| Congresso Brasileiro de Microbiologia 2023 | Resumo: 410-1 | ||||
Resumo:Mastitis is a harmful inflammatory condition in dairy cows, leading to health problems and financial losses. Chronic mastitis and antibiotic resistance are heavily influenced by biofilms. The efficacy of antibiotic treatments varies, and overuse can promote resistance, therefore, there is a pressing need to explore new alternatives. This research aims to evaluate the ability of the rumen derived antimicrobial peptide AMP_1 to hinder adhesion and invasion of biofilm-forming mastitis-causing pathogens, such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus chromogenes. Biofilm formation ability of 45 bovine mastitis isolates was assessed using the crystal violet assay on 0.4% glucose enriched BHI media after 48 hours. Biofilm production was measured at optical density of 570 nm (OD570). The most proficient biofilm producers (n=7) were chosen as targets. Adhesion and invasion assays were conducted on bovine mammary alveolar cells after preincubating 1.5 x 105 cells.ml-1 with AMP_1 at 128 μg.ml-1 for 30 minutes in antibiotic-free Dulbecco’s Modified Eagle’s Medium enriched with 10% Fetal Bovine Serum. Mastitis-causing bacteria were introduced at 105 and 107 cells.ml-1, resulting in multiplicities of infection (MOIs) of 1:1 and 100:1, respectively. Controls with no antimicrobial addition were included. Cocultures were incubated at 37°C with 5% CO2 for 3 hours. Invasion assays involved treatment with gentamicin (100 μg.ml-1) to remove extracellular bacteria after 3 hours incubation, followed by cell lysis using Triton X-100 (0.1% vol/vol). Adhered bacteria and cell lysates were quantified using dilution plate technique. Fluorescence images were taken using live/dead bacterial stain kit. Data normality was assessed with the Shapiro-Wilk test, and pairwise inferences were made using multiple paired t-tests. It was identified two strains each of E. coli, K. pneumoniae, S. aureus, and one strain of S. chromogenes as the highest biofilm producers. These strains exhibited OD570 readings ranging from 0.82 to 0.46 and were selected as representative targets for prevalent mastitis-associated species. The mean log10 values of CFU.ml-1 was analyzed for all seven strains and found a significant reduction in both adhesion and invasion rates at both infection concentrations (p < 0.05). There was a 100-fold decrease in invasion at both MOIs and adhesion at MOI 100:1. Additionally, there was an 10,000-fold reduction in adhesion at MOI of 1:1. Upon analyzing the data for each strain separately, AMP_1 demonstrated effectiveness in reducing the number of K. pneumoniae strain 158 cells, the highest biofilm producer, in all scenarios tested. There was no significant difference observed between the treatment and control groups for both invasion and adhesion of S. chromogenes strain 196 and E. coli strain 219 at MOI 100:1. AMP_1 demonstrated itself to be an effective treatment capable of significantly reducing infection by mastitis pathogens on mammalian gland epithelial cells. Fluorescence microscopy revealed red dead adhered bacterial cells surrounding green live eukaryotic cells. This finding highlights the potential of AMP_1 as a potent and safe alternative option for mastitis prevention and treatment. Further investigations into the efficacy of combinatorial therapies will be conducted through in vitro studies. Palavras-chave: antimicrobial peptide, bovine mastitis, adhesion and invasion, biofilm Agência de fomento:DAIRY INNOVATION HUB, FAPEMIG, CAPES, CNPq | |||||