Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Population Health
Non Technical Summary
Avian pathogenicEscherichia coli(APEC) is the etiologic agent of colibacillosis, an important cause of morbidity and mortality affecting all facets of the poultry industry worldwide. Though APEC's abilities to persist in the poultry environment, resist the effects of antimicrobial agents, and acquire genetic information from other microbes are well known, the role of biofilms, known to contribute to these traits in related bacteria, is poorly understood for APEC. Here, we propose to fill the gap in knowledge of APEC biofilms in order to better assist producers on how to prevent this disease and the emergence and persistence of resistant APEC in their flocks. In general, biofilms, which are microbial communities that adhere to one another, foster bacterial survival and provide an environment for genetic exchange. Here, we willanalyze the competition for biofilm suppression against mutant and WT; and analyze thecompetition forbiofilm suppression against other very common poultry pathogens. With the reduction of biofilm formation, we anticipate that most of the APEC accumulation and other common poultry pathogen persistence on the poultry farm will be significantly reduced. This project will provide critical information for the development of novel, broad-spectrum, low-cost, practical, and efficacious control against avian pathogens, thereby improving overall bird health and quality.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
Our long-term research goal seeks to understand the biology and population ecology of Avian PathogenicEscherichia coli(APEC), including antibiotic-resistant strains, and risk factors for infection in commercial poultry operations.The long-term goalof our researchto which this project contributes is tounderstand the molecular basis of biofilm formation in APEC and use that knowledge to design effective strategies for colibacillosis control, thereby significantly improving bird health, and welfare and industry performance. In addition, we wish to ascertain what factors during production favor selection of APEC. A potential secondary impact of our work is a reduction in the zoonotic risk of APEC for humans.Biofilms are microbial communities that adhere to one another, ensuring their survival and providing anenvironment for genetic exchange.Resistance of biofilm-forming bacteria to antimicrobial drugs and detergents complicates biofilm eliminationduring cleaning and disinfectionon poultry farms and in industrial settings.To date, little information has been available on the genetic determinants of biofilm production by APEC.Our primary hypotheses are: (i) Biofilms are critical to APEC's environmental persistence and transfer of large plasmids to avirulentE. colistrains conferring upon them virulence and multidrug resistance. (ii)Resistance of biofilm-forming bacteria to antimicrobial drugs and detergents complicates biofilm eliminationduring cleaning and disinfectionon poultry farms and in industrial settingsand facilitates the widespread distribution of APEC.Ourmain goalwill be toidentifynew targets forcompetition for biofilm suppression:Oursupporting objectiveswill be to:Assess the effects of competition on APEC and select poultry pathogens during growth,Assess the effects of competition on APEC and select poultry pathogens on biofilm formationAssess inhibition of APEC and select poultry pathogens on biofilm formation using inhibitory supernatant content (no cell-cell contact)Identify potential candidate agents in supernatant content.To test these hypotheses, ourapproachwill be to use thebiofilm screening that was already performed by our labthat allowed us to identify over 2000 candidate mutants for further investigation.Herethe candidate mutants will be grown in combination with the WT strain to evaluate potential inhibitory growth of the mutant strain against the WT to produce biofilm. In order to test if the use of these new candidates can affect other pathogens, we will also perform inhibition growth usingplanktonic bacterialcells and biofilm suppression against the most common poultry pathogens. We plan to test Gram positives including: biofilm formingClostridium,Staphylococus,Streptococcus,ListeriaandEnterococcus. For the Gram negatives, we expect to test: biofilm formingSalmonella,Campylobacter,Pasteurella,Ornithobacterium,Riemerella anatipestiferandBordetella spp.
Project Methods
Our approach to our hypothesis follows.Specific Aim 1: Assess the effects of competition on APEC and select poultry pathogens during growthTo identify the candidates for biofilm suppression in poultry, we select approximately 100 hypothetical genes that are present in over 80% of the APEC collection, and present in less than 20 % of the avian fecal (AFEC) collection for further analysis.For APEC: The candidate mutants will be grown in combination with the wild-type APEC strain in order to evaluate the potential inhibition of growth of the mutant strain. Competitivegrowth of the WT and mutant strains will be carried out at 37°C in minimal medium M9. Briefly, strains will be incubated overnight in LB with chloramphenicol (10 µg/mL) at37°C. Next, OD600of the cultures will be measured and cultures diluted to an OD600 of 0.05 in M9. Cultures will be incubated at37°C with shaking at 220 rpm, and OD600growth measured every 30 minutes. To measure OD600, 300 µL of culture will be dispensed in a well of a 96 well plate, and the absorbance read using an ELX 808 Ultra microplate reader (Bio-Tek instruments). Absorbance data will be plotted against time to build the growth curves.The co-culture will be performed using the procedure as described, but every 30 min the co-culture will be sampled and plated on LB media with or without Kanamycin (selective for mutant), the plates will be incubated at 37°C overnight and counted and data plotted against time to build the growth curves.For Select Poultry Pathogens: The candidate mutants will be grown in combination with the pathogenic strains indicated and one of the candidate mutants in order to evaluate the potential of the mutant to inhibit the growth of the test strains.The co-culture will be performed using the same approach as described previously. Briefly, every 30 min the co-culture will be plated on LB agar (or selective media for the target poultry pathogen) with or without Kanamycin (selective for the mutant, or other specific antimicrobials to kill the pathogen strain), the plates will be incubated at 37°C or 42°C overnight and plates will be counted and data will be plotted against time to build the growth curves.Specific Aim 2: Assess the effects of competition on APEC and select poultry pathogens on biofilm formation.For APEC:The ability of the APEC WT x mutants to form biofilm will be screened according to Skyberget al.,2006(18). Mutants and APEC WT will be incubated overnight in LB broth at 37°C. They will be diluted 1:100 and a 1:1 mixture of the WT and tested mutant in M63 and loaded into 6 wells of a 48 well flat bottom plate. Following incubation at 37°C for 48 h, the media will be poured off and the plates will be washed with sterile MiliQ water and biofilm will be scraped in order to resuspend the bacterial cells. Resuspended bacteria will be serially diluted and plated on LB media with or without kanamycin (selective for mutant), the plates will be incubated at 37°C overnight and plates will be counted and data will be plotted. The difference between the bacterial growth in LB with or without kanamycin indicates the ability of the mutant to reduce the APEC WT biofilm.For Select Poultry Pathogens:The ability of the pathogen strain x mutants to form biofilm will be screened according to Skyberget al.,2006(18)as described previously. Briefly, mutants and pathogens will be incubated overnight in broth media at 37°C. They will be diluted 1:100 and mix at a 1:1 concentration of pathogen and tested mutant in minimal media and loaded into 6 wells of a 48 well flat bottom plate. Following incubation at 37°C for 48 h, the media will be poured off and the plates will be washed with sterile MiliQ water and biofilm will be scraped for resuspension. The resuspended bacteria will be serially diluted and plated on LB media (or selective media for the target poultry pathogen) with or without kanamycin (selective for the mutant, or other specific antimicrobials to kill the pathogen strain), the plates will be incubated at 37°C overnight and plates will be counted and data will be plotted. The difference between the bacterial growth in LB with or without kanamycin indicates the ability of the mutant to reduce the pathogen's ability to form biofilm. These data will be compared to the biofilm formation of the pathogen strain by itself also.Specific Aim 3: Assess inhibition of APEC and select poultry pathogens on biofilm formation using inhibitory supernatant content (no cell-cell contact)An alternative way to measure the biofilm suppression is to perform biofilm growth of the WT using the supernatant broth of the biofilm formed by the mutant (biofilm concentrate media).Briefly, mutants will be incubated overnight in LB broth at 37°C and diluted 1:100 in M63, and loaded into 6 wells of a 48 well flat bottom plate. Following incubation at 37°C for 48 h, the media will be filter sterilized (0.22um filter) and used as a media for biofilm formation of the APEC WT strains and for the other pathogens.For APEC:WT will be incubated overnight in LB broth at 37°C and diluted 1:100 in mutant filtered media and loaded into 7 wells of a 96 well flat bottom plate. The media will be poured off and the plates will be washed with sterile MiliQ water. Plates will be stained with 500 ul of 0.1% Crystal Violet for 30 min, washed with MiliQ water, and air-dried for 1 h. 500 ul of an 80:20 solution of ethanol and acetone will be added to solubilize the cells. 150 ul of the resulting solution will be transferred to a new microtiter plate, and the biofilm density determined by measuring the OD600.For Select Poultry Pathogens:Each pathogen tested will be incubated overnight in broth media at 37°C and diluted 1:100 in mutant filtered media (biofilm concentrate media) and loaded into 7 wells of a 96 well flat bottom plate. The media will be poured off and the plates will be washed with sterile MiliQ water. Plates will be stained with 500 ul of 0.1% Crystal Violet for 30 min, washed with MiliQ water, and air-dried for 1 h. 500 ul of an 80:20 solution of ethanol and acetone will be added to solubilize the cells. 150 ul of the resulting solution will be transferred to a new microtiter plate, and the biofilm density will be determined by measuring the OD600.This specific assay will allow us to identify if the mutant is secreting some type of biofilm inhibitory factor that affects other bacterial pathogens.Specific Aim 4: Identify potential candidate agents in supernatant content.In order to evaluate the presence of the secretion inhibition molecules produced by the biofilm suppression candidates, the candidates will be inoculated as inAim 3, and we will use here the supernatant broth of the biofilm formed by the mutant (biofilm concentrate media). Biofilm supernatant containing secreted proteins and sugars will be filtered through a 0.22 μm pore filter. One fraction will be TCA protein precipitated and proteins will be subjected to polyacrylamide gel electrophoresis and submitted to the UGA protein analysis facility that will process the Protein ID by LC-MS/MS (60 min/run) from gel digestion. Another fraction will be quantified to determine the total amounts of sugars by phenol-sulfuric acid methods, using glucose as a standard. Extract active fractions will be purified and submitted to either sizing chromatography or composition analysis as described previously(24, 35)at UGA Complex Carbohydrate Research Center.The APEC WT (biofilm concentrate media) will be used for content comparison evaluation with candidate mutants.