Inhibitory effects of p-coumaric acid against avian pathogenic Escherichia coli O2:K1 in vitro and in vivo.

Avian colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is a severe bacterial disease in the poultry industry. With the increasing problem of antibiotic resistance, safe and effective alternatives are urgently needed. p-Coumaric acid (pCA) is a natural phenolic compound with reported antimicrobial activity; however, systematic data regarding its effects against APEC O2:K1 both in vitro and in vivo remain limited. In this study, the minimum inhibitory concentration (MIC) of pCA against APEC O2:K1 was determined by the broth microdilution method. The in vitro antibacterial activity was evaluated by growth curve analysis, crystal violet staining, drug-containing plate diffusion assay, nucleic acid and protein leakage tests, and the effect on virulence gene expression was examined by quantitative real-time PCR (qPCR). An infection model was established by intraperitoneal injection of APEC O2:K1 into 14-day-old chicks. The chicks were randomly divided into blank control group, model group, low, medium, and high-dose pCA groups (50, 100, and 200 mg/kg), and enrofloxacin positive control group (100 mg/kg). Chicks received the respective treatments via oral gavage once daily for three consecutive days post-challenge. Survival rate was observed for seven days, and organ indices, bacterial loads, serum inflammatory cytokines, and oxidative stress markers were measured. Heart and liver tissues were collected for histopathological examination. The MIC of pCA against APEC O2:K1 was 3.125 mg/mL. pCA inhibited bacterial growth in a concentration-dependent manner. At 1/2 MIC, the inhibition rates of biofilm formation and colony spreading were 71.4% and 47.6%, respectively. pCA significantly disrupted bacterial cell membrane integrity, leading to leakage of intracellular nucleic acids and proteins. At sub-inhibitory concentrations, pCA significantly down-regulated the expression of virulence genes, including luxS, fimH, ompA, iss, iutA, tsh, and ibeA. In the in vivo experiment, the seven-day survival rate of the model group was 45.83%, whereas that of the medium-dose pCA group (100 mg/kg) was 83.33% (P = 0.007). Compared with the model group, the medium-dose pCA group showed significantly reduced heart, liver, lung, and spleen indices, and recovered thymus and bursa of Fabricius indices. Bacterial loads in blood and liver were significantly decreased. Serum levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), endotoxin LPS, and infection marker PCT were significantly reduced, while the anti-inflammatory cytokine IL-10 level was elevated. Immunoglobulin IgY and IgM levels returned to near normal ranges. The activities of antioxidant enzymes SOD, CAT, and GSH-Px in serum and liver were significantly increased, and the lipid peroxidation product MDA content was significantly decreased. Histopathological examination confirmed that pericarditis and perihepatitis lesions were markedly alleviated in the pCA treatment group. These results indicate that p-coumaric acid exerts significant therapeutic effects against avian colibacillosis through direct antibacterial activity and regulation of host inflammatory and oxidative stress responses, demonstrating its potential for development as a novel anti-APEC agent.