Source: UNIVERSITY OF WYOMING submitted to
ENTERIC DISEASES OF FOOD ANIMALS: ENHANCED PREVENTION, CONTROL AND FOOD SAFETY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1015650
Grant No.
(N/A)
Project No.
WYO-596-18
Proposal No.
(N/A)
Multistate No.
NC-_old1202
Program Code
(N/A)
Project Start Date
Feb 14, 2018
Project End Date
Sep 30, 2022
Grant Year
(N/A)
Project Director
Bisha, BL.
Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000
Performing Department
Animal Science
Non Technical Summary
The long-term goal of this collaborative project is to develop strategies to prevent and control enteric diseases of cattle, swine, and poultry, ultimately to decrease theincidence of enteric diseases in food animals, and decrease zoonotic food and water-borne illnesses in the USA. Despite many concerted efforts to control enteric pathogens as well as zoonotic pathogens in food animals at both on-farm (pre-harvest) and food processing (post-harvest) environments, theincidence of diseases caused by enteric pathogens, and food and water-borne pathogens remains high and some diseases are increasing. At the University of Wyoming we will focus our efforts on helping improve understanding of the persistance, resistance, and dissemination of foodborne pathogens in the natural environment (including wildlife), food animals, and food processing enviroment. Additionally we will improve diagnostic techniques and sample preparation methods to improve diagnostics of foodborne pathogens and indicator bacteria. We will utilize methods including phenotypic (culture-based, MALDI-TOF MS), cytomic (flow cytometry, microscopy), and molecular (whole genome sequencing, metagenomics) to better understand and characterize microbial resistance, stress and survival in the environment, food, and animals.
Animal Health Component
5%
Research Effort Categories
Basic
25%
Applied
65%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7223399110025%
3113999110025%
7123910110025%
7223910110025%
Knowledge Area
311 - Animal Diseases; 722 - Zoonotic Diseases and Parasites Affecting Humans; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3999 - Animal research, general; 3910 - Cross-commodity research--multiple animal species; 3399 - Beef cattle, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
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. 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. 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. 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.
Project Methods
We will develop diagnostic methods for foodborne bacterial and viral pathogens and indicator organisms, based on novel principles/platforms such as microfluidics, mass spectrometry, flow cytometry, and associated technologies. We will improve the ability of Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) for rapid and specific identification and characterization of foodborne bacterial pathogens and antibiotic resistant (AMR) bacteria by enhancing sample preparation, analytical procedures, and database entries. We will utilize 'cytomics' tools such as flow cytometry to characterize microbial responses at the single cell level, in order to better understand microbial resistance, stress and injury. We will develop inexpensive microfluidics-based diagnostics in the form of paper-based analytical devices (μPADs) amenable to field- based detection of bacterial pathogens and indicator bacteria, using enzymatic and molecular detection approaches. We will develop effective methods for concentration and purification of bacterial and viral targets from a variety of matrices (water, bioaerosols, food, biological fluids). We will employ a 'multi-omics' approach for determination and characterization of AMR, including phenotypic (culture-based, biochemical testing, and MALDI-TOF MS) and genotypic (whole genome sequencing and metagenomics). We will focus our examinations to AMR derived from wildlife, food production animals, and associated environment. By combining the data outputs of these methods, the acquired information will provide needed insight into the magnitude and causation of the AMR problem, and help determine what actions should be taken to mitigate this risk.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Undergraduate and graduate students, state and federal government employees, consumers, academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate and two undergraduate students were provided opportunities to conduct research on these topics. How have the results been disseminated to communities of interest?Peer-reviewed journal publications and direct communications with stakeholders and public health officials. 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. 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. Antimicrobial Resistance (AMR) A real-time PCR method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. We assessed the role of European starling in the dissemination of antimicrobial resistance in cattle feedlots, with phylogenetic and subtyping assessments showing that highly similar isolates (≥99.4% shared core genome, ≥99.6% shared coding sequence) with priority AMR were found in birds on feedlots separated by distances exceeding 150 km, suggesting that European starlings could be involved in the interstate dissemination of priority AMR bacteria. 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 and foodborne and waterborne pathogens. We evaluated targeted control of invasive European starlings in feedlots to control wildlife-mediated spread of antimicrobial resistance. Bird numbers were reduced on feedlots by 70.4%, but decreasing populations of European starlings was not associated with corresponding reductions in bovine fecal prevalence of ciprofloxacin-resistant E. coli.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chandler JC, Anders JE, Blouin NA, Carlson JC, LeJeune JT, Goodridge LD, Wang B, Day LA, Mangan AM, Reid DA, Coleman SM, Hopken MW, Bisha B. Author Correction: The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations. Sci Rep. 2020 Jul 15;10(1):11978. doi: 10.1038/s41598-020-68365-9. PMID: 32669574 Free PMC article.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Carlson JC, Chandler JC, Bisha B, LeJeune JT, Wittum TE. Bird-livestock interactions associated with increased cattle fecal shedding of ciprofloxacin-resistant Escherichia coli within feedlots in the United States. Sci Rep. 2020 Jun 23;10(1):10174. doi: 10.1038/s41598-020-66782-4. PMID: 32576851 Free PMC article.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chandler JC, Anders JE, Blouin NA, Carlson JC, LeJeune JT, Goodridge LD, Wang B, Day LA, Mangan AM, Reid DA, Coleman SM, Hopken MW, Bisha B. The role of European starlings (Sturnus vulgaris) in the dissemination of multidrug-resistant Escherichia coli among concentrated animal feeding operations. Sci Rep. 2020 May 15;10(1):8093. doi: 10.1038/s41598-020-64544-w. PMID: 32415136 Free PMC article.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chandler JC, Franklin AB, Bevins SN, Bentler KT, Bonnedahl J, Ahlstrom CA, Bisha B, Shriner SA. Validation of a screening method for the detection of colistin-resistant E. coli containing mcr-1 in feral swine feces. J Microbiol Methods. 2020 May;172:105892. doi: 10.1016/j.mimet.2020.105892. Epub 2020 Mar 14. PMID: 32184162.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Stahl RS, Bisha B, Mahapatra S, Chandler JC. A model for the prediction of antimicrobial resistance in Escherichia coli based on a comparative evaluation of fatty acid profiles. Diagn Microbiol Infect Dis. 2020 Mar;96(3):114966. doi: 10.1016/j.diagmicrobio.2019.114966. Epub 2019 Dec 18. PMID: 31948696


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Food scientists, microbiologists, food safety professionals, government officials, food industry, graduate and undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided trainining opportunities for three graduate and two undergraduate students. How have the results been disseminated to communities of interest?Results have been disseminated via journal articles and presentations in scientific meetings. 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. 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. Antimicrobial Resistance (AMR) We evaluated a machine learning classifier in conjunction with similarity searches against identification libraries for the analysis of mass spectral fingerprints obtained by MALDI-TOF MS in order to improve microbial identification of select foodborne bacteria. MALDI-TOF MS coupled with library-based identification for all strains tested correctly identified 86% of the MALDI-TOF mass spectra at the species level. The machine learning neural net evaluated resulted in a significantly higher accuracy of discrimination of 95%. We developed a method to predict antimicrobial sensitive/resistant (S/R) phenotypes and host sources of Escherichia coli based on differential fatty acid abundance. Myristic, pentadecanoic acid, palmitic, elaidic and steric acid were significantly different for predicting nalidixic acid, ciprofloxacin, aztreonam, cefotaxime, and ceftazidime S/R phenotypes. Linear regression models of fatty acid abundance made using a replicated-randomized subsampling and modelling approach predicted S/R phenotype with 79% and 81% accuracy for nalidixic acid and ciprofloxacin, respectively. The isolate host source was predicted with 63% accuracy.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Maus A, Bisha B, Fagerquist C, Basile F. Detection and identification of a protein biomarker in antibiotic-resistant Escherichia coli using intact protein LC offline MALDI-MS and MS/MS. J Appl Microbiol. 2020 Mar;128(3):697-709. doi: 10.1111/jam.14507. Epub 2019 Dec 9.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Stahl RS, Bisha B, Mahapatra S, Chandler JC. A model for the prediction of antimicrobial resistance in Escherichia coli based on a comparative evaluation of fatty acid profiles. Diagn Microbiol Infect Dis. 2020 Mar;96(3):114966. doi: 10.1016/j.diagmicrobio.2019.114966. Epub 2019 Dec 18.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 1. Broten CJ, Wydallis JB, Reilly T III, Bisha B. Colorimetric Detection of Clostridium perfringens in a Model Meat System Using Paper-Based Microfluidics. International Association for Food Protection Annual Meeting. July 21 - July 24, 2019, Louisville, KY.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Colavecchio A, Chandler J, Bisha B, Coleman S, Emond-Rheault J, Hamel J, Kukavica-Ibrulj I, Levesque R, Fanning S, Goodridge LD. Phage-like Plasmids Transfer Antibiotic and Heavy Metal Resistance Genes by Transduction, Transformation and Conjugation. International Association for Food Protection Annual Meeting. July 21 - July 24, 2019, Louisville, KY.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Chandler J, Hamel J, Emond-Rheault J-G, Boyele B, Aljasir S, Shriner S, Root J, Mooers N, Bentler K, Ellis J, Russell M, Robeson M, Goodridge L, Franklin A, L�vesque R, Bisha B. Oceanobacillus spp., Sources of Antimicrobial Resistance at the Wildlife-Livestock Interface. American Society for Microbiology Microbe. June 20 - June 24, 2019, San Francisco, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bisha B, Blouin N, Bono J, Franklin A, Goodridge L, Root J, Shriner S, Chandler J. Oceanobacillus spp., Wildlife Sources of Antimicrobial Resistance in Agricultural Production. American Society for Microbiology Microbe. June 20 - June 24, 2019, San Francisco, CA.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lindsey B, Rowley C, Young B, DeWolf E, Bisha B. Machine Leaning Approaches for Improved MALDI-ToF MS Identification of Foodborne Bacteria. American Society for Microbiology Microbe. June 20 - June 24, 2019, San Francisco, CA.


Progress 02/14/18 to 09/30/18

Outputs
Target Audience: Target audiences comprised livestock producers, federal and state governmental public health officials, graduate and undergraduate students in food microbiology/animal science/veterinary science, food safety and quality professionals, and extension educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training has been provided for two graduate students and one undergraduate student. How have the results been disseminated to communities of interest?Peer-reviwed publications, presentations, symposia. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The focus of research at the University of Wyoming has been on antibiotic resistant (AMR) bacteria with special emphasis on the role of wildlife in the dissemination of AMR to livestock operations. We have investigated the genetic context of phenotypically similar AMR Escherichia coli harboring priority AMR phenotypes collected from cattle and raccoons at six livestock facilities. Additionally we have developed and optimized LC-MALDI-ToF-MS protocols in conjunction advanced data analysis to identify protein biomarkers for β-lactam resistance in E. coli. Furthermore, we have conducted the first nationwide survey of antimicrobial resistance phenotypes in indicator bacteria collected from dairy farms in the newly-formed country of Kosovo. 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. Antimicrobial Resistance (AMR) 1) E. coli isolates (n = 39) resistant to cephems and cephems/fluoroquinolones were subjected to whole genome sequencing analyses on the basis of identical antibiograms (18 different antibiotics tested) and presence in the feces of both raccoons and cattle on multiple farms. Complete closed and polished genomes (PacBio/HiSeq sequencing) were acquired for each isolate, and in silico (Parsnp, CARD-RGI, etc.) analyses were used to evaluate phylogenetic relationships and AMR phenotypes. Seven main clades of AMR E. coli isolates were identified, with the most highly conserved clade containing isolates from both cattle and raccoons. The variable genomes of isolates were primarily attributable to differential repertoires of Inc-like plasmids and prophage mosaics. Similarities between cattle and wildlife isolates were further highlighted by conserved AMR determinants. In total, 72 chromosomal AMR determinants were detected, with 45 of these found in all isolates tested. Plasmid-encoded CMY-2 AmpC β-lactamases were present in 11 isolates. Similarly, conserved mutations in gyrA and parC were linked to fluoroquinolone resistance. The genetic conservation between AMR isolates from cattle and wildlife suggest a complex AMR livestock ecology that has inputs from multiple sources. 2) We have expanded the detectable proteome in antibiotic-resistant bacteria by developing an offline LC protein separation/fractionation prior to MALDI-ToF-MS analysis and applied it for the analysis of antibiotic-resistant E. coli isolates. Using the developed LC-MALDI-ToF-MS protocol in conjunction with supervised principal components analysis (sup-PCA), we identified protein biomarkers which exhibited the strongest correlation to β-lactam resistance among the E. coli tested, namely resistance mediated by the blaCMY-2 gene (encoding AmpC-type β-lactamase) in the incompatibility plasmid complex A/C (IncA/C). Our results demonstrate the utility of LC-MALDI-MS and MS/MS to extend the number of proteins detected and perform MALDI-accessible biomarker discovery in microorganisms. 3) We conducted the first nationwide survey of antimicrobial resistance phenotypes in indicator bacteria collected from dairy farms in Kosovo. Composite fecal samples were collected from 52 farms located within all 7 administrative districts of Kosovo. Isolation and characterization of the indicator bacteria Escherichia coli (n = 165) and Enterococcus spp. (n = 153). E. coli and Enterococcus spp. isolated using sub-minimum inhibitory concentrations of cefoxitin, ciprofloxacin, or erythromycin were typically resistant to at least one and often multiple antibiotic types, which primarily consisted of certain β-lactams, quinolones, sulfonamides, phenicols, and tetracyclines for E. coli isolates and macrolides, tetracyclines, and rifamycins for enterococci isolates. 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. Dr. Bledar Bisha provided an overview of packing plant food safety interventions and provided educational materials to over 40 sheep producers during the Wyoming Wool Growers Association meeting on August 7, 2018.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 1. Schaeffer JW, Chandler JC, Davidson M, Magzamen SL, P�rez-M�ndez A, Reynolds SJ, Goodridge LD, Volckens J, Franklin AB, Shriner SA, Bisha B. Detection of Viruses from Bioaerosols Using Anion Exchange Resin. J Vis Exp. 2018 Aug 22;(138). doi: 10.3791/58111.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 2. Chandler JC, Aljasir SF, Hamidi A, Sylejmani D, Gerow KG, Bisha B. Short communication: A countrywide survey of antimicrobial-resistant indicator bacteria in Kosovo's dairy farms. J Dairy Sci. 2018 Aug;101(8):6982-6989. doi: 10.3168/jds.2017-14091. Epub 2018 May 30.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 1. Chandler J, Blouin N, Bono J, Franklin A, Goodridge L, Root J, Shriner S, Bisha B. Genetic Context of Antimicrobial-Resistant Escherichia coli at the Livestock-Wildlife Interface. International Association for Food Protection Annual Meeting. July 8 - July 11, 2018, Salt Lake City, UT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 2. Anders J, Chandler J, Carlson J, LeJeune J, Goodridge L, Wang B, Day L, Mangan A, Reid D, Coleman S, Bisha B. Antimicrobial Resistance Profiles of Escherichia coli from European Starlings (Sturnus vulgaris) Associated with Concentrated Animal Feeding Operations International Association for Food Protection Annual Meeting. July 8  July 11, 2018, Salt Lake City, UT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 1. Bisha B. Colorimetric and Electrochemical Bacteria Detection Using Printed Paper- and Transparency-Based Analytic Devices. Fifth Annual Rapid Detection for Food Safety Conference, June 27, 2018, Bethesda, MD (invited talk).