Innovative essential oil formulations for in vitro inhibition of Biofilm-Forming Extended-Spectrum β-Lactamase-Producing Escherichia coli isolated from canine infectious diarrhea.

BACKGROUND: The emergence of bacteria producing extended-spectrum β-lactamases (ESBLs) has presented a substantial challenge to veterinary and human medicine. ESBL-producing Escherichia coli exhibits biofilm-forming capabilities that enhance its persistence in domestic animals, contribute to chronic infectious diarrhea, and increase the risk of treatment failure. This study investigated ESBL-producing E. coli in dogs and evaluated the antimicrobial and antibiofilm activities of NHK-EO, an innovative essential oil formulation derived from Coleus amboinicus (N), Ocimum basilicum (H), and Ocimum tenuiflorum (K). METHODS: The chemical composition of NHK-EO was analyzed using gas chromatography-mass spectrometry (GC-MS). Fifty-six rectal swab samples were randomly collected from dogs aged 3 months to 10 years, including 30 clinically healthy and 26 diarrheic animals of both sexes. Bacterial isolation and identification were conducted using conventional biochemical tests and the VITEK automated system. ESBL production was confirmed through genotypic detection of blaCTX-M using polymerase chain reaction, and antimicrobial susceptibility was evaluated to determine multidrug resistance profiles. Twenty isolates (10 from healthy and 10 from diarrheic dogs) were selected for further analyses. The antimicrobial activity of NHK-EO was assessed using the broth microdilution method, and biofilm formation and inhibition were evaluated using crystal violet staining. Data are presented as mean ± standard deviation from three independent experiments. RESULTS: GC-MS analysis of NHK-EO revealed three major constituents: carvacrol (16.37%), eugenol (15.04%), and linalool (13.97%). In total, 78 Gram-negative bacterial isolates were identified, with E. coli being the predominant species (56 isolates, 71.79%). Among these, four blaCTX-M-positive E. coli isolates (three from healthy dogs and one from a diarrheic dog) exhibited strong biofilm-forming ability. The minimum inhibitory concentration (MIC) of NHK-EO against the isolates was 1024-2048 µg/mL. The MIC of NHK-EO against the four blaCTX-M-positive E. coli isolates was 1024 µg/mL. NHK-EO at 1/4× MIC (256 µg/mL) exerted significant inhibitory effects on both prebiofilm and postbiofilm formation, as confirmed using scanning electron microscopy. Treated samples exhibited notably reduced biofilm mass and decreased bacterial density compared with untreated controls. CONCLUSIONS: NHK-EO possesses significant antimicrobial and antibiofilm activities against E. coli, including ESBL-producing strains, indicating its potential as an alternative therapeutic agent to mitigate antimicrobial resistance. Further research is warranted to explore its clinical applications in veterinary and human medicine.