Unlocking the Antibacterial Potential of <i>Eucalyptus citriodora</i> Hook: Essential Oil on Some Bacterial Pathogens with a Focus on Antiadhesive, Antibiofilm, and Mechanisms of Action Against <i>Pseudomonas aeruginosa</i>

<i>Eucalyptus citriodora</i> (lemon eucalyptus) is a medicinal plant commonly used as an antiseptic agent to prevent infection and promote healing. The aim of this exploration was to investigate for the first time the antibacterial mode of action of a gas chromatography-mass spectrometry chemically characterized <i>E. citriodora</i> essential oil (EO) obtained by microwave-assisted hydrodistillation. First, the chemical investigation revealed citronellal (30.67%) and citronellol (19.14%) as the major components. <i>E. citriodora</i> EO represented remarkable antibacterial properties, as demonstrated by significant zone of inhibition in the disc-diffusion test against all tested bacterial pathogens, including <i>Listeria innocua</i> ATCC 33090, <i>L. monocytogenes</i> ATCC 13932, <i>Proteus mirabilis</i> ATCC 25933, <i>Salmonella typhimurium</i> ATCC 700408, and <i>Pseudomonas aeruginosa</i> ATCC 27853. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values confirmed these findings showing antibacterial efficacy at low concentrations ranging 5.75-92 µg/mL for MICs and 11.5-184 µg/mL for MBCs. These results were comparable with gentamicin. The MIC_index revealed that <i>E. citriodora</i> EO has bactericidal effects. We evaluated the antibacterial properties of <i>E. citriodora</i> EO and determined its mode of action using advanced tests with <i>P. aeruginosa</i> as a typical pathogen. The time-kill test revealed dose-dependent inhibitory effects on the cell proliferation of <i>P. aeruginosa</i>, affecting its membrane permeability based on the time and concentration of the EO. <i>E. citriodora</i> EO also caused damage to the cell membrane, resulting in leakage of cellular constituents, including RNA and DNA. Anti-quorum sensing activity was tested by inhibiting biofilm formation, showing that <i>E. citriodora</i> EO effectively inhibited biofilm formation. The UV-visible investigation on the release of cellular materials absorbed at 260 nm showed significant leakage of cytoplasmic substances, including proteins, DNA, and RNA, indicating devastation to bacterial cell membrane integrity. In addition, the use of <i>E. citriodora</i> EO significantly inhibited <i>P. aeruginosa</i> adhesion on 304 L stainless-steel surface, making it more suitable for applications requiring better wetting ability and surface interactions, particularly in food production. [Figure: see text].