Recipient Organization
EASTERN SCIENTIFIC LLC
316 N ESTILL AVE
RICHMOND,KY 40475
Performing Department
(N/A)
Non Technical Summary
Existing methods for measuring agricultural water quality that has been impacted by fecal bacteria from livestock, wildlife, and humans is not easily performed on the farm and requires laboratory resources. With an increased frequency of extreme weather events inundating agricultural lands and potentially impacting private groundwater systems and surface waters, their remains a need for water monitoring to inform pollution mitigation and conservation practices while also safeguarding food systems and public health. Additionally, the USDA, FDA, and CDC have joined efforts as part of the National Antimicrobial Resistance Monitoring System to better understand how antibiotic resistant bacteria move in the environment between livestock, humans, and wildlife. Water and fecal indicator bacteria are both important for assessing this movement of antibiotic-resistant bacteria. Accordingly, farmers, conservationists, and even citizen scientists who desire to contribute to monitoring and improving our nation's waters may have the opportunity to take action if affordable, practical, field portable kits become available that provide rapid and scientifically defensible results. The research proposed seeks to enhance and validate an affordable handheld quantitative fluorometer and colorimeter for enumeratingE. coliand antibiotic-resistantE. coli from agricultural waters . The results obtained will be compared to the standard methods, which are slower, more expensive and require laboratory resources. For comparing methods, over 96 water samples will be obtained from eight different locations, with many locations having some degree of agricultural impact, including a stream adjacent to a university farm. Statistical approaches will be used to compare the standard approach versus the more rapid novel approaches proposed. These research efforts presumably would benefit conservation-minded agricultural operations, conservation districts, watershed organizations, educators, and citizen scientists by leading to a kit that enables their participation in water quality monitoring and antibiotic resistance surveillance efforts. Added benefits may include improved public health protections of leafy vegetables and more rapid and affordable testing of irrigation waters by agricultural producers.
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Goals / Objectives
Based upon our engagement with watershed organizations and agricultural partners concerned about fecal contamination of natural and agricultural waters, we have two major project goalsrelative to enhancing our existing ColiGlowmethod forE. colidensity enumeration.Goal1 is to develop a rapid ColiGlow test that works with a handheld fluorometer or handheld colorimeter for enumeratingE. colidensity within nine hours rather than current methodologies requiring18-24 hours. Specifically, the project aims to be able to use a handheld instrument and a cuvette or series of cuvettes to determine an amount of fluorescence or absorbance fromtests of water samples and be able to link that result to the actual density ofE. colias determined by a standard method. Using 96 water samples (eight locations over 12 weeks) inclusive of waterways impacted by agriculture, the project willenumerateE. coliusing a standard method and the rapid method for informing the predictive relationship. For Goal2, the project willassess the discrimination of modified (addition of antibiotic) and rapid modified ColiGlow kits for detecting and quantifying antibiotic resistantE. colifrom natural waters.Isolated colonies (at least 90)will be obtainedfrom fluorescing tubes on selective differentialE. colimedia, and then be evaluated for antibiotic susceptibility and species identification using standard methods. Based upon these goals, promising results will inform efforts fora potentialPhase 2 SBIR/STTR submission.
Project Methods
Goal 1 will rely upon experiential learning opportunities for university students using 96 water samples from eight locations over 12 weeks, in which they will determine the amount of E. coliand antibiotic-resistantE. coli through a standard method that enumerates these bacteria. Samples will come from waterways that are inclusive of agricultural waters and natural waters with less agricultural impact in Central Kentucky.Samples will also be given to Eastern Scientific for their research team to use, whereby a novel media formulation coupled with fluorometric and colorimetric methods will be used to measure the fluorescence and color absorbance, respectively, for determining the abundance of E. coli and antibiotic-resistant E. coli in the various samples within nine hours. Analyses will then be done to determine the presence/absence of correlation between the standard method and the proposed rapid fluorometric and colorimetric methods. Modeling will also be performed to optimize the relationship between the standard method and innovative methods. Key evaluation milestones for Goal 1 include the successful collection and analysis of 96 water samples using the standard method and the proposed methods. Statistically significant (p < 0.05) predictive regression models with R-square values greater than 70% will be indicative of success, and greater than 90% will be indicative of excellent performance.For Goal 2, the project will provide experiential learning opportunities to two undergraduate students and one graduate student from the partner public academic institution/university for isolating bacterial colonies from the presumed positive fluorometric and colorometric samples provided by Eastern Scientific. The students and their faculty mentors will be using analytical instrumentation for the microbial identification of bacteria using MALDI-TOF-MSand for antibiotic susceptibility determination using a MicroScan.The project will evaluate 90 isolates. Chi-square tests, logistic regression, and sensitivity/specificity analyses will be used to evaluate the new fluorometric and colorimetric methods' discrimination ability for E. coli species identification and tetracycline-resistance. Key evaluation milestones for Goal 2 include the successful evaluation of 90 isolates from the proposed methods. Acceptable discrimination for species identification and tetracycline-resistance determination will be evaluated using the area under the receiver-operator-characteristic curve (AUC). An AUC greater than 70% will be deemed acceptable discrimination and over 90% will be indicative of excellent discrimination.