rRNA sequencing. Linto Antony, DVM, MS student: Performed experiments to develop humanized pig model for human gut microbiome. Sudeep Ghimire, DVM, PhD student: Cultured and identified Salmonella samples from poultry and swine. Learned whole genome sequencing and analysis methods. Supapit Wongkuna, visiting scholar: Learned S. enterica typing and genome sequencing methods. Andrew Foley, BS student: Learned anaerobic bacteriology methods and MALDI-TOF based identification of bacteria. Alex Thomas Fonder, BS student: Learned to isolate Salmonella enterica from cattle and swine. How have the results been disseminated to communities of interest?Research community: We have published three peer reviewed manuscripts and two conference abstract during this reporting period. Salmonella and E. coli genome data has been released for public access through NCBI SRA. Producer groups: PI has presented the results on the Salmonella genome sequencing and identification of beneficial bacteria that reduced Salmonella colonization in SD Beef producers meeting and in SD poultry producers meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 1) Focus on emerging diseases We plan to expand our whole genome based surveillance of foodborne pathogens and sequence at least 400 Salmonella genomes. Data from this will be released for public access through NCBI SRA database. Objective 2) Focus on preventions and interventions We plan to determine the molecular mechanisms behind the emergence of more invasive strains of S. enterica. We use bioreactor based assays to determine whether the newly emerged variant strains have more metabolic fitness than wild type strains. Objective 3) Focus on disseminating knowledge We plan to publish at least 3 peer reviewed manuscripts based on the current results.
Impacts What was accomplished under these goals?
Objective 1) Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals. For the first time, we identified a new serotype of Salmonella knows as Salmonella enterica Lubbock from the liver abscess in feedlot cattle. This new serotype is found to be more invasive and likely to migrate to lymph nodes. We did a whole genome sequencing based comparison of 75 Salmonella Lubbock isolates. One manuscript was published based on this study. Our results will help in improving pre-harvest food safety in beef operations. We determined the prevalence of Salmonella enterica in poultry sources in Nigeria. Transcontinental transmission of multi-drug resistant Salmonella is problem compromising the food safety of the United States. Our study and the availability of whole genome data from Nigeria isolates will improve the United States Food and Drug Administration's trace back efforts in imported food items. One manuscript was published based on the results from this study. Objective 2) Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric pathogens of livestock and foodborne and waterborne pathogens. We collected E. coli 0157 strains from feedlots in Midwestern states and sequenced the genomes of 100 isolates with the goal of establishing a reference point for tracking foodborne infections of E. coli 0157 likely originating from beef. Genome data for these 100 reference isolates have been made publicly available through NCBI SRA database (https://www.ncbi.nlm.nih.gov/bioproject/299490 ). We developed a humanized pig model to study the function of human gut microbiota and dietary effects. Piglets were delivered by C-section and were maintained in sterile isolators. Piglets were then implanted with pathogen free human gut microbiota. Microbiome Colonization dynamics was determined using metagenome sequencing. Effect of diet on microbiome and host health was determined feeding different treatment groups with western or Mediterranean diets. Immunological, microbiological and metabolic parameters were then assayed to quantify the effects of these two diets. Development of this humanized pig model was instrumental in obtaining a planning grant from SD Governor's office. The goal of this planning grant is to develop research teams that would develop biologics that would improve the enteric health of humans and animals. Results obtained from the humanized pig model will also improve our understanding about human and swine gut function. Objective 3) Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures. Our expertise in bacterial genomics, bioinformatics and the infrastructure investments in next generation sequencing helped us to become a regional whole genome sequencing lab for food borne pathogen trace back analysis in FDA network labs. Currently we are one of the four regional labs in the US and will sequence foodborne pathogen samples for the states of North Carolina, Oklahoma, Minnesota, Iowa and Michigan. In collaboration with state diagnostic labs in New York and Ohio, we standardized a whole genome sequencing based diagnostic method for the detection of Salmonella and E. coli. Objective 4) Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings. Dr. Scaria presented the progress of the work done at SD AES in the NC1202 Annual meeting, Dec 3-4, 2016, Chicago, IL . We also discussed submission of joint grant applications with researchers from Washington State University and Kansas State University. One peer reviwed manuscript was published jointly with Dr. TG Nagaraja from Kansas State University
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Draft Genome Sequences of Three Flavobacterium psychrophilum Strains Isolated from Coldwater Disease Outbreaks at Three Production Hatcheries. Neiger R, Thomas M, Das S, Barnes M, Fletcher B, Snekvik K, Thompson J, Scaria J*. GenomeA. 2016 Mar 10;4(2). pii: e00035-16 PMID: 26966210
- 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:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Draft genome sequences of 37 Salmonella enterica strains isolated from poultry sources in Nigeria. Useh NM, Ngbede EO, Akange N, Thomas M,Foley A, Keena M, Nelson EA,
Christopher-Hennings J, Tomita M, Suzuki H and Scaria J*. GenomeA, 2016 May 5;4(3). pii: e00315-16.PMID: 27151793
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
M. Thomas, S. Ghimire, A. Fonder, A. Khoruts, M. Sadowsky, D. Francis, and J. Scaria. Development of a humanized piglet model for the integrative analysis of microbiome and metabolome of the gut. Metabolomics symposium, Mayo Clinic, MN. October 15-16, 2016.
- 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 09/15/15 to 09/30/15
Outputs Target Audience:The target groups for this project are swine and cattle producer groups. Most of the work done in the past year were realted to swine and cattle infections caused by enteric pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two undergraduate students, one graduate student and Postdoctoral fellow was trained on molecular biology mehtods, next generation genome sequencing and bioinformatics. 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?In the next year, we will focus on developing competitive exclusion based bacterial mixes for controlling and treating enteric infection in swine and cattle. We will also focus on developing new diagnostic assays for emerging enteric infections.
Impacts What was accomplished under these goals?
A. Development and validation of whole genome sequencing method for Porcine Epidemic Diarrhea virus (PEDV). In collaboration with National Veterinary Services Laboratory (NVSL) Ames, Iowa, we developed and validated new method for whole genome sequencing of PEDV directly from fecal samples. We optimized this protocol by depleting all RNA types except PEDV and other viral RNA in the samples. With our enriched protocol, the virus reads were between 10-30% of the total reads obtained from Illumina MiSeq platform. This allowed us multiplex more samples in a single run. This reduces the sequencing cost for the samples. A total of 56 samples were tested using our method. Results were submitted to NVSL for comparison with the sequencing data obtained at NVSL and other labs. B. Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus. Recombinant North American nucleoprotein (NP) based iELISA was developed and validated along with a bELISA using newly developed PEDV-NP specific biotinylated monoclonal antibodies (mAbs) and an FMIA using magnetic beads coupled with expressed NA PEDV-NP. Receiver operating characteristic (ROC) analysis was performed using swine serum samples (iELISA n = 1486, bELISA n = 1186, FMIA n = 1420). The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. The iELISA and bELISA showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. Inter-rater (kappa) agreement was calculated to be 0.941 between iELISA and IFA, 0.945 between bELISA and IFA and 0.932 between FMIA and IFA. Similar comparative kappa values were observed between the iELISA, bELISA and FMIA, which demonstrated a significant level of testing agreement among the three assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted with these assays. All three assays detected seroconversion of naïve animals within 6-9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples. C. Genomic and phenomic characterization of Clostridium difficile and Clostridium sordellii. Clostridium difficile and C. sordellii are two anaerobic, spore forming, gram positive pathogens that cause infection in cattle and swine. Despite strong similarities between the two Clostridial species, differences in their host tissue preference place C. difficile infections in the gastrointestinal tract and C. sordellii infections in soft tissues. To improve our understanding of C. sordellii and C. difficile virulence and pathogenesis, we performed a comparative genomic and phenomic analysis of the two. The global phenomes of C. difficile and C. sordellii were compared using Biolog Phenotype microarrays. When compared to C. difficile, C. sordellii was found to better utilize more complex sources of carbon and nitrogen, including peptides. Phenotype microarray comparison also revealed that C. sordellii was better able to grow in acidic pH conditions. Using next generation sequencing technology, we determined the draft genome of C. sordellii strain 8483 and performed comparative genome analysis with C. difficile and other Clostridial genomes. Comparative genome analysis revealed the presence of several enzymes, including the urease gene cluster, specific to the C. sordellii genome that confer the ability of expanded peptide utilization and survival in acidic pH. The identified phenotypes of C. sordellii might be important in causing wound and vaginal infections respectively. Proteins involved in the metabolic differences between C. sordellii and C. difficile should be targets for further studies aimed at understanding C. difficile and C. sordellii infection site specificity and pathogenesis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Scaria J, Suzuki H, Ptak CP, Chen JW, Zhu Y, Guo XK, Chang YF. Comparative genomic and phenomic analysis of Clostridium difficile and Clostridium sordellii, two related pathogens with differing host tissue preference. BMC Genomics. 2015 Jun 10;16:448.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Okda F, Liu X, Singrey A, Clement T, Nelson J, Christopher-Hennings J, Nelson EA, Lawson S. Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus. BMC Vet Res. 2015 Aug 1;11:180.
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