Location: Poultry Research
Project Number: 6064-13000-014-007-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 15, 2022
End Date: Sep 14, 2024
Objective:
To characterize and quantify bacterial pathogens on surface materials commonly associated within commercial poultry hatcheries.
Approach:
To investigate the occurrence of biofilms, the Cooperator will immerse material coupons in cultures of bacterial pathogens. The plates will be incubated aerobically for various time intervals (48-120 h) at 37°C. Biofilm density on the coupons will be determined using a crystal violet assay and the biofilm cells will be quantified by agar plating. Biofilms will also be visualized using scanning electron microscopy. Expression levels of biofilm specific genes will be determined using RT-PCR. Data will be analyzed by the GLIMMIX procedure of SAS 9.4 at a significance level of 0.05.
Crystal Violet Assay: A modified crystal violet assay will be established based on several previously described protocols (Obe et al., 2021; Ma et al., 2019; Corcoran et al., 2014; Garcia et al., 2012). Coupons of each surface material will be placed in 24-well tissue culture plates with sterile forceps. One milliliter of prepared inoculum (~106 CFU/mL) in TSB and negative controls containing only TSB will be then pipetted into individual wells containing the coupons. Well plates will be incubated at 37°C for 48hrs. After incubation, coupons will be gently rinsed with sterile deionized water to remove loosely attached cells. The coupons will be then placed in new sterile 24 well plates containing 1mL of crystal violet solution and incubated at room temperature for 45min (0.41% w/v dye, AC447570500, ACROS Organics). After incubation, the coupons will be gently rinsed again with sterile deionized water and decolorized using (80% isopropyl alcohol, 20% acetone). The optical density (OD600) will be measured using a spectrophotometer. Based on the O.D. produced by the biofilms, the strains will be classified in the following categories: no biofilm forming ability, weak biofilm former, moderate biofilm former, and strong biofilm former, as previously described (Stepanovic et al., 2000).
Quantification of Biofilm Attached Cells: For the enumeration of biofilms formed on each coupon surface type, coupons will be prepared similarly to the crystal violet assay. The coupons with biofilms will be rinsed with sterile deionized water to remove planktonic cells. Each coupon will be placed in a 12mL snap cap tube containing 10mL of TSB and vortexed for 1 minute to remove the attached biofilm from the coupon surface. Serial dilutions will be then prepared in TSB and the biofilm cells will be enumerated on tryptic soy agar media plates (TSA) by the spread plate method.
Scanning Electron Microscopy: Scanning electron microscopy (SEM) will be performed to verify the attachment of cells and formation of a biofilm extracellular matrix. For each experimental replication, three additional coupons will be prepared for SEM. The coupons will be placed in a new 24 well plate and 1mL of fixative (2.5 glutaraldehyde, 2% paraformaldehyde, 0.1M sodium cacodylate) will be pipetted into each well. The coupons will be incubated at room temperature for 45min in the fixative and then placed in a new sterile 24 well plate to dry. Each coupon will be sputter coated with 30µg of platinum and imaged under SEM.
