Recipient Organization
INDIANA UNIVERSITY
JORDAN HALL 142
BLOOMINGTON,IN 47401
Performing Department
(N/A)
Non Technical Summary
Coprophagous flies are uniquely suited to be used as one health biomonitors of terrestrial antimicrobial resistance. Where multiple sources of fecal material are accessible to them, coprophagous flies act as composite samplers of localized terrestrial AMR by repeatedly feeding on uncontained wastes. In agricultural settings, flies also pose a risk of transporting ARBs and ARGs from fecal wastes to agricultural products. We propose further study of coprophagous flies to better understand the persistence and emission of ARB and ARGs as part of a novel approach to monitor and mitigate AMR in terrestrial environments. Our three research objectives include, (1) Determine the rate of ingestion and persistence of ARB and ARGs in coprophagous flies under a range of environmental conditions; (2) Determine the excretion of ARB and ARGs from coprophagous flies to surfaces under a range of environmental conditions; and (3) Determine the relationship between ARB and ARGs in wild caught coprophagous flies with temporally and geographically matched agricultural products and irrigation water. We will conduct controlled feeding studies using coprophagous flies to accomplish Objectives 1 and 2. We will incorporate Objective 3 into our ongoing field work in Maputo, Mozambique. This fundamental work advances our understanding how coprophagous flies can be used as one health biomonitors of terrestrial antimicrobial resistance. The valuable insights into the spread and prevalence of AMR, will enable targeted interventions to reduce its impact in the food chain and help break the transmission cycle of resistant bacteria, improving public health.
Animal Health Component
33%
Research Effort Categories
Basic
67%
Applied
33%
Developmental
0%
Goals / Objectives
Objective 1: Determine the rate of ingestion and persistence of ARB and potential mobilization ARGs in coprophagous flies under a range of environmental conditions.Objective 2: Determine the excretion of ARB and ARGs from coprophagous flies to surfaces under a range of environmental conditions.Objective 3: Determine the relationship between ARB and ARGs in wild caught coprophagous flies with temporally and geographically matched agricultural products and irrigation water.
Project Methods
Methods Aim 1. We will conduct controlled feeding studies with the common blow fly (species Phormia regina). Blow flies will be collected from local areas and a colony will be established at the Capone Lab using standard methods8,26. As part of the standard process for maintaining flies, eggs will be reared to adults, and 5 cages maintained. A series of controlled feeding experiments will be conducted. Flies will be acclimatized under each unique combination of temperature (15, 25, 35°C) and relative humidity (30, 50, 70%) during the 12-hour starvation period, as described in Figure 6 and Table 1. Following starvation, individual flies will be placed in sterile cup and permitted to feed on fresh bovine fecal material spiked with 108 CFU of ESBL E. coli for four hours (type species and strain: E. coli NC11). Afterwards, flies will be returned to cages and maintained at the pre-specified temperature and humidity conditions in incubators (Percival I-36VL). Flies will be removed in triplicate at eight time points (0, 24, 48, 96, 120, 240 hours) to undergo aseptic dissection. Controls will be flies that are only exposed to water. Four flies (three treatment + one control) will be removed, dissected aseptically and the alimentary canal removed for culture-dependent and independent analysis at each time point (i.e., 8 flies per time point).Culture Dependent and Independent Analysis. Culture-dependent and culture-independent methods will be used to quantify fecal indicators (Table 2). For culture-dependent analysis, individual flies will be surfaced washed with a 10% bleach solution, and then the alimentary canals of the flies dissected. Tissue will be crushed in sterile phosphate buffer saline (PBS) and shaken for 30 seconds. The solids will be permitted to settle for 1 minute, and then serial dilution of the supernatant will be performed. Supernatant from these dilutions will be plated according to the WHO Tricycle Protocol, including plating onto Tryptone Bile X-glucuronide Agar (TBX) for E. coli and TBX supplemented with Cefotaxime (CTX) at 4 µg/mL (TBX-CTX) for ESBL E. coli5. Plates will be incubated for 24 hours and CFUs counted. A positive and negative control will be run on each day that culture-dependent work is performed 27.Digital PCR. For culture-independent analysis, again, the flies will be surface sterilized with a 10% bleach solution dip, dissected, and then DNA will be extracted from each alimentary canal using a commercial kit, like our previous methods16. Purified nucleic acids will be assayed for the blaCTX-Mgene using digital PCR with a QIAcuity 4 instrument (Qiagen, Hilden, Germany). We will include a DNA extraction control and run negative controls.Methods Objective 2. As part of Objective 1, flies will be exposed to bovine feces spiked with ESBL E. coli and will be placed in an incubator at pre-specified temperature and humidity conditions. Following the removal of flies from their individual containers - where flies will be used for Objective 1 - we will retain the individual containers for Objective 2. We will wash each container using a phosphate buffered saline solution containing 0.1% Tween 80 by manual shaking for two minutes. Wash water will be concentrated using vacuum membrane filtration with a 0.45 µm filter. Half of the filters from each combination of temperature and humidity will be cultured for ESBL E. coli using the methods described in Objective 1. We will extract nucleic acids from the other half of filters using commercial kits, which include a bead beating step31. Purified nucleic acids from the filters will also be assayed for the blaCTX-Mgene using digital PCR with a QIAcuity 4 instrument (Qiagen, Hilden, Germany). Positive and negative controls will be included27.Emission Rates. At each time point we will quantify the total culturable ESBL E. coli and the number of ARGs transferred or excreted from each individual fly to the container that housed the fly during the controlled feeding study. The sum of these values will be taken as the total emissions and plotted as a cumulative distribution from 0 to 10 days. We expect emissions from flies to be best described by an exponential distribution, with the highest rate of emission occurring within the first 24 hours. We will determine the best fit exponential line to the data for each combination of temperature and humidity to estimate emission rates per fly per day.Approach. Patógenos, meio Ambiente, Resistência Antimicrobiana e águas Residuais (PARAR). The PARAR study in Maputo, Mozambique aims to evaluate the impact of a World Bank funded rehabilitation of Maputo's wastewater treatment plant (WWTP) on pathogens and AMR in downstream river water used for agricultural irrigation and agricultural products irrigated with river water receiving WWTP effluent. It is a collaboration between the Capone Lab and the Mozambican National Institute of Health, who have worked together since 2017. The study team received internal pilot funding from the Indiana University Faculty Support for Research Program (FSRP) in 2024, which will be used to fund collection and culture-based analysis of wastewater, irrigation water and agricultural products starting in Q1 2025. In this study, we will leverage USDA funding to collect coprophagous flies geographically and temporally matched to the planned collection of irrigation water and agricultural products.Sampling sites will include 50 individual farm plots upstream and downstream of Maputo's WWTP along the Infulene River (totaling 100 plots). Flies will be caught using our 3D printed baited trap and sweep net methodology (n=300 for analysis). Irrigation water (i.e., 6-12 L of river water) will be concentrated with the bag mediated filtration system32-34 (n=100) and 30 grams of collected agricultural products (i.e., lettuce or spinach, n=300) will be homogenized in 330 mL of PBS using an Interscience BagMixer 400 with the resulting supernatant concentrated via membrane filtration (Figure 7). In Maputo all samples will be assayed for E. coli and ESBL E. coli using the methods described above (with funding from IU). Samples will be shipped on dry ice with the appropriate permit to Indiana University. We will extract total nucleic acids from these samples and quantify the blaCTX-Mand intI1 gene using digital PCR (with funding from USDA)5,35.Education: We will broaden the scientific literacy of students and the public regarding coprophagous flies and AMR. First, we will train undergraduates and graduate students at the interface of engineering, public health, and microbiology. We will train 1 PhD student and each summer a group of 3-4 undergraduates will contribute to the project as part of Indiana University's STEM Summer Research Program, which provides a stipend and housing. We will develop and implement a "Maggot Art" activity where participants use fly maggots to paint art, and we will implement this activity at the annual Celebrate Science Indiana festival.Extension. Wewill form a steering committee that includes local public health departments (see LOS), policy focused organizations (see LOS), and the Indiana Department of Environmental Management. We will meet quarterly to discuss how our findings can inform policy and practice for AMR mitigation in Indiana and nationally.