Recipient Organization
OHIO STATE UNIVERSITY - VET MED
1900 COFFEY ROAD, 127L VMAB
COLUMBUS,OH 43210
Performing Department
Preventive Medicine
Non Technical Summary
AR threatens public health care system by compromising its ability to treat common infectious diseases. Bacteria has the ability to share their genetic information including ARGs contributing to an increased spread of AR. Salmonella is a common AR foodborne pathogen found in livestock surroundings with the capacity to form biofilms. Biofilms are group of microorganisms embedded in a slimy layer that offers protection against antimicrobials. In livestock production systems, cleaning and disinfection are common practices used to control foodborne pathogens that are free living as well as those residing in biofilms. Inadequate disinfectant concentration could cause stress adaptation in bacteria and aid in their survival in challenging conditions. The proposed study aims to characterize the role played by the disinfectant stress adapted bacteria in environmental dissemination of antibiotic resistance. Our goal is to outline the consequences of improper disinfectant usage in livestock environment and its impact on enhanced AR dissemination.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Goals / Objectives
Evolution and spread of antibiotic resistance (AR) has become a global challenge. Horizontal gene transfer (HGT) promotes the exchange of genetic materials (including AR genes) among diverse bacterial species, enhancing the spread of ARGs in the environment. Among various AR foodborne pathogens, Salmonella is associated with more than 1 million infections per year in the United States. Livestock farms are the most important source for Salmonella, with animals acting as silent shedders. Currently, cleaning and disinfection using biocidal agents are standard procedures adopted in livestock farms to control infectious diseases. However, the use of biocidal agents risks increasing AR when not used in appropriate concentrations. Continuous exposure to sublethal concentrations of biocides can result in stress adaptation and selection of biocide resistant mutants. Moreover, there is an increasing concern that biocide resistant mutants can promote horizontal transfer of ARGs. Additionally, biofilm forming capacity of Salmonella further contributes to this burden by acting as a continuous source of ARGs. Under disinfectant selective pressure, chances of increased HGT of ARGs to commensal bacteria such as E. coli in biofilms could result in detrimental ecological consequences. However, biocide tolerance and AR dissemination were studied in planktonic cells, and is limited to laboratory isolates. Targeted research on biocide tolerance and HGT in foodborne pathogen is necessary to mitigate AR dissemination in food production environments. Therefore, the overall goal of this proposal is to assess stress adaptation in pathogenic and environmental bacteria during biocidal exposure and how it impacts HGT in planktonic and biofilm-associated cells. To do this, we will utilize specific microbiological techniques to determine sub-inhibitory concentrations of disinfectants and to understand the molecular basis for increased HGT under disinfectant stress response. This research will emphasize the need for better guidance on disinfection practices in livestock farms and contribute to prevention of AR dissemination.
Project Methods
Specific Aim 1: Create biocide adapted strains of Salmonella and E. coli by exposing to sub-inhibitory concentrations (SIC) and minimum-inhibitory concentrations (MIC) of commonly used biocides for planktonic and biofilm forming environmentally isolated MDR-Salmonella Agona (donor) and E. coli (recipient) cells.Research Approach: Salmonella Agona (containing blaTEM gene) that was previously isolated from the livestock environment will be used as the donor bacterium. SIC and MIC of biocides against both MDR-Salmonella andE. coli will be determined using a 96-well microtiter plates. For the planktonic cells, overnight cultures of Salmonella and E. coli will be used for the assay while for the biofilms, both the donor and the recipient cells will be allowed to grow for 48 hours to form mature biofilms followed by the addition of increasing concentrations of various biocides. Biocides commonly used on the farm for disinfection will be tested and will include chlorine based oxidizing agents, quaternary ammonium compounds, products containing glutaraldehyde, peroxy based compounds, iodine-based compounds and bisbiguanides. Once the SIC and MIC is determined, both the donor and recipient cells will be grown in increasing concentrations of biocides. This will lead to stress adaptation in bacteria and the adapted cells will be stored and used for specific Aim 2 and 3. Further, the adaptive stability of the bacteria will be tested by determining the MIC of the stress adapted cells in 96-well microtiter cells.Specific Aim 2: Determine the HGT frequency of the stress adapted and non-stress adapted donor and recipient bacteria in biofilms and in planktonic phase.Research Approach: Before performing HGT experiments, antibiotic resistance marker for the donor and recipient bacteria will be determined by antibiotic disc diffusion. HGT experiments will be conducted by mixing equal concentrations of stress adapted donor and recipient bacteria from SA1, and will be incubated for 24 hours for planktonic cells and 48 hours for biofilms allowing for mature biofilm formation. Following incubation, the donor, recipient and transconjugant colonies will be separately enumerated using agar plates containing respective antibiotic markers. Transconjugants that has gained the blaTEM gene will be able to grow in the presence of ampicillin as well as the resistance marker for the recipient. HGT frequency will be calculated as a ratio of the number of transconjugants (/CFU/ml) over the number if recipients (CFU/ml) (Welsh et al., 2008). All the experiments will be done in duplicates and replicated thrice.?Specific Aim 3: Characterize the responses of donor and recipient bacterial cells to sublethal exposure of biocidal agents by: a) Investigating mRNA expression of conjugation associated genes after exposure to biocidal agents b) evaluating the cell membrane permeability after biocide exposure using flow cytometry c) assessing oxidative stress induced by sublethal exposure of biocides by measuring ROS production.Research Approach: For mRNA investigation, total RNA will be isolated from stress adapted bacteria cells using RNA isolation kit. The isolated RNA will be transcribed into cDNA using cDNA synthesis kit. The expression of conjugation associated genes, outer membrane protein encoding genes and oxidative stress regulatory genes will be quantified using real-time PCR. The changes in membrane permeability of donor and recipient bacteria after biocide stress adaptation will be evaluated by propidium iodide staining (DNA-intercalating fluorescent dye) followed by flow cytometry. In order to test whether oxidative stress play any role in increasing HGT, intracellular ROS production will be measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA, fluorescent reporter dye) and a microplate reader.Data Analysis: The data will be analyzed using Student's t-test, and means ± standard error of means (SEM) will be considered significantly different at p ≤ 0.05. All statistical analysis will be done using Prism 8 (GraphPad Software Inc., San Diego, CA).Pitfalls and Limitations: This area of research is critical for controlling AR dissemination in livestock production environments. Even though the results from this research would open a profound and intriguing question regarding the impact of current disinfection protocols on AR transmission, our study is limited to few of the environmental contaminants. Hence, a detailed delineation of complex interactions between multiple environmental components such as presence of multispecies bacteria, and presence of organic materials is needed to provide a more cohesive understanding of the persistence and transmission of AR in farm environment.